RELATED APPLICATIONS
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This application is a continuation of U.S. patent application Ser. No. 10/241,220, filed Sep. 11, 2002, which claims priority to U.S. Provisional Patent Application No. 60/323,268, filed Sep. 18, 2001, 60/339,227, filed Oct. 19, 2001, 60/336,827, filed Nov. 7, 2001, 60/331,906, filed Nov. 20, 2001, 60/345,444, filed Jan. 2, 2002, 60/369,724, filed Apr. 3, 2002 and 60/404,809, filed Aug. 19, 2002, the entirety of which are incorporated herein by reference.[0001]
FIELD OF THE INVENTION
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The present invention is directed to compositions of matter useful for the diagnosis and treatment of tumor in mammals and to methods of using those compositions of matter for the same. [0002]
BACKGROUND OF THE INVENTION
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Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., C A [0003] Cancel J. Clin. 43:7(1993)). Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
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In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise membrane-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such membrane-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies. In this regard, it is noted that antibody-based therapy has proved very effective in the treatment of certain cancers. For example, HERCEPTIN® and RITUXAN® (both from Genentech Inc., South San Francisco, Calif.) are antibodies that have been used successfully to treat breast cancer and non-Hodgkin's lymphoma, respectively. More specifically, HERCEPTIN® is a recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 (HER2) proto-oncogene. HER2 protein overexpression is observed in 25-30% of primary breast cancers. RITUXAN® is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Both these antibodies are recombinantly produced in CHO cells. [0004]
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In other attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify (1) non-membrane-associated polypeptides that are specifically produced by one or more particular type(s) of cancer cell(s) as compared to by one or more particular type(s) of non-cancerous normal cell(s), (2) polypeptides that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous cell(s), or (3) polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both the cancerous and non-cancerous state (e.g., normal prostate and prostate tumor tissue). Such polypeptides may remain intracellularly located or may be secreted by the cancer cell. Moreover, such polypeptides may be expressed not by the cancer cell itself, but rather by cells which produce and/or secrete polypeptides having a potentiating or growth-enhancing effect on cancer cells. Such secreted polypeptides are often proteins that provide cancer cells with a growth advantage over normal cells and include such things as, for example, angiogenic factors, cellular adhesion factors, growth factors, and the like. Identification of antagonists of such non-membrane associated polypeptides would be expected to serve as effective therapeutic agents for the treatment of such cancers. Furthermore, identification of the expression pattern of such polypeptides would be useful for the diagnosis of particular cancers in mammals. [0005]
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Despite the above identified advances in mammalian cancer therapy, there is a great need for additional diagnostic and therapeutic agents capable of detecting the presence of tumor in a mammal and for effectively inhibiting neoplastic cell growth, respectively. Accordingly, it is an objective of the present invention to identify: (1) cell membrane-associated polypeptides that are more abundantly expressed on one or more type(s) of cancer cell(s) as compared to on normal cells or on other different cancer cells, (2) non-membrane-associated polypeptides that are specifically produced by one or more particular type(s) of cancer cell(s) (or by other cells that produce polypeptides having a potentiating effect on the growth of cancer cells) as compared to by one or more particular type(s) of non-cancerous normal cell(s), (3) non-membrane-associated polypeptides that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous cell(s), or (4) polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both a cancerous and non-cancerous state (e.g., normal prostate and prostate tumor tissue), and to use those polypeptides, and their encoding nucleic acids, to produce compositions of matter useful in the therapeutic treatment and diagnostic detection of cancer in mammals. It is also an objective of the present invention to identify cell membrane-associated, secreted or intracellular polypeptides whose expression is limited to a single or very limited number of tissues, and to use those polypeptides, and their encoding nucleic acids, to produce compositions of matter useful in the therapeutic treatment and diagnostic detection of cancer in mammals. [0006]
SUMMARY OF THE INVENTION
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A. Embodiments [0007]
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In the present specification, Applicants describe for the first time the identification of various cellular polypeptides (and their encoding nucleic acids or fragments thereof) which are expressed to a greater degree on the surface of or by one or more types of cancer cell(s) as compared to on the surface of or by one or more types of normal non-cancer cells. Alternatively, such polypeptides are expressed by cells which produce and/or secrete polypeptides having a potentiating or growth-enhancing effect on cancer cells. Again alternatively, such polypeptides may not be overexpressed by tumor cells as compared to normal cells of the same tissue type, but rather may be specifically expressed by both tumor cells and normal cells of only a single or very limited number of tissue types (preferably tissues which are not essential for life, e.g., prostate, etc.). All of the above polypeptides are herein referred to as Tumor-associated Antigenic Target polypeptides (“TAT” polypeptides) and are expected to serve as effective targets for cancer therapy and diagnosis in mammals. [0008]
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Accordingly, in one embodiment of the present invention, the invention provides an isolated nucleic acid molecule having a nucleotide sequence that encodes a tumor-associated antigenic target polypeptide or fragment thereof (a “TAT” polypeptide). [0009]
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In certain aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule encoding a full-length TAT polypeptide having an amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a). [0010]
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In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule comprising the coding sequence of a full-length TAT polypeptide cDNA as disclosed herein, the coding sequence of a TAT polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a). [0011]
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In further aspects, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule that encodes the same mature polypeptide encoded by the full-length coding region of any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a). [0012]
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Another aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a TAT polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide(s) are disclosed herein. Therefore, soluble extracellular domains of the herein described TAT polypeptides are contemplated. [0013]
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In other aspects, the present invention is directed to isolated nucleic acid molecules which hybridize to (a) a nucleotide sequence encoding a TAT polypeptide having a full-length amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the nucleotide sequence of (a). In this regard, an embodiment of the present invention is directed to fragments of a full-length TAT polypeptide coding sequence, or the complement thereof, as disclosed herein, that may find use as, for example, hybridization probes useful as, for example, diagnostic probes, antisense oligonucleotide probes, or for encoding fragments of a full-length TAT polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-TAT polypeptide antibody, a TAT binding oligopeptide or other small organic molecule that binds to a TAT polypeptide. Such nucleic acid fragments are usually at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term “about” means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a TAT polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the TAT polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which TAT polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such novel fragments of TAT polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the TAT polypeptide fragments encoded by these nucleotide molecule fragments, preferably those TAT polypeptide fragments that comprise a binding site for an anti-TAT antibody, a TAT binding oligopeptide or other small organic molecule that binds to a TAT polypeptide. [0014]
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In another embodiment, the invention provides isolated TAT polypeptides encoded by any of the isolated nucleic acid sequences hereinabove identified. [0015]
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In a certain aspect, the invention concerns an isolated TAT polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, to a TAT polypeptide having a full-length amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide protein, with or without the signal peptide, as disclosed herein, an amino acid sequence encoded by any of the nucleic acid sequences disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein. [0016]
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In a further aspect, the invention concerns an isolated TAT polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein. [0017]
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In a specific aspect, the invention provides an isolated TAT polypeptide without the N-terminal signal sequence and/or without the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide and recovering the TAT polypeptide from the cell culture. [0018]
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Another aspect of the invention provides an isolated TAT polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide and recovering the TAT polypeptide from the cell culture. [0019]
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In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides. Host cells comprising any such vector are also provided. By way of example, the host cells may be CHO cells, [0020] E. coli cells, or yeast cells. A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
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In other embodiments, the invention provides isolated chimeric polypeptides comprising any of the herein described TAT polypeptides fused to a heterologous (non-TAT) polypeptide. Example of such chimeric molecules comprise any of the herein described TAT polypeptides fused to a heterologous polypeptide such as, for example, an epitope tag sequence or a Fc region of an immunoglobulin. [0021]
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In another embodiment, the invention provides an antibody which binds, preferably specifically, to any of the above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single-chain antibody or antibody that competitively inhibits the binding of an anti-TAT polypeptide antibody to its respective antigenic epitope. Antibodies of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies of the present invention may optionally be produced in CHO cells or bacterial cells and preferably induce death of a cell to which they bind. For diagnostic purposes, the antibodies of the present invention may be detectably labeled, attached to a solid support, or the like. [0022]
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In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described antibodies. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells, [0023] E. coli cells, or yeast cells. A process for producing any of the herein described antibodies is further provided and comprises culturing host cells under conditions suitable for expression of the desired antibody and recovering the desired antibody from the cell culture.
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In another embodiment, the invention provides oligopeptides (“TAT binding oligopeptides”) which bind, preferably specifically, to any of the above or below described TAT polypeptides. Optionally, the TAT binding oligopeptides of the present invention may be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The TAT binding oligopeptides of the present invention may optionally be produced in CHO cells or bacterial cells and preferably induce death of a cell to which they bind. For diagnostic purposes, the TAT binding oligopeptides of the present invention may be detectably labeled, attached to a solid support, or the like. [0024]
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In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described TAT binding oligopeptides. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells, [0025] E. coli cells, or yeast cells. A process for producing any of the herein described TAT binding oligopeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired oligopeptide and recovering the desired oligopeptide from the cell culture.
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In another embodiment, the invention provides small organic molecules (“TAT binding organic molecules”) which bind, preferably specifically, to any of the above or below described TAT polypeptides. Optionally, the TAT binding organic molecules of the present invention may be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The TAT binding organic molecules of the present invention preferably induce death of a cell to which they bind. For diagnostic purposes, the TAT binding organic molecules of the present invention may be detectably labeled, attached to a solid support, or the like. [0026]
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In a still further embodiment, the invention concerns a composition of matter comprising a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier. [0027]
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In yet another embodiment, the invention concerns an article of manufacture comprising a container and a composition of matter contained within the container, wherein the composition of matter may comprise a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT bindinng oligopeptide as described herein, or a TAT binding organic molecule as described herein. The article may further optionally comprise a label affixed to the container, or a package insert included with the container, that refers to the use of the composition of matter for the therapeutic treatment or diagnostic detection of a tumor. [0028]
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Another embodiment of the present invention is directed to the use of a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT polypeptide antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein, for the preparation of a medicament useful in the treatment of a condition which is responsive to the TAT polypeptide, chimeric TAT polypeptide, anti-TAT polypeptide antibody, TAT binding oligopeptide, or TAT binding organic molecule. [0029]
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B. Additional Embodiments [0030]
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Another embodiment of the present invention is directed to a method for inhibiting the growth of a cell that expresses a TAT polypeptide, wherein the method comprises contacting the cell with an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, and wherein the binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes inhibition of the growth of the cell expressing the TAT polypeptide. In preferred embodiments, the cell is a cancer cell and binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes death of the cell expressing the TAT polypeptide. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and TAT binding oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells. [0031]
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Yet another embodiment of the present invention is directed to a method of therapeutically treating a mammal having a cancerous tumor comprising cells that express a TAT polypeptide, wherein the method comprises administering to the mammal a therapeutically effective amount of an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby resulting in the effective therapeutic treatment of the tumor. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells. [0032]
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Yet another embodiment of the present invention is directed to a method of determining the presence of a TAT polypeptide in a sample suspected of containing the TAT polypeptide, wherein the method comprises exposing the sample to an antibody, oligopeptide or small organic molecule that binds to the TAT polypeptide and determining binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide in the sample, wherein the presence of such binding is indicative of the presence of the TAT polypeptide in the sample. Optionally, the sample may contain cells (which may be cancer cells) suspected of expressing the TAT polypeptide. The antibody, TAT binding oligopeptide or TAT binding organic molecule employed in the method may optionally be detectably labeled, attached to a solid support, or the like. [0033]
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A further embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a mammal, wherein the method comprises detecting the level of expression of a gene encoding a TAT polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal non-cancerous cells of the same tissue origin or type, wherein a higher level of expression of the TAT polypeptide in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test sample was obtained. [0034]
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Another embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a mammal, wherein the method comprises (a) contacting a test sample comprising tissue cells obtained from the mammal with an antibody, oligopeptide or small organic molecule that binds to a TAT polypeptide and (b) detecting the formation of a complex between the antibody, oligopeptide or small organic molecule and the TAT polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in the mammal. Optionally, the antibody, TAT binding oligopeptide or TAT binding organic molecule employed is detectably labeled, attached to a solid support, or the like, and/or the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor. [0035]
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Yet another embodiment of the present invention is directed to a method for treating or preventing a cell proliferative disorder associated with altered, preferably increased, expression or activity of a TAT polypeptide, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a TAT polypeptide. Preferably, the cell proliferative disorder is cancer and the antagonist of the TAT polypeptide is an anti-TAT polypeptide antibody, TAT binding oligopeptide, TAT binding organic molecule or antisense oligonucleotide. Effective treatment or prevention of the cell proliferative disorder may be a result of direct killing or growth inhibition of cells that express a TAT polypeptide or by antagonizing the cell growth potentiating activity of a TAT polypeptide. [0036]
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Yet another embodiment of the present invention is directed to a method of binding an antibody, oligopeptide or small organic molecule to a cell that expresses a TAT polypeptide, wherein the method comprises contacting a cell that expresses a TAT polypeptide with said antibody, oligopeptide or small organic molecule under conditions which are suitable for binding of the antibody, oligopeptide or small organic molecule to said TAT polypeptide and allowing binding therebetween. [0037]
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Other embodiments of the present invention are directed to the use of (a) a TAT polypeptide, (b) a nucleic acid encoding a TAT polypeptide or a vector or host cell comprising that nucleic acid, (c) an anti-TAT polypeptide antibody, (d) a TAT-binding oligopeptide, or (e) a TAT-binding small organic molecule in the preparation of a medicament useful for (i) the therapeutic treatment or diagnostic detection of a cancer or tumor, or (ii) the therapeutic treatment or prevention of a cell proliferative disorder. [0038]
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Another embodiment of the present invention is directed to a method for inhibiting the growth of a cancer cell, wherein the growth of said cancer cell is at least in part dependent upon the growth potentiating effect(s) of a TAT polypeptide (wherein the TAT polypeptide may be expressed either by the cancer cell itself or a cell that produces polypeptide(s) that have a growth potentiating effect on cancer cells), wherein the method comprises contacting the TAT polypeptide with an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth-potentiating activity of the TAT polypeptide and, in turn, inhibiting the growth of the cancer cell. Preferably the growth of the cancer cell is completely inhibited. Even more preferably, binding of the antibody, oligopeptide or small organic molecule to the TAT polypeptide induces the death of the cancer cell. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and TAT binding oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells. [0039]
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Yet another embodiment of the present invention is directed to a method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor is at least in part dependent upon the growth potentiating effect(s) of a TAT polypeptide, wherein the method comprises administering to the mammal a therapeutically effective amount of an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth potentiating activity of said TAT polypeptide and resulting in the effective therapeutic treatment of the tumor. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells. [0040]
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C. Further Additional Embodiments [0041]
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In yet further embodiments, the invention is directed to the following set of potential claims for this application: [0042]
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1. Isolated nucleic acid having a nucleotide sequence that has at least 80% nucleic acid sequence identity to: [0043]
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(a) a DNA molecule encoding the amino acid sequence shown in any one of FIGS. 57-112, [0044] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
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(b) a DNA molecule encoding the amino acid sequence shown in any one of FIGS. 57-112, [0045] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
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(c) a DNA molecule encoding an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0046] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
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(d) a DNA molecule encoding an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0047] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
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(e) the nucleotide sequence shown in any one of FIGS. 1-56, [0048] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
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(f) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0049] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
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(g) the complement of (a), (b), (c), (d), (e) or (f). [0050]
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2. Isolated nucleic acid having: [0051]
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(a) a nucleotide sequence that encodes the amino acid sequence shown in any one of FIGS. 57-112, [0052] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
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(b) a nucleotide sequence that encodes the amino acid sequence shown in any one of FIGS. 57-112, [0053] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
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(c) a nucleotide sequence that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0054] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
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(d) a nucleotide sequence that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0055] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
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(e) the nucleotide sequence shown in any one of FIGS. 1-56, [0056] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
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(f) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0057] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
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(g) the complement of (a), (b), (c), (d), (e) or (f). [0058]
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3. Isolated nucleic acid that hybridizes to: [0059]
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(a) a nucleic acid that encodes the amino acid sequence shown in any one of FIGS. 57-112, [0060] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
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(b) a nucleic acid that encodes the amino acid sequence shown in any one of FIGS. 57-112, [0061] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
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(c) a nucleic acid that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0062] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
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(d) a nucleic acid that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0063] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
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(e) the nucleotide sequence shown in any one of FIGS. 1-56, [0064] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
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(f) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0065] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
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(g) the complement of (a), (b), (c), (d), (e) or (f). [0066]
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4. The nucleic acid of [0067] claim 3, wherein the hybridization occurs under stringent conditions.
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5. The nucleic acid of [0068] claim 3 which is at least about 5 nucleotides in length.
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6. An expression vector comprising the nucleic acid of [0069] claim 1, 2 or 3.
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7. The expression vector of claim [0070] 6, wherein said nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.
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8. A host cell comprising the expression vector of [0071] claim 7.
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9. The host cell of claim [0072] 8 which is a CHO cell, an E. coli cell or a yeast cell.
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10. A process for producing a polypeptide comprising culturing the host cell of claim [0073] 8 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.
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11. An isolated polypeptide having at least 80% amino acid sequence identity to: [0074]
-
(a) the polypeptide shown in any one of FIGS. 57-112, [0075] 114, 116, 118 or 120 (SEQ ID NOS:57-112,114, 116, 118 or 120);
-
(b) the polypeptide shown in any one of FIGS. 57-112, [0076] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0077] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0078] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0079] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0080] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
12. An isolated polypeptide having: [0081]
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0082] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0083] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0084] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0085] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0086] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0087] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
13. A chimeric polypeptide comprising the polypeptide of claim [0088] 11 or 12 fused to a heterologous polypeptide.
-
14. The chimeric polypeptide of claim [0089] 13, wherein said heterologous polypeptide is an epitope tag sequence or an Fc region of an immunoglobulin.
-
15. An isolated antibody that binds to a polypeptide having at least 80% amino acid sequence identity to: [0090]
-
(a) the polypeptide shown in any one of FIGS. 57-112, [0091] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in any one of FIGS. 57-112, [0092] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0093] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0094] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0095] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0096] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
16. An isolated antibody that binds to a polypeptide having: [0097]
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0098] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0099] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0100] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0101] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0102] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0103] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
17. The antibody of claim [0104] 15 or 16 which is a monoclonal antibody.
-
18. The antibody of claim [0105] 15 or 16 which is an antibody fragment.
-
19. The antibody of claim [0106] 15 or 16 which is a chimeric or a humanized antibody.
-
20. The antibody of claim [0107] 15 or 16 which is conjugated to a growth inhibitory agent.
-
21. The antibody of claim [0108] 15 or 16 which is conjugated to a cytotoxic agent.
-
22. The antibody of claim [0109] 21, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
-
23. The antibody of claim [0110] 21, wherein the cytotoxic agent is a toxin.
-
24. The antibody of claim [0111] 23, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
-
25. The antibody of claim [0112] 23, wherein the toxin is a maytansinoid.
-
26. The antibody of claim [0113] 15 or 16 which is produced in bacteria.
-
27. The antibody of claim [0114] 15 or 16 which is produced in CHO cells.
-
28. The antibody of claim [0115] 15 or 16 which induces death of a cell to which it binds.
-
29. The antibody of claim [0116] 15 or 16 which is detectably labeled.
-
30. An isolated nucleic acid having a nucleotide sequence that encodes the antibody of claim [0117] 15 or 16.
-
31. An expression vector comprising the nucleic acid of claim [0118] 30 operably linked to control sequences recognized by a host cell transformed with the vector.
-
32. A host cell comprising the expression vector of claim [0119] 31.
-
33. The host cell of claim [0120] 32 which is a CHO cell, an E. coli cell or a yeast cell.
-
34. A process for producing an antibody comprising culturing the host cell of claim [0121] 32 under conditions suitable for expression of said antibody and recovering said antibody from the cell culture.
-
35. An isolated oligopeptide that binds to a polypeptide having at least 80% amino acid sequence identity to: [0122]
-
(a) the polypeptide shown in anyone of FIGS. 57-112, [0123] 114, 116, 118 or 120(SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in anyone of FIGS. 57-112, [0124] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0125] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0126] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0127] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0128] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
36. An isolated oligopeptide that binds to a polypeptide having: [0129]
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0130] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0131] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0132] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0133] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0134] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
37. The oligopeptide of claim [0135] 35 or 36 which is conjugated to a growth inhibitory agent.
-
38. The oligopeptide of claim [0136] 35 or 36 which is conjugated to a cytotoxic agent.
-
39. The oligopeptide of claim [0137] 38, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
-
40. The oligopeptide of claim [0138] 38, wherein the cytotoxic agent is a toxin.
-
41. The oligopeptide of claim [0139] 40, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
-
42. The oligopeptide of claim [0140] 40, wherein the toxin is a maytansinoid.
-
43. The oligopeptide of claim [0141] 35 or 36 which induces death of a cell to which it binds.
-
44. The oligopeptide of claim [0142] 35 or 36 which is detectably labeled.
-
45. A TAT binding organic molecule that binds to a polypeptide having at least 80% amino acid sequence identity to: [0143]
-
(a) the polypeptide shown in anyone of FIGS. 57-112, [0144] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in anyone of FIGS. 57-112, [0145] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0146] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0147] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0148] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0149] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
46. The organic molecule of claim [0150] 45 that binds to a polypeptide having:
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0151] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0152] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0153] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0154] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0155] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0156] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
47. The organic molecule of claim [0157] 45 or 46 which is conjugated to a growth inhibitory agent.
-
48. The organic molecule of claim [0158] 45 or 46 which is conjugated to a cytotoxic agent.
-
49. The organic molecule of claim [0159] 48, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
-
50. The organic molecule of claim [0160] 48, wherein the cytotoxic agent is a toxin.
-
51. The organic molecule of claim [0161] 50, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
-
52. The organic molecule of claim [0162] 50, wherein the toxin is a maytansinoid.
-
53. The organic molecule of claim [0163] 45 or 46 which induces death of a cell to which it binds.
-
54. The organic molecule of claim [0164] 45 or 46 which is detectably labeled.
-
55. A composition of matter comprising: [0165]
-
(a) the polypeptide of claim [0166] 11;
-
(b) the polypeptide of claim [0167] 12;
-
(c) the chimeric polypeptide of claim [0168] 13;
-
(d) the antibody of claim [0169] 15;
-
(e) the antibody of claim [0170] 16;
-
(f) the oligopeptide of claim [0171] 35;
-
(g) the oligopeptide of claim [0172] 36;
-
(h) the TAT binding organic molecule of claim [0173] 45; or
-
(i) the TAT binding organic molecule of claim [0174] 46; in combination with a carrier.
-
56. The composition of matter of claim [0175] 55, wherein said carrier is a pharmaceutically acceptable carrier.
-
57. An article of manufacture comprising: [0176]
-
(a) a container; and [0177]
-
(b) the composition of matter of claim [0178] 55 contained within said container.
-
58. The article of manufacture of claim [0179] 57 further comprising a label affixed to said container, or a package insert included with said container, referring to the use of said composition of matter for the therapeutic treatment of or the diagnostic detection of a cancer.
-
59. A method of inhibiting the growth of a cell that expresses a protein having at least 80% amino acid sequence identity to: [0180]
-
(a) the polypeptide shown in any one of FIGS. 57-112, [0181] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in anyone of FIGS. 7-112, [0182] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0183] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0184] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0185] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0186] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said cell with an antibody, oligopeptide or organic molecule that binds to said protein, the binding of said antibody, oligopeptide or organic molecule to said protein thereby causing an inhibition of growth of said cell.
-
60. The method of claim [0187] 59, wherein said antibody is a monoclonal antibody.
-
61. The method of claim [0188] 59, wherein said antibody is an antibody fragment.
-
62. The method of claim [0189] 59, wherein said antibody is a chimeric or a humanized antibody.
-
63. The method of claim [0190] 59, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
-
64. The method of claim [0191] 59, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
-
65. The method of claim [0192] 64, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
-
66. The method of claim [0193] 64, wherein the cytotoxic agent is a toxin.
-
67. The method of claim [0194] 66, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
-
68. The method of claim [0195] 66, wherein the toxin is a maytansinoid.
-
69. The method of claim [0196] 59, wherein said antibody is produced in bacteria.
-
70. The method of claim [0197] 59, wherein said antibody is produced in CHO cells.
-
71. The method of claim [0198] 59, wherein said cell is a cancer cell.
-
72. The method of claim [0199] 71, wherein said cancer cell is further exposed to radiation treatment or a chemotherapeutic agent.
-
73. The method of claim [0200] 71, wherein said cancer cell is selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, an ovarian cancer cell, a central nervous system cancer cell, a liver cancer cell, a bladder cancer cell, a pancreatic cancer cell, a cervical cancer cell, a melanoma cell and a leukemia cell.
-
74. The method of claim [0201] 71, wherein said protein is more abundantly expressed by said cancer cell as compared to a normal cell of the same tissue origin.
-
75. The method of claim [0202] 59 which causes the death of said cell.
-
76. The method of claim [0203] 59, wherein said protein has:
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0204] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0205] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0206] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0207] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0208] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0209] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
77. A method of therapeutically treating a mammal having a cancerous tumor comprising cells that express a protein having at least 80% amino acid sequence identity to: [0210]
-
(a) the polypeptide shown in any one of FIGS. 57-112, [0211] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in any one of FIGS. 57-112, [0212] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0213] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0214] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0215] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0216] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising administering to said mammal a therapeutically effective amount of an antibody, oligopeptide or organic molecule that binds to said protein, thereby effectively treating said mammal.
-
78. The method of claim [0217] 77, wherein said antibody is a monoclonal antibody.
-
79. The method of claim [0218] 77, wherein said antibody is an antibody fragment.
-
80. The method of claim [0219] 77, wherein said antibody is a chimeric or a humanized antibody.
-
81. The method of claim [0220] 77, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
-
82. The method of claim [0221] 77, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
-
83. The method of claim [0222] 82, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
-
84. The method of claim [0223] 82, wherein the cytotoxic agent is a toxin.
-
85. The method of claim [0224] 84, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
-
86. The method of claim [0225] 84, wherein the toxin is a maytansinoid.
-
87. The method of claim [0226] 77, wherein said antibody is produced in bacteria.
-
88. The method of claim [0227] 77, wherein said antibody is produced in CHO cells.
-
89. The method of claim [0228] 77, wherein said tumor is further exposed to radiation treatment or a chemotherapeutic agent.
-
90. The method of claim [0229] 77, wherein said tumor is a breast tumor, a colorectal tumor, a lung tumor, an ovarian tumor, a central nervous system tumor, a liver tumor, a bladder tumor, a pancreatic tumor, or a cervical tumor.
-
91. The method of claim [0230] 77, wherein said protein is more abundantly expressed by the cancerous cells of said tumor as compared to a normal cell of the same tissue origin.
-
92. The method of claim [0231] 77, wherein said protein has:
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0232] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0233] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0234] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0235] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0236] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0237] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
93. A method of determining the presence of a protein in a sample suspected of containing said protein, wherein said protein has at least 80% amino acid sequence identity to: [0238]
-
(a) the polypeptide shown in anyone of FIGS. 57-112, [0239] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in any one of FIGS. 57-112, [0240] 114, 116, 118 or 120 (SEQ ID NOS:57-112,114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0241] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0242] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0243] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0244] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising exposing said sample to an antibody, oligopeptide or organic molecule that binds to said protein and determining binding of said antibody, oligopeptide or organic molecule to said protein in said sample, wherein binding of the antibody, oligopeptide or organic molecule to said protein is indicative of the presence of said protein in said sample.
-
94. The method of claim [0245] 93, wherein said sample comprises a cell suspected of expressing said protein.
-
95. The method of claim [0246] 94, wherein said cell is a cancer cell.
-
96. The method of claim [0247] 93, wherein said antibody, oligopeptide or organic molecule is detectably labeled.
-
97. The method of claim [0248] 93, wherein said protein has:
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0249] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0250] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0251] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0252] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0253] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0254] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
98. A method of diagnosing the presence of a tumor in a mammal, said method comprising determining the level of expression of a gene encoding a protein having at least 80% amino acid sequence identity to: [0255]
-
(a) the polypeptide shown in anyone of FIGS. 57-112, [0256] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in any one of FIGS. 57-112, [0257] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0258] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0259] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0260] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0261] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), in a test sample of tissue cells obtained from said mammal and in a control sample of known normal cells of the same tissue origin, wherein a higher level of expression of said protein in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test sample was obtained.
-
99. The method of claim [0262] 98, wherein the step of determining the level of expression of a gene encoding said protein comprises employing an oligonucleotide in an in situ hybridization or RT-PCR analysis.
-
100. The method of claim [0263] 98, wherein the step determining the level of expression of a gene encoding said protein comprises employing an antibody in an immunohistochemistry or Western blot analysis.
-
101. The method of claim [0264] 98, wherein said protein has:
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0265] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0266] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0267] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0268] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0269] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0270] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
102. A method of diagnosing the presence of a tumor in a mammal, said method comprising contacting a test sample of tissue cells obtained from said mammal with an antibody, oligopeptide or organic molecule that binds to a protein having at least 80% amino acid sequence identity to: [0271]
-
(a) the polypeptide shown in any one of FIGS. 57-112, [0272] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in any one of FIGS. 57-112, [0273] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0274] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0275] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0276] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0277] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), and detecting the formation of a complex between said antibody, oligopeptide or organic molecule and said protein in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal.
-
103. The method of claim [0278] 102, wherein said antibody, oligopeptide or organic molecule is detectably labeled.
-
104. The method of claim [0279] 102, wherein said test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.
-
105. The method of claim [0280] 102, wherein said protein has:
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0281] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0282] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0283] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0284] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0285] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0286] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
106. A method for treating or preventing a cell proliferative disorder associated with increased expression or activity of a protein having at least 80% amino acid sequence identity to: [0287]
-
(a) the polypeptide shown in anyone of FIGS. 57-112, [0288] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in anyone of FIGS. 57-112, [0289] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0290] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0291] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0292] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0293] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising administering to a subject in need of such treatment an effective amount of an antagonist of said protein, thereby effectively treating or preventing said cell proliferative disorder.
-
107. The method of claim [0294] 106, wherein said cell proliferative disorder is cancer.
-
108. The method of claim [0295] 106, wherein said antagonist is an anti-TAT polypeptide antibody, TAT binding oligopeptide, TAT binding organic molecule or antisense oligonucleotide.
-
109. A method of binding an antibody, oligopeptide or organic molecule to a cell that expresses a protein having at least 80% amino acid sequence identity to: [0296]
-
(a) the polypeptide shown in any one of FIGS. 57-112, [0297] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in any one of FIGS. 57-112, [0298] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0299] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0300] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0301] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0302] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said cell with an antibody, oligopeptide or organic molecule that binds to said protein and allowing the binding of the antibody, oligopeptide or organic molecule to said protein to occur, thereby binding said antibody, oligopeptide or organic molecule to said cell.
-
110. The method of claim [0303] 109, wherein said antibody is a monoclonal antibody.
-
111. The method of claim [0304] 109, wherein said antibody is an antibody fragment.
-
112. The method of claim [0305] 109, wherein said antibody is a chimeric or a humanized antibody.
-
113. The method of claim [0306] 109, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
-
114. The method of claim [0307] 109, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
-
115. The method of claim [0308] 114, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
-
116. The method of claim [0309] 114, wherein the cytotoxic agent is a toxin.
-
117. The method of claim [0310] 116, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
-
118. The method of claim [0311] 116, wherein the toxin is a maytansinoid.
-
119. The method of claim [0312] 109, wherein said antibody is produced in bacteria.
-
120. The method of claim [0313] 109, wherein said antibody is produced in CHO cells.
-
121. The method of claim [0314] 109, wherein said cell is a cancer cell.
-
122. The method of claim [0315] 121, wherein said cancer cell is further exposed to radiation treatment or a chemotherapeutic agent.
-
123. The method of claim [0316] 121, wherein said cancer cell is selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, an ovarian cancer cell, a central nervous system cancer cell, a liver cancer cell, a bladder cancer cell, a pancreatic cancer cell, a cervical cancer cell, a melanoma cell and a leukemia cell.
-
124. The method of claim [0317] 123, wherein said protein is more abundantly expressed by said cancer cell as compared to a normal cell of the same tissue origin.
-
125. The method of claim [0318] 109 which causes the death of said cell.
-
126. Use of a nucleic acid as claimed in any of [0319] claims 1 to 5 or 30 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
-
127. Use of a nucleic acid as claimed in any of [0320] claims 1 to 5 or 30 in the preparation of a medicament for treating a tumor.
-
128. Use of a nucleic acid as claimed in any of [0321] claims 1 to 5 or 30 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
-
129. Use of an expression vector as claimed in any of [0322] claims 6, 7 or 31 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
-
130. Use of an expression vector as claimed in any of [0323] claims 6, 7 or 31 in the preparation of medicament for treating a tumor.
-
131. Use of an expression vector as claimed in any of [0324] claims 6, 7 or 31 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
-
132. Use of a host cell as claimed in any of claims [0325] 8, 9, 32, or 33 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
-
133. Use of a host cell as claimed in any of claims [0326] 8, 9, 32 or 33 in the preparation of a medicament for treating a tumor.
-
134. Use of a host cell as claimed in any of claims [0327] 8, 9, 32 or 33 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
-
135. Use of a polypeptide as claimed in any of claims [0328] 11 to 14 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
-
136. Use of a polypeptide as claimed in any of claims [0329] 11 to 14 in the preparation of a medicament for treating a tumor.
-
137. Use of a polypeptide as claimed in any of claims [0330] 11 to 14 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
-
138. Use of an antibody as claimed in any of claims [0331] 15 to 29 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
-
139. Use of an antibody as claimed in any of claims [0332] 15 to 29 in the preparation of a medicament for treating a tumor.
-
140. Use of an antibody as claimed in any of claims [0333] 15 to 29 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
-
141. Use of an oligopeptide as claimed in any of claims [0334] 35 to 44 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
-
142. Use of an oligopeptide as claimed in any of claims [0335] 35 to 44 in the preparation of a medicament for treating a tumor.
-
143. Use of an oligopeptide as claimed in any of claims [0336] 35 to 44 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
-
144. Use of a TAT binding organic molecule as claimed in any of claims [0337] 45 to 54 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
-
145. Use of a TAT binding organic molecule as claimed in any of claims [0338] 45 to 54 in the preparation of a medicament for treating a tumor.
-
146. Use of a TAT binding organic molecule as claimed in any of claims [0339] 45 to 54 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
-
147. Use of a composition of matter as claimed in any of claims [0340] 55 or 56 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
-
148. Use of a composition of matter as claimed in any of claims [0341] 55 or 56 in the preparation of a medicament for treating a tumor.
-
149. Use of a composition of matter as claimed in any of claims [0342] 55 or 56 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
-
150. Use of an article of manufacture as claimed in any of claims [0343] 57 or 58 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
-
151. Use of an article of manufacture as claimed in any of claims [0344] 57 or 58 in the preparation of a medicament for treating a tumor.
-
152. Use of an article of manufacture as claimed in any of claims [0345] 57 or 58 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
-
153. A method for inhibiting the growth of a cell, wherein the growth of said cell is at least in part dependent upon a growth potentiating effect of a protein having at least 80% amino acid sequence identity to: [0346]
-
(a) the polypeptide shown in any one of FIGS. 57-112, [0347] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in any one of FIGS. 57-112, [0348] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0349] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0350] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0351] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0352] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said protein with an antibody, oligopeptide or organic molecule that binds to said protein, there by inhibiting the growth of said cell.
-
154. The method of claim [0353] 153, wherein said cell is a cancer cell.
-
155. The method of claim [0354] 153, wherein said protein is expressed by said cell.
-
156. The method of claim [0355] 153, wherein the binding of said antibody, oligopeptide or organic molecule to said protein antagonizes a cell growth-potentiating activity of said protein.
-
157. The method of claim [0356] 153, wherein the binding of said antibody, oligopeptide or organic molecule to said protein induces the death of said cell.
-
158. The method of claim [0357] 153, wherein said antibody is a monoclonal antibody.
-
159. The method of claim [0358] 153, wherein said antibody is an antibody fragment.
-
160. The method of claim [0359] 153, wherein said antibody is a chimeric or a humanized antibody.
-
161. The method of claim [0360] 153, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
-
162. The method of claim [0361] 153, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
-
163. The method of claim [0362] 162, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
-
164. The method of claim [0363] 162, wherein the cytotoxic agent is a toxin.
-
165. The method of claim [0364] 164, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
-
166. The method of claim [0365] 164, wherein the toxin is a maytansinoid.
-
167. The method of claim [0366] 153, wherein said antibody is produced in bacteria.
-
168. The method of claim [0367] 153, wherein said antibody is produced in CHO cells.
-
169. The method of claim [0368] 153, wherein said protein has:
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0369] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0370] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0371] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0372] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0373] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0374] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
170. A method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor is at least in part dependent upon a growth potentiating effect of a protein having at least 80% amino acid sequence identity to: [0375]
-
(a) the polypeptide shown in any one of FIGS. 57-112, [0376] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the polypeptide shown in any one of FIGS. 57-112, [0377] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0378] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
-
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0379] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
-
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0380] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0381] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said protein with an antibody, oligopeptide or organic molecule that binds to said protein, thereby effectively treating said tumor.
-
171. The method of claim [0382] 170, wherein said protein is expressed by cells of said tumor.
-
172. The method of claim [0383] 170, wherein the binding of said antibody, oligopeptide or organic molecule to said protein antagonizes a cell growth-potentiating activity of said protein.
-
173. The method of claim [0384] 170, wherein said antibody is a monoclonal antibody.
-
174. The method of claim [0385] 170, wherein said antibody is an antibody fragment.
-
175. The method of claim [0386] 170, wherein said antibody is a chimeric or a humanized antibody.
-
176. The method of claim [0387] 170, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
-
177. The method of claim [0388] 170, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
-
178. The method of claim [0389] 177, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
-
179. The method of claim [0390] 177, wherein the cytotoxic agent is a toxin.
-
180. The method of claim [0391] 179, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
-
181. The method of claim [0392] 179, wherein the toxin is a maytansinoid.
-
182. The method of claim [0393] 170, wherein said antibody is produced in bacteria.
-
183. The method of claim [0394] 170, wherein said antibody is produced in CHO cells.
-
184. The method of claim [0395] 170, wherein said protein has:
-
(a) the amino acid sequence shown in any one of FIGS. 57-112, [0396] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
-
(b) the amino acid sequence shown in any one of FIGS. 57-112, [0397] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0398] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
-
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112, [0399] 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
-
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56, [0400] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
-
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56, [0401] 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
-
Yet further embodiments of the present invention will be evident to the skilled artisan upon a reading of the present specification.[0402]
BRIEF DESCRIPTION OF THE DRAWINGS
-
FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a TAT207 cDNA, wherein SEQ ID NO:1 is a clone designated herein as “DNA67962”. [0403]
-
FIG. 2 shows a nucleotide sequence (SEQ ID NO:2) of a TAT177 cDNA, wherein SEQ ID NO:2 is a clone designated herein as “DNA77507”. [0404]
-
FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a TAT235 cDNA, wherein SEQ ID NO:3 is a clone designated herein as “DNA87993”. [0405]
-
FIG. 4 shows a nucleotide sequence (SEQ ID NO:4) of a TAT234 cDNA, wherein SEQ ID NO:4 is a clone designated herein as “DNA92980”. [0406]
-
FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a TAT239 cDNA, wherein SEQ ID NO:5 is a clone designated herein as “DNA96792”. [0407]
-
FIG. 6 shows a nucleotide sequence (SEQ ID NO:6) of a TAT193 cDNA, wherein SEQ ID NO:6 is a clone designated herein as “DNA96964”. [0408]
-
FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a TAT233 cDNA, wherein SEQ ID NO:7 is a clone designated herein as “DNA105792”. [0409]
-
FIG. 8 shows a nucleotide sequence (SEQ ID NO:8) of a TAT226 cDNA, wherein SEQ ID NO:8 is a clone designated herein as “DNA 119474”. [0410]
-
FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a TAT199 cDNA, wherein SEQ ID NO:9 is a clone designated herein as “DNA142915”. [0411]
-
FIGS. [0412] 10A-B show a nucleotide sequence (SEQ ID NO:10) of a TAT204 cDNA, wherein SEQ ID NO:10 is a clone designated herein as “DNA150491”.
-
FIGS. [0413] 11A-B show a nucleotide sequence (SEQ ID NO:11) of a TAT248 cDNA, wherein SEQ ID NO:11 is a clone designated herein as “DNA280351”.
-
FIG. 12 shows a nucleotide sequence (SEQ ID NO:12) of a TAT232 cDNA, wherein SEQ ID NO:12 is a clone designated herein as “DNA150648”. [0414]
-
FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a TAT219 cDNA, wherein SEQ ID NO:13 is a clone designated herein as “DNA172500”. [0415]
-
FIG. 14 shows a nucleotide sequence (SEQ ID NO:14) of a TAT224 cDNA, wherein SEQ ID NO:14 is a clone designated herein as “DNA179651”. [0416]
-
FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a TAT237 cDNA, wherein SEQ ID NO:15 is a clone designated herein as “DNA207698”. [0417]
-
FIG. 16 shows a nucleotide sequence (SEQ ID NO:16) of a TAT178 cDNA, wherein SEQ ID NO:16 is a clone designated herein as “DNA208551”. [0418]
-
FIGS. [0419] 17A-B show a nucleotide sequence (SEQ ID NO:17) of a TAT198 cDNA, wherein SEQ ID NO:17 is a clone designated herein as “DNA210159”.
-
FIGS. [0420] 18A-B show a nucleotide sequence (SEQ ID NO:18) of a TAT194 cDNA, wherein SEQ ID NO:18 is a clone designated herein as “DNA225706”.
-
FIGS. [0421] 19A-B show a nucleotide sequence (SEQ ID NO:19) of a TAT223 cDNA, wherein SEQ ID NO:19 is a clone designated herein as “DNA225793”.
-
FIG. 20 shows a nucleotide sequence (SEQ ID NO:20) of a TAT196 cDNA, wherein SEQ ID NO:20 is a clone designated herein as “DNA225796”. [0422]
-
FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a TAT236 cDNA, wherein SEQ ID NO:21 is a clone designated herein as “DNA225886”. [0423]
-
FIG. 22 shows a nucleotide sequence (SEQ ID NO:22) of a TAT195 cDNA, wherein SEQ ID NO:22 is a clone designated herein as “DNA225943”. [0424]
-
FIG. 23 shows a nucleotide sequence (SEQ ID NO:23) of a TAT203 cDNA, wherein SEQ ID NO:23 is a clone designated herein as “DNA226283”. [0425]
-
FIGS. [0426] 24A-B show a nucleotide sequence (SEQ ID NO:24) of a TAT200 cDNA, wherein SEQ ID NO:24 is a clone designated herein as “DNA226589”.
-
FIGS. [0427] 25A-B show a nucleotide sequence (SEQ ID NO:25) of a TAT205 cDNA, wherein SEQ ID NO:25 is a clone designated herein as “DNA226622”.
-
FIGS. [0428] 26A-B show a nucleotide sequence (SEQ ID NO:26) of a TAT185 cDNA, wherein SEQ ID NO:26 is a clone designated herein as “DNA226717”.
-
FIGS. [0429] 27A-B show a nucleotide sequence (SEQ ID NO:27) of a TAT225 cDNA, wherein SEQ ID NO:27 is a clone designated herein as “DNA227162”.
-
FIG. 28 shows a nucleotide sequence (SEQ ID NO:28) of a TAT247 cDNA, wherein SEQ ID NO:28 is a clone designated herein as “DNA277804”. [0430]
-
FIG. 29 shows a nucleotide sequence (SEQ ID NO:29) of a TAT197 cDNA, wherein SEQ ID NO:29 is a clone designated herein as “DNA227545”. [0431]
-
FIG. 30 shows a nucleotide sequence (SEQ ID NO:30) of a TAT175 cDNA, wherein SEQ ID NO:30 is a clone designated herein as “DNA227611”. [0432]
-
FIG. 31 shows a nucleotide sequence (SEQ ID NO:31) of a TAT208 cDNA, wherein SEQ ID NO:31 is a clone designated herein as “DNA261021”. [0433]
-
FIG. 32 shows a nucleotide sequence (SEQ ID NO:32) of a TAT174 cDNA, wherein SEQ ID NO:32 is a clone designated herein as “DNA233034”. [0434]
-
FIG. 33 shows a nucleotide sequence (SEQ ID NO:33) of a TAT214 cDNA, wherein SEQ ID NO:33 is a clone designated herein as “DNA266920”. [0435]
-
FIG. 34 shows a nucleotide sequence (SEQ ID NO:34) of a TAT220 cDNA, wherein SEQ ID NO:34 is a clone designated herein as “DNA266921”. [0436]
-
FIG. 35 shows a nucleotide sequence (SEQ ID NO:35) of a TAT221 cDNA, wherein SEQ ID NO:35 is a clone designated herein as “DNA266922”. [0437]
-
FIG. 36 shows a nucleotide sequence (SEQ ID NO:36) of a TAT201 cDNA, wherein SEQ ID NO:36 is a clone designated herein as “DNA234441”. FIGS. [0438] 37A-B show a nucleotide sequence (SEQ ID NO:37) of a TAT179 cDNA, wherein SEQ ID NO:37 is a clone designated herein as “DNA234834”.
-
FIG. 38 shows a nucleotide sequence (SEQ ID NO:38) of a TAT216 cDNA, wherein SEQ ID NO:38 is a clone designated herein as “DNA247587”. [0439]
-
FIG. 39 shows a nucleotide sequence (SEQ ID NO:39) of a TAT218 cDNA, wherein SEQ ID NO:39 is a clone designated herein as “DNA255987”. [0440]
-
FIG. 40 shows a nucleotide sequence (SEQ ID NO:40) of a TAT206 cDNA, wherein SEQ ID NO:40 is a clone designated herein as “DNA56041”. [0441]
-
FIGS. [0442] 41A-B show a nucleotide sequence (SEQ ID NO:41) of a TAT374 cDNA, wherein SEQ ID NO:41 is a clone designated herein as “DNA257845”.
-
FIG. 42 shows a nucleotide sequence (SEQ ID NO:42) of a TAT209 cDNA, wherein SEQ ID NO:42 is a clone designated herein as “DNA260655”. [0443]
-
FIG. 43 shows a nucleotide sequence (SEQ ID NO:43) of a TAT192 cDNA, wherein SEQ ID NO:43 is a clone designated herein as “DNA260945”. [0444]
-
FIG. 44 shows a nucleotide sequence (SEQ ID NO:44) of a TAT180 cDNA, wherein SEQ ID NO:44 is a clone designated herein as “DNA247476”. [0445]
-
FIG. 45 shows a nucleotide sequence (SEQ ID NO:45) of a TAT375 cDNA, wherein SEQ ID NO:45 is a clone designated herein as “DNA260990”. [0446]
-
FIG. 46 shows a nucleotide sequence (SEQ ID NO:46) of a TAT181 cDNA, wherein SEQ ID NO:46 is a clone designated herein as “DNA261001”. [0447]
-
FIG. 47 shows a nucleotide sequence (SEQ ID NO:47) of a TAT176 cDNA, wherein SEQ ID NO:47 is a clone designated herein as “DNA261013”. [0448]
-
FIG. 48 shows a nucleotide sequence (SEQ ID NO:48) of a TAT184 cDNA, wherein SEQ ID NO:48 is a clone designated herein as “DNA262144”. [0449]
-
FIG. 49 shows a nucleotide sequence (SEQ ID NO:49) of a TAT182 cDNA, wherein SEQ ID NO:49 is a clone designated herein as “DNA266928”. [0450]
-
FIGS. [0451] 50A-B show a nucleotide sequence (SEQ ID NO:50) of a TAT213 cDNA, wherein SEQ ID NO:50 is a clone designated herein as “DNA267342”.
-
FIGS. [0452] 51A-C show a nucleotide sequence (SEQ ID NO:51) of a TAT217 cDNA, wherein SEQ ID NO:51 is a clone designated herein as “DNA267626”.
-
FIG. 52 shows a nucleotide sequence (SEQ ID NO:52) of a TAT222 cDNA, wherein SEQ ID NO:52 is a clone designated herein as “DNA268035”. [0453]
-
FIG. 53 shows a nucleotide sequence (SEQ ID NO:53) of a TAT202 cDNA, wherein SEQ ID NO:53 is a clone designated herein as “DNA268334”. [0454]
-
FIG. 54 shows a nucleotide sequence (SEQ ID NO:54) of a TAT215 cDNA, wherein SEQ ID NO:54 is a clone designated herein as “DNA269238”. [0455]
-
FIG. 55 shows a nucleotide sequence (SEQ ID NO:55) of a TAT238 cDNA, wherein SEQ ID NO:55 is a clone designated herein as “DNA272578”. [0456]
-
FIG. 56 shows a nucleotide sequence (SEQ ID NO:56) of a TAT212 cDNA, wherein SEQ ID NO:56 is a clone designated herein as “DNA277797”. [0457]
-
FIG. 57 shows the amino acid sequence (SEQ ID NO:57) derived from the coding sequence of SEQ ID NO:1 shown in FIG. 1. [0458]
-
FIG. 58 shows the amino acid sequence (SEQ ID NO:58) derived from the coding sequence of SEQ ID NO:2 shown in FIG. 2. [0459]
-
FIG. 59 shows the amino acid sequence (SEQ ID NO:59) derived from the coding sequence of SEQ ID NO:3 shown in FIG. 3. [0460]
-
FIG. 60 shows the amino acid sequence (SEQ ID NO:60) derived from the coding sequence of SEQ ID NO:4 shown in FIG. 4. [0461]
-
FIG. 61 shows the amino acid sequence (SEQ ID NO:61) derived from the coding sequence of SEQ ID NO:5 shown in FIG. 5. [0462]
-
FIG. 62 shows the amino acid sequence (SEQ ID NO:62) derived from the coding sequence of SEQ ID NO:6 shown in FIG. 6. [0463]
-
FIG. 63 shows the amino acid sequence (SEQ ID NO:63) derived from the coding sequence of SEQ ID NO:7 shown in FIG. 7. [0464]
-
FIG. 64 shows the amino acid sequence (SEQ ID NO:64) derived from the coding sequence of SEQ ID NO:8 shown in FIG. 8. [0465]
-
FIG. 65 shows the amino acid sequence (SEQ ID NO:65) derived from the coding sequence of SEQ ID NO:9 shown in FIG. 9. [0466]
-
FIG. 66 shows the amino acid sequence (SEQ ID NO:66) derived from the coding sequence of SEQ ID NO:10 shown in FIGS. [0467] 10A-B.
-
FIG. 67 shows the amino acid sequence (SEQ ID NO:67) derived from the coding sequence of SEQ ID NO:11 shown in FIGS. [0468] 11A-B.
-
FIG. 68 shows the amino acid sequence (SEQ ID NO:68) derived from the coding sequence of SEQ ID NO:12 shown in FIG. 12. [0469]
-
FIG. 69 shows the amino acid sequence (SEQ ID NO:69) derived from the coding sequence of SEQ ID NO:13 shown in FIG. 13. [0470]
-
FIG. 70 shows the amino acid sequence (SEQ ID NO:70) derived from the coding sequence of SEQ ID NO:14 shown in FIG. 14. [0471]
-
FIG. 71 shows the amino acid sequence (SEQ ID NO:71) derived from the coding sequence of SEQ ID NO:15 shown in FIG. 15. [0472]
-
FIG. 72 shows the amino acid sequence (SEQ ID NO:72) derived from the coding sequence of SEQ ID NO:16 shown in FIG. 16. [0473]
-
FIG. 73 shows the amino acid sequence (SEQ ID NO:73) derived from the coding sequence of SEQ ID NO:17 shown in FIGS. [0474] 17A-B.
-
FIG. 74 shows the amino acid sequence (SEQ ID NO:74) derived from the coding sequence of SEQ ID NO:18 shown in FIGS. [0475] 18A-B.
-
FIG. 75 shows the amino acid sequence (SEQ ID NO:75) derived from the coding sequence of SEQ ID NO:19 shown in FIGS. [0476] 19A-B.
-
FIG. 76 shows the amino acid sequence (SEQ ID NO:76) derived from the coding sequence of SEQ ID NO:20 shown in FIG. 20. [0477]
-
FIG. 77 shows the amino acid sequence (SEQ ID NO:77) derived from the coding sequence of SEQ ID NO:21 shown in FIG. 21. [0478]
-
FIG. 78 shows the amino acid sequence (SEQ ID NO:78) derived from the coding sequence of SEQ ID NO:22 shown in FIG. 22. [0479]
-
FIG. 79 shows the amino acid sequence (SEQ ID NO:79) derived from the coding sequence of SEQ ID NO:23 shown in FIG. 23. [0480]
-
FIG. 80 shows the amino acid sequence (SEQ ID NO:80) derived from the coding sequence of SEQ ID NO:24 shown in FIGS. [0481] 24A-B.
-
FIG. 81 shows the amino acid sequence (SEQ ID NO:81) derived from the coding sequence of SEQ ID NO:25 shown in FIGS. [0482] 25A-B.
-
FIG. 82 shows the amino acid sequence (SEQ ID NO:82) derived from the coding sequence of SEQ ID NO:26 shown in FIGS. [0483] 26A-B.
-
FIG. 83 shows the amino acid sequence (SEQ ID NO:83) derived from the coding sequence of SEQ ID NO:27 shown in FIGS. [0484] 27A-B.
-
FIG. 84 shows the amino acid sequence (SEQ ID NO:84) derived from the coding sequence of SEQ ID NO:28 shown in FIG. 28. [0485]
-
FIG. 85 shows the amino acid sequence (SEQ ID NO:85) derived from the coding sequence of SEQ ID NO:29 shown in FIG. 29. [0486]
-
FIG. 86 shows the amino acid sequence (SEQ ID NO:86) derived from the coding sequence of SEQ ID NO:30 shown in FIG. 30. [0487]
-
FIG. 87 shows the amino acid sequence (SEQ ID NO:87) derived from the coding sequence of SEQ ID NO:31 shown in FIG. 31. [0488]
-
FIG. 88 shows the amino acid sequence (SEQ ID NO:88) derived from the coding sequence of SEQ ID NO:32 shown in FIG. 32. [0489]
-
FIG. 89 shows the amino acid sequence (SEQ ID NO:89) derived from the coding sequence of SEQ ID NO:33 shown in FIG. 33. [0490]
-
FIG. 90 shows the amino acid sequence (SEQ ID NO:90) derived from the coding sequence of SEQ ID NO:34 shown in FIG. 34. [0491]
-
FIG. 91 shows the amino acid sequence (SEQ ID NO:91) derived from the coding sequence of SEQ ID NO:35 shown in FIG. 35. [0492]
-
FIG. 92 shows the amino acid sequence (SEQ ID NO:92) derived from the coding sequence of SEQ ID NO:36 shown in FIG. 36. [0493]
-
FIG. 93 shows the amino acid sequence (SEQ ID NO:93) derived from the coding sequence of SEQ ID NO:37 shown in FIGS. [0494] 37A-B.
-
FIG. 94 shows the amino acid sequence (SEQ ID NO:94) derived from the coding sequence of SEQ ID NO:38 shown in FIG. 38. [0495]
-
FIG. 95 shows the amino acid sequence (SEQ ID NO:95) derived from the coding sequence of SEQ ID NO:39 shown in FIG. 39. [0496]
-
FIG. 96 shows the amino acid sequence (SEQ ID NO:96) derived from the coding sequence of SEQ ID NO:40 shown in FIG. 40. [0497]
-
FIG. 97 shows the amino acid sequence (SEQ ID NO:97) derived from the coding sequence of SEQ ID NO:41 shown in FIGS. [0498] 41A-B.
-
FIG. 98 shows the amino acid sequence (SEQ ID NO:98) derived from the coding sequence of SEQ ID NO:42 shown in FIG. 42. [0499]
-
FIG. 99 shows the amino acid sequence (SEQ ID NO:99) derived from the coding sequence of SEQ ID NO:43 shown in FIG. 43. [0500]
-
FIG. 100 shows the amino acid sequence (SEQ ID NO:100) derived from the coding sequence of SEQ ID NO:44 shown in FIG. 44. [0501]
-
FIG. 101 shows the amino acid sequence (SEQ ID NO:101) derived from the coding sequence of SEQ ID NO:45 shown in FIG. 45. [0502]
-
FIG. 102 shows the amino acid sequence (SEQ ID NO:102) derived from the coding sequence of SEQ ID NO:46 shown in FIG. 46. [0503]
-
FIG. 103 shows the amino acid sequence (SEQ ID NO:103) derived from the coding sequence of SEQ ID NO:47 shown in FIG. 47. [0504]
-
FIG. 104 shows the amino acid sequence (SEQ ID NO:104) derived from the coding sequence of SEQ ID NO:48 shown in FIG. 48. [0505]
-
FIG. 105 shows the amino acid sequence (SEQ ID NO:105) derived from the coding sequence of SEQ ID NO:49 shown in FIG. 49. [0506]
-
FIG. 106 shows the amino acid sequence (SEQ ID NO:106) derived from the coding sequence of SEQ ID NO:50 shown in FIGS. [0507] 50A-B.
-
FIGS. [0508] 107A-B show the amino acid sequence (SEQ ID NO:107) derived from the coding sequence of SEQ ID NO:51 shown in FIGS. 51A-C.
-
FIG. 108 shows the amino acid sequence (SEQ ID NO:108) derived from the coding sequence of SEQ ID NO:52 shown in FIG. 52. [0509]
-
FIG. 109 shows the amino acid sequence (SEQ ID NO:109) derived from the coding sequence of SEQ ID NO:53 shown in FIG. 53. [0510]
-
FIG. 110 shows the amino acid sequence (SEQ ID NO:110) derived from the coding sequence of SEQ ID NO:54 shown in FIG. 54. [0511]
-
FIG. 111 shows the amino acid sequence (SEQ ID NO:111) derived from the coding sequence of SEQ ID NO:55 shown in FIG. 55. [0512]
-
FIG. 112 shows the amino acid sequence (SEQ ID NO:112) derived from the coding sequence of SEQ ID NO:56 shown in FIG. 56. [0513]
-
FIG. 113 shows a nucleotide sequence (SEQ ID NO:113) of a TAT376 cDNA, wherein SEQ ID NO:113 is a clone designated herein as “DNA304853”. [0514]
-
FIG. 114 shows the amino acid sequence (SEQ ID NO:114) derived from the coding sequence of SEQ ID NO:113 shown in FIG. 113. [0515]
-
FIG. 115 shows a nucleotide sequence (SEQ ID NO:115) of a TAT377 cDNA, wherein SEQ ID NO:115 is a clone designated herein as “DNA304854”. [0516]
-
FIG. 116 shows the amino acid sequence (SEQ ID NO:116) derived from the coding sequence of SEQ ID NO:115 shown in FIG. 115. [0517]
-
FIG. 117 shows a nucleotide sequence (SEQ ID NO:117) of a TAT378 cDNA, wherein SEQ ID NO:117 is a clone designated herein as “DNA304855”. [0518]
-
FIG. 118 shows the amino acid sequence (SEQ ID NO:118) derived from the coding sequence of SEQ ID NO:117 shown in FIG. 117. [0519]
-
FIGS. [0520] 119A-B show a nucleotide sequence (SEQ ID NO:119) of a TAT379 cDNA, wherein SEQ ID NO: 119 is a clone designated herein as “DNA287971”.
-
FIG. 120 shows the amino acid sequence (SEQ ID NO:120) derived from the coding sequence of SEQ ID NO:119 shown in FIGS. [0521] 119A-B.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
-
I. Definitions [0522]
-
The terms “TAT polypeptide” and “TAT” as used herein and when immediately followed by a numerical designation, refer to various polypeptides, wherein the complete designation (i.e., TAT/number) refers to specific polypeptide sequences as described herein. The terms “TAT/number polypeptide” and “TAT/number” wherein the term “number” is provided as an actual numerical designation as used herein encompass native sequence polypeptides, polypeptide variants and fragments of native sequence polypeptides and polypeptide variants (which are further defined herein). The TAT polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. The term “TAT polypeptide” refers to each individual TAT/number polypeptide disclosed herein. All disclosures in this specification which refer to the “TAT polypeptide” refer to each of the polypeptides individually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against, formation of TAT binding oligopeptides to or against, formation of TAT binding organic molecules to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the invention individually. The term “TAT polypeptide” also includes variants of the TAT/number polypeptides disclosed herein. [0523]
-
A “native sequence TAT polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding TAT polypeptide derived from nature. Such native sequence TAT polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term “native sequence TAT polypeptide” specifically encompasses naturally-occurring truncated or secreted forms of the specific TAT polypeptide (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In certain embodiments of the invention, the native sequence TAT polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop codons (if indicated) are shown in bold font and underlined in the figures. Nucleic acid residues indicated as “N” in the accompanying figures are any nucleic acid residue. However, while the TAT polypeptides disclosed in the accompanying figures are shown to begin with methionine residues designated herein as [0524] amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue for the TAT polypeptides.
-
The TAT polypeptide “extracellular domain” or “ECD” refers to a form of the TAT polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a TAT polypeptide ECD will have less than 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the TAT polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but mqst likely by no more than about 5 amino acids at either end of the domain as initially identified herein. Optionally, therefore, an extracellular domain of a TAT polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are contemplated by the present invention. [0525]
-
The approximate location of the “signal peptides” of the various TAT polypeptides disclosed herein may be shown in the present specification and/or the accompanying figures. It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al., [0526] Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
-
“TAT polypeptide variant” means a TAT polypeptide, preferably an active TAT polypeptide, as defined herein having at least about 80% amino acid sequence identity with a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein (such as those encoded by a nucleic acid that represents only a portion of the complete coding sequence for a full-length TAT polypeptide). Such TAT polypeptide variants include, for instance, TAT polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a TAT polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide sequence as disclosed herein. Ordinarily, TAT variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 amino acids in length, or more. Optionally, TAT variant polypeptides will have no more than one conservative amino acid substitution as compared to the native TAT polypeptide sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared to the native TAT polypeptide sequence. [0527]
-
“Percent (%) amino acid sequence identity” with respect to the TAT polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific TAT polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. [0528]
-
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: [0529]
-
100 times the fraction X/Y
-
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations using this method, Tables 2 and 3 demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated “Comparison Protein” to the amino acid sequence designated “TAT”, wherein “TAT” represents the amino acid sequence of a hypothetical TAT polypeptide of interest, “Comparison Protein” represents the amino acid sequence of a polypeptide against which the “TAT” polypeptide of interest is being compared, and “X, “Y” and “Z” each represent different hypothetical amino acid residues. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. [0530]
-
“TAT variant polynucleotide” or “TAT variant nucleic acid sequence” means a nucleic acid molecule which encodes a TAT polypeptide, preferably an active TAT polypeptide, as defined herein and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence TAT polypeptide sequence as disclosed herein, a full-length native sequence TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein (such as those encoded by a nucleic acid that represents only a portion of the complete coding sequence for a full-length TAT polypeptide). Ordinarily, a TAT variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence TAT polypeptide sequence as disclosed herein, a full-length native sequence TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein. Variants do not encompass the native nucleotide sequence. [0531]
-
Ordinarily, TAT variant polynucleotides are at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term “about” means the referenced nucleotide sequence length plus or minus 10% of that referenced length. [0532]
-
“Percent (%) nucleic acid sequence identity” with respect to TAT-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the TAT nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes herein, however, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. [0533]
-
In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: [0534]
-
100 times the fraction W/Z
-
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated “Comparison DNA” to the nucleic acid sequence designated “TAT-DNA”, wherein “TAT-DNA” represents a hypothetical TAT-encoding nucleic acid sequence of interest, “Comparison DNA” represents the nucleotide sequence of a nucleic acid molecule against which the “TAT-DNA” nucleic acid molecule of interest is being compared, and “N”, “L” and “V” each represent different hypothetical nucleotides. Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. [0535]
-
In other embodiments, TAT variant polynucleotides are nucleic acid molecules that encode a TAT polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length TAT polypeptide as disclosed herein. TAT variant polypeptides may be those that are encoded by a TAT variant polynucleotide. [0536]
-
The term “full-length coding region” when used in reference to a nucleic acid encoding a TAT polypeptide refers to the sequence of nucleotides which encode the full-length TAT polypeptide of the invention (which is often shown between start and stop codons, inclusive thereof, in the accompanying figures). The term “full-length coding region” when used in reference to an ATCC deposited nucleic acid refers to the TAT polypeptide-encoding portion of the cDNA that is inserted into the vector deposited with the ATCC (which is often shown between start and stop codons, inclusive thereof, in the accompanying figures). [0537]
-
“Isolated,” when used to describe the various TAT polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the TAT polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step. [0538]
-
An “isolated” TAT polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells. [0539]
-
The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. [0540]
-
Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. [0541]
-
“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., [0542] Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
-
“Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) overnight hybridization in a solution that employs 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10 minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) followed by a 10 minute high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. [0543]
-
“Moderately stringent conditions” may be identified as described by Sambrook et al., [0544] Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
-
The term “epitope tagged” when used herein refers to a chimeric polypeptide comprising a TAT polypeptide or anti-TAT antibody fused to a “tag polypeptide”. The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues). [0545]
-
“Active” or “activity” for the purposes herein refers to form(s) of a TAT polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring TAT, wherein “biological” activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring TAT other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring TAT and an “immunological” activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring TAT. [0546]
-
The term “antagonist” is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native TAT polypeptide disclosed herein. In a similar manner, the term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native TAT polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native TAT polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying agonists or antagonists of a TAT polypeptide may comprise contacting a TAT polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the TAT polypeptide. [0547]
-
“Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully “treated” for a TAT polypeptide-expressing cancer if, after receiving a therapeutic amount of an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. To the extent the anti-TAT antibody or TAT binding oligopeptide may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or symptoms may also be felt by the patient. [0548]
-
The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR). Metastasis can be determined by staging tests and by bone scan and tests for calcium level and other enzymes to determine spread to the bone. CT scans can also be done to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastasis to the lungs and liver, respectively. Other routine methods for monitoring the disease include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB). [0549]
-
For bladder cancer, which is a more localized cancer, methods to determine progress of disease include urinary cytologic evaluation by cystoscopy, monitoring for presence of blood in the urine, visualization of the urothelial tract by sonography or an intravenous pyelogram, computed tomography (CT) and magnetic resonance imaging (MRI). The presence of distant metastases can be assessed by CT of the abdomen, chest x-rays, or radionuclide imaging of the skeleton. [0550]
-
“Chronic” administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature. [0551]
-
“Mammal” for purposes of the treatment of, alleviating the symptoms of or diagnosis of a cancer refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human. [0552]
-
Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. [0553]
-
“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. [0554]
-
By “solid phase” or “solid support” is meant a non-aqueous matrix to which an antibody, TAT binding oligopeptide or TAT binding organic molecule of the present invention can adhere or attach. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149. [0555]
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A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a TAT polypeptide, an antibody thereto or a TAT binding oligopeptide) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. [0556]
-
A “small” molecule or “small” organic molecule is defined herein to have a molecular weight below about 500 Daltons. [0557]
-
An “effective amount” of a polypeptide, antibody, TAT binding oligopeptide, TAT binding organic molecule or an agonist or antagonist thereof as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” may be determined empirically and in a routine manner, in relation to the stated purpose. [0558]
-
The term “therapeutically effective amount” refers to an amount of an antibody, polypeptide, TAT binding oligopeptide, TAT binding organic molecule or other drug effective to “treat” a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of “treating”. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. [0559]
-
A “growth inhibitory amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A “growth inhibitory amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner. [0560]
-
A “cytotoxic amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of causing the destruction of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A “cytotoxic amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner. [0561]
-
The term “antibody” is used in the broadest sense and specifically covers, for example, single anti-TAT monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-TAT antibody compositions with polyepitopic specificity, polyclonal antibodies, single chain anti-TAT antibodies, and fragments of anti-TAT antibodies (see below) as long as they exhibit the desired biological or immunological activity. The term “immunoglobulin” (Ig) is used interchangeable with antibody herein. [0562]
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An “isolated antibody” is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (I) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. [0563]
-
The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain). In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (V[0564] H) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.
-
The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C[0565] H), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated (α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
-
The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and define specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., [0566] Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
-
The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V[0567] L, and around about 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
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The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., [0568] Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
-
The monoclonal antibodies herein include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., [0569] Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, Ape etc), and human constant region sequences.
-
An “intact” antibody is one which comprises an antigen-binding site as well as a C[0570] L and at least heavy chain constant domains, C H1, C H2 and C H3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.
-
“Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′) 2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., [0571] Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
-
Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V[0572] H), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab′) 2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab′ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the C H1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them.
-
Other chemical couplings of antibody fragments are also known. [0573]
-
The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells. [0574]
-
“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and—binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. [0575]
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“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the V[0576] H and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacologe of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.
-
The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the V[0577] H and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404, 097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
-
“Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., [0578] Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
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A “species-dependent antibody,” e.g., a mammalian anti-human IgE antibody, is an antibody which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody “bind specifically” to a human antigen (i.e., has a binding affinity (Kd) value of no more than about 1×10[0579] −7 M, preferably no more than about 1×10−8 and most preferably no more than about 1×10−9 M) but has a binding affinity for a homologue of the antigen from a second non-human mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be of any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.
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A “TAT binding oligopeptide” is an oligopeptide that binds, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. TAT binding oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such oligopeptides that are capable of binding, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Pat. Nos. 5,556,762,5,750,373,4,708,871,4,833,092, 5,223,409, 5,403,484,5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668). [0580]
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A “TAT binding organic molecule” is an organic molecule other than an oligopeptide or antibody as defined herein that binds, preferably specifically, to a TAT polypeptide as described herein. TAT binding organic molecules may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are capable of binding, preferably specifically, to a TAT polypeptide as described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g., PCT Publication Nos. WO0/00823 and WO00/39585). [0581]
-
An antibody, oligopeptide or other organic molecule “which binds” an antigen of interest, e.g. a tumor-associated polypeptide antigen target, is one that binds the antigen with sufficient affinity such that the antibody, oligopeptide or other organic molecule is useful as a diagnostic and/or therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody, oligopeptide or other organic molecule to a “non-target” protein will be less than about 10% of the binding of the antibody, oligopeptide or other organic molecule to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With regard to the binding of an antibody, oligopeptide or other organic molecule to a target molecule, the term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 10[0582] −4 M, alternatively at least about 10−5 M, alternatively at least about 10−6 M, alternatively at least about 10−7 M, alternatively at least about 10−8 M, alternatively at least about 10−9 M, alternatively at least about 10−10 M, alternatively at least about 10−11 M, alternatively at least about 10−12 M, or greater. In one embodiment, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
-
An antibody, oligopeptide or other organic molecule that “inhibits the growth of tumor cells expressing a TAT polypeptide” or a “growth inhibitory” antibody, oligopeptide or other organic molecule is one which results in measurable growth inhibition of cancer cells expressing or overexpressing the appropriate TAT polypeptide. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferred growth inhibitory anti-TAT antibodies, oligopeptides or organic molecules inhibit growth of TAT-expressing tumor cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the antibody, oligopeptide or other organic molecule being tested. In one embodiment, growth inhibition can be measured at an antibody concentration of about 0.1 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. Growth inhibition of tumor cells in vivo can be determined in various ways such as is described in the Experimental Examples section below. The antibody is growth inhibitory in vivo if administration of the anti-TAT antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days. [0583]
-
An antibody, oligopeptide or other organic molecule which “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). The cell is usually one which overexpresses a TAT polypeptide. Preferably the cell is a tumor cell, e.g., a prostate, breast, ovarian, stomach, endometrial, lung, kidney, colon, bladder cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the antibody, oligopeptide or other organic molecule which induces apoptosis is one which results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay. [0584]
-
Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation. [0585]
-
“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, [0586] Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. (USA) 95:652-656 (1998).
-
“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see review M. in Daëron, [0587] Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).
-
“Human effector cells” are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood. [0588]
-
“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al. [0589] Immunol. Methods 202:163 (1996), may be performed.
-
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid mal ignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases. [0590]
-
The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative-disorder is cancer. [0591]
-
“Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. [0592]
-
An antibody, oligopeptide or other organic molecule which “induces cell death” is one which causes a viable cell to become nonviable. The cell is one which expresses a TAT polypeptide, preferably a cell that overexpresses a TAT polypeptide as compared to a normal cell of the same tissue type. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferably, the cell is a cancer cell, e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Cell death in vitro may be determined in the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay for cell death may be performed using heat inactivated serum (i.e., in the absence of complement) and in the absence of immune effector cells. To determine whether the antibody, oligopeptide or other organic molecule is able to induce cell death, loss of membrane integrity as evaluated by uptake of propidium iodide (PI), trypan blue (see Moore et al. [0593] Cytotechnology 17:1-11 (1995)) or 7AAD can be assessed relative to untreated cells. Preferred cell death-inducing antibodies, oligopeptides or other organic molecules are those which induce PI uptake in the PI uptake assay in BT474 cells.
-
A “TAT-expressing cell” is a cell which expresses an endogenous or transfected TAT polypeptide either on the cell surface or in a secreted form. A “TAT-expressing cancer” is a cancer comprising cells that have a TAT polypeptide present on the cell surface or that produce and secrete a TAT polypeptide. A “TAT-expressing cancer” optionally produces sufficient levels of TAT polypeptide on the surface of cells thereof, such that an anti-TAT antibody, oligopeptide ot other organic molecule can bind thereto and have a therapeutic effect with respect to the cancer. In another embodiment, a “TAT-expressing cancer” optionally produces and secretes sufficient levels of TAT polypeptide, such that an anti-TAT antibody, oligopeptide ot other organic molecule antagonist can bind thereto and have a therapeutic effect with respect to the cancer. With regard to the latter, the antagonist may be an antisense oligonucleotide which reduces, inhibits or prevents production and secretion of the secreted TAT polypeptide by tumor cells. A cancer which “overexpresses” a TAT polypeptide is one which has significantly higher levels of TAT polypeptide at the cell surface thereof, or produces and secretes, compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. TAT polypeptide overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the TAT protein present on the surface of a cell, or secreted by the cell (e.g., via an immunohistochemistry assay using anti-TAT antibodies prepared against an isolated TAT polypeptide which may be prepared using recombinant DNA technology from an isolated nucleic acid encoding the TAT polypeptide; FACS analysis, etc.). Alternatively, or additionally, one may measure levels of TAT polypeptide-encoding nucleic acid or mRNA in the cell, e.g., via fluorescent in situ hybridization using a nucleic acid based probe corresponding to a TAT-encoding nucleic acid or the complement thereof; (FISH; see WO98/45479 published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). One may also study TAT polypeptide overexpression by measuring shed antigen in a biological fluid such as serum, e.g, using antibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et al., [0594] J. Immunol. Methods 132:73-80 (1990)). Aside from the above assays, various in vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.
-
As used herein, the term “immunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is “heterologous”), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. [0595]
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The word “label” when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody, oligopeptide or other organic molecule so as to generate a “labeled” antibody, oligopeptide or other organic molecule. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. [0596]
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The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At[0597] 211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.
-
A “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially a TAT-expressing cancer cell, either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of TAT-expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in [0598] The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (W B Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
-
“Doxorubicin” is an anthracycline antibiotic. The full chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione. [0599]
-
The term “cytokine” is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and —II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-αor TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. [0600]
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The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
[0601] TABLE 2 |
|
|
TAT | XXXXXXXXXXXXXXX | (Length = 15 amino acids) |
Comparison | XXXXXYYYYYYY | (Length = 12 amino acids) |
Protein |
|
|
-
[0602] TABLE 3 |
|
|
TAT | XXXXXXXXXX | (Length = 10 amino acids) |
Comparison | XXXXXYYYYYYZZYZ | (Length = 15 amino acids) |
Protein |
|
|
-
[0603] TABLE 4 |
|
|
TAT-DNA | NNNNNNNNNNNNNN | (Length = 14 nucleotides) |
Comparison | NNNNNNLLLLLLLLLL | (Length = 16 nucleotides) |
DNA |
|
|
-
[0604] TABLE 5 |
|
|
TAT-DNA | NNNNNNNNNNNN | (Length = 12 nucleotides) |
Comparison DNA | NNNNLLLVV | (Length = 9 nucleotides) |
|
|
-
II. Compositions and Methods of the Invention [0605]
-
A. Anti-TAT Antibodies [0606]
-
In one embodiment, the present invention provides anti-TAT antibodies which may find use herein as therapeutic and/or diagnostic agents. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies. [0607]
-
1. Polyclonal Antibodies [0608]
-
Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl[0609] 2, or R1N═C═NR, where R and R1 are different alkyl groups.
-
Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with ⅕ to {fraction (1/10)} the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response. [0610]
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2. Monoclonal Antibodies [0611]
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Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., [0612] Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
-
In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, [0613] Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).
-
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. [0614]
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Preferred fusion partner myelomacells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, [0615] J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
-
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). [0616]
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The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., [0617] Anal. Biochem., 107:220 (1980).
-
Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, [0618] Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g, by i.p. injection of the cells into mice.
-
The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc. [0619]
-
DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as [0620] E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).
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In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., [0621] Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
-
The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (C[0622] H and CL) sequences for the homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
-
3. Human and Humanized Antibodies [0623]
-
The anti-TAT antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)[0624] 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
-
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., [0625] Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
-
The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and HAMA response (human anti-mouse antibody) when the antibody is intended for human therapeutic use. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human V domain sequence which is closest to that of the rodent is identified and the human framework region (FR) within it accepted for the humanized antibody (Sims et al., [0626] J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).
-
It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding. [0627]
-
Various forms of a humanized anti-TAT antibody are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) in order to generate an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody. [0628]
-
As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J[0629] H) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852.
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Alternatively, phage display technology (McCafferty et al., [0630] Nature 348:552-553 [1990]) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al. Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.
-
As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275). [0631]
-
4. Antibody Fragments [0632]
-
In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors. [0633]
-
Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., [0634] Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′)2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab′) 2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab′) fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
-
5. Bispecific Antibodies [0635]
-
Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a TAT protein as described herein. Other such antibodies may combine a TAT binding site with a binding site for another protein. Alternatively, an anti-TAT arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as to focus and localize cellular defense mechanisms to the TAT-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express TAT. These antibodies possess a TAT-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab′)[0636] 2 bispecific antibodies).
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WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fc α antibody is shown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody. [0637]
-
Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., [0638] Nature 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).
-
According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, [0639] C H2, and C H3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.
-
In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., [0640] Methods in Enzymology 121:210 (1986).
-
According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the [0641] C H3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
-
Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques. [0642]
-
Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., [0643] Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
-
Recent progress has facilitated the direct recovery of Fab′-SH fragments from [0644] E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
-
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., [0645] J. Immunol. 147:60 (1991).
-
6. Heteroconjugate Antibodies [0646]
-
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980. [0647]
-
7. Multivalent Antibodies [0648]
-
A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1)[0649] n-VD2-(X2)nFc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
-
8. Effector Function Engineering [0650]
-
It may be desirable to modify the antibody of the invention with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., [0651] J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
-
9. Immunoconjugates [0652]
-
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). [0653]
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Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from [0654] Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as-N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
-
Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein. [0655]
-
Mavtansine and Maytansinoids [0656]
-
In one preferred embodiment, an anti-TAT antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules. [0657]
-
Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub [0658] Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269;4,309,428;4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the disclosures of which are hereby expressly incorporated by reference.
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Maytansinoid-Antibody Conjugates [0659]
-
In an attempt to improve their therapeutic index, maytansine and maytansinoids have been conjugated to antibodies specifically binding to tumor cell antigens. Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 [0660] B 1, the disclosures of which are hereby expressly incorporated by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52:127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3×105 HER-2 surface antigens per cell. The drug conjugate achieved a degree of cytotoxicity similar to the free maytansonid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.
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Anti-TAT Polypeptide Antibody-Maytansinoid Conjugates (Immunoconiugates) [0661]
-
Anti-TAT antibody-maytansinoid conjugates are prepared by chemically linking an Anti-TAT antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove. Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters. [0662]
-
There are many linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 [0663] B 1, and Chari et al., Cancer Research 52:127-131 (1992). The linking groups include disufide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.
-
Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyidithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as [0664] toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 [1978]) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.
-
The linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hyrdoxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue. [0665]
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Calicheamicin [0666]
-
Another immunoconjugate of interest comprises an anti-TAT antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586,5,739,116,5,767,285,5,770,701,5,770,710,5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues of calicheamicin which may be used include, but are not limited to, γ[0667] 1 I,α2 I, α3 I, N-acetyl-γ1 I, PSAG and θI 1, (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents to American Cyanamid). Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.
-
Other Cytotoxic Agents [0668]
-
Other antitumor agents that can be conjugated to the anti-TAT antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296). [0669]
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Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from [0670] Pseudoinonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.
-
The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase). [0671]
-
For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated anti-TAT antibodies. Examples include At[0672] 211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
-
The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc[0673] 99m or I123, Re186, Re188 and In111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes other methods in detail.
-
Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyidithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as [0674] tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a “cleavable linker” facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
-
Alternatively, a fusion protein comprising the anti-TAT antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate. [0675]
-
In yet another embodiment, the antibody may be conjugated to a “receptor” (such streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide). [0676]
-
10. Immunoliposomes [0677]
-
The anti-TAT antibodies disclosed herein may also be formulated as immunoliposomes. A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., [0678] Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
-
Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. [0679]
-
Fab′ fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., [0680] J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81(19):1484 (1989).
-
B. TAT Binding Oligopeptides [0681]
-
TAT binding oligopeptides of the present invention are oligopeptides that bind, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. TAT binding oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such oligopeptides that are capable of binding, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., [0682] J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352:624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).
-
In this regard, bacteriophage (phage) display is one well known technique which allows one to screen large oligopeptide libraries to identify member(s) of those libraries which are capable of specifically binding to a polypeptide target. Phage display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been used for screening millions of polypeptides or oligopeptides for ones with specific binding properties (Smith, G. P. (1991) Current Opin. Biotechnol., 2:668). Sorting phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments. U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,689, and 5,663,143. [0683]
-
Although most phage display methods have used filamentous phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024), T4 phage display systems (Ren, Z-J. et al. (1998) Gene 215:439; Zhu, Z. (1997) CAN 33:534; Jiang, J. et al. (1997) can 128:44380; Ren, Z-J. et al. (1997) CAN 127:215644; Ren, Z-J. (1996) Protein Sci. 5:1833; Efimov, V. P. et al. (1995) Virus Genes 10:173) and T7 phage display systems (Smith, G. P. and Scott, J. K. (1993) Methods in Enzymology, 217, 228-257; U.S. Pat. No. 5,766,905) are also known. [0684]
-
Many other improvements and variations of the basic phage display concept have now been developed. These improvements enhance the ability of display systems to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO 98/14277) and phage display libraries have been used to analyze and control bimolecular interactions (WO 98/20169; WO 98/20159) and properties of constrained helical peptides (WO 98/20036). WO 97/35196 describes a method of isolating an affinity ligand in which a phage display library is contacted with one solution in which the ligand will bind to a target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands. WO 97/46251 describes a method of biopanning a random phage display library with an affinity purified antibody and then isolating binding phage, followed by a micropanning process using microplate wells to isolate high affinity binding phage. The use of [0685] Staphlylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol Biotech., 9:187). WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library which may be a phage display library. A method for selecting enzymes suitable for use in detergents using phage display is described in WO 97/09446. Additional methods of selecting specific binding proteins are described in U.S. Pat. Nos. 5,498,538, 5,432,018, and WO 98/15833.
-
Methods of generating peptide libraries and screening these libraries are also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530; 5,770,434, 5,734,018, 5,698,426, 5,763,192, and 5,723,323. [0686]
-
C. TAT Binding Organic Molecules [0687]
-
TAT binding organic molecules are organic molecules other than oligopeptides or antibodies as defined herein that bind, preferably specifically, to a TAT polypeptide as described herein. TAT binding organic molecules may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are capable of binding, preferably specifically, to a TAT polypeptide as described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules may be, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo compounds, acid chlorides, or the like. [0688]
-
D. Screening for Anti-TAT Antibodies, TAT Binding Oligopeptides and TAT Binding Organic Molecules With the Desired Properties [0689]
-
Techniques for generating antibodies, oligopeptides and organic molecules that bind to TAT polypeptides have been described above. One may further select antibodies, oligopeptides or other organic molecules with certain biological characteristics, as desired. [0690]
-
The growth inhibitory effects of an anti-TAT antibody, oligopeptide or other organic molecule of the invention may be assessed by methods known in the art, e.g., using cells which express a TAT polypeptide either endogenously or following transfection with the TAT gene. For example, appropriate tumor cell lines and TAT-transfected cells may treated with an anti-TAT monoclonal antibody, oligopeptide or other organic molecule of the invention at various concentrations for a few days (e.g., 2-7) days and stained with crystal violet or MTT or analyzed by some other colorimetric assay. Another method of measuring proliferation would be by comparing 3H-thymidine uptake by the cells treated in the presence or absence an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule of the invention. After treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter. Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line. Growth inhibition of tumor cells in vivo can be determined in various ways known in the art. Preferably, the tumor cell is one that overexpresses a TAT polypeptide. Preferably, the anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule will inhibit cell proliferation of a TAT-expressing tumor cell in vitro or in vivo by about 25-100% compared to the untreated tumor cell, more preferably, by about 30-100%, and even more preferably by about 50-100% or 70-100%, in one embodiment, at an antibody concentration of about 0.5 to 30 μg/ml. Growth inhibition can be measured at an antibody concentration of about 0.5 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. The antibody is growth inhibitory in vivo if administration of the anti-TAT antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or reduction of tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days. [0691]
-
To select for an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule which induces cell death, loss of membrane integrity as indicated by, e.g., propidium iodide (PI), trypan blue or 7AAD uptake may be assessed relative to control. A PI uptake assay can be performed in the absence of complement and immune effector cells. TAT polypeptide-expressing tumor cells are incubated with medium alone or medium containing the appropriate anti-TAT antibody (e.g, at about 10 μg/ml), TAT binding oligopeptide or TAT binding organic molecule. The cells are incubated for a 3 day time period. Following each treatment, cells are washed and aliquoted into 35 mm strainer-capped 12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). Those anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules that induce statistically significant levels of cell death as determined by PI uptake may be selected as cell death-inducing anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules. [0692]
-
To screen for antibodies, oligopeptides or other organic molecules which bind to an epitope on a TAT polypeptide bound by an antibody of interest, a routine cross-blocking assay such as that described in [0693] Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if a test antibody, oligopeptide or other organic molecule binds the same site or epitope as a known anti-TAT antibody. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized such as by alanine scanning, to identify contact residues. The mutant antibody is initailly tested for binding with polyclonal antibody to ensure proper folding. In a different method, peptides corresponding to different regions of a TAT polypeptide can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.
-
E. Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT) [0694]
-
The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278. [0695]
-
The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form. [0696]
-
Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as [0697] serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuramimidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs.
-
Alternatively, antibodies with enzymatic activity, also known in the art as “abzymes”, can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, [0698] Nature 328:457-458(1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
-
The enzymes of this invention can be covalently bound to the anti-TAT antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., [0699] Nature 312:604-608 (1984).
-
F. Full-Length TAT Polyyeptides [0700]
-
The present invention also provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as TAT polypeptides. In particular, cDNAs (partial and full-length) encoding various TAT polypeptides have been identified and isolated, as disclosed in further detail in the Examples below. [0701]
-
As disclosed in the Examples below, various cDNA clones have been deposited with the ATCC. The actual nucleotide sequences of those clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill. For the TAT polypeptides and encoding nucleic acids described herein, in some cases, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time. [0702]
-
G. Anti-TAT Antibody and TAT Polypeptide Variants [0703]
-
In addition to the anti-TAT antibodies and full-length native sequence TAT polypeptides described herein, it is contemplated that anti-TAT antibody and TAT polypeptide variants can be prepared. Anti-TAT antibody and TAT polypeptide variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the anti-TAT antibody or TAT polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics. [0704]
-
Variations in the anti-TAT antibodies and TAT polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the anti-TAT antibody or TAT polypeptide. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the anti-TAT antibody or TAT polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. [0705]
-
Anti-TAT antibody and TAT polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native antibody or protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the anti-TAT antibody or TAT polypeptide. [0706]
-
Anti-TAT antibody and TAT polypeptide fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating antibody or polypeptide fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired antibody or polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5′ and 3′ primers in the PCR. Preferably, anti-TAT antibody and TAT polypeptide fragments share at least one biological and/or immunological activity with the native anti-TAT antibody or TAT polypeptide disclosed herein. [0707]
-
In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened.
[0708] | TABLE 6 |
| |
| |
| Original | Exemplary | Preferred |
| Residue | Substitutions | Substitutions |
| |
| Ala (A) | val; leu; ile | val |
| Arg (R) | lys; gln; asn | lys |
| Asn (N) | gln; his; lys; arg | gln |
| Asp (D) | glu | glu |
| Cys (C) | ser | ser |
| Gln (Q) | asn | asn |
| Glu (E) | asp | asp |
| Gly (G) | pro; ala | ala |
| His (H) | asn; gln; lys; arg | arg |
| Ile (I) | leu; val; met; ala; phe; | leu |
| | norleucine |
| Leu (L) | norleucine; ile; val; | ile |
| | met; ala; phe |
| Lys (K) | arg; gln; asn | arg |
| Met (M) | leu; phe; ile | leu |
| Phe (F) | leu; val; ile; ala; tyr | leu |
| Pro (P) | ala | ala |
| Ser (S) | thr | thr |
| Thr (T) | ser | ser |
| Trp (W) | tyr; phe | tyr |
| Tyr (Y) | trp; phe; thr; ser | phe |
| Val (V) | ile; leu; met; phe; | leu |
| | ala; norleucine |
| |
-
Substantial modifications in function or immunological identity of the anti-TAT antibody or TAT polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: [0709]
-
(1) hydrophobic: norleucine, met, ala, val, leu, ile; [0710]
-
(2) neutral hydrophilic: cys, ser, thr; [0711]
-
(3) acidic: asp, glu; [0712]
-
(4) basic: asn, gin, his, lys, arg; [0713]
-
(5) residues that influence chain orientation: gly, pro; and [0714]
-
(6) aromatic: trp, tyr, phe. [0715]
-
Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites. [0716]
-
The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., [0717] Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the anti-TAT antibody or TAT polypeptide variant DNA.
-
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, [0718] Science, 244:1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
-
Any cysteine residue not involved in maintaining the proper conformation of the anti-TAT antibody or TAT polypeptide also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the anti-TAT antibody or TAT polypeptide to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment). [0719]
-
A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human TAT polypeptide. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development. [0720]
-
Nucleic acid molecules encoding amino acid sequence variants of the anti-TAT antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-TAT antibody. [0721]
-
H. Modifications of Anti-TAT Antibodies and TAT Polypeptides [0722]
-
Covalent modifications of anti-TAT antibodies and TAT polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of an anti-TAT antibody or TAT polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the anti-TAT antibody or TAT polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking anti-TAT antibody or TAT polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-TAT antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate. [0723]
-
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains [T. E. Creighton, [0724] Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
-
Another type of covalent modification of the anti-TAT antibody or TAT polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the antibody or polypeptide. “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence anti-TAT antibody or TAT polypeptide (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence anti-TAT antibody or TAT polypeptide. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present. [0725]
-
Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. [0726]
-
Addition of glycosylation sites to the anti-TAT antibody or TAT polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original anti-TAT antibody or TAT polypeptide (for O-linked glycosylation sites). The anti-TAT antibody or TAT polypeptide amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the anti-TAT antibody or TAT polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids. [0727]
-
Another means of increasing the number of carbohydrate moieties on the anti-TAT antibody or TAT polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston, [0728] CRC Crit. Rev. Biochem., pp. 259-306 (1981).
-
Removal of carbohydrate moieties present on the anti-TAT antibody or TAT polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., [0729] Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
-
Another type of covalent modification of anti-TAT antibody or TAT polypeptide comprises linking the antibody or polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The antibody or polypeptide also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in [0730] Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).
-
The anti-TAT antibody or TAT polypeptide of the present invention may also be modified in a way to form chimeric molecules comprising an anti-TAT antibody or TAT polypeptide fused to another, heterologous polypeptide or amino acid sequence. [0731]
-
In one embodiment, such a chimeric molecule comprises a fusion of the anti-TAT antibody or TAT polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the anti-TAT antibody or TAT polypeptide. The presence of such epitope-tagged forms of the anti-TAT antibody or TAT polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the anti-TAT antibody or TAT polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., [0732] Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7,6E10, G4, B7 and 9E 10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et a BioTechnology, 6:1204-1210(1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
-
In an alternative embodiment, the chimeric molecule may comprise a fusion of the anti-TAT antibody or TAT polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of an anti-TAT antibody or TAT polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH[0733] 2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
-
I. Preparation of Anti-TAT Antibodies and TAT Polypeptides [0734]
-
The description below relates primarily to production of anti-TAT antibodies and TAT polypeptides by culturing cells transformed or transfected with a vector containing anti-TAT antibody- and TAT polypeptide-encoding nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare anti-TAT antibodies and TAT polypeptides. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., [0735] Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions. Various portions of the anti-TAT antibody or TAT polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-TAT antibody or TAT polypeptide.
-
1. Isolation of DNA Encoding Anti-TAT Antibody or TAT Polypeptide [0736]
-
DNA encoding anti-TAT antibody or TAT polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the anti-TAT antibody or TAT polypeptide mRNA and to express it at a detectable level. Accordingly, human anti-TAT antibody or TAT polypeptide DNA can be conveniently obtained from a cDNA library prepared from human tissue. The anti-TAT antibody- or TAT polypeptide-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis). [0737]
-
Libraries can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., [0738] Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding anti-TAT antibody or TAT polypeptide is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
-
Techniques for screening a cDNA library are well known in the art. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like [0739] 32P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
-
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein. [0740]
-
Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA. [0741]
-
2. Selection and Transformation of Host Cells [0742]
-
Host cells are transfected or transformed with expression or cloning vectors described herein for anti-TAT antibody or TAT polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in [0743] Mammalian Cell Biotechnology: a Practical Approach M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
-
Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl[0744] 2, CaPO4, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene; 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Pat. No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).
-
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as [0745] E. coli. Various E. coli strains are publicly available, such as E. coli K 12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhinurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1 A2, which has the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3, E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT karn, E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG karn, E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
-
Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) and the immunoconjugate by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half life in circulation. Production in [0746] E. coli is faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), and U.S. Pat. No. 5,840,523 (Simmons et al.) which describes translation initiation regio (TIR) and signal sequences for optimizing expression and secretion, these patents incorporated herein by reference. After expression, the antibody is isolated from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g, in CHO cells.
-
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-TAT antibody- or TAT polypeptide-encoding vectors. [0747] Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology,9:968-975(1991)) such as, e.g., K. lactis(MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichodernia reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomnyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
-
Suitable host cells for the expression of glycosylated anti-TAT antibody or TAT polypeptide are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as [0748] Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
-
However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., [0749] J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
-
Host cells are transformed with the above-described expression or cloning vectors for anti-TAT antibody or TAT polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. [0750]
-
3. Selection and Use of a Replicable Vector [0751]
-
The nucleic acid (e.g., cDNA or genomic DNA) encoding anti-TAT antibody or TAT polypeptide may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. [0752]
-
The TAT may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the anti-TAT antibody- or TAT polypeptide-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including [0753] Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the signal described in WO 90/13646 published 15 Nov. 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
-
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. [0754]
-
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for [0755] Bacilli.
-
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-TAT antibody- or TAT polypeptide-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., [0756] Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the rp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
-
Expression and cloning vectors usually contain a promoter operably linked to the anti-TAT antibody- or TAT polypeptide-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., [0757] Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding anti-TAT antibody or TAT polypeptide.
-
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., [0758] J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
-
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for [0759] alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
-
Anti-TAT antibody or TAT polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems. [0760]
-
Transcription of a DNA encoding the anti-TAT antibody or TAT polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a [0761] position 5′ or 3′ to the anti-TAT antibody or TAT polypeptide coding sequence, but is preferably located at a site 5′ from the promoter.
-
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-TAT antibody or TAT polypeptide. [0762]
-
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of anti-TAT antibody or TAT polypeptide in recombinant vertebrate cell culture are described in Gething et al., [0763] Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
-
4. Culturing the Host Cells [0764]
-
The host cells used to produce the anti-TAT antibody or TAT polypeptide of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., [0765] Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. No. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
-
5. Detecting Gene Amplification/Expression [0766]
-
Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, [0767] Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
-
Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence TAT polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to TAT DNA and encoding a specific antibody epitope. [0768]
-
6. Purification of Anti-TAT Antibody and TAT Polyyeptide Forms of anti-TAT antibody and TAT polypeptide may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of anti-TAT antibody and TAT polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents. [0769]
-
It may be desired to purify anti-TAT antibody and TAT polypeptide from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the anti-TAT antibody and TAT polypeptide. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, [0770] Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular anti-TAT antibody or TAT polypeptide produced.
-
When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., [0771] Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
-
The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., [0772] J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
-
Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt). [0773]
-
J. Pharmaceutical Formulations Therapeutic formulations of the anti-TAT antibodies, TAT binding oligopeptides, TAT binding organic molecules and/or TAT polypeptides used in accordance with the present invention are prepared for storage by mixing the antibody, polypeptide, oligopeptide or organic molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers [0774] Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG). The antibody preferably comprises the antibody at a concentration of between 5-200 mg/ml, preferably between 10-100 mg/ml.
-
The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to an anti-TAT antibody, TAT binding oligopeptide, or TAT binding organic molecule, it may be desirable to include in the one formulation, an additional antibody, e.g., a second anti-TAT antibody which binds a different epitope on the TAT polypeptide, or an antibody to some other target such as a growth factor that affects the growth of the particular cancer. Alternatively, or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. [0775]
-
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in [0776] Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).
-
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. [0777]
-
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. [0778]
-
K. Diagnosis and Treatment with Anti-TAT Antibodies. TAT Binding Oligopeptides and TAT Binding Organic Molecules [0779]
-
To determine TAT expression in the cancer, various diagnostic assays are available. In one embodiment, TAT polypeptide overexpression may be analyzed by immunohistochemistry (IHC). Parrafin embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded a TAT protein staining intensity criteria as follows: [0780]
-
Score 0— no staining is observed or membrane staining is observed in less than 10% of tumor cells. [0781]
-
[0782] Score 1+—a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells. The cells are only stained in part of their membrane.
-
[0783] Score 2+—a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells.
-
[0784] Score 3+—a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells.
-
Those tumors with 0 or 1+scores for TAT polypeptide expression may be characterized as not overexpressing TAT, whereas those tumors with 2+ or 3+scores may be characterized as overexpressing TAT. [0785]
-
Alternatively, or additionally, FISH assays such as the INFORM® (sold by Ventana, Arizona) or PATHVISION® (Vysis, Ill.) may be carried out on formalin-fixed, paraffin-embedded tumor tissue to determine the extent (if any) of TAT overexpression in the tumor. [0786]
-
TAT overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g., by administering a molecule (such as an antibody, oligopeptide or organic molecule) which binds the molecule to be detected and is tagged with a detectable label (e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label. [0787]
-
As described above, the anti-TAT antibodies, oligopeptides and organic molecules of the invention have various non-therapeutic applications. The anti-TAT antibodies, oligopeptides and organic molecules of the present invention can be useful for diagnosis and staging of TAT polypeptide-expressing cancers (e.g., in radioimaging). The antibodies, oligopeptides and organic molecules are also useful for purification or immunoprecipitation of TAT polypeptide from cells, for detection and quantitation of TAT polypeptide in vitro, e.g., in an ELISA or a Western blot, to kill and eliminate TAT-expressing cells from a population of mixed cells as a step in the purification of other cells. [0788]
-
Currently, depending on the stage of the cancer, cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy. Anti-TAT antibody, oligopeptide or organic molecule therapy may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well and in metastatic disease where radiation therapy has limited usefulness. The tumor targeting anti-TAT antibodies, oligopeptides and organic molecules of the invention are useful to alleviate TAT-expressing cancers upon initial diagnosis of the disease or during relapse. For therapeutic applications, the anti-TAT antibody, oligopeptide or organic molecule can be used alone, or in combination therapy with, e.g., hormones, antiangiogens, or radiolabelled compounds, or with surgery, cryotherapy, and/or radiotherapy. Anti-TAT antibody, oligopeptide or organic molecule treatment can be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post-conventional therapy. Chemotherapeutic drugs such as TAXOTERE® (docetaxel), TAXOL® (palictaxel), estramustine and mitoxantrone are used in treating cancer, in particular, in good risk patients. In the present method of the invention for treating or alleviating cancer, the cancer patient can be administered anti-TAT antibody, oligopeptide or organic molecule in conjuction with treatment with the one or more of the preceding chemotherapeutic agents. In particular, combination therapy with palictaxel and modified derivatives (see, e.g., EP0600517) is contemplated. The anti-TAT antibody, oligopeptide or organic molecule will be administered with a therapeutically effective dose of the chemotherapeutic agent. In another embodiment, the anti-TAT antibody, oligopeptide or organic molecule is administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference (PDR) discloses dosages of these agents that have been used in treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician. [0789]
-
In one particular embodiment, a conjugate comprising an anti-TAT antibody, oligopeptide or organic molecule conjugated with a cytotoxic agent is administered to the patient. Preferably, the immunoconjugate bound to the TAT protein is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds. In a preferred embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the cancer cell. Examples of such cytotoxic agents are described above and include maytansinoids, calicheamicins, ribonucleases and DNA endonucleases. [0790]
-
The anti-TAT antibodies, oligopeptides, organic molecules or toxin conjugates thereof are administered to a human patient, in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of the antibody, oligopeptide or organic molecule is preferred. [0791]
-
Other therapeutic regimens may be combined with the administration of the anti-TAT antibody, oligopeptide or organic molecule. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preferably such combined therapy results in a synergistic therapeutic effect. [0792]
-
It may also be desirable to combine administration of the anti-TAT antibody or antibodies, oligopeptides or organic molecules, with administration of an antibody directed against another tumor antigen associated with the particular cancer. [0793]
-
In another embodiment, the therapeutic treatment methods of the present invention involves the combined administration of an anti-TAT antibody (or antibodies), oligopeptides or organic molecules and one or more chemotherapeutic agents or growth inhibitory agents, including co-administration of cocktails of different chemotherapeutic agents. Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992). [0794]
-
The antibody, oligopeptide or organic molecule may be combined with an anti-hormonal compound; e.g., an anti-estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (see, EP 616 812); or an anti-androgen such as flutamide, in dosages known for such molecules. Where the cancer to be treated is androgen independent cancer, the patient may previously have been subjected to anti-androgen therapy and, after the cancer becomes androgen independent, the anti-TAT antibody, oligopeptide or organic molecule (and optionally other agents as described herein) may be administered to the patient. [0795]
-
Sometimes, it may be beneficial to also co-administer a cardioprotectant (to prevent or reduce myocardial dysfunction associated with the therapy) or one or more cytokines to the patient. In addition to the above therapeutic regimes, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy, before, simultaneously with, or post antibody, oligopeptide or organic molecule therapy. Suitable dosages for any of the above co-administered agents are those presently used and may be lowered due to the combined action (synergy) of the agent and anti-TAT antibody, oligopeptide or organic molecule. [0796]
-
For the prevention or treatment of disease, the dosage and mode of administration will be chosen by the physician according to known criteria. The appropriate dosage of antibody, oligopeptide or organic molecule will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody, oligopeptide or organic molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, oligopeptide or organic molecule, and the discretion of the attending physician. The antibody, oligopeptide or organic molecule is suitably administered to the patient at one time or over a series of treatments. Preferably, the antibody, oligopeptide or organic molecule is administered by intravenous infusion or by subcutaneous injections. Depending on the type and severity of the disease, about 1 μg/kg to about 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A dosing regimen can comprise administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the anti-TAT antibody. However, other dosage regimens may be useful. A typical daily dosage might range from about μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art. [0797]
-
Aside from administration of the antibody protein to the patient, the present application contemplates administration of the antibody by gene therapy. Such administration of nucleic acid encoding the antibody is encompassed by the expression “administering a therapeutically effective amount of an antibody”. See, for example, WO96/07321 published Mar. 14, 1996 concerning the use of gene therapy to generate intracellular antibodies. [0798]
-
There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells; in vivo and ex vivo. Forin vivo delivery the nucleic acid is injected directly into the patient, usually at the site where the antibody is required. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retroviral vector. [0799]
-
The currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, for example). For review of the currently known gene marking and gene therapy protocols see Anderson et al., [0800] Science 256:808-813 (1992). See also WO 93/25673 and the references cited therein.
-
The anti-TAT antibodies of the invention can be in the different forms encompassed by the definition of “antibody” herein. Thus, the antibodies include full length or intact antibody, antibody fragments, native sequence antibody or amino acid variants, humanized, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof. In fusion antibodies an antibody sequence is fused to a heterologous polypeptide sequence. The antibodies can be modified in the Fc region to provide desired effector functions. As discussed in more detail in the sections herein, with the appropriate Fc regions, the naked antibody bound on the cell surface can induce cytotoxicity, e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by recruiting complement in complement dependent cytotoxicity, or some other mechanism. Alternatively, where it is desirable to eliminate or reduce effector function, so as to minimize side effects or therapeutic complications, certain other Fc regions may be used. [0801]
-
In one embodiment, the antibody competes for binding or bind substantially to, the same epitope as the antibodies of the invention. Antibodies having the biological characteristics of the present anti-TAT antibodies of the invention are also contemplated, specifically including the in vivo tumor targeting and any cell proliferation inhibition or cytotoxic characteristics. [0802]
-
Methods of producing the above antibodies are described in detail herein. [0803]
-
The present anti-TAT antibodies, oligopeptides and organic molecules are useful for treating a TAT-expressing cancer or alleviating one or more symptoms of the cancer in a mammal. Such a cancer includes prostate cancer, cancer of the urinary tract, lung cancer, breast cancer, colon cancer and ovarian cancer, more specifically, prostate adenocarcinoma, renal cell carcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lung squamous cell carcinomas, and pleural mesothelioma. The cancers encompass metastatic cancers of any of the preceding. The antibody, oligopeptide or organic molecule is able to bind to at least a portion of the cancer cells that express TAT polypeptide in the mammal. In a preferred embodiment, the antibody, oligopeptide or organic molecule is effective to destroy or kill TAT-expressing tumor cells or inhibit the growth of such tumor cells, in vitro or in vivo, upon binding to TAT polypeptide on the cell. Such an antibody includes a naked anti-TAT antibody (not conjugated to any agent). Naked antibodies that have cytotoxic or cell growth inhibition properties can be further harnessed with a cytotoxic agent to render them even more potent in tumor cell destruction. Cytotoxic properties can be conferred to an anti-TAT antibody by, e.g., conjugating the antibody with a cytotoxic agent, to form an immunoconjugate as described herein. The cytotoxic agent or a growth inhibitory agent is preferably a small molecule. Toxins such as calicheamicin or a maytansinoid and analogs or derivatives thereof, are preferable. [0804]
-
The invention provides a composition comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention, and a carrier. For the purposes of treating cancer, compositions can be administered to the patient in need of such treatment, wherein the composition can comprise one or more anti-TAT antibodies present as an immunoconjugate or as the naked antibody. In a further embodiment, the compositions can comprise these antibodies, oligopeptides or organic molecules in combination with other therapeutic agents such as cytotoxic or growth inhibitory agents, including chemotherapeutic agents. The invention also provides formulations comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention, and a carrier. In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier. [0805]
-
Another aspect of the invention is isolated nucleic acids encoding the anti-TAT antibodies. Nucleic acids encoding both the H and L chains and especially the hypervariable region residues, chains which encode the native sequence antibody as well as variants, modifications and humanized versions of the antibody, are encompassed. [0806]
-
The invention also provides methods useful for treating a TAT polypeptide-expressing cancer or alleviating one or more symptoms of the cancer in a mammal, comprising administering a therapeutically effective amount of an anti-TAT antibody, oligopeptide or organic molecule to the mammal. The antibody, oligopeptide or organic molecule therapeutic compositions can be administered short term (acute) or chronic, or intermittent as directed by physician. Also provided are methods of inhibiting the growth of, and killing a TAT polypeptide-expressing cell. [0807]
-
The invention also provides kits and articles of manufacture comprising at least one anti-TAT antibody, oligopeptide or organic molecule. Kits containing anti-TAT antibodies, oligopeptides or organic molecules find use, e.g., for TAT cell killing assays, for purification or immunoprecipitation of TAT polypeptide from cells. For example, for isolation and purification of TAT, the kit can contain an anti-TAT antibody, oligopeptide or organic molecule coupled to beads (e.g., sepharose beads). Kits can be provided which contain the antibodies, oligopeptides or organic molecules for detection and quantitation of TAT in vitro, e.g., in an ELISA or a Western blot. Such antibody, oligopeptide or organic molecule useful for detection may be provided with a label such as a fluorescent or radiolabel. [0808]
-
L. Articles of Manufacture and Kits [0809]
-
Another embodiment of the invention is an article of manufacture containing materials useful for the treatment of anti-TAT expressing cancer. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-TAT antibody, oligopeptide or organic molecule of the invention. The label or package insert indicates that the composition is used for treating cancer. The label or package insert will further comprise instructions for administering the antibody, oligopeptide or organic molecule composition to the cancer patient. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. [0810]
-
Kits are also provided that are useful for various purposes, e.g., for TAT-expressing cell killing assays, for purification or immunoprecipitation of TAT polypeptide from cells. For isolation and purification of TAT polypeptide, the kit can contain an anti-TAT antibody, oligopeptide or organic molecule coupled to beads (e.g., sepharose beads). Kits can be provided which contain the antibodies, oligopeptides or organic molecules for detection and quantitation of TAT polypeptide in vitro, e.g., in an ELISA or a Western blot. As with the article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one anti-TAT antibody, oligopeptide or organic molecule of the invention. Additional containers may be included that contain, e.g., diluents and buffers, control antibodies. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use. [0811]
-
M. Uses for TAT Polypeptides and TAT-Polypeptide Encoding Nucleic Acids [0812]
-
Nucleotide sequences (or their complement) encoding TAT polypeptides have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA probes. TAT-encoding nucleic acid will also be useful for the preparation of TAT polypeptides by the recombinant techniques described herein, wherein those TAT polypeptides may find use, for example, in the preparation of anti-TAT antibodies as described herein. [0813]
-
The full-length native sequence TAT gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length TAT cDNA or to isolate still other cDNAs (for instance, those encoding naturally-occurring variants of TAT or TAT from other species) which have a desired sequence identity to the native TAT sequence disclosed herein. Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherein those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of native sequence TAT. By way of example, a screening method will comprise isolating the coding region of the TAT gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as [0814] 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the TAT gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below. Any EST sequences disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.
-
Other useful fragments of the TAT-encoding nucleic acids include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target TAT mRNA (sense) or TAT DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of TAT DNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen ([0815] Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniclues 6:958, 1988).
-
Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. Such methods are encompassed by the present invention. The antisense oligonucleotides thus may be used to block expression of TAT proteins, wherein those TAT proteins may play a role in the induction of cancer in mammals. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. [0816]
-
Preferred intragenic sites for antisense binding include the region incorporating the translation initiation/start codon (5′-AUG/5′-ATG) or termination/stop codon (5′-UAA, 5′-UAG and 5-UGA/5′-TAA, 5′-TAG and 5′-TGA) of the open reading frame (ORF) of the gene. These regions refer to a portion of the mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation or termination codon. Other preferred regions for antisense binding include: introns; exons; intron-exon junctions; the open reading frame (ORF) or “coding region,” which is the region between the translation initiation codon and the translation termination codon; the 5′ cap of an mRNA which comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage and includes 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap; the 5′ untranslated region (5′UTR), the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene; and the 3′ untranslated region (3′UTR), the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. [0817]
-
Specific examples of preferred antisense compounds useful for inhibiting expression of TAT proteins include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of which is herein incorporated by reference. [0818]
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Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts. Representative United States patents that teach the preparation of such oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of which is herein incorporated by reference. [0819]
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In other preferred antisense oligonucleotides, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500. [0820]
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Preferred antisense oligonucleotides incorporate phosphorothioate backbones and/or heteroatom backbones, and in particular —CH[0821] 2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —O—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] described in the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are antisense oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
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Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkeynyl, or N-alkenyl; O-alkynyl, S-alkynyl or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C[0822] 1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred antisense oligonucleotides comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
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A preferred modification includes 2′-methoxyethoxy(2′-O—CH[0823] 2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2).
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A further prefered modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH[0824] 2—)a group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
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Other preferred modifications include 2′-methoxy(2′-O—CH[0825] 3),2′-aminopropoxy(2′-OCH2CH2CH2 NH2), 2′-allyl(2′-CH2—CH═CH2),2′-O-allyl(2′-O—CH2—CH═CH2) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of which is herein incorporated by reference in its entirety.
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Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH[0826] 3 or —CH2—C≡CH) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi et al, Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. Representative United States patents that teach the preparation of modified nucleobases include, but are not limited to: U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,681,941 and 5,750,692, each of which is herein incorporated by reference.
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Another modification of antisense oligonucleotides chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, cation lipids, phospholipids, cationic phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-[0827] ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) and U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.
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It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Preferred chimeric antisense oligonucleotides incorporate at least one 2′ modified sugar (preferably 2′-O-(CH[0828] 2)2—O—CH3) at the 3′ terminal to confer nuclease resistance and a region with at least 4 contiguous 2′-H sugars to confer RNase H activity. Such compounds have also been referred to in the art as hybrids or gapmers. Preferred gapmers have a region of 2′ modified sugars (preferably 2′-O—(CH2)2—O—CH3) at the 3′-terminal and at the 5′ terminal separated by at least one region having at least 4 contiguous 2′-H sugars and preferably incorporate phosphorothioate backbone linkages. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.
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The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330;4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference. [0829]
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Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence. [0830]
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Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPo[0831] 4 mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. In a preferred procedure, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
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Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. [0832]
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Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase. [0833]
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Antisense or sense RNA or DNA molecules are generally at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term “about” means the referenced nucleotide sequence length plus or minus 10% of that referenced length. [0834]
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The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related TAT coding sequences. [0835]
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Nucleotide sequences encoding a TAT can also be used to construct hybridization probes for mapping the gene which encodes that TAT and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries. [0836]
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When the coding sequences for TAT encode a protein which binds to another protein (example, where the TAT is a receptor), the TAT can be used in assays to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of the receptor/ligand binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Also, the receptor TAT can be used to isolate correlative ligand(s). Screening assays can be designed to find lead compounds that mimic the biological activity of a native TAT or a receptor for TAT. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art. [0837]
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Nucleic acids which encode TAT or its modified forms can also be used to generate either transgenic animals or “knock out” animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding TAT can be used to clone genomic DNA encoding TAT in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding TAT. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for TAT transgene incorporation with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding TAT introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding TAT. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition. [0838]
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Alternatively, non-human homologues of TAT can be used to construct a TAT “knock out” animal which has a defective or altered gene encoding TAT as a result of homologous recombination between the endogenous gene encoding TAT and altered genomic DNA encoding TAT introduced into an embryonic stem cell of the animal. For example, cDNA encoding TAT can be used to clone genomic DNA encoding TAT in accordance with established techniques. A portion of the genomic DNA encoding TAT can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector [see e.g., Thomas and Capecchi, [0839] Cell, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embronic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the TAT polypeptide.
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Nucleic acid encoding the TAT polypeptides may also be used in gene therapy. In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. “Gene therapy” includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al., [0840] Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
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There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., [0841] Trends in Biotechnology 11, 205-210 [1993]). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and gene therapy protocols see Anderson et al., Science 256, 808-813 (1992).
-
The nucleic acid molecules encoding the TAT polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there exists an ongoing need to identify new chromosome markers, since relatively few chromosome marking reagents, based upon actual sequence data are presently available. Each TAT nucleic acid molecule of the present invention can be used as a chromosome marker. [0842]
-
The TAT polypeptides and nucleic acid molecules of the present invention may also be used diagnostically for tissue typing, wherein the TAT polypeptides of the present invention may be differentially expressed in one tissue as compared to another, preferably in a diseased tissue as compared to a normal tissue of the same tissue type. TAT nucleic acid molecules will find use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis. [0843]
-
This invention encompasses methods of screening compounds to identify those that mimic the TAT polypeptide (agonists) or prevent the effect of the TAT polypeptide (antagonists). Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the TAT polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins, including e.g., inhibiting the expression of TAT polypeptide from cells. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. [0844]
-
The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art. [0845]
-
All assays for antagonists are common in that they call for contacting the drug candidate with a TAT polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact. [0846]
-
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the TAT polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the TAT polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the TAT polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex. [0847]
-
If the candidate compound interacts with but does not bind to a particular TAT polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, [0848] Nature (London), 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the “two-hybrid system”) takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GAL 1-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
-
Compounds that interfere with the interaction of a gene encoding a TAT polypeptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner. [0849]
-
To assay for antagonists, the TAT polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the TAT polypeptide indicates that the compound is an antagonist to the TAT polypeptide. Alternatively, antagonists may be detected by combining the TAT polypeptide and a potential antagonist with membrane-bound TAT polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The TAT polypeptide can be labeled, such as by radioactivity, such that the number of TAT polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Coligan et al., [0850] Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the TAT polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the TAT polypeptide. Transfected cells that are grown on glass slides are exposed to labeled TAT polypeptide. The TAT polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
-
As an alternative approach for receptor identification, labeled TAT polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro-sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor. [0851]
-
In another assay for antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled TAT polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured. [0852]
-
More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with TAT polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the TAT polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the TAT polypeptide. [0853]
-
Another potential TAT polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes the mature TAT polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., [0854] Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science, 251:1360 (1991)), thereby preventing transcription and the production of the TAT polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the TAT polypeptide (antisense-Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, Fla., 1988). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the TAT polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about −10 and +10 positions of the target gene nucleotide sequence, are preferred.
-
Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the TAT polypeptide, thereby blocking the normal biological activity of the TAT polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds. [0855]
-
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, [0856] Current Biology, 4:469-471 (1994), and PCT publication No. WO 97/33551 (published Sep. 18, 1997).
-
Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551, supra. [0857]
-
These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art. [0858]
-
Isolated TAT polypeptide-encoding nucleic acid can be used herein for recombinantly producing TAT polypeptide using techniques well known in the art and as described herein. In turn, the produced TAT polypeptides can be employed for generating anti-TAT antibodies using techniques well known in the art and as described herein. [0859]
-
Antibodies specifically binding a TAT polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders, including cancer, in the form of pharmaceutical compositions. [0860]
-
If the TAT polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., [0861] Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
-
The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. [0862]
-
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. [0863]
-
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. [0864]
EXAMPLES
-
Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, Va. [0865]
Example 1
Tissue Expression Profiling Using GeneExgress®
-
A proprietary database containing gene expression information (GeneExpress®, Gene Logic Inc., Gaithersburg, Md.) was analyzed in an attempt to identify polypeptides (and their encoding nucleic acids) whose expression is significantly upregulated in a particular tumor tissue(s) of interest as compared to other tumor(s) and/or normal tissues. Specifically, analysis of the GeneExpress® database was conducted using either software available through Gene Logic Inc., Gaithersburg, Md., for use with the GeneExpress® database or with proprietary software written and developed at Genentech, Inc. for use with the GeneExpress® database. The rating of positive hits in the analysis is based upon several criteria including, for example, tissue specificity, tumor specificity and expression level in normal essential and/or normal proliferating tissues. The following is a list of molecules whose tissue expression profile as determined from an analysis of the GeneExpress® database evidences high tissue expression and significant upregulation of expression in a specific tumor or tumors as compared to other tumor(s) and/or normal tissues and optionally relatively low expression in normal essential and/or normal proliferating tissues. As such, the molecules listed below are excellent polypeptide targets for the diagnosis and therapy of cancer in mammals.
[0866] |
|
Molecule | upregulation of expression in: | as compared to: |
|
DNA96792 (TAT239) | colon tumor | normal colon tissue |
DNA96792 (TAT239) | rectum tumor | normal rectum tissue |
DNA96792 (TAT239) | pancreas tumor | normal pancreas tissue |
DNA96792 (TAT239) | lung tumor | normal lung tissue |
DNA96792 (TAT239) | stomach tumor | normal stomach tissue |
DNA96792 (TAT239) | esophagus tumor | normal esophagus tissue |
DNA96792 (TAT239) | breast tumor | normal breast tissue |
DNA96792 (TAT239) | uterus tumor | normal uterus tissue |
DNA225793 (TAT223) | ovarian tumor | normal ovarian tissue |
DNA225793 (TAT223) | kidney tumor | normal kidney tissue |
DNA227611 (TAT175) | prostate tumor | normal prostate tissue |
DNA227611 (TAT175) | colon tumor | normal colon tissue |
DNA227611 (TAT175) | breast tumor | normal breast tissue |
DNA261021 (TAT208) | breast tumor | normal breast tissue |
DNA260655 (TAT209) | lung tumor | normal lung tissue |
DNA260655 (TAT209) | colon tumor | normal colon tissue |
DNA260655 (TAT209) | breast tumor | normal breast tissue |
DNA260655 (TAT209) | liver tumor | normal liver tissue |
DNA260655 (TAT209) | ovarian tumor | normal ovarian tissue |
DNA260655 (TAT209) | skin tumor | normal skin tissue |
DNA260655 (TAT209) | spleen tumor | normal spleen tissue |
DNA260655 (TAT209) | myeloid tumor | normal myeloid tissue |
DNA260655 (TAT209) | muscle tumor | normal muscle tissue |
DNA260655 (TAT209) | bone tumor | normal bone tissue |
DNA261001 (TAT181) | bone tumor | normal bone tissue |
DNA261001 (TAT181) | lung tumor | normal lung tissue |
DNA266928 (TAT182) | bone tumor | normal bone tissue |
DNA266928 (TAT182) | lung tumor | normal lung tissue |
DNA268035 (TAT222) | breast tumor | normal breast tissue |
DNA268035 (TAT222) | colon tumor | normal colon tissue |
DNA268035 (TAT222) | ovarian tumor | normal ovarian tissue |
DNA268035 (TAT222) | uterine tumor | normal uterine tissue |
DNA77509 (TAT177) | colon tumor | normal colon tissue |
DNA87993 (TAT235) | breast tumor | normal breast tissue |
DNA87993 (TAT235) | pancreatic tumor | normal pancreatic tissue |
DNA87993 (TAT235) | lung tumor | normal lung tissue |
DNA87993 (TAT235) | colon tumor | normal colon tissue |
DNA87993 (TAT235) | rectum tumor | normal rectum tissue |
DNA87993 (TAT235) | gallbladder tumor | normal gallbladder tissue |
DNA92980 (TAT234) | bone tumor | normal bone tissue |
DNA92980 (TAT234) | breast tumor | normal breast tissue |
DNA92980 (TAT234) | cervical tumor | normal cervical tissue |
DNA92980 (TAT234) | colon tumor | normal colon tissue |
DNA92980 (TAT234) | rectum tumor | normal rectum tissue |
DNA92980 (TAT234) | endometrial tumor | normal endometrial tissue |
DNA92980 (TAT234) | liver tumor | normal liver tissue |
DNA92980 (TAT234) | lung tumor | normal lung tissue |
DNA92980 (TAT234) | ovarian tumor | normal ovarian tissue |
DNA92980 (TAT234) | pancreatic tumor | normal pancreatic tissue |
DNA92980 (TAT234) | skin tumor | normal skin tissue |
DNA92980 (TAT234) | soft tissue tumor | normal soft tissue |
DNA92980 (TAT234) | stomach tumor | normal stomach tissue |
DNA92980 (TAT234) | bladder tumor | normal bladder tissue |
DNA92980 (TAT234) | thyroid tumor | normal thyroid tissue |
DNA105792 (TAT233) | bone tumor | normal bone tissue |
DNA105792 (TAT233) | breast tumor | normal breast tissue |
DNA105792 (TAT233) | endometrial tumor | normal endometrial tissue |
DNA105792 (TAT233) | esophagus tumor | normal esophagus tissue |
DNA105792 (TAT233) | kidney tumor | normal kidney tissue |
DNA105792 (TAT233) | lung tumor | normal lung tissue |
DNA105792 (TAT233) | ovarian tumor | normal ovarian tissue |
DNA105792 (TAT233) | pancreatic tumor | normal pancreatic tissue |
DNA105792 (TAT233) | prostate tumor | normal prostate tissue |
DNA105792 (TAT233) | soft tissue tumor | normal soft tissue |
DNA105792 (TAT233) | stomach tumor | normal stomach tissue |
DNA105792 (TAT233) | thyroid tumor | normal thyroid tissue |
DNA105792 (TAT233) | bladder tumor | normal bladder tissue |
DNA105792 (TAT233) | brain tumor | normal brain tissue |
DNA105792 (TAT233) | Wilm's tumor | normal associated tissue |
DNA119474 (TAT228) | uterine tumor | normal uterine tissue |
DNA119474 (TAT228) | ovarian tumor | normal ovarian tissue |
DNA280351 (TAT248) | squamous cell lung tumor | normal squamous cell lung |
| | tissue |
DNA280351 (TAT248) | colon tumor | normal colon tissue |
DNA150648 (TAT232) | liver tumor | normal liver tissue |
DNA150648 (TAT232) | breast tumor | normal breast tissue |
DNA150648 (TAT232) | brain tumor | normal brain tissue |
DNA150648 (TAT232) | lung tumor | normal lung tissue |
DNA150648 (TAT232) | colon tumor | normal colon tissue |
DNA150648 (TAT232) | rectum tumor | normal rectum tissue |
DNA150648 (TAT232) | kidney tumor | normal kidney tissue |
DNA150648 (TAT232) | bladder tumor | normal bladder tissue |
DNA179651 (TAT224) | breast tumor | normal breast tissue |
DNA179651 (TAT224) | cervical tumor | normal cervical tissue |
DNA179651 (TAT224) | colon tumor | normal colon tissue |
DNA179651 (TAT224) | rectum tumor | normal rectum tissue |
DNA179651 (TAT224) | uterine tumor | normal uterine tissue |
DNA179651 (TAT224) | lung tumor | normal lung tissue |
DNA179651 (TAT224) | ovarian tumor | normal ovarian tissue |
DNA207698 (TAT237) | breast tumor | normal breast tissue |
DNA207698 (TAT237) | colon tumor | normal colon tissue |
DNA207698 (TAT237) | ovarian tumor | normal ovarian tissue |
DNA207698 (TAT237) | pancreatic tumor | normal pancreatic tissue |
DNA207698 (TAT237) | stomach tumor | normal stomach tissue |
DNA225886 (TAT236) | breast tumor | normal breast tissue |
DNA225886 (TAT236) | colon tumor | normal colon tissue |
DNA225886 (TAT236) | rectum tumor | normal rectum tissue |
DNA225886 (TAT236) | endometrial tumor | normal endometrial tissue |
DNA225886 (TAT236) | lung tumor | normal lung tissue |
DNA225886 (TAT236) | ovarian tumor | normal ovarian tissue |
DNA225886 (TAT236) | pancreas tumor | normal pancreas tissue |
DNA225886 (TAT236) | prostate tumor | normal prostate tissue |
DNA225886 (TAT236) | bladder tumor | normal bladder tissue |
DNA226717 (TAT185) | glioma | normal glial tissue |
DNA226717 (TAT185) | brain tumor | normal brain tissue |
DNA227162 (TAT225) | breast tumor | normal breast tissue |
DNA227162 (TAT225) | endometrial tumor | normal endometrial tissue |
DNA227162 (TAT225) | lung tumor | normal lung tissue |
DNA227162 (TAT225) | ovarian tumor | normal ovarian tissue |
DNA277804 (TAT247) | breast tumor | normal breast tissue |
DNA277804 (TAT247) | endometrial tumor | normal endometrial tissue |
DNA277804 (TAT247) | lung tumor | normal lung tissue |
DNA277804 (TAT247) | ovarian tumor | normal ovarian tissue |
DNA233034 (TAT174) | glioma | normal glial tissue |
DNA233034 (TAT174) | brain tumor | normal brain tissue |
DNA266920 (TAT214) | glioma | normal glial tissue |
DNA266920 (TAT214) | brain tumor | normal brain tissue |
DNA266921 (TAT220) | glioma | normal glial tissue |
DNA266921 (TAT220) | brain tumor | normal brain tissue |
DNA266922 (TAT221) | glioma | normal glial tissue |
DNA266922 (TAT221) | brain tumor | normal brain tissue |
DNA234441 (TAT201) | colon tumor | normal colon tissue |
DNA234441 (TAT201) | rectum tumor | normal rectum tissue |
DNA234834 (TAT179) | breast tumor | normal breast tissue |
DNA234834 (TAT179) | colon tumor | normal colon tissue |
DNA234834 (TAT179) | rectum tumor | normal rectum tissue |
DNA234834 (TAT179) | prostate tumor | normal prostate tissue |
DNA234834 (TAT179) | pancreatic tumor | normal pancreatic tissue |
DNA234834 (TAT179) | endometrial tumor | normal endometrial tissue |
DNA234834 (TAT179) | lung tumor | normal lung tissue |
DNA234834 (TAT179) | ovarian tumor | normal ovarian tissue |
DNA247587 (TAT216) | breast tumor | normal breast tissue |
DNA247587 (TAT216) | lung tumor | normal lung tissue |
DNA247587 (TAT216) | ovarian tumor | normal ovarian tissue |
DNA247587 (TAT216) | pancreatic tumor | normal pancreatic tissue |
DNA247587 (TAT216) | stomach tumor | normal stomach tissue |
DNA247587 (TAT216) | urinary tumor | normal urinary tissue |
DNA255987 (TAT218) | breast tumor | normal breast tissue |
DNA56041 (TAT206) | lymphoid tumor | normal lymphoid tissue |
DNA257845 (TAT374) | lymphoid tumor | normal lymphoid tissue |
DNA247476 (TAT180) | bone tumor | normal bone tissue |
DNA247476 (TAT180) | breast tumor | normal breast tissue |
DNA247476 (TAT180) | colon tumor | normal colon tissue |
DNA247476 (TAT180) | rectum tumor | normal rectum tissue |
DNA247476 (TAT180) | kidney tumor | normal kidney tissue |
DNA247476 (TAT180) | lung tumor | normal lung tissue |
DNA247476 (TAT180) | pancreatic tumor | normal pancreatic tissue |
DNA247476 (TAT180) | prostate tumor | normal prostate tissue |
DNA247476 (TAT180) | skin tumor | normal skin tissue |
DNA247476 (TAT180) | soft tissue tumor | normal soft tissue |
DNA247476 (TAT180) | stomach tumor | normal stomach tissue |
DNA260990 (TAT375) | bone tumor | normal bone tissue |
DNA260990 (TAT375) | breast tumor | normal breast tissue |
DNA260990 (TAT375) | colon tumor | normal colon tissue |
DNA260990 (TAT375) | rectum tumor | normal rectum tissue |
DNA260990 (TAT375) | kidney tumor | normal kidney tissue |
DNA260990 (TAT375) | lung tumor | normal lung tissue |
DNA260990 (TAT375) | pancreatic tumor | normal pancreatic tissue |
DNA260990 (TAT375) | prostate tumor | normal prostate tissue |
DNA260990 (TAT375) | skin tumor | normal skin tissue |
DNA260990 (TAT375) | soft tissue tumor | normal soft tissue |
DNA260990 (TAT375) | stomach tumor | normal stomach tissue |
DNA261013 (TAT176) | breast tumor | normal breast tissue |
DNA261013 (TAT176) | colon tumor | normal colon tissue |
DNA261013 (TAT176) | rectum tumor | normal rectum tissue |
DNA261013 (TAT176) | lung tumor | normal lung tissue |
DNA261013 (TAT176) | ovarian tumor | normal ovarian tissue |
DNA261013 (TAT176) | stomach tumor | normal stomach tissue |
DNA262144 (TAT184) | breast tumor | normal breast tissue |
DNA262144 (TAT184) | colon tumor | normal colon tissue |
DNA262144 (TAT184) | rectum tumor | normal rectum tissue |
DNA262144 (TAT184) | endometrial tumor | normal endometrial tissue |
DNA262144 (TAT184) | kidney tumor | normal kidney tissue |
DNA262144 (TAT184) | lung tumor | normal lung tissue |
DNA262144 (TAT184) | ovarian tumor | normal ovarian tissue |
DNA267342 (TAT213) | stroma associated with the following | normal associated tissues, |
| tumors: bone, breast, colon, rectum, | respectively |
| lung, ovarian, pancreas, soft tissue, |
| bladder |
DNA267626 (TAT217) | breast tumor | normal breast tissue |
DNA267626 (TAT217) | colon tumor | normal colon tissue |
DNA267626 (TAT217) | rectum tumor | normal rectum tissue |
DNA267626 (TAT217) | endometrial tumor | normal endometrial tissue |
DNA267626 (TAT217) | lung tumor | normal lung tissue |
DNA267626 (TAT217) | pancreatic tumor | normal pancreatic tissue |
DNA268334 (TAT202) | kidney tumor | normal kidney tissue |
DNA269238 (TAT215) | kidney tumor | normal kidney tissue |
DNA272578 (TAT238) | liver tumor | normal liver tissue |
DNA272578 (TAT238) | lung tumor | normal lung tissue |
DNA272578 (TAT238) | ovarian tumor | normal ovarian tissue |
DNA304853 (TAT376) | breast tumor | normal breast tissue |
DNA304853 (TAT376) | colon tumor | normal colon tissue |
DNA304853 (TAT376) | rectum tumor | normal rectum tissue |
DNA304853 (TAT376) | prostate tumor | normal prostate tissue |
DNA304853 (TAT376) | pancreatic tumor | normal pancreatic tissue |
DNA304853 (TAT376) | endometrial tumor | normal endometrial tissue |
DNA304853 (TAT376) | lung tumor | normal lung tissue |
DNA304853 (TAT376) | ovarian tumor | normal ovarian tissue |
DNA304854 (TAT377) | breast tumor | normal breast tissue |
DNA304854 (TAT377) | colon tumor | normal colon tissue |
DNA304854 (TAT377) | rectum tumor | normal rectum tissue |
DNA304854 (TAT377) | prostate tumor | normal prostate tissue |
DNA304854 (TAT377) | pancreatic tumor | normal pancreatic tissue |
DNA304854 (TAT377) | endometrial tumor | normal endometrial tissue |
DNA304854 (TAT377) | lung tumor | normal lung tissue |
DNA304854 (TAT377) | ovarian tumor | normal ovarian tissue |
DNA304855 (TAT378) | breast tumor | normal breast tissue |
DNA304855 (TAT378) | colon tumor | normal colon tissue |
DNA304855 (TAT378) | rectum tumor | normal rectum tissue |
DNA304855 (TAT378) | prostate tumor | normal prostate tissue |
DNA304855 (TAT378) | pancreatic tumor | normal pancreatic tissue |
DNA304855 (TAT378) | endometrial tumor | normal endometrial tissue |
DNA304855 (TAT378) | lung tumor | normal lung tissue |
DNA304855 (TAT378) | ovarian tumor | normal ovarian tissue |
DNA287971 (TAT379) | bone tumor | normal bone tissue |
DNA287971 (TAT379) | breast tumor | normal breast tissue |
DNA287971 (TAT379) | colon tumor | normal colon tissue |
DNA287971 (TAT379) | rectum tumor | normal rectum tissue |
DNA287971 (TAT379) | kidney tumor | normal kidney tissue |
DNA287971 (TAT379) | lung tumor | normal lung tissue |
DNA287971 (TAT379) | pancreatic tumor | normal pancreatic tissue |
DNA287971 (TAT379) | prostate tumor | normal prostate tissue |
DNA287971 (TAT379) | skin tumor | normal skin tissue |
DNA287971 (TAT379) | soft tissue tumor | normal soft tissue |
DNA287971 (TAT379) | stomach tumor | normal stomach tissue |
|
Example 2
Microarray Analysis to Detect Upregulation of TAT Polypeptides in Cancerous Tumors
-
Nucleic acid microarrays, often containing thousands of gene sequences, are useful for identifying differentially expressed genes in diseased tissues as compared to their normal counterparts. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The cDNA probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes known to be expressed in certain disease states may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. If the hybridization signal of a probe from a test (disease tissue) sample is greater than hybridization signal of a probe from a control (normal tissue) sample, the gene or genes overexpressed in the disease tissue are identified. The implication of this result is that an overexpressed protein in a diseased tissue is useful not only as a diagnostic marker for the presence of the disease condition, but also as a therapeutic target for treatment of the disease condition. [0867]
-
The methodology of hybridization of nucleic acids and microarray technology is well known in the art. In one example, the specific preparation of nucleic acids for hybridization and probes, slides, and hybridization conditions are all detailed in PCT Patent Application Serial No. PCT/US01/10482, filed on Mar. 30, 2001 and which is herein incorporated by reference. [0868]
-
In the present example, cancerous tumors derived from various human tissues were studied for upregulated gene expression relative to cancerous tumors from different tissue types and/or non-cancerous human tissues in an attempt to identify those polypeptides which are overexpressed in a particular cancerous tumor(s). In certain experiments, cancerous human tumor tissue and non-cancerous human tumor tissue of the same tissue type (often from the same patient) were obtained and analyzed for TAT polypeptide expression. Additionally, cancerous human tumor tissue from any of a variety of different human tumors was obtained and compared to a “universal” epithelial control sample which was prepared by pooling non-cancerous human tissues of epithelial origin, including liver, kidney, and lung. mRNA isolated from the pooled tissues represents a mixture of expressed gene products from these different tissues. Microarray hybridization experiments using the pooled control samples generated a linear plot in a 2-color analysis. The slope of the line generated in a 2-color analysis was then used to normalize the ratios of (test:control detection) within each experiment. The normalized ratios from various experiments were then compared and used to identify clustering of gene expression. Thus, the pooled “universal control” sample not only allowed effective relative gene expression determinations in a simple 2-sample comparison, it also allowed multi-sample comparisons across several experiments. [0869]
-
In the present experiments, nucleic acid probes derived from the herein described TAT polypeptide-encoding nucleic acid sequences were used in the creation of the microarray and RNA from various tumor tissues were used for the hybridization thereto. Below is shown the results of these experiments, demonstrating that various TAT polypeptides of the present invention are significantly overexpressed in various human tumor tissues as compared to their normal counterpart tissue(s). Moreover, all of the molecules shown below are significantly overexpressed in their specific tumor tissue(s) as compared to in the “universal” epithelial control. As described above, these data demonstrate that the TAT polypeptides of the present invention are useful not only as diagnostic markers for the presence of one or more cancerous tumors, but also serve as therapeutic targets for the treatment of those tumors.
[0870] |
|
| upregulation of | |
Molecule | expression in: | as compared to: |
|
DNA172500 | renal cell carcinoma | normal kidney (renal cell) |
(TAT219) | | tissue |
|
Example 3
Quantitative Analysis of TAT mRNA Expression
-
In this assay, a 5′ nuclease assay (for example, TaqMan®) and real-time quantitative PCR (for example, ABI Prizm 7700 Sequence Detection System® (Perkin Elmer, Applied Biosystems Division, Foster City, Calif.)), were used to find genes that are significantly overexpressed in a cancerous tumor or tumors as compared to other cancerous tumors or normal non-cancerous tissue. The 5′ nuclease assay reaction is a fluorescent PCR-based technique which makes use of the 5′ exonuclease activity of Taq DNA polymerase enzyme to monitor gene expression in real time. Two oligonucleotide primers (whose sequences are based upon the gene or EST sequence of interest) are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the PCR amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data. [0871]
-
The 5′ nuclease procedure is run on a real-time quantitative PCR device such as the ABI Prism 7700™ Sequence Detection. The system consists of a thermocycler, laser, charge-coupled device (CCD) camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data. [0872]
-
The starting material for the screen was mRNA isolated from a variety of different cancerous tissues. The mRNA is quantitated precisely, e.g., fluorometrically. As a negative control, RNA was isolated from various normal tissues of the same tissue type as the cancerous tissues being tested. [0873]
-
5′ nuclease assay data are initially expressed as Ct, or the threshold cycle. This is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence. The ΔCt values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer mRNA results to normal human mRNA results. As one Ct unit corresponds to 1 PCR cycle or approximately a 2-fold relative increase relative to normal, two units corresponds to a 4-fold relative increase, 3 units corresponds to an 8-fold relative increase and so on, one can quantitatively measure the relative fold increase in mRNA expression between two or more different tissues. Using this technique, the molecules listed below have been identified as being significantly overexpressed in a particular tumor(s) as compared to their normal non-cancerous counterpart tissue(s) (from both the same and different tissue donors) and thus, represent excellent polypeptide targets for the diagnosis and therapy of cancer in mammals.
[0874] |
|
| upregulation of | |
Molecule | expression in: | as compared to: |
|
DNA261021 (TAT208) | lung tumor | normal lung tissue |
DNA77509 (TAT177) | colon tumor | normal colon tissue |
DNA119474 (TAT226) | ovarian tumor | normal ovarian tissue |
DNA179651 (TAT224) | ovarian tumor | normal ovarian tissue |
DNA226717 (TAT185) | glioma | normal glial/brain tissue |
DNA227162 (TAT225) | ovarian tumor | normal ovarian tissue |
DNA277804 (TAT247) | ovarian tumor | normal ovarian tissue |
DNA233034 (TAT174) | glioma | normal glial/brain tissue |
DNA266920 (TAT214) | glioma | normal glial/brain tissue |
DNA266921 (TAT220) | glioma | normal glial/brain tissue |
DNA266922 (TAT221) | glioma | normal glial/brain tissue |
DNA234441 (TAT201) | colon tumor | normal colon tissue |
DNA234834 (TAT179) | colon tumor | normal colon tissue |
DNA247587 (TAT216) | squamous cell | normal squamous |
| lung tumor | cell lung tissue |
DNA255987 (TAT218) | breast tumor | normal breast tissue |
DNA247476 (TAT180) | colon tumor | normal colon tissue |
DNA260990 (TAT375) | colon tumor | normal colon tissue |
DNA261013 (TAT176) | breast tumor | normal breast tissue |
DNA262144 (TAT184) | kidney tumor | normal kidney tissue |
DNA267342 (TAT213) | breast tumor | normal breast tissue |
DNA267626 (TAT217) | breast tumor | normal breast tissue |
DNA268334 (TAT202) | kidney tumor | normal kidney tissue |
DNA269238 (TAT215) | kidney tumor | normal kidney tissue |
DNA87993 (TAT235) | lung tumor | normal lung tissue |
DNA92980 (TAT234) | ovarian tumor | normal ovarian tissue |
DNA105792 (TAT233) | lung tumor | normal lung tissue |
DNA207698 (TAT237) | colon tumor | normal colon tissue |
DNA225886 (TAT236) | colon tumor | normal colon tissue |
DNA272578 (TAT238) | ovarian tumor | normal ovarian tissue |
DNA304853 (TAT376) | colon tumor | normal colon tissue |
DNA304854 (TAT377) | colon tumor | normal colon tissue |
DNA304855 (TAT378) | colon tumor | normal colon tissue |
DNA287971 (TAT379) | colon tumor | normal colon tissue |
|
Example 4
In Situ Hybridization
-
In situ hybridization is apowerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping. [0875]
-
In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, [0876] Cell Vision 1:169-176 (1994), using PCR-generated 33P-labeled riboprobes. Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., and further processed for in situ hybridization as described by Lu and Gillett, supra. A [33-P] UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55° C. overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
-
[0877] 33P-Riboprobe synthesis
-
6.0 μl (125 mCi) of [0878] 33P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed vac dried. To each tube containing dried 33P-UTP, the following ingredients were added:
-
2.0 [0879] μl 5× transcription buffer
-
1.0 μl DTT (100 mM) [0880]
-
2.0 μl NTP mix (2.5 mM: 10μ; each of 10 mM GTP, CTP & ATP+10 μl H[0881] 2O)
-
1.0 μl UTP (50 μM) [0882]
-
1.0 μl Rnasin [0883]
-
1.0 μDNA template (1 μg) [0884]
-
1.0 μl H[0885] 2O
-
1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually) [0886]
-
The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNase were added, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a Microcon-50 ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 μl TE were added. 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of Biofluor II. [0887]
-
The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNA Mrk III were added to 3 μl of loading buffer. After heating on a 95° C. heat block for three minutes, the probe was immediately placed on ice. The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes. The gel was wrapped in saran wrap and exposed to XAR film with an intensifying screen in −70° C. freezer one hour to overnight. [0888]
-
[0889] 33P-Hybridization
-
A. Pretreatment of Frozen Sections [0890]
-
The slides were removed from the freezer, placed on aluminium trays and thawed at room temperature for 5 minutes. The trays were placed in 55° C. incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975 ml SQ H[0891] 2O). After deproteination in 0.5 μg/ml proteinase K for 10 minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the sections were washed in 0.5×SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, 100% ethanol, 2 minutes each.
-
B. Pretreatment of Paraffin-Embedded Sections [0892]
-
The slides were deparaffinized, placed in SQ H[0893] 2O, and rinsed twice in 2×SSC at room temperature, for 5 minutes each time. The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml RNase-free RNase buffer; 37° C., 15 minutes)-human embryo, or 8× proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30 minutes)-formalin tissues. Subsequent rinsing in 0.5×SSC and dehydration were performed as described above.
-
C. Prehybridization [0894]
-
The slides were laid out in a plastic box lined with Box buffer (4×SSC, 50% formamide)-saturated filter paper. [0895]
-
D. Hybridization [0896]
-
1.0×10[0897] 6 cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heated at 95° C. for 3 minutes. The slides were cooled on ice, and 48 μl hybridization buffer were added per slide. After vortexing, 50 μl 33P mix were added to 50 μl prehybridization on slide. The slides were incubated overnight at 55° C.
-
E. Washes [0898]
-
Washing was done 2×10 minutes with 2×SSC, EDTA at room temperature (400 ml 20×SSC+16 ml 0.25M EDTA, V[0899] f=4 L), followed by RNaseA treatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer=20 μg/ml), The slides were washed 2×10 minutes with 2×SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, Vf=4 L).
-
F. Oligonucleotides [0900]
-
In situ analysis was performed on a variety of DNA sequences disclosed herein. The oligonucleotides employed for these analyses were obtained so as to be complementary to the nucleic acids (or the complements thereof) as shown in the accompanying figures. [0901]
-
G. Results [0902]
-
In situ analysis was performed on a variety of DNA sequences disclosed herein. The results from these analyses are as follows. [0903]
-
(1) DNA 19474 (TAT226) [0904]
-
Positive expression is observed in 2 of 3 non-small cell lung carcinomsa, 2 of 3 pancreatic adenocarcinomas, 1 of 2 hepatocellular carcinomas and 2 of 3 endometrial adenocarcinomas. In a separate analysis, 10 of 16 ovarian adenocarcinomas are positive and 3 of 9 endometrial adenocarcinomas are positive. All normal tissues examined are negative for expression. [0905]
-
(2) DNA179651 (TAT224) [0906]
-
In one analysis, expression is seen in 5 of 7 uterine adenocarcinomas and in 7 of 16 ovarian adenocarcinomas. Two cases of dysgerminoma are positive as is one case of a Brenner's tumor. [0907]
-
In another analysis, 33 of 68 ovarian adenocarcinomas (serous, mucinous, endometrioid, clear cell) are positive for expression. Moderate to strong expression is seen in normal endometrium (no other normal tissues) and normal ovarian stroma is negative. [0908]
-
In yet another analysis, positive:expression is seen in {fraction (3/3)} endometrial, {fraction (2/2)} colorectal, ⅓ transitional cell, {fraction (3/3)} lung and ½ ovarian cancers. [0909]
-
(3) DNA227162 (TAT225) [0910]
-
Expression is seen in the following tumors: 1 of 3 lung cancers, 1 of 2 colon cancers, 1 of 1 pancreatic cancer, 2 of 3 transitional cell carcinomas, 3 of 3 endometrial carcinomas, 2 of 2 ovarian carcinomas and 2 of 3 malignant melanomas. [0911]
-
In a separate analysis, positive expression is seen in 6 of 9 uterine adenocarcinomas and 6 of 14 ovarian tumors. [0912]
-
With regard to expression in normal tissues, weak expression is seen in one core of urothelium (superficial cell layer positive) and one core of gall bladder mucosa. All other normal tissues are negative for expression. [0913]
-
(4) DNA277804 (TAT247) [0914]
-
Expression is seen in the following tumors: 1 of 3 lung cancers, 1 of 2 colon cancers, 1 of 1 pancreatic cancer, 2 of 3 transitional cell carcinomas, 3 of 3 endometrial carcinomas, 2 of 2 ovarian carcinomas and 2 of 3 malignant melanomas. [0915]
-
In a separate analysis, positive expression is seen in 6 of 9 uterine adenocarcinomas and 6 of 14 ovarian tumors. [0916]
-
With regard to expression in normal tissues, weak expression is seen in one core of urothelium (superficial cell layer positive) and one core of gall bladder mucosa. All other normal tissues are negative for expression. [0917]
-
(5) DNA234441 (TAT201) [0918]
-
Weak (and inconsistent) expression is seen in normal kidney, normal colon mucosa and normal gallbladder. Weak to moderate, though somewhat inconsistent expression is seen in normal gastrointestinal mucosa (esophagus, stomach, small intestine, colon, anus). Significant expression in tumors is seen as follows: 11 of 12 colorectal adenocarcinomas, 4 of 4 gastric adenocarcinomas, 6 of 8 metastatic adenocarcinomas, 4 of 4 esophageal cancers and 1 of 2 pancreatic adenocarcinomas. [0919]
-
(6) DNA234834 (TAT179) [0920]
-
With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas. [0921]
-
In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues. [0922]
-
(7) DNA247587 (TAT216) [0923]
-
Expression is seen in 13 of 16 non-small cell lung carcinomas. Expression is also seen in benign bronchial mucosa and occasional activated pneumocytes. Moreover, 65 of 89 cases of invasive breast cancer are positive for expression. Strong expression is seen in normal skin and normal urothelium. Moderate expression is seen in normal mammary epithelium and trophoblasts of the placenta, weak expression in normal prostate and normal gall bladder epithelium and distal renal tubules. [0924]
-
(8) DNA56041 (TAT206) [0925]
-
In non-malignant lymphoid tissue expression is seen in occasional larger lymphoid cells within germinal centers and in interfollicular regions. Positive cells account for less than 5% of all lymphoid cells. In section of spleen scattered positive cells are seen within the periarteriolar lymphoid sheath and in the marginal zone. [0926]
-
In four cases of Hodgkin's disease Reed-Sternberg cells are negative, positive signal is observed in scattered lymphocytes. Three of four cases of follicular lymphoma are positive (weak to moderate), four of six cases of diffuse large cell lymphoma are positive (weak to moderate). Two cases of small lymphocytic lymphoma show a weak signal in variable proportion of cells. [0927]
-
(9) DNA257845 (TAT374) [0928]
-
In non-malignant lymphoid tissue expression is seen in occasional larger lymphoid cells within germinal centers and in interfollicular regions. Positive cells account for less than 5% of all lymphoid cells. In section of spleen scattered positive cells are seen within the periarteriolar lymphoid sheath and in the marginal zone. [0929]
-
In four cases of Hodgkin's disease Reed-Sternberg cells are negative, positive signal is observed in scattered lymphocytes. Three of four cases of follicular lymphoma are positive (weak to moderate), four of six cases of diffuse large cell lymphoma are positive (weak to moderate). Two cases of small lymphocytic lymphoma show a weak signal in variable proportion of cells. [0930]
-
(10) DNA247476 (TAT180) [0931]
-
With regard to normal tissues, strong expression is seen in prostatic epithelium and in a section of peripheral nerve. Moderate expression is seen in renal glomeruli. Weak expression is seen in bile duct epithelium and mammary epithelium. Two sections of stomach show weak expression in a subset of gastric glands. Sections of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatic epithelium. [0932]
-
In a separate analysis, 42 of 77 breast tumors are positive (55%) for expression. [0933]
-
In yet another analysis, 8 of 11 breast cancers are positive for expression. [0934]
-
In yet another analysis, expression is seen in ½ non-small cell lung carcinomas, ⅓ colorectal adenocarcinomas, ⅔ pancreatic adenocarcinomas, {fraction (1/1)} prostate cancers, ⅓ transitional cell carcinomas, {fraction (3/3)} renal cell carcinomas, {fraction (3/3)} endometrial adenocarcinomas, ½ ovarian adenocarcinomas and ⅓ malignant melanomas. [0935]
-
In yet another analysis, expression is seen in 42 of 45 (93%) prostate cancers. [0936]
-
In yet another analysis, expression is seen in all of 23 primary and in 12 of 15 (80%) metastatic prostate cancers analyzed. [0937]
-
In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocarcinoma—2 of 2 cases are positive; colorectal adenocarcinoma—12 of 14 cases are positive; gastric adenocarcinoma—6 of 8 cases are positive; esophageal carcinoma—2 of 3 cases are positive; cholangiocarcinoma —1 of 1 case is positive; metastatic adenocarcinoma (ovary, liver, lymph node, diaphragm)—8 of 12 cases are positive. [0938]
-
(11) DNA260990 (TAT375) [0939]
-
With regard to normal tissues, strong expression is seen in prostatic epithelium and in a section of peripheral nerve. Moderate expression is seen in renal glomeruli. Weak expression is seen in bile duct epithelium and mammary epithelium. Two sections of stomach show weak expression in a subset of gastric glands. Sections of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatic epithelium. [0940]
-
In a separate analysis, 42 of 77 breast tumors are positive (55%) for expression. [0941]
-
In yet another analysis, 8 of 11 breast cancers are positive for expression. [0942]
-
In yet another analysis, expression is seen in ½non-small cell lung carcinomas, ⅓ colorectal adenocarcinomas, ⅔ pancreatic adenocarcinomas, {fraction (1/1)} prostate cancers, ⅓ transitional cell carcinomas, {fraction (3/3)} renal cell carcinomas, {fraction (3/3)} endometrial adenocarcinomas, ½ ovarian adenocarcinomas and ⅓ malignant melanomas. [0943]
-
In yet another analysis, expression is seen in 42 of 45 (93%) prostate cancers. [0944]
-
In yet another analysis, expression is seen in all of 23 primary and in 12 of 15 (80%) metastatic prostate cancers analyzed. [0945]
-
In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocarcinoma—2 of 2 cases are positive; colorectal adenocarcinoma—12 of 14 cases are positive; gastric adenocarcinoma—6 of 8 cases are positive; esophageal carcinoma—2 of 3 cases are positive; cholangiocarcinoma —1 of 1 case is positive; metastatic adenocarcinoma (ovary, liver, lymph node, diaphragm)—8 of 12 cases are positive. [0946]
-
(12) DNA261013 (TAT176) [0947]
-
With regard to normal tissues, prostate epithelium shows a weak positive signal. Also, one core of colonic mucosa shows a weak signal in mucosal epithelium. Two cores of a testicular neoplasm are positive. [0948]
-
In another analysis, 87 cases of infiltrating ductal breast cancer are available for review. 40 cases are positive for expression. Additionally, all tested cell lines (A549, SK-MES, SKBR3, MDA231, MDA453, MDA 175, MCF7) are positive for expression. [0949]
-
In another analysis, there is no consistent expression in benign colon, small intestinal, liver, pancreatic, gastric or esophageal tissue. In malignant tumors expression is observed as follows: colorectal adenocarcinoma: 10 of 14 cases are positive, gastric adenocarcinoma: 4 of 8 cases are positive, esophageal carcinoma: 3 of 4 cases are positive and metastatic adenocarcinoma: 8 of 11 cases are positive. [0950]
-
(13) DNA262144 (TAT184) [0951]
-
Two of 4 cases of non-small cell lung carcinoma are positive for expression while no signal is observed in non-neoplastic lung. In a separate analysis, three cases of non-small cell lung carcinoma are positive (14) DNA267342 (TAT213) [0952]
-
Expression is not observed in any of the normal adult tissues tested. Seventy four cases of breast cancer are available for review and 30 cases give a positive signal Expression localizes to tumor-associated stroma. [0953]
-
In a separate analysis, expression is seen in a minority of sarcomas; moderate and occasionally strong expression is seen in a case of a synovial sarcoma, angiosarcoma, fibrosarcoma, gliosarcoma and malignant fibrohistiocytoma. In most cases expression appears to localize to the malignant cell population. [0954]
-
(15) DNA267626 (TAT217) [0955]
-
Expression is seen in 6 of 9 invasive breast cancers. Expression is in most cases of moderate intensity, expression is also seen in benign mammary epithelium and fibroadenoma. The large sections included in this study show expression in 1 of 1 endometrial adenocarcinomas, in 2 of 3 invasive ductal breast cancers, in benign renal tubules, in normal breast epithelium and in epidermis. Sections of lung, brain, myometrium and eye are negative. [0956]
-
(16) DNA268334 (TAT202) [0957]
-
No expression is seen in any of the adult, normal tissues tested while expression is observed in 3 of 3 renal cell carcinomas. [0958]
-
(17) DNA269238 (TAT215) [0959]
-
Tumor-associated vasculature was strongly positive in all renal cell carcinomas tested (n=6), in all hepatocellular carcinomas tested (n=3), in all gastric adenocarcinomas tested (n=5), in all endometrial adenocarcinomas tested (n=3), in all malignant melanomas tested (n=3), in all malignant lymphomas tested (n=3), in all pancreatic adenocarcinomas tested (n=1), in all esophageal carcinomas tested (n=4), in all cholangiocarcinomas tested (n=2), in 93% of all non-small cell lung cancers tested (n=15), in 86% of all invasive ductal breast cancers tested (n=88), in 83% of all colorectal adenocarcinomas tested (n=12), in 67% of all metastatic adenocarcinomas tested (n=6), in 75% of all transitional cell carcinomas tested (n=4). While TAT215 expression is also observed in endothelial components of various normal non-cancerous tissues, the expression level is significantly lower in these non-cancerous tissues as compared to their cancerous counterparts and the expression pattern in the tumor tissues was distinct from that in the normal tissues, thereby providing a means for both therapy and diagnosis of the cancerous condition. [0960]
-
(18) DNA304853 (TAT376) [0961]
-
With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas. [0962]
-
In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues. [0963]
-
(19) DNA304854 (TAT377) [0964]
-
With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas. [0965]
-
In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues. [0966]
-
(20) DNA304855 (TAT378) [0967]
-
With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas. [0968]
-
In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues. [0969]
-
(21) DNA287971 (TAT379) [0970]
-
With regard to normal tissues, strong expression is seen in prostatic epithelium and in a section of peripheral nerve. Moderate expression is seen in renal glomeruli. Weak expression is seen in bile duct epithelium and mammary epithelium. Two sections of stomach show weak expression in a subset of gastric glands. Sections of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatic epithelium. [0971]
-
In a separate analysis, 42 of 77 breast tumors are positive (55%) for expression. [0972]
-
In yet another analysis, 8 of 11 breast cancers are positive for expression. [0973]
-
In yet another analysis, expression is seen in ½ non-small cell lung carcinomas, ⅓ colorectal adenocarcinomas, ⅔ pancreatic adenocarcinomas, {fraction (1/1)} prostate cancers, ⅓ transitional cell carcinomas, {fraction (3/3)} renal cell carcinomas, {fraction (3/3)} endometrial adenocarcinomas, ½ ovarian adenocarcinomas and ⅓ malignant melanomas. [0974]
-
In yet another analysis, expression is seen in 42 of 45 (93%) prostate cancers. [0975]
-
In yet another analysis, expression is seen in all of 23 primary and in 12 of 15 (80%) metastatic prostate cancers analyzed. [0976]
-
In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocarcinoma—2 of 2 cases are positive; colorectal adenocarcinoma—12 of 14 cases are positive; gastric adenocarcinoma—6 of 8 cases are positive; esophageal carcinoma—2 of 3 cases are positive; cholangiocarcinoma —1 of 1 case is positive; metastatic adenocarcinoma (ovary, liver, lymph node, diaphragm)-8 of 12 cases are positive. [0977]
Example 5
Verification and Analysis of Differential TAT Polypeptide Expression by GEPIS
-
TAT polypeptides which may have been identified as a tumor antigen as described in one or more of the above Examples were analyzed and verified as follows. An expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and interesting EST sequences were identified by GEPIS. Gene expression profilingin silico (GEPIS) is a bioinformatics tool developed at Genentech, Inc. that characterizes genes of interest for new cancer therapeutic targets. GEPIS takes advantage of large amounts of EST sequence and library information to determine gene expression profiles. GEPIS is capable of determining the expression profile of a gene based upon its proportional correlation with the number of its occurrences in EST databases, and it works by integrating the LIFESEQ® EST relational database and Genentech proprietary information in a stringent and statistically meaningful way. In this example, GEPIS is used to identify and cross-validate novel tumor antigens, although GEPIS can be configured to perform either very specific analyses or broad screening tasks. For the initial screen, GEPIS is used to identify EST sequences from the LIFESEQ® database that correlate to expression in a particular tissue or tissues of interest (often a tumor tissue of interest). The EST sequences identified in this initial screen (or consensus sequences obtained from aligning multiple related and overlapping EST sequences obtained from the initial screen) were then subjected to a screen intended to identify the presence of at least one transmembrane domain in the encoded protein. Finally, GEPIS was employed to generate a complete tissue expression profile for the various sequences of interest. Using this type of screening bioinformatics, various TAT polypeptides (and their encoding nucleic acid molecules) were identified as being significantly overexpressed in a particular type of cancer or certain cancers as compared to other cancers and/or normal non-cancerous tissues. The rating of GEPIS hits is based upon several criteria including, for example, tissue specificity, tumor specificity and expression level in normal essential and/or normal proliferating tissues. The following is a list of molecules whose tissue expression profile as determined by GEPIS evidences high tissue expression and significant upregulation of expression in a specific tumor or tumors as compared to other tumor(s) and/or normal tissues and optionally relatively low expression in normal essential and/or normal proliferating tissues. As such, the molecules listed below are excellent polypeptide targets for the diagnosis and therapy of cancer in mammals.
[0978] |
|
Molecule | upregulation of expression in: | as compared to: |
|
DNA67962 (TAT207) | colon tumor | normal colon tissue |
DNA67962 (TAT207) | uterus tumor | normal uterus tissue |
DNA67962 (TAT207) | lung tumor | normal lung tissue |
DNA67962 (TAT207) | prostate tumor | normal prostate tissue |
DNA67962 (TAT207) | breast tumor | normal breast tissue |
DNA96792 (TAT239) | colon tumor | normal colon tissue |
DNA96792 (TAT239) | rectum tumor | normal rectum tissue |
DNA96792 (TAT239) | pancreas tumor | normal pancreas tissue |
DNA96792 (TAT239) | lung tumor | normal lung tissue |
DNA96792 (TAT239) | stomach tumor | normal stomach tissue |
DNA96792 (TAT239) | esophagus tumor | normal esophagus tissue |
DNA96792 (TAT239) | breast tumor | normal breast tissue |
DNA96792 (TAT239) | uterus tumor | normal uterus tissue |
DNA96964 (TAT193) | breast tumor | normal breast tissue |
DNA96964 (TAT193) | brain tumor | normal brain tissue |
DNA142915 (TAT199) | breast tumor | normal breast tissue |
DNA142915 (TAT199) | ovary tumor | normal ovary tissue |
DNA142915 (TAT199) | brain tumor | normal brain tissue |
DNA208551 (TAT178) | prostate tumor | normal prostate tissue |
DNA208551 (TAT178) | colon tumor | normal colon tissue |
DNA210159 (TAT198) | prostate tumor | normal prostate tissue |
DNA210159 (TAT198) | uterus tumor | normal uterus tissue |
DNA210159 (TAT198) | breast tumor | normal breast tissue |
DNA210159 (TAT198) | ovarian tumor | normal ovarian tissue |
DNA225706 (TAT194) | adrenal tumor | normal adrenal tissue |
DNA225706 (TAT194) | prostate tumor | normal prostate tissue |
DNA225706 (TAT194) | breast tumor | normal breast tissue |
DNA225706 (TAT194) | connective tissue tumor | normal connective tissue |
DNA225793 (TAT223) | ovarian tumor | normal ovarian tissue |
DNA225793 (TAT223) | fallopian tube tumor | normal fallopian tube tissue |
DNA225793 (TAT223) | kidney tumor | normal kidney tissue |
DNA225796 (TAT196) | breast tumor | normal breast tissue |
DNA225943 (TAT195) | liver tumor | normal liver tissue |
DNA225943 (TAT195) | lung tumor | normal lung tissue |
DNA225943 (TAT195) | breast tumor | normal breast tissue |
DNA226283 (TAT203) | uterine tumor | normal uterine tissue |
DNA226283 (TAT203) | breast tumor | normal breast tissue |
DNA226283 (TAT203) | squamous cell lung tumor | normal squamous cell lung |
| | tissue |
DNA226283 (TAT203) | colon tumor | normal colon tissue |
DNA226283 (TAT203) | ovarian tumor | normal ovarian tissue |
DNA226589 (TAT200) | brain tumor | normal brain tissue |
DNA226589 (TAT200) | colon tumor | normal colon tissue |
DNA226589 (TAT200) | breast tumor | normal breast tissue |
DNA226589 (TAT200) | prostate tumor | normal prostate tissue |
DNA226622 (TAT205) | squamous cell lung tumor | normal squamous cell lung |
| | tissue |
DNA226622 (TAT205) | kidney tumor | normal kidney tissue |
DNA226622 (TAT205) | uterine tumor | normal uterine tissue |
DNA226622 (TAT205) | breast tumor | normal breast tissue |
DNA226622 (TAT205) | colon tumor | normal colon tissue |
DNA227545 (TAT197) | breast tumor | normal breast tissue |
DNA227611 (TAT175) | prostate tumor | normal prostate tissue |
DNA227611 (TAT175) | colon tumor | normal colon tissue |
DNA227611 (TAT175) | breast tumor | normal breast tissue |
DNA227611 (TAT175) | uterine tumor | normal uterine tissue |
DNA261021 (TAT208) | prostate tumor | normal prostate tissue |
DNA261021 (TAT208) | colon tumor | normal colon tissue |
DNA261021 (TAT208) | breast tumor | normal breast tissue |
DNA261021 (TAT208) | uterine tumor | normal uterine tissue |
DNA260655 (TAT209) | lung tumor | normal lung tissue |
DNA260655 (TAT209) | colon tumor | normal colon tissue |
DNA260655 (TAT209) | breast tumor | normal breast tissue |
DNA260655 (TAT209) | liver tumor | normal liver tissue |
DNA260655 (TAT209) | ovarian tumor | normal ovarian tissue |
DNA260655 (TAT209) | skin tumor | normal skin tissue |
DNA260655 (TAT209) | spleen tumor | normal spleen tissue |
DNA260655 (TAT209) | myeloid tumor | normal myeloid tissue |
DNA260655 (TAT209) | muscle tumor | normal muscle tissue |
DNA260655 (TAT209) | bone tumor | normal bone tissue |
DNA260945 (TAT192) | brain tumor | normal brain tissue |
DNA260945 (TAT192) | breast tumor | normal breast tissue |
DNA260945 (TAT192) | colon tumor | normal colon tissue |
DNA260945 (TAT192) | ovarian tumor | normal ovarian tissue |
DNA260945 (TAT192) | pancreatic tumor | normal pancreatic tissue |
DNA261001 (TAT181) | bone tumor | normal bone tissue |
DNA261001 (TAT181) | lung tumor | normal lung tissue |
DNA266928 (TAT182) | bone tumor | normal bone tissue |
DNA266928 (TAT182) | lung tumor | normal lung tissue |
DNA268035 (TAT222) | ovarian tumor | normal ovarian tissue |
DNA277797 (TAT212) | breast tumor | normal breast tissue |
DNA277797 (TAT212) | pancreatic tumor | normal pancreatic tissue |
DNA77509 (TAT177) | colon tumor | normal colon tissue |
DNA77509 (TAT177) | testis tumor | normal testis tissue |
DNA87993 (TAT235) | breast tumor | normal breast tissue |
DNA87993 (TAT235) | prostate tumor | normal prostate tissue |
DNA87993 (TAT235) | colon tumor | normal colon tissue |
DNA87993 (TAT235) | ovarian tumor | normal ovarian tissue |
DNA92980 (TAT234) | bone tumor | normal bone tissue |
DNA92980 (TAT234) | breast tumor | normal breast tissue |
DNA92980 (TAT234) | cervical tumor | normal cervical tissue |
DNA92980 (TAT234) | colon tumor | normal colon tissue |
DNA92980 (TAT234) | rectum tumor | normal rectum tissue |
DNA92980 (TAT234) | endometrial tumor | normal endometrial tissue |
DNA92980 (TAT234) | liver tumor | normal liver tissue |
DNA92980 (TAT234) | lung tumor | normal lung tissue |
DNA92980 (TAT234) | ovarian tumor | normal ovarian tissue |
DNA92980 (TAT234) | pancreatic tumor | normal pancreatic tissue |
DNA92980 (TAT234) | skin tumor | normal skin tissue |
DNA92980 (TAT234) | soft tissue tumor | normal soft tissue |
DNA92980 (TAT234) | stomach tumor | normal stomach tissue |
DNA92980 (TAT234) | bladder tumor | normal bladder tissue |
DNA92980 (TAT234) | thyroid tumor | normal thyroid tissue |
DNA92980 (TAT234) | esophagus tumor | normal esophagus tissue |
DNA92980 (TAT234) | testis tumor | normal testis tissue |
DNA105792 (TAT233) | adrenal tumor | normal adrenal tissue |
DNA105792 (TAT233) | breast tumor | normal breast tissue |
DNA105792 (TAT233) | endometrial tumor | normal endometrial tissue |
DNA105792 (TAT233) | esophagus tumor | normal esophagus tissue |
DNA105792 (TAT233) | kidney tumor | normal kidney tissue |
DNA105792 (TAT233) | lung tumor | normal lung tissue |
DNA105792 (TAT233) | ovarian tumor | normal ovarian tissue |
DNA105792 (TAT233) | pancreatic tumor | normal pancreatic tissue |
DNA105792 (TAT233) | prostate tumor | normal prostate tissue |
DNA105792 (TAT233) | soft tissue tumor | normal soft tissue |
DNA105792 (TAT233) | myeloid tumor | normal myeloid tissue |
DNA105792 (TAT233) | thyroid tumor | normal thyroid tissue |
DNA105792 (TAT233) | bladder tumor | normal bladder tissue |
DNA105792 (TAT233) | brain tumor | normal brain tissue |
DNA105792 (TAT233) | testis tumor | normal testis tissue |
DNA119474 (TAT226) | kidney tumor | normal kidney tissue |
DNA119474 (TAT226) | adrenal tumor | normal adrenal tissue |
DNA119474 (TAT226) | uterine tumor | normal uterine tissue |
DNA119474 (TAT226) | ovarian tumor | normal ovarian tissue |
DNA150491 (TAT204) | squamous cell lung tumor | normal squamous cell lung |
| | tissue |
DNA150491 (TAT204) | colon tumor | normal colon tissue |
DNA280351 (TAT248) | squamous cell lung tumor | normal squamous cell lung |
| | tissue |
DNA280351 (TAT248) | colon tumor | normal colon tissue |
DNA150648 (TAT232) | liver tumor | normal liver tissue |
DNA150648 (TAT232) | breast tumor | normal breast tissue |
DNA150648 (TAT232) | brain tumor | normal brain tissue |
DNA150648 (TAT232) | lung tumor | normal lung tissue |
DNA150648 (TAT232) | colon tumor | normal colon tissue |
DNA150648 (TAT232) | rectum tumor | normal rectum tissue |
DNA150648 (TAT232) | kidney tumor | normal kidney tissue |
DNA150648 (TAT232) | bladder tumor | normal bladder tissue |
DNA179651 (TAT224) | colon tumor | normal colon tissue |
DNA179651 (TAT224) | uterine tumor | normal uterine tissue |
DNA179651 (TAT224) | lung tumor | normal lung tissue |
DNA179651 (TAT224) | kidney tumor | normal kidney tissue |
DNA225886 (TAT236) | breast tumor | normal breast tissue |
DNA225886 (TAT236) | colon tumor | normal colon tissue |
DNA225886 (TAT236) | rectum tumor | normal rectum tissue |
DNA225886 (TAT236) | ovarian tumor | normal ovarian tissue |
DNA225886 (TAT236) | pancreas tumor | normal pancreas tissue |
DNA225886 (TAT236) | prostate tumor | normal prostate tissue |
DNA225886 (TAT236) | bladder tumor | normal bladder tissue |
DNA225886 (TAT236) | testis tumor | normal testis tissue |
DNA226717 (TAT185) | glioma | normal glial tissue |
DNA226717 (TAT185) | brain tumor | normal brain tissue |
DNA227162 (TAT225) | myeloid tumor | normal myeloid tissue |
DNA227162 (TAT225) | uterine tumor | normal uterine tissue |
DNA227162 (TAT225) | prostate tumor | normal prostate tissue |
DNA277804 (TAT247) | myeloid tumor | normal myeloid tissue |
DNA277804 (TAT247) | uterine tumor | normal uterine tissue |
DNA277804 (TAT247) | prostate tumor | normal prostate tissue |
DNA233034 (TAT174) | glioma | normal glial tissue |
DNA233034 (TAT174) | brain tumor | normal brain tissue |
DNA233034 (TAT174) | kidney tumor | normal kidney tissue |
DNA233034 (TAT174) | adrenal tumor | normal adrenal tissue |
DNA266920 (TAT214) | glioma | normal glial tissue |
DNA266920 (TAT214) | brain tumor | normal brain tissue |
DNA266920 (TAT214) | kidney tumor | normal kidney tissue |
DNA266920 (TAT214) | adrenal tumor | normal adrenal tissue |
DNA266921 (TAT220) | glioma | normal glial tissue |
DNA266921 (TAT220) | brain tumor | normal brain tissue |
DNA266921 (TAT220) | kidney tumor | normal kidney tissue |
DNA266921 (TAT220) | adrenal tumor | normal adrenal tissue |
DNA266922 (TAT221) | glioma | normal glial tissue |
DNA266922 (TAT221) | brain tumor | normal brain tissue |
DNA266922 (TAT221) | kidney tumor | normal kidney tissue |
DNA266922 (TAT221) | adrenal tumor | normal adrenal tissue |
DNA234834 (TAT179) | colon tumor | normal colon tissue |
DNA234834 (TAT179) | uterine tumor | normal uterine tissue |
DNA234834 (TAT179) | breast tumor | normal breast tissue |
DNA234834 (TAT179) | prostate tumor | normal prostate tissue |
DNA247587 (TAT216) | breast tumor | normal breast tissue |
DNA247587 (TAT216) | prostate tumor | normal prostate tissue |
DNA247587 (TAT216) | bladder tumor | normal bladder tissue |
DNA247587 (TAT216) | lymphoid tumor | normal lymphoid tissue |
DNA255987 (TAT218) | brain tumor | normal brain tissue |
DNA255987 (TAT218) | breast tumor | normal breast tissue |
DNA247476 (TAT180) | prostate tumor | normal prostate tissue |
DNA247476 (TAT180) | pancreas tumor | normal pancreas tissue |
DNA247476 (TAT180) | brain tumor | normal brain tissue |
DNA247476 (TAT180) | stomach tumor | normal stomach tissue |
DNA247476 (TAT180) | bladder tumor | normal bladder tissue |
DNA247476 (TAT180) | soft tissue tumor | normal soft tissue |
DNA247476 (TAT180) | skin tumor | normal skin tissue |
DNA247476 (TAT180) | kidney tumor | normal kidney tissue |
DNA260990 (TAT375) | prostate tumor | normal prostate tissue |
DNA260990 (TAT375) | pancreas tumor | normal pancreas tissue |
DNA260990 (TAT375) | brain tumor | normal brain tissue |
DNA260990 (TAT375) | stomach tumor | normal stomach tissue |
DNA260990 (TAT375) | bladder tumor | normal bladder tissue |
DNA260990 (TAT375) | soft tissue tumor | normal soft tissue |
DNA260990 (TAT375) | skin tumor | normal skin tissue |
DNA260990 (TAT375) | kidney tumor | normal kidney tissue |
DNA261013 (TAT176) | prostate tumor | normal prostate tissue |
DNA261013 (TAT176) | colon tumor | normal colon tissue |
DNA261013 (TAT176) | small intestine tumor | normal small intestine tissue |
DNA261013 (TAT176) | pancreatic tumor | normal pancreatic tissue |
DNA261013 (TAT176) | uterine tumor | normal uterine tissue |
DNA261013 (TAT176) | ovarian tumor | normal ovarian tissue |
DNA261013 (TAT176) | bladder tumor | normal bladder tissue |
DNA261013 (TAT176) | stomach tumor | normal stomach tissue |
DNA267342 (TAT213) | breast tumor | normal breast tissue |
DNA267342 (TAT213) | uterine tumor | normal uterine tissue |
DNA267342 (TAT213) | colon tumor | normal colon tissue |
DNA267342 (TAT213) | kidney tumor | normal kidney tissue |
DNA267342 (TAT213) | bladder tumor | normal bladder tissue |
DNA267342 (TAT213) | bone tumor | normal bone tissue |
DNA267342 (TAT213) | ovarian tumor | normal ovarian tissue |
DNA267342 (TAT213) | pancreatic tumor | normal pancreatic tissue |
DNA267626 (TAT217) | breast tumor | normal breast tissue |
DNA267626 (TAT217) | colon tumor | normal colon tissue |
DNA267626 (TAT217) | pancreatic tumor | normal pancreatic tissue |
DNA267626 (TAT217) | ovarian tumor | normal ovarian tissue |
DNA268334 (TAT202) | kidney tumor | normal kidney tissue |
DNA269238 (TAT215) | colon tumor | normal colon tissue |
DNA269238 (TAT215) | kidney tumor | normal kidney tissue |
DNA269238 (TAT215) | adrenal tumor | normal adrenal tissue |
DNA269238 (TAT215) | bladder tumor | normal bladder tissue |
DNA272578 (TAT238) | adrenal tumor | normal adrenal tissue |
DNA272578 (TAT238) | lung tumor | normal lung tissue |
DNA272578 (TAT238) | ovarian tumor | normal ovarian tissue |
DNA272578 (TAT238) | uterine tumor | normal uterine tissue |
DNA304853 (TAT376) | colon tumor | normal colon tissue |
DNA304853 (TAT376) | uterine tumor | normal uterine tissue |
DNA304853 (TAT376) | breast tumor | normal breast tissue |
DNA304853 (TAT376) | prostate tumor | normal prostate tissue |
DNA304854 (TAT377) | colon tumor | normal colon tissue |
DNA304854 (TAT377) | uterine tumor | normal uterine tissue |
DNA304854 (TAT377) | breast tumor | normal breast tissue |
DNA304854 (TAT377) | prostate tumor | normal prostate tissue |
DNA304855 (TAT378) | colon tumor | normal colon tissue |
DNA304855 (TAT378) | uterine tumor | normal uterine tissue |
DNA304855 (TAT378) | breast tumor | normal breast tissue |
DNA304855 (TAT378) | prostate tumor | normal prostate tissue |
DNA287971 (TAT379) | prostate tumor | normal prostate tissue |
DNA287971 (TAT379) | pancreas tumor | normal pancreas tissue |
DNA287971 (TAT379) | brain tumor | normal brain tissue |
DNA287971 (TAT379) | stomach tumor | normal stomach tissue |
DNA287971 (TAT379) | bladder tumor | normal bladder tissue |
DNA287971 (TAT379) | soft tissue tumor | normal soft tissue |
DNA287971 (TAT379) | skin tumor | normal skin tissue |
DNA287971 (TAT379) | kidney tumor | normal kidney tissue |
|
Example 6
Use of TAT as a Hybridization Probe
-
The following method describes use of a nucleotide sequence encoding TAT as a hybridization probe for, i.e., diagnosis of the presence of a tumor in a mammal. [0979]
-
DNA comprising the coding sequence of full-length or mature TAT as disclosed herein can also be employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of TAT) in human tissue cDNA libraries or human tissue genomic libraries. [0980]
-
Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled TAT-derived probe to the filters is performed in a solution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2× Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1×SSC and 0.1% SDS at 42° C. [0981]
-
DNAs having a desired sequence identity with the DNA encoding full-length native sequence TAT can then be identified using standard techniques known in the art. [0982]
Example 7
Expression of TAT in E. Coli
-
This example illustrates preparation of an unglycosylated form of TAT by recombinant expression in [0983] E. coli.
-
The DNA sequence encoding TAT is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from [0984] E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the TAT coding region, lambda transcriptional terminator, and an argu gene.
-
The ligation mixture is then used to transform a selected [0985] E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.
-
Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on. [0986]
-
After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized TAT protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein. [0987]
-
TAT may be expressed in [0988] E. coli in a poly-His tagged form, using the following procedure. The DNA encoding TAT is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) cIpP(lacIq). Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH4)2SO4, 0.71 g sodium citrate-2H20, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO4) and grown for approximately 20-30 hours at 30° C. with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
-
[0989] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1 M and 0.02 M, respectively, and the solution is stirred overnight at 4° C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4° C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
-
The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml. The refolding solution is stirred gently at 4° C. for 12-36 hours. The refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final concentration. The refolded protein is chromatographed on a Poros R1/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples. [0990]
-
Fractions containing the desired folded TAT polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered. [0991]
-
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s). [0992]
Example 8
Expression of TAT in Mammalian Cells
-
This example illustrates preparation of a potentially glycosylated form of TAT by recombinant expression in mammalian cells. [0993]
-
The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employed as the expression vector. Optionally, the TAT DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the TAT DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRK5-TAT. [0994]
-
In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 μg pRK5-TAT DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al., [0995] Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO4, and a precipitate is allowed to form for 10 minutes at 25° C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37° C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.
-
Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/n d [0996] 35S-cysteine and 200 μCi/ml 35S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of TAT polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
-
In an alternative technique, TAT may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., [0997] Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-TAT DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed TAT can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
-
In another embodiment, TAT can be expressed in CHO cells. The pRK5-TAT can be transfected into CHO cells using known reagents such as CaPO[0998] 4 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 3S-methionine. After determining the presence of TAT polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed TAT can then be concentrated and purified by any selected method.
-
Epitope-tagged TAT may also be expressed in host CHO cells. The TAT may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged TAT insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged TAT can then be concentrated and purified by any selected method, such as by Ni[0999] 2+-chelate affinity chromatography.
-
TAT may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure. [1000]
-
Stable expression in CHO cells is performed using the following procedure. The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins are fused to an IgG1 constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form. [1001]
-
Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al., [1002] Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites 5′ and 3′ of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used expression in CHO cells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.
-
Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Quiagen), Dosper® or Fugene® (Boehringer Mannheim). The cells are grown as described in Lucas et al., supra. Approximately 3×10[1003] 7 cells are frozen in an ampule for further growth and production as described below.
-
The ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37° C. After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3×10[1004] 5 cells/mL. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be used. A 3 L production spinner is seeded at 1.2×106 cells/mL. On day 0, the cell number pH ie determined. On day 1, the spinner is sampled and sparging with filtered air is commenced. On day 2, the spinner is sampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture is harvested by centrifugation and filtering through a 0.22 μm filter. The filtrate was either stored at 4° C. or immediately loaded onto columns for purification.
-
For the poly-His tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4° C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at −80° C. [1005]
-
Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation. [1006]
-
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s). [1007]
Example 9
Expression of TAT in Yeast
-
The following method describes recombinant expression of TAT in yeast. [1008]
-
First, yeast expression vectors are constructed for intracellular production or secretion of TAT from the ADH2/GAPDH promoter. DNA encoding TAT and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of TAT. For secretion, DNA encoding TAT can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native TAT signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of TAT. [1009]
-
Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain. [1010]
-
Recombinant TAT can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing TAT may further be purified using selected column chromatography resins. [1011]
-
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s). [1012]
Example 10
Expression of TAT in Baculovirus-Infected Insect Cells
-
The following method describes recombinant expression of TAT in Baculovirus-infected insect cells. [1013]
-
The sequence coding for TAT is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding TAT or the desired portion of the coding sequence of TAT such as the sequence encoding an extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5′ and 3′ regions. The 5′ primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector. [1014]
-
Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold™ virus DNA (Pharmingen) into [1015] Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28° C., the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
-
Expressed poly-his tagged TAT can then be purified, for example, by Ni[1016] 2+-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni2+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline A280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching A280 baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni2+NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His10-tagged TAT are pooled and dialyzed against loading buffer.
-
Alternatively, purification of the IgG tagged (or Fc tagged) TAT can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography. [1017]
-
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s). [1018]
Example 11
Preparation of Antibodies that Bind TAT
-
This example illustrates preparation of monoclonal antibodies which can specifically bind TAT. [1019]
-
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified TAT, fusion proteins containing TAT, and cells expressing recombinant TAT on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation. [1020]
-
Mice, such as Balb/c, are immunized with the TAT immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-TAT antibodies. [1021]
-
After a suitable antibody titer has been detected, the animals “positive” for antibodies can be injected with a final intravenous injection of TAT. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids. [1022]
-
The hybridoma cells will be screened in an ELISA for reactivity against TAT. Determination of “positive” hybridoma cells secreting the desired monoclonal antibodies against TAT is within the skill in the art. [1023]
-
The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-TAT monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed. [1024]
Example 12
Purification of TAT Polypeptides Using Specific Antibodies
-
Native or recombinant TAT polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-TAT polypeptide, mature TAT polypeptide, or pre-TAT polypeptide is purified by immunoaffinity chromatography using antibodies specific for the TAT polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-TAT polypeptide antibody to an activated chromatographic resin. [1025]
-
Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions. [1026]
-
Such an immunoaffinity column is utilized in the purification of TAT polypeptide by preparing a fraction from cells containing TAT polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble TAT polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown. [1027]
-
A soluble TAT polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of TAT polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/TAT polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and TAT polypeptide is collected. [1028]
Example 13
In Vitro Tumor Cell Killing Assay
-
Mammalian cells expressing the TAT polypeptide of interest may be obtained using standard expression vector and cloning techniques. Alternatively, many tumor cell lines expressing TAT polypeptides of interest are publicly available, for example, through the ATCC and can be routinely identified using standard ELISA or FACS analysis. Anti-TAT polypeptide monoclonal antibodies (and toxin conjugated derivatives thereof) may then be employed in assays to determine the ability of the antibody to kill TAT polypeptide expressing cells in vitro. [1029]
-
For example, cells expressing the TAT polypeptide of interest are obtained as described above and plated into 96 well dishes. In one analysis, the antibody/toxin conjugate (or naked antibody) is included throughout the cell incubation for a period of 4 days. In a second independent analysis, the cells are incubated for 1 hour with the antibody/toxin conjugate (or naked antibody) and then washed and incubated in the absence of antibody/toxin conjugate for a period of 4 days. Cell viability is then measured using the CellTiter-Glo Luminescent Cell Viability Assay from Promega (Cat# G7571). Untreated cells serve as a negative control. [1030]
Example 14
In Vivo Tumor Cell Killing Assay
-
To test the efficacy of conjugated or unconjugated anti-TAT polypeptide monoclonal antibodies, anti-TAT antibody is injected intraperitoneally into nude mice 24 hours prior to receiving tumor promoting cells subcutaneously in the flank. Antibody injections continue twice per week for the remainder of the study. Tumor volume is then measured twice per week. [1031]
-
The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. [1032]
-
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. [1033]
-
1
120
1
3781
DNA
Homo Sapien
1
ctccgggtcc ccaggggctg cgccgggccg gcctggcaag ggggacgagt 50
cagtggacac tccaggaaga gcggccccgc ggggggcgat gaccgtgcgc 100
tgaccctgac tcactccagg tccggaggcg ggggcccccg gggcgactcg 150
ggggcggacc gcggggcgga gctgccgccc gtgagtccgg ccgagccacc 200
tgagcccgag ccgcgggaca ccgtcgctcc tgctctccga atgctgcgca 250
ccgcgatggg cctgaggagc tggctcgccg ccccatgggg cgcgctgccg 300
cctcggccac cgctgctgct gctcctgctg ctgctgctcc tgctgcagcc 350
gccgcctccg acctgggcgc tcagcccccg gatcagcctg cctctgggct 400
ctgaagagcg gccattcctc agattcgaag ctgaacacat ctccaactac 450
acagcccttc tgctgagcag ggatggcagg accctgtacg tgggtgctcg 500
agaggccctc tttgcactca gtagcaacct cagcttcctg ccaggcgggg 550
agtaccagga gctgctttgg ggtgcagacg cagagaagaa acagcagtgc 600
agcttcaagg gcaaggaccc acagcgcgac tgtcaaaact acatcaagat 650
cctcctgccg ctcagcggca gtcacctgtt cacctgtggc acagcagcct 700
tcagccccat gtgtacctac atcaacatgg agaacttcac cctggcaagg 750
gacgagaagg ggaatgtcct cctggaagat ggcaagggcc gttgtccctt 800
cgacccgaat ttcaagtcca ctgccctggt ggttgatggc gagctctaca 850
ctggaacagt cagcagcttc caagggaatg acccggccat ctcgcggagc 900
caaagccttc gccccaccaa gaccgagagc tccctcaact ggctgcaaga 950
cccagctttt gtggcctcag cctacattcc tgagagcctg ggcagcttgc 1000
aaggcgatga tgacaagatc tactttttct tcagcgagac tggccaggaa 1050
tttgagttct ttgagaacac cattgtgtcc cgcattgccc gcatctgcaa 1100
gggcgatgag ggtggagagc gggtgctaca gcagcgctgg acctccttcc 1150
tcaaggccca gctgctgtgc tcacggcccg acgatggctt ccccttcaac 1200
gtgctgcagg atgtcttcac gctgagcccc agcccccagg actggcgtga 1250
cacccttttc tatggggtct tcacttccca gtggcacagg ggaactacag 1300
aaggctctgc cgtctgtgtc ttcacaatga aggatgtgca gagagtcttc 1350
agcggcctct acaaggaggt gaaccgtgag acacagcagt ggtacaccgt 1400
gacccacccg gtgcccacac cccggcctgg agcgtgcatc accaacagtg 1450
cccgggaaag gaagatcaac tcatccctgc agctcccaga ccgcgtgctg 1500
aacttcctca aggaccactt cctgatggac gggcaggtcc gaagccgcat 1550
gctgctgctg cagccccagg ctcgctacca gcgcgtggct gtacaccgcg 1600
tccctggcct gcaccacacc tacgatgtcc tcttcctggg cactggtgac 1650
ggccggctcc acaaggcagt gagcgtgggc ccccgggtgc acatcattga 1700
ggagctgcag atcttctcat cgggacagcc cgtgcagaat ctgctcctgg 1750
acacccacag ggggctgctg tatgcggcct cacactcggg cgtagtccag 1800
gtgcccatgg ccaactgcag cctgtaccgg agctgtgggg actgcctcct 1850
cgcccgggac ccctactgtg cttggagcgg ctccagctgc aagcacgtca 1900
gcctctacca gcctcagctg gccaccaggc cgtggatcca ggacatcgag 1950
ggagccagcg ccaaggacct ttgcagcgcg tcttcggttg tgtccccgtc 2000
ttttgtacca acaggggaga agccatgtga gcaagtccag ttccagccca 2050
acacagtgaa cactttggcc tgcccgctcc tctccaacct ggcgacccga 2100
ctctggctac gcaacggggc ccccgtcaat gcctcggcct cctgccacgt 2150
gctacccact ggggacctgc tgctggtggg cacccaacag ctgggggagt 2200
tccagtgctg gtcactagag gagggcttcc agcagctggt agccagctac 2250
tgcccagagg tggtggagga cggggtggca gaccaaacag atgagggtgg 2300
cagtgtaccc gtcattatca gcacatcgcg tgtgagtgca ccagctggtg 2350
gcaaggccag ctggggtgca gacaggtcct actggaagga gttcctggtg 2400
atgtgcacgc tctttgtgct ggccgtgctg ctcccagttt tattcttgct 2450
ctaccggcac cggaacagca tgaaagtctt cctgaagcag ggggaatgtg 2500
ccagcgtgca ccccaagacc tgccctgtgg tgctgccccc tgagacccgc 2550
ccactcaacg gcctagggcc ccctagcacc ccgctcgatc accgagggta 2600
ccagtccctg tcagacagcc ccccgggggc ccgagtcttc actgagtcag 2650
agaagaggcc actcagcatc caagacagct tcgtggaggt atccccagtg 2700
tgcccccggc cccgggtccg ccttggctcg gagatccgtg actctgtggt 2750
gtgagagctg acttccagag gacgctgccc tggcttcagg ggctgtgaat 2800
gctcggagag ggtcaactgg acctcccctc cgctctgctc ttcgtggaac 2850
acgaccgtgg tgcccggccc ttgggagcct tggagccagc tggcctgctg 2900
ctctccagtc aagtagcgaa gctcctacca cccagacacc caaacagccg 2950
tggccccaga ggtcctggcc aaatatgggg gcctgcctag gttggtggaa 3000
cagtgctcct tatgtaaact gagccctttg tttaaaaaac aattccaaat 3050
gtgaaactag aatgagaggg aagagatagc atggcatgca gcacacacgg 3100
ctgctccagt tcatggcctc ccaggggtgc tggggatgca tccaaagtgg 3150
ttgtctgaga cagagttgga aaccctcacc aactggcctc ttcaccttcc 3200
acattatccc gctgccaccg gctgccctgt ctcactgcag attcaggacc 3250
agcttgggct gcgtgcgttc tgccttgcca gtcagccgag gatgtagttg 3300
ttgctgccgt cgtcccacca cctcagggac cagagggcta ggttggcact 3350
gcggccctca ccaggtcctg ggctcggacc caactcctgg acctttccag 3400
cctgtatcag gctgtggcca cacgagagga cagcgcgagc tcaggagaga 3450
tttcgtgaca atgtacgcct ttccctcaga attcagggaa gagactgtcg 3500
cctgccttcc tccgttgttg cgtgagaacc cgtgtgcccc ttcccaccat 3550
atccaccctc gctccatctt tgaactcaaa cacgaggaac taactgcacc 3600
ctggtcctct ccccagtccc cagttcaccc tccatccctc accttcctcc 3650
actctaaggg atatcaacac tgcccagcac aggggccctg aatttatgtg 3700
gtttttatac attttttaat aagatgcact ttatgtcatt ttttaataaa 3750
gtctgaagaa ttactgttta aaaaaaaaaa a 3781
2
2010
DNA
Homo Sapien
2
ggaaaggctg agtctccagc tcaaggtcaa aacgtccaag gccgaaagcc 50
ctccagtttc ccctggacgc cttgctcctg cttctgctac gaccttctgg 100
ggaaaacgaa tttctcattt tcttcttaaa ttgccatttt cgctttagga 150
gatgaatgtt ttcctttggc tgttttggca atgactctga attaaagcga 200
tgctaacgcc tcttttcccc ctaattgtta aaagctatgg actgcaggaa 250
gatggcccgc ttctcttaca gtgtgatttg gatcatggcc atttctaaag 300
tctttgaact gggattagtt gccgggctgg gccatcagga atttgctcgt 350
ccatctcggg gatacctggc cttcagagat gacagcattt ggccccagga 400
ggagcctgca attcggcctc ggtcttccca gcgtgtgccg cccatgggga 450
tacagcacag taaggagcta aacagaacct gctgcctgaa tgggggaacc 500
tgcatgctgg ggtccttttg tgcctgccct ccctccttct acggacggaa 550
ctgtgagcac gatgtgcgca aagagaactg tgggtctgtg ccccatgaca 600
cctggctgcc caagaagtgt tccctgtgta aatgctggca cggtcagctc 650
cgctgctttc ctcaggcatt tctacccggc tgtgatggcc ttgtgatgga 700
tgagcacctc gtggcttcca ggactccaga actaccaccg tctgcacgta 750
ctaccacttt tatgctagtt ggcatctgcc tttctataca aagctactat 800
taatcgacat tgacctattt ccagaaatac aattttagat atcatgcaaa 850
tttcatgacc agtaaaggct gctgctacaa tgtcctaact gaaagatgat 900
catttgtagt tgccttaaaa taatgaatac atttccaaaa tggtctctaa 950
catttcctta cagaactact tcttacttct ttgccctgcc ctctcccaaa 1000
aaactacttc ttttttcaaa agaaagtcag ccatatctcc attgtgccta 1050
agtccagtgt ttcttttttt tttttttttg agacggagtc tcactctgtc 1100
acccaggctg gactgcaatg acgcgatctt ggttcactgc aacctccgca 1150
tccggggttc aagccattct cctgcctcag cctcccaagt aactgggatt 1200
acaggcatgt gtcaccatgc ccagctaatt tttttgtatt tttagtagag 1250
atgggggttt caccatattg gccagtctgg tctcgaactc ctgaccttgt 1300
gatccactcg cctcagcctc tcgaagtgct gagattacac acgtgagcaa 1350
ctgtgcaagg cctggtgttt cttgatacat gtaattctac caaggtcttc 1400
ttaatatgtt cttttaaatg attgaattat atgttcagat tattggagac 1450
taattctaat gtggacctta gaatacagtt ttgagtagag ttgatcaaaa 1500
tcaattaaaa tagtctcttt aaaaggaaag aaaacatctt taaggggagg 1550
aaccagagtg ctgaaggaat ggaagtccat ctgcgtgtgt gcagggagac 1600
tgggtaggaa agaggaagca aatagaagag agaggttgaa aaacaaaatg 1650
ggttacttga ttggtgatta ggtggtggta gagaagcaag taaaaaggct 1700
aaatggaagg gcaagtttcc atcatctata gaaagctata taagacaaga 1750
actccccttt ttttcccaaa ggcattataa aaagaatgaa gcctccttag 1800
aaaaaaaatt atacctcaat gtccccaaca agattgctta ataaattgtg 1850
tttcctccaa gctattcaat tcttttaact gttgtagaag acaaaatgtt 1900
cacaatatat ttagttgtaa accaagtgat caaactacat attgtaaagc 1950
ccatttttaa aatacattgt atatatgtgt atgcacagta aaaatggaaa 2000
ctatattgaa 2010
3
549
DNA
Homo Sapien
3
gccaggaggg agagccttcc ccaagcaaac aatccagagc agctgtgcaa 50
acaacggtgc ataaatgagg cctcctggac catgaagcga gtcctgagct 100
gcgtcccgga gcccacggtg gtcatggctg ccagagcgct ctgcatgctg 150
gggctggtcc tggccttgct gtcctccagc tctgctgagg agtacgtggg 200
cctgtctgca aaccagtgtg ccgtgccagc caaggacagg gtggactgcg 250
gctaccccca tgtcaccccc aaggagtgca acaaccgggg ctgctgcttt 300
gactccagga tccctggagt gccttggtgt ttcaagcccc tgcaggaagc 350
agaatgcacc ttctgaggca cctccagctg cccccggccg ggggatgcga 400
ggctcggagc acccttgccc ggctgtgatt gctgccaggc actgttcatc 450
tcagcttttc tgtccctttg ctcccggcaa gcgcttctgc tgaaagttca 500
tatctggagc ctgatgtctt aacgaataaa ggtcccatgc tccacccga 549
4
1424
DNA
Homo Sapien
4
gaccagactc gtctcaggcc agttgcagcc ttctcagcca aacgccgacc 50
aaggaaaact cactaccatg agaattgcag tgatttgctt ttgcctccta 100
ggcatcacct gtgccatacc agttaaacag gctgattctg gaagttctga 150
ggaaaagcag ctttacaaca aatacccaga tgctgtggcc acatggctaa 200
accctgaccc atctcagaag cagaatctcc tagccccaca gaatgctgtg 250
tcctctgaag aaaccaatga ctttaaacaa gagacccttc caagtaagtc 300
caacgaaagc catgaccaca tggatgatat ggatgatgaa gatgatgatg 350
accatgtgga cagccaggac tccattgact cgaacgactc tgatgatgta 400
gatgacactg atgattctca ccagtctgat gagtctcacc attctgatga 450
atctgatgaa ctggtcactg attttcccac ggacctgcca gcaaccgaag 500
ttttcactcc agttgtcccc acagtagaca catatgatgg ccgaggtgat 550
agtgtggttt atggactgag gtcaaaatct aagaagtttc gcagacctga 600
catccagtac cctgatgcta cagacgagga catcacctca cacatggaaa 650
gcgaggagtt gaatggtgca tacaaggcca tccccgttgc ccaggacctg 700
aacgcgcctt ctgattggga cagccgtggg aaggacagtt atgaaacgag 750
tcagctggat gaccagagtg ctgaaaccca cagccacaag cagtccagat 800
tatataagcg gaaagccaat gatgagagca atgagcattc cgatgtgatt 850
gatagtcagg aactttccaa agtcagccgt gaattccaca gccatgaatt 900
tcacagccat gaagatatgc tggttgtaga ccccaaaagt aaggaagaag 950
ataaacacct gaaatttcgt atttctcatg aattagatag tgcatcttct 1000
gaggtcaatt aaaaggagaa aaaatacaat ttctcacttt gcatttagtc 1050
aaaagaaaaa atgctttata gcaaaatgaa agagaacatg aaatgcttct 1100
ttctcagttt attggttgaa tgtgtatcta tttgagtctg gaaataacta 1150
atgtgtttga taattagttt agtttgtggc ttcatggaaa ctccctgtaa 1200
actaaaagct tcagggttat gtctatgttc attctataga agaaatgcaa 1250
actatcactg tattttaata tttgttattc tctcatgaat agaaatttat 1300
gtagaagcaa acaaaatact tttacccact taaaaagaga atataacatt 1350
ttatgtcact ataatctttt gttttttaag ttagtgtata ttttgttgtg 1400
attatctttt tgtggtgtga ataa 1424
5
1166
DNA
Homo Sapien
unsure
721-761
unknown base
5
cggacgcgtg ggcggaggga agaggaccgc aaaccaaccc aggacccgct 50
cagttccacg cgcggcagcc ctccgtgcgc gcaggctcgg tatgagccgc 100
acagcctaca cggtgggagc cctgcttctc ctcttgggga ccctgctgcc 150
ggctgctgaa gggaaaaaga aagggtccca aggtgccatc cccccgccag 200
acaaggccca gcacaatgac tcagagcaga ctcagtcgcc ccagcagcct 250
ggctccagga accgggggcg gggccaaggg cggggcactg ccatgcccgg 300
ggaggaggtg ctggagtcca gccaagaggc cctgcatgtg acggagcgca 350
aatacctgaa gcgagactgg tgcaaaaccc agccgcttaa gcagaccatc 400
cacgaggaag gctgcaacag tcgcaccatc atcaaccgct tctgttacgg 450
ccagtgcaac tctttctaca tccccaggca catccggaag gaggaaggtt 500
cctttcagtc ctgctccttc tgcaagccca agaaattcac taccatgatg 550
gtcacactca actgccctga actacagcca cctaccaaga agaagagagt 600
cacacgtgtg aagcagtgtc gttgcatatc catcgatttg gattaagcca 650
aatccaggtg cacccagcat gtcctaggaa tgcagcccca ggaagtccca 700
gacctaaaac aaccagattc nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 750
nnnnnnnnnn nagacttacg atgcatgtat acaaacgaat agcagataat 800
gatgactagt tcacacataa agtcctttta aggagaaaat ctaaaatgaa 850
aagtggataa acagaacatt tataagtgat cagttaatgc ctaagagtga 900
aagtagttct attgacattc ctcaagatat ttaatatcaa ctgcattatg 950
tattatgtct gcttaaatca tttaaaaacg gcaaagaatt atatagacta 1000
tgaggtacct tgctgtgtag gaggatgaaa ggggagttga tagtctcata 1050
aaactaattt ggcttcaagt ttcatgaatc tgtaactaga atttaatttt 1100
caccccaata atgttctata tagcctttgc taaagagcaa ctaataaatt 1150
aaacctattc tttcaa 1166
6
2279
DNA
Homo Sapien
6
cggacctgaa cccctaaaag cggaaccgcc tcccgccctc gccatcgcgg 50
agctgagtcg ccggcggcgg tggctgctgc cagacccgga gtttcctctt 100
tcactggatg gagctgaact ttgggcggcc agagcagcac agctgtccgg 150
ggatcgctgc atgctgagct ccctcggcaa gacccagcgg cggctcggga 200
tttttttggg ggggcgggga ccagccccgc gccggcacca tgttcctggc 250
gaccctgtac ttcgcgctgc cgctcttgga cttgctcctg tcggccgaag 300
tgagcggcgg agaccgcctg gattgcgtga aagccagtga tcagtgcctg 350
aaggagcaga gctgcagcac caagtaccgc acgctaaggc agtgcgtggc 400
gggcaaggag accaacttca gcctggcatc cggcctggag gccaaggatg 450
agtgccgcag cgccatggag gccctgaagc agaagtcgct ctacaactgc 500
cgctgcaagc ggggtatgaa gaaggagaag aactgcctgc gcatttactg 550
gagcatgtac cagagcctgc agggaaatga tctgctggag gattccccat 600
atgaaccagt taacagcaga ttgtcagata tattccgggt ggtcccattc 650
atatcagtgg agcacattcc caaagggaac aactgcctgg atgcagcgaa 700
ggcctgcaac ctcgacgaca tttgcaagaa gtacaggtcg gcgtacatca 750
ccccgtgcac caccagcgtg tccaatgatg tctgcaaccg ccgcaagtgc 800
cacaaggccc tccggcagtt ctttgacaag gtcccggcca agcacagcta 850
cggaatgctc ttctgctcct gccgggacat cgcctgcaca gagcggaggc 900
gacagaccat cgtgcctgtg tgctcctatg aagagaggga gaagcccaac 950
tgtttgaatt tgcaggactc ctgcaagacg aattacatct gcagatctcg 1000
ccttgcggat ttttttacca actgccagcc agagtcaagg tctgtcagca 1050
gctgtctaaa ggaaaactac gctgactgcc tcctcgccta ctcggggctt 1100
attggcacag tcatgacccc caactacata gactccagta gcctcagtgt 1150
ggccccatgg tgtgactgca gcaacagtgg gaacgaccta gaagagtgct 1200
tgaaattttt gaatttcttc aaggacaata catgtcttaa aaatgcaatt 1250
caagcctttg gcaatggctc cgatgtgacc gtgtggcagc cagccttccc 1300
agtacagacc accactgcca ctaccaccac tgccctccgg gttaagaaca 1350
agcccctggg gccagcaggg tctgagaatg aaattcccac tcatgttttg 1400
ccaccgtgtg caaatttaca ggcacagaag ctgaaatcca atgtgtcggg 1450
caatacacac ctctgtattt ccaatggtaa ttatgaaaaa gaaggtctcg 1500
gtgcttccag ccacataacc acaaaatcaa tggctgctcc tccaagctgt 1550
ggtctgagcc cactgctggt cctggtggta accgctctgt ccaccctatt 1600
atctttaaca gaaacatcat agctgcatta aaaaaataca atatggacat 1650
gtaaaaagac aaaaaccaag ttatctgttt cctgttctct tgtatagctg 1700
aaattccagt ttaggagctc agttgagaaa cagttccatt caactggaac 1750
attttttttt tttcctttta agaaagcttc ttgtgatcct tcggggcttc 1800
tgtgaaaaac ctgatgcagt gctccatcca aactcagaag gctttgggat 1850
atgctgtatt ttaaagggac agtttgtaac ttgggctgta aagcaaactg 1900
gggctgtgtt ttcgatgatg atgatgatca tgatgatgat catcatgatc 1950
atgatgatga tcatcatgat catgatgatg attttaacag ttttacttct 2000
ggcctttcct agctagagaa ggagttaata tttctaaggt aactcccata 2050
tctcctttaa tgacattgat ttctaatgat ataaatttca gcctacattg 2100
atgccaagct tttttgccac aaagaagatt cttaccaaga gtgggctttg 2150
tggaaacagc tggtactgat gttcaccttt atatatgtac tagcattttc 2200
cacgctgatg tttatgtact gtaaacagtt ctgcactctt gtacaaaaga 2250
aaaaacacct gtcacatcca aatataaaa 2279
7
562
DNA
Homo Sapien
7
atgcagcacc gaggcttcct cctcctcacc ctcctcgccc tgctggcgct 50
cacctccgcg gtcgccaaaa agaaagataa ggtgaagaag ggcggcccgg 100
ggagcgagtg cgctgagtgg gcctgggggc cctgcacccc cagcagcaag 150
gattgcggcg tgggtttccg cgagggcacc tgcggggccc agacccagcg 200
catccggtgc agggtgccct gcaactggaa gaaggagttt ggagccgact 250
gcaagtacaa gtttgagaac tggggtgcgt gtgatggggg cacaggcacc 300
aaagtccgcc aaggcaccct gaagaaggcg cgctacaatg ctcagtgcca 350
ggagaccatc cgcgtcacca agccctgcac ccccaagacc aaagcaaagg 400
ccaaagccaa gaaagggaag ggaaaggact agacgccaag cctggatgcc 450
aaggagcccc tggtgtcaca tggggcctgg cccacgccct ccctctccca 500
ggcccgagat gtgacccacc agtgccttct gtctgctcgt tagctttaat 550
caatcatgcc cc 562
8
1524
DNA
Homo Sapien
8
gcggcagcag cgcgggcccc agcagcctcg gcagccacag ccgctgcagc 50
cggggcagcc tccgctgctg tcgcctcctc tgatgcgctt gccctctccc 100
ggccccggga ctccgggaga atgtgggtcc taggcatcgc ggcaactttt 150
tgcggattgt tcttgcttcc aggctttgcg ctgcaaatcc agtgctacca 200
gtgtgaagaa ttccagctga acaacgactg ctcctccccc gagttcattg 250
tgaattgcac ggtgaacgtt caagacatgt gtcagaaaga agtgatggag 300
caaagtgccg ggatcatgta ccgcaagtcc tgtgcatcat cagcggcctg 350
tctcatcgcc tctgccgggt accagtcctt ctgctcccca gggaaactga 400
actcagtttg catcagctgc tgcaacaccc ctctttgtaa cgggccaagg 450
cccaagaaaa ggggaagttc tgcctcggcc ctcaggccag ggctccgcac 500
caccatcctg ttcctcaaat tagccctctt ctcggcacac tgctgaagct 550
gaaggagatg ccaccccctc ctgcattgtt cttccagccc tcgcccccaa 600
ccccccacct ccctgagtga gtttcttctg ggtgtccttt tattctgggt 650
agggagcggg agtccgtgtt ctcttttgtt cctgtgcaaa taatgaaaga 700
gctcggtaaa gcattctgaa taaattcagc ctgactgaat tttcagtatg 750
tacttgaagg aaggaggtgg agtgaaagtt cacccccatg tctgtgtaac 800
cggagtcaag gccaggctgg cagagtcagt ccttagaagt cactgaggtg 850
ggcatctgcc ttttgtaaag cctccagtgt ccattccatc cctgatgggg 900
gcatagtttg agactgcaga gtgagagtga cgttttctta gggctggagg 950
gccagttccc actcaaggct ccctcgcttg acattcaaac ttcatgctcc 1000
tgaaaaccat tctctgcagc agaattggct ggtttcgcgc ctgagttggg 1050
ctctagtgac tcgagactca atgactggga cttagactgg ggctcggcct 1100
cgctctgaaa agtgcttaag aaaatcttct cagttctcct tgcagaggac 1150
tggcgccggg acgcgaagag caacgggcgc tgcacaaagc gggcgctgtc 1200
ggtggtggag tgcgcatgta cgcgcaggcg cttctcgtgg ttggcgtgct 1250
gcagcgacag gcggcagcac agcacctgca cgaacacccg ccgaaactgc 1300
tgcgaggaca ccgtgtacag gagcgggttg atgaccgagc tgaggtagaa 1350
aaacgtctcc gagaagggga ggaggatcat gtacgcccgg aagtaggacc 1400
tcgtccagtc gtgcttgggt ttggccgcag ccatgatcct ccgaatctgg 1450
ttgggcatcc agcatacggc caatgtcaca acaatcagcc ctgggcagac 1500
acgagcagga gggagagaca gaga 1524
9
1253
DNA
Homo Sapien
9
caccctccgt ggcaaggcga ggccccgggg gcgggccggg gtcaccacgc 50
ctgccccagg gaaccgcaca gacggtactc acccttcttg cgatgatgtg 100
agatgataaa atgcctacat gatgagatga agtgagatga aaaacatagg 150
ccttgtgatg gaatgggaaa ttccagagat aatttgcacg tgcgctaagc 200
tgcggctacc cccgcaagca accttccaag tccttcgtgg caatggtgct 250
tccgtgggga ccgtgctcat gttccgctgc ccctccaacc accagatggt 300
ggggtctggg ctcctcacct gcacctggaa ggggagcatc gctgagtggt 350
cttcagggtc cccagtgtgc aaactggtgc caccacacga gacctttggc 400
ttcaaggtgg ccgtgatcgc ctccattgtg agctgtgcca tcatcctgct 450
catgtccatg gccttcctca cctgctgcct cctcaagtgc gtgaagaaga 500
gcaagcggcg gcgctccaac aggtcagccc agctgtggtc ccagctgaaa 550
gatgaggact tggagacggt gcaggccgca taccttggcc tcaagcactt 600
caacaaaccc gtgagcgggc ccagccaggc gcacgacaac cacagcttca 650
ccacagacca tggtgagagc accagcaagc tggccagtgt gacccgcagc 700
gtggacaagg accctgggat ccccagagct ctaagcctca gtggctcctc 750
cagctcaccc caagcccagg tgatggtgca catggcaaac cccagacagc 800
ccctgcctgc ctctgggctg gccacaggaa tgccacaaca gcccgcagca 850
tatgccctag ggtgaccacg cagtgaggct ggtgcccatg ctccacactg 900
ggaggccagg ctgaccccac cagccagtca gctacaactc cacatcaact 950
ccacatgcgc ccagctcgag actgatgagt ggaatcagct tccaggtgta 1000
gggacccctt gaggggccga gctgacatcc aaggctgagg accccagtgg 1050
ggagtgttct gttccggcat atcctggccg taacgatttt tatagttatg 1100
gactacttga aaccactact gagggtaatt tactagctgt ggcctcccac 1150
taactagcat tcctttaaag agactgggaa atgttttaag caaatctagt 1200
tttgtataat aaaataagaa aatagcaata aacttctttt cagcaactac 1250
aaa 1253
10
5542
DNA
Homo Sapien
10
ctgactgcac tggtgatggt ccctggcaat ccaacctggc accatcgcag 50
ttggagtact atgcatcttc accagatgaa aaggctctag tagaagctgc 100
tgcaaggatt ggtattgtgt ttattggcaa ttctgaagaa actatggagg 150
ttaaaactct tggaaaactg gaacggtaca aactgcttca tattctggaa 200
tttgattcag atcgtaggag aatgagtgta attgttcagg caccttcagg 250
tgagaagtta ttatttgcta aaggagctga gtcatcaatt ctccctaaat 300
gtataggtgg agaaatagaa aaaaccagaa ttcatgtaga tgaatttgct 350
ttgaaagggc taagaactct gtgtatagca tatagaaaat ttacatcaaa 400
agagtatgag gaaatagata aacgcatatt tgaagccagg actgccttgc 450
agcagcggga agagaaattg gcagctgttt tccagttcat agagaaagac 500
ctgatattac ttggagccac agcagtagaa gacagactac aagataaagt 550
tcgagaaact attgaagcat tgagaatggc tggtatcaaa gtatgggtac 600
ttactgggga taaacatgaa acagctgtta gtgtgagttt atcatgtggc 650
cattttcata gaaccatgaa catccttgaa cttataaacc agaaatcaga 700
cagcgagtgt gctgaacaat tgaggcagct tgccagaaga attacagagg 750
atcatgtgat tcagcatggg ctggtagtgg atgggaccag cctatctctt 800
gcactcaggg agcatgaaaa actatttatg gaagtttgca gaaattgttc 850
agctgtatta tgctgtcgta tggctccact gcagaaagca aaagtaataa 900
gactaataaa aatatcacct gagaaaccta taacattggc tgttggtgat 950
ggtgctaatg acgtaagcat gatacaagaa gcccatgttg gcataggaat 1000
catgggtaaa gaaggaagac aggctgcaag aaacagtgac tatgcaatag 1050
ccagatttaa gttcctctcc aaattgcttt ttgttcatgg tcatttttat 1100
tatattagaa tagctaccct tgtacagtat tttttttata agaatgtgtg 1150
ctttatcaca ccccagtttt tatatcagtt ctactgtttg ttttctcagc 1200
aaacattgta tgacagcgtg tacctgactt tatacaatat ttgttttact 1250
tccctaccta ttctgatata tagtcttttg gaacagcatg tagaccctca 1300
tgtgttacaa aataagccca ccctttatcg agacattagt aaaaaccgcc 1350
tcttaagtat taaaacattt ctttattgga ccatcctggg cttcagtcat 1400
gcctttattt tcttttttgg atcctattta ctaataggga aagatacatc 1450
tctgcttgga aatggccaga tgtttggaaa ctggacattt ggcactttgg 1500
tcttcacagt catggttatt acagtcacag taaagatggc tctggaaact 1550
catttttgga cttggatcaa ccatctcgtt acctggggat ctattatatt 1600
ttattttgta ttttccttgt tttatggagg gattctctgg ccatttttgg 1650
gctcccagaa tatgtatttt gtgtttattc agctcctgtc aagtggttct 1700
gcttggtttg ccataatcct catggttgtt acatgtctat ttcttgatat 1750
cataaagaag gtctttgacc gacacctcca ccctacaagt actgaaaagg 1800
cacagcttac tgaaacaaat gcaggtatca agtgcttgga ctccatgtgc 1850
tgtttcccgg aaggagaagc agcgtgtgca tctgttggaa gaatgctgga 1900
acgagttata ggaagatgta gtccaaccca catcagcaga tcatggagtg 1950
catcggatcc tttctatacc aacgacagga gcatcttgac tctctccaca 2000
atggactcat ctacttgtta aaggggcagt agtactttgt gggagccagt 2050
tcacctcctt tcctaaaatt cagtgtgatc accctgttaa tggccacact 2100
agctctgaaa ttaatttcca aaatctttgt agtagttcat acccactcag 2150
agttataatg gcaaacaaac agaaagcatt agtacaagcc cctcccaaca 2200
cccttaattt gaatctgaac atgttaaaat ttgagaataa agagacattt 2250
ttcatctctt tgtctggttt gtcccttgtg cttatgggac tcctaatggc 2300
atttcagtct gttgctgagg ccattatatt ttaatataaa tgtagaaaaa 2350
agagagaaat cttagtaaag agtatttttt agtattagct tgattattga 2400
ctcttctatt taaatctgct tctgtaaatt atgctgaaag tttgccttga 2450
gaactctatt tttttattag agttatattt aaagcttttc atgggaaaag 2500
ttaatgtgaa tactgaggaa ttttggtccc tcagtgacct gtgttgttaa 2550
ttcattaatg cattctgagt tcacagagca aattaggaga atcatttcca 2600
accattattt actgcagtat ggggagtaaa tttataccaa ttcctctaac 2650
tgtactgtaa cacagcctgt aaagttagcc atataaatgc aagggtatat 2700
catatataca aatcaggaat caggtccgtt caccgaactt caaattgatg 2750
tttactaata tttttgtgac agagtataaa gaccctatag tgggtaaatt 2800
agatactatt agcatattat taatttaatg tctttatcat tggatctttt 2850
gcatgcttta atctggttaa catatttaaa tttgcttttt ttctctttac 2900
ctgaaggctc tgtgtatagt atttcatgac atcgttgtac agtttaacta 2950
tcaataaaaa gtttggacag tatttaaata ttgcaaatat gtttaattat 3000
acaaatcaga atagtatggg taattaaatg aatacaaaaa gaagagcctc 3050
tttctgcagc cgacttagac atgctcttcc ctttctataa gctagatttt 3100
agaataaagg gtttcagtta ataatcttat tttcaggtta tgtcatctaa 3150
cttatagcaa actaccacaa tacagtgagt tctgccagtg tcccagtaca 3200
aggcatattt caggtgtggc tgtggaatgt aaaaatgctc aacttgtatc 3250
aggtaatgtt agcaataaat taaatgctaa gaatgattaa tcgggtacat 3300
gttactgtaa ttaactcatt gcacttcaaa acctaacttc catcctgaat 3350
ttatcaagta gttcagtatt gtcatttgtt tttgttttat tgaaaagtaa 3400
tgttgtctta agatttagaa gtgattatta gcttgagaac tattacccag 3450
ctctaagcaa ataatgattg tatacatatt aagataatgg ttaaatgcgg 3500
ttttaccaag ttttcccttg aaaatgtaat tcctttatgg agatttattg 3550
tgcagcccta agcttccttc ccatttcatg aatataaggc ttctagaatt 3600
ggactggcag gggaaagaat ggtagagaca gaaattaaga ctttatcctt 3650
gtttgcttgt aaactattat tttcttgcta atgtaacatt tgtctgttcc 3700
agtgatgtaa ggatattaag ttattaagct aaatattaat tttcaaaaat 3750
agtccttctt taacttagat atttcatagc tggatttagg aagatctgtt 3800
attctggaag tactaaaaag aataatacaa cgtacaatgt ctgcattcac 3850
taattcatgt tccagaagag gaaataatga agatatactc agtagagtac 3900
taggtgggag gatatggaaa tttgctcata aaatctctta taaaacgtgc 3950
atataacaaa atgacaccca gtaggcctgc attacattta catgaccgtg 4000
tttatttgcc atcaaataaa ctgagtactg acaccagaca aagactccaa 4050
agtcataaaa tagcctatga ccaactgcag caagacagga ggtcagctcg 4100
cctataatgg tgcttaaagt gtgattgatg taattttctg tactcaccat 4150
ttgaagttag ttaaggagaa ctttattttt ttaaaaaaag taaatggcaa 4200
ccactagtgt gctcatcctg aactgttact ccaaatccac tccgttttta 4250
aagcaaaatt atcttgtgat tttaagaaaa gagttttcta tttatttaag 4300
aaagtaacaa tgcagtctgc aagctttcag tagttttcta gtgctatatt 4350
catcctgtaa aactcttact acgtaaccag taatcacaag gaaagtgtcc 4400
cctttgcata tttctttaaa attctttctt tggaaagtat gatgttgata 4450
attaacttac ccttatctgc caaaaccaga gcaaaatgct aaatacgtta 4500
ttgctaatca gtggtctcaa atcgatttgc ctccctttgc ctcgtctgag 4550
ggctgtaagc ctgaagatag tggcaagcac caagtcagtt tccaaaattg 4600
cccctcagct gctttaagtg actcagcacc ctgcctcagc ttcagcaggc 4650
gtaggctcac cctgggcgga gcaaagtatg ggccagggag aactacagct 4700
acgaagacct gctgtcgagt tgagaaaagg ggagaattta tggtctgaat 4750
tttctaactg tcctctttct tgggtctaaa gctcataata cacaaaggct 4800
tccagacctg agccacaccc aggccctatc ctgaacagga gactaaacag 4850
aggcaaatca accctaggaa atacttgcat tctgccctac ggttagtacc 4900
aggactgagg tcatttctac tggaaaagat tgtgagattg aacttatctg 4950
atcgcttgag actcctaata ggcaggagtc aaggccacta gaaaattgac 5000
agttaagagc caaaagtttt taaaatatgc tactctgaaa aatctcgtga 5050
aggctgtagg aaaagggaga atcttccatg ttggtgtttt tcctgtaaag 5100
atcagtttgg ggtatgatat aagcaggtat taataaaaat aacacaccaa 5150
agagttacgt aaaacatgtt ttattaattt tggtccccac gtacagacat 5200
tttatttcta ttttgaaatg agttatctat tttcataaaa gtaaaacact 5250
attaaagtgc tgttttatgt gaaataactt gaatgttgtt cctataaaaa 5300
atagatcata actcatgata tgtttgtaat catggtaatt tagattttta 5350
tgaggaatga gtatctggaa atattgtagc aatacttggt ttaaaatttt 5400
ggacctgaga cactgtggct gtctaatgta atcctttaaa aattctctgc 5450
attgtcagta aatgtagtat attattgtac agctactcat aattttttaa 5500
agtttatgaa gttatattta tcaaataaaa actttcctat at 5542
11
6155
DNA
Homo Sapien
11
atgtgggaag aagaagacat tgctattctg ttcaataaag aaccaggaaa 50
aacagagaat attgaaaata atctaagttc caaccataga agaagctgca 100
gaagaagtga agaaagtgat gatgatttgg attttgatat tggtttagaa 150
aacacaggag gagaccctca aattctgaga tttatttcag acttccttgc 200
ttttttggtt ctctacaatt tcatcattcc aatttcatta tatgtgacag 250
tcgaaatgca gaaatttctt ggatcatttt ttattggctg ggatcttgat 300
ctgtatcatg aagaatcaga tcagaaagct caagtcaata cttccgatct 350
gaatgaagag cttggacagg tagagtacgt gtttacagat aaaactggta 400
cactgacaga aaatgagatg cagtttcggg aatgttcaat taatggcatg 450
aaataccaag aaattaatgg tagacttgta cccgaaggac caacaccaga 500
ctcttcagaa ggaaacttat cttatcttag tagtttatcc catcttaaca 550
acttatccca tcttacaacc agttcctctt tcagaaccag tcctgaaaat 600
gaaactgaac taattaaaga acatgatctc ttctttaaag cagtcagtct 650
ctgtcacact gtacagatta gcaatgttca aactgactgc actggtgatg 700
gtccctggca atccaacctg gcaccatcgc agttggagta ctatgcatct 750
tcaccagatg aaaaggctct agtagaagct gctgcaaggt acaaactgct 800
tcatattctg gaatttgatt cagatcgtag gagaatgagt gtaattgttc 850
aggcaccttc aggtgagaag ttattatttg ctaaaggagc tgagtcatca 900
attctcccta aatgtatagg tggagaaata gaaaaaacca gaattcatgt 950
agatgaattt gctttgaaag ggctaagaac tctgtgtata gcatatagaa 1000
aatttacatc aaaagagtat gaggaaatag ataaacgcat atttgaagcc 1050
aggactgcct tgcagcagcg ggaagagaaa ttggcagctg ttttccagtt 1100
catagagaaa gacctgatat tacttggagc cacagcagta gaagacagac 1150
tacaagataa agttcgagaa actattgaag cattgagaat ggctggtatc 1200
aaagtatggg tacttactgg ggataaacat gaaacagctg ttagtgtgag 1250
tttatcatgt ggccattttc atagaaccat gaacatcctt gaacttataa 1300
accagaaatc agacagcgag tgtgctgaac aattgaggca gcttgccaga 1350
agaattacag aggatcatgt gattcagcat gggctggtag tggatgggac 1400
cagcctatct cttgcactca gggagcatga aaaactattt atggaagttt 1450
gcagaaattg ttcagctgta ttatgctgtc gtatggctcc actgcagaaa 1500
gcaaaagtaa taagactaat aaaaatatca cctgagaaac ctataacatt 1550
ggctgttggt gatggtgcta atgacgtaag catgatacaa gaagcccatg 1600
ttggcatagg aatcatgggt aaagaaggaa gacaggctgc aagaaacagt 1650
gactatgcaa tagccagatt taagttcctc tccaaattgc tttttgttca 1700
tggtcatttt tattatatta gaatagctac ccttgtacag tatttttttt 1750
ataagaatgt gtgctttatc acaccccagt ttttatatca gttctactgt 1800
ttgttttctc agcaaacatt gtatgacagc gtgtacctga ctttatacaa 1850
tatttgtttt acttccctac ctattctgat atatagtctt ttggaacagc 1900
atgtagaccc tcatgtgtta caaaataagc ccacccttta tcgagacatt 1950
agtaaaaacc gcctcttaag tattaaaaca tttctttatt ggaccatcct 2000
gggcttcagt catgccttta ttttcttttt tggatcctat ttactaatag 2050
ggaaagatac atctctgctt ggaaatggcc agatgtttgg aaactggaca 2100
tttggcactt tggtcttcac agtcatggtt attacagtca cagtaaagat 2150
ggctctggaa actcattttt ggacttggat caaccatctc gttacctggg 2200
gatctattat attttatttt gtattttcct tgttttatgg agggattctc 2250
tggccatttt tgggctccca gaatatgtat tttgtgttta ttcagctcct 2300
gtcaagtggt tctgcttggt ttgccataat cctcatggtt gttacatgtc 2350
tatttcttga tatcataaag aaggtctttg accgacacct ccaccctaca 2400
agtactgaaa aggcacagct tactgaaaca aatgcaggta tcaagtgctt 2450
ggactccatg tgctgtttcc cggaaggaga agcagcgtgt gcatctgttg 2500
gaagaatgct ggaacgagtt ataggaagat gtagtccaac ccacatcagc 2550
agatcatgga gtgcatcgga tcctttctat accaacgaca ggagcatctt 2600
gactctctcc acaatggact catctacttg ttaaaggggc agtagtactt 2650
tgtgggagcc agttcacctc ctttcctaaa attcagtgtg atcaccctgt 2700
taatggccac actagctctg aaattaattt ccaaaatctt tgtagtagtt 2750
catacccact cagagttata atggcaaaca aacagaaagc attagtacaa 2800
gcccctccca acacccttaa tttgaatctg aacatgttaa aatttgagaa 2850
taaagagaca tttttcatct ctttgtctgg tttgtccctt gtgcttatgg 2900
gactcctaat ggcatttcag tctgttgctg aggccattat attttaatat 2950
aaatgtagaa aaaagagaga aatcttagta aagagtattt tttagtatta 3000
gcttgattat tgactcttct atttaaatct gcttctgtaa attatgctga 3050
aagtttgcct tgagaactct atttttttat tagagttata tttaaagctt 3100
ttcatgggaa aagttaatgt gaatactgag gaattttggt ccctcagtga 3150
cctgtgttgt taattcatta atgcattctg agttcacaga gcaaattagg 3200
agaatcattt ccaaccatta tttactgcag tatggggagt aaatttatac 3250
caattcctct aactgtactg taacacagcc tgtaaagtta gccatataaa 3300
tgcaagggta tatcatatat acaaatcagg aatcaggtcc gttcaccgaa 3350
cttcaaattg atgtttacta atatttttgt gacagagtat aaagacccta 3400
tagtgggtaa attagatact attagcatat tattaattta atgtctttat 3450
cattggatct tttgcatgct ttaatctggt taacatattt aaatttgctt 3500
tttttctctt tacctgaagg ctctgtgtat agtatttcat gacatcgttg 3550
tacagtttaa ctatcaataa aaagtttgga cagtatttaa atattgcaaa 3600
tatgtttaat tatacaaatc agaatagtat gggtaattaa atgaatacaa 3650
aaagaagagc ctctttctgc agccgactta gacatgctct tccctttcta 3700
taagctagat tttagaataa agggtttcag ttaataatct tattttcagg 3750
ttatgtcatc taacttatag caaactacca caatacagtg agttctgcca 3800
gtgtcccagt acaaggcata tttcaggtgt ggctgtggaa tgtaaaaatg 3850
ctcaacttgt atcaggtaat gttagcaata aattaaatgc taagaatgat 3900
taatcgggta catgttactg taattaactc attgcacttc aaaacctaac 3950
ttccatcctg aatttatcaa gtagttcagt attgtcattt gtttttgttt 4000
tattgaaaag taatgttgtc ttaagattta gaagtgatta ttagcttgag 4050
aactattacc cagctctaag caaataatga ttgtatacat attaagataa 4100
tggttaaatg cggttttacc aagttttccc ttgaaaatgt aattccttta 4150
tggagattta ttgtgcagcc ctaagcttcc ttcccatttc atgaatataa 4200
ggcttctaga attggactgg caggggaaag aatggtagag acagaaatta 4250
agactttatc cttgtttgct tgtaaactat tattttcttg ctaatgtaac 4300
atttgtctgt tccagtgatg taaggatatt aagttattaa gctaaatatt 4350
aattttcaaa aatagtcctt ctttaactta gatatttcat agctggattt 4400
aggaagatct gttattctgg aagtactaaa aagaataata caacgtacaa 4450
tgtctgcatt cactaattca tgttccagaa gaggaaataa tgaagatata 4500
ctcagtagag tactaggtgg gaggatatgg aaatttgctc ataaaatctc 4550
ttataaaacg tgcatataac aaaatgacac ccagtaggcc tgcattacat 4600
ttacatgacc gtgtttattt gccatcaaat aaactgagta ctgacaccag 4650
acaaagactc caaagtcata aaatagccta tgaccaactg cagcaagaca 4700
ggaggtcagc tcgcctataa tggtgcttaa agtgtgattg atgtaatttt 4750
ctgtactcac catttgaagt tagttaagga gaactttatt tttttaaaaa 4800
aagtaaatgg caaccactag tgtgctcatc ctgaactgtt actccaaatc 4850
cactccgttt ttaaagcaaa attatcttgt gattttaaga aaagagtttt 4900
ctatttattt aagaaagtaa caatgcagtc tgcaagcttt cagtagtttt 4950
ctagtgctat attcatcctg taaaactctt actacgtaac cagtaatcac 5000
aaggaaagtg tcccctttgc atatttcttt aaaattcttt ctttggaaag 5050
tatgatgttg ataattaact tacccttatc tgccaaaacc agagcaaaat 5100
gctaaatacg ttattgctaa tcagtggtct caaatcgatt tgcctccctt 5150
tgcctcgtct gagggctgta agcctgaaga tagtggcaag caccaagtca 5200
gtttccaaaa ttgcccctca gctgctttaa gtgactcagc accctgcctc 5250
agcttcagca ggcgtaggct caccctgggc ggagcaaagt atgggccagg 5300
gagaactaca gctacgaaga cctgctgtcg agttgagaaa aggggagaat 5350
ttatggtctg aattttctaa ctgtcctctt tcttgggtct aaagctcata 5400
atacacaaag gcttccagac ctgagccaca cccaggccct atcctgaaca 5450
ggagactaaa cagaggcaaa tcaaccctag gaaatacttg cattctgccc 5500
tacggttagt accaggactg aggtcatttc tactggaaaa gattgtgaga 5550
ttgaacttat ctgatcgctt gagactccta ataggcagga gtcaaggcca 5600
ctagaaaatt gacagttaag agccaaaagt ttttaaaata tgctactctg 5650
aaaaatctcg tgaaggctgt aggaaaaggg agaatcttcc atgttggtgt 5700
ttttcctgta aagatcagtt tggggtatga tataagcagg tattaataaa 5750
aataacacac caaagagtta cgtaaaacat gttttattaa ttttggtccc 5800
cacgtacaga cattttattt ctattttgaa atgagttatc tattttcata 5850
aaagtaaaac actattaaag tgctgtttta tgtgaaataa cttgaatgtt 5900
gttcctataa aaaatagatc ataactcatg atatgtttgt aatcatggta 5950
atttagattt ttatgaggaa tgagtatctg gaaatattgt agcaatactt 6000
ggtttaaaat tttggacctg agacactgtg gctgtctaat gtaatccttt 6050
aaaaattctc tgcattgtca gtaaatgtag tatattattg tacagctact 6100
cataattttt taaagtttat gaagttatat ttatcaaata aaaactttcc 6150
tatat 6155
12
1372
DNA
Homo Sapien
12
gcacgagggc gcttttgtct ccggtgagtt ttgtggcggg aagcttctgc 50
gctggtgctt agtaaccgac tttcctccgg actcctgcac gacctgctcc 100
tacagccggc gatccactcc cggctgttcc cccggagggt ccagaggcct 150
ttcagaagga gaaggcagct ctgtttctct gcagaggagt agggtccttt 200
cagccatgaa gcatgtgttg aacctctacc tgttaggtgt ggtactgacc 250
ctactctcca tcttcgttag agtgatggag tccctagaag gcttactaga 300
gagcccatcg cctgggacct cctggaccac cagaagccaa ctagccaaca 350
cagagcccac caagggcctt ccagaccatc catccagaag catgtgataa 400
gacctccttc catactggcc atattttgga acactgacct agacatgtcc 450
agatgggagt cccattccta gcagacaagc tgagcaccgt tgtaaccaga 500
gaactattac taggccttga agaacctgtc taactggatg ctcattgcct 550
gggcaaggcc tgtttaggcc ggttgcggtg gctcatgcct gtaatcctag 600
cactttggga ggctgaggtg ggtggatcac ctgaggtcag gagttcgaga 650
ccagcctcgc caacatggcg aaaccccatc tctactaaaa atacaaaagt 700
tagctgggtg tggtggcaga ggcctgtaat cccagttcct tgggaggctg 750
aggcgggaga attgcttgaa cccggggacg gaggttgcag tgaaccgaga 800
tcgcactgct gtacccagcc tgggccacag tgcaagactc catctcaaaa 850
aaaaaaagaa aagaaaaagc ctgtttaatg cacaggtgtg agtggattgc 900
ttatggctat gagataggtt gatctcgccc ttaccccggg gtctggtgta 950
tgctgtgctt tcctcagcag tatggctctg acatctctta gatgtcccaa 1000
cttcagctgt tgggagatgg tgatattttc aaccctactt cctaaacatc 1050
tgtctggggt tcctttagtc ttgaatgtct tatgctcaat tatttggtgt 1100
tgagcctctc ttccacaaga gctcctccat gtttggatag cagttgaaga 1150
ggttgtgtgg gtgggctgtt gggagtgagg atggagtgtt cagtgcccat 1200
ttctcatttt acattttaaa gtcgttcctc caacatagtg tgtattggtc 1250
tgaagggggt ggtgggatgc caaagcctgc tcaagttatg gacattgtgg 1300
ccaccatgtg gcttaaatga ttttttctaa ctaataaagt ggaatatata 1350
tttcaaaaaa aaaaaaaaaa aa 1372
13
770
DNA
Homo Sapien
unsure
45, 611, 715
unknown base
13
atacgactca ctatagggcg aattgggtac cgggcccccc ctcgngtcga 50
cggtatcgat aagcttgata tcgaattcgg ccacactggc cggatcctct 100
agagatccct cgacctcgac ccacgcgtcc gcccacgcgt ccgatgtgcc 150
tctgggcaaa gaagcagagc taacgaggaa agggatttaa agagtttttc 200
ttgggtgttt gtcaaacttt tattccctgt ctgtgtgcag aggggattca 250
acttcaattt ttctgcagtg gctctgagtc cagcccctta cttaaagatc 300
tggaaagcat gaagactggg ctttttttcc tatgtctctt gggaactgca 350
gctgcaatcc cgacaaatgc aagattatta tctgatcatt ccaaaccaac 400
tgctgaaacg gtagcacccg acaacactgc aatccccagt ttaagggctg 450
aagatgaaga aaatgaaaaa gaaacagcag tatccacaga agacgattcc 500
caccataagg ctgaaaaatc atcagtacta aagtcaaaag aggaaagcca 550
tgaacagtca gcagaacagg gcaagagttc tagccaagag ctgggattga 600
aggatcaaga ngacagtgat ggtgacttaa gtgtgaattt ggagtatgca 650
ccaactgaag gtacattgga cataaaagaa gatatgagtg agcctcagga 700
gaaaaactct caganacact gattttttgg ctcctggggt agttccttcc 750
agattctacc acagaagttt 770
14
1187
DNA
Homo Sapien
14
cgcgggccat ggctccctgg gcggaggccg agcactcggc gctgaacccg 50
ctgcgcgcgg tgtggctcac gctgaccgcc gccttcctgc tgaccctact 100
gctgcagctc ctgccgcccg gcctgctccc gggctgcgcg atcttccagg 150
acctgatccg ctatgggaaa accaagtgtg gggagccgtc gcgccccgcc 200
gcctgccgag cctttgatgt ccccaagaga tatttttccc acttttatat 250
catctcagtg ctgtggaatg gcttcctgct ttggtgcctt actcaatctc 300
tgttcctggg agcacctttt ccaagctggc ttcatggttt gctcagaatt 350
ctcggggcgg cacagttcca gggaggggag ctggcactgt ctgcattctt 400
agtgctagta tttctgtggc tgcacagctt acgaagactc ttcgagtgcc 450
tctacgtcag tgtcttctcc aatgtcatga ttcacgtcgt gcagtactgt 500
tttggacttg tctattatgt ccttgttggc ctaactgtgc tgagccaagt 550
gccaatggat ggcaggaatg cctacataac agggaaaaat ctattgatgc 600
aagcacggtg gttccatatt cttgggatga tgatgttcat ctggtcatct 650
gcccatcagt ataagtgcca tgttattctc ggcaatctca ggaaaaataa 700
agcaggagtg gtcattcact gtaaccacag gatcccattt ggagactggt 750
ttgaatatgt ttcttcccct aactacttag cagagctgat gatctacgtt 800
tccatggccg tcacctttgg gttccacaac ttaacttggt ggctagtggt 850
gacaaatgtc ttctttaatc aggccctgtc tgcctttctc agccaccaat 900
tctacaaaag caaatttgtc tcttacccga agcataggaa agctttccta 950
ccatttttgt tttaagttaa cctcagtcat gaagaatgca aaccaggtga 1000
tggtttcaat gcctaaggac agtgaagtct ggagcccaaa gtacagtttc 1050
agcaaagctg tttgaaactc tccattccat ttctataccc cacaagtttt 1100
cactgaatga gcatggcagt gccactcaat aaaatgaatc tccaaagtat 1150
cttcaaagaa taaatactaa tggcaaaaaa aaaaaaa 1187
15
1840
DNA
Homo Sapien
15
tccacacaca caaaaaacct gcgcgtgagg ggggaggaaa agcagggcct 50
ttaaaaaggc aatcacaaca acttttgctg ccaggatgcc cttgctttgg 100
ctgagaggat ttctgttggc aagttgctgg attatagtga ggagttcccc 150
caccccagga tccgaggggc acagcgcggc ccccgactgt ccgtcctgtg 200
cgctggccgc cctcccaaag gatgtaccca actctcagcc agagatggtg 250
gaggccgtca agaagcacat tttaaacatg ctgcacttga agaagagacc 300
cgatgtcacc cagccggtac ccaaggcggc gcttctgaac gcgatcagaa 350
agcttcatgt gggcaaagtc ggggagaacg ggtatgtgga gatagaggat 400
gacattggaa ggagggcaga aatgaatgaa cttatggagc agacctcgga 450
gatcatcacg tttgccgagt caggaacagc caggaagacg ctgcacttcg 500
agatttccaa ggaaggcagt gacctgtcag tggtggagcg tgcagaagtc 550
tggctcttcc taaaagtccc caaggccaac aggaccagga ccaaagtcac 600
catccgcctc ttccagcagc agaagcaccc gcagggcagc ttggacacag 650
gggaagaggc cgaggaagtg ggcttaaagg gggagaggag tgaactgttg 700
ctctctgaaa aagtagtaga cgctcggaag agcacctggc atgtcttccc 750
tgtctccagc agcatccagc ggttgctgga ccagggcaag agctccctgg 800
acgttcggat tgcctgtgag cagtgccagg agagtggcgc cagcttggtt 850
ctcctgggca agaagaagaa gaaagaagag gagggggaag ggaaaaagaa 900
gggcggaggt gaaggtgggg caggagcaga tgaggaaaag gagcagtcgc 950
acagaccttt cctcatgctg caggcccggc agtctgaaga ccaccctcat 1000
cgccggcgtc ggcggggctt ggagtgtgat ggcaaggtca acatctgctg 1050
taagaaacag ttctttgtca gtttcaagga catcggctgg aatgactgga 1100
tcattgctcc ctctggctat catgccaact actgcgaggg tgagtgcccg 1150
agccatatag caggcacgtc cgggtcctca ctgtccttcc actcaacagt 1200
catcaaccac taccgcatgc ggggccatag cccctttgcc aacctcaaat 1250
cgtgctgtgt gcccaccaag ctgagaccca tgtccatgtt gtactatgat 1300
gatggtcaaa acatcatcaa aaaggacatt cagaacatga tcgtggagga 1350
gtgtgggtgc tcatagagtt gcccagccca gggggaaagg gagcaagagt 1400
tgtccagaga agacagtggc aaaatgaaga aatttttaag gtttctgagt 1450
taaccagaaa aatagaaatt aaaaacaaaa caaaacaaaa aaaaaaacaa 1500
aaaaaaacaa aagtaaatta aaaacaaacc tgatgaaaca gatgaaacag 1550
atgaaggaag atgtggaaat cttagcctgc cttagccagg gctcagagat 1600
gaagcagtga agagacagat tgggagggaa agggagaatg gtgtaccctt 1650
tatttcttct gaaatcacac tgatgacatc agttgtttaa acggggtatt 1700
gtcctttccc cccttgaggt tcccttgtga gcttgaatca accaatctga 1750
tctgcagtag tgtggactag aacaacccaa atagcatcta gaaagccatg 1800
agtttgaaag ggcccatcac aggcactttc ctagcctaat 1840
16
1771
DNA
Homo Sapien
16
gcggagaagc cgggagcgcg gggctcagtc ggggggcggc ggcggcggcg 50
gctccgggga tggcggcggc tccgctgctg ctgctgctgc tgctcgtgcc 100
cgtgccgctg ctgccgctgc tggcccaagg gcccggaggg gcgctgggaa 150
accggcatgc ggtgtactgg aacagctcca accagcacct gcggcgagag 200
ggctacaccg tgcaggtgaa cgtgaacgac tatctggata tttactgccc 250
gcactacaac agctcggggg tgggccccgg ggcgggaccg gggcccggag 300
gcggggcaga gcagtacgtg ctgtacatgg tgagccgcaa cggctaccgc 350
acctgcaacg ccagccaggg cttcaagcgc tgggagtgca accggccgca 400
cgccccgcac agccccatca agttctcgga gaagttccag cgctacagcg 450
ccttctctct gggctacgag ttccacgccg gccacgagta ctactacatc 500
tccacgccca ctcacaacct gcactggaag tgtctgagga tgaaggtgtt 550
cgtctgctgc gcctccacat cgcactccgg ggagaagccg gtccccactc 600
tcccccagtt caccatgggc cccaatgtga agatcaacgt gctggaagac 650
tttgagggag agaaccctca ggtgcccaag cttgagaaga gcatcagcgg 700
gaccagcccc aaacgggaac acctgcccct ggccgtgggc atcgccttct 750
tcctcatgac gttcttggcc tcctagctct gccccctccc ctgggggggg 800
agagatgggg cggggcttgg aaggagcagg gagcctttgg cctctccaag 850
ggaagcctag tgggcctaga cccctcctcc catggctaga agtggggcct 900
gcaccataca tctgtgtccg ccccctctac cccttccccc cacgtagggc 950
actgtagtgg accaagcacg gggacagcca tgggtcccgg gcggccttgt 1000
ggctctggta atgtttggta ccaaacttgg gggccaaaaa gggcagtgct 1050
caggactccc tggcccctgg tacctttccc tgactcctgg tgccctctcc 1100
ctttgtcccc ccagagagac atatgccccc agagagagca aatcgaagcg 1150
tgggaggcac ccccattgct ctcctccagg ggcagaacat ggggagggga 1200
ctagatgggc aaggggcagc actgcctgct gcttccttcc cctgtttaca 1250
gcaataaagc acgtcctcct cccccactcc cacttccagg attgtggttt 1300
ggattgaaac caagtttaca agtagacacc cctggggggg cgggcagtgg 1350
acaaggatgg caaggggtgg gcattggggt gccaggcagg catgtacaga 1400
ctctatatct ctatatataa tgtacagaca gacagagtcc cttccctctt 1450
taaccccctg acctttcttg acttcccctt cagcttcaga ccccttcccc 1500
accaggctta ggccccccca caccttgggg ggacccccct ggcccctctt 1550
ttgtcttctg tgaagacagg acctatgcaa cgcacagaca cttttggaga 1600
ccgtaaaaca acagcgcccc ctcccttcca gccctgagcc gggaaccatc 1650
tcccaggacc ttgccctgct caccctatgt ggtcccacct atcctcctgg 1700
gcctttttca agtgctttgg ctgtgacttt catactctgc tcttagtcta 1750
aaaaaaataa actggagata a 1771
17
4126
DNA
Homo sapien
17
cgctcgccat gggccactcc ccacctgtcc tgcctttgtg tgcctctgtg 50
tctttgctgg gtggcctgac ctttggttat gaactggcag tcatatcagg 100
tgccctgctg ccactgcagc ttgactttgg gctaagctgc ttggagcagg 150
agttcctggt gggcagcctg ctcctggggg ctctcctcgc ctccctggtt 200
ggtggcttcc tcattgactg ctatggcagg aagcaagcca tcctcgggag 250
caacttggtg ctgctggcag gcagcctgac cctgggcctg gctggttccc 300
tggcctggct ggtcctgggc cgcgctgtgg ttggcttcgc catttccctc 350
tcctccatgg cttgctgtat ctacgtgtca gagctggtgg ggccacggca 400
gcggggagtg ctggtgtccc tctatgaggc aggcatcacc gtgggcatcc 450
tgctctccta tgccctcaac tatgcactgg ctggtacccc ctggggatgg 500
aggcacatgt tcggctgggc cactgcacct gctgtcctgc aatccctcag 550
cctcctcttc ctccctgctg gtacagatga gactgcaaca cacaaggacc 600
tcatcccact ccagggaggt gaggccccca agctgggccc ggggaggcca 650
cggtactcct ttctggacct cttcagggca cgcgataaca tgcgaggccg 700
gaccacagtg ggcctggggc tggtgctctt ccagcaacta acagggcagc 750
ccaacgtgct gtgctatgcc tccaccatct tcagctccgt tggtttccat 800
gggggatcct cagccgtgct ggcctctgtg gggcttggcg cagtgaaggt 850
ggcagctacc ctgaccgcca tggggctggt ggaccgtgca ggccgcaggg 900
ctctgttgct agctggctgt gccctcatgg ccctgtccgt cagtggcata 950
ggcctcgtca gctttgccgt gcccatggac tcaggcccaa gctgtctggc 1000
tgtgcccaat gccaccgggc agacaggcct ccctggagac tctggcctgc 1050
tgcaggactc ctctctacct cccattccaa ggaccaatga ggaccaaagg 1100
gagccaatct tgtccactgc taagaaaacc aagccccatc ccagatctgg 1150
agacccctca gcccctcctc ggctggccct gagctctgcc ctccctgggc 1200
cccctctgcc cgctcggggg catgcactgc tgcgctggac cgcactgctg 1250
tgcctgatgg tctttgtcag tgccttctcc tttgggtttg ggccagtgac 1300
ctggcttgtc ctcagcgaga tctaccctgt ggagatacga ggaagagcct 1350
tcgccttctg caacagcttc aactgggcgg ccaacctctt catcagcctc 1400
tccttcctcg atctcattgg caccatcggc ttgtcctgga ccttcctgct 1450
ctacggactg accgctgtcc tcggcctggg cttcatctat ttatttgttc 1500
ctgaaacaaa aggccagtcg ttggcagaga tagaccagca gttccagaag 1550
agacggttca ccctgagctt tggccacagg cagaactcca ctggcatccc 1600
gtacagccgc atcgagatct ctgcggcctc ctgaggaatc cgtctgcctg 1650
gaaattctgg aactgtggct ttggcagacc atctccagca tcctgcttcc 1700
taggccccag agcacaagtt ccagctggtc ttttgggagt ggcccctgcc 1750
cccaaaggtg gtctgctttt gctggggtaa aaaggatgaa agtctgagaa 1800
tgcccaactc ttcattttga gtctcaggcc ctgaaggttc ctgaggatct 1850
agcttcatgc ctcagtttcc ccattgactt gcacatctct gcagtattta 1900
taagaagaat attctatgaa gtctttgttg caccatggac ttttctcaaa 1950
gaatctcaag ggtaccaatc ctggcaggaa gtctctcccg atatcacccc 2000
taaatccaaa tgaggatatc atcttttcta atctcttttt tcaactggct 2050
gggacatttt cggaaggggg aagtctcttt ttttactctt atcatttttt 2100
ttttttgagg tggagtctca ttctgttgcc caggctggcc tgatcttggc 2150
tcactgcaac ctccacctcc tgagttcaag cgattcttgt gcctcagcct 2200
cctaagcagc tgggactaca ggcgcatgca accataccca gctaatttat 2250
ttttagcaga gatggggttt cactgtgttg gccaggctgg tcgtgaactc 2300
ctgagctcaa gtgatccacc cacctcagcc tcccagagtg ctaggattac 2350
aggccttttg actcttttat ctgagtttta ttgacccctc taattctctt 2400
acccagaata tttatccttc accagcaact ctgactcttt gacgggaggc 2450
ctcagttcta gtccttggtc tgctggtgtc attgctgtag gaatgaccac 2500
gggcctcagt ttccccattt gtataatggg aagcctgtac caggtcattc 2550
ttaagatttc tcctgactcc agtgagctgg aattctaaat gctggtctag 2600
gagctgtctc caggatggtg caggatggct ttgcggaaag gagatgggtt 2650
tggaggccaa caaacctgct tgtcaatatt gcctttgcct cttggcagcc 2700
cttgaacttg agtaaataac aactccctga acctcagttt cctcatctgc 2750
agaatgggga taattatgtc ccaggggtat atttagaccc tgtttccttt 2800
caggagggtc cccagctggt ccagggcctg ggaaatttct acttatcctc 2850
attacccagg tccctccttt ggaccctgta aagggtcagg gtgaatcaga 2900
tgggggactg agcaagtagc tatgactgca gatcatgtaa ggaagggact 2950
gacaagaagc tcccagatgc tggggagaat gaagagctaa aatagatcct 3000
aggtgctgga tgctttgtca tccatgcgtg cacatatggg tgctggcaga 3050
gcccccaagg actctggcct ctcgagttct cctatcttct ccattctaga 3100
tgcttccctt gtatccagtg atgtgctgga gctggctttg ccaagcttgt 3150
gagagctggt tgctacattt tcaggatttt tacaagttgg taaacacagc 3200
cattataaaa aattaaatga tttaaattta taattaagta aattacatta 3250
aaacaaaaaa attatactca aaattcatta cttaatttta ctacctgtta 3300
ctattatctg tgcttttgag gctatttcta catagtaact cttatggaga 3350
cctaggggag acaccgcgca tctcttcctg attccccact caatgacatc 3400
atgttagtct ttggttgctt aactggctgt ggggagtgtt tttgtatcac 3450
aaagattaga gaggactaca catcagggct tgatttattg tttgttgatt 3500
ttctagactt cagaacatgc tggataaaat gtcagtaatg caaattaaac 3550
tttaaagtat gtcttgtttg tagccaatac atggtgtata gcaccaaaaa 3600
atggagggat tattcttcca gtagttgaac actgtcatcc gtttcagctg 3650
acagctgctc aaatcattta agaaggagtt ctgacattca ttttcattgt 3700
tttacttttg tcttcctcac tagtgtaaac aaaaatttca accagcattc 3750
atgccgaacc tatacccatt cttcagtgcc tagctgtaca gttatcaggg 3800
atttttattt gtagtctaat tttgtcaaat catggccaaa tcgcagtgat 3850
agttgacttt ggatacaagg tttggcaaaa aaaaaaatat taacaaaata 3900
ttctgtaaga atcaattgtc tatatggaat ttaggataaa gaatatttac 3950
aataaagaat atttacaata aagagtttat tattatttgt aagttgtgtg 4000
caacaaacat accctttatc tctgtaaaat ttatacacac aaaaattaac 4050
aaaagattct gtaagaatta attggctata tggaatttag gatagaatat 4100
ttacaataaa gagtatttac aataaa 4126
18
5615
DNA
Homo Sapien
unsure
429
unknown base
18
gcttcagtcc cgcgaccgaa gcagggcgcg cagcagcgct gagtgccccg 50
gaacgtgcgt cgcgccccca gtgtccgtcg cgtccgccgc gccccgggcg 100
gggatggggc ggccagactg agcgccgcac ccgccatcca gacccgccgg 150
ccctagccgc agtccctcca gccgtggccc cagcgcgcac gggcgatggc 200
gaaggcgacg tccggtgccg cggggctgcg tctgctgttg ctgctgctgc 250
tgccgctgct aggcaaagtg gcattgggcc tctacttctc gagggatgct 300
tactgggaga agctgtatgt ggaccaggcg gccggcacgc ccttgctgta 350
cgtccatgcc ctgcgggacg cccctgagga ggtgcccagc ttccgcctgg 400
gccagcatct ctacggcacg taccgcacnc ggctgcatga gaacaactgg 450
atctgcatcc aggaggacac cggcctcctc taccttaacc ggagcctgga 500
ccatagctcc tgggagaagc tcagtgtccg caaccgcggc tttcccctgc 550
tcaccgtcta cctcaaggtc ttcctgtcac ccacatccct tcgtgagggc 600
gagtgccagt ggccaggctg tgcccgcgta tacttctcct tcttcaacac 650
ctcctttcca gcctgcagct ccctcaagcc ccgggagctc tgcttcccag 700
agacaaggcc ctccttccgc attcgggaga accgaccccc aggcaccttc 750
caccagttcc gcctgctgcc tgtgcagttc ttgtgcccca acatcagcgt 800
ggcctacagg ctcctggagg gtgagggtct gcccttccgc tgcgccccgg 850
acagcctgga ggtgagcacg cgctgggccc tggaccgcga gcagcgggag 900
aagtacgagc tggtggccgt gtgcaccgtg cacgccggcg cgcgcgagga 950
ggtggtgatg gtgcccttcc cggtgaccgt gtacgacgag gacgactcgg 1000
cgcccacctt ccccgcgggc gtcgacaccg ccagcgccgt ggtggagttc 1050
aagcggaagg aggacaccgt ggtggccacg ctgcgtgtct tcgatgcaga 1100
cgtggtacct gcatcagggg agctggtgag gcggtacaca agcacgctgc 1150
tccccgggga cacctgggcc cagcagacct tccgggtgga acactggccc 1200
aacgagacct cggtccaggc caacggcagc ttcgtgcggg cgaccgtaca 1250
tgactatagg ctggttctca accggaacct ctccatctcg gagaaccgca 1300
ccatgcagct ggcggtgctg gtcaatgact cagacttcca gggcccagga 1350
gcgggcgtcc tcttgctcca cttcaacgtg tcggtgctgc cggtcagcct 1400
gcacctgccc agtacctact ccctctccgt gagcaggagg gctcgccgat 1450
ttgcccagat cgggaaagtc tgtgtggaaa actgccaggc gttcagtggc 1500
atcaacgtcc agtacaagct gcattcctct ggtgccaact gcagcacgct 1550
aggggtggtc acctcagccg aggacacctc ggggatcctg tttgtgaatg 1600
acaccaaggc cctgcggcgg cccaagtgtg ccgaacttca ctacatggtg 1650
gtggccaccg accagcagac ctctaggcag gcccaggccc agctgcttgt 1700
aacagtggag gggtcatatg tggccgagga ggcgggctgc cccctgtcct 1750
gtgcagtcag caagagacgg ctggagtgtg aggagtgtgg cggcctgggc 1800
tccccaacag gcaggtgtga gtggaggcaa ggagatggca aagggatcac 1850
caggaacttc tccacctgct ctcccagcac caagacctgc cccgacggcc 1900
actgcgatgt tgtggagacc caagacatca acatttgccc tcaggactgc 1950
ctccggggca gcattgttgg gggacacgag cctggggagc cccgggggat 2000
taaagctggc tatggcacct gcaactgctt ccctgaggag gagaagtgct 2050
tctgcgagcc cgaagacatc caggatccac tgtgcgacga gctgtgccgc 2100
acggtgatcg cagccgctgt cctcttctcc ttcatcgtct cggtgctgct 2150
gtctgccttc tgcatccact gctaccacaa gtttgcccac aagccaccca 2200
tctcctcagc tgagatgacc ttccggaggc ccgcccaggc cttcccggtc 2250
agctactcct cttccggtgc ccgccggccc tcgctggact ccatggagaa 2300
ccaggtctcc gtggatgcct tcaagatcct ggaggatcca aagtgggaat 2350
tccctcggaa gaacttggtt cttggaaaaa ctctaggaga aggcgaattt 2400
ggaaaagtgg tcaaggcaac ggccttccat ctgaaaggca gagcagggta 2450
caccacggtg gccgtgaaga tgctgaaaga gaacgcctcc ccgagtgagc 2500
ttcgagacct gctgtcagag ttcaacgtcc tgaagcaggt caaccaccca 2550
catgtcatca aattgtatgg ggcctgcagc caggatggcc cgctcctcct 2600
catcgtggag tacgccaaat acggctccct gcggggcttc ctccgcgaga 2650
gccgcaaagt ggggcctggc tacctgggca gtggaggcag ccgcaactcc 2700
agctccctgg accacccgga tgagcgggcc ctcaccatgg gcgacctcat 2750
ctcatttgcc tggcagatct cacaggggat gcagtatctg gccgagatga 2800
agctcgttca tcgggacttg gcagccagaa acatcctggt agctgagggg 2850
cggaagatga agatttcgga tttcggcttg tcccgagatg tttatgaaga 2900
ggattcctac gtgaagagga gccagggtcg gattccagtt aaatggatgg 2950
caattgaatc cctttttgat catatctaca ccacgcaaag tgatgtatgg 3000
tcttttggtg tcctgctgtg ggagatcgtg accctagggg gaaaccccta 3050
tcctgggatt cctcctgagc ggctcttcaa ccttctgaag accggccacc 3100
ggatggagag gccagacaac tgcagcgagg agatgtaccg cctgatgctg 3150
caatgctgga agcaggagcc ggacaaaagg ccggtgtttg cggacatcag 3200
caaagacctg gagaagatga tggttaagag gagagactac ttggaccttg 3250
cggcgtccac tccatctgac tccctgattt atgacgacgg cctctcagag 3300
gaggagacac cgctggtgga ctgtaataat gcccccctcc ctcgagccct 3350
cccttccaca tggattgaaa acaaactcta tggcatgtca gacccgaact 3400
ggcctggaga gagtcctgta ccactcacga gagctgatgg cactaacact 3450
gggtttccaa gatatccaaa tgatagtgta tatgctaact ggatgctttc 3500
accctcagcg gcaaaattaa tggacacgtt tgatagttaa catttctttg 3550
tgaaaggtaa tggactcaca aggggaagaa acatgctgag aatggaaagt 3600
ctaccggccc tttctttgtg aacgtcacat tggccgagcc gtgttcagtt 3650
cccaggtggc agactcgttt ttggtagttt gttttaactt ccaaggtggt 3700
tttacttctg atagccggtg attttccctc ctagcagaca tgccacaccg 3750
ggtaagagct ctgagtctta gtggttaagc attcctttct cttcagtgcc 3800
cagcagcacc cagtgttggt ctgtgtccat cagtgaccac caacattctg 3850
tgttcacatg tgtgggtcca acacttacta cctggtgtat gaaattggac 3900
ctgaactgtt ggatttttct agttgccgcc aaacaaggca aaaaaattta 3950
aacatgaagc acacacacaa aaaaggcagt aggaaaaatg ctggccctga 4000
tgacctgtcc ttattcagaa tgagagactg cggggggggc ctgggggtag 4050
tgtcaatgcc cctccagggc tggaggggaa gaggggcccc gaggatgggc 4100
ctgggctcag cattcgagat cttgagaatg atttttttta aatcatgcaa 4150
cctttcctta ggaagacatt tggttttcat catgattaag atgattccta 4200
gatttagcac aatggagaga ttccatgcca tctttactat gtggatggtg 4250
gtatcaggga agagggctca caagacacat ttgtcccccg ggcccaccac 4300
atcatcctca cgtgttcggt actgagcagc cactacccct gatgagaaca 4350
gtatgaagaa agggggctgt tggagtccca gaattgctga cagcagaggc 4400
tttgctgctg tgaatcccac ctgccaccag cctgcagcac accccacagc 4450
caagtagagg cgaaacgagt ggctcatcct acctgttagg agcaggtagg 4500
gcttgtactc actttaattt gaatcttatc aacttactca taaagggaca 4550
ggctagctag ctgtgtcaga agtagcaatg acaatgacca aggactgcta 4600
cacctctgat tacaattctg atgtgaaaaa gatggtgttt ggctcttata 4650
gagcctgtgt gaaaggccca tggatcagct cttcctgtgt ttgtaattta 4700
atgctgctac aagatgtttc tgtttcttag attctgacca tgactcataa 4750
gcttcttgtc attcttcatt gcttgtttgt ggtcacagat gcacaacact 4800
cctccagtct tgtgggggca gcttttggga agtctcagca gctcttctgg 4850
ctgtgttgtc agcactgtaa cttcgcagaa aagagtcgga ttaccaaaac 4900
actgcctgct cttcagactt aaagcactga taggacttaa aatagtctca 4950
ttcaaatact gtattttata taggcatttc acaaaaacag caaaattgtg 5000
gcattttgtg aggccaaggc ttggatgcgt gtgtaataga gccttatggt 5050
gtgtgcgcac acacccagag gagagtttga aaaatgctta ttggacacgt 5100
aacctggctc taatttgggc tgtttttcag atacactgtg ataagttctt 5150
ttacaaatat ctatagacat ggtaaacttt tggttttcag atatgcttaa 5200
tgatagtctt actaaatgca gaaataagaa taaactttct caaattatta 5250
aaaatgccta cacagtaagt gtgaattgct gcaacaggtt tgttctcagg 5300
agggtaagaa ctccaggtct aaacagctga cccagtgatg gggaatttat 5350
ccttgaccaa tttatccttg accaataacc taattgtcta ttcctgagtt 5400
ataaaggtcc ccatccttat tagctctact ggaattttca tacacgtaaa 5450
tgcagaagtt actaagtatt aagtattact gagtattaag tagtaatctg 5500
tcagttatta aaatttgtaa aatctattta tgaaaggtca ttaaaccaga 5550
tcatgttcct ttttttgtaa tcaaggtgac taagaaaatc agttgtgtaa 5600
ataaaatcat gtatc 5615
19
4315
DNA
Homo Sapien
19
tgagagccaa gcaaagaaca ttaaggaagg aaggaggaat gaggctggat 50
acggtgcagt gaaaaaggca cttccaagag tggggcactc actacgcaca 100
gactcgacgg tgccatcagc atgagaactt accgctactt cttgctgctc 150
ttttgggtgg gccagcccta cccaactctc tcaactccac tatcaaagag 200
gactagtggt ttcccagcaa agaaaagggc cctggagctc tctggaaaca 250
gcaaaaatga gctgaaccgt tcaaaaagga gctggatgtg gaatcagttc 300
tttctcctgg aggaatacac aggatccgat tatcagtatg tgggcaagtt 350
acattcagac caggatagag gagatggatc acttaaatat atcctttcag 400
gagatggagc aggagatctc ttcattatta atgaaaacac aggcgacata 450
caggccacca agaggctgga cagggaagaa aaacccgttt acatccttcg 500
agctcaagct ataaacagaa ggacagggag acccgtggag cccgagtctg 550
aattcatcat caagatccat gacatcaatg acaatgaacc aatattcacc 600
aaggaggttt acacagccac tgtccctgaa atgtctgatg tcggtacatt 650
tgttgtccaa gtcactgcga cggatgcaga tgatccaaca tatgggaaca 700
gtgctaaagt tgtctacagt attctacagg gacagcccta tttttcagtt 750
gaatcagaaa caggtattat caagacagct ttgctcaaca tggatcgaga 800
aaacagggag cagtaccaag tggtgattca agccaaggat atgggcggcc 850
agatgggagg attatctggg accaccaccg tgaacatcac actgactgat 900
gtcaacgaca accctccccg attcccccag agtacatacc agtttaaaac 950
tcctgaatct tctccaccgg ggacaccaat tggcagaatc aaagccagcg 1000
acgctgatgt gggagaaaat gctgaaattg agtacagcat cacagacggt 1050
gaggggctgg atatgtttga tgtcatcacc gaccaggaaa cccaggaagg 1100
gattataact gtcaaaaagc tcttggactt tgaaaagaag aaagtgtata 1150
cccttaaagt ggaagcctcc aatccttatg ttgagccacg atttctctac 1200
ttggggcctt tcaaagattc agccacggtt agaattgtgg tggaggatgt 1250
agatgagcca cctgtcttca gcaaactggc ctacatctta caaataagag 1300
aagatgctca gataaacacc acaataggct ccgtcacagc ccaagatcca 1350
gatgctgcca ggaatcctgt caagtactct gtagatcgac acacagatat 1400
ggacagaata ttcaacattg attctggaaa tggttcgatt tttacatcga 1450
aacttcttga ccgagaaaca ctgctatggc acaacattac agtgatagca 1500
acagagatca ataatccaaa gcaaagtagt cgagtacctc tatatattaa 1550
agttctagat gtcaatgaca acgccccaga atttgctgag ttctatgaaa 1600
cttttgtctg tgaaaaagca aaggcagatc agttgattca gaccctgcat 1650
gctgttgaca aggatgaccc ttatagtgga caccaatttt cgttttcctt 1700
ggcccctgaa gcagccagtg gctcaaactt taccattcaa gacaacaaag 1750
acaacacggc gggaatctta actcggaaaa atggctataa tagacacgag 1800
atgagcacct atctcttgcc tgtggtcatt tcagacaacg actacccagt 1850
tcaaagcagc actgggacag tgactgtccg ggtctgtgca tgtgaccacc 1900
acgggaacat gcaatcctgc catgcggagg cgctcatcca ccccacggga 1950
ctgagcacgg gggctctggt tgccatcctt ctgtgcatcg tgatcctact 2000
agtgacagtg gtgctgtttg cagctctgag gcggcagcga aaaaaagagc 2050
ctttgatcat ttccaaagag gacatcagag ataacattgt cagttacaac 2100
gacgaaggtg gtggagagga ggacacccag gcttttgata tcggcaccct 2150
gaggaatcct gaagccatag aggacaacaa attacgaagg gacattgtgc 2200
ccgaagccct tttcctaccc cgacggactc caacagctcg cgacaacacc 2250
gatgtcagag atttcattaa ccaaaggtta aaggaaaatg acacggaccc 2300
cactgccccg ccatacgact ccttggccac ttacgcctat gaaggcactg 2350
gctccgtggc ggattccctg agctcgctgg agtcagtgac cacggatgca 2400
gatcaagact atgattacct tagtgactgg ggacctcgat tcaaaaagct 2450
tgcagatatg tatggaggag tggacagtga caaagactcc taatctgttg 2500
cctttttcat tttccaatac gacactgaaa tatgtgaagt ggctatttct 2550
ttatatttat ccactactcc gtgaaggctt ctctgttcta cccgttccaa 2600
aagccaatgg ctgcagtccg tgtggatcca atgttagaga cttttttcta 2650
gtacactttt atgagcttcc aaggggcaaa tttttatttt ttagtgcatc 2700
cagttaacca agtcagccca acaggcaggt gccggagggg aggacaggga 2750
acagtatttc cacttgttct cagggcagcg tgcccgcttc cgctgtcctg 2800
gtgttttact acactccatg tcaggtcagc caactgccct aactgtacat 2850
ttcacaggct aatgggataa aggactgtgc tttaaagata aaaatatcat 2900
catagtaaaa gaaatgaggg catatcggct cacaaagaga taaactacat 2950
aggggtgttt atttgtgtca caaagaattt aaaataacac ttgcccatgc 3000
tatttgttct tcaagaactt tctctgccat caactactat tcaaaacctc 3050
aaatccaccc atatgttaaa attctcatta ctcttaagga atagaagcaa 3100
attaaacggt aacatccaaa agcaaccaca aacctagtac gacttcattc 3150
cttccactaa ctcatagttt gttatatcct agactagaca tgcgaaagtt 3200
tgcctttgta ccatataaag ggggagggaa atagctaata atgttaacca 3250
aggaaatata ttttaccata catttaaagt tttggccacc acatgtatca 3300
cgggtcactt gaaattcttt cagctatcag taggctaatg tcaaaattgt 3350
ttaaaaattc ttgaaagaat tttcctgaga caaattttaa cttcttgtct 3400
atagttgtca gtattattct actatactgt acatgaaagt agcagtgtga 3450
agtacaataa ttcatattct tcatatcctt cttacacgac taagttgaat 3500
tagtaaagtt agattaaata aaacttaaat ctcactctag gagttcagtg 3550
gagaggttag agccagccac acttgaacct aataccctgc ccttgacatc 3600
tggaaacctc tacatattta tataacgtga tacatttgga taaacaacat 3650
tgagattatg atgaaaacct acatattcca tgtttggaag acccttggaa 3700
gaggaaaatt ggattccctt aaacaaaagt gtttaagatt gtaattaaaa 3750
tgatagttga ttttcaaaag cattaatttt ttttcattgt ttttaacttt 3800
gctttcatga ccatcctgcc atccttgact ttgaactaat gataaagtaa 3850
tgatctcaaa ctatgacaga aaagtaatgt aaaatccatc caatctatta 3900
tttctctaat tatgcaatta gcctcatagt tattatccag aggacccaac 3950
tgaactgaac taatccttct ggcagattca aatcgtttat ttcacacgct 4000
gttctaatgg cacttatcat tagaatctta ccttgtgcag tcatcagaaa 4050
ttccagcgta ctataatgaa aacatccttg ttttgaaaac ctaaaagaca 4100
ggctctgtat atatatatac ttaagaatat gctgacttca cttattagtc 4150
ttagggattt attttcaatt aatattaatt ttctacaaat aattttagtg 4200
tcatttccat ttggggatat tgtcatatca gcacatattt tctgtttgga 4250
aacacactgt tgtttagtta agttttaaat aggtgtatta cccaagaagt 4300
aaagatggaa acgtt 4315
20
2521
DNA
Homo Sapien
20
cggtggaggc cacagacacc tcaaacctgg attccacaat tctacgttaa 50
gtgttggagt ttttattact ctgctgtagg aaagcctttg ccaatgctta 100
caaggaactg tttatccctg cttctctggg ttctgtttga tggaggtctc 150
ctaacaccac tacaaccaca gccacagcag actttagcca cagagccaag 200
agaaaatgtt atccatctgc caggacaacg gtcacatttc caacgtgtta 250
aacgtggctg ggtatggaat caattttttg tgctggaaga atacgtgggc 300
tccgagcctc agtatgtggg aaagctccat tccgacttag acaagggaga 350
gggcactgtg aaatacaccc tctcaggaga tggcgctggc accgttttta 400
ccattgatga aaccacaggg gacattcatg caataaggag cctagataga 450
gaagagaaac ctttctacac tcttcgtgct caggctgtgg acatagaaac 500
cagaaagccc ctggagcctg aatcagaatt catcatcaaa gtgcaggata 550
ttaatgataa tgagccaaag tttttggatg gaccttatgt tgctactgtt 600
ccagaaatgt ctcctgtggg tgcatatgta ctccaggtca aggccacaga 650
tgcagatgac ccgacctatg gaaacagtgc cagagtcgtt tacagcattc 700
ttcagggaca accttatttc tctattgatc ccaagacagg tgttattaga 750
acagctttgc caaacatgga cagagaagtc aaagaacaat atcaagtact 800
catccaagcc aaggatatgg gaggacagct tggaggatta gccggaacaa 850
caatagtcaa catcactctc accgatgtca atgacaatcc acctcgattc 900
cccaaaagca tcttccactt gaaagttcct gagtcttccc ctattggttc 950
agctattgga agaataagag ctgtggatcc tgattttgga caaaatgcag 1000
aaattgaata caatattgtt ccaggagatg ggggaaattt gtttgacatc 1050
gtcacagatg aggatacaca agagggagtc atcaaattga aaaagccttt 1100
agattttgaa acaaagaagg catacacttt caaagttgag gcttccaacc 1150
ttcaccttga ccaccggttt cactcggcgg gccctttcaa agacacagct 1200
acggtgaaga tcagcgtgct ggacgtagat gagccaccgg ttttcagcaa 1250
gccgctctac accatggagg tttatgaaga cactccggta gggaccatca 1300
ttggcgctgt cactgctcaa gacctggatg taggcagcgg tgctgttagg 1350
tacttcatag attggaagag tgatggggac agctacttta caatagatgg 1400
aaatgaagga accatcgcca ctaatgaatt actagacaga gaaagcactg 1450
cgcagtataa tttctccata attgcgagta aagttagtaa ccctttattg 1500
accagcaaag tcaatatact gattaatgtc ttagatgtaa atgaatttcc 1550
tccagaaata tctgtgccat atgagacagc cgtgtgtgaa aatgccaagc 1600
caggacagat aattcagata gtcagtgctg cagaccgaga tctttcacct 1650
gctgggcaac aattctcctt tagattatca cctgaggctg ctatcaaacc 1700
aaattttaca gttcgtgact tcagaaacaa cacagcgggg attgaaaccc 1750
gaagaaatgg atacagccgc aggcagcaag agttgtattt cctccctgtt 1800
gtaatagaag acagcagcta ccctgtccag agcagcacaa acacaatgac 1850
tattcgagtc tgtagatgtg actctgatgg caccatcctg tcttgtaatg 1900
tggaagcaat ttttctacct gtaggactta gcactggggc gttgattgca 1950
attctactat gcattgttat actcttagcc atagttgtac tgtatgtagc 2000
actgcgaagg cagaagaaaa agcacaccct gatgacctct aaagaagaca 2050
tcagagacaa cgtcatccat tacgatgatg aaggaggtgg ggaggaagat 2100
acccaggctt tcgacatcgg ggctctgaga aacccaaaag tgattgagga 2150
gaacaaaatt cgcagggata taaaaccaga ctctctctgt ttacctcgtc 2200
agagaccacc catggaagat aacacagaca taagggattt cattcatcaa 2250
aggctacagg aaaatgatgt agatccaact gccccaccaa tcgattcact 2300
ggccacatat gcctacgaag ggagtgggtc cgtggcagag tccctcagct 2350
ctatagactc tctcaccaca gaagccgacc aggactatga ctatctgaca 2400
gactggggac cccgctttaa agtcttggca gacatgtttg gcgaagaaga 2450
gagttataac cctgataaag tcacttaagg gagtcgtgga ggctaaaata 2500
caaccgagag gggagatttt t 2521
21
736
DNA
Homo Sapien
21
ggctctcacc ctcctctcct gcagctccag ctctgtgctc tgcctctgag 50
gagaccatgg cccggcctct gtgtaccctg ctactcctga tggctaccct 100
ggctggggct ctggcctcga gctccaagga ggagaatagg ataatcccag 150
gtggcatcta tgatgcagac ctcaatgatg agtgggtaca gcgtgccctt 200
cacttcgcca tcagcgagta caacaaggcc accgaagatg agtactacag 250
acgcccgctg caggtgctgc gagccaggga gcagaccttt gggggggtga 300
attacttctt cgacgtagag gtgggccgca ccatatgtac caagtcccag 350
cccaacttgg acacctgtgc cttccatgaa cagccagaac tgcagaagaa 400
acagttatgc tctttcgaga tctacgaagt tccctgggag gacagaatgt 450
ccctggtgaa ttccaggtgt caagaagcct aggggtctgt gccaggccag 500
tcacaccgac caccacccac tcccaccccc tgtagtgctc ccacccctgg 550
actggtggcc cccaccctgc gggaggcctc cccatgtgcc tgtgccaaga 600
gacagacaga gaaggctgca ggagtccttt gttgctcagc agggcgctct 650
gccctccctc cttccttctt gcttctaata gacctggtac atggtacaca 700
cacccccacc tcctgcaatt aaacagtagc atcgcc 736
22
2025
DNA
Homo Sapien
22
ggcagcggtg gcaggggctg caggagcaag tgaccaggag caggactggg 50
gacaggcctg atcgcccctg cacgaaccag acccttcgcc gccctcacga 100
tgactacctc tccgatcctg cagctgctgc tgcggctctc actgtgcggg 150
ctgctgctcc agagggcgga gacaggctct aaggggcaga cggcggggga 200
gctgtaccag cgctgggaac ggtaccgcag ggagtgccag gagaccttgg 250
cagccgcgga accgccttca ggcctcgcct gtaacgggtc cttcgatatg 300
tacgtctgct gggactatgc tgcacccaat gccactgccc gtgcgtcctg 350
cccctggtac ctgccctggc accaccatgt ggctgcaggt ttcgtcctcc 400
gccagtgtgg cagtgatggc caatggggac tttggagaga ccatacacaa 450
tgtgagaacc cagagaagaa tgaggccttt ctggaccaaa ggctcatctt 500
ggagcggttg caggtcatgt acactgtcgg ctactccctg tctctcgcca 550
cactgctgct agccctgctc atcttgagtt tgttcaggcg gctacattgc 600
actagaaact atatccacat caacctgttc acgtctttca tgctgcgagc 650
tgcggccatt ctcagccgag accgtctgct acctcgacct ggcccctacc 700
ttggggacca ggcccttgcg ctgtggaacc aggccctcgc tgcctgccgc 750
acggcccaga tcgtgaccca gtactgcgtg ggtgccaact acacgtggct 800
gctggtggag ggcgtctacc tgcacagtct cctggtgctc gtgggaggct 850
ccgaggaggg ccacttccgc tactacctgc tcctcggctg gggggccccc 900
gcgcttttcg tcattccctg ggtgatcgtc aggtacctgt acgagaacac 950
gcagtgctgg gagcgcaacg aagtcaaggc catttggtgg attatacgga 1000
cccccatcct catgaccatc ttgattaatt tcctcatttt tatccgcatt 1050
cttggcattc tcctgtccaa gctgaggaca cggcaaatgc gctgccggga 1100
ttaccggctg aggctggctc gctccacgct gacgctggtg cccctgctgg 1150
gtgtccacga ggtggtgttt gctcccgtga cagaggaaca ggcccggggc 1200
gccctgcgct tcgccaagct cggctttgag atcttcctca gctccttcca 1250
gggcttcctg gtcagcgtcc tctactgctt catcaacaag gaggtgcagt 1300
cggagatccg ccgtggctgg caccactgcc gcctgcgccg cagcctgggc 1350
gaggagcaac gccagctccc ggagcgcgcc ttccgggccc tgccctccgg 1400
ctccggcccg ggcgaggtcc ccaccagccg cggcttgtcc tcggggaccc 1450
tcccagggcc tgggaatgag gccagccggg agttggaaag ttactgctag 1500
ggggcgggat ccccgtgtct gttcagttag catggattta ttgagtgcca 1550
actgcgtgcc aggcccagta cggaggacgc tggggaaatg gtgaaggaaa 1600
cagaaaaaag gtccctgccc ttctggagat gacaactgag tggggaaaac 1650
agaccgtgaa cacaaaacat caagttccac acacgctatg gaatggttat 1700
gaagggaagc gagaaggggg cctagggtgg tctgggaggc gtctccaagg 1750
aggtgacact taagccatcc ccgaaagagg tgaaagagat cactttgggg 1800
agagctggag aacaggattc taggcggaag cgatagcata ggcaaaggcc 1850
cttgggcagg aaggcgctca gccttggctg gagtagaatt aagtcagagc 1900
caacaggttg gggagagaca gagaagtggg caggggcacc caagttggga 1950
tttcatttca ggtgcattgg agattcttag gagtgtctct tgggggtaat 2000
attttatttt ttaaaaaatg aggat 2025
23
3168
DNA
Homo Sapien
23
gccagagcgt gagccgcgac ctccgcgcag gtggtcgcgc cggtctccgc 50
ggaaatgttg tccaaagttc ttccagtcct cctaggcatc ttattgatcc 100
tccagtcgag ggtcgaggga cctcagactg aatcaaagaa tgaagcctct 150
tcccgtgatg ttgtctatgg cccccagccc cagcctctgg aaaatcagct 200
cctctctgag gaaacaaagt caactgagac tgagactggg agcagagttg 250
gcaaactgcc agaagcctct cgcatcctga acactatcct gagtaattat 300
gaccacaaac tgcgccctgg cattggagag aagcccactg tggtcactgt 350
tgagatcgcc gtcaacagcc ttggtcctct ctctatccta gacatggaat 400
acaccattga catcatcttc tcccagacct ggtacgacga acgcctctgt 450
tacaacgaca cctttgagtc tcttgttctg aatggcaatg tggtgagcca 500
gctatggatc ccggacacct tttttaggaa ttctaagagg acccacgagc 550
atgagatcac catgcccaac cagatggtcc gcatctacaa ggatggcaag 600
gtgttgtaca caattaggat gaccattgat gccggatgct cactccacat 650
gctcagattt ccaatggatt ctcactcttg ccctctatct ttctctagct 700
tttcctatcc tgagaatgag atgatctaca agtgggaaaa tttcaagctt 750
gaaatcaatg agaagaactc ctggaagctc ttccagtttg attttacagg 800
agtgagcaac aaaactgaaa taatcacaac cccagttggt gacttcatgg 850
tcatgacgat tttcttcaat gtgagcaggc ggtttggcta tgttgccttt 900
caaaactatg tcccttcttc cgtgaccacg atgctctcct gggtttcctt 950
ttggatcaag acagagtctg ctccagcccg gacctctcta gggatcacct 1000
ctgttctgac catgaccacg ttgggcacct tttctcgtaa gaatttcccg 1050
cgtgtctcct atatcacagc cttggatttc tatatcgcca tctgcttcgt 1100
cttctgcttc tgcgctctgt tggagtttgc tgtgctcaac ttcctgatct 1150
acaaccagac aaaagcccat gcttctccta aactccgcca tcctcgtatc 1200
aatagccgtg cccatgcccg tacccgtgca cgttcccgag cctgtgcccg 1250
ccaacatcag gaagcttttg tgtgccagat tgtcaccact gagggaagtg 1300
atggagagga gcgcccgtct tgctcagccc agcagccccc tagcccaggt 1350
agccctgagg gtccccgcag cctctgctcc aagctggcct gctgtgagtg 1400
gtgcaagcgt tttaagaagt acttctgcat ggtccccgat tgtgagggca 1450
gtacctggca gcagggccgc ctctgcatcc atgtctaccg cctggataac 1500
tactcgagag ttgttttccc agtgactttc ttcttcttca atgtgctcta 1550
ctggcttgtt tgccttaact tgtaggtacc agctggtacc ctgtggggca 1600
acctctccag ttccccagga ggtccaagcc ccttgccaag ggagttgggg 1650
gaaagcagca gcagcagcag gagcgactag agtttttcct gccccattcc 1700
ccaaacagaa gcttgcagag ggtttgtctt tgctgcccct ctcccctacc 1750
tggcccattc actgagtctt ctcagcagac catttcaaat tattaataaa 1800
tgggccacct ccctcttctt caaggagcat ccgtgatgct cagtgttcaa 1850
aaccacagcc acttagtgat cagctcccta aaaccatgcc taagtacagg 1900
cggattagct atcttccaac aatgctgacc accagacaat tactgcattt 1950
ttccagaagc ccactattgc ctttgtagtg ctttcggccc agttctggcc 2000
tcagcctcaa agtgcaccga ctagttgctt gcctatacct ggcacctcat 2050
taagatgctg ggcagcagta taacaggagg aagagatccc tctcctttgg 2100
tcagattatt atgttctcag ttctctctcc ctgctacccc tttctctgca 2150
gatagataga cactggcatt atccctttag gaagaggggg gggcagcaag 2200
agagcctatt tgggacagca ttcctctctc tctgctgctg tgacatctcc 2250
ctctccttgc tggctccatc tttcgtctgc actaccaatt caatgccctt 2300
catccaatgg gtatctattt ttgtgtgtga ttatagtaac tactccctgc 2350
tttatatgcc accctcttcc ttctctttga cccctgtgac tctttctgta 2400
actttcccag tgacttcccc tagccctgac ccaggcacta ggccttggtg 2450
acttcctggg gccaagaaac taaggaaact cggctttgca acaggcatta 2500
ctcgccattg attggtgccc acccagggca cactgtcgga gttctatcac 2550
ttgcttgacc cctggaccca taaaccagtc cactgttata cccggggcac 2600
tctaaccatc acaatcaatc aatcaaattc ccttaaattt gtatggcact 2650
ggaactttgg caaagcactt ttgacaagtt gtgtctgatt ggagcttcat 2700
gatagccttg tgacatcttt agggcaggat tcttatcccc attttgcaga 2750
tgaaaaccct gagtcacaga tttctgtggg actgtggatc tcactggaag 2800
ctatccaaga gcccactgtc accttctaga ccacatgata gggctagaca 2850
gctcagttca ccatgattct cttctgtcac ctctgctggc acaccagtgg 2900
caaggcccag aatggcgacc tctctttagc tcaatttctg ggcctgaggt 2950
gctcagactg cccccaagat caaatctctc ctggctgtag taacccagtg 3000
gaatgaattt ggacatgccc caatgcttct atatgctaag tgaaatctgt 3050
gtctgtaatt tgttgggggg tggatagggt ggggtctcca tctacttttt 3100
gtcaccatca tctgaaatgg ggaaatatgt aaataaatat atcagcaaag 3150
caaaaagaaa aaaaaaaa 3168
24
2837
DNA
Homo Sapien
24
tatcacagga ttgtctactt cagcaatagc aactaatgga tttgtaagag 50
gaggaggagc atattattta atatctagaa gtctagggcc agaatttggt 100
ggtgcaattg gtctaatctt cgcctttgcc aacgctgttg cagttgctat 150
gtatgtggtt ggatttgcag aaaccgtggt ggagttgctt aaggaacatt 200
ccatacttat gatagatgaa atcaatgata tccgaattat tggagccatt 250
acagtcgtga ttcttttagg tatctcagta gctggaatgg agtgggaagc 300
aaaagctcag attgttcttt tggtgatcct acttcttgct attggtgatt 350
tcgtcatagg aacatttatc ccactggaga gcaagaagcc aaaagggttt 400
tttggttata aatctgaaat atttaatgag aactttgggc ccgattttcg 450
agaggaagag actttctttt ctgtatttgc catctttttt cctgctgcaa 500
ctggtattct ggctggagca aatatctcag gtgatcttgc agatcctcag 550
tcagccatac ccaaaggaac actcctagcc attttaatta ctacattggt 600
ttacgtagga attgcagtat ctgtaggttc ttgtgttgtt cgagatgcca 650
ctggaaacgt taatgacact atcgtaacag agctaacaaa ctgtacttct 700
gcagcctgca aattaaactt tgatttttca tcttgtgaaa gcagtccttg 750
ttcctatggc ctaatgaaca acttccaggt aatgagtatg gtgtcaggat 800
ttacaccact aatttctgca ggtatatttt cagccactct ttcttcagca 850
ttagcatccc tagtgagtgc tcccaaaata tttcaggctc tatgtaagga 900
caacatctac ccagctttcc agatgtttgc taaaggttat gggaaaaata 950
atgaacctct tcgtggctac atcttaacat tcttaattgc acttggattc 1000
atcttaattg ctgaactgaa tgttattgca ccaattatct caaacttctt 1050
ccttgcatca tatgcattga tcaatttttc agtattccat gcatcacttg 1100
caaaatctcc aggatggcgt cctgcattca aatactacaa catgtggata 1150
tcacttcttg gagcaattct ttgttgcata gtaatgttcg tcattaactg 1200
gtgggctgca ttgctaacat atgtgatagt ccttgggctg tatatttatg 1250
ttacctacaa aaaaccagat gtgaattggg gatcctctac acaagccctg 1300
acttacctga atgcactgca gcattcaatt cgtctttctg gagtggaaga 1350
ccacgtgaaa aactttaggc cacagtgtct tgttatgaca ggtgctccaa 1400
actcacgtcc agctttactt catcttgttc atgatttcac aaaaaatgtt 1450
ggtttgatga tctgtggcca tgtacatatg ggtcctcgaa gacaagccat 1500
gaaagagatg tccatcgatc aagccaaata tcagcgatgg cttattaaga 1550
acaaaatgaa ggcattttat gctccagtac atgcagatga cttgagagaa 1600
ggtgcacagt atttgatgca ggctgctggt cttggtcgta tgaagccaaa 1650
cacacttgtc cttggattta agaaagattg gttgcaagca gatatgaggg 1700
atgtggatat gtatataaac ttatttcatg atgcttttga catacaatat 1750
ggagtagtgg ttattcgcct aaaagaaggt ctggatatat ctcatcttca 1800
aggacaagaa gaattattgt catcacaaga gaaatctcct ggcaccaagg 1850
atgtggtagt aagtgtggaa tatagtaaaa agtccgattt agatacttcc 1900
aaaccactca gtgaaaaacc aattacacac aaagttgagg aagaggatgg 1950
caagactgca actcaaccac tgttgaaaaa agaatccaaa ggccctattg 2000
tgcctttaaa tgtagctgac caaaagcttc ttgaagctag tacacagttt 2050
cagaaaaaac aaggaaagaa tactattgat gtctggtggc tttttgatga 2100
tggaggtttg accttattga taccttacct tctgacgacc aagaaaaaat 2150
ggaaagactg taagatcaga gtattcattg gtggaaagat aaacagaata 2200
gaccatgacc ggagagcgat ggctactttg cttagcaagt tccggataga 2250
cttttctgat atcatggttc taggagatat caataccaaa ccaaagaaag 2300
aaaatattat agcttttgag gaaatcattg agccatacag acttcatgaa 2350
gatgataaag agcaagatat tgcagataaa atgaaagaag atgaaccatg 2400
gcgaataaca gataatgagc ttgaacttta taagaccaag acataccggc 2450
agatcaggtt aaatgagtta ttaaaggaac attcaagcac agctaatatt 2500
attgtcatga gtctcccagt tgcacgaaaa ggtgctgtgt ctagtgctct 2550
ctacatggca tggttagaag ctctatctaa ggacctacca ccaatcctcc 2600
tagttcgtgg gaatcatcag agtgtcctta ccttctattc ataaatgttc 2650
tatacagtgg acagccctcc agaatggtac ttcagtgcct agtgtagtaa 2700
cctgaaatct tcaatgacac attaacatca caatggcgaa tggtgacttt 2750
tctttcacga tttcattaat ttgaaagcac acaggaaagc ttgctccatt 2800
gataacgtgt atggagactt cggttttagt caattcc 2837
25
4709
DNA
Homo Sapien
25
gagcttgtcc agacgaagcc tcgcagggat gggttggagc ctgggccgtg 50
cttcgctcag gcagcgtttg aggcagaccc agcagggtcc tcctggggcc 100
ttcctgcctt tgaactgcgg tggcgggcgg gcgcacggtc tcctgtacgc 150
cctagactag gggccgccat ctccatggcc acggccgtga gccggccctg 200
cgccggcagg tcgcgggaca tactgtggcg cgttttgggc tggaggatag 250
ttgcaagtat tgtttggtca gtgctatttc tacccatctg caccacagta 300
tttataattt tcagcaggat tgatttgttt catcctatac agtggctgtc 350
tgattctttc agtgacctgt atagttccta tgtaatcttt tacttcctgc 400
tgctgtcagt ggtaataata ataataagta ttttcaatgt ggagttctat 450
gcagttgtgc cttctattcc ttgctccaga ctagctctga tagggaagat 500
cattcatcct cagcaactca tgcactcatt tattcatgct gcaatgggaa 550
tggtgatggc ctggtgtgct gcagtgataa cccagggcca gtacagcttt 600
cttgtggttc cctgcactgg tactaacagc tttggtagcc ctgctgcgca 650
aacctgctta aatgaatatc atcttttttt cctactgact ggagcattta 700
tgggctatag ctatagcctc ctgtattttg ttaacaacat gaactatctt 750
ccatttccca tcatacagca atacaagttc ttgcgtttta ggagatctct 800
gctcttatta gttaaacaca gttgtgtgga atcactgttc ctggttagaa 850
atttctgcat tttatattat tttcttggct atattcccaa agcttggatt 900
agcactgcta tgaaccttca catagatgag caggttcata ggccacttga 950
cacagtgagt ggcctcttaa atctctcgtt actctaccat gtctggctgt 1000
gtggtgtctt tctcctgacg acttggtatg tctcatggat actcttcaaa 1050
atctatgcca cagaggctca tgtgtttcct gttcaaccac catttgcaga 1100
agggtcagat gagtgccttc caaaagtgtt aaatagcaat cctcccccca 1150
tcataaagta tttagccttg caggacctga tgttgctttc tcaatattct 1200
ccttcacgaa gacaagaagt tttcagcctc agccaaccag gtggacatcc 1250
ccacaattgg acagccattt caagggagtg tttgaatctt ttaaatggta 1300
tgactcagaa actgattctc tatcaagaag ctgctgctac gaatgggaga 1350
gtgtcttcat cttacccagt ggaacctaag aaattaaatt ctccagaaga 1400
aactgctttt cagacaccaa aatctagcca gatgcctcgg ccttcagtgc 1450
caccattagt taaaacatca ctgttttctt caaaattatc tacacctgat 1500
gttgtgagcc catttgggac cccatttggc tctagtgtaa tgaatcggat 1550
ggctggaatt tttgatgtaa acacctgcta tgggtcaccg caaagtcctc 1600
agctaataag aagggggcca agattgtgga catcagcttc tgatcagcaa 1650
atgactgaat tttctaatcc ttctccatct acctctatta gtgctgaggg 1700
taagacaatg agacaaccca gtgtgattta ttcatggatt cagaataaac 1750
gtgaacagat taagaatttc ttgtcaaaac gggtgctgat aatgtatttt 1800
ttcagtaagc acccagaggc ctccattcag gctgtttttt cagatgccca 1850
aatgcatatt tgggcattag aaggtctgtc gcacttagta gcagcatcat 1900
ttacagagga tagatttgga gttgtccaga cgacactacc agctatcctt 1950
aatactttgt tgacactgca agaggcagtc gacaagtact ttaagcttcc 2000
tcatgcttcc agtaaaccac cccggatttc aggaagcctt gtggacactt 2050
catataaaac attaagattt gcattcagag catcactgaa aactgccatc 2100
tatcgaataa ctactacatt tggtgaacat ctgaatgctg tgcaagcatc 2150
tgcagaacat cagaaaagac ttcaacagtt cttggagttc aaagaatagt 2200
taagtaatat aaactgtgtt cattacactg ctgatacaac tacagatggg 2250
acagtaaatg ttcagcattc ttggatcaga agaaaacgga ctaattagat 2300
gcttcctttg tcgtggtggt tgctttgaaa actatacttt aatgggagaa 2350
atcatggaaa gaaattctca acagaataac tgaaaactgc cttttctgta 2400
ccgattgctt tttgtgtgtg tggtataata aaatctttat tcaattttac 2450
agaagcattg atggcagtcg aaatgtctct agctcatata acttaatagt 2500
aataactaaa aaacttttag aatttacttt tgaaaggagg gaagccagtt 2550
ctgaaatgag tataggttga tttcatagtc ttcttaatta agagtttagc 2600
tctttgtaaa ctcaaaatac ataaactttt taagtgtagt ttcatttact 2650
gaaggataaa aatggtaaca gtgcagcaat attcacaaaa aatattgtct 2700
aacggacata ttttgttaat ctgttaggtt gggtttttgt ttccagggac 2750
aaattaaatt tgtatgatta cccaaaaaag ggtctcagtt tacagatgct 2800
aactctatat aaaggaatgt ggaaaaactc agttcttaag ttacaagatt 2850
aaaaattcac atttggtctt taagaaacaa ttgactgaca tctatgaatt 2900
tattttgtat catgctagta aacacgaagt attaatgtat gggtattttc 2950
ccagctagtt ttgctttctt tttctggagc aaaacattaa gtgattgcag 3000
agtttttcaa gcaagagaaa aaggtttgca aaaaaaccca ggaaatgttc 3050
ccttttttcc ccaccattca tcttcattag atcaaattct gtgaaacttg 3100
tctggtctct caaagggagc agcctctgta gtgttaaatg gctaattaaa 3150
ataggaagat ctttatagcc agaaacaact tagtcatcaa atagcaagtg 3200
aaaccaaaac gtcagaggga ttactgtact tggaagtatg ttgtgtgtcc 3250
caaatgtgaa cgaagtattg ttagaattta ttagatcagc ttctttggag 3300
atcaaagatt ggaaatccta gtcatagata ttcactggac tggctttgga 3350
ctgaaatgct cctttgtaat tcttttccta ttgtcttttc cttctagtgt 3400
cccaaaatat tttctttaaa gtcagcacag tactgtatat gaatctttaa 3450
tgtggtatca tatatgtcta cttttgtctg attcatcgat gtattatatc 3500
tttataattg aatattttag ctccgggtcc tgttgcccct tcaagcagta 3550
catgccaaat tataaatagg tgctactggc cttgagcata tcactgtggg 3600
acagttcccc aattgtcaag tgtttagata tgtagactat tgccatttgt 3650
ttttttgttt tggttttgct ttgtgtctga agctgaattg atttcttttt 3700
tttgaatgtg aaagttgaat ttcaaacgta gtcatttctt acagatggcc 3750
aagacagaaa attgtggcta ggttgactga gaactgttgt cttccatgta 3800
ttaacacaat taagcttttt atattccact ctctgtgctg accctggctg 3850
aggcattttg ggagacaagg actctgaatc ttctgcttcc attaaagaag 3900
aactgtgata ttcaacattg gatttctgag aataaagata ggatgattcc 3950
tttgaacttt gacttacttg tataaaatgt ccagctaggt taggtttttg 4000
ccatttccta tatactttgg gtaaagctac atttgatgag caatgtgaat 4050
gtttctgaga atgttcattc ctgttttctc ttaagagaat gtgctgtgta 4100
ctaaatacag gccacatagt gtctgcctgt tgaagatctg gaaactgcct 4150
ccccagatct gtattgtatt tggtaggtaa gggggtcagt ttctttttct 4200
cattgtgtgt tgataatcta cacaccatct gttggaacca gggtgttatt 4250
atggggaact cctcctgtgt actaggagga ggaccttagg gagaccaaga 4300
ggagagaagc atttcctttg atgaagtcac atcctgtcta tgagcccact 4350
aatgctgtaa cattggcctg aaagagagtg ttctttaaaa gcctttctcg 4400
gctgttagta taaaaacatg atggtatcag ctcttagcat gtttgcttga 4450
cccttatgga aggtataaat ccacagaact tccttcccag agaactggga 4500
aattgtccta gaaataaacc ttgtacagtt gagtggacat ggataagcaa 4550
caatttgtta ctttgcagga tttgttcctt ggtaattgtt tggtgtgtca 4600
tcctgtaaat attcatgata gtctgtttat atccttttgt atatcgttga 4650
tactggattg ggtagaaaaa taaattggca atttaaaaaa atggaacagt 4700
taattgaaa 4709
26
6310
DNA
Homo Sapien
26
gatgggggcc ccgtttgtct gggccttggg ccttttgatg ctgcagatgc 50
tgctctttgt ggctggggaa cagggcacac aggatatcac cgatgccagc 100
gaaagggggc tccacatgca gaagctgggg tctgggtcag tgcaggctgc 150
gctggcggag ctggtggccc tgccctgtct ctttaccctg cagccacggc 200
caagcgcagc ccgagatgcc cctcggataa agtggaccaa ggtgcggact 250
gcgtcgggcc agcgacagga cttgcccatc ctggtggcca aggacaatgt 300
cgtgagggtg gccaaaagct ggcagggacg agtgtcactg ccttcctacc 350
cccggcgccg agccaacgcc acgctacttc tggggccact gagggccagt 400
gactctgggc tgtaccgctg ccaggtggtg aggggcatcg aggatgagca 450
ggacctggtg cccttggagg tgacaggtgt tgtgttccac taccgatcag 500
cccgggaccg ctatgcactg accttcgctg aggcccagga ggcctgccgt 550
ctcagctcag ccatcattgc agcccctcgg catctacagg ctgcctttga 600
ggatggcttt gacaactgtg atgctggctg gctctctgac cgcactgttc 650
ggtatcctat cacccagtcc cgtcctggtt gctatggcga ccgtagcagc 700
cttccagggg ttcggagcta tgggaggcgc aacccacagg aactctacga 750
tgtgtattgc tttgcccggg agctgggggg cgaggtcttc tacgtgggcc 800
cggcccgccg cctgacactg gccggcgcgc gtgcacagtg ccgccgccag 850
ggtgccgcgc tggcctcggt gggacagctg cacctggcct ggcatgaggg 900
cctggaccag tgcgacccgg gctggctggc cgacggcagc gtgcgctacc 950
cgatccagac gccgcgccgg cgctgcgggg gcccagcccc gggcgtgcgc 1000
accgtctacc gcttcgctaa ccggaccggc ttcccctcac ccgccgagcg 1050
cttcgacgcc tactgcttcc gagctcatca ccccacgtca caacatggag 1100
acctagagac cccatcctct ggggatgagg gggagattct gtcagcagag 1150
gggcccccag ttagagaact ggagcccacc ctggaggagg aagaggtggt 1200
cacccctgac ttccaggagc ctctggtgtc cagtggggaa gaagaaaccc 1250
tgattttgga ggagaagcag gagtctcaac agaccctcag ccctacccct 1300
ggggacccca tgctggcctc atggcccact ggggaagtgt ggctaagcac 1350
ggtggccccc agccctagcg acatgggggc aggcactgca gcaagttcac 1400
acacggaggt ggccccaact gaccctatgc ctaggagaag ggggcgcttc 1450
aaagggttga atgggcgcta cttccagcag caggaaccgg agccggggct 1500
gcaagggggg atggaggcca gcgcccagcc ccccacctca gaggctgcag 1550
tgaaccaaat ggagcctccg ttggccatgg cagtcacaga gatgttgggc 1600
agtggccaga gccggagccc ctgggctgat ctgaccaatg aggtggatat 1650
gcctggagct ggttctgctg gtggcaagag ctccccagag ccctggctgt 1700
ggccccctac catggtccca cccagcatct caggccacag cagggcccct 1750
gtcctggagc tagagaaagc cgagggcccc agtgccaggc cagccacccc 1800
agacctgttt tggtccccct tggaggccac tgtctcagct cccagccctg 1850
ccccctggga ggcattccct gtggccacct ccccagatct ccctatgatg 1900
gccatgctgc gtggtcccaa agagtggatg ctaccacacc ccacccccat 1950
ctccaccgag gccaatagag ttgaggcaca tggtgaggcc accgccacgg 2000
ctccaccctc ccctgctgca gagaccaagg tgtattccct gcctctctct 2050
ttgaccccaa caggacaggg tggagaggcc atgcccacaa cacctgagtc 2100
ccccagggca gacttcagag aaactgggga gaccagccct gctcaggtca 2150
acaaagctga gcactccagc tccagcccat ggccttctgt aaacaggaat 2200
gtggctgtag gttttgtccc cactgagact gccactgagc caacgggcct 2250
caggggtatc ccggggtctg agtctggggt cttcgacaca gcagaaagcc 2300
ccacttctgg cttgcaggcc actgtagatg aggtgcagga cccctggccc 2350
tcagtgtaca gcaaagggct ggatgcaagt tccccatctg cccccctggg 2400
gagccctgga gtcttcttgg tacccaaagt caccccaaat ttggagcctt 2450
gggttgctac agatgaagga cccactgtga atcccatgga ttccacagtc 2500
acgccggccc ccagtgatgc tagtggaatt tgggaacctg gatcccaggt 2550
gtttgaagaa gccgaaagca ccaccttgag ccctcaggtg gccctggata 2600
caagcattgt gacgcccctc acgaccctgg agcaggggga caaggttgga 2650
gttccagcca tgtctacact gggctcctca agctcccaac cccacccaga 2700
gccagaggat caggtggaga cccagggaac atcaggagct tcagtgcctc 2750
cgcatcagag cagtccccta gggaaaccgg ctgttcctcc tgggacaccg 2800
actgcagcca gtgtgggcga gtctgcctca gtttcctcag gggagcctac 2850
ggtaccgtgg gacccctcca gcaccctgct gcctgtcacc ctgggcatag 2900
aggacttcga actggaggtc ctggcaggga gcccgggtgt agagagcttc 2950
tgggaggagg tggcaagtgg agaggagcca gccctgccag ggacccctat 3000
gaatgcaggt gcggaggagg tgcactcaga tccctgtgag aacaaccctt 3050
gtcttcatgg agggacatgt aatgccaatg gcaccatgta tggctgtagc 3100
tgtgatcagg gcttcgccgg ggagaactgt gagattgaca ttgatgactg 3150
cctctgcagc ccctgtgaga atggaggcac ctgtattgat gaggtcaatg 3200
gctttgtctg cctttgcctc cccagctatg ggggcagctt ttgtgagaaa 3250
gacaccgagg gctgtgaccg cggctggcat aagttccagg gccactgtta 3300
ccgctatttt gcccaccgga gggcatggga agatgccgag aaggactgcc 3350
gccgccgctc cggccacctg accagcgtcc actcaccgga ggaacacagc 3400
ttcattaata gctttgggca tgaaaacacg tggatcggcc tgaacgacag 3450
gatcgtggag agagatttcc agtggacgga caacaccggg ctgcaatttg 3500
agaactggcg agagaaccag ccggacaatt tcttcgcggg tggcgaggac 3550
tgtgtggtga tggtggcgca tgaaagcggg cgctggaacg atgtcccctg 3600
caactacaac ctaccctatg tctgcaagaa gggcacagtg ctctgtggtc 3650
cccctccggc agtggagaat gcctcactca tcggtgcccg caaggccaag 3700
aacaatgtcc atgccactgt aaggtaccag tgcaatgaag gatttgccca 3750
gcaccatgtg gtcaccattc gatgccggag caatggcaag tgggacaggc 3800
cccaaattgt ctgcaccaaa cccagacgtt cacatcggat gcggggacac 3850
caccaccacc accaacacca ccaccagcat caccaccaca aatcccgcaa 3900
ggagcgcaga aaacacaaga aacacccaac ggaggactgg gagaaggacg 3950
aagggaattt ttgctgaaga accagaaaaa agaaagcaca acacctttcc 4000
catgcctcct ctggagcctt cgcctgggga gacagaaccc agagagaaac 4050
aagagagtcc agaagtccct gaaccccaaa ctgttctcgc aaaaaaaata 4100
ttcctttgaa caaaggtctt cttttccttt ttttacatac acaagatctt 4150
cttggcaggt ggagccaggt gtctgaaaag ttcattctcg tctggctgaa 4200
ctctgggagt gtgtcccagc tgagggaagc acaagtagca aagctcattg 4250
gtctggtctc ttgtttgcca ggctgattga agcaggcctt gatgagggtg 4300
catgagtgta tgtttgcatt cacatgaagg aattgctttt cacaccagaa 4350
attcagactt agtcaatgtt ggctgaattc ctaaatccag gaagaagcct 4400
ggacgtaggg tcattagctt tgggaataga aggctacaca gaagcacact 4450
gtttttgaac ttgacaacag ctctcccttt accctggact tcagcccaag 4500
ttccgtcttt ggtcttggtg gataaacaca cagtgtggag atcccacgta 4550
ctgcatttta gggatgtttt taggacaacc tccctccatg ccttcagagt 4600
taggagtgag aatgatcaaa gcaatatgta ggtgatggag ggagagtgta 4650
ttgctaaccc ttccaggtct agtccagcgc tgagatttgg tggttctgca 4700
tgtgtgatga atctctttca cacaaataga cgagaggata tttagggcta 4750
gatgagccca gatttcttcc ccctccatct ctcagggaga caaagaacct 4800
ccttcctgga ccaaggaggt gctgccaagt tttctagccc agtgcacata 4850
cccagtcctt aagcagacat tggtagtgcc cctgccctgg gtcccactcc 4900
tgccccaccc cacccttgtc cctggccatt gcctggtggt ctagaaacac 4950
ttaaaacttg aagtagtgac acctacctgc ggtcatattg tagagagatg 5000
ctcagtgtta aaactgaaac acacaaacac acacacacac acatttttct 5050
cttgtagatt ttaatttttt aagtgggaaa gaactcacct tgccttcctc 5100
ccccaaatgt gcaacctgta aaaggtctct ccacaccagg ggccaggatc 5150
cagttccctc atctctggca ggaaagatcc acagcttttc ctccatgtct 5200
gttactcact ttcagcagtc cgggtaaaat ctgtggatca gggttaaaaa 5250
agcaccgtgg agaatggccc tcttcaggaa agaaaaataa gcaaatgaat 5300
ggtccaccta ggggttcagt aaagaaagaa atgtgttaac tgagcctgaa 5350
tcccttctgg gaagtaataa tgaccattga caactaagaa gtagacacca 5400
tgctaaagac ttacatacaa tctccttgaa tcttctcaat agcccattga 5450
cttagaaact gttactttcc cattttacac acagtgaaac tgaggctcag 5500
atataaagga aaggtactgg cttgaagtca caaccacgac aggagtaagg 5550
atttggaata aggatttggt cctgttttct ggaccaaatc cttactctgg 5600
ctctgcttac actttctctc catcaccaaa tccttactcc aaatccagaa 5650
gtcagagcca actcccatct tggttctgac ccaaatcctg ctctggactc 5700
tggagaggag attgaaatat aattgcaccc tcatacacat ttaggaaatg 5750
gttaagaagt gtaaactgaa cccttatcct tgtcttcaat cttcctccct 5800
gtagacatct atcttattat ggttattatt cagaaaaccc agggatacag 5850
gtttgtcttc ttactttgat aactcttctt agtttaaaat aataataata 5900
acacatcttt ggtcatctat gtcacacaaa aattttcctt tgtttgcggg 5950
gggctgggga tgcagtgttt tttggggggt cttggtttat gctccctgcc 6000
cttgagcccc tcagccgttt gccctgcccc cacctcggct ccatggtggg 6050
agggggctct ggtcttttct aaagtgggcg gtttgtcttt tgatctttcc 6100
cttttggatg tgcgtgtgtg tctgcgtgtg ccatgtgcgt ggcacgcata 6150
tgagtgtgtg tgcgtgtgaa cggctttggg tcctgctggt tttgctgtga 6200
gctgcagtgt tctgtgggtc tgtggtatct gacactgtgg acattaatgt 6250
acttcttgga cattttaata aattttttaa cagttcaaaa aaaaaaaaaa 6300
aaaaaaaaaa 6310
27
4577
DNA
Homo Sapien
27
actagagatg gcgggcgggc tgctctgaag agacctcggc ggcggcggag 50
gaggagagaa gcgcagcgcc gcgccgcgcc ggggcccatg tggggaggag 100
tcggagtcgc tgttgccgcc gccgcctgta gctgctggac ccgagtggga 150
gtgaggggga aacggcagga tgaagttcgc cgagcacctc tccgcgcaca 200
tcactcccga gtggaggaag caatacatcc agtatgaggc tttcaaggat 250
atgctgtatt cagctcagga ccaggcacct tctgtggaag ttacagatga 300
ggacacagta aagaggtatt ttgccaagtt tgaagagaag tttttccaaa 350
cctgtgaaaa agaacttgcc aaaatcaaca cattttattc agagaagctc 400
gcagaggctc agcgcaggtt tgctacactt cagaatgagc ttcagtcatc 450
actggatgca cagaaagaaa gcactggtgt tactacgctg cgacaacgca 500
gaaagccagt cttccacttg tcccatgagg aacgtgtcca acatagaaat 550
attaaagacc ttaaactggc cttcagtgag ttctacctca gtctaatcct 600
gctgcagaac tatcagaatc tgaattttac agggtttcga aaaatcctga 650
aaaagcatga caagatcctg gaaacatctc gtggagcaga ttggcgagtg 700
gctcacgtag aggtggcccc attttataca tgcaagaaaa tcaaccagct 750
tatctctgaa actgaggctg tagtgaccaa tgaacttgaa gatggtgaca 800
gacaaaaggc tatgaagcgt ttacgtgtcc cccctttggg agctgctcag 850
cctgcaccag catggactac ttttagagtt ggcctatttt gtggaatatt 900
cattgtactg aatattaccc ttgtgcttgc cgctgtattt aaacttgaaa 950
cagatagaag tatatggccc ttgataagaa tctatcgggg tggctttctt 1000
ctgattgaat tcctttttct actgggcatc aacacgtatg gttggagaca 1050
ggctggagta aaccatgtac tcatctttga acttaatccg agaagcaatt 1100
tgtctcatca acatctcttt gagattgctg gattcctcgg gatattgtgg 1150
tgcctgagcc ttctggcatg cttctttgct ccaattagtg tcatccccac 1200
atatgtgtat ccacttgccc tttatggatt tatggttttc ttccttatca 1250
accccaccaa aactttctac tataaatccc ggttttggct gcttaaactg 1300
ctgtttcgag tatttacagc ccccttccat aaggtaggct ttgctgattt 1350
ctggctggcg gatcagctga acagcctgtc agtgatactg atggacctgg 1400
aatatatgat ctgcttctac agtttggagc tcaaatggga tgaaagtaag 1450
ggcctgttgc caaataattc agaagaatca ggaatttgcc acaaatatac 1500
atatggtgtg cgggccattg ttcagtgcat tcctgcttgg cttcgcttca 1550
tccagtgcct gcgccgatat cgagacacaa aaagggcctt tcctcattta 1600
gttaatgctg gcaagtactc cacaactttc ttcatggtgg cgtttgcagc 1650
cctttacagc actcacaaag aacgaggtca ctcggacact atggtgttct 1700
tttacctgtg gattgtcttt tatatcatca gttcctgcta taccctcatc 1750
tgggatctca agatggactg gggtctcttc gataagaatg ctggagagaa 1800
cactttcctc cgggaagaga ttgtataccc ccaaaaagcc tactactact 1850
gtgccataat agaggatgtg attctgcgct ttgcttggac tatccaaatc 1900
tcgattacct ctacaacttt gttgcctcat tctggggaca tcattgctac 1950
tgtctttgcc ccacttgagg ttttccggcg atttgtgtgg aacttcttcc 2000
gcctggagaa tgaacatctg aataactgtg gtgaattccg tgctgtgcgg 2050
gacatctctg tggcccccct gaacgcagat gatcagactc tcctagaaca 2100
gatgatggac caggatgatg gggtacgaaa ccgccagaag aatcggtcat 2150
ggaagtacaa ccagagcata tccctgcgcc ggcctcgcct cgcttctcaa 2200
tccaaggctc gtgacactaa ggtattgata gaagacacag atgatgaagc 2250
taacacttga attttctgaa gtctagctta acatctttgg ttttcctact 2300
ctacaatcct ttcctcgacc aacgcaacct ctagtacctt tccagccgaa 2350
aacaggagaa aacacataac acattttccg agctcttccg gatcggatcc 2400
tatggactcc aaacaagctc actgtgtttc ttttcttttc ttctggttta 2450
attttaattt tctattttca aaacaagtat ttacttcatt tgccaatcag 2500
aggatgtttt aagaaacaaa acatagtatc ttatggattg tttacaatca 2550
caaggacata gatacctatc aggatgaaga acaggcattg caaggaccct 2600
ctgatgggac ggtactgaga tatctcggct tccgctcagc ccggttttga 2650
atggttgaaa ccggacattg gtttttaaat tttttgtcag tttatgtgga 2700
gaattttttt ctttccttca tacccagcgc aaaggcactg gccgcacttg 2750
caggaaaagt gcaacttaaa gcagtacctt cattcatgaa gctacttttt 2800
aatttgatgt aacttttctt attttgggaa gggttgctgg gtgggtggga 2850
aatatgatgt atttgttaca catagttttc tcattattta tgaaacttaa 2900
ccatacagaa tgatataact cctgtgcaat gaaggtgata acagtaaaag 2950
tgatataact cctgtgcaat gaaggtgata acagtaaaag aaggcagggg 3000
aaacttacgt tggatgacat ttatgagggt cagtcccaca tacctctttc 3050
aggagacaac ttgcaccagt ttgacctttt cttttctttg tttttatttt 3100
aagccaaagt ttcattgcta acttcttaag ttgctgctgc tttagagtcc 3150
tgagcatatc tctcataaca aggaatccca cacttcacac caccggctga 3200
atttcatgga agaggttctg ataatttttt taacttttta aggaacagat 3250
gtggaataca ctggcccata tttcaacctt aacagctgaa gctatgcctt 3300
attatgcatc cacatgtatg gtccctgtag cgtgaccttt actagctctg 3350
aatcagaaga cagagctatt tcagaggctc tgtgtgccct cactagatag 3400
tttttcttct gggttcaacc actttagcca gaatttgatc aaattaaaag 3450
tctgtcatgg ggaaactata tttttgagca catggaacaa attatacttc 3500
ctcattcata ttatgttgat acaaaagacc ttggcagcca tttctcccag 3550
cagttttaaa ggatgaacat tggatttcat gccatcccat agaaaacctg 3600
ttttaaaatt ttagggatct ttacttggtc atacatgaaa agtacactgc 3650
ttagaaatta tagactatta tgatctgtcc acagtgccca ttgtcacttc 3700
tttgtctcat ttcttccctt tgttccttag tcatccaaat aagcctgaaa 3750
accataagag atattacttt attgaatatg gttggcatta aatttagcat 3800
ttcattatct aacaaaatta atataaattc caggacatgg taaaatgtgt 3850
tttaataacc cccagaccca aatgaaaatt tcaaagtcaa taccagcaga 3900
ttcatgaaag taaatttagt cctataattt tcagcttaat tataaacaaa 3950
ggaacaaata agtggaaggg cagctattac cattcgctta gtcaaaacat 4000
tcggttactg ccctttaata cactcctatc atcagcactt ccaccatgta 4050
ttacaagtct tgacccatcc ctgtcgtaac tccagtaaaa gttactgtta 4100
ctagaaaatt tttatcaatt aactgacaaa tagtttcttt ttaaagtagt 4150
ttcttccatc tttattctga ctagcttcca aaatgtgttc cctttttgaa 4200
tcgaggtttt tttgttttgt tttgttttct gaaaaaatca tacaactttg 4250
tgcttctatt gcttttttgt gttttgttaa gcatgtccct tggcccaaat 4300
ggaagaggaa atgtttaatt aatgcttttt agtttaaata aattgaatca 4350
tttataataa tcagtgttaa caatttagtg acccttggta ggttaaaggt 4400
tgcattattt atacttgaga tttttttccc ctaactattc tgttttttgt 4450
actttaaaac tatgggggaa atatcactgg tctgtcaaga aacagcagta 4500
attattactg agttaaattg aaaagtccag tggaccaggc atttcttata 4550
taaataaaat tggtggtact aatgtgt 4577
28
2203
DNA
Homo Sapien
28
cccttgctgg acccgagtgg gagtgagggg gaaacggcag gatgaagttc 50
gccgagcacc tctccgcgca catcactccc gagtggagga agcaatacat 100
ccagtatgag gctttcaagg atatgctgta ttcagctcag gaccaggcac 150
cttctgtgga agttacagat gaggacacag taaagaggta ttttgccaag 200
tttgaagaga agtttttcca aacctgtgaa aaagaacttg ccaaaatcaa 250
cacattttat tcagagaagc tcgcagaggc tcagcgcagg tttgctacac 300
ttcagaatga gcttcagtca tcactggatg cacagaaaga aagcactggt 350
gttactacgc tgcgacaacg cagaaagcca gtcttccact tgtcccatga 400
ggaacgtgtc caacatagaa atattaaaga ccttaaactg gccttcagtg 450
agttctacct cagtctaatc ctgctgcaga actatcagaa tctgaatttt 500
acagggtttc gaaaaatcct gaaaaagcat gacaagatcc tggaaacatc 550
tcgtggagca gattggcgag tggctcacgt agaggtggcc ccattttata 600
catgcaagaa aatcaaccag cttatctctg aaactgaggc tgtagtgacc 650
aatgaacttg aagatggtga cagacaaaag gctatgaagc gtttacgtgt 700
cccccctttg ggagctgctc agcctgcacc agcatggact acttttagag 750
ttggcctatt ttgtggaata ttcattgtac tgaatattac ccttgtgctt 800
gccgctgtat ttaaacttga aacagataga agtatatggc ccttgataag 850
aatctatcgg ggtggctttc ttctgattga attccttttt ctactgggca 900
tcaacacgta tggttggaga caggctggag taaaccatgt actcatcttt 950
gaacttaatc cgagaagcaa tttgtctcat caacatctct ttgagattgc 1000
tggattcctc gggatattgt ggtgcctgag ccttctggca tgcttctttg 1050
ctccaattag tgtcatcccc acatatgtgt atccacttgc cctttatgga 1100
tttatggttt tcttccttat caaccccacc aaaactttct actataaatc 1150
ccggttttgg ctgcttaaac tgctgtttcg agtatttaca gcccccttcc 1200
ataaggtagg ctttgctgat ttctggctgg cggatcagct gaacagcctg 1250
tcagtgatac tgatggacct ggaatatatg atctgcttct acagtttgga 1300
gctcaaatgg gatgaaagta agggcctgtt gccaaataat tcagaagaat 1350
caggaatttg ccacaaatat acatatggtg tgcgggccat tgttcagtgc 1400
attcctgctt ggcttcgctt catccagtgc ctgcgccgat atcgagacac 1450
aaaaagggcc tttcctcatt tagttaatgc tggcaaatac tccacaactt 1500
tcttcatggt gacgtttgca gccctttaca gcactcacaa agaacgaggt 1550
cactcggaca ctatggtgtt cttttacctg tggattgtct tttatatcat 1600
cagttcctgc tataccctca tctgggatct caagatggac tggggtctct 1650
tcgataagaa tgctggagag aacactttcc tccgggaaga gattgtatac 1700
ccccaaaaag cctactacta ctgtgccata atagaggatg tgattctgcg 1750
ctttgcttgg actatccaaa tctcgattac ctctacaact ttgttgcctc 1800
attctgggga catcattgct actgtctttg ccccacttga ggttttccgg 1850
cgatttgtgt ggaacttctt ccgcctggag aatgaacatc tgaataactg 1900
tggtgaattc cgtgctgtgc gggacatctc tgtggccccc ctgaacgcag 1950
atgatcagac tctcctagaa cagatgatgg accaggatga tggggtacga 2000
aaccgccaga agaatcggtc atggaagtac aaccagagca tatccctgcg 2050
ccggcctcgc ctcgcttctc aatccaaggc tcgtgacact aaggtattga 2100
tagaagacac agatgatgaa gctaacactt gaattttctg aagtctagct 2150
taacatcttt ggttttccta ctctacaatc ctttcctcga ccaacgcaag 2200
ggc 2203
29
3162
DNA
Homo Sapien
29
gcgccctagc cctctttcgg ggatactggc cgaccccctc ttccttttcc 50
cctttagtga aggcctcccc cgtcgccgcg cggcttcccg gagccgactg 100
cagactccct cagcccggtg ttccccgcgt ccggacgccg aggtcgcggc 150
ttcgcagaaa ctcgggcccc tccatccgcc ctcagaaaag ggagcgatgt 200
tgatctcagg aagcacaaag ggaccttcct agctctgact gaaccacgga 250
gctcaccctg gacagtatca ctccgtggag gaagactgtg agactgtggc 300
tggaagccag attgtagcca cacatccgcc cctgccctac cccagagccc 350
tggagcagca actggctgca gatcacagac acagtgagga tatgagtgta 400
ggggtgagca cctcagcccc tctttcccca acctcgggca caagcgtggg 450
catgtctacc ttctccatca tggactatgt ggtgttcgtc ctgctgctgg 500
ttctctctct tgccattggg ctctaccatg cttgtcgtgg ctggggccgg 550
catactgttg gtgagctgct gatggcggac cgcaaaatgg gctgccttcc 600
ggtggcactg tccctgctgg ccaccttcca gtcagccgtg gccatcctgg 650
gtgtgccgtc agagatctac cgatttggga cccaatattg gttcctgggc 700
tgctgctact ttctggggct gctgatacct gcacacatct tcatccccgt 750
tttctaccgc ctgcatctca ccagtgccta tgagtacctg gagcttcgat 800
tcaataaaac tgtgcgagtg tgtggaactg tgaccttcat ctttcagatg 850
gtgatctaca tgggagttgt gctctatgct ccgtcattgg ctctcaatgc 900
agtgactggc tttgatctgt ggctgtccgt gctggccctg ggcattgtct 950
gtaccgtcta tacagctctg ggtgggctga aggccgtcat ctggacagat 1000
gtgttccaga cactggtcat gttcctcggg cagctggcag ttatcatcgt 1050
ggggtcagcc aaggtgggcg gcttggggcg tgtgtgggcc gtggcttccc 1100
agcacggccg catctctggg tttgagctgg atccagaccc ctttgtgcgg 1150
cacaccttct ggaccttggc cttcgggggt gtcttcatga tgctctcctt 1200
atacggggtg aaccaggctc aggtgcagcg gtacctcagt tcccgcacgg 1250
agaaggctgc tgtgctctcc tgttatgcag tgttcccctt ccagcaggtg 1300
tccctctgcg tgggctgcct cattggcctg gtcatgttcg cgtattacca 1350
ggagtatccc atgagcattc agcaggctca ggcagcccca gaccagttcg 1400
tcctgtactt tgtgatggat ctcctgaagg gcctgccagg cctgccaggg 1450
ctcttcattg cctgcctctt cagcggctct ctcagcacta tatcctctgc 1500
ttttaattca ttggcaactg ttacgatgga agacctgatt cgaccttggt 1550
tccctgagtt ctctgaagcc cgggccatca tgctttccag aggccttgcc 1600
tttggctatg ggctgctttg tctaggaatg gcctatattt cctcccagat 1650
gggacctgtg ctgcaggcag caatcagcat ctttggcatg gttgggggac 1700
cgctgctggg actcttctgc cttggaatgt tctttccatg tgctaaccct 1750
cctggtgctg ttgtgggcct gttggctggg ctcgtcatgg ccttctggat 1800
tggcatcggg agcatcgtga ccagcatggg cttcagcatg ccaccctctc 1850
cctctaatgg gtccagcttc tccctgccca ccaatctaac cgttgccact 1900
gtgaccacac tgatgccctt gactaccttc tccaagccca cagggctgca 1950
gcggttctat tccttgtctt acttatggta cagtgctcac aactccacca 2000
cagtgattgt ggtgggcctg attgtcagtc tactcactgg gagaatgcga 2050
ggccggtccc tgaaccctgc aaccatttac ccagtgttgc caaagctcct 2100
gtccctcctt ccgttgtcct gtcagaagcg gctccactgc aggagctacg 2150
gccaggacca cctcgacact ggcctgtttc ctgagaagcc gaggaatggt 2200
gtgctggggg acagcagaga caaggaggcc atggccctgg atggcacagc 2250
ctatcagggg agcagctcca cctgcatcct ccaggagacc tccctgtgat 2300
gttgactcag gaccccgcct ctgtcctcac tgtgccaggc catagccaga 2350
ggccaccctg tagtacaggg atgagtcttg gtgtgttctg cagggacagg 2400
cctggatgat ctagctcata ccaaaggacc ttgttctgag aggttcttgc 2450
ctgcaggaga agctgtcaca tctcaagcat gtgaggcacc gtttttctcg 2500
tcgcttgcca atctgttttt taaaggatca ggctcgtagg gagcaggatc 2550
atgccagaaa tagggatgga agtgcatcct ctgggaaaaa gataatggct 2600
tctgattcaa catagccata gtcctttgaa gtaagtggct agaaacagca 2650
ctctggttat aattgcccca gggcctgatt caggactgac tctccaccat 2700
aaaactggaa gctgcttccc ctgtagtccc catttcagta ccagttctgc 2750
cagccacagt gagcccctat tattactttc agattgtctg tgacactcaa 2800
gcccctctca tttttatctg tctacctcca ttctgaagag ggaggttttg 2850
gtgtccctgg tcctctggga atagaagatc catttgtctt tgtgtagagc 2900
aagcacgttt tccacctcac tgtctccatc ctccacctct gagatggaca 2950
cttaagagac ggggcaaatg tggatccaag aaaccagggc catgaccagg 3000
tccactgtgg agcagccatc tatctacctg actcctgagc caggctgccg 3050
tggtgtcatt tctgtcatcc gtgctctgtt tccttttgga gtttcttctc 3100
cacattatct ttgttcctgg ggaataaaaa ctaccattgg acctaaaaaa 3150
aaaaaaaaaa aa 3162
30
1432
DNA
Homo Sapien
30
gcgggcgccc agtgcaccgg aggaggtgag cgccaggtcg ccttcgcggc 50
ccggggacac aggcagggac gcgggagctg atgcggctgg accggccggg 100
gaaacagtat tttctggaag ggggcccctc tgaagcggtc caggatcctg 150
cacatggcgc tgaccggggc ctcagacccc tctgcagagg cagaggccaa 200
cggggagaag ccctttctgc tgcgggcatt gcagatcgcg ctggtggtct 250
ccctctactg ggtcacctcc atctccatgg tgttccttaa taagtacctg 300
ctggacagcc cctccctgcg gctggacacc cccatcttcg tcaccttcta 350
ccagtgcctg gtgaccacgc tgctgtgcaa aggcctcagc gctctggccg 400
cctgctgccc tggtgccgtg gacttcccca gcttgcgcct ggacctcagg 450
gtggcccgca gcgtcctgcc cctgtcggtg gtcttcatcg gcatgatcac 500
cttcaataac ctctgcctca agtacgtcgg tgtggccttc tacaatgtgg 550
gccgctcact caccaccgtc ttcaacgtgc tgctctccta cctgctgctc 600
aagcagacca cctccttcta tgccctgctc acctgcggta tcatcatcgg 650
gggcttctgg cttggtgtgg accaggaggg ggcagaaggc accctgtcgt 700
ggctgggcac cgtcttcggc gtgctggcta gcctctgtgt ctcgctcaac 750
gccatctaca ccacgaaggt gctcccggcg gtggacggca gcatctggcg 800
cctgactttc tacaacaacg tcaacgcctg catcctcttc ctgcccctgc 850
tcctgctgct cggggagctt caggccctgc gtgaccttgc ccagctgggc 900
agtgcccact tctgggggat gatgacgctg ggcggcctgt ttggctttgc 950
catcggctac gtgacaggac tgcagatcaa gttcaccagt ccgctgaccc 1000
acaatgtgtc gggcacggcc aaggcctgtg cccagacagt gctggccgtg 1050
ctctactacg aggagaccaa gagcttcctc tggtggacga gcaacatgat 1100
ggtgctgggc ggctcctccg cctacacctg ggtcaggggc tgggagatga 1150
agaagactcc ggaggagccc agccccaaag acagcgagaa gagcgccatg 1200
ggggtgtgag caccacaggc accctggatg gcccggcccc ggggcccgta 1250
cacaggcggg gccagcacag tagtgaaggc ggtctcctgg accccagaag 1300
cgtgctgtgg tgtggactgg gtgctactta tagacccaat cagaatacgg 1350
tggttgagaa ggaaccagtg tttacaagta atatcagaaa gttgaaggaa 1400
ccagtgttta caagtaatac cagaaagttg cc 1432
31
1094
DNA
Homo Sapien
31
gcccttatcc tgcacatggc gctgaccggg gcctcagacc cctctgcaga 50
ggcagaggcc aacggggaga agccctttct gctgcgggca ttgcagatcg 100
cgctggtggt ctccctctac tgggtcacct ccatctccat ggtgttcctt 150
aataagtacc tgctggacag cccctccctg cggctggaca cccccatctt 200
cgtcaccttc taccagtgcc tggtgaccac gctgctgtgc aaaggcctca 250
gcgctctggc cgcctgctgc cctggtgccg tggacttccc cagcttgcgc 300
ctggacctca gggtggcccg cagcgtcctg cccctgtcgg tggtcttcat 350
cggcatgatc accttcaata acctctgcct caagtacgtc ggtgtggcct 400
tctacaatgt gggccgctca ctcaccaccg tcttcaacgt gctgctctcc 450
tacctgctgc tcaagcagac cacctccttc tatgccctgc tcacctgcgg 500
tatcatcatc gggggcttct ggcttggtgt ggaccaggag ggggcagaag 550
gcaccctgtc gtggctgggc accgtcttcg gcgtgctggc tagcctctgt 600
gtctcgctca acgccatcta caccacgaag gtgctcccgg cggtggacgg 650
cagcatctgg cgcctgactt tctacaacaa cgtcaacgcc tgcatcctct 700
tcctgcccct gctcctgctg ctcggggagc ttcaggccct gcgtgacttt 750
gcccagctgg gcagtgccca cttctggggg atgatgacgc tgggcggcct 800
gtttggcttt gccatcggct acgtgacagg actgcagatc aagttcacca 850
gtccgctgac ccacaatgtg tcgggcacgg ccaaggcctg tgcccagaca 900
gtgctggccg tgctctacta cgaggagacc aagagcttcc tctggtggac 950
gagcaacatg atggtgctgg gcggctcctc cgcctacacc tgggtcaggg 1000
gctgggagat gaagaagact ccggaggagc ccagccccaa agacagcgag 1050
aagagcgcca tgggggtgtg agcaccacag gcaccctgaa gggc 1094
32
900
DNA
Homo Sapien
32
ccgagcgcgg ggcaccgggg gcctcctgta taggcgggca ccatgggctc 50
ctgctccggc cgctgcgcgc tcgtcgtcct ctgcgctttt cagctggtcg 100
ccgccctgga gaggcaggtg tttgacttcc tgggctacca gtgggcgccc 150
atcctggcca actttgtcca catcatcatc gtcatcctgg gactcttcgg 200
caccatccag taccggctgc gctacgtcat ggtgtacacg ctgtgggcag 250
ccgtctgggt cacctggaac gtcttcatca tctgcttcta cctggaagtc 300
ggtggcctct tacaggacag cgagctactg accttcagcc tctcccggca 350
tcgctcctgg tggcgtgagc gctggccagg ctgtctgcat gaggaggtgc 400
cagcagtggg cctcggggcc ccccatggcc aggccctggt gtcaggtgct 450
ggctgtgccc tggagcccag ctatgtggag gccctacaca gtggcctgca 500
gatcctgatc gcgcttctgg gctttgtctg tggctgccag gtggtcagcg 550
tgtttacgga ggaagaggac agctttgatt tcattggtgg atttgatcca 600
tttcctctct accatgtcaa tgaaaagcca tccagtctct tgtccaagca 650
ggtgtacttg cctgcgtaag tgaggaaaca gctgatcctg ctcctgtggc 700
ctccagcctc agcgaccgac cagtgacaat gacaggagct cccaggcctt 750
gggacgcgcc cccacccagc accccccagg cggccggcag cacctgccct 800
gggttctaag tactggacac cagccagggc ggcagggcag tgccacggct 850
ggctgcagcg tcaagagagt ttgtaatttc ctttctctta aaaaaaaaaa 900
33
666
DNA
Homo Sapien
33
ctcctgtata ggcgggcacc atgggctcct gctccggccg ctgcgcgctc 50
gtcgtcctct gcgcttttca gctggtcgcc gccctggaga ggcaggtgtt 100
tgacttcctg ggctaccagt gggcgcccat cctggccaac tttgtccaca 150
tcatcatcgt catcctggga ctcttcggca ccatccagta ccggctgcgc 200
tatgtcatgg tgtacacgct gtgggcagcc gtctgggtca cctggaacgt 250
cttcatcatc tgcttctacc tggaagtcgg tggcctctta aaggacagcg 300
agctactgac cttcagcctc tcccggcatc gctcctggtg gcgtgagcgc 350
tggccaggct gtctgcatga ggaggtgcca gcagtgggcc tcggggcccc 400
ccatggccag gccctggtgt caggtgctgg ctgtgccctg gagcccagct 450
atgtggaggc cctacacagt tgcctgcaga tcctgatcgc gcttctgggc 500
tttgtctgtg gctgccaggt ggtcagcgtg tttacggagg aagaggacag 550
ctttgatttc attggtggat ttgatccatt tcctctctac catgtcaatg 600
aaaagccatc cagtctcttg tccaagcagg tgtacttgcc tgcgtaagtg 650
aggaaacagc tgatcc 666
34
582
DNA
Homo sapien
34
ctcctgtata ggcgggcacc atgggctcct gctccggccg ctgcgcgctc 50
gtcgtcctct gcgcttttca gctggtcgcc gccctggaga ggcaggtgtt 100
tgacttcctg ggctaccagt gggcgcccat cctggccaac tttgtccaca 150
tcatcatcgt catcctggga ctcttcggca ccatccagta ccggctgcgc 200
tatgtcatgg tgtacacgct gtgggcagcc gtctgggtca cctggaacgt 250
cttcatcatc tgcttctacc tggaagtcgg tggcctctta aaggacagcg 300
agctactgac cttcagcctc tcccggcatc gctcctggtg gcgtgagcgc 350
tggccaggct gtctgcatga ggaggtgcca gcagtgggcc tcggggcccc 400
ccatggccag gccctggtgt caggtgctgg ctgtgccctg gagcccagct 450
atgtggaggc cctacacagt tgcctgcaga tcctgatcgc gcttctgggc 500
tttgtctgtg gctgccaggt ggtcagcgtg tttacggagg aagaggacag 550
ctgcctgcgt aagtgaggaa acagctgatc ca 582
35
582
DNA
Homo Sapien
35
ctcctgtata ggcgggcacc atgggctcct gctccggccg ctgcgcgctc 50
gtcgtcctct gcgcttttca gctggtcgcc gccctggaga ggcaggtgtt 100
tgacttcctg ggctaccagt gggcgcccat cctggccaac tttgtccaca 150
tcatcatcgt catcctggga ctcttcggca ccatccagta ccggctgcgc 200
tacgtcatgg tgtacacgct gtgggcagcc gtctgggtca cctggaacgt 250
cttcatcatc tgcttctacc tggaagtcgg tggcctctta caggacagcg 300
agctactgac cttcagcctc tcccggcatc gctcctggtg gcgtgagcgc 350
tggccaggct gtctgcatga ggaggtgcca gcagtgggcc tcggggcccc 400
ccatggccag gccctggtgt caggtgctgg ctgtgccctg gagcccagct 450
atgtggaggc cctacacagt ggcctgcaga tcctgatcgc gcttctgggc 500
tttgtctgtg gctgccaggt ggtcagcgtg tttacggagg aagaggacag 550
ctgcctgcgt aagtgaggaa acagctgatc ca 582
36
1546
DNA
Homo sapien
36
gcatggaaag tctttatttg agccccttag ctgatgtgga atcagaagag 50
caaaaaggtc atcttcagag tggcctgggc tgggtccttt tctctccagg 100
atagaaaagt ggtggtcact ttatccctag tagacatgct gctgggcttt 150
atcgccccag cattcccatc ccctccagag ccccttgtca ctccagacca 200
gcgagtgtgg gcctttatct ggactctgct tcctccctgg ggacaccagg 250
tcttggagca agagaacttg gcaggctctc cccatggcag tcttattcct 300
cctcctgttc ctatgtggaa ctccccaggc tgcagacaac atgcaggcca 350
tctatgtggc cttgggggag gcagtagagc tgccatgtcc ctcaccacct 400
actctacatg gggacgaaca cctgtcatgg ttctgcagcc ctgcagcagg 450
ctccttcacc accctggtag cccaagtcca agtgggcagg ccagccccag 500
accctggaaa accaggaagg gaatccaggc tcagactgct ggggaactat 550
tctttgtggt tggagggatc caaagaggaa gatgccgggc ggtactggtg 600
cgctgtgcta ggtcagcacc acaactacca gaactggagg gtgtacgacg 650
tcttggtgct caaaggatcc cagttatctg caagggctgc agatggatcc 700
ccctgcaatg tcctcctgtg ctctgtggtc cccagcagac gcatggactc 750
tgtgacctgg caggaaggga agggtcccgt gaggggccgt gttcagtcct 800
tctggggcag tgaggctgcc ctgctcttgg tgtgtcctgg ggaggggctt 850
tctgagccca ggagccgaag accaagaatc atccgctgcc tcatgactca 900
caacaaaggg gtcagcttta gcctggcagc ctccatcgat gcttctcctg 950
ccctctgtgc cccttccacg ggctgggaca tgccttggat tctgatgctg 1000
ctgctcacaa tgggccaggg agttgtcatc ctggccctca gcatcgtgct 1050
ctggaggcag agggtccgtg gggctccagg cagaggaaac cgaatgcggt 1100
gctacaactg tggtggaagc cccagcagtt cttgcaaaga ggccgtgacc 1150
acctgtggcg agggcagacc ccagccaggc ctggaacaga tcaagctacc 1200
tggaaacccc ccagtgacct tgattcacca acatccagcc tgcgtcgcag 1250
cccatcattg caatcaagtg gagacagagt cggtgggaga cgtgacttat 1300
ccagcccaca gggactgcta cctgggagac ctgtgcaaca gcgccgtggc 1350
aagccatgtg gcccctgcag gcattttggc tgcagcagct accgccctga 1400
cctgtctctt gccaggactg tggagcggat agggggagta ggagtagaga 1450
agggaacaag ggagcaaggg aacaagggac atctgaacat ctaatgtgag 1500
aagagaaaca tccttctgtg agtcattaaa atctatgaac cactct 1546
37
4619
DNA
Homo Sapien
37
ctttagagaa aggaagggcc aaaactacga cttggctttc tgaaacggaa 50
gcataaatgt tcttttcctc catttgtctg gatctgagaa cctgcatttg 100
gtattagcta gtggaagcag tatgtatggt tgaagtgcat tgctgcagct 150
ggtagcatga gtggtggcca ccagctgcag ctggctgccc tctggccctg 200
gctgctgatg gctaccctgc aggcaggctt tggacgcaca ggactggtac 250
tggcagcagc ggtggagtct gaaagatcag cagaacagaa agctgttatc 300
agagtgatcc ccttgaaaat ggaccccaca ggaaaactga atctcacttt 350
ggaaggtgtg tttgctggtg ttgctgaaat aactccagca gaaggaaaat 400
taatgcagtc ccacccactg tacctgtgca atgccagtga tgacgacaat 450
ctggagcctg gattcatcag catcgtcaag ctggagagtc ctcgacgggc 500
cccccgcccc tgcctgtcac tggctagcaa ggctcggatg gcgggtgagc 550
gaggagccag tgctgtcctc tttgacatca ctgaggatcg agctgctgct 600
gagcagctgc agcagccgct ggggctgacc tggccagtgg tgttgatctg 650
gggtaatgac gctgagaagc tgatggagtt tgtgtacaag aaccaaaagg 700
cccatgtgag gattgagctg aaggagcccc cggcctggcc agattatgat 750
gtgtggatcc taatgacagt ggtgggcacc atctttgtga tcatcctggc 800
ttcggtgctg cgcatccggt gccgcccccg ccacagcagg ccggatccgc 850
ttcagcagag aacagcctgg gccatcagcc agctggccac caggaggtac 900
caggccagct gcaggcaggc ccggggtgag tggccagact cagggagcag 950
ctgcagctca gcccctgtgt gtgccatctg tctggaggag ttctctgagg 1000
ggcaggagct acgggtcatt tcctgcctcc atgagttcca tcgtaactgt 1050
gtggacccct ggttacatca gcatcggact tgccccctct gcgtgttcaa 1100
catcacagag ggagattcat tttcccagtc cctgggaccc tctcgatctt 1150
accaagaacc aggtcgaaga ctccacctca ttcgccagca tcccggccat 1200
gcccactacc acctccctgc tgcctacctg ttgggccctt cccggagtgc 1250
agtggctcgg cccccacgac ctggtccctt cctgccatcc caggagccag 1300
gcatgggccc tcggcatcac cgcttcccca gagctgcaca tccccgggct 1350
ccaggagagc agcagcgcct ggcaggagcc cagcacccct atgcacaagg 1400
ctggggaatg agccacctcc aatccacctc acagcaccct gctgcttgcc 1450
cagtgcccct acgccgggcc aggccccctg acagcagtgg atctggagaa 1500
agctattgca cagaacgcag tgggtacctg gcagatgggc cagccagtga 1550
ctccagctca gggccctgtc atggctcttc cagtgactct gtggtcaact 1600
gcacggacat cagcctacag ggggtccatg gcagcagttc tactttctgc 1650
agctccctaa gcagtgactt tgacccccta gtgtactgca gccctaaagg 1700
ggatccccag cgagtggaca tgcagcctag tgtgacctct cggcctcgtt 1750
ccttggactc ggtggtgccc acaggggaaa cccaggtttc cagccatgtc 1800
cactaccacc gccaccggca ccaccactac aaaaagcggt tccagtggca 1850
tggcaggaag cctggcccag aaaccggagt cccccagtcc aggcctccta 1900
ttcctcggac acagccccag ccagagccac cttctcctga tcagcaagtc 1950
accggatcca actcagcagc cccttcgggg cggctctcta acccacagtg 2000
ccccagggcc ctccctgagc cagcccctgg cccagttgac gcctccagca 2050
tctgccccag taccagcagt ctgttcaact tgcaaaaatc cagcctctct 2100
gcccgacacc cacagaggaa aaggcggggg ggtccctccg agcccacccc 2150
tggctctcgg ccccaggatg caactgtgca cccagcttgc cagatttttc 2200
cccattacac ccccagtgtg gcatatcctt ggtccccaga ggcacacccc 2250
ttgatctgtg gacctccagg cctggacaag aggctgctac cagaaacccc 2300
aggcccctgt tactcaaatt cacagccagt gtggttgtgc ctgactcctc 2350
gccagcccct ggaaccacat ccacctgggg aggggccttc tgaatggagt 2400
tctgacaccg cagagggcag gccatgccct tatccgcact gccaggtgct 2450
gtcggcccag cctggctcag aggaggaact cgaggagctg tgtgaacagg 2500
ctgtgtgaga tgttcaggcc tagctccaac caagagtgtg ctccagatgt 2550
gtttgggccc tacctggcac agagtcctgc tcctgggaaa ggaaaggacc 2600
acagcaaaca ccattctttt tgccgtactt cctagaagca ctggaagagg 2650
actggtgatg gtggagggtg agagggtgcc gtttcctgct ccagctccag 2700
accttgtctg cagaaaacat ctgcagtgca gcaaatccat gtccagccag 2750
gcaaccagct gctgcctgtg gcgtgtgtgg gctggatccc ttgaaggctg 2800
agtttttgag ggcagaaagc tagctatggg tagccaggtg ttacaaaggt 2850
gctgctcctt ctccaacccc tacttggttt ccctcacccc aagcctcatg 2900
ttcataccag ccagtgggtt cagcagaacg catgacacct tatcacctcc 2950
ctccttgggt gagctctgaa caccagcttt ggcccctcca cagtaaggct 3000
gctacatcag gggcaaccct ggctctatca ttttcctttt ttgccaaaag 3050
gaccagtagc ataggtgagc cctgagcact aaaaggaggg gtccctgaag 3100
ctttcccact atagtgtgga gttctgtccc tgaggtgggt acagcagcct 3150
tggttcctct gggggttgag aataagaata gtggggaggg aaaaactcct 3200
ccttgaagat ttcctgtctc agagtcccag agaggtagaa aggaggaatt 3250
tctgctggac ttcatctggg cagaggaagg atggaatgaa ggtagaaaag 3300
gcagaattac agctgagcgg ggacaacaaa gagttcttct ctgggaaaag 3350
ttttgtctta gagcaaggat ggaaaatggg gacaacaaag gaaaagcaaa 3400
gtgtgaccct tgggtttgga cagcccagag gcccagctcc ccagtataag 3450
ccatacaggc cagggaccca caggagagtg gattagagca caagtctggc 3500
ctcactgagt ggacaagagc tgatgggcct catcagggtg acattcaccc 3550
cagggcagcc tgaccactct tggcccctca ggcattatcc catttggaat 3600
gtgaatgtgg tggcaaagtg ggcagaggac cccacctggg aacctttttc 3650
cctcagttag tggggagact agcacctagg tacccacatg ggtatttata 3700
tctgaaccag acagacgctt gaatcaggca ctatgttaag aaatatattt 3750
atttgctaat atatttatcc acaaatgtgg tctggtcttg tggttttgtt 3800
ctgtcgtgac tgtcactcag ggtaacaacg tcatctcttt ctacatcaag 3850
agaagtaaat tatttatgtt atcagaggct aggctccgat tcatgaaagg 3900
atagggtaga gtagagggct tggcaataag aactggtttg taagccccta 3950
aaagtgtggc ttagtgagat cagggaagga gaaagcatga ctggattctt 4000
actgtgcttc agtcattatt attatactgt tcacttcaca cattatcata 4050
cttcagtgac tyagaccttg ggcaaatact ctgtgcctcg ctttttcagt 4100
ccataaaatg ggcctactta atagttgttg caggacttac atgagataat 4150
agagtgtaga aaatatgttc caaagtggaa agttttattc agtgatagaa 4200
aacatccaaa cctgtcacag agcccatctg aacacagcat gggaccgcca 4250
acaagaagaa agcccgcccg gaagcagctc aatcaggagg ctgggctgga 4300
atgacagcgc agcggggcct gaaactattt atatcccaaa gctcctctca 4350
gataaacaca aatgactgcg ttctgcctgc actcgggcta ttgcgaggac 4400
agagagctgg tgctccattg gcgtgaagtc tccagggcca gaaggggcct 4450
ttgtcgcttc ctcacaaggc acaagttccc cttctgcttc cccgagaaag 4500
gtttggtagg ggtggtggtt tagtgcctat agaacaaggc atttcgcttc 4550
ctagacggtg aaatgaaagg gaaaaaaagg acacctaatc tcctacaaat 4600
ggtctttagt aaaggaacc 4619
38
3510
DNA
Homo Sapien
38
gcagctctgg gggagctcgg agctcccgat cacggcttct tgggggtagc 50
tacggctggg tgtgtagaac ggggccgggg ctggggctgg gtcccctagt 100
ggagacccaa gtgcgagagg caagaactct gcagcttcct gccttctggg 150
tcagttcctt attcaagtct gcagccggct cccagggaga tctcggtgga 200
acttcagaaa cgctgggcag tctgcctttc aaccatgccc ctgtccctgg 250
gagccgagat gtgggggcct gaggcctggc tgctgctgct gctactgctg 300
gcatcattta caggccggtg ccccgcgggt gagctggaga cctcagacgt 350
ggtaactgtg gtgctgggcc aggacgcaaa actgccctgc ttctaccgag 400
gggactccgg cgagcaagtg gggcaagtgg catgggctcg ggtggacgcg 450
ggcgaaggcg cccaggaact agcgctactg cactccaaat acgggcttca 500
tgtgagcccg gcttacgagg gccgcgtgga gcagccgccg cccccacgca 550
accccctgga cggctcagtg ctcctgcgca acgcagtgca ggcggatgag 600
ggcgagtacg agtgccgggt cagcaccttc cccgccggca gcttccaggc 650
gcggctgcgg ctccgagtgc tggtgcctcc cctgccctca ctgaatcctg 700
gtccagcact agaagagggc cagggcctga ccctggcagc ctcctgcaca 750
gctgagggca gcccagcccc cagcgtgacc tgggacacgg aggtcaaagg 800
cacaacgtcc agccgttcct tcaagcactc ccgctctgct gccgtcacct 850
cagagttcca cttggtgcct agccgcagca tgaatgggca gccactgact 900
tgtgtggtgt cccatcctgg cctgctccag gaccaaagga tcacccacat 950
cctccacgtg tccttccttg ctgaggcctc tgtgaggggc cttgaagacc 1000
aaaatctgtg gcacattggc agagaaggag ctatgctcaa gtgcctgagt 1050
gaagggcagc cccctccctc atacaactgg acacggctgg atgggcctct 1100
gcccagtggg gtacgagtgg atggggacac tttgggcttt cccccactga 1150
ccactgagca cagcggcatc tacgtctgcc atgtcagcaa tgagttctcc 1200
tcaagggatt ctcaggtcac tgtggatgtt cttgaccccc aggaagactc 1250
tgggaagcag gtggacctag tgtcagcctc ggtggtggtg gtgggtgtga 1300
tcgccgcact cttgttctgc cttctggtgg tggtggtggt gctcatgtcc 1350
cgataccatc ggcgcaaggc ccagcagatg acccagaaat atgaggagga 1400
gctgaccctg accagggaga actccatccg gaggctgcat tcccatcaca 1450
cggaccccag gagccagccg gaggagagtg tagggctgag agccgagggc 1500
caccctgata gtctcaagga caacagtagc tgctctgtga tgagtgaaga 1550
gcccgagggc cgcagttact ccacgctgac cacggtgagg gagatagaaa 1600
cacagactga actgctgtct ccaggctctg ggcgggccga ggaggaggaa 1650
gatcaggatg aaggcatcaa acaggccatg aaccattttg ttcaggagaa 1700
tgggacccta cgggccaagc ccacgggcaa tggcatctac atcaatgggc 1750
ggggacacct ggtctgaccc aggcctgcct cccttcccta ggcctggctc 1800
cttctgttga catgggagat tttagctcat cttgggggcc tccttaaaca 1850
cccccatttc ttgcggaaga tgctccccat cccactgact gcttgacctt 1900
tacctccaac ccttctgttc atcgggaggg ctccaccaat tgagtctctc 1950
ccaccatgca tgcaggtcac tgtgtgtgtg catgtgtgcc tgtgtgagtg 2000
ttgactgact gtgtgtgtgt ggaggggtga ctgtccgtgg aggggtgact 2050
gtgtccgtgg tgtgtattat gctgtcatat cagagtcaag tgaactgtgg 2100
tgtatgtgcc acgggatttg agtggttgcg tgggcaacac tgtcagggtt 2150
tggcgtgtgt gtcatgtggc tgtgtgtgac ctctgcctga aaaagcaggt 2200
attttctcag accccagagc agtattaatg atgcagaggt tggaggagag 2250
aggtggagac tgtggctcag acccaggtgt gcgggcatag ctggagctgg 2300
aatctgcctc cggtgtgagg gaacctgtct cctaccactt cggagccatg 2350
ggggcaagtg tgaagcagcc agtccctggg tcagccagag gcttgaactg 2400
ttacagaagc cctctgccct ctggtggcct ctgggcctgc tgcatgtaca 2450
tattttctgt aaatatacat gcgccgggag cttcttgcag gaatactgct 2500
ccgaatcact tttaattttt ttcttttttt tttcttgccc tttccattag 2550
ttgtattttt tatttatttt tatttttatt tttttttaga gatggagtct 2600
cactatgttg ctcaggctgg ccttgaactc ctgggctcaa gcaatcctcc 2650
tgcctcagcc tccctagtag ctgggacttt aagtgtacac cactgtgcct 2700
gctttgaatc ctttacgaag agaaaaaaaa aattaaagaa agcctttaga 2750
tttatccaat gtttactact gggattgctt aaagtgaggc ccctccaaca 2800
ccagggggtt aattcctgtg attgtgaaag gggctacttc caaggcatct 2850
tcatgcaggc agccccttgg gagggcacct gagagctggt agagtctgaa 2900
attagggatg tgagcctcgt ggttactgag taaggtaaaa ttgcatccac 2950
cattgtttgt gataccttag ggaattgctt ggacctggtg acaagggctc 3000
ctgttcaata gtggtgttgg ggagagagag agcagtgatt atagaccgag 3050
agagtaggag ttgaggtgag gtgaaggagg tgctgggggt gagaatgtcg 3100
cctttccccc tgggttttgg atcactaatt caaggctctt ctggatgttt 3150
ctctgggttg gggctggagt tcaatgaggt ttatttttag ctggcccacc 3200
cagatacact cagccagaat acctagattt agtacccaaa ctcttcttag 3250
tctgaaatct gctggatttc tggcctaagg gagaggctcc catccttcgt 3300
tccccagcca gcctaggact tcgaatgtgg agcctgaaga tctaagatcc 3350
taacatgtac attttatgta aatatgtgca tatttgtaca taaaatgata 3400
ttctgttttt aaataaacag acaaaacttg aaaaaaaaaa aaaaaaaaaa 3450
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3500
aaaaaaaaaa 3510
39
2211
DNA
Homo sapien
39
ttgggggttt attctcttcc cttctaactt gacagggtct tgctctgtca 50
ttcaggcaag agtgcagtag tgtgatcact tcttactgcc gcctcaagct 100
tccagcctca actcaagcaa tcctcccacc tcagccaccc aagtggctgg 150
gactacagat taagaatgac ccaaaataaa ttaaagcttt gttccaaagc 200
caatgtgtat actgaagtgc ctgatggagg atggggctgg gcggtagctg 250
tttcattttt cttcgttgaa gtcttcacct acggcatcat caagacattt 300
ggtgtcttct ttaatgactt aatggacagt tttaatgaat ccaatagcag 350
gatctcatgg ataatctcaa tctgtgtgtt tgtcttaaca ttttcagctc 400
ccctcgccac agtcctgagc aatcgtttcg gacaccgtct ggtagtgatg 450
ttgggggggc tacttgtcag caccgggatg gtggccgcct ccttctcaca 500
agaggtttct catatgtacg tcgccatcgg catcatctct ggtctgggat 550
actgctttag ttttctccca actgtaacca tcctatcaca atattttggc 600
aaaagacgtt ccatagtcac tgcagttgct tccacaggag aatgtttcgc 650
tgtgtttgct ttcgcaccag caatcatggc tctgaaggag cgcattggct 700
ggagatacag cctcctcttc gtgggcctac tacagttaaa cattgtcatc 750
ttcggagcac tgctcagacc catcattatc agaggaccag cgtcaccgaa 800
aatagtcatc caggaaaatc ggaaagaagc gcagtatatg cttgaaaatg 850
agaaaacacg aacctcaata gactccattg actcaggagt agaactaact 900
acctcaccta aaaatgtgcc tactcacact aacctggaac tggagccgaa 950
ggccgacatg cagcaggtcc tggtgaagac cagccccagg ccaagcgaaa 1000
agaaagcccc gctattagac ttctccattt tgaaagagaa aagttttatt 1050
tgttatgcat tatttggtct ctttgcaaca ctgggattct ttgcaccttc 1100
cttgtacatc attcctctgg gcattagtct gggcattgac caggaccgcg 1150
ctgctttttt attatctacg atggccattg cagaagtttt cggaaggatc 1200
ggagctggtt ttgtcctcaa cagggagccc attcgtaaga tttacattga 1250
gctcatctgc gtcatcttat tgactgtgtc tctgtttgcc tttacttttg 1300
ctactgaatt ctggggtcta atgtcatgca gcatattttt tgggtttatg 1350
gttggaacaa taggaggact cacattccac tgcttgctga agatgatgtc 1400
gtgggcattg cagaagatgt cttctgcagc tggggtctac atcttcattc 1450
agagcatagc aggactggct ggaccgcccc ttgcaggttt gttggtggac 1500
caaagtaaga tctacagcag ggccttctac tcctgcgcag ctggcatggc 1550
cctggctgct gtgtgcctcg ccctggtgag accgtgtaag atgggactgt 1600
gccagcgtca tcactcaggt gaaacaaagg tagtgagcca tcgtgggaag 1650
actttacagg acatacctga agactttctg gaaatggatc ttgcaaaaaa 1700
tgagcacaga gttcacgtgc aaatggagcc ggtatgacac actttcttac 1750
aacaacagcc actgtgttgg ctggagaggg atggggtggg cccaacgggg 1800
acacaaggag gcagaggagc taacccctct actccacttt caaaactaca 1850
ttttaaaggg aatgtgtatg tgaagagcac taccaacatc gcttttgttt 1900
tgttttgttt tgttttaagc tttttttttt tgcttgtttt taaagccaaa 1950
acaaaaaaca accaagcact cttccatata taaatctggc tgtattcagt 2000
agcaatacaa gagatatgta gaaagactct ttggttcaca ttccgatatt 2050
aaaatagtga catgaactgg caaagtggtt ttaaaagctt tcacgtggga 2100
taaatgattt tctttttttc ttttctttct tcctatggtc ttgtctgaat 2150
aaactactct cctgaataaa acaacatcca acccaggtca ttgaaatgaa 2200
attggccagt c 2211
40
685
DNA
Homo Sapien
40
gatgtgctcc ttggagctgg tgtgcagtgt cctgactgta agatcaagtc 50
caaacctgtt ttggaattga ggaaacttct cttttgatct cagcccttgg 100
tggtccaggt cttcatgctg ctgtgggtga tattactggt cctggctcct 150
gtcagtggac agtttgcaag gacacccagg cccattattt tcctccagcc 200
tccatggacc acagtcttcc aaggagagag agtgaccctc acttgcaagg 250
gatttcgctt ctactcacca cagaaaacaa aatggtacca tcggtacctt 300
gggaaagaaa tactaagaga aaccccagac aatatccttg aggttcagga 350
atctggagag tacagatgcc aggcccaggg ctcccctctc agtagccctg 400
tgcacttgga tttttcttca gagatgggat ttcctcatgc tgcccaggct 450
aatgttgaac tcctgggctc aagtgatctg ctcacctagg cctctcaaag 500
cgctgggatt acagcttcgc tgatcctgca agctccactt tctgtgtttg 550
aaggagactc tgtggttctg aggtgccggg caaaggcgga agtaacactg 600
aataatacta tttacaagaa tgataatgtc ctggcattcc ttaataaaag 650
aactgacttc caaaaaaaaa aaaaaaaaaa aaaaa 685
41
5392
DNA
Homo Sapien
41
aattcactaa tgcattctgc tctttttgag agcacagctt ctcagatgtg 50
ctccttggag ctggtgtgca gtgtcctgac tgtaagatca agtccaaacc 100
tgttttggaa ttgaggaaac ttctcttttg atctcagccc ttggtggtcc 150
aggtcttcat gctgctgtgg gtgatattac tggtcctggc tcctgtcagt 200
ggacagtttg caaggacacc caggcccatt attttcctcc agcctccatg 250
gaccacagtc ttccaaggag agagagtgac cctcacttgc aagggatttc 300
gcttctactc accacagaaa acaaaatggt accatcggta cctcgggaaa 350
gaaatactaa gagaaacccc agacaatatc cttgaggttc aggaatctgg 400
agagtacaga tgccaggccc agggctcccc tctcagtagc cctgtgcact 450
tggatttttc ttcagcttcg ctgatcctgc aagctccact ttctgtgttt 500
gaaggagact ctgtggttct gaggtgccgg gcaaaggcgg aagtaacact 550
gaataatact atttacaaga atgataatgt cctggcattc cttaataaaa 600
gaactgactt ccatattcct catgcatgtc tcaaggacaa tggtgcatat 650
cgctgtactg gatataagga aagttgttgc cctgtttctt ccaatacagt 700
caaaatccaa gtccaagagc catttacacg tccagtgctg agagccagct 750
ccttccagcc catcagcggg aacccagtga ccctgacctg tgagacccag 800
ctctctctag agaggtcaga tgtcccgctc cggttccgct tcttcagaga 850
tgaccagacc ctgggattag gctggagtct ctccccgaat ttccagatta 900
ctgccatgtg gagtaaagat tcagggttct actggtgtaa ggcagcaaca 950
atgcctcaca gcgtcatatc tgacagcccg agatcctgga tacaggtgca 1000
gatccctgca tctcatcctg tcctcactct cagccctgaa aaggctctga 1050
attttgaggg aaccaaggtg acacttcact gtgaaaccca ggaagattct 1100
ctgcgcactt tgtacaggtt ttatcatgag ggtgtccccc tgaggcacaa 1150
gtcagtccgc tgtgaaaggg gagcatccat cagcttctca ctgactacag 1200
agaattcagg gaactactac tgcacagctg acaatggcct tggcgccaag 1250
cccagtaagg ctgtgagcct ctcagtcact gttcccgtgt ctcatcctgt 1300
cctcaacctc agctctcctg aggacctgat ttttgaggga gccaaggtga 1350
cacttcactg tgaagcccag agaggttcac tccccatcct gtaccagttt 1400
catcatgagg atgctgccct ggagcgtagg tcggccaact ctgcaggagg 1450
agtggccatc agcttctctc tgactgcaga gcattcaggg aactactact 1500
gcacagctga caatggcttt ggcccccagc gcagtaaggc ggtgagcctc 1550
tccatcactg tccctgtgtc tcatcctgtc ctcaccctca gctctgctga 1600
ggccctgact tttgaaggag ccactgtgac acttcactgt gaagtccaga 1650
gaggttcccc acaaatccta taccagtttt atcatgagga catgcccctg 1700
tggagcagct caacaccctc tgtgggaaga gtgtccttca gcttctctct 1750
gactgaagga cattcaggga attactactg cacagctgac aatggctttg 1800
gtccccagcg cagtgaagtg gtgagccttt ttgtcactgt tccagtgtct 1850
cgccccatcc tcaccctcag ggttcccagg gcccaggctg tggtggggga 1900
cctgctggag cttcactgtg aggccccgag aggctctccc ccaatcctgt 1950
actggtttta tcatgaggat gtcaccctgg ggagcagctc agccccctct 2000
ggaggagaag cttctttcaa cctctctctg actgcagaac attctggaaa 2050
ctactcatgt gaggccaaca atggcctagt ggcccagcac agtgacacaa 2100
tatcactcag tgttatagtt ccagtatctc gtcccatcct caccttcagg 2150
gctcccaggg cccaggctgt ggtgggggac ctgctggagc ttcactgtga 2200
ggccctgaga ggctcctccc caatcctgta ctggttttat catgaagatg 2250
tcaccctggg taagatctca gccccctctg gaggaggggc ctccttcaac 2300
ctctctctga ctacagaaca ttctggaatc tactcctgtg aggcagacaa 2350
tggtccggag gcccagcgca gtgagatggt gacactgaaa gttgcagttc 2400
cggtgtctcg cccggtcctc accctcaggg ctcccgggac ccatgctgcg 2450
gtgggggacc tgctggagct tcactgtgag gccctgagag gctctcccct 2500
gatcctgtac cggttttttc atgaggatgt caccctagga aataggtcgt 2550
ccccctctgg aggagcgtcc ttaaacctct ctctgactgc agagcactct 2600
ggaaactact cctgtgaggc cgacaatggc ctcggggccc agcgcagtga 2650
gacagtgaca ctttatatca cagggctgac cgcgaacaga agtggccctt 2700
ttgccacagg agtcgccggg ggcctgctca gcatagcagg ccttgctgcg 2750
ggggcactgc tgctctactg ctggctctcg agaaaagcag ggagaaagcc 2800
tgcctctgac cccgccagga gccctccaga ctcggactcc caagagccca 2850
cctatcacaa tgtaccagcc tgggaagagc tgcaaccagt gtacactaat 2900
gcaaatccta gaggagaaaa tgtggtttac tcagaagtac ggatcatcca 2950
agagaaaaag aaacatgcag tggcctctga ccccaggcat ctcaggaaca 3000
agggttcccc tatcatctac tctgaagtta aggtggcgtc aaccccggtt 3050
tccggatccc tgttcttggc ttcctcagct cctcacagat gagtccacac 3100
gtctctccaa ctgctgtttc agcctctgca ccccaaagtt ccccttgggg 3150
gagaagcagc attgaagtgg gaagatttag gctgccccag accatatcta 3200
ctggcctttg tttcacatgt cctcattctc agtctgacca gaatgcaggg 3250
ccctgctgga ctgtcacctg tttcccagtt aaagccctga ctggcaggtt 3300
ttttaatcca gtggcaaggt gctcccactc cagggcccag cacatctcct 3350
ggattcctta gtgggcttca gctgtgattg ctgttctgag tactgctctc 3400
atcacacccc cacagagggg gtcttaccac acaaagggag agtgggcctt 3450
caggagatgc cgggctggcc taacagctca ggtgctccta aactccgaca 3500
cagagttcct gctttgggtg gatgcatttc tcaattgtca tcagcctggt 3550
ggggctactg cagtgtgctg ccaaatggga cagcacacag cctgtgcaca 3600
tgggacatgt gatgggtctc cccacggggg ctgcatttca cactcctcca 3650
cctgtctcaa actctaaggt cggcacttga caccaaggta acttctctcc 3700
tgctcatgtg tcagtgtcta cctgcccaag taagtggctt tcatacacca 3750
agtcccaagt tcttcccatc ctaacagaag taacccagca agtcaaggcc 3800
aggaggacca ggggtgcaga cagaacacat actggaacac aggaggtgct 3850
caattactat ttgactgact gactgaatga atgaatgaat gaggaagaaa 3900
actgtgggta atcaaactgg cataaaatcc agtgcactcc ctaggaaatc 3950
cgggaggtat tctggcttcc ctaagaaaca acggaagaga aggagcttgg 4000
atgaggaaac tgttcagcaa gaggaagggc ttctcacact ttcatgtgct 4050
tgtggatcac ctgaggatcc tgtgaaaata cagatactga ttcagtgggt 4100
ctgtgtagag cctgagactg ccattctaac atgttcccag gggatgctga 4150
tgctgctggc cctgggactg cactgcatgc atgtgaagcc ctataggtct 4200
cagcagaggc ccatggagag ggaatgtgtg gctctggctg cccagggccc 4250
aactcggttc acacggatcg tgctgctccc tggccagcct ttggccacag 4300
caccaccagc tgctgttgct gagagagctt cttctctgtg acatgttggc 4350
tttcatcagc caccctggga agcggaaagt agctgccact atctttgttt 4400
ccccacctca ggcctcacac tttcccatga aaagggtgaa tgtatataac 4450
ctgagccctc tccattcaga gttgttctcc catctctgag caatgggatg 4500
ttctgttccg cttttatgat atccatcaca tcttatcttg atctttgctc 4550
ccagtggatt gtacagtgat gacttttaag ccccacggcc ctgaaataaa 4600
atccttccaa gggcattgga agctctctcc acctgaacca tggcttttca 4650
tgcttccaag tgtcagggcc ttgcccagat agacagggct gactctgctg 4700
ccccaacctt tcaaggagga aaccagacac ctgagacagg agcctgtatg 4750
cagcccagtg cagccttgca gaggacaagg ctggaggcat ttgtcatcac 4800
tacagatatg caactaaaat agacgtggag caagagaaat gcattcccac 4850
cgaggccgct tttttaggcc tagttgaaag tcaagaagga cagcagcaag 4900
cataggctca ggattaaaga aaaaaatctg ctcacagttt gttctggagg 4950
tcacatcacc aacaaagctc acgccctatg cagttctgag aaggtggagg 5000
caccaggctc aaaagaggaa atttagaatt tctcattggg agagtaaggt 5050
acccccatcc cagaatgata actgcacagt ggcagaacaa actccaccct 5100
aatgtgggtg gaccccatcc agtctgttga aggcctgagt gtaacaaaag 5150
ggcttattct tcctcaagta agggggaact cctgctttgg gctgggacat 5200
aagtttttct gctttcagac gcaaactgaa aaatggctct tcttgggtct 5250
tgagcttgct ggcatatgga ctgaaagaaa ctatgctatt ggatctcctg 5300
gatctccagc ttgctgactg cagatcttga gatatgtcag cctctacagt 5350
cacaagagct aattcattct aataaaccaa tctttctgta aa 5392
42
626
DNA
Homo Sapien
42
ggacctggga aggagcatag gacagggcaa ggcgggataa ggaggggcac 50
cacagccctt aaggcacgag ggaacctcac tgcgcatgct cctttggtgc 100
ccacctcagt gcgcatgttc actgggcgtc ttcccatcgg ccccttcgcc 150
agtgtgggga acgcggcgga gctgtgagcc ggcgactcgg gtccctgagg 200
tctggattct ttctccgcta ctgagacacg gcggacacac acaaacacag 250
aaccacacag ccagtcccag gagcccagta atggagagcc ccaaaaagaa 300
gaaccagcag ctgaaagtcg ggatcctaca cctgggcagc agacagaaga 350
agatcaggat acagctgaga tcccagtgcg cgacatggaa ggtgatctgc 400
aagagctgca tcagtcaaac accggggata aatctggatt tgggttccgg 450
cgtcaaggtg aagataatac ctaaagagga acactgtaaa atgccagaag 500
caggtgaaga gcaaccacaa gtttaaatga agacaagctg aaacaacgca 550
agctggtttt atattagata tttgacttaa actatctcaa taaagttttg 600
cagctttcac caaaaaaaaa aaaaaa 626
43
1505
DNA
Homo Sapien
43
agcggctggc gagccggcgc cggccgagct gcgggagccg cggagagcac 50
cagctgtcgc cgcgggagct gctccggccg caccatgcgg gagctggcca 100
ttgagatcgg ggtgcgagcc ctgctcttcg gagtcttcgt ttttacagag 150
tttttggatc cgttccagag agtcatccag ccagaagaga tctggctcta 200
taaaaatcct ttggtgcaat cagataacat acctacccgc ctcatgtttg 250
caatttcttt cctcacaccc ctggctgtta tttgtgtggt gaaaattatc 300
cggcgaacag acaagactga aattaaggaa gccttcttag cggtgtcctt 350
ggctcttgct ttgaatggag tctgcacaaa cactattaaa ttaatagtgg 400
gaagacctcg cgccgatttc ttttaccgct gctttccaga tggagtgatg 450
aactcggaaa tgcattgcac aggtgacccc gatctggtgt ccgagggccg 500
caaaagcttc cccagcatcc attcctcctt tgccttttcg ggccttggct 550
tcacgacgtt ctacttggcg ggcaagctgc actgcttcac cgagagtggg 600
cggggaaaga gctggcggct ctgtgctgcc atcctgccct tgtactgcgc 650
catgatgatt gccctgtccc gcatgtgcga ctacaagcat cactggcaag 700
attcctttgt gggtggagtc atcgcgctca tttttgcata catttgctac 750
agacagcact atcctcctct gggccaacac agcttgccat aaaccctacg 800
ttagtctgcg agtttgccat aaaccctacg ttagtctgcg agtcccagcc 850
tcactgaaga aagaggagag gcccacagct gacagcgcac ccagcttgcc 900
tctggagggg atcaccgaag gcccggtatg accagtgtcc tgggaggatg 950
gacactaagc cctgggcaca tctgccaccc tgacatcata acacaataga 1000
aatggttttc tgtagtgtat ttttcatcag ttgtttctca aagtcatcgt 1050
acttctgctt ctgtttcact gatggtgttc ctgctacttt aaatgtctac 1100
ttccaacatc cttgaatttg caagtgaagg acaacaatct ctgagagacg 1150
tgtggaagag gctgcgaagg tggggtttgg ggagcttcgc cgattcgtct 1200
atctgaaatg tttgctgtaa cagccacctt cctatgtttt catggttagt 1250
aaacataata aaacctccca tcgggaaaaa atacaaaatt cattgattta 1300
ggaatatata tataatattc acatgtgtaa ttccccccct ccctttagtg 1350
agggtaattc aagatccttc tcaactgctt tgtgcgactt agactttatg 1400
ttgcagcaga cttttttatt ttacttatag cgcggaatcc gtgtttcctc 1450
agaatcaggg aatccgcccg aaaatctgtt acaaaggccg ccaagtgaca 1500
taact 1505
44
1850
DNA
Homo Sapien
44
tccttgggtt cgggtgaaag cgcctggggg ttcgtggcca tgatccccga 50
gctgctggag aactgaaggc ggacagtctc ctgcgaaacc aggcaatggc 100
ggagctggag tttgttcaga tcatcatcat cgtggtggtg atgatggtga 150
tggtggtggt gatcacgtgc ctgctgagcc actacaagct gtctgcacgg 200
tccttcatca gccggcacag ccaggggcgg aggagagaag atgccctgtc 250
ctcagaagga tgcctgtggc cctcggagag cacagtgtca ggcaacggaa 300
tcccagagcc gcaggtctac gccccgcctc ggcccaccga ccgcctggcc 350
gtgccgccct tcgcccagcg ggagcgcttc caccgcttcc agcccaccta 400
tccgtacctg cagcacgaga tcgacctgcc acccaccatc tcgctgtcag 450
acggggagga gcccccaccc taccagggcc cctgcaccct ccagcttcgg 500
gaccccgagc agcagctgga actgaaccgg gagtcggtgc gcgcaccccc 550
aaacagaacc atcttcgaca gtgacctgat ggatagtgcc aggctgggcg 600
gcccctgccc ccccagcagt aactcgggca tcagcgccac gtgctacggc 650
agcggcgggc gcatggaggg gccgccgccc acctacagcg aggtcatcgg 700
ccactacccg gggtcctcct tccagcacca gcagagcagt gggccgccct 750
ccttgctgga ggggacccgg ctccaccaca cacacatcgc gcccctagag 800
agcgcagcca tctggagcaa agagaaggat aaacagaaag gacaccctct 850
ctagggtccc caggggggcc gggctggggc tgcgtaggtg aaaaggcaga 900
acactccgcg cttcttagaa gaggagtgag aggaaggcgg ggggcgcagc 950
aacgcatcgt gtggccctcc cctcccacct ccctgtgtat aaatatttac 1000
atgtgatgtc tggtctgaat gcacaagcta agagagcttg caaaaaaaaa 1050
aagaaaaaag aaaaaaaaaa accacgtttc tttgttgagc tgtgtcttga 1100
aggcaaaaga aaaaaaattt ctacagtagt ctttcttgtt tctagttgag 1150
ctgcgtgcgt gaatgcttat tttcttttgt ttatgataat ttcacttaac 1200
tttaaagaca tatttgcaca aaacctttgt ttaaagatct gcaatattat 1250
atatataaat atatataaga taagagaaac tgtatgtgcg agggcaggag 1300
tatttttgta ttagaagagg cctattaaaa aaaaaagttg ttttctgaac 1350
tagaagagga aaaaaatggc aatttttgag tgccaagtca gaaagtgtgt 1400
attaccttgt aaagaaaaaa attacaaagc aggggtttag agttatttat 1450
ataaatgttg agattttgca ctatttttta atataaatat gtcagtgctt 1500
gcttgatgga aacttctctt gtgtctgttg agactttaag ggagaaatgt 1550
cggaatttca gagtcgcctg acggcagagg gtgagccccc gtggagtctg 1600
cagagaggcc ttggccagga gcggcgggct ttcccgaggg gccactgtcc 1650
ctgcagagtg gatgcttctg cctagtgaca ggttatcacc acgttatata 1700
ttccctaccg aaggagacac cttttccccc ctgacccaga acagccttta 1750
aatcacaagc aaaataggaa agttaaccac ggaggcaccg agttccaggt 1800
agtggttttg cctttcccaa aaatgaaaat aaactgttac cgaaggaatt 1850
45
806
DNA
Homo Sapien
45
gcccttcgga cagtctcctg cgaaaccagg caatggcgga gctggagttt 50
gttcagatca tcatcatcgt ggtggtgatg atggtgatgg tggtggtgat 100
cacgtgcctg ctgagccact acaagctgtc tgcacggtcc ttcatcagcc 150
ggcacagcca ggggcggagg agagaagatg ccctgtcctc agaaggatgc 200
ctgtggccct cggagagcac agtgtcaggc aacggaatcc cagagccgca 250
ggtctacgcc ccgcctcggc ccaccgaccg cctggccgtg ccgcccttcg 300
cccagcggga gcgcttccac cgcttccagc ccacctatcc gtacctgcag 350
cacgagatcg acctgccgcc caccatctcg ctgtcagacg gggaggagcc 400
cccaccctac cagggcccct gcaccctcca gcttcgggac cccgagcagc 450
agctggaact gaaccgggag tcggtgcgcg cacccccaaa cagaaccatc 500
ttcgacagtg acctgatgga tagtgccagg ctgggcggcc cctgcccccc 550
cagcagtaac tcgggcatca gcgccacgtg ctacggcagc ggcgggcgca 600
tggaggggcc gccgcccacc tacagcgagg tcatcggcca ctacccgggg 650
tcctccttcc agcaccagca gagcagtggg ccgccctcct tgctggaggg 700
gacccggctc caccacacac acatcgcgcc cctagagagc gcagccatct 750
ggagcaaaga gaaggataaa cagaaaggac accctctcta gggtccccag 800
aagggc 806
46
1982
DNA
Homo Sapien
46
ggcgagaggc gggctgaggc ggcccagcgg cggcaggtga ggcggaacca 50
accctcctgg ccatgggagg ggccgtggtg gacgagggcc ccacaggcgt 100
caaggcccct gacggcggct ggggctgggc cgtgctcttc ggctgtttcg 150
tcatcactgg cttctcctac gccttcccca aggccgtcag tgtcttcttc 200
aaggagctca tacaggagtt tgggatcggc tacagcgaca cagcctggat 250
ctcctccatc ctgctggcca tgctctacgg gacaggtccg ctctgcagtg 300
tgtgcgtgaa ccgctttggc tgccggcccg tcatgcttgt ggggggtctc 350
tttgcgtcgc tgggcatggt ggctgcgtcc ttttgccgga gcatcatcca 400
ggtctacctc accactgggg tcatcacggg gttgggtttg gcactcaact 450
tccagccctc gctcatcatg ctgaaccgct acttcagcaa gcggcgcccc 500
atggccaacg ggctggcggc agcaggtagc cctgtcttcc tgtgtgccct 550
gagcccgctg gggcagctgc tgcaggaccg ctacggctgg cggggcggct 600
tcctcatcct gggcggcctg ctgctcaact gctgcgtgtg tgccgcactc 650
atgaggcccc tggtggtcac ggcccagccg ggctcggggc cgccgcgacc 700
ctcccggcgc ctgctagacc tgagcgtctt ccgggaccgc ggctttgtgc 750
tttacgccgt ggccgcctcg gtcatggtgc tggggctctt cgtcccgccc 800
gtgttcgtgg tgagctacgc caaggacctg ggcgtgcccg acaccaaggc 850
cgccttcctg ctcaccatcc tgggcttcat tgacatcttc gcgcggccgg 900
ccgcgggctt cgtggcgggg cttgggaagg tgcggcccta ctccgtctac 950
ctcttcagct tctccatgtt cttcaacggc ctcgcggacc tggcgggctc 1000
tacggcgggc gactacggcg gcctcgtggt cttctgcatc ttctttggca 1050
tctcctacgg catggtgggg gccctgcagt tcgaggtgct catggccatc 1100
gtgggcaccc acaagttctc cagtgccatt ggcctggtgc tgctgatgga 1150
ggcggtggcc gtgctcgtcg ggcccccttc gggaggcaaa ctcctggatg 1200
cgacccacgt ctacatgtac gtgttcatcc tggcgggggc cgaggtgctc 1250
acctcctccc tgattttgct gctgggcaac ttcttctgca ttaggaagaa 1300
gcccaaagag ccacagcctg aggtggcggc cgcggaggag gagaagctcc 1350
acaagcctcc tgcagactcg ggggtggact tgcgggaggt ggagcatttc 1400
ctgaaggctg agcctgagaa aaacggggag gtggttcaca ccccggaaac 1450
aagtgtctga gtggctgggc ggggccggca ggcacaggga ggaggtacag 1500
aagccggcaa cgcttgctat ttattttaca aactggactg gctcaggcag 1550
ggccacggct gggctccagc tgccggccca gcggatcgtc gcccgatcag 1600
tgttttgagg gggaaggtgg cggggtggga accgtgtcat tccagagtgg 1650
atctgcggtg aagccaagcc gcaaggttac aaggcatcct caccaggggc 1700
cccgcctgct gctcccaggt ggcctgcggc cactgctatg ctcaaggacc 1750
tggaaaccca tgcttcgaga caacgtgact ttaatgggag ggtgggtggg 1800
ccgcagacag gctggcaggg caggtgctgc gtggggccct ctccagcccg 1850
tcctaccctg ggctcacatg gggcctgtgc ccacccctct tgagtgtctt 1900
ggggacagct ctttccaccc ctggaagatg gaaataaacc tgcgtgtggg 1950
tggagtgttc tcgtgccgaa ttcaaaaagc tt 1982
47
2171
DNA
Homo Sapien
47
cccacgcgtc cgcccacgcg tccgccgggt cctgcgcgct ccggactgag 50
gtggcgtccc tgggccggac ggcggtgtcc cggcgtggcg ggaagccggc 100
actggagcgg gagcgcactg ggcgcgggac cgggaggcgc agggaccgga 150
cggctcccga gtcgcccacc tgacggtacc gagagggcgg cgcccctccg 200
agcagagccg tcccggccac tcccctggga tctgacttgg ctcttgcggt 250
cgcgggcacc gtgaagccct ggggtgtgcg tggctcctcc tggtaggcgc 300
cctttcccgg cgtccggctt ggggtggtgg tggcgttgac tccagccccg 350
cctctccctg gagaggaggg ctccactcgc tccttcggcc tcctcccctg 400
gggccgcagc gactcgggcc ggcttcctgc ttccctgcct gccggcggtc 450
ccgctggcta gaagaagtct tcacttccca ggagagccaa agcgtgtctg 500
gccctaggtg ggaaaagaac tggctgtgac ctttgccctg acctggaagg 550
gcccagcctt gggctgaatg gcagcaccca cgcccgcccg tccggtgctg 600
acccacctgc tggtggctct cttcggcatg ggctcctggg ctgcggtcaa 650
tgggatctgg gtggagctac ctgtggtggt caaagagctt ccagagggtt 700
ggagcctccc ctcttacgtc tctgtgcttg tggctctggg gaacctgggt 750
ctgctggtgg tgaccctctg gaggaggctg gccccaggaa aggacgagca 800
ggtccccatc cgggtggtgc aggtgctggg catggtgggc acagccctgc 850
tggcctctct gtggcaccat gtggccccag tggcaggaca gttgcattct 900
gtggccttct tagcactggc ctttgtgctg gcactggcat gctgtgcctc 950
gaatgtcact ttcctgccct tcttgagcca cctgccacct cgcttcttac 1000
ggtcattctt cctgggtcaa ggcctgagtg ccctgctgcc ctgcgtgctg 1050
gccctagtgc agggtgtggg ccgcctcgag tgcccgccag cccccatcaa 1100
cggcacccct ggccccccgc tcgacttcct tgagcgtttt cccgccagca 1150
ccttcttctg ggcactgact gcccttctgg tcgcttcagc tgctgccttc 1200
cagggtcttc tgctgctgtt gccgccacca ccatctgtac ccacagggga 1250
gttaggatca ggcctccagg tgggagcccc aggagcagag gaagaggtgg 1300
aagagtcctc accactgcaa gagccaccaa gccaggcagc aggcaccacc 1350
cctggtccag accctaaggc ctatcagctt ctatcagccc gcagtgcctg 1400
cctgctgggc ctgttggccg ccaccaacgc gctgaccaat ggcgtgctgc 1450
ctgccgtgca gagcttttcc tgcttaccct acgggcgtct ggcctaccac 1500
ctggctgtgg tgctgggcag tgctgccaat cccctggcct gcttcctggc 1550
catgggtgtg ctgtgcaggt ccttggcagg gctgggcggc ctctctctgc 1600
tgggcgtgtt ctgtgggggc tacctgatgg cgctggcagt cctgagcccc 1650
tgcccgcccc tggtgggcac ctcggcgggg gtggtcctcg tggtgctgtc 1700
gtgggtgctg tgtcttggcg tgttctccta cgtgaaggtg gcagccagct 1750
ccctgctgca tggcgggggc cggccggcat tgctggcagc cggcgtggcc 1800
atccaggtgg gctctctgct cggcgctgtt gctatgttcc ccccgaccag 1850
catctatcac gtgttccaca gcagaaagga ctgtgcagac ccctgtgact 1900
cctgagcctg ggcaggtggg gaccccgctc cccaacacct gtctttccct 1950
caatgctgcc accatgcctg agtgcctgca gcccaggagg cccgcacacc 2000
ggtacactcg tggacaccta cacactccat aggagatcct ggctttccag 2050
ggtgggcaag ggcaaggagc aggcttggag ccagggacca gtgggggctg 2100
tagggtaagc ccctgagcct gggacctaca tgtggtttgc gtaataaaac 2150
atttgtattt aaaaaaaaaa a 2171
48
1617
DNA
Homo Sapien
48
gccagcacag ctgccctctg gaccctgcgg accccagccg agccccttcc 50
tgagttccac aggcgcagcc cccgggcggt cgggcggagg ggtccccggg 100
gcggtgccag gcgcaatcct ggagggcggc cgggaggagg aggtgcgcgc 150
ggccatgcac accgtggcta cgtccggacc caacgcgtcc tggggggcac 200
cggccaacgc ctccggctgc ccgggctgtg gcgccaacgc ctcggacggc 250
ccagtccctt cgccgcgggc cgtggacgcc tggctcgtgc cgctcttctt 300
cgcggcgctg atgctgctgg gcctggtggg gaactcgctg gtcatctacg 350
tcatctgccg ccacaagccg atgcggaccg tgaccaactt ctacatcgcc 400
aacctggcgg ccacggacgt gaccttcctc ctgtgctgcg tccccttcac 450
ggccctgctg tacccgctgc ccggctgggt gctgggcgac ttcatgtgca 500
agttcgtcaa ctacatccag caggtctcgg tgcaggccac gtgtgccact 550
ctgaccgcca tgagtgtgga ccgctggtac gtgacggtgt tcccgttgcg 600
cgccctgcac cgccgcacgc cccgcctggc gctggctgtc agcctcagca 650
tctgggtagg ctctgcggcg gtgtctgcgc cggtgctcgc cctgcaccgc 700
ctgtcacccg ggccgcgcgc ctactgcagt gaggccttcc ccagccgcgc 750
cctggagcgc gccttcgcac tgtacaacct gctggcgctg tacctgctgc 800
cgctgctcgc cacctgcgcc tgctatgcgg ccatgctgcg ccacctgggc 850
cgggtcgccg tgcgccccgc gcccgccgat agcgccctgc aggggcaggt 900
gctggcagag cgcgcaggcg ccgtgcgggc caaggtctcg cggctggtgg 950
cggccgtggt cctgctcttc gccgcctgct ggggccccat ccagctgttc 1000
ctggtgctgc aggcgctggg ccccgcgggc tcctggcacc cacgcagcta 1050
cgccgcctac gcgcttaaga cctgggctca ctgcatgtcc tacagcaact 1100
ccgcgctgaa cccgctgctc tacgccttcc tgggctcgca cttccgacag 1150
gccttccgcc gcgtctgccc ctgcgcgccg cgccgccccc gccgcccccg 1200
ccggcccgga ccctcggacc ccgcagcccc acacgcggag ctgcaccgcc 1250
tggggtccca cccggccccc gccagggcgc agaagccagg gagcagtggg 1300
ctggccgcgc gcgggctgtg cgtcctgggg gaggacaacg cccctctttg 1350
agcggacccg gtgggaatcc gagcggctcc ctcgggagcg gggactgctg 1400
gaacagcggc tattcttctg ttattagtat tttttttact gtccaagatc 1450
aactgtggaa atattttggt ctcttgtgac gttcggtgca gtttcgttgt 1500
gaagtttgct attgatattg aaattatgac ttctgtgttt cctgaaatta 1550
aacatgtgtc aacacaggac tttttggatc attccagaaa gtgtcagacg 1600
tttaaaaaaa aaaaaaa 1617
49
3095
DNA
Homo Sapien
49
ggcgcggggc gccatggcac accgagcggc tccgtcttct gctcctcaga 50
gagcccggct ggcggcctgg gatgacaaga tgtctggact gcaatcctgc 100
acagttttga gagggagatg acttgagtgg ttggctttta tctccacaac 150
aatgtccatg aacaattcca aacagctagt gtctcctgca gctgcgcttc 200
tttcaaacac aacctgccag acggaaaacc ggctttccgt atttttttca 250
gtaatcttca tgacagtggg aatcttgtca aacagccttg ccatcgccat 300
tctcatgaag gcatatcaga gatttagaca gaagtccaag gcatcgtttc 350
tgcttttggc cagcggcctg gtaatcactg atttctttgg ccatctcatc 400
aatggagcca tagcagtatt tgtatatgct tctgataaag aatggatccg 450
ctttgaccaa tcaaatgtcc tttgcagtat ttttggtatc tgcatggtgt 500
tttctggtct gtgcccactt cttctaggca gtgtgatggc cattgagcgg 550
tgtattggag tcacaaaacc aatatttcat tctacgaaaa ttacatccaa 600
acatgtgaaa atgatgttaa gtggtgtgtg cttgtttgct gttttcatag 650
ctttgctgcc catccttgga catcgagact ataaaattca ggcgtcgagg 700
acctggtgtt tctacaacac agaagacatc aaagactggg aagatagatt 750
ttatcttcta cttttttctt ttctggggct cttagccctt ggtgtttcat 800
tgttgtgcaa tgcaatcaca ggaattacac ttttaagagt taaatttaaa 850
agtcagcagc acagacaagg cagatctcat catttggaaa tggtaatcca 900
gctcctggcg ataatgtgtg tctcctgtat ttgttggagc ccatttctgg 950
ttacaatggc caacattgga ataaatggaa atcattctct ggaaacctgt 1000
gaaacaacac tttttgctct ccgaatggca acatggaatc aaatcttaga 1050
tccttgggta tatattcttc tacgaaaggc tgtccttaag aatctctata 1100
agcttgccag tcaatgctgt ggagtgcatg tcatcagctt acatatttgg 1150
gagcttagtt ccattaaaaa ttccttaaag gttgctgcta tttctgagtc 1200
accagttgca gagaaatcag caagcaccta gcttaatagg acagtaaatc 1250
tgtgtggggc tagaacaaaa attaagacat gtttggcaat atttcagtta 1300
gttaaatacc tgtagcctaa ctggaaaatt caggcttcat catgtagttt 1350
gaagatacta ttgtcagatt caggttttga aatttgtcaa ataaacagga 1400
taactgtaca ttttcaactt gtttttgcca atgggaggta gacacaataa 1450
aataatgcca tgggagtcac actgaaagca attttgagct tatctgtctt 1500
atttatgctt tgagtgaatc atctgttgag gtctaatgcc tctacttggc 1550
ctatttgcca gagaacatct taatgcagcc tgcatagtga aatggttatt 1600
ttgagatcac cgctctgtag ctaaccctta taaactaggc tcagtaaaat 1650
aaagcactct tattttttga tctggcctat tttgcccctc attgtgtagc 1700
ctcaattaac acatgcatgg tcatgacacc cagaattcat gatggtttgt 1750
tataacaacc tctgcatatt ccaggtctgg cagacaggtt gcctgaccct 1800
gcaatcctat ctagaatggg cccattcttg tcacatttga caaataggac 1850
tgcctacatt tattattatg aaggtcgatt gttgttggaa gtgttttttc 1900
atgtcataga ttagcaattt tcaaataatt attttttctc tgaaaatttt 1950
gtgtgtgatt gcacaataaa taatttttag agaaacaaag gctctttctc 2000
agcacattga tgggcaacta gaattacagc agtttcaaac tctaccatgg 2050
ataatgcaaa caaaccgaag ctacatgcca atgataggtg caaagaatat 2100
tggcaaaagg tgctttacct tgagccatta tttgtgtcag agaacaaaag 2150
aaacagaatc aatatataaa ttcaaagact atctgcagct agtgtgtttc 2200
ttctttacac acatatacac acagacatca gaaaattctg ttgagagcag 2250
gttcattaaa tttgtaagat ggcatattct aaagcctgtg ctaccagtac 2300
taagagggga agactggcaa tttgccaagc acttggggat tattataaca 2350
attaactagg agatcaagag ataataatct ctccccaaat tttccaataa 2400
taattgagac tttttctttg cttgtttgtg taattcaacc aaaagaattt 2450
caatacccat tcaaattgtc ctaggtctat cagaaattag ggaaggtagt 2500
cctgctttat aataggaaaa tgtatttctg tataagattt ctttgctttc 2550
attaaaaatg ggattcattt aaaaattaat ctttccctgt taggctgatt 2600
tcagattctc taggaaatct ggtgaagtaa ccagaagact ttcagatggt 2650
ttatttgctt tcagcagaga atttatttca tacagttact taagagtgtt 2700
gatgtcttgt gaacagagat ataaggaacc attctccatc cttccttatc 2750
atgctgggta caatgcttct atgaatattt ccatgtattt tgactgggga 2800
gaggcatgga gaagaaactc tcattcaggg gctccaggat ccttctcctt 2850
gaggcttcta aataaatggc agaattcttg ctgtattgcc atgatgtcac 2900
cctggccatg tgtactgact tgaggagatc ttgcaacatg gccatgtgca 2950
aggctttaag gagtgagaga gatgtgtaca tatcttagga gggttatcta 3000
tgttatctga gtatatgttt gggtaaccaa attggtctta aaaatgatgt 3050
taacccaaga agtagacatc aaaaattaaa aaaaaaaaaa aaaaa 3095
50
6476
DNA
Homo Sapien
50
atgtcacgca tgagccggca tccagacaag gacctggccc agggtccctt 50
caacacctgc tgtggctgca ccttaatggc tagtcctgct aatctccctc 100
cgaacactca agcagctgca gaaagggccc tttcccagag caggtggaag 150
agggtgcaag tgcccgcccc ggcatccctg tcccctttcc cactggccat 200
ggcttcagtt gccttctgga tcagcatcct gattggctgc gaggaacaga 250
ctctctgcag aggctggcgt agcccagtcg gggatggctg tgctcatgtg 300
cctccccagg agcgagcgac cgcagaggca gaccctccag ggcggtgcag 350
cacctccacg gcgtcgtcta ccatctgtgg cctgtggcat ttgtccccac 400
ggctgcagct cctcccacct ctgcattcca ggcagggaga agagtcgggc 450
aaaactgaga aggtgcttct ctggggaaga gagggcctcc atgtgtggaa 500
acccggagtc ctgcagcccg atgtccacgg cacctccaac ctggggaact 550
gctccttcct gcacggcctg gttacggctc cctcttgtcc acggcgggcg 600
ggcgccgagc tgctgaattc tttaggaagt cagtttgcca ttagcctttt 650
tgaagttcag agtggaactg agcccagcat tacaggtgtg gccacgtcag 700
ggcagtgcag ggctatgcca ctgaagcatt atctcctttt gctggtgggc 750
tgccaagcct ggggtgcagg gttggcctac catggctgcc ctagcgagtg 800
tacctgctcc agggcctccc aggtggagtg caccggggca cgcattgtgg 850
cggtgcccac ccctctgccc tggaacgcca tgagcctgca gatcctcaac 900
acgcacatca ctgaactcaa tgagtccccg ttcctcaata tttcagccct 950
catcgccctg aggattgaga agaatgagct gtcgcgcatc acgcctgggg 1000
ccttccgaaa cctgggctcg ctgcgctatc tcagcctcgc caacaacaag 1050
ctgcaggttc tgcccatcgg cctcttccag ggcctggaca gccttgagtc 1100
tctccttctg tccagtaacc agctgttgca gatccagccg gcccacttct 1150
cccagtgcag caacctcaag gagctgcagt tgcacggcaa ccacctggaa 1200
tacatccctg acggagcctt cgaccacctg gtaggactca cgaagctcaa 1250
tctgggcaag aatagcctca cccacatctc acccagggtc ttccagcacc 1300
tgggcaatct ccaggtcctc cggctgtatg agaacaggct cacggatatc 1350
cccatgggca cttttgatgg gcttgttaac ctgcaggaac tggctctaca 1400
gcagaaccag attggactgc tctcccctgg tctcttccac aacaaccaca 1450
acctccagag actctacctg tccaacaacc acatctccca gctgccaccc 1500
agcatcttca tgcagctgcc ccagctcaac cgtcttactc tctttgggaa 1550
ttccctgaag gagctctctc tggggatctt cgggcccatg cccaacctgc 1600
gggagctttg gctctatgac aaccacatct cttctctacc cgacaatgtc 1650
ttcagcaacc tccgccagtt gcaggtcctg attcttagcc gcaatcagat 1700
cagcttcatc tccccgggtg ccttcaacgg gctaacggag cttcgggagc 1750
tgtccctcca caccaacgca ctgcaggacc tggacgggaa tgtcttccgc 1800
atgttggcca acctgcagaa catctccctg cagaacaatc gcctcagaca 1850
gctcccaggg aatatcttcg ccaacgtcaa tggcctcatg gccatccagc 1900
tgcagaacaa ccagctggag aacttgcccc tcggcatctt cgatcacctg 1950
gggaaactgt gtgagctgcg gctgtatgac aatccctgga ggtgtgactc 2000
agacatcctt ccgctccgca actggctcct gctcaaccag cctaggttag 2050
ggacggacac tgtacctgtg tgtttcagcc cagccaatgt ccgaggccag 2100
tccctcatta tcatcaatgt caacgttgct gttccaagcg tccatgtacc 2150
tgaggtgcct agttacccag aaacaccatg gtacccagac acacccagtt 2200
accctgacac cacatccgtc tcttctacca ctgagctaac cagccctgtg 2250
gaagactaca ctgatctgac taccattcag gtcactgatg accgcagcgt 2300
ttggggcatg acccatgccc atagcgggct ggccattgcc gccattgtaa 2350
ttggcattgt cgccctggcc tgctccctgg ctgcctgcgt cggctgttgc 2400
tgctgcaaga agaggagcca agctgtcctg atgcagatga aggcacccaa 2450
tgagtgttaa agaggcaggc tggagcaggg ctggggaatg atgggactgg 2500
aggacctggg aatttcatct ttctgcctcc acccctgggt ccatggagct 2550
ttcccgtgat tgctctttct ggccctagat aaaggtgtgc ctacctcttc 2600
ctgacttgcc tgattctccc gtagagaagc aggtcgtgcc ggaccttcct 2650
acaatcagga agatagatcc aactggccat ggcaaaagcc ctggggattt 2700
ccgattcata cccctgggct tccttcgaga gggctcttcc tccaaatcct 2750
ccccacctgt cctccaagaa cagccttccc tgcgcccagg ccccctccgg 2800
gcctctgtag actcagttag tccacagcct gctcacttcg tgggaatagt 2850
tctccgctga gatagcccct ctcgcctaag tattatgtaa gttgatttcc 2900
cttcttttgt ttctcttgtt tgtgctatgg cttgacccag catgtcccct 2950
caaatgaaag ttctcccctt gattttctgc tcctgaaggc agggtgagtt 3000
ctctcctcaa agaagacttc aaaccattta actggtttct taagagccgt 3050
caatcagcct ggttttgggg atgctatgaa agagagaagg aaaatcatgc 3100
cgctcagttc ctggagacag aagagccgtc atcagtgtct cacttgtgat 3150
ttttatctgg aaaaggaaga aacaccccag cacagcaagc tcagcctttt 3200
agagaaggat atttccaaac tgcaaacttt gctttgaaaa gtttagccct 3250
ttaaggaatg aaatcatgta gaattttgga cttctaaaaa cattaaaatc 3300
agcttattaa tacgggatag agaaagaaat ctggtgcctg ggggtccctg 3350
tgttcacccc tagagtttgt tttaaaattt ttaattgaag catgtgaagt 3400
gtacctgcag aaaagtggga acatgatagt gtatggcttg gtggattttc 3450
acaaactgaa catacctgtg taatcagcat ctagacccag acccagagcg 3500
tcacaaatat cccccatcct gggcttttcc cagaggagat gggggcttct 3550
gaagatggac ttacctggga cctgcccccc atgagccagg acggtccccc 3600
cacagtcagc ctgtgcaaag gccccgtggc caggggtgga ggagaatatg 3650
tgggtgtgga caggatggga gactgtggcc tgaacaggag attttattat 3700
atctggagac cctgagagac cctgagacct ggggcaccct ggctggccag 3750
gtcagaagca tcctgactgc agaggtccgt gcagccacac cctcttccct 3800
gccagcaagc tgtctgcggc tcatcggagg cccctccgcc tggagccttc 3850
tatggacgtg atatgcctgt atctgttttt aattttcatt cttcacttag 3900
gggaagtgaa atcgctcaga gatgagatcc tttaattgaa aacgaagtgt 3950
aacggaatct agtgtctttc taatgtggta aaattctcca tcaacatcac 4000
agtcagctgg cagctgaact tcagaatctc acttacagca ggcgacacgg 4050
gggtacaccg atgggtcaca ctgggtctgg gggctccctg gagctcctcc 4100
tgcgtgtggt ctggttagga gttgagttgt ttgctccagg gttattctcc 4150
tcctcgagtc acagtcacac gaatacctgc cttctctggc tttcctgcta 4200
tacacatatt cacatggcgc tcaagaagtt aggctcatgg caacgtgtgt 4250
ctttctctgg acaactggcc cagtttacag tgaaatggag aatttcaggt 4300
ctccacgtct gcccaggaaa gaacttcagc tgactccacg gggatctgga 4350
aatccacgac caatcccgat cggctcttat tagctccccg ctccacaaga 4400
cacctgtgct ttggaaatcc accaccaatc ccgatcggct cttattagct 4450
ccccgctcca caagacacct gtgatctgga aatctaccac caatcccgat 4500
cggctcttat tagctccccg ctccacaaga cacctgtgac atcctccagg 4550
gccacaggag cacgtgctga ccagttttcc cttccagttc ctgcacaaaa 4600
agtgtccaga gggctgtttg caaacactag tgcactttgt agcttttcac 4650
cctctgtccc agggaatcta ggagagatga ggcccgtcag agtcaagaga 4700
tgtcatcccc ccagggtctc caaggcattt ccacactatt ggtggcacct 4750
ggaggacatg caccaaggct tgccagagcc aacaggaagt gagcccagag 4800
catggcacat gagcatcacc cgctgatggt ggcctgctgt gcctggtgcc 4850
aacaggggca tcccggccca tacccctcca gacaggaagc atgggtttgc 4900
ccacagacct gtcgggtgct cctgtgagtg gcctccagat gtctttgtgc 4950
ataggcacaa gtgggccagg gctggaggga ggtgggaaac ctcatcatcc 5000
ggtgggccct gccaatctta acccagaacc cttaggtatt cctggcagta 5050
gccatgacat tggagcacct tcctctccag ccagaggctg acctgagggc 5100
cactgtcctc agatgacacc acccaggagc accctaggtg aggggtgagg 5150
gcccccttat gtgaacctct tgcctcttcc tttctcccat cagagtggtt 5200
ggatggagcc attggcctcc ttttcttcag cgggcccttc aacctctctg 5250
caccatgttg tctggctgag gagctactag aaaagctgag tggagtctcc 5300
tttccaacag gatgatgcat ttgctcaatt ctcagggctg gaatgagccg 5350
gctggtcccc cagaaagctg gagtggggta cagagttcag ttttcctctc 5400
tgtttacagc tccttgacag tcccacgccc atctggagtg ggagctggga 5450
gtcagtgttg gagaagaaac aacaaaagcc aattagaacc actattttta 5500
aaaagtgctt actgtgcaca gatactcttc aagcactgga cgtggattct 5550
ctctctagcc ctcagcaccc ctgcggtagg agtgccgcct ctacccactt 5600
gtgatggggt acagaggcac ttgctcttct gcatggtgtt caataggctg 5650
ggagttttat ttatctcttc aaactttgta caagagctca tggcttgtct 5700
tgggctttcg tcattaaacc aaaggaaatg gaagccattc ccctgttgct 5750
ctccttagtc ttggtcatca gaacctcact tggtaccata tagatcaaaa 5800
gctttgtaac cacaggaaaa aataaactct tccatccctt aaagaataga 5850
atagtttgtc cctctcatgg gaattgggct gtatgtatat tgttcttcct 5900
ccttagaatt tagagataca agagttctac ttagaacttt tcatggacac 5950
aatttccaca acctttcaga tgctgatgta gagctattgg gaaagaactt 6000
ccaaactcag gaagtttgca gagagcagac agctagagat aactcgggac 6050
ccagagttgg tcgacagatg ttagatgtat cctagctttt agctataaac 6100
cactcaaaga ttcagccccc agatcccaca gtcagaactg aatctgcgtt 6150
gttgggaagc cagcagtggc cttgggaagg aagccatggc tgtggttcag 6200
agagggtggg ctggcaagcc acttccgggg aaaactcctt ccgccccagg 6250
tttcttcttc tcttaaggag agattattct caccaacccg ctgccttcat 6300
gctgccttca aagctagatc atgtttgcct tgcttagaga attactgcaa 6350
atcagcccca gtgcttggcg atgcatttac agatttctag gccctcaggg 6400
ttttgtagag tgtgagccct ggtgggcagg gttggggggt ctgtcttctg 6450
ctggatgctg cttgtaatcc atttgg 6476
51
11389
DNA
Homo sapien
51
atggcgccgc cgccgccgcc cgtgctgccc gtgctgctgc tcctggccgc 50
cgccgccgcc ctgccggcga tggggctgcg agcggccgcc tgggagccgc 100
gcgtacccgg cgggacccgc gccttcgccc tccggcccgg ctgtacctac 150
gcggtgggcg ccgcttgcac gccccgggcg ccgcgggagc tgctggacgt 200
gggccgcgat gggcggctgg caggacgtcg gcgcgtctcg ggcgcggggc 250
gcccgctgcc gctgcaagtc cgcttggtgg cccgcagtgc cccgacggcg 300
ctgagccgcc gcctgcgggc gcgcacgcac cttcccggct gcggagcccg 350
tgcccggctc tgcggaaccg gtgcccggct ctgcggggcg ctctgcttcc 400
ccgtccccgg cggctgcgcg gccgcgcagc attcggcgct cgcagctccg 450
accaccttac ccgcctgccg ctgcccgccg cgccccaggc cccgctgtcc 500
cggccgtccc atctgcctgc cgccgggcgg ctcggtccgc ctgcgtctgc 550
tgtgcgccct gcggcgcgcg gctggcgccg tccgggtggg actggcgctg 600
gaggccgcca ccgcggggac gccctccgcg tcgccatccc catcgccgcc 650
cctgccgccg aacttgcccg aagcccgggc ggggccggcg cgacgggccc 700
ggcggggcac gagcggcaga gggagcctga agtttccgat gcccaactac 750
caggtggcgt tgtttgagaa cgaaccggcg ggcaccctca tcctccagct 800
gcacgcgcac tacaccatcg agggcgagga ggagcgcgtg agctattaca 850
tggaggggct gttcgacgag cgctcccggg gctacttccg aatcgactct 900
gccacgggcg ccgtgagcac ggacagcgta ctggaccgcg agaccaagga 950
gacgcacgtc ctcagggtga aagccgtgga ctacagtacg ccgccgcgct 1000
cggccaccac ctacatcact gtcttggtca aagacaccaa cgaccacagc 1050
ccggtcttcg agcagtcgga gtaccgcgag cgcgtgcggg agaacctgga 1100
ggtgggctac gaggtgctga ccatccgcgc cagcgaccgc gactcgccca 1150
tcaacgccaa cttgcgttac cgcgtgttgg ggggcgcgtg ggacgtcttc 1200
cagctcaacg agagctctgg cgtggtgagc acacgggcgg tgctggaccg 1250
ggaggaggcg gccgagtacc agctcctggt ggaggccaac gaccaggggc 1300
gcaatccggg cccgctcagt gccacggcca ccgtgtacat cgaggtggag 1350
gacgagaacg acaactaccc ccagttcagc gagcagaact acgtggtcca 1400
ggtgcccgag gacgtggggc tcaacacggc tgtgctgcga gtgcaggcca 1450
cggaccggga ccagggccag aacgcggcca ttcactacag catcctcagc 1500
gggaacgtgg ccggccagtt ctacctgcac tcgctgagcg ggatcctgga 1550
tgtgatcaac cccttggatt tcgaggatgt ccagaaatac tcgctgagca 1600
ttaaggccca ggatgggggc cggcccccgc tcatcaattc ttcaggggtg 1650
gtgtctgtgc aggtgctgga tgtcaacgac aacgagccta tctttgtgag 1700
cagccccttc caggccacgg tgctggagaa tgtgcccctg ggctaccccg 1750
tggtgcacat tcaggcggtg gacgcggact ctggagagaa cgcccggctg 1800
cactatcgcc tggtggacac ggcctccacc tttctggggg gcggcagcgc 1850
tgggcctaag aatcctgccc ccacccctga cttccccttc cagatccaca 1900
acagctccgg ttggatcaca gtgtgtgccg agctggaccg cgaggaggtg 1950
gagcactaca gcttcggggt ggaggcggtg gaccacggct cgccccccat 2000
gagctcctcc accagcgtgt ccatcacggt gctggacgtg aatgacaacg 2050
acccggtgtt cacgcagccc acctacgagc ttcgtctgaa tgaggatgcg 2100
gccgtgggga gcagcgtgct gaccctgcag gcccgcgacc gtgacgccaa 2150
cagtgtgatt acctaccagc tcacaggcgg caacacccgg aaccgctttg 2200
cactcagcag ccagagaggg ggcggcctca tcaccctggc gctacctctg 2250
gactacaagc aggagcagca gtacgtgctg gcggtgacag catccgacgg 2300
cacacggtcg cacactgcgc atgtcctaat caacgtcact gatgccaaca 2350
cccacaggcc tgtctttcag agctcccatt acacagtgag tgtcagtgag 2400
gacaggcctg tgggcacctc cattgctacc ctcagtgcca acgatgagga 2450
cacaggagag aatgcccgca tcacctacgt gattcaggac cccgtgccgc 2500
agttccgcat tgaccccgac agtggcacca tgtacaccat gatggagctg 2550
gactatgaga accaggtcgc ctacacgctg accatcatgg cccaggacaa 2600
cggcatcccg cagaaatcag acaccaccac cctagagatc ctcatcctcg 2650
atgccaatga caatgcaccc cagttcctgt gggatttcta ccagggttcc 2700
atctttgagg atgctccacc ctcgaccagc atcctccagg tctctgccac 2750
ggaccgggac tcaggtccca atgggcgtct gctgtacacc ttccagggtg 2800
gggacgacgg cgatggggac ttctacatcg agcccacgtc cggtgtgatt 2850
cgcacccagc gccggctgga ccgggagaat gtggccgtgt acaacctttg 2900
ggctctggct gtggatcggg gcagtcccac tccccttagc gcctcggtag 2950
aaatccaggt gaccatcttg gacattaatg acaatgcccc catgtttgag 3000
aaggacgaac tggagctgtt tgttgaggag aacaacccag tggggtcggt 3050
ggtggcaaag attcgtgcta acgaccctga tgaaggccct aatgcccaga 3100
tcatgtatca gattgtggaa ggggacatgc ggcatttctt ccagctggac 3150
ctgctcaacg gggacctgcg tgccatggtg gagctggact ttgaggtccg 3200
gcgggagtat gtgctggtgg tgcaggccac gtcggctccg ctggtgagcc 3250
gagccacggt gcacatcctt ctcgtggacc agaatgacaa cccgcctgtg 3300
ctgcccgact tccagatcct cttcaacaac tatgtcacca acaagtccaa 3350
cagtttcccc accggcgtga tcggctgcat cccggcccat gaccccgacg 3400
tgtcagacag cctcaactac accttcgtgc agggcaacga gctgcgcctg 3450
ttgctgctgg accccgccac gggcgaactg cagctcagcc gcgacctgga 3500
caacaaccgg ccgctggagg cgctcatgga ggtgtctgtg tctgatggca 3550
tccacagcgt cacggccttc tgcaccctgc gtgtcaccat catcacggac 3600
gacatgctga ccaacagcat cactgtccgc ctggagaaca tgtcccagga 3650
gaagttcctg tccccgctgc tggccctctt cgtggagggg gtggccgccg 3700
tgctgtccac caccaaggac gacgtcttcg tcttcaacgt ccagaacgac 3750
accgacgtca gctccaacat cctgaacgtg accttctcgg cgctgctgcc 3800
tggcggcgtc cgcggccagt tcttcccgtc ggaggacctg caggagcaga 3850
tctacctgaa tcggacgctg ctgaccacca tctccacgca gcgcgtgctg 3900
cccttcgacg acaacatctg cctgcgcgag ccctgcgaga actacatgaa 3950
gtgcgtgtcc gttctgcgat tcgacagctc cgcgcccttc ctcagctcca 4000
ccaccgtgct cttccggccc atccacccca tcaacggcct gcgctgccgc 4050
tgcccgcccg gcttcaccgg cgactactgc gagacggaga tcgacctctg 4100
ctactccgac ccgtgcggcg ccaacggccg ctgccgcagc cgcgagggcg 4150
gctacacctg cgagtgcttc gaggacttca ctggagagca ctgtgaggtg 4200
gatgcccgct caggccgctg tgccaacggg gtgtgcaaga acgggggcac 4250
ctgcgtgaac ctgctcatcg gcggcttcca ctgcgtgtgt cctcctggcg 4300
agtatgagag gccctactgt gaggtgacca ccaggagctt cccgccccag 4350
tccttcgtca ccttccgggg cctgagacag cgcttccact tcaccatctc 4400
cctcacgttt gccactcagg aaaggaacgg cttgcttctc tacaacggcc 4450
gcttcaatga gaagcacgac ttcatcgccc tggagatcgt ggacgagcag 4500
gtgcagctca ccttctctgc aggcgagaca acaacgaccg tggcaccgaa 4550
ggttcccagt ggtgtgagtg acgggcggtg gcactctgtg caggtgcagt 4600
actacaacaa gcccaatatt ggccacctgg gcctgcccca tgggccgtcc 4650
ggggaaaaga tggccgtggt gacagtggat gattgtgaca caaccatggc 4700
tgtgcgcttt ggaaaggaca tcgggaacta cagctgcgct gcccagggca 4750
ctcagaccgg ctccaagaag tccctggatc tgaccggccc tctactcctg 4800
gggggtgtcc ccaacctgcc agaagacttc ccagtgcaca accggcagtt 4850
cgtgggctgc atgcggaacc tgtcagtcga cggcaaaaat gtggacatgg 4900
ccggattcat cgccaacaat ggcacccggg aaggctgcgc tgctcggagg 4950
aacttctgcg atgggaggcg gtgtcagaat ggaggcacct gtgtcaacag 5000
gtggaatatg tatctgtgtg agtgtccact ccgattcggc gggaagaact 5050
gtgagcaagc catgcctcac ccccagctct tcagcggtga gagcgtcgtg 5100
tcctggagtg acctgaacat catcatctct gtgccctggt acctggggct 5150
catgttccgg acccggaagg aggacagcgt tctgatggag gccaccagtg 5200
gtgggcccac cagctttcgc ctccagatcc tgaacaacta cctccagttt 5250
gaggtgtccc acggcccctc cgatgtggag tccgtgatgc tgtccgggtt 5300
gcgggtgacc gacggggagt ggcaccacct gctgatcgag ctgaagaatg 5350
ttaaggagga cagtgagatg aagcacctgg tcaccatgac cttggactat 5400
gggatggacc agaacaaggc agatatcggg ggcatgcttc ccgggctgac 5450
ggtaaggagc gtggtggtcg gaggcgcctc tgaagacaag gtctccgtgc 5500
gccgtggatt ccgaggctgc atgcagggag tgaggatggg ggggacgccc 5550
accaacgtcg ccaccctgaa catgaacaac gcactcaagg tcagggtgaa 5600
ggacggctgt gatgtggacg acccctgtac ctcgagcccc tgtcccccca 5650
atagccgctg ccacgacgcc tgggaggact acagctgcgt ctgtgacaaa 5700
gggtaccttg gaataaactg tgtggatgcc tgtcacctga acccctgcga 5750
gaacatgggg gcctgcgtgc gctcccccgg ctccccgcag ggctacgtgt 5800
gcgagtgtgg gcccagtcac tacgggccgt actgtgagaa caaactcgac 5850
cttccgtgcc ccagaggctg gtgggggaac cccgtctgtg gaccctgcca 5900
ctgtgccgtc agcaaaggct ttgatcccga ctgtaataag accaacggcc 5950
agtgccaatg caaggagaat tactacaagc tcctagccca ggacacctgt 6000
ctgccctgcg actgcttccc ccatggctcc cacagccgca cttgcgacat 6050
ggccaccggg cagtgtgcct gcaagcccgg cgtcatcggc cgccagtgca 6100
accgctgcga caacccgttt gccgaggtca ccacgctcgg ctgtgaagtg 6150
atctacaatg gctgtcccaa agcatttgag gccggcatct ggtggccaca 6200
gaccaagttc gggcagccgg ctgcggtgcc atgccctaag ggatccgttg 6250
gaaatgcggt ccgacactgc agcggggaga agggctggct gcccccagag 6300
ctctttaact gtaccaccat ctccttcgtg gacctcaggg ccatgaatga 6350
gaagctgagc cgcaatgaga cgcaggtgga cggcgccagg gccctgcagc 6400
tggtgagggc gctgcgcagt gctacacagc acacgggcac gctctttggc 6450
aatgacgtgc gcacggccta ccagctgctg ggccacgtcc ttcagcacga 6500
gagctggcag cagggcttcg acctggcagc cacgcaggac gccgactttc 6550
acgaggacgt catccactcg ggcagcgccc tcctggcccc agccaccagg 6600
gcggcgtggg agcagatcca gcggagcgag ggcggcacgg cacagctgct 6650
ccggcgcctc gagggctact tcagcaacgt ggcacgcaac gtgcggcgga 6700
cgtacctgcg gcccttcgtc atcgtcaccg ccaacatgat tcttgctgtc 6750
gacatctttg acaagttcaa ctttacggga gccagggtcc cgcgattcga 6800
caccatccat gaagagttcc ccagggagct ggagtcctcc gtctccttcc 6850
cagccgactt cttcagacca cctgaagaaa aagaaggccc cctgctgagg 6900
ccggctggcc ggaggaccac cccgcagacc acgcgcccgg ggcctggcac 6950
cgagagggag gccccgatca gcaggcggag gcgacaccct gatgacgctg 7000
gccagttcgc cgtcgctctg gtcatcattt accgcaccct ggggcagctc 7050
ctgcccgagc gctacgaccc cgaccgtcgc agcctccggt tgcctcaccg 7100
gcccatcatt aataccccga tggtgagcac gctggtgtac agcgaggggg 7150
ctccgctccc gagacccctg gagaggcccg tcctggtgga gttcgccctg 7200
ctggaggtgg aggagcgaac caagcctgtc tgcgtgttct ggaaccactc 7250
cctggccgtt ggtgggacgg gagggtggtc tgcccggggc tgcgagctcc 7300
tgtccaggaa ccggacacat gtcgcctgcc agtgcagcca cacagccagc 7350
tttgcggtgc tcatggatat ctccaggcgt gagaacgggg aggtcctgcc 7400
tctgaagatt gtcacctatg ccgctgtgtc cttgtcactg gcagccctgc 7450
tggtggcctt cgtcctcctg agcctggtcc gcatgctgcg ctccaacctg 7500
cacagcattc acaagcacct cgccgtggcg ctcttcctct ctcagctggt 7550
gttcgtgatt gggatcaacc agacggaaaa cccgtttctg tgcacagtgg 7600
ttgccatcct cctccactac atctacatga gcacctttgc ctggaccctc 7650
gtggagagcc tgcatgtcta ccgcatgctg accgaggtgc gcaacatcga 7700
cacggggccc atgcggttct actacgtcgt gggctggggc atcccggcca 7750
ttgtcacagg actggcggtc ggcctggacc cccagggcta cgggaacccc 7800
gacttctgct ggctgtcgct tcaagacacc ctgatttgga gctttgcggg 7850
gcccatcgga gctgttataa tcatcaacac agtcacttct gtcctatctg 7900
caaaggtttc ctgccaaaga aagcaccatt attatgggaa aaaagggatc 7950
gtctccctgc tgaggaccgc attcctcctg ctgctgctca tcagcgccac 8000
ctggctgctg gggctgctgg ctgtgaaccg cgatgcactg agctttcact 8050
acctcttcgc catcttcagc ggcttacagg gccccttcgt cctccttttc 8100
cactgcgtgc tcaaccagga ggtccggaag cacctgaagg gcgtgctcgg 8150
cgggaggaag ctgcacctgg aggactccgc caccaccagg gccaccctgc 8200
tgacgcgctc cctcaactgc aacaccacct tcggtgacgg gcctgacatg 8250
ctgcgcacag acttgggcga gtccaccgcc tcgctggaca gcatcgtcag 8300
ggatgaaggg atccagaagc tcggcgtgtc ctctgggctg gtgaggggca 8350
gccacggaga gccagacgcg tccctcatgc ccaggagctg caaggatccc 8400
cctggccacg attccgactc agatagcgag ctgtccctgg atgagcagag 8450
cagctcttac gcctcctcac actcgtcaga cagcgaggac gatggggtgg 8500
gagctgagga aaaatgggac ccggccaggg gcgccgtcca cagcaccccc 8550
aaaggggacg ctgtggccaa ccacgttccg gccggctggc ccgaccagag 8600
cctggctgag agtgacagtg aggaccccag cggcaagccc cgcctgaagg 8650
tggagaccaa ggtcagcgtg gagctgcacc gcgaggagca gggcagtcac 8700
cgtggagagt accccccgga ccaggagagc gggggcgcag ccaggcttgc 8750
tagcagccag cccccagagc agaggaaagg catcttgaaa aataaagtca 8800
cctacccgcc gccgctgacg ctgacggagc agacgctgaa gggccggctc 8850
cgggagaagc tggccgactg tgagcagagc cccacatcct cgcgcacgtc 8900
ttccctgggc tctggcggcc ccgactgcgc catcacagtc aagagccctg 8950
ggagggagcc ggggcgtgac cacctcaacg gggtggccat gaatgtgcgc 9000
actgggagcg cccaggccga tggctccgac tctgagaaac cgtgaggcaa 9050
gcccgtcacc ccacacaggc tgcggcatca ccctcagacc ttggagccca 9100
aggggccact gcccttgaag tggagtgggc ccagagtgtg gcggtcccca 9150
tggtggcagc cccccgactg atcatccaga cacaaaggtc ttggttctcc 9200
caggagctca gggcctgtca gacctggtga caagtgccaa aggccacagg 9250
catgagggag gcgtggacca ctgggccagc accgctgagt cctaagactg 9300
cagtcaaagc cagaactgag aggggacccc agactgggcc cagaggctgg 9350
ccagagttca ggaacgccgg gcacagacca aagaccgcgg tccagccccg 9400
cccaggcggg catctcatgg cagtgcggac ccgtggctgg cagcccgggc 9450
agtcctttgc aaaggcaccc cttgtcttaa aatcacttcg ctatgtggga 9500
aaggtggaga tacttttata tatttgtatg ggactctgag gaggtgcaac 9550
ctgtatatat attgcattcg tgctgacttt gttatcccga gagatccatg 9600
caatgatctc ttgctgtctt ctctgtcaag attgcacagt tgtacttgaa 9650
tctggcatgt gttgacgaaa ctggtgcccc agcagatcaa aggtgggaaa 9700
tacgtcagca gtggggctaa aaccaagcgg ctagaagccc tacagctgcc 9750
ttcggccagg aagtgaggat ggtgtgggcc ctccccgccg gccccctggg 9800
tccccagtgt tcgctgtgtg tgcgtttgtc ctctgctgcc atctgccccg 9850
gctgtgtgaa ttcaagacag ggcagtgcag cactaggcag gtgtgaggag 9900
ccctgctgag gtcactgtgg ggcacggttg ccacacggct gtcatttttc 9950
acctggtcat tctgtgacca ccaccccctc ccctcaccgc ctcccaggtg 10000
gcccgggagc tgcaggtggg gatggctttg tcctttgctc ctgctccccg 10050
tgggacctgg gaccttaaag cgttgcaggt tcctgatttg gacagaggtg 10100
tggggccttc caggccgtta catacctcct gccaattctc taactctctg 10150
agactgcgag gatctccagg cagggttctc ccctctggag tctgaccaat 10200
tacttcattt tgcttcaaat ggccaattgt gcagagggac aaagccacag 10250
ccacactctt caacggttac caaactgttt ttggaaattc acaccaaggt 10300
cgggcccact gcaggcagct ggcacagcgt ggcccgaggg gctgtggaac 10350
gggtcccgga actgtcagac atgtttgatt ttagcgtttc ctttgttctt 10400
caaatcaggt gcccaaataa gtgatcagca cagctgcttc caaataggag 10450
aaaccataaa ataggatgaa aatcaagtaa aatgcaaaga tgtccacact 10500
gttttaaact tgaccctgat gaaaatgtga gcactgttag cagatgccta 10550
tgggagagga aaagcgtatc tgaaaatggt ccaggacagg aggatgaaat 10600
gagatcccag agtcctcaca cctgaatgaa ttatacatgt gccttaccag 10650
gtgagtggtc tttcgaagat aaaaaactct agtcccttta aacgtttgcc 10700
cctggcgttt cctaagtacg aaaaggtttt taagtcttcg aacagtctcc 10750
tttcatgact ttaacaggat tctgccccct gaggtgtaat ttttttgttc 10800
tatttttttc cacgtactcc acagccaaca tcacgaggtg taatttttaa 10850
tttgatcaga actgttacca aaaaacaact gtcagtttta ttgagatggg 10900
aaaaatgtaa acctattttt attacttaag actttatggg agagattaga 10950
cactggaggt ttttaacaga acgtgtattt attaatgttc aaaacactgg 11000
aattacaaat gagaagagtc tacaataaat taagattttt gaatttgtac 11050
ttctgcggtg ctggtttttc tccacaaaca cccccgcccc tccccatgcc 11100
cagggtggcc gtggaaggga cggtttacgg acgtgcagct gagctgtccg 11150
tgtcccatgc tccctcagcc agtggaacgt gccggaactt tttgtccatt 11200
ccctagtagg cctgccacag cctagatggg cagtttttgt ctttcaccaa 11250
atttgaggac tttttttttt tgccattatt tcttcagttt tcttttcttg 11300
cactgatctt tctcctctcc ttctgtgact ccagtgactc agacgttaga 11350
cctcttgatg ttttcccact ggtccctgag gctctgttc 11389
52
1107
DNA
Homo Sapien
unsure
170-208
unknown base
52
cggcctaagg tagcgacggg actggccggg ggcggcagga cccgaaggcg 50
ctaggcggat tcaccggatg ggagttgaat cgcgtcccgg tctttctagc 100
tgtgcccgga aatcgggcgt gcgggcagct acagcagaga atcggacaag 150
gagggaagaa agagatggtn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 200
nnnnnnnnga agtgagtgca agaggagccg gcttagcatc taaactgatt 250
ctaccatcag aaaagaggcc aaacttctat catcatggtg gatgtgaagt 300
gtctgagtga ctgtaaattg cagaaccaac ttgagaagct tggattttca 350
cctggcccaa tactaccttc caccagaaag ttgtatgaaa aaaagttagt 400
acagttgttg gtctcacctc cctgtgcacc acctgtgatg aatggaccca 450
gagagctgga tggagcgcag gacagtgatg acagcgaaga gcttaatatc 500
attttgcaag gaaatatcat actctcaaca gaaaaaagca agaaactcaa 550
aaaatggcct gaggcttcca ccactaaacg caaagctgta gatacctatt 600
gcttggatta taagccttcc aagggaagaa ggtgggctgc aagagcacca 650
agcaccagaa tcacatatgg gactatcacc aaagagagag actactgcgc 700
ggaagaccag actatcgaga gctggagaga agaaggtttc ccagtgggct 750
tgaagcttgc tgtgcttggt attttcatca ttgtggtgtt tgtctacctg 800
actgtggaaa ataagtcgct gtttggttaa gtaatttagg agcaaagcaa 850
tgctccaagc gaggcctcct gcttcaggaa agaaccaaaa cactaccctg 900
aagggccagc ctagcctgca gccctccctt gcagggagcc ttcccttgca 950
ctgtgctgct ctcacagatc ggtgtctggg ctcagccagg tggaaggaac 1000
ctgcctaacc aggcacctgt gttaagagca tgatggttag gaaatccccc 1050
aagtcatgtc aactctcatt aaaggtgctt ccatatttga gcaggcgtca 1100
aacaagg 1107
53
3946
DNA
Homo Sapien
53
accgctccgg agcgggaggg gaggcttcgc ggaacgctct cggcgccagg 50
actcgcgtgc aaagcccagg cccgggcggc cagaccaaga gggaagaagc 100
acagaattcc tcaactccca gtgtgcccat gagtaagagc aaatgctccg 150
tgggactcat gtcttccgtg gtggccccgg ctaaggagcc caatgccgtg 200
ggcccgaagg aggtggagct catccttgtc aaggagcaga acggagtgca 250
gctcaccagc tccaccctca ccaacccgcg gcagagcccc gtggaggccc 300
aggatcggga gacctggggc aagaagatcg actttctcct gtccgtcatt 350
ggctttgctg tggacctggc caacgtctgg cggttcccct acctgtgcta 400
caaaaatggt ggcggtgcct tcctggtccc ctacctgctc ttcatggtca 450
ttgctgggat gccacttttc tacatggagc tggccctcgg ccagttcaac 500
agggaagggg ccgctggtgt ctggaagatc tgccccatac tgaaaggtgt 550
gggcttcacg gtcatcctca tctcactgta tgtcggcttc ttctacaacg 600
tcatcatcgc ctgggcgctg cactatctct tctcctcctt caccacggag 650
ctcccctgga tccactgcaa caactcctgg aacagcccca actgctcgga 700
tgcccatcct ggtgactcca gtggagacag ctcgggcctc aacgacactt 750
ttgggaccac acctgctgcc gagtactttg aacgtggcgt gctgcacctc 800
caccagagcc atggcatcga cgacctgggg cctccgcggt ggcagctcac 850
agcctgcctg gtgctggtca tcgtgctgct ctacttcagc ctctggaagg 900
gcgtgaagac ctcagggaag gtggtatgga tcacagccac catgccatac 950
gtggtcctca ctgccctgct cctgcgtggg gtcaccctcc ctggagccat 1000
agacggcatc agagcatacc tgagcgttga cttctaccgg ctctgcgagg 1050
cgtctgtttg gattgacgcg gccacccagg tgtgcttctc cctgggcgtg 1100
gggttcgggg tgctgatcgc cttctccagc tacaacaagt tcaccaacaa 1150
ctgctacagg gacgcgattg tcaccacctc catcaactcc ctgacgagct 1200
tctcctccgg cttcgtcgtc ttctccttcc tggggtacat ggcacagaag 1250
cacagtgtgc ccatcgggga cgtggccaag gacgggccag ggctgatctt 1300
catcatctac ccggaagcca tcgccacgct ccctctgtcc tcagcctggg 1350
ccgtggtctt cttcatcatg ctgctcaccc tgggtatcga cagcgccatg 1400
ggtggtatgg agtcagtgat caccgggctc atcgatgagt tccagctgct 1450
gcacagacac cgtgagctct tcacgctctt catcgtcctg gcgaccttcc 1500
tcctgtccct gttctgcgtc accaacggtg gcatctacgt cttcacgctc 1550
ctggaccatt ttgcagccgg cacgtccatc ctctttggag tgctcatcga 1600
agccatcgga gtggcctggt tctatggtgt tgggcagttc agcgacgaca 1650
tccagcagat gaccgggcag cggcccagcc tgtactggcg gctgtgctgg 1700
aagctggtca gcccctgctt tctcctgttc gtggtcgtgg tcagcattgt 1750
gaccttcaga cccccccact acggagccta catcttcccc gactgggcca 1800
acgcgctggg ctgggtcatc gccacatcct ccatggccat ggtgcccatc 1850
tatgcggcct acaagttctg cagcctgcct gggtcctttc gagagaaact 1900
ggcctacgcc attgcacccg agaaggaccg tgagctggtg gacagagggg 1950
aggtgcgcca gttcacgctc cgccactggc tcaaggtgta gagggagcag 2000
agacgaagac cccaggaagt catcctgcaa tgggagagac acgaacaaac 2050
caaggaaatc taagtttcga gagaaaggag ggcaacttct actcttcaac 2100
ctctactgaa aacacaaaca acaaagcaga agactcctct cttctgactg 2150
tttacacctt tccgtgccgg gagcgcacct cgccgtgtct tgtgttgctg 2200
taataacgac gtagatctgt gcagcgaggt ccaccccgtt gttgtccctg 2250
cagggcagaa aaacgtctaa cttcatgctg tctgtgtgag gctccctccc 2300
tccctgctcc ctgctcccgg ctctgaggct gccccagggg cactgtgttc 2350
tcaggcgggg atcacgatcc ttgtagacgc acctgctgag aatccccgtg 2400
ctcacagtag cttcctagac catttacttt gcccatatta aaaagccaag 2450
tgtcctgctt ggtttagctg tgcagaaggt gaaatggagg aaaccacaaa 2500
ttcatgcaaa gtcctttccc gatgcgtggc tcccagcaga ggccgtaaat 2550
tgagcgttca gttgacacat tgcacacaca gtctgttcag aggcattgga 2600
ggatgggggt cctggtatgt ctcaccagga aattctgttt atgttcttgc 2650
agcagagaga aataaaactc cttgaaacca gctcaggcta ctgccactca 2700
ggcagcctgt gggtccttgt ggtgtaggga acggcctgag aggagcgtgt 2750
cctatccccg gacgcatgca gggcccccac aggagcgtgt cctatccccg 2800
gacgcatgca gggcccccac aggagcatgt cctatccctg gacgcatgca 2850
gggcccccac aggagcgtgt actaccccag aacgcatgca gggcccccac 2900
aggagcgtgt actaccccag gacgcatgca gggcccccac tggagcgtgt 2950
actaccccag gacgcatgca gggcccccac aggagcgtgt cctatccccg 3000
gaccggacgc atgcagggcc cccacaggag cgtgtactac cccaggacgc 3050
atgcagggcc cccacaggag cgtgtactac cccaggatgc atgcagggcc 3100
cccacaggag cgtgtactac cccaggacgc atgcagggcc cccatgcagg 3150
cagcctgcag accaacactc tgcctggcct tgagccgtga cctccaggaa 3200
gggaccccac tggaatttta tttctctcag gtgcgtgcca catcaataac 3250
aacagttttt atgtttgcga atggcttttt aaaatcatat ttacctgtga 3300
atcaaaacaa attcaagaat gcagtatccg cgagcctgct tgctgatatt 3350
gcagtttttg tttacaagaa taattagcaa tactgagtga aggatgttgg 3400
ccaaaagctg ctttccatgg cacactgccc tctgccactg acaggaaagt 3450
ggatgccata gtttgaattc atgcctcaag tcggtgggcc tgcctacgtg 3500
ctgcccgagg gcaggggccg tgcagggcca gtcatggctg tcccctgcaa 3550
gtggacgtgg gctccaggga ctggagtgta atgctcggtg ggagccgtca 3600
gcctgtgaac tgccaggcag ctgcagttag cacagaggat ggcttcccca 3650
ttgccttctg gggagggaca cagaggacgg cttccccatc gccttctggc 3700
cgctgcagtc agcacagaga gcggcttccc cattgccttc tggggaggga 3750
cacagaggac agtttcccca tcgccttctg gttgttgaag acagcacaga 3800
gagcggcttc cccatcgcct tctggggagg ggctccgtgt agcaacccag 3850
gtgttgtccg tgtctgttga ccaatctcta ttcagcatcg tgtgggtccc 3900
taagcacaat aaaagacatc cacaatggaa aaaaaaaaag gaattc 3946
54
2317
DNA
Homo Sapien
54
cggacgcgtg ggtgagcagg gacggtgcac cggacggcgg gatcgagcaa 50
atgggtctgg ccatggagca cggagggtcc tacgctcggg cggggggcag 100
ctctcggggc tgctggtatt acctgcgcta cttcttcctc ttcgtctccc 150
tcatccaatt cctcatcatc ctggggctcg tgctcttcat ggtctatggc 200
aacgtgcacg tgagcacaga gtccaacctg caggccaccg agcgccgagc 250
cgagggccta tacagtcagc tcctagggct cacggcctcc cagtccaact 300
tgaccaagga gctcaacttc accacccgcg ccaaggatgc catcatgcag 350
atgtggctga atgctcgccg cgacctggac cgcatcaatg ccagcttccg 400
ccagtgccag ggtgaccggg tcatctacac gaacaatcag aggtacatgg 450
ctgccatcat cttgagtgag aagcaatgca gagatcaatt caaggacatg 500
aacaagagct gcgatgcctt gctcttcatg ctgaatcaga aggtgaagac 550
gctggaggtg gagatagcca aggagaagac catttgcact aaggataagg 600
aaagcgtgct gctgaacaaa cgcgtggcgg aggaacagct ggttgaatgc 650
gtgaaaaccc gggagctgca gcaccaagag cgccagctgg ccaaggagca 700
actgcaaaag gtgcaagccc tctgcctgcc cctggacaag gacaagtttg 750
agatggacct tcgtaacctg tggagggact ccattatccc acgcagcctg 800
gacaacctgg gttacaacct ctaccatccc ctgggctcgg aattggcctc 850
catccgcaga gcctgcgacc acatgcccag cctcatgagc tccaaggtgg 900
aggagctggc ccggagcctc cgggcggata tcgaacgcgt ggcccgcgag 950
aactcagacc tccaacgcca gaagctggaa gcccagcagg gcctgcgggc 1000
cagtcaggag gcgaaacaga aggtggagaa ggaggctcag gcccgggagg 1050
ccaagctcca agctgaatgc tcccggcaga cccagctagc gctggaggag 1100
aaggcggtgc tgcggaagga acgagacaac ctggccaagg agctggaaga 1150
gaagaagagg gaggcggagc agctcaggat ggagctggcc atcagaaact 1200
cagccctgga cacctgcatc aagaccaagt cgcagccgat gatgccagtg 1250
tcaaggccca tgggccctgt ccccaacccc cagcccatcg acccagctag 1300
cctggaggag ttcaagagga agatcctgga gtcccagagg ccccctgcag 1350
gcatccctgt agccccatcc agtggctgag gaggctccag gcctgaggac 1400
caagggatgg cccgactcgg cggtttgcgg aggatgcagg gatatgctca 1450
cagcgcccga cacaaccccc tcccgccgcc cccaaccacc cagggccacc 1500
atcagacaac tccctgcatg caaaccccta gtaccctctc acacccgcac 1550
ccgcgcctca cgatccctca cccagagcac acggccgcgg agatgacgtc 1600
acgcaagcaa cggcgctgac gtcacatatc accgtggtga tggcgtcacg 1650
tggccatgta gacgtcacga agagatatag cgatggcgtc gtgcagatgc 1700
agcacgtcgc acacagacat ggggaacttg gcatgacgtc acaccgagat 1750
gcagcaacga cgtcacgggc catgtcgacg tcacacatat taatgtcaca 1800
cagacgcggc gatggcatca cacagacggt gatgatgtca cacacagaca 1850
cagtgacaac acacaccatg acaacgacac ctatagatat ggcaccaaca 1900
tcacatgcac gcatgccctt tcacacacac tttctaccca attctcacct 1950
agtgtcacgt tcccccgacc ctggcacacg ggccaaggta cccacaggat 2000
cccatcccct cccgcacagc cctgggcccc agcacctccc ctcctccagc 2050
ttcctggcct cccagccact tcctcacccc cagtgcctgg acccggaggt 2100
gagaacagga agccattcac ctccgctcct tgagcgtgag tgtttccagg 2150
accccctcgg ggccctgagc cgggggtgag ggtcacctgt tgtcgggagg 2200
ggagccactc cttctccccc aactcccagc cctgcctgtg gcccgttgaa 2250
atgttggtgg cacttaataa atattagtaa atccttaaaa aaaaaaaaaa 2300
aaaaaaaaaa aaaaaaa 2317
55
756
DNA
Homo Sapien
55
cggacttggc ttgttagaag gctgaaagat gatggcagga atgaaaatcc 50
agcttgtatg catgctactc ctggctttca gctcctggag tctgtgctca 100
gattcagaag aggaaatgaa agcattagaa gcagatttct tgaccaatat 150
gcatacatca aagattagta aagcacatgt tccctcttgg aagatgactc 200
tgctaaatgt ttgcagtctt gtaaataatt tgaacagccc agctgaggaa 250
acaggagaag ttcatgaaga ggagcttgtt gcaagaagga aacttcctac 300
tgctttagat ggctttagct tggaagcaat gttgacaata taccagctcc 350
acaaaatctg tcacagcagg gcttttcaac actgggagtt aatccaggaa 400
gatattcttg atactggaaa tgacaaaaat ggaaaggaag aagtcataaa 450
gagaaaaatt ccttatattc tgaaacggca gctgtatgag aataaaccca 500
gaagacccta catactcaaa agagattctt actattactg agagaataaa 550
tcatttattt acatgtgatt gtgattcatc atcccttaat taaatatcaa 600
attatatttg tgtgaaaatg tgacaaacac acttatctgt ctcttctaca 650
attgtggttt attgaatgtg tttttctgca ctaatagaaa ttagactaag 700
tgttttcaaa taaatctaaa tcttcaaaaa aaaaaaaaaa aaatggggcc 750
gcaatt 756
56
3722
DNA
Homo Sapien
56
cgcggggcgc ggagtcggcg gggcctcgcg ggacgcgggc agtgcggaga 50
ccgcggcgct gaggacgcgg gagccgggag cgcacgcgcg gggtggagtt 100
cagcctactc tttcttagat gtgaaaggaa aggaagatca tttcatgcct 150
tgttgataaa ggttcagact tctgctgatt cataaccatt tggctctgag 200
ctatgacaag agaggaaaca aaaagttaaa cttacaagcc tgccataagt 250
gagaagcaaa cttccttgat aacatgcttt tgcgaagtgc aggaaaatta 300
aatgtgggca ccaagaaaga ggatggtgag agtacagccc ccaccccccg 350
tccaaaggtc ttgcgttgta aatgccacca ccattgtcca gaagactcag 400
tcaacaatat ttgcagcaca gacggatatt gtttcacgat gatagaagag 450
gatgactctg ggttgcctgt ggtcacttct ggttgcctag gactagaagg 500
ctcagatttt cagtgtcggg acactcccat tcctcatcaa agaagatcaa 550
ttgaatgctg cacagaaagg aacgaatgta ataaagacct acaccctaca 600
ctgcctccat tgaaaaacag agattttgtt gatggaccta tacaccacag 650
ggctttactt atatctgtga ctgtctgtag tttgctcttg gtccttatca 700
tattattttg ttacttccgg tataaaagac aagaaaccag acctcgatac 750
agcattgggt tagaacagga tgaaacttac attcctcctg gagaatccct 800
gagagactta attgagcagt ctcagagctc aggaagtgga tcaggcctcc 850
ctctgctggt ccaaaggact atagctaagc agattcagat ggtgaaacag 900
attggaaaag gtcgctatgg ggaagtttgg atgggaaagt ggcgtggcga 950
aaaggtagct gtgaaagtgt tcttcaccac agaggaagcc agctggttca 1000
gagagacaga aatatatcag acagtgttga tgaggcatga aaacattttg 1050
ggtttcattg ctgcagatat caaagggaca gggtcctgga cccagttgta 1100
cctaatcaca gactatcatg aaaatggttc cctttatgat tatctgaagt 1150
ccaccaccct agacgctaaa tcaatgctga agttagccta ctcttctgtc 1200
agtggcttat gtcatttaca cacagaaatc tttagtactc aaggcaaacc 1250
agcaattgcc catcgagatc tgaaaagtaa aaacattctg gtgaagaaaa 1300
atggaacttg ctgtattgct gacctgggcc tggctgttaa atttattagt 1350
gatacaaatg aagttgacat accacctaac actcgagttg gcaccaaacg 1400
ctatatgcct ccagaagtgt tggacgagag cttgaacaga aatcacttcc 1450
agtcttacat catggctgac atgtatagtt ttggcctcat cctttgggag 1500
gttgctagga gatgtgtatc aggaggtata gtggaagaat accagcttcc 1550
ttatcatgac ctagtgccca gtgacccctc ttatgaggac atgagggaga 1600
ttgtgtgcat caagaagtta cgcccctcat tcccaaaccg gtggagcagt 1650
gatgagtgtc taaggcagat gggaaaactc atgacagaat gctgggctca 1700
caatcctgca tcaaggctga cagccctgcg ggttaagaaa acacttgcca 1750
aaatgtcaga gtcccaggac attaaactct gataggagag gaaaagtaag 1800
catctctgca gaaagccaac aggtactctt ctgtttgtgg gcagagcaaa 1850
agacatcaaa taagcatcca cagtacaagc cttgaacatc gtcctgcttc 1900
ccagtgggtt cagacctcac ctttcaggga gcgacctggg caaagacaga 1950
gaagctccca gaaggagaga ttgatccatg tctgtttgta ggacggagaa 2000
accgcttggg taacttgttc aagatatgat gcatgttgct ttctaagaaa 2050
gccctgtatt ttgtgattgc cttttttttt ttttaagatg ctttcatttt 2100
gccaaaataa aacagataat gtggatggtt taagggttat agtattatag 2150
tttaaataat aacaacaaaa ttcttcccag gaactctgct ggaaggtaaa 2200
ttaaaatact tgtttttcca ttggtaaaat attgttgcac tctgtgaacc 2250
aaaagacagt ctaagttgga ggacatagaa cggaactcat cttaaacata 2300
ctccccaccc cgtcttggcc tcctcagacc actttggcca tccctgcatt 2350
tggggccgct atggtaatgt gaatgcactg ggtacaaaca ccgcctgtct 2400
aggaccacat ttggaattcc tgcaggtggc cttttgcagc ttcaggcaat 2450
atggaacaaa tgaaggttta tgtgactcta atagaagtaa ttgttgatag 2500
gtgtttttca gatccacttc tgtttctgat tgagttaggc atctctttca 2550
tggtaaaacc cttttcatta aacacaaaaa aagctttttt tttttttttt 2600
tttttttttt ttttttaatg tgcagaggat tgacctgtgc atgcttttga 2650
tctctcattc aaaggatcaa tattaaataa aattgtcatg agctgtgttg 2700
aagacagggt gctttcaaat agaggtaatt tgctcttgtg ttgtaagagg 2750
aacatgtcaa caaagatagg aaatgagggt gatcgtgcag atggcttgta 2800
tcttatatat gcaaaggagc caatctcaga agcacaaaga aaaaagtgtg 2850
cataccttat tttgtacaga taaagatgat gtctttttgt tattgtctgt 2900
ctgttttgta tgtgtctgag ataagggata gagaggaaac atccgtcagg 2950
ctaatttaac tacattttat tttaaaaata gagaaacata acctctagat 3000
gggacagcag aggacagtta gtagaggcca caaactgtta tgggctgctg 3050
tgttttgttc taaaatcaat atggttggag catgtatatc ttaggtgatc 3100
atttcacatc ttaggaatgc ctactcattt tattttattc tagtgatgct 3150
caattcacta tttaatttat tatattttct cttctgtggc acttatacaa 3200
aatatctctt cacctactta gttctacagg gttttaactt tggagcaaca 3250
tgaataaaat catcgagaag gccaatattg tttagcaaca tgaatacaat 3300
acagtttaaa gttgtacaca tcctgctcaa ctttattcat atacatttcc 3350
tttctgtggt tttcttttgc ttcttagaaa ttctgttagt ggttagtaaa 3400
gaatttgaaa gtactttctc cttgctgttt tttttttttt ttaagacatt 3450
cctcccagaa tactccaggg ggcagtgttt tataacacat tttccccact 3500
gggtgattga aggatggagg atttttgaaa atttgacagc tacatgaaac 3550
atgagaaaac attttcctca cttctgaagt cggtttgcag ctggtaactt 3600
gttcatccag aaaacattct aaagcaatga gactttgtga gctgtgctta 3650
cagtttggga gaatcatgaa gattctttct atattttgca tttacttccc 3700
agtgcttcat agctgcattt tg 3722
57
837
PRT
Homo Sapien
57
Met Leu Arg Thr Ala Met Gly Leu Arg Ser Trp Leu Ala Ala Pro
1 5 10 15
Trp Gly Ala Leu Pro Pro Arg Pro Pro Leu Leu Leu Leu Leu Leu
20 25 30
Leu Leu Leu Leu Leu Gln Pro Pro Pro Pro Thr Trp Ala Leu Ser
35 40 45
Pro Arg Ile Ser Leu Pro Leu Gly Ser Glu Glu Arg Pro Phe Leu
50 55 60
Arg Phe Glu Ala Glu His Ile Ser Asn Tyr Thr Ala Leu Leu Leu
65 70 75
Ser Arg Asp Gly Arg Thr Leu Tyr Val Gly Ala Arg Glu Ala Leu
80 85 90
Phe Ala Leu Ser Ser Asn Leu Ser Phe Leu Pro Gly Gly Glu Tyr
95 100 105
Gln Glu Leu Leu Trp Gly Ala Asp Ala Glu Lys Lys Gln Gln Cys
110 115 120
Ser Phe Lys Gly Lys Asp Pro Gln Arg Asp Cys Gln Asn Tyr Ile
125 130 135
Lys Ile Leu Leu Pro Leu Ser Gly Ser His Leu Phe Thr Cys Gly
140 145 150
Thr Ala Ala Phe Ser Pro Met Cys Thr Tyr Ile Asn Met Glu Asn
155 160 165
Phe Thr Leu Ala Arg Asp Glu Lys Gly Asn Val Leu Leu Glu Asp
170 175 180
Gly Lys Gly Arg Cys Pro Phe Asp Pro Asn Phe Lys Ser Thr Ala
185 190 195
Leu Val Val Asp Gly Glu Leu Tyr Thr Gly Thr Val Ser Ser Phe
200 205 210
Gln Gly Asn Asp Pro Ala Ile Ser Arg Ser Gln Ser Leu Arg Pro
215 220 225
Thr Lys Thr Glu Ser Ser Leu Asn Trp Leu Gln Asp Pro Ala Phe
230 235 240
Val Ala Ser Ala Tyr Ile Pro Glu Ser Leu Gly Ser Leu Gln Gly
245 250 255
Asp Asp Asp Lys Ile Tyr Phe Phe Phe Ser Glu Thr Gly Gln Glu
260 265 270
Phe Glu Phe Phe Glu Asn Thr Ile Val Ser Arg Ile Ala Arg Ile
275 280 285
Cys Lys Gly Asp Glu Gly Gly Glu Arg Val Leu Gln Gln Arg Trp
290 295 300
Thr Ser Phe Leu Lys Ala Gln Leu Leu Cys Ser Arg Pro Asp Asp
305 310 315
Gly Phe Pro Phe Asn Val Leu Gln Asp Val Phe Thr Leu Ser Pro
320 325 330
Ser Pro Gln Asp Trp Arg Asp Thr Leu Phe Tyr Gly Val Phe Thr
335 340 345
Ser Gln Trp His Arg Gly Thr Thr Glu Gly Ser Ala Val Cys Val
350 355 360
Phe Thr Met Lys Asp Val Gln Arg Val Phe Ser Gly Leu Tyr Lys
365 370 375
Glu Val Asn Arg Glu Thr Gln Gln Trp Tyr Thr Val Thr His Pro
380 385 390
Val Pro Thr Pro Arg Pro Gly Ala Cys Ile Thr Asn Ser Ala Arg
395 400 405
Glu Arg Lys Ile Asn Ser Ser Leu Gln Leu Pro Asp Arg Val Leu
410 415 420
Asn Phe Leu Lys Asp His Phe Leu Met Asp Gly Gln Val Arg Ser
425 430 435
Arg Met Leu Leu Leu Gln Pro Gln Ala Arg Tyr Gln Arg Val Ala
440 445 450
Val His Arg Val Pro Gly Leu His His Thr Tyr Asp Val Leu Phe
455 460 465
Leu Gly Thr Gly Asp Gly Arg Leu His Lys Ala Val Ser Val Gly
470 475 480
Pro Arg Val His Ile Ile Glu Glu Leu Gln Ile Phe Ser Ser Gly
485 490 495
Gln Pro Val Gln Asn Leu Leu Leu Asp Thr His Arg Gly Leu Leu
500 505 510
Tyr Ala Ala Ser His Ser Gly Val Val Gln Val Pro Met Ala Asn
515 520 525
Cys Ser Leu Tyr Arg Ser Cys Gly Asp Cys Leu Leu Ala Arg Asp
530 535 540
Pro Tyr Cys Ala Trp Ser Gly Ser Ser Cys Lys His Val Ser Leu
545 550 555
Tyr Gln Pro Gln Leu Ala Thr Arg Pro Trp Ile Gln Asp Ile Glu
560 565 570
Gly Ala Ser Ala Lys Asp Leu Cys Ser Ala Ser Ser Val Val Ser
575 580 585
Pro Ser Phe Val Pro Thr Gly Glu Lys Pro Cys Glu Gln Val Gln
590 595 600
Phe Gln Pro Asn Thr Val Asn Thr Leu Ala Cys Pro Leu Leu Ser
605 610 615
Asn Leu Ala Thr Arg Leu Trp Leu Arg Asn Gly Ala Pro Val Asn
620 625 630
Ala Ser Ala Ser Cys His Val Leu Pro Thr Gly Asp Leu Leu Leu
635 640 645
Val Gly Thr Gln Gln Leu Gly Glu Phe Gln Cys Trp Ser Leu Glu
650 655 660
Glu Gly Phe Gln Gln Leu Val Ala Ser Tyr Cys Pro Glu Val Val
665 670 675
Glu Asp Gly Val Ala Asp Gln Thr Asp Glu Gly Gly Ser Val Pro
680 685 690
Val Ile Ile Ser Thr Ser Arg Val Ser Ala Pro Ala Gly Gly Lys
695 700 705
Ala Ser Trp Gly Ala Asp Arg Ser Tyr Trp Lys Glu Phe Leu Val
710 715 720
Met Cys Thr Leu Phe Val Leu Ala Val Leu Leu Pro Val Leu Phe
725 730 735
Leu Leu Tyr Arg His Arg Asn Ser Met Lys Val Phe Leu Lys Gln
740 745 750
Gly Glu Cys Ala Ser Val His Pro Lys Thr Cys Pro Val Val Leu
755 760 765
Pro Pro Glu Thr Arg Pro Leu Asn Gly Leu Gly Pro Pro Ser Thr
770 775 780
Pro Leu Asp His Arg Gly Tyr Gln Ser Leu Ser Asp Ser Pro Pro
785 790 795
Gly Ala Arg Val Phe Thr Glu Ser Glu Lys Arg Pro Leu Ser Ile
800 805 810
Gln Asp Ser Phe Val Glu Val Ser Pro Val Cys Pro Arg Pro Arg
815 820 825
Val Arg Leu Gly Ser Glu Ile Arg Asp Ser Val Val
830 835
58
188
PRT
Homo Sapien
58
Met Asp Cys Arg Lys Met Ala Arg Phe Ser Tyr Ser Val Ile Trp
1 5 10 15
Ile Met Ala Ile Ser Lys Val Phe Glu Leu Gly Leu Val Ala Gly
20 25 30
Leu Gly His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu Ala
35 40 45
Phe Arg Asp Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile Arg
50 55 60
Pro Arg Ser Ser Gln Arg Val Pro Pro Met Gly Ile Gln His Ser
65 70 75
Lys Glu Leu Asn Arg Thr Cys Cys Leu Asn Gly Gly Thr Cys Met
80 85 90
Leu Gly Ser Phe Cys Ala Cys Pro Pro Ser Phe Tyr Gly Arg Asn
95 100 105
Cys Glu His Asp Val Arg Lys Glu Asn Cys Gly Ser Val Pro His
110 115 120
Asp Thr Trp Leu Pro Lys Lys Cys Ser Leu Cys Lys Cys Trp His
125 130 135
Gly Gln Leu Arg Cys Phe Pro Gln Ala Phe Leu Pro Gly Cys Asp
140 145 150
Gly Leu Val Met Asp Glu His Leu Val Ala Ser Arg Thr Pro Glu
155 160 165
Leu Pro Pro Ser Ala Arg Thr Thr Thr Phe Met Leu Val Gly Ile
170 175 180
Cys Leu Ser Ile Gln Ser Tyr Tyr
185
59
80
PRT
Homo Sapien
59
Met Ala Ala Arg Ala Leu Cys Met Leu Gly Leu Val Leu Ala Leu
1 5 10 15
Leu Ser Ser Ser Ser Ala Glu Glu Tyr Val Gly Leu Ser Ala Asn
20 25 30
Gln Cys Ala Val Pro Ala Lys Asp Arg Val Asp Cys Gly Tyr Pro
35 40 45
His Val Thr Pro Lys Glu Cys Asn Asn Arg Gly Cys Cys Phe Asp
50 55 60
Ser Arg Ile Pro Gly Val Pro Trp Cys Phe Lys Pro Leu Gln Glu
65 70 75
Ala Glu Cys Thr Phe
80
60
314
PRT
Homo Sapien
60
Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys
1 5 10 15
Ala Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys
20 25 30
Gln Leu Tyr Asn Lys Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn
35 40 45
Pro Asp Pro Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn Ala
50 55 60
Val Ser Ser Glu Glu Thr Asn Asp Phe Lys Gln Glu Thr Leu Pro
65 70 75
Ser Lys Ser Asn Glu Ser His Asp His Met Asp Asp Met Asp Asp
80 85 90
Glu Asp Asp Asp Asp His Val Asp Ser Gln Asp Ser Ile Asp Ser
95 100 105
Asn Asp Ser Asp Asp Val Asp Asp Thr Asp Asp Ser His Gln Ser
110 115 120
Asp Glu Ser His His Ser Asp Glu Ser Asp Glu Leu Val Thr Asp
125 130 135
Phe Pro Thr Asp Leu Pro Ala Thr Glu Val Phe Thr Pro Val Val
140 145 150
Pro Thr Val Asp Thr Tyr Asp Gly Arg Gly Asp Ser Val Val Tyr
155 160 165
Gly Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg Pro Asp Ile Gln
170 175 180
Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr Ser His Met Glu Ser
185 190 195
Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro Val Ala Gln Asp
200 205 210
Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys Asp Ser Tyr
215 220 225
Glu Thr Ser Gln Leu Asp Asp Gln Ser Ala Glu Thr His Ser His
230 235 240
Lys Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser Asn
245 250 255
Glu His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser Lys Val Ser
260 265 270
Arg Glu Phe His Ser His Glu Phe His Ser His Glu Asp Met Leu
275 280 285
Val Val Asp Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe
290 295 300
Arg Ile Ser His Glu Leu Asp Ser Ala Ser Ser Glu Val Asn
305 310
61
184
PRT
Homo Sapien
61
Met Ser Arg Thr Ala Tyr Thr Val Gly Ala Leu Leu Leu Leu Leu
1 5 10 15
Gly Thr Leu Leu Pro Ala Ala Glu Gly Lys Lys Lys Gly Ser Gln
20 25 30
Gly Ala Ile Pro Pro Pro Asp Lys Ala Gln His Asn Asp Ser Glu
35 40 45
Gln Thr Gln Ser Pro Gln Gln Pro Gly Ser Arg Asn Arg Gly Arg
50 55 60
Gly Gln Gly Arg Gly Thr Ala Met Pro Gly Glu Glu Val Leu Glu
65 70 75
Ser Ser Gln Glu Ala Leu His Val Thr Glu Arg Lys Tyr Leu Lys
80 85 90
Arg Asp Trp Cys Lys Thr Gln Pro Leu Lys Gln Thr Ile His Glu
95 100 105
Glu Gly Cys Asn Ser Arg Thr Ile Ile Asn Arg Phe Cys Tyr Gly
110 115 120
Gln Cys Asn Ser Phe Tyr Ile Pro Arg His Ile Arg Lys Glu Glu
125 130 135
Gly Ser Phe Gln Ser Cys Ser Phe Cys Lys Pro Lys Lys Phe Thr
140 145 150
Thr Met Met Val Thr Leu Asn Cys Pro Glu Leu Gln Pro Pro Thr
155 160 165
Lys Lys Lys Arg Val Thr Arg Val Lys Gln Cys Arg Cys Ile Ser
170 175 180
Ile Asp Leu Asp
62
460
PRT
Homo Sapien
62
Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu
1 5 10 15
Leu Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val
20 25 30
Lys Ala Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys
35 40 45
Tyr Arg Thr Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe
50 55 60
Ser Leu Ala Ser Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala
65 70 75
Met Glu Ala Leu Lys Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys
80 85 90
Arg Gly Met Lys Lys Glu Lys Asn Cys Leu Arg Ile Tyr Trp Ser
95 100 105
Met Tyr Gln Ser Leu Gln Gly Asn Asp Leu Leu Glu Asp Ser Pro
110 115 120
Tyr Glu Pro Val Asn Ser Arg Leu Ser Asp Ile Phe Arg Val Val
125 130 135
Pro Phe Ile Ser Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu
140 145 150
Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp Ile Cys Lys Lys Tyr
155 160 165
Arg Ser Ala Tyr Ile Thr Pro Cys Thr Thr Ser Val Ser Asn Asp
170 175 180
Val Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe
185 190 195
Asp Lys Val Pro Ala Lys His Ser Tyr Gly Met Leu Phe Cys Ser
200 205 210
Cys Arg Asp Ile Ala Cys Thr Glu Arg Arg Arg Gln Thr Ile Val
215 220 225
Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn
230 235 240
Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys Arg Ser Arg Leu
245 250 255
Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg Ser Val Ser
260 265 270
Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala Tyr Ser
275 280 285
Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile Asp Ser Ser
290 295 300
Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn
305 310 315
Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn
320 325 330
Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp
335 340 345
Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala
350 355 360
Thr Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro
365 370 375
Ala Gly Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys
380 385 390
Ala Asn Leu Gln Ala Gln Lys Leu Lys Ser Asn Val Ser Gly Asn
395 400 405
Thr His Leu Cys Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly Leu
410 415 420
Gly Ala Ser Ser His Ile Thr Thr Lys Ser Met Ala Ala Pro Pro
425 430 435
Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val Thr Ala Leu
440 445 450
Ser Thr Leu Leu Ser Leu Thr Glu Thr Ser
455 460
63
143
PRT
Homo Sapien
63
Met Gln His Arg Gly Phe Leu Leu Leu Thr Leu Leu Ala Leu Leu
1 5 10 15
Ala Leu Thr Ser Ala Val Ala Lys Lys Lys Asp Lys Val Lys Lys
20 25 30
Gly Gly Pro Gly Ser Glu Cys Ala Glu Trp Ala Trp Gly Pro Cys
35 40 45
Thr Pro Ser Ser Lys Asp Cys Gly Val Gly Phe Arg Glu Gly Thr
50 55 60
Cys Gly Ala Gln Thr Gln Arg Ile Arg Cys Arg Val Pro Cys Asn
65 70 75
Trp Lys Lys Glu Phe Gly Ala Asp Cys Lys Tyr Lys Phe Glu Asn
80 85 90
Trp Gly Ala Cys Asp Gly Gly Thr Gly Thr Lys Val Arg Gln Gly
95 100 105
Thr Leu Lys Lys Ala Arg Tyr Asn Ala Gln Cys Gln Glu Thr Ile
110 115 120
Arg Val Thr Lys Pro Cys Thr Pro Lys Thr Lys Ala Lys Ala Lys
125 130 135
Ala Lys Lys Gly Lys Gly Lys Asp
140
64
141
PRT
Homo sapien
64
Met Trp Val Leu Gly Ile Ala Ala Thr Phe Cys Gly Leu Phe Leu
1 5 10 15
Leu Pro Gly Phe Ala Leu Gln Ile Gln Cys Tyr Gln Cys Glu Glu
20 25 30
Phe Gln Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe Ile Val Asn
35 40 45
Cys Thr Val Asn Val Gln Asp Met Cys Gln Lys Glu Val Met Glu
50 55 60
Gln Ser Ala Gly Ile Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala
65 70 75
Ala Cys Leu Ile Ala Ser Ala Gly Tyr Gln Ser Phe Cys Ser Pro
80 85 90
Gly Lys Leu Asn Ser Val Cys Ile Ser Cys Cys Asn Thr Pro Leu
95 100 105
Cys Asn Gly Pro Arg Pro Lys Lys Arg Gly Ser Ser Ala Ser Ala
110 115 120
Leu Arg Pro Gly Leu Arg Thr Thr Ile Leu Phe Leu Lys Leu Ala
125 130 135
Leu Phe Ser Ala His Cys
140
65
242
PRT
Homo Sapien
65
Met Lys Asn Ile Gly Leu Val Met Glu Trp Glu Ile Pro Glu Ile
1 5 10 15
Ile Cys Thr Cys Ala Lys Leu Arg Leu Pro Pro Gln Ala Thr Phe
20 25 30
Gln Val Leu Arg Gly Asn Gly Ala Ser Val Gly Thr Val Leu Met
35 40 45
Phe Arg Cys Pro Ser Asn His Gln Met Val Gly Ser Gly Leu Leu
50 55 60
Thr Cys Thr Trp Lys Gly Ser Ile Ala Glu Trp Ser Ser Gly Ser
65 70 75
Pro Val Cys Lys Leu Val Pro Pro His Glu Thr Phe Gly Phe Lys
80 85 90
Val Ala Val Ile Ala Ser Ile Val Ser Cys Ala Ile Ile Leu Leu
95 100 105
Met Ser Met Ala Phe Leu Thr Cys Cys Leu Leu Lys Cys Val Lys
110 115 120
Lys Ser Lys Arg Arg Arg Ser Asn Arg Ser Ala Gln Leu Trp Ser
125 130 135
Gln Leu Lys Asp Glu Asp Leu Glu Thr Val Gln Ala Ala Tyr Leu
140 145 150
Gly Leu Lys His Phe Asn Lys Pro Val Ser Gly Pro Ser Gln Ala
155 160 165
His Asp Asn His Ser Phe Thr Thr Asp His Gly Glu Ser Thr Ser
170 175 180
Lys Leu Ala Ser Val Thr Arg Ser Val Asp Lys Asp Pro Gly Ile
185 190 195
Pro Arg Ala Leu Ser Leu Ser Gly Ser Ser Ser Ser Pro Gln Ala
200 205 210
Gln Val Met Val His Met Ala Asn Pro Arg Gln Pro Leu Pro Ala
215 220 225
Ser Gly Leu Ala Thr Gly Met Pro Gln Gln Pro Ala Ala Tyr Ala
230 235 240
Leu Gly
66
672
PRT
Homo sapien
66
Asp Cys Thr Gly Asp Gly Pro Trp Gln Ser Asn Leu Ala Pro Ser
1 5 10 15
Gln Leu Glu Tyr Tyr Ala Ser Ser Pro Asp Glu Lys Ala Leu Val
20 25 30
Glu Ala Ala Ala Arg Ile Gly Ile Val Phe Ile Gly Asn Ser Glu
35 40 45
Glu Thr Met Glu Val Lys Thr Leu Gly Lys Leu Glu Arg Tyr Lys
50 55 60
Leu Leu His Ile Leu Glu Phe Asp Ser Asp Arg Arg Arg Met Ser
65 70 75
Val Ile Val Gln Ala Pro Ser Gly Glu Lys Leu Leu Phe Ala Lys
80 85 90
Gly Ala Glu Ser Ser Ile Leu Pro Lys Cys Ile Gly Gly Glu Ile
95 100 105
Glu Lys Thr Arg Ile His Val Asp Glu Phe Ala Leu Lys Gly Leu
110 115 120
Arg Thr Leu Cys Ile Ala Tyr Arg Lys Phe Thr Ser Lys Glu Tyr
125 130 135
Glu Glu Ile Asp Lys Arg Ile Phe Glu Ala Arg Thr Ala Leu Gln
140 145 150
Gln Arg Glu Glu Lys Leu Ala Ala Val Phe Gln Phe Ile Glu Lys
155 160 165
Asp Leu Ile Leu Leu Gly Ala Thr Ala Val Glu Asp Arg Leu Gln
170 175 180
Asp Lys Val Arg Glu Thr Ile Glu Ala Leu Arg Met Ala Gly Ile
185 190 195
Lys Val Trp Val Leu Thr Gly Asp Lys His Glu Thr Ala Val Ser
200 205 210
Val Ser Leu Ser Cys Gly His Phe His Arg Thr Met Asn Ile Leu
215 220 225
Glu Leu Ile Asn Gln Lys Ser Asp Ser Glu Cys Ala Glu Gln Leu
230 235 240
Arg Gln Leu Ala Arg Arg Ile Thr Glu Asp His Val Ile Gln His
245 250 255
Gly Leu Val Val Asp Gly Thr Ser Leu Ser Leu Ala Leu Arg Glu
260 265 270
His Glu Lys Leu Phe Met Glu Val Cys Arg Asn Cys Ser Ala Val
275 280 285
Leu Cys Cys Arg Met Ala Pro Leu Gln Lys Ala Lys Val Ile Arg
290 295 300
Leu Ile Lys Ile Ser Pro Glu Lys Pro Ile Thr Leu Ala Val Gly
305 310 315
Asp Gly Ala Asn Asp Val Ser Met Ile Gln Glu Ala His Val Gly
320 325 330
Ile Gly Ile Met Gly Lys Glu Gly Arg Gln Ala Ala Arg Asn Ser
335 340 345
Asp Tyr Ala Ile Ala Arg Phe Lys Phe Leu Ser Lys Leu Leu Phe
350 355 360
Val His Gly His Phe Tyr Tyr Ile Arg Ile Ala Thr Leu Val Gln
365 370 375
Tyr Phe Phe Tyr Lys Asn Val Cys Phe Ile Thr Pro Gln Phe Leu
380 385 390
Tyr Gln Phe Tyr Cys Leu Phe Ser Gln Gln Thr Leu Tyr Asp Ser
395 400 405
Val Tyr Leu Thr Leu Tyr Asn Ile Cys Phe Thr Ser Leu Pro Ile
410 415 420
Leu Ile Tyr Ser Leu Leu Glu Gln His Val Asp Pro His Val Leu
425 430 435
Gln Asn Lys Pro Thr Leu Tyr Arg Asp Ile Ser Lys Asn Arg Leu
440 445 450
Leu Ser Ile Lys Thr Phe Leu Tyr Trp Thr Ile Leu Gly Phe Ser
455 460 465
His Ala Phe Ile Phe Phe Phe Gly Ser Tyr Leu Leu Ile Gly Lys
470 475 480
Asp Thr Ser Leu Leu Gly Asn Gly Gln Met Phe Gly Asn Trp Thr
485 490 495
Phe Gly Thr Leu Val Phe Thr Val Met Val Ile Thr Val Thr Val
500 505 510
Lys Met Ala Leu Glu Thr His Phe Trp Thr Trp Ile Asn His Leu
515 520 525
Val Thr Trp Gly Ser Ile Ile Phe Tyr Phe Val Phe Ser Leu Phe
530 535 540
Tyr Gly Gly Ile Leu Trp Pro Phe Leu Gly Ser Gln Asn Met Tyr
545 550 555
Phe Val Phe Ile Gln Leu Leu Ser Ser Gly Ser Ala Trp Phe Ala
560 565 570
Ile Ile Leu Met Val Val Thr Cys Leu Phe Leu Asp Ile Ile Lys
575 580 585
Lys Val Phe Asp Arg His Leu His Pro Thr Ser Thr Glu Lys Ala
590 595 600
Gln Leu Thr Glu Thr Asn Ala Gly Ile Lys Cys Leu Asp Ser Met
605 610 615
Cys Cys Phe Pro Glu Gly Glu Ala Ala Cys Ala Ser Val Gly Arg
620 625 630
Met Leu Glu Arg Val Ile Gly Arg Cys Ser Pro Thr His Ile Ser
635 640 645
Arg Ser Trp Ser Ala Ser Asp Pro Phe Tyr Thr Asn Asp Arg Ser
650 655 660
Ile Leu Thr Leu Ser Thr Met Asp Ser Ser Thr Cys
665 670
67
877
PRT
Homo Sapien
67
Met Trp Glu Glu Glu Asp Ile Ala Ile Leu Phe Asn Lys Glu Pro
1 5 10 15
Gly Lys Thr Glu Asn Ile Glu Asn Asn Leu Ser Ser Asn His Arg
20 25 30
Arg Ser Cys Arg Arg Ser Glu Glu Ser Asp Asp Asp Leu Asp Phe
35 40 45
Asp Ile Gly Leu Glu Asn Thr Gly Gly Asp Pro Gln Ile Leu Arg
50 55 60
Phe Ile Ser Asp Phe Leu Ala Phe Leu Val Leu Tyr Asn Phe Ile
65 70 75
Ile Pro Ile Ser Leu Tyr Val Thr Val Glu Met Gln Lys Phe Leu
80 85 90
Gly Ser Phe Phe Ile Gly Trp Asp Leu Asp Leu Tyr His Glu Glu
95 100 105
Ser Asp Gln Lys Ala Gln Val Asn Thr Ser Asp Leu Asn Glu Glu
110 115 120
Leu Gly Gln Val Glu Tyr Val Phe Thr Asp Lys Thr Gly Thr Leu
125 130 135
Thr Glu Asn Glu Met Gln Phe Arg Glu Cys Ser Ile Asn Gly Met
140 145 150
Lys Tyr Gln Glu Ile Asn Gly Arg Leu Val Pro Glu Gly Pro Thr
155 160 165
Pro Asp Ser Ser Glu Gly Asn Leu Ser Tyr Leu Ser Ser Leu Ser
170 175 180
His Leu Asn Asn Leu Ser His Leu Thr Thr Ser Ser Ser Phe Arg
185 190 195
Thr Ser Pro Glu Asn Glu Thr Glu Leu Ile Lys Glu His Asp Leu
200 205 210
Phe Phe Lys Ala Val Ser Leu Cys His Thr Val Gln Ile Ser Asn
215 220 225
Val Gln Thr Asp Cys Thr Gly Asp Gly Pro Trp Gln Ser Asn Leu
230 235 240
Ala Pro Ser Gln Leu Glu Tyr Tyr Ala Ser Ser Pro Asp Glu Lys
245 250 255
Ala Leu Val Glu Ala Ala Ala Arg Tyr Lys Leu Leu His Ile Leu
260 265 270
Glu Phe Asp Ser Asp Arg Arg Arg Met Ser Val Ile Val Gln Ala
275 280 285
Pro Ser Gly Glu Lys Leu Leu Phe Ala Lys Gly Ala Glu Ser Ser
290 295 300
Ile Leu Pro Lys Cys Ile Gly Gly Glu Ile Glu Lys Thr Arg Ile
305 310 315
His Val Asp Glu Phe Ala Leu Lys Gly Leu Arg Thr Leu Cys Ile
320 325 330
Ala Tyr Arg Lys Phe Thr Ser Lys Glu Tyr Glu Glu Ile Asp Lys
335 340 345
Arg Ile Phe Glu Ala Arg Thr Ala Leu Gln Gln Arg Glu Glu Lys
350 355 360
Leu Ala Ala Val Phe Gln Phe Ile Glu Lys Asp Leu Ile Leu Leu
365 370 375
Gly Ala Thr Ala Val Glu Asp Arg Leu Gln Asp Lys Val Arg Glu
380 385 390
Thr Ile Glu Ala Leu Arg Met Ala Gly Ile Lys Val Trp Val Leu
395 400 405
Thr Gly Asp Lys His Glu Thr Ala Val Ser Val Ser Leu Ser Cys
410 415 420
Gly His Phe His Arg Thr Met Asn Ile Leu Glu Leu Ile Asn Gln
425 430 435
Lys Ser Asp Ser Glu Cys Ala Glu Gln Leu Arg Gln Leu Ala Arg
440 445 450
Arg Ile Thr Glu Asp His Val Ile Gln His Gly Leu Val Val Asp
455 460 465
Gly Thr Ser Leu Ser Leu Ala Leu Arg Glu His Glu Lys Leu Phe
470 475 480
Met Glu Val Cys Arg Asn Cys Ser Ala Val Leu Cys Cys Arg Met
485 490 495
Ala Pro Leu Gln Lys Ala Lys Val Ile Arg Leu Ile Lys Ile Ser
500 505 510
Pro Glu Lys Pro Ile Thr Leu Ala Val Gly Asp Gly Ala Asn Asp
515 520 525
Val Ser Met Ile Gln Glu Ala His Val Gly Ile Gly Ile Met Gly
530 535 540
Lys Glu Gly Arg Gln Ala Ala Arg Asn Ser Asp Tyr Ala Ile Ala
545 550 555
Arg Phe Lys Phe Leu Ser Lys Leu Leu Phe Val His Gly His Phe
560 565 570
Tyr Tyr Ile Arg Ile Ala Thr Leu Val Gln Tyr Phe Phe Tyr Lys
575 580 585
Asn Val Cys Phe Ile Thr Pro Gln Phe Leu Tyr Gln Phe Tyr Cys
590 595 600
Leu Phe Ser Gln Gln Thr Leu Tyr Asp Ser Val Tyr Leu Thr Leu
605 610 615
Tyr Asn Ile Cys Phe Thr Ser Leu Pro Ile Leu Ile Tyr Ser Leu
620 625 630
Leu Glu Gln His Val Asp Pro His Val Leu Gln Asn Lys Pro Thr
635 640 645
Leu Tyr Arg Asp Ile Ser Lys Asn Arg Leu Leu Ser Ile Lys Thr
650 655 660
Phe Leu Tyr Trp Thr Ile Leu Gly Phe Ser His Ala Phe Ile Phe
665 670 675
Phe Phe Gly Ser Tyr Leu Leu Ile Gly Lys Asp Thr Ser Leu Leu
680 685 690
Gly Asn Gly Gln Met Phe Gly Asn Trp Thr Phe Gly Thr Leu Val
695 700 705
Phe Thr Val Met Val Ile Thr Val Thr Val Lys Met Ala Leu Glu
710 715 720
Thr His Phe Trp Thr Trp Ile Asn His Leu Val Thr Trp Gly Ser
725 730 735
Ile Ile Phe Tyr Phe Val Phe Ser Leu Phe Tyr Gly Gly Ile Leu
740 745 750
Trp Pro Phe Leu Gly Ser Gln Asn Met Tyr Phe Val Phe Ile Gln
755 760 765
Leu Leu Ser Ser Gly Ser Ala Trp Phe Ala Ile Ile Leu Met Val
770 775 780
Val Thr Cys Leu Phe Leu Asp Ile Ile Lys Lys Val Phe Asp Arg
785 790 795
His Leu His Pro Thr Ser Thr Glu Lys Ala Gln Leu Thr Glu Thr
800 805 810
Asn Ala Gly Ile Lys Cys Leu Asp Ser Met Cys Cys Phe Pro Glu
815 820 825
Gly Glu Ala Ala Cys Ala Ser Val Gly Arg Met Leu Glu Arg Val
830 835 840
Ile Gly Arg Cys Ser Pro Thr His Ile Ser Arg Ser Trp Ser Ala
845 850 855
Ser Asp Pro Phe Tyr Thr Asn Asp Arg Ser Ile Leu Thr Leu Ser
860 865 870
Thr Met Asp Ser Ser Thr Cys
875
68
63
PRT
Homo Sapien
68
Met Lys His Val Leu Asn Leu Tyr Leu Leu Gly Val Val Leu Thr
1 5 10 15
Leu Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Glu Gly Leu
20 25 30
Leu Glu Ser Pro Ser Pro Gly Thr Ser Trp Thr Thr Arg Ser Gln
35 40 45
Leu Ala Asn Thr Glu Pro Thr Lys Gly Leu Pro Asp His Pro Ser
50 55 60
Arg Ser Met
69
137
PRT
Homo Sapien
unsure
101, 136
unknown amino acid
69
Met Lys Thr Gly Leu Phe Phe Leu Cys Leu Leu Gly Thr Ala Ala
1 5 10 15
Ala Ile Pro Thr Asn Ala Arg Leu Leu Ser Asp His Ser Lys Pro
20 25 30
Thr Ala Glu Thr Val Ala Pro Asp Asn Thr Ala Ile Pro Ser Leu
35 40 45
Arg Ala Glu Asp Glu Glu Asn Glu Lys Glu Thr Ala Val Ser Thr
50 55 60
Glu Asp Asp Ser His His Lys Ala Glu Lys Ser Ser Val Leu Lys
65 70 75
Ser Lys Glu Glu Ser His Glu Gln Ser Ala Glu Gln Gly Lys Ser
80 85 90
Ser Ser Gln Glu Leu Gly Leu Lys Asp Gln Xaa Asp Ser Asp Gly
95 100 105
Asp Leu Ser Val Asn Leu Glu Tyr Ala Pro Thr Glu Gly Thr Leu
110 115 120
Asp Ile Lys Glu Asp Met Ser Glu Pro Gln Glu Lys Asn Ser Gln
125 130 135
Xaa His
70
318
PRT
Homo Sapien
70
Met Ala Pro Trp Ala Glu Ala Glu His Ser Ala Leu Asn Pro Leu
1 5 10 15
Arg Ala Val Trp Leu Thr Leu Thr Ala Ala Phe Leu Leu Thr Leu
20 25 30
Leu Leu Gln Leu Leu Pro Pro Gly Leu Leu Pro Gly Cys Ala Ile
35 40 45
Phe Gln Asp Leu Ile Arg Tyr Gly Lys Thr Lys Cys Gly Glu Pro
50 55 60
Ser Arg Pro Ala Ala Cys Arg Ala Phe Asp Val Pro Lys Arg Tyr
65 70 75
Phe Ser His Phe Tyr Ile Ile Ser Val Leu Trp Asn Gly Phe Leu
80 85 90
Leu Trp Cys Leu Thr Gln Ser Leu Phe Leu Gly Ala Pro Phe Pro
95 100 105
Ser Trp Leu His Gly Leu Leu Arg Ile Leu Gly Ala Ala Gln Phe
110 115 120
Gln Gly Gly Glu Leu Ala Leu Ser Ala Phe Leu Val Leu Val Phe
125 130 135
Leu Trp Leu His Ser Leu Arg Arg Leu Phe Glu Cys Leu Tyr Val
140 145 150
Ser Val Phe Ser Asn Val Met Ile His Val Val Gln Tyr Cys Phe
155 160 165
Gly Leu Val Tyr Tyr Val Leu Val Gly Leu Thr Val Leu Ser Gln
170 175 180
Val Pro Met Asp Gly Arg Asn Ala Tyr Ile Thr Gly Lys Asn Leu
185 190 195
Leu Met Gln Ala Arg Trp Phe His Ile Leu Gly Met Met Met Phe
200 205 210
Ile Trp Ser Ser Ala His Gln Tyr Lys Cys His Val Ile Leu Gly
215 220 225
Asn Leu Arg Lys Asn Lys Ala Gly Val Val Ile His Cys Asn His
230 235 240
Arg Ile Pro Phe Gly Asp Trp Phe Glu Tyr Val Ser Ser Pro Asn
245 250 255
Tyr Leu Ala Glu Leu Met Ile Tyr Val Ser Met Ala Val Thr Phe
260 265 270
Gly Phe His Asn Leu Thr Trp Trp Leu Val Val Thr Asn Val Phe
275 280 285
Phe Asn Gln Ala Leu Ser Ala Phe Leu Ser His Gln Phe Tyr Lys
290 295 300
Ser Lys Phe Val Ser Tyr Pro Lys His Arg Lys Ala Phe Leu Pro
305 310 315
Phe Leu Phe
71
426
PRT
Homo sapien
71
Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp
1 5 10 15
Ile Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser
20 25 30
Ala Ala Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys
35 40 45
Asp Val Pro Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys
50 55 60
His Ile Leu Asn Met Leu His Leu Lys Lys Arg Pro Asp Val Thr
65 70 75
Gln Pro Val Pro Lys Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu
80 85 90
His Val Gly Lys Val Gly Glu Asn Gly Tyr Val Glu Ile Glu Asp
95 100 105
Asp Ile Gly Arg Arg Ala Glu Met Asn Glu Leu Met Glu Gln Thr
110 115 120
Ser Glu Ile Ile Thr Phe Ala Glu Ser Gly Thr Ala Arg Lys Thr
125 130 135
Leu His Phe Glu Ile Ser Lys Glu Gly Ser Asp Leu Ser Val Val
140 145 150
Glu Arg Ala Glu Val Trp Leu Phe Leu Lys Val Pro Lys Ala Asn
155 160 165
Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe Gln Gln Gln Lys
170 175 180
His Pro Gln Gly Ser Leu Asp Thr Gly Glu Glu Ala Glu Glu Val
185 190 195
Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val
200 205 210
Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser Ser
215 220 225
Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val
230 235 240
Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val
245 250 255
Leu Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys
260 265 270
Lys Lys Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys
275 280 285
Glu Gln Ser His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser
290 295 300
Glu Asp His Pro His Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp
305 310 315
Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe Phe Val Ser Phe
320 325 330
Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro Ser Gly Tyr
335 340 345
His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile Ala Gly
350 355 360
Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val Ile Asn His
365 370 375
Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys
380 385 390
Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp
395 400 405
Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val
410 415 420
Glu Glu Cys Gly Cys Ser
425
72
238
PRT
Homo Sapien
72
Met Ala Ala Ala Pro Leu Leu Leu Leu Leu Leu Leu Val Pro Val
1 5 10 15
Pro Leu Leu Pro Leu Leu Ala Gln Gly Pro Gly Gly Ala Leu Gly
20 25 30
Asn Arg His Ala Val Tyr Trp Asn Ser Ser Asn Gln His Leu Arg
35 40 45
Arg Glu Gly Tyr Thr Val Gln Val Asn Val Asn Asp Tyr Leu Asp
50 55 60
Ile Tyr Cys Pro His Tyr Asn Ser Ser Gly Val Gly Pro Gly Ala
65 70 75
Gly Pro Gly Pro Gly Gly Gly Ala Glu Gln Tyr Val Leu Tyr Met
80 85 90
Val Ser Arg Asn Gly Tyr Arg Thr Cys Asn Ala Ser Gln Gly Phe
95 100 105
Lys Arg Trp Glu Cys Asn Arg Pro His Ala Pro His Ser Pro Ile
110 115 120
Lys Phe Ser Glu Lys Phe Gln Arg Tyr Ser Ala Phe Ser Leu Gly
125 130 135
Tyr Glu Phe His Ala Gly His Glu Tyr Tyr Tyr Ile Ser Thr Pro
140 145 150
Thr His Asn Leu His Trp Lys Cys Leu Arg Met Lys Val Phe Val
155 160 165
Cys Cys Ala Ser Thr Ser His Ser Gly Glu Lys Pro Val Pro Thr
170 175 180
Leu Pro Gln Phe Thr Met Gly Pro Asn Val Lys Ile Asn Val Leu
185 190 195
Glu Asp Phe Glu Gly Glu Asn Pro Gln Val Pro Lys Leu Glu Lys
200 205 210
Ser Ile Ser Gly Thr Ser Pro Lys Arg Glu His Leu Pro Leu Ala
215 220 225
Val Gly Ile Ala Phe Phe Leu Met Thr Phe Leu Ala Ser
230 235
73
541
PRT
Homo Sapien
73
Met Gly His Ser Pro Pro Val Leu Pro Leu Cys Ala Ser Val Ser
1 5 10 15
Leu Leu Gly Gly Leu Thr Phe Gly Tyr Glu Leu Ala Val Ile Ser
20 25 30
Gly Ala Leu Leu Pro Leu Gln Leu Asp Phe Gly Leu Ser Cys Leu
35 40 45
Glu Gln Glu Phe Leu Val Gly Ser Leu Leu Leu Gly Ala Leu Leu
50 55 60
Ala Ser Leu Val Gly Gly Phe Leu Ile Asp Cys Tyr Gly Arg Lys
65 70 75
Gln Ala Ile Leu Gly Ser Asn Leu Val Leu Leu Ala Gly Ser Leu
80 85 90
Thr Leu Gly Leu Ala Gly Ser Leu Ala Trp Leu Val Leu Gly Arg
95 100 105
Ala Val Val Gly Phe Ala Ile Ser Leu Ser Ser Met Ala Cys Cys
110 115 120
Ile Tyr Val Ser Glu Leu Val Gly Pro Arg Gln Arg Gly Val Leu
125 130 135
Val Ser Leu Tyr Glu Ala Gly Ile Thr Val Gly Ile Leu Leu Ser
140 145 150
Tyr Ala Leu Asn Tyr Ala Leu Ala Gly Thr Pro Trp Gly Trp Arg
155 160 165
His Met Phe Gly Trp Ala Thr Ala Pro Ala Val Leu Gln Ser Leu
170 175 180
Ser Leu Leu Phe Leu Pro Ala Gly Thr Asp Glu Thr Ala Thr His
185 190 195
Lys Asp Leu Ile Pro Leu Gln Gly Gly Glu Ala Pro Lys Leu Gly
200 205 210
Pro Gly Arg Pro Arg Tyr Ser Phe Leu Asp Leu Phe Arg Ala Arg
215 220 225
Asp Asn Met Arg Gly Arg Thr Thr Val Gly Leu Gly Leu Val Leu
230 235 240
Phe Gln Gln Leu Thr Gly Gln Pro Asn Val Leu Cys Tyr Ala Ser
245 250 255
Thr Ile Phe Ser Ser Val Gly Phe His Gly Gly Ser Ser Ala Val
260 265 270
Leu Ala Ser Val Gly Leu Gly Ala Val Lys Val Ala Ala Thr Leu
275 280 285
Thr Ala Met Gly Leu Val Asp Arg Ala Gly Arg Arg Ala Leu Leu
290 295 300
Leu Ala Gly Cys Ala Leu Met Ala Leu Ser Val Ser Gly Ile Gly
305 310 315
Leu Val Ser Phe Ala Val Pro Met Asp Ser Gly Pro Ser Cys Leu
320 325 330
Ala Val Pro Asn Ala Thr Gly Gln Thr Gly Leu Pro Gly Asp Ser
335 340 345
Gly Leu Leu Gln Asp Ser Ser Leu Pro Pro Ile Pro Arg Thr Asn
350 355 360
Glu Asp Gln Arg Glu Pro Ile Leu Ser Thr Ala Lys Lys Thr Lys
365 370 375
Pro His Pro Arg Ser Gly Asp Pro Ser Ala Pro Pro Arg Leu Ala
380 385 390
Leu Ser Ser Ala Leu Pro Gly Pro Pro Leu Pro Ala Arg Gly His
395 400 405
Ala Leu Leu Arg Trp Thr Ala Leu Leu Cys Leu Met Val Phe Val
410 415 420
Ser Ala Phe Ser Phe Gly Phe Gly Pro Val Thr Trp Leu Val Leu
425 430 435
Ser Glu Ile Tyr Pro Val Glu Ile Arg Gly Arg Ala Phe Ala Phe
440 445 450
Cys Asn Ser Phe Asn Trp Ala Ala Asn Leu Phe Ile Ser Leu Ser
455 460 465
Phe Leu Asp Leu Ile Gly Thr Ile Gly Leu Ser Trp Thr Phe Leu
470 475 480
Leu Tyr Gly Leu Thr Ala Val Leu Gly Leu Gly Phe Ile Tyr Leu
485 490 495
Phe Val Pro Glu Thr Lys Gly Gln Ser Leu Ala Glu Ile Asp Gln
500 505 510
Gln Phe Gln Lys Arg Arg Phe Thr Leu Ser Phe Gly His Arg Gln
515 520 525
Asn Ser Thr Gly Ile Pro Tyr Ser Arg Ile Glu Ile Ser Ala Ala
530 535 540
Ser
74
1114
PRT
Homo Sapien
74
Met Ala Lys Ala Thr Ser Gly Ala Ala Gly Leu Arg Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Pro Leu Leu Gly Lys Val Ala Leu Gly Leu Tyr
20 25 30
Phe Ser Arg Asp Ala Tyr Trp Glu Lys Leu Tyr Val Asp Gln Ala
35 40 45
Ala Gly Thr Pro Leu Leu Tyr Val His Ala Leu Arg Asp Ala Pro
50 55 60
Glu Glu Val Pro Ser Phe Arg Leu Gly Gln His Leu Tyr Gly Thr
65 70 75
Tyr Arg Thr Arg Leu His Glu Asn Asn Trp Ile Cys Ile Gln Glu
80 85 90
Asp Thr Gly Leu Leu Tyr Leu Asn Arg Ser Leu Asp His Ser Ser
95 100 105
Trp Glu Lys Leu Ser Val Arg Asn Arg Gly Phe Pro Leu Leu Thr
110 115 120
Val Tyr Leu Lys Val Phe Leu Ser Pro Thr Ser Leu Arg Glu Gly
125 130 135
Glu Cys Gln Trp Pro Gly Cys Ala Arg Val Tyr Phe Ser Phe Phe
140 145 150
Asn Thr Ser Phe Pro Ala Cys Ser Ser Leu Lys Pro Arg Glu Leu
155 160 165
Cys Phe Pro Glu Thr Arg Pro Ser Phe Arg Ile Arg Glu Asn Arg
170 175 180
Pro Pro Gly Thr Phe His Gln Phe Arg Leu Leu Pro Val Gln Phe
185 190 195
Leu Cys Pro Asn Ile Ser Val Ala Tyr Arg Leu Leu Glu Gly Glu
200 205 210
Gly Leu Pro Phe Arg Cys Ala Pro Asp Ser Leu Glu Val Ser Thr
215 220 225
Arg Trp Ala Leu Asp Arg Glu Gln Arg Glu Lys Tyr Glu Leu Val
230 235 240
Ala Val Cys Thr Val His Ala Gly Ala Arg Glu Glu Val Val Met
245 250 255
Val Pro Phe Pro Val Thr Val Tyr Asp Glu Asp Asp Ser Ala Pro
260 265 270
Thr Phe Pro Ala Gly Val Asp Thr Ala Ser Ala Val Val Glu Phe
275 280 285
Lys Arg Lys Glu Asp Thr Val Val Ala Thr Leu Arg Val Phe Asp
290 295 300
Ala Asp Val Val Pro Ala Ser Gly Glu Leu Val Arg Arg Tyr Thr
305 310 315
Ser Thr Leu Leu Pro Gly Asp Thr Trp Ala Gln Gln Thr Phe Arg
320 325 330
Val Glu His Trp Pro Asn Glu Thr Ser Val Gln Ala Asn Gly Ser
335 340 345
Phe Val Arg Ala Thr Val His Asp Tyr Arg Leu Val Leu Asn Arg
350 355 360
Asn Leu Ser Ile Ser Glu Asn Arg Thr Met Gln Leu Ala Val Leu
365 370 375
Val Asn Asp Ser Asp Phe Gln Gly Pro Gly Ala Gly Val Leu Leu
380 385 390
Leu His Phe Asn Val Ser Val Leu Pro Val Ser Leu His Leu Pro
395 400 405
Ser Thr Tyr Ser Leu Ser Val Ser Arg Arg Ala Arg Arg Phe Ala
410 415 420
Gln Ile Gly Lys Val Cys Val Glu Asn Cys Gln Ala Phe Ser Gly
425 430 435
Ile Asn Val Gln Tyr Lys Leu His Ser Ser Gly Ala Asn Cys Ser
440 445 450
Thr Leu Gly Val Val Thr Ser Ala Glu Asp Thr Ser Gly Ile Leu
455 460 465
Phe Val Asn Asp Thr Lys Ala Leu Arg Arg Pro Lys Cys Ala Glu
470 475 480
Leu His Tyr Met Val Val Ala Thr Asp Gln Gln Thr Ser Arg Gln
485 490 495
Ala Gln Ala Gln Leu Leu Val Thr Val Glu Gly Ser Tyr Val Ala
500 505 510
Glu Glu Ala Gly Cys Pro Leu Ser Cys Ala Val Ser Lys Arg Arg
515 520 525
Leu Glu Cys Glu Glu Cys Gly Gly Leu Gly Ser Pro Thr Gly Arg
530 535 540
Cys Glu Trp Arg Gln Gly Asp Gly Lys Gly Ile Thr Arg Asn Phe
545 550 555
Ser Thr Cys Ser Pro Ser Thr Lys Thr Cys Pro Asp Gly His Cys
560 565 570
Asp Val Val Glu Thr Gln Asp Ile Asn Ile Cys Pro Gln Asp Cys
575 580 585
Leu Arg Gly Ser Ile Val Gly Gly His Glu Pro Gly Glu Pro Arg
590 595 600
Gly Ile Lys Ala Gly Tyr Gly Thr Cys Asn Cys Phe Pro Glu Glu
605 610 615
Glu Lys Cys Phe Cys Glu Pro Glu Asp Ile Gln Asp Pro Leu Cys
620 625 630
Asp Glu Leu Cys Arg Thr Val Ile Ala Ala Ala Val Leu Phe Ser
635 640 645
Phe Ile Val Ser Val Leu Leu Ser Ala Phe Cys Ile His Cys Tyr
650 655 660
His Lys Phe Ala His Lys Pro Pro Ile Ser Ser Ala Glu Met Thr
665 670 675
Phe Arg Arg Pro Ala Gln Ala Phe Pro Val Ser Tyr Ser Ser Ser
680 685 690
Gly Ala Arg Arg Pro Ser Leu Asp Ser Met Glu Asn Gln Val Ser
695 700 705
Val Asp Ala Phe Lys Ile Leu Glu Asp Pro Lys Trp Glu Phe Pro
710 715 720
Arg Lys Asn Leu Val Leu Gly Lys Thr Leu Gly Glu Gly Glu Phe
725 730 735
Gly Lys Val Val Lys Ala Thr Ala Phe His Leu Lys Gly Arg Ala
740 745 750
Gly Tyr Thr Thr Val Ala Val Lys Met Leu Lys Glu Asn Ala Ser
755 760 765
Pro Ser Glu Leu Arg Asp Leu Leu Ser Glu Phe Asn Val Leu Lys
770 775 780
Gln Val Asn His Pro His Val Ile Lys Leu Tyr Gly Ala Cys Ser
785 790 795
Gln Asp Gly Pro Leu Leu Leu Ile Val Glu Tyr Ala Lys Tyr Gly
800 805 810
Ser Leu Arg Gly Phe Leu Arg Glu Ser Arg Lys Val Gly Pro Gly
815 820 825
Tyr Leu Gly Ser Gly Gly Ser Arg Asn Ser Ser Ser Leu Asp His
830 835 840
Pro Asp Glu Arg Ala Leu Thr Met Gly Asp Leu Ile Ser Phe Ala
845 850 855
Trp Gln Ile Ser Gln Gly Met Gln Tyr Leu Ala Glu Met Lys Leu
860 865 870
Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Ala Glu Gly
875 880 885
Arg Lys Met Lys Ile Ser Asp Phe Gly Leu Ser Arg Asp Val Tyr
890 895 900
Glu Glu Asp Ser Tyr Val Lys Arg Ser Gln Gly Arg Ile Pro Val
905 910 915
Lys Trp Met Ala Ile Glu Ser Leu Phe Asp His Ile Tyr Thr Thr
920 925 930
Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Val
935 940 945
Thr Leu Gly Gly Asn Pro Tyr Pro Gly Ile Pro Pro Glu Arg Leu
950 955 960
Phe Asn Leu Leu Lys Thr Gly His Arg Met Glu Arg Pro Asp Asn
965 970 975
Cys Ser Glu Glu Met Tyr Arg Leu Met Leu Gln Cys Trp Lys Gln
980 985 990
Glu Pro Asp Lys Arg Pro Val Phe Ala Asp Ile Ser Lys Asp Leu
995 1000 1005
Glu Lys Met Met Val Lys Arg Arg Asp Tyr Leu Asp Leu Ala Ala
1010 1015 1020
Ser Thr Pro Ser Asp Ser Leu Ile Tyr Asp Asp Gly Leu Ser Glu
1025 1030 1035
Glu Glu Thr Pro Leu Val Asp Cys Asn Asn Ala Pro Leu Pro Arg
1040 1045 1050
Ala Leu Pro Ser Thr Trp Ile Glu Asn Lys Leu Tyr Gly Met Ser
1055 1060 1065
Asp Pro Asn Trp Pro Gly Glu Ser Pro Val Pro Leu Thr Arg Ala
1070 1075 1080
Asp Gly Thr Asn Thr Gly Phe Pro Arg Tyr Pro Asn Asp Ser Val
1085 1090 1095
Tyr Ala Asn Trp Met Leu Ser Pro Ser Ala Ala Lys Leu Met Asp
1100 1105 1110
Thr Phe Asp Ser
75
790
PRT
Homo Sapien
75
Met Arg Thr Tyr Arg Tyr Phe Leu Leu Leu Phe Trp Val Gly Gln
1 5 10 15
Pro Tyr Pro Thr Leu Ser Thr Pro Leu Ser Lys Arg Thr Ser Gly
20 25 30
Phe Pro Ala Lys Lys Arg Ala Leu Glu Leu Ser Gly Asn Ser Lys
35 40 45
Asn Glu Leu Asn Arg Ser Lys Arg Ser Trp Met Trp Asn Gln Phe
50 55 60
Phe Leu Leu Glu Glu Tyr Thr Gly Ser Asp Tyr Gln Tyr Val Gly
65 70 75
Lys Leu His Ser Asp Gln Asp Arg Gly Asp Gly Ser Leu Lys Tyr
80 85 90
Ile Leu Ser Gly Asp Gly Ala Gly Asp Leu Phe Ile Ile Asn Glu
95 100 105
Asn Thr Gly Asp Ile Gln Ala Thr Lys Arg Leu Asp Arg Glu Glu
110 115 120
Lys Pro Val Tyr Ile Leu Arg Ala Gln Ala Ile Asn Arg Arg Thr
125 130 135
Gly Arg Pro Val Glu Pro Glu Ser Glu Phe Ile Ile Lys Ile His
140 145 150
Asp Ile Asn Asp Asn Glu Pro Ile Phe Thr Lys Glu Val Tyr Thr
155 160 165
Ala Thr Val Pro Glu Met Ser Asp Val Gly Thr Phe Val Val Gln
170 175 180
Val Thr Ala Thr Asp Ala Asp Asp Pro Thr Tyr Gly Asn Ser Ala
185 190 195
Lys Val Val Tyr Ser Ile Leu Gln Gly Gln Pro Tyr Phe Ser Val
200 205 210
Glu Ser Glu Thr Gly Ile Ile Lys Thr Ala Leu Leu Asn Met Asp
215 220 225
Arg Glu Asn Arg Glu Gln Tyr Gln Val Val Ile Gln Ala Lys Asp
230 235 240
Met Gly Gly Gln Met Gly Gly Leu Ser Gly Thr Thr Thr Val Asn
245 250 255
Ile Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Arg Phe Pro Gln
260 265 270
Ser Thr Tyr Gln Phe Lys Thr Pro Glu Ser Ser Pro Pro Gly Thr
275 280 285
Pro Ile Gly Arg Ile Lys Ala Ser Asp Ala Asp Val Gly Glu Asn
290 295 300
Ala Glu Ile Glu Tyr Ser Ile Thr Asp Gly Glu Gly Leu Asp Met
305 310 315
Phe Asp Val Ile Thr Asp Gln Glu Thr Gln Glu Gly Ile Ile Thr
320 325 330
Val Lys Lys Leu Leu Asp Phe Glu Lys Lys Lys Val Tyr Thr Leu
335 340 345
Lys Val Glu Ala Ser Asn Pro Tyr Val Glu Pro Arg Phe Leu Tyr
350 355 360
Leu Gly Pro Phe Lys Asp Ser Ala Thr Val Arg Ile Val Val Glu
365 370 375
Asp Val Asp Glu Pro Pro Val Phe Ser Lys Leu Ala Tyr Ile Leu
380 385 390
Gln Ile Arg Glu Asp Ala Gln Ile Asn Thr Thr Ile Gly Ser Val
395 400 405
Thr Ala Gln Asp Pro Asp Ala Ala Arg Asn Pro Val Lys Tyr Ser
410 415 420
Val Asp Arg His Thr Asp Met Asp Arg Ile Phe Asn Ile Asp Ser
425 430 435
Gly Asn Gly Ser Ile Phe Thr Ser Lys Leu Leu Asp Arg Glu Thr
440 445 450
Leu Leu Trp His Asn Ile Thr Val Ile Ala Thr Glu Ile Asn Asn
455 460 465
Pro Lys Gln Ser Ser Arg Val Pro Leu Tyr Ile Lys Val Leu Asp
470 475 480
Val Asn Asp Asn Ala Pro Glu Phe Ala Glu Phe Tyr Glu Thr Phe
485 490 495
Val Cys Glu Lys Ala Lys Ala Asp Gln Leu Ile Gln Thr Leu His
500 505 510
Ala Val Asp Lys Asp Asp Pro Tyr Ser Gly His Gln Phe Ser Phe
515 520 525
Ser Leu Ala Pro Glu Ala Ala Ser Gly Ser Asn Phe Thr Ile Gln
530 535 540
Asp Asn Lys Asp Asn Thr Ala Gly Ile Leu Thr Arg Lys Asn Gly
545 550 555
Tyr Asn Arg His Glu Met Ser Thr Tyr Leu Leu Pro Val Val Ile
560 565 570
Ser Asp Asn Asp Tyr Pro Val Gln Ser Ser Thr Gly Thr Val Thr
575 580 585
Val Arg Val Cys Ala Cys Asp His His Gly Asn Met Gln Ser Cys
590 595 600
His Ala Glu Ala Leu Ile His Pro Thr Gly Leu Ser Thr Gly Ala
605 610 615
Leu Val Ala Ile Leu Leu Cys Ile Val Ile Leu Leu Val Thr Val
620 625 630
Val Leu Phe Ala Ala Leu Arg Arg Gln Arg Lys Lys Glu Pro Leu
635 640 645
Ile Ile Ser Lys Glu Asp Ile Arg Asp Asn Ile Val Ser Tyr Asn
650 655 660
Asp Glu Gly Gly Gly Glu Glu Asp Thr Gln Ala Phe Asp Ile Gly
665 670 675
Thr Leu Arg Asn Pro Glu Ala Ile Glu Asp Asn Lys Leu Arg Arg
680 685 690
Asp Ile Val Pro Glu Ala Leu Phe Leu Pro Arg Arg Thr Pro Thr
695 700 705
Ala Arg Asp Asn Thr Asp Val Arg Asp Phe Ile Asn Gln Arg Leu
710 715 720
Lys Glu Asn Asp Thr Asp Pro Thr Ala Pro Pro Tyr Asp Ser Leu
725 730 735
Ala Thr Tyr Ala Tyr Glu Gly Thr Gly Ser Val Ala Asp Ser Leu
740 745 750
Ser Ser Leu Glu Ser Val Thr Thr Asp Ala Asp Gln Asp Tyr Asp
755 760 765
Tyr Leu Ser Asp Trp Gly Pro Arg Phe Lys Lys Leu Ala Asp Met
770 775 780
Tyr Gly Gly Val Asp Ser Asp Lys Asp Ser
785 790
76
794
PRT
Homo Sapien
76
Met Leu Thr Arg Asn Cys Leu Ser Leu Leu Leu Trp Val Leu Phe
1 5 10 15
Asp Gly Gly Leu Leu Thr Pro Leu Gln Pro Gln Pro Gln Gln Thr
20 25 30
Leu Ala Thr Glu Pro Arg Glu Asn Val Ile His Leu Pro Gly Gln
35 40 45
Arg Ser His Phe Gln Arg Val Lys Arg Gly Trp Val Trp Asn Gln
50 55 60
Phe Phe Val Leu Glu Glu Tyr Val Gly Ser Glu Pro Gln Tyr Val
65 70 75
Gly Lys Leu His Ser Asp Leu Asp Lys Gly Glu Gly Thr Val Lys
80 85 90
Tyr Thr Leu Ser Gly Asp Gly Ala Gly Thr Val Phe Thr Ile Asp
95 100 105
Glu Thr Thr Gly Asp Ile His Ala Ile Arg Ser Leu Asp Arg Glu
110 115 120
Glu Lys Pro Phe Tyr Thr Leu Arg Ala Gln Ala Val Asp Ile Glu
125 130 135
Thr Arg Lys Pro Leu Glu Pro Glu Ser Glu Phe Ile Ile Lys Val
140 145 150
Gln Asp Ile Asn Asp Asn Glu Pro Lys Phe Leu Asp Gly Pro Tyr
155 160 165
Val Ala Thr Val Pro Glu Met Ser Pro Val Gly Ala Tyr Val Leu
170 175 180
Gln Val Lys Ala Thr Asp Ala Asp Asp Pro Thr Tyr Gly Asn Ser
185 190 195
Ala Arg Val Val Tyr Ser Ile Leu Gln Gly Gln Pro Tyr Phe Ser
200 205 210
Ile Asp Pro Lys Thr Gly Val Ile Arg Thr Ala Leu Pro Asn Met
215 220 225
Asp Arg Glu Val Lys Glu Gln Tyr Gln Val Leu Ile Gln Ala Lys
230 235 240
Asp Met Gly Gly Gln Leu Gly Gly Leu Ala Gly Thr Thr Ile Val
245 250 255
Asn Ile Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Arg Phe Pro
260 265 270
Lys Ser Ile Phe His Leu Lys Val Pro Glu Ser Ser Pro Ile Gly
275 280 285
Ser Ala Ile Gly Arg Ile Arg Ala Val Asp Pro Asp Phe Gly Gln
290 295 300
Asn Ala Glu Ile Glu Tyr Asn Ile Val Pro Gly Asp Gly Gly Asn
305 310 315
Leu Phe Asp Ile Val Thr Asp Glu Asp Thr Gln Glu Gly Val Ile
320 325 330
Lys Leu Lys Lys Pro Leu Asp Phe Glu Thr Lys Lys Ala Tyr Thr
335 340 345
Phe Lys Val Glu Ala Ser Asn Leu His Leu Asp His Arg Phe His
350 355 360
Ser Ala Gly Pro Phe Lys Asp Thr Ala Thr Val Lys Ile Ser Val
365 370 375
Leu Asp Val Asp Glu Pro Pro Val Phe Ser Lys Pro Leu Tyr Thr
380 385 390
Met Glu Val Tyr Glu Asp Thr Pro Val Gly Thr Ile Ile Gly Ala
395 400 405
Val Thr Ala Gln Asp Leu Asp Val Gly Ser Gly Ala Val Arg Tyr
410 415 420
Phe Ile Asp Trp Lys Ser Asp Gly Asp Ser Tyr Phe Thr Ile Asp
425 430 435
Gly Asn Glu Gly Thr Ile Ala Thr Asn Glu Leu Leu Asp Arg Glu
440 445 450
Ser Thr Ala Gln Tyr Asn Phe Ser Ile Ile Ala Ser Lys Val Ser
455 460 465
Asn Pro Leu Leu Thr Ser Lys Val Asn Ile Leu Ile Asn Val Leu
470 475 480
Asp Val Asn Glu Phe Pro Pro Glu Ile Ser Val Pro Tyr Glu Thr
485 490 495
Ala Val Cys Glu Asn Ala Lys Pro Gly Gln Ile Ile Gln Ile Val
500 505 510
Ser Ala Ala Asp Arg Asp Leu Ser Pro Ala Gly Gln Gln Phe Ser
515 520 525
Phe Arg Leu Ser Pro Glu Ala Ala Ile Lys Pro Asn Phe Thr Val
530 535 540
Arg Asp Phe Arg Asn Asn Thr Ala Gly Ile Glu Thr Arg Arg Asn
545 550 555
Gly Tyr Ser Arg Arg Gln Gln Glu Leu Tyr Phe Leu Pro Val Val
560 565 570
Ile Glu Asp Ser Ser Tyr Pro Val Gln Ser Ser Thr Asn Thr Met
575 580 585
Thr Ile Arg Val Cys Arg Cys Asp Ser Asp Gly Thr Ile Leu Ser
590 595 600
Cys Asn Val Glu Ala Ile Phe Leu Pro Val Gly Leu Ser Thr Gly
605 610 615
Ala Leu Ile Ala Ile Leu Leu Cys Ile Val Ile Leu Leu Ala Ile
620 625 630
Val Val Leu Tyr Val Ala Leu Arg Arg Gln Lys Lys Lys His Thr
635 640 645
Leu Met Thr Ser Lys Glu Asp Ile Arg Asp Asn Val Ile His Tyr
650 655 660
Asp Asp Glu Gly Gly Gly Glu Glu Asp Thr Gln Ala Phe Asp Ile
665 670 675
Gly Ala Leu Arg Asn Pro Lys Val Ile Glu Glu Asn Lys Ile Arg
680 685 690
Arg Asp Ile Lys Pro Asp Ser Leu Cys Leu Pro Arg Gln Arg Pro
695 700 705
Pro Met Glu Asp Asn Thr Asp Ile Arg Asp Phe Ile His Gln Arg
710 715 720
Leu Gln Glu Asn Asp Val Asp Pro Thr Ala Pro Pro Ile Asp Ser
725 730 735
Leu Ala Thr Tyr Ala Tyr Glu Gly Ser Gly Ser Val Ala Glu Ser
740 745 750
Leu Ser Ser Ile Asp Ser Leu Thr Thr Glu Ala Asp Gln Asp Tyr
755 760 765
Asp Tyr Leu Thr Asp Trp Gly Pro Arg Phe Lys Val Leu Ala Asp
770 775 780
Met Phe Gly Glu Glu Glu Ser Tyr Asn Pro Asp Lys Val Thr
785 790
77
141
PRT
Homo Sapien
77
Met Ala Arg Pro Leu Cys Thr Leu Leu Leu Leu Met Ala Thr Leu
1 5 10 15
Ala Gly Ala Leu Ala Ser Ser Ser Lys Glu Glu Asn Arg Ile Ile
20 25 30
Pro Gly Gly Ile Tyr Asp Ala Asp Leu Asn Asp Glu Trp Val Gln
35 40 45
Arg Ala Leu His Phe Ala Ile Ser Glu Tyr Asn Lys Ala Thr Glu
50 55 60
Asp Glu Tyr Tyr Arg Arg Pro Leu Gln Val Leu Arg Ala Arg Glu
65 70 75
Gln Thr Phe Gly Gly Val Asn Tyr Phe Phe Asp Val Glu Val Gly
80 85 90
Arg Thr Ile Cys Thr Lys Ser Gln Pro Asn Leu Asp Thr Cys Ala
95 100 105
Phe His Glu Gln Pro Glu Leu Gln Lys Lys Gln Leu Cys Ser Phe
110 115 120
Glu Ile Tyr Glu Val Pro Trp Glu Asp Arg Met Ser Leu Val Asn
125 130 135
Ser Arg Cys Gln Glu Ala
140
78
466
PRT
Homo Sapien
78
Met Thr Thr Ser Pro Ile Leu Gln Leu Leu Leu Arg Leu Ser Leu
1 5 10 15
Cys Gly Leu Leu Leu Gln Arg Ala Glu Thr Gly Ser Lys Gly Gln
20 25 30
Thr Ala Gly Glu Leu Tyr Gln Arg Trp Glu Arg Tyr Arg Arg Glu
35 40 45
Cys Gln Glu Thr Leu Ala Ala Ala Glu Pro Pro Ser Gly Leu Ala
50 55 60
Cys Asn Gly Ser Phe Asp Met Tyr Val Cys Trp Asp Tyr Ala Ala
65 70 75
Pro Asn Ala Thr Ala Arg Ala Ser Cys Pro Trp Tyr Leu Pro Trp
80 85 90
His His His Val Ala Ala Gly Phe Val Leu Arg Gln Cys Gly Ser
95 100 105
Asp Gly Gln Trp Gly Leu Trp Arg Asp His Thr Gln Cys Glu Asn
110 115 120
Pro Glu Lys Asn Glu Ala Phe Leu Asp Gln Arg Leu Ile Leu Glu
125 130 135
Arg Leu Gln Val Met Tyr Thr Val Gly Tyr Ser Leu Ser Leu Ala
140 145 150
Thr Leu Leu Leu Ala Leu Leu Ile Leu Ser Leu Phe Arg Arg Leu
155 160 165
His Cys Thr Arg Asn Tyr Ile His Ile Asn Leu Phe Thr Ser Phe
170 175 180
Met Leu Arg Ala Ala Ala Ile Leu Ser Arg Asp Arg Leu Leu Pro
185 190 195
Arg Pro Gly Pro Tyr Leu Gly Asp Gln Ala Leu Ala Leu Trp Asn
200 205 210
Gln Ala Leu Ala Ala Cys Arg Thr Ala Gln Ile Val Thr Gln Tyr
215 220 225
Cys Val Gly Ala Asn Tyr Thr Trp Leu Leu Val Glu Gly Val Tyr
230 235 240
Leu His Ser Leu Leu Val Leu Val Gly Gly Ser Glu Glu Gly His
245 250 255
Phe Arg Tyr Tyr Leu Leu Leu Gly Trp Gly Ala Pro Ala Leu Phe
260 265 270
Val Ile Pro Trp Val Ile Val Arg Tyr Leu Tyr Glu Asn Thr Gln
275 280 285
Cys Trp Glu Arg Asn Glu Val Lys Ala Ile Trp Trp Ile Ile Arg
290 295 300
Thr Pro Ile Leu Met Thr Ile Leu Ile Asn Phe Leu Ile Phe Ile
305 310 315
Arg Ile Leu Gly Ile Leu Leu Ser Lys Leu Arg Thr Arg Gln Met
320 325 330
Arg Cys Arg Asp Tyr Arg Leu Arg Leu Ala Arg Ser Thr Leu Thr
335 340 345
Leu Val Pro Leu Leu Gly Val His Glu Val Val Phe Ala Pro Val
350 355 360
Thr Glu Glu Gln Ala Arg Gly Ala Leu Arg Phe Ala Lys Leu Gly
365 370 375
Phe Glu Ile Phe Leu Ser Ser Phe Gln Gly Phe Leu Val Ser Val
380 385 390
Leu Tyr Cys Phe Ile Asn Lys Glu Val Gln Ser Glu Ile Arg Arg
395 400 405
Gly Trp His His Cys Arg Leu Arg Arg Ser Leu Gly Glu Glu Gln
410 415 420
Arg Gln Leu Pro Glu Arg Ala Phe Arg Ala Leu Pro Ser Gly Ser
425 430 435
Gly Pro Gly Glu Val Pro Thr Ser Arg Gly Leu Ser Ser Gly Thr
440 445 450
Leu Pro Gly Pro Gly Asn Glu Ala Ser Arg Glu Leu Glu Ser Tyr
455 460 465
Cys
79
506
PRT
Homo Sapien
79
Met Leu Ser Lys Val Leu Pro Val Leu Leu Gly Ile Leu Leu Ile
1 5 10 15
Leu Gln Ser Arg Val Glu Gly Pro Gln Thr Glu Ser Lys Asn Glu
20 25 30
Ala Ser Ser Arg Asp Val Val Tyr Gly Pro Gln Pro Gln Pro Leu
35 40 45
Glu Asn Gln Leu Leu Ser Glu Glu Thr Lys Ser Thr Glu Thr Glu
50 55 60
Thr Gly Ser Arg Val Gly Lys Leu Pro Glu Ala Ser Arg Ile Leu
65 70 75
Asn Thr Ile Leu Ser Asn Tyr Asp His Lys Leu Arg Pro Gly Ile
80 85 90
Gly Glu Lys Pro Thr Val Val Thr Val Glu Ile Ala Val Asn Ser
95 100 105
Leu Gly Pro Leu Ser Ile Leu Asp Met Glu Tyr Thr Ile Asp Ile
110 115 120
Ile Phe Ser Gln Thr Trp Tyr Asp Glu Arg Leu Cys Tyr Asn Asp
125 130 135
Thr Phe Glu Ser Leu Val Leu Asn Gly Asn Val Val Ser Gln Leu
140 145 150
Trp Ile Pro Asp Thr Phe Phe Arg Asn Ser Lys Arg Thr His Glu
155 160 165
His Glu Ile Thr Met Pro Asn Gln Met Val Arg Ile Tyr Lys Asp
170 175 180
Gly Lys Val Leu Tyr Thr Ile Arg Met Thr Ile Asp Ala Gly Cys
185 190 195
Ser Leu His Met Leu Arg Phe Pro Met Asp Ser His Ser Cys Pro
200 205 210
Leu Ser Phe Ser Ser Phe Ser Tyr Pro Glu Asn Glu Met Ile Tyr
215 220 225
Lys Trp Glu Asn Phe Lys Leu Glu Ile Asn Glu Lys Asn Ser Trp
230 235 240
Lys Leu Phe Gln Phe Asp Phe Thr Gly Val Ser Asn Lys Thr Glu
245 250 255
Ile Ile Thr Thr Pro Val Gly Asp Phe Met Val Met Thr Ile Phe
260 265 270
Phe Asn Val Ser Arg Arg Phe Gly Tyr Val Ala Phe Gln Asn Tyr
275 280 285
Val Pro Ser Ser Val Thr Thr Met Leu Ser Trp Val Ser Phe Trp
290 295 300
Ile Lys Thr Glu Ser Ala Pro Ala Arg Thr Ser Leu Gly Ile Thr
305 310 315
Ser Val Leu Thr Met Thr Thr Leu Gly Thr Phe Ser Arg Lys Asn
320 325 330
Phe Pro Arg Val Ser Tyr Ile Thr Ala Leu Asp Phe Tyr Ile Ala
335 340 345
Ile Cys Phe Val Phe Cys Phe Cys Ala Leu Leu Glu Phe Ala Val
350 355 360
Leu Asn Phe Leu Ile Tyr Asn Gln Thr Lys Ala His Ala Ser Pro
365 370 375
Lys Leu Arg His Pro Arg Ile Asn Ser Arg Ala His Ala Arg Thr
380 385 390
Arg Ala Arg Ser Arg Ala Cys Ala Arg Gln His Gln Glu Ala Phe
395 400 405
Val Cys Gln Ile Val Thr Thr Glu Gly Ser Asp Gly Glu Glu Arg
410 415 420
Pro Ser Cys Ser Ala Gln Gln Pro Pro Ser Pro Gly Ser Pro Glu
425 430 435
Gly Pro Arg Ser Leu Cys Ser Lys Leu Ala Cys Cys Glu Trp Cys
440 445 450
Lys Arg Phe Lys Lys Tyr Phe Cys Met Val Pro Asp Cys Glu Gly
455 460 465
Ser Thr Trp Gln Gln Gly Arg Leu Cys Ile His Val Tyr Arg Leu
470 475 480
Asp Asn Tyr Ser Arg Val Val Phe Pro Val Thr Phe Phe Phe Phe
485 490 495
Asn Val Leu Tyr Trp Leu Val Cys Leu Asn Leu
500 505
80
1212
PRT
Homo Sapien
80
Met Glu Pro Arg Pro Thr Ala Pro Ser Ser Gly Ala Pro Gly Leu
1 5 10 15
Ala Gly Val Gly Glu Thr Pro Ser Ala Ala Ala Leu Ala Ala Ala
20 25 30
Arg Val Glu Leu Pro Gly Thr Ala Val Pro Ser Val Pro Glu Asp
35 40 45
Ala Ala Pro Ala Ser Arg Asp Gly Gly Gly Val Arg Asp Glu Gly
50 55 60
Pro Ala Ala Ala Gly Asp Gly Leu Gly Arg Pro Leu Gly Pro Thr
65 70 75
Pro Ser Gln Ser Arg Phe Gln Val Asp Leu Val Ser Glu Asn Ala
80 85 90
Gly Arg Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
95 100 105
Ala Ala Gly Ala Gly Ala Gly Ala Lys Gln Thr Pro Ala Asp Gly
110 115 120
Glu Ala Ser Gly Glu Ser Glu Pro Ala Lys Gly Ser Glu Glu Ala
125 130 135
Lys Gly Arg Phe Arg Val Asn Phe Val Asp Pro Ala Ala Ser Ser
140 145 150
Ser Ala Glu Asp Ser Leu Ser Asp Ala Ala Gly Val Gly Val Asp
155 160 165
Gly Pro Asn Val Ser Phe Gln Asn Gly Gly Asp Thr Val Leu Ser
170 175 180
Glu Gly Ser Ser Leu His Ser Gly Gly Gly Gly Gly Ser Gly His
185 190 195
His Gln His Tyr Tyr Tyr Asp Thr His Thr Asn Thr Tyr Tyr Leu
200 205 210
Arg Thr Phe Gly His Asn Thr Met Asp Ala Val Pro Arg Ile Asp
215 220 225
His Tyr Arg His Thr Ala Ala Gln Leu Gly Glu Lys Leu Leu Arg
230 235 240
Pro Ser Leu Ala Glu Leu His Asp Glu Leu Glu Lys Glu Pro Phe
245 250 255
Glu Asp Gly Phe Ala Asn Gly Glu Glu Ser Thr Pro Thr Arg Asp
260 265 270
Ala Val Val Thr Tyr Thr Ala Glu Ser Lys Gly Val Val Lys Phe
275 280 285
Gly Trp Ile Lys Gly Val Leu Val Arg Cys Met Leu Asn Ile Trp
290 295 300
Gly Val Met Leu Phe Ile Arg Leu Ser Trp Ile Val Gly Gln Ala
305 310 315
Gly Ile Gly Leu Ser Val Leu Val Ile Met Met Ala Thr Val Val
320 325 330
Thr Thr Ile Thr Gly Leu Ser Thr Ser Ala Ile Ala Thr Asn Gly
335 340 345
Phe Val Arg Gly Gly Gly Ala Tyr Tyr Leu Ile Ser Arg Ser Leu
350 355 360
Gly Pro Glu Phe Gly Gly Ala Ile Gly Leu Ile Phe Ala Phe Ala
365 370 375
Asn Ala Val Ala Val Ala Met Tyr Val Val Gly Phe Ala Glu Thr
380 385 390
Val Val Glu Leu Leu Lys Glu His Ser Ile Leu Met Ile Asp Glu
395 400 405
Ile Asn Asp Ile Arg Ile Ile Gly Ala Ile Thr Val Val Ile Leu
410 415 420
Leu Gly Ile Ser Val Ala Gly Met Glu Trp Glu Ala Lys Ala Gln
425 430 435
Ile Val Leu Leu Val Ile Leu Leu Leu Ala Ile Gly Asp Phe Val
440 445 450
Ile Gly Thr Phe Ile Pro Leu Glu Ser Lys Lys Pro Lys Gly Phe
455 460 465
Phe Gly Tyr Lys Ser Glu Ile Phe Asn Glu Asn Phe Gly Pro Asp
470 475 480
Phe Arg Glu Glu Glu Thr Phe Phe Ser Val Phe Ala Ile Phe Phe
485 490 495
Pro Ala Ala Thr Gly Ile Leu Ala Gly Ala Asn Ile Ser Gly Asp
500 505 510
Leu Ala Asp Pro Gln Ser Ala Ile Pro Lys Gly Thr Leu Leu Ala
515 520 525
Ile Leu Ile Thr Thr Leu Val Tyr Val Gly Ile Ala Val Ser Val
530 535 540
Gly Ser Cys Val Val Arg Asp Ala Thr Gly Asn Val Asn Asp Thr
545 550 555
Ile Val Thr Glu Leu Thr Asn Cys Thr Ser Ala Ala Cys Lys Leu
560 565 570
Asn Phe Asp Phe Ser Ser Cys Glu Ser Ser Pro Cys Ser Tyr Gly
575 580 585
Leu Met Asn Asn Phe Gln Val Met Ser Met Val Ser Gly Phe Thr
590 595 600
Pro Leu Ile Ser Ala Gly Ile Phe Ser Ala Thr Leu Ser Ser Ala
605 610 615
Leu Ala Ser Leu Val Ser Ala Pro Lys Ile Phe Gln Ala Leu Cys
620 625 630
Lys Asp Asn Ile Tyr Pro Ala Phe Gln Met Phe Ala Lys Gly Tyr
635 640 645
Gly Lys Asn Asn Glu Pro Leu Arg Gly Tyr Ile Leu Thr Phe Leu
650 655 660
Ile Ala Leu Gly Phe Ile Leu Ile Ala Glu Leu Asn Val Ile Ala
665 670 675
Pro Ile Ile Ser Asn Phe Phe Leu Ala Ser Tyr Ala Leu Ile Asn
680 685 690
Phe Ser Val Phe His Ala Ser Leu Ala Lys Ser Pro Gly Trp Arg
695 700 705
Pro Ala Phe Lys Tyr Tyr Asn Met Trp Ile Ser Leu Leu Gly Ala
710 715 720
Ile Leu Cys Cys Ile Val Met Phe Val Ile Asn Trp Trp Ala Ala
725 730 735
Leu Leu Thr Tyr Val Ile Val Leu Gly Leu Tyr Ile Tyr Val Thr
740 745 750
Tyr Lys Lys Pro Asp Val Asn Trp Gly Ser Ser Thr Gln Ala Leu
755 760 765
Thr Tyr Leu Asn Ala Leu Gln His Ser Ile Arg Leu Ser Gly Val
770 775 780
Glu Asp His Val Lys Asn Phe Arg Pro Gln Cys Leu Val Met Thr
785 790 795
Gly Ala Pro Asn Ser Arg Pro Ala Leu Leu His Leu Val His Asp
800 805 810
Phe Thr Lys Asn Val Gly Leu Met Ile Cys Gly His Val His Met
815 820 825
Gly Pro Arg Arg Gln Ala Met Lys Glu Met Ser Ile Asp Gln Ala
830 835 840
Lys Tyr Gln Arg Trp Leu Ile Lys Asn Lys Met Lys Ala Phe Tyr
845 850 855
Ala Pro Val His Ala Asp Asp Leu Arg Glu Gly Ala Gln Tyr Leu
860 865 870
Met Gln Ala Ala Gly Leu Gly Arg Met Lys Pro Asn Thr Leu Val
875 880 885
Leu Gly Phe Lys Lys Asp Trp Leu Gln Ala Asp Met Arg Asp Val
890 895 900
Asp Met Tyr Ile Asn Leu Phe His Asp Ala Phe Asp Ile Gln Tyr
905 910 915
Gly Val Val Val Ile Arg Leu Lys Glu Gly Leu Asp Ile Ser His
920 925 930
Leu Gln Gly Gln Glu Glu Leu Leu Ser Ser Gln Glu Lys Ser Pro
935 940 945
Gly Thr Lys Asp Val Val Val Ser Val Glu Tyr Ser Lys Lys Ser
950 955 960
Asp Leu Asp Thr Ser Lys Pro Leu Ser Glu Lys Pro Ile Thr His
965 970 975
Lys Val Glu Glu Glu Asp Gly Lys Thr Ala Thr Gln Pro Leu Leu
980 985 990
Lys Lys Glu Ser Lys Gly Pro Ile Val Pro Leu Asn Val Ala Asp
995 1000 1005
Gln Lys Leu Leu Glu Ala Ser Thr Gln Phe Gln Lys Lys Gln Gly
1010 1015 1020
Lys Asn Thr Ile Asp Val Trp Trp Leu Phe Asp Asp Gly Gly Leu
1025 1030 1035
Thr Leu Leu Ile Pro Tyr Leu Leu Thr Thr Lys Lys Lys Trp Lys
1040 1045 1050
Asp Cys Lys Ile Arg Val Phe Ile Gly Gly Lys Ile Asn Arg Ile
1055 1060 1065
Asp His Asp Arg Arg Ala Met Ala Thr Leu Leu Ser Lys Phe Arg
1070 1075 1080
Ile Asp Phe Ser Asp Ile Met Val Leu Gly Asp Ile Asn Thr Lys
1085 1090 1095
Pro Lys Lys Glu Asn Ile Ile Ala Phe Glu Glu Ile Ile Glu Pro
1100 1105 1110
Tyr Arg Leu His Glu Asp Asp Lys Glu Gln Asp Ile Ala Asp Lys
1115 1120 1125
Met Lys Glu Asp Glu Pro Trp Arg Ile Thr Asp Asn Glu Leu Glu
1130 1135 1140
Leu Tyr Lys Thr Lys Thr Tyr Arg Gln Ile Arg Leu Asn Glu Leu
1145 1150 1155
Leu Lys Glu His Ser Ser Thr Ala Asn Ile Ile Val Met Ser Leu
1160 1165 1170
Pro Val Ala Arg Lys Gly Ala Val Ser Ser Ala Leu Tyr Met Ala
1175 1180 1185
Trp Leu Glu Ala Leu Ser Lys Asp Leu Pro Pro Ile Leu Leu Val
1190 1195 1200
Arg Gly Asn His Gln Ser Val Leu Thr Phe Tyr Ser
1205 1210
81
674
PRT
Homo Sapien
81
Met Ala Thr Ala Val Ser Arg Pro Cys Ala Gly Arg Ser Arg Asp
1 5 10 15
Ile Leu Trp Arg Val Leu Gly Trp Arg Ile Val Ala Ser Ile Val
20 25 30
Trp Ser Val Leu Phe Leu Pro Ile Cys Thr Thr Val Phe Ile Ile
35 40 45
Phe Ser Arg Ile Asp Leu Phe His Pro Ile Gln Trp Leu Ser Asp
50 55 60
Ser Phe Ser Asp Leu Tyr Ser Ser Tyr Val Ile Phe Tyr Phe Leu
65 70 75
Leu Leu Ser Val Val Ile Ile Ile Ile Ser Ile Phe Asn Val Glu
80 85 90
Phe Tyr Ala Val Val Pro Ser Ile Pro Cys Ser Arg Leu Ala Leu
95 100 105
Ile Gly Lys Ile Ile His Pro Gln Gln Leu Met His Ser Phe Ile
110 115 120
His Ala Ala Met Gly Met Val Met Ala Trp Cys Ala Ala Val Ile
125 130 135
Thr Gln Gly Gln Tyr Ser Phe Leu Val Val Pro Cys Thr Gly Thr
140 145 150
Asn Ser Phe Gly Ser Pro Ala Ala Gln Thr Cys Leu Asn Glu Tyr
155 160 165
His Leu Phe Phe Leu Leu Thr Gly Ala Phe Met Gly Tyr Ser Tyr
170 175 180
Ser Leu Leu Tyr Phe Val Asn Asn Met Asn Tyr Leu Pro Phe Pro
185 190 195
Ile Ile Gln Gln Tyr Lys Phe Leu Arg Phe Arg Arg Ser Leu Leu
200 205 210
Leu Leu Val Lys His Ser Cys Val Glu Ser Leu Phe Leu Val Arg
215 220 225
Asn Phe Cys Ile Leu Tyr Tyr Phe Leu Gly Tyr Ile Pro Lys Ala
230 235 240
Trp Ile Ser Thr Ala Met Asn Leu His Ile Asp Glu Gln Val His
245 250 255
Arg Pro Leu Asp Thr Val Ser Gly Leu Leu Asn Leu Ser Leu Leu
260 265 270
Tyr His Val Trp Leu Cys Gly Val Phe Leu Leu Thr Thr Trp Tyr
275 280 285
Val Ser Trp Ile Leu Phe Lys Ile Tyr Ala Thr Glu Ala His Val
290 295 300
Phe Pro Val Gln Pro Pro Phe Ala Glu Gly Ser Asp Glu Cys Leu
305 310 315
Pro Lys Val Leu Asn Ser Asn Pro Pro Pro Ile Ile Lys Tyr Leu
320 325 330
Ala Leu Gln Asp Leu Met Leu Leu Ser Gln Tyr Ser Pro Ser Arg
335 340 345
Arg Gln Glu Val Phe Ser Leu Ser Gln Pro Gly Gly His Pro His
350 355 360
Asn Trp Thr Ala Ile Ser Arg Glu Cys Leu Asn Leu Leu Asn Gly
365 370 375
Met Thr Gln Lys Leu Ile Leu Tyr Gln Glu Ala Ala Ala Thr Asn
380 385 390
Gly Arg Val Ser Ser Ser Tyr Pro Val Glu Pro Lys Lys Leu Asn
395 400 405
Ser Pro Glu Glu Thr Ala Phe Gln Thr Pro Lys Ser Ser Gln Met
410 415 420
Pro Arg Pro Ser Val Pro Pro Leu Val Lys Thr Ser Leu Phe Ser
425 430 435
Ser Lys Leu Ser Thr Pro Asp Val Val Ser Pro Phe Gly Thr Pro
440 445 450
Phe Gly Ser Ser Val Met Asn Arg Met Ala Gly Ile Phe Asp Val
455 460 465
Asn Thr Cys Tyr Gly Ser Pro Gln Ser Pro Gln Leu Ile Arg Arg
470 475 480
Gly Pro Arg Leu Trp Thr Ser Ala Ser Asp Gln Gln Met Thr Glu
485 490 495
Phe Ser Asn Pro Ser Pro Ser Thr Ser Ile Ser Ala Glu Gly Lys
500 505 510
Thr Met Arg Gln Pro Ser Val Ile Tyr Ser Trp Ile Gln Asn Lys
515 520 525
Arg Glu Gln Ile Lys Asn Phe Leu Ser Lys Arg Val Leu Ile Met
530 535 540
Tyr Phe Phe Ser Lys His Pro Glu Ala Ser Ile Gln Ala Val Phe
545 550 555
Ser Asp Ala Gln Met His Ile Trp Ala Leu Glu Gly Leu Ser His
560 565 570
Leu Val Ala Ala Ser Phe Thr Glu Asp Arg Phe Gly Val Val Gln
575 580 585
Thr Thr Leu Pro Ala Ile Leu Asn Thr Leu Leu Thr Leu Gln Glu
590 595 600
Ala Val Asp Lys Tyr Phe Lys Leu Pro His Ala Ser Ser Lys Pro
605 610 615
Pro Arg Ile Ser Gly Ser Leu Val Asp Thr Ser Tyr Lys Thr Leu
620 625 630
Arg Phe Ala Phe Arg Ala Ser Leu Lys Thr Ala Ile Tyr Arg Ile
635 640 645
Thr Thr Thr Phe Gly Glu His Leu Asn Ala Val Gln Ala Ser Ala
650 655 660
Glu His Gln Lys Arg Leu Gln Gln Phe Leu Glu Phe Lys Glu
665 670
82
1321
PRT
Homo Sapien
82
Met Gly Ala Pro Phe Val Trp Ala Leu Gly Leu Leu Met Leu Gln
1 5 10 15
Met Leu Leu Phe Val Ala Gly Glu Gln Gly Thr Gln Asp Ile Thr
20 25 30
Asp Ala Ser Glu Arg Gly Leu His Met Gln Lys Leu Gly Ser Gly
35 40 45
Ser Val Gln Ala Ala Leu Ala Glu Leu Val Ala Leu Pro Cys Leu
50 55 60
Phe Thr Leu Gln Pro Arg Pro Ser Ala Ala Arg Asp Ala Pro Arg
65 70 75
Ile Lys Trp Thr Lys Val Arg Thr Ala Ser Gly Gln Arg Gln Asp
80 85 90
Leu Pro Ile Leu Val Ala Lys Asp Asn Val Val Arg Val Ala Lys
95 100 105
Ser Trp Gln Gly Arg Val Ser Leu Pro Ser Tyr Pro Arg Arg Arg
110 115 120
Ala Asn Ala Thr Leu Leu Leu Gly Pro Leu Arg Ala Ser Asp Ser
125 130 135
Gly Leu Tyr Arg Cys Gln Val Val Arg Gly Ile Glu Asp Glu Gln
140 145 150
Asp Leu Val Pro Leu Glu Val Thr Gly Val Val Phe His Tyr Arg
155 160 165
Ser Ala Arg Asp Arg Tyr Ala Leu Thr Phe Ala Glu Ala Gln Glu
170 175 180
Ala Cys Arg Leu Ser Ser Ala Ile Ile Ala Ala Pro Arg His Leu
185 190 195
Gln Ala Ala Phe Glu Asp Gly Phe Asp Asn Cys Asp Ala Gly Trp
200 205 210
Leu Ser Asp Arg Thr Val Arg Tyr Pro Ile Thr Gln Ser Arg Pro
215 220 225
Gly Cys Tyr Gly Asp Arg Ser Ser Leu Pro Gly Val Arg Ser Tyr
230 235 240
Gly Arg Arg Asn Pro Gln Glu Leu Tyr Asp Val Tyr Cys Phe Ala
245 250 255
Arg Glu Leu Gly Gly Glu Val Phe Tyr Val Gly Pro Ala Arg Arg
260 265 270
Leu Thr Leu Ala Gly Ala Arg Ala Gln Cys Arg Arg Gln Gly Ala
275 280 285
Ala Leu Ala Ser Val Gly Gln Leu His Leu Ala Trp His Glu Gly
290 295 300
Leu Asp Gln Cys Asp Pro Gly Trp Leu Ala Asp Gly Ser Val Arg
305 310 315
Tyr Pro Ile Gln Thr Pro Arg Arg Arg Cys Gly Gly Pro Ala Pro
320 325 330
Gly Val Arg Thr Val Tyr Arg Phe Ala Asn Arg Thr Gly Phe Pro
335 340 345
Ser Pro Ala Glu Arg Phe Asp Ala Tyr Cys Phe Arg Ala His His
350 355 360
Pro Thr Ser Gln His Gly Asp Leu Glu Thr Pro Ser Ser Gly Asp
365 370 375
Glu Gly Glu Ile Leu Ser Ala Glu Gly Pro Pro Val Arg Glu Leu
380 385 390
Glu Pro Thr Leu Glu Glu Glu Glu Val Val Thr Pro Asp Phe Gln
395 400 405
Glu Pro Leu Val Ser Ser Gly Glu Glu Glu Thr Leu Ile Leu Glu
410 415 420
Glu Lys Gln Glu Ser Gln Gln Thr Leu Ser Pro Thr Pro Gly Asp
425 430 435
Pro Met Leu Ala Ser Trp Pro Thr Gly Glu Val Trp Leu Ser Thr
440 445 450
Val Ala Pro Ser Pro Ser Asp Met Gly Ala Gly Thr Ala Ala Ser
455 460 465
Ser His Thr Glu Val Ala Pro Thr Asp Pro Met Pro Arg Arg Arg
470 475 480
Gly Arg Phe Lys Gly Leu Asn Gly Arg Tyr Phe Gln Gln Gln Glu
485 490 495
Pro Glu Pro Gly Leu Gln Gly Gly Met Glu Ala Ser Ala Gln Pro
500 505 510
Pro Thr Ser Glu Ala Ala Val Asn Gln Met Glu Pro Pro Leu Ala
515 520 525
Met Ala Val Thr Glu Met Leu Gly Ser Gly Gln Ser Arg Ser Pro
530 535 540
Trp Ala Asp Leu Thr Asn Glu Val Asp Met Pro Gly Ala Gly Ser
545 550 555
Ala Gly Gly Lys Ser Ser Pro Glu Pro Trp Leu Trp Pro Pro Thr
560 565 570
Met Val Pro Pro Ser Ile Ser Gly His Ser Arg Ala Pro Val Leu
575 580 585
Glu Leu Glu Lys Ala Glu Gly Pro Ser Ala Arg Pro Ala Thr Pro
590 595 600
Asp Leu Phe Trp Ser Pro Leu Glu Ala Thr Val Ser Ala Pro Ser
605 610 615
Pro Ala Pro Trp Glu Ala Phe Pro Val Ala Thr Ser Pro Asp Leu
620 625 630
Pro Met Met Ala Met Leu Arg Gly Pro Lys Glu Trp Met Leu Pro
635 640 645
His Pro Thr Pro Ile Ser Thr Glu Ala Asn Arg Val Glu Ala His
650 655 660
Gly Glu Ala Thr Ala Thr Ala Pro Pro Ser Pro Ala Ala Glu Thr
665 670 675
Lys Val Tyr Ser Leu Pro Leu Ser Leu Thr Pro Thr Gly Gln Gly
680 685 690
Gly Glu Ala Met Pro Thr Thr Pro Glu Ser Pro Arg Ala Asp Phe
695 700 705
Arg Glu Thr Gly Glu Thr Ser Pro Ala Gln Val Asn Lys Ala Glu
710 715 720
His Ser Ser Ser Ser Pro Trp Pro Ser Val Asn Arg Asn Val Ala
725 730 735
Val Gly Phe Val Pro Thr Glu Thr Ala Thr Glu Pro Thr Gly Leu
740 745 750
Arg Gly Ile Pro Gly Ser Glu Ser Gly Val Phe Asp Thr Ala Glu
755 760 765
Ser Pro Thr Ser Gly Leu Gln Ala Thr Val Asp Glu Val Gln Asp
770 775 780
Pro Trp Pro Ser Val Tyr Ser Lys Gly Leu Asp Ala Ser Ser Pro
785 790 795
Ser Ala Pro Leu Gly Ser Pro Gly Val Phe Leu Val Pro Lys Val
800 805 810
Thr Pro Asn Leu Glu Pro Trp Val Ala Thr Asp Glu Gly Pro Thr
815 820 825
Val Asn Pro Met Asp Ser Thr Val Thr Pro Ala Pro Ser Asp Ala
830 835 840
Ser Gly Ile Trp Glu Pro Gly Ser Gln Val Phe Glu Glu Ala Glu
845 850 855
Ser Thr Thr Leu Ser Pro Gln Val Ala Leu Asp Thr Ser Ile Val
860 865 870
Thr Pro Leu Thr Thr Leu Glu Gln Gly Asp Lys Val Gly Val Pro
875 880 885
Ala Met Ser Thr Leu Gly Ser Ser Ser Ser Gln Pro His Pro Glu
890 895 900
Pro Glu Asp Gln Val Glu Thr Gln Gly Thr Ser Gly Ala Ser Val
905 910 915
Pro Pro His Gln Ser Ser Pro Leu Gly Lys Pro Ala Val Pro Pro
920 925 930
Gly Thr Pro Thr Ala Ala Ser Val Gly Glu Ser Ala Ser Val Ser
935 940 945
Ser Gly Glu Pro Thr Val Pro Trp Asp Pro Ser Ser Thr Leu Leu
950 955 960
Pro Val Thr Leu Gly Ile Glu Asp Phe Glu Leu Glu Val Leu Ala
965 970 975
Gly Ser Pro Gly Val Glu Ser Phe Trp Glu Glu Val Ala Ser Gly
980 985 990
Glu Glu Pro Ala Leu Pro Gly Thr Pro Met Asn Ala Gly Ala Glu
995 1000 1005
Glu Val His Ser Asp Pro Cys Glu Asn Asn Pro Cys Leu His Gly
1010 1015 1020
Gly Thr Cys Asn Ala Asn Gly Thr Met Tyr Gly Cys Ser Cys Asp
1025 1030 1035
Gln Gly Phe Ala Gly Glu Asn Cys Glu Ile Asp Ile Asp Asp Cys
1040 1045 1050
Leu Cys Ser Pro Cys Glu Asn Gly Gly Thr Cys Ile Asp Glu Val
1055 1060 1065
Asn Gly Phe Val Cys Leu Cys Leu Pro Ser Tyr Gly Gly Ser Phe
1070 1075 1080
Cys Glu Lys Asp Thr Glu Gly Cys Asp Arg Gly Trp His Lys Phe
1085 1090 1095
Gln Gly His Cys Tyr Arg Tyr Phe Ala His Arg Arg Ala Trp Glu
1100 1105 1110
Asp Ala Glu Lys Asp Cys Arg Arg Arg Ser Gly His Leu Thr Ser
1115 1120 1125
Val His Ser Pro Glu Glu His Ser Phe Ile Asn Ser Phe Gly His
1130 1135 1140
Glu Asn Thr Trp Ile Gly Leu Asn Asp Arg Ile Val Glu Arg Asp
1145 1150 1155
Phe Gln Trp Thr Asp Asn Thr Gly Leu Gln Phe Glu Asn Trp Arg
1160 1165 1170
Glu Asn Gln Pro Asp Asn Phe Phe Ala Gly Gly Glu Asp Cys Val
1175 1180 1185
Val Met Val Ala His Glu Ser Gly Arg Trp Asn Asp Val Pro Cys
1190 1195 1200
Asn Tyr Asn Leu Pro Tyr Val Cys Lys Lys Gly Thr Val Leu Cys
1205 1210 1215
Gly Pro Pro Pro Ala Val Glu Asn Ala Ser Leu Ile Gly Ala Arg
1220 1225 1230
Lys Ala Lys Asn Asn Val His Ala Thr Val Arg Tyr Gln Cys Asn
1235 1240 1245
Glu Gly Phe Ala Gln His His Val Val Thr Ile Arg Cys Arg Ser
1250 1255 1260
Asn Gly Lys Trp Asp Arg Pro Gln Ile Val Cys Thr Lys Pro Arg
1265 1270 1275
Arg Ser His Arg Met Arg Gly His His His His His Gln His His
1280 1285 1290
His Gln His His His His Lys Ser Arg Lys Glu Arg Arg Lys His
1295 1300 1305
Lys Lys His Pro Thr Glu Asp Trp Glu Lys Asp Glu Gly Asn Phe
1310 1315 1320
Cys
83
696
PRT
Homo Sapien
83
Met Lys Phe Ala Glu His Leu Ser Ala His Ile Thr Pro Glu Trp
1 5 10 15
Arg Lys Gln Tyr Ile Gln Tyr Glu Ala Phe Lys Asp Met Leu Tyr
20 25 30
Ser Ala Gln Asp Gln Ala Pro Ser Val Glu Val Thr Asp Glu Asp
35 40 45
Thr Val Lys Arg Tyr Phe Ala Lys Phe Glu Glu Lys Phe Phe Gln
50 55 60
Thr Cys Glu Lys Glu Leu Ala Lys Ile Asn Thr Phe Tyr Ser Glu
65 70 75
Lys Leu Ala Glu Ala Gln Arg Arg Phe Ala Thr Leu Gln Asn Glu
80 85 90
Leu Gln Ser Ser Leu Asp Ala Gln Lys Glu Ser Thr Gly Val Thr
95 100 105
Thr Leu Arg Gln Arg Arg Lys Pro Val Phe His Leu Ser His Glu
110 115 120
Glu Arg Val Gln His Arg Asn Ile Lys Asp Leu Lys Leu Ala Phe
125 130 135
Ser Glu Phe Tyr Leu Ser Leu Ile Leu Leu Gln Asn Tyr Gln Asn
140 145 150
Leu Asn Phe Thr Gly Phe Arg Lys Ile Leu Lys Lys His Asp Lys
155 160 165
Ile Leu Glu Thr Ser Arg Gly Ala Asp Trp Arg Val Ala His Val
170 175 180
Glu Val Ala Pro Phe Tyr Thr Cys Lys Lys Ile Asn Gln Leu Ile
185 190 195
Ser Glu Thr Glu Ala Val Val Thr Asn Glu Leu Glu Asp Gly Asp
200 205 210
Arg Gln Lys Ala Met Lys Arg Leu Arg Val Pro Pro Leu Gly Ala
215 220 225
Ala Gln Pro Ala Pro Ala Trp Thr Thr Phe Arg Val Gly Leu Phe
230 235 240
Cys Gly Ile Phe Ile Val Leu Asn Ile Thr Leu Val Leu Ala Ala
245 250 255
Val Phe Lys Leu Glu Thr Asp Arg Ser Ile Trp Pro Leu Ile Arg
260 265 270
Ile Tyr Arg Gly Gly Phe Leu Leu Ile Glu Phe Leu Phe Leu Leu
275 280 285
Gly Ile Asn Thr Tyr Gly Trp Arg Gln Ala Gly Val Asn His Val
290 295 300
Leu Ile Phe Glu Leu Asn Pro Arg Ser Asn Leu Ser His Gln His
305 310 315
Leu Phe Glu Ile Ala Gly Phe Leu Gly Ile Leu Trp Cys Leu Ser
320 325 330
Leu Leu Ala Cys Phe Phe Ala Pro Ile Ser Val Ile Pro Thr Tyr
335 340 345
Val Tyr Pro Leu Ala Leu Tyr Gly Phe Met Val Phe Phe Leu Ile
350 355 360
Asn Pro Thr Lys Thr Phe Tyr Tyr Lys Ser Arg Phe Trp Leu Leu
365 370 375
Lys Leu Leu Phe Arg Val Phe Thr Ala Pro Phe His Lys Val Gly
380 385 390
Phe Ala Asp Phe Trp Leu Ala Asp Gln Leu Asn Ser Leu Ser Val
395 400 405
Ile Leu Met Asp Leu Glu Tyr Met Ile Cys Phe Tyr Ser Leu Glu
410 415 420
Leu Lys Trp Asp Glu Ser Lys Gly Leu Leu Pro Asn Asn Ser Glu
425 430 435
Glu Ser Gly Ile Cys His Lys Tyr Thr Tyr Gly Val Arg Ala Ile
440 445 450
Val Gln Cys Ile Pro Ala Trp Leu Arg Phe Ile Gln Cys Leu Arg
455 460 465
Arg Tyr Arg Asp Thr Lys Arg Ala Phe Pro His Leu Val Asn Ala
470 475 480
Gly Lys Tyr Ser Thr Thr Phe Phe Met Val Ala Phe Ala Ala Leu
485 490 495
Tyr Ser Thr His Lys Glu Arg Gly His Ser Asp Thr Met Val Phe
500 505 510
Phe Tyr Leu Trp Ile Val Phe Tyr Ile Ile Ser Ser Cys Tyr Thr
515 520 525
Leu Ile Trp Asp Leu Lys Met Asp Trp Gly Leu Phe Asp Lys Asn
530 535 540
Ala Gly Glu Asn Thr Phe Leu Arg Glu Glu Ile Val Tyr Pro Gln
545 550 555
Lys Ala Tyr Tyr Tyr Cys Ala Ile Ile Glu Asp Val Ile Leu Arg
560 565 570
Phe Ala Trp Thr Ile Gln Ile Ser Ile Thr Ser Thr Thr Leu Leu
575 580 585
Pro His Ser Gly Asp Ile Ile Ala Thr Val Phe Ala Pro Leu Glu
590 595 600
Val Phe Arg Arg Phe Val Trp Asn Phe Phe Arg Leu Glu Asn Glu
605 610 615
His Leu Asn Asn Cys Gly Glu Phe Arg Ala Val Arg Asp Ile Ser
620 625 630
Val Ala Pro Leu Asn Ala Asp Asp Gln Thr Leu Leu Glu Gln Met
635 640 645
Met Asp Gln Asp Asp Gly Val Arg Asn Arg Gln Lys Asn Arg Ser
650 655 660
Trp Lys Tyr Asn Gln Ser Ile Ser Leu Arg Arg Pro Arg Leu Ala
665 670 675
Ser Gln Ser Lys Ala Arg Asp Thr Lys Val Leu Ile Glu Asp Thr
680 685 690
Asp Asp Glu Ala Asn Thr
695
84
696
PRT
Homo Sapien
84
Met Lys Phe Ala Glu His Leu Ser Ala His Ile Thr Pro Glu Trp
1 5 10 15
Arg Lys Gln Tyr Ile Gln Tyr Glu Ala Phe Lys Asp Met Leu Tyr
20 25 30
Ser Ala Gln Asp Gln Ala Pro Ser Val Glu Val Thr Asp Glu Asp
35 40 45
Thr Val Lys Arg Tyr Phe Ala Lys Phe Glu Glu Lys Phe Phe Gln
50 55 60
Thr Cys Glu Lys Glu Leu Ala Lys Ile Asn Thr Phe Tyr Ser Glu
65 70 75
Lys Leu Ala Glu Ala Gln Arg Arg Phe Ala Thr Leu Gln Asn Glu
80 85 90
Leu Gln Ser Ser Leu Asp Ala Gln Lys Glu Ser Thr Gly Val Thr
95 100 105
Thr Leu Arg Gln Arg Arg Lys Pro Val Phe His Leu Ser His Glu
110 115 120
Glu Arg Val Gln His Arg Asn Ile Lys Asp Leu Lys Leu Ala Phe
125 130 135
Ser Glu Phe Tyr Leu Ser Leu Ile Leu Leu Gln Asn Tyr Gln Asn
140 145 150
Leu Asn Phe Thr Gly Phe Arg Lys Ile Leu Lys Lys His Asp Lys
155 160 165
Ile Leu Glu Thr Ser Arg Gly Ala Asp Trp Arg Val Ala His Val
170 175 180
Glu Val Ala Pro Phe Tyr Thr Cys Lys Lys Ile Asn Gln Leu Ile
185 190 195
Ser Glu Thr Glu Ala Val Val Thr Asn Glu Leu Glu Asp Gly Asp
200 205 210
Arg Gln Lys Ala Met Lys Arg Leu Arg Val Pro Pro Leu Gly Ala
215 220 225
Ala Gln Pro Ala Pro Ala Trp Thr Thr Phe Arg Val Gly Leu Phe
230 235 240
Cys Gly Ile Phe Ile Val Leu Asn Ile Thr Leu Val Leu Ala Ala
245 250 255
Val Phe Lys Leu Glu Thr Asp Arg Ser Ile Trp Pro Leu Ile Arg
260 265 270
Ile Tyr Arg Gly Gly Phe Leu Leu Ile Glu Phe Leu Phe Leu Leu
275 280 285
Gly Ile Asn Thr Tyr Gly Trp Arg Gln Ala Gly Val Asn His Val
290 295 300
Leu Ile Phe Glu Leu Asn Pro Arg Ser Asn Leu Ser His Gln His
305 310 315
Leu Phe Glu Ile Ala Gly Phe Leu Gly Ile Leu Trp Cys Leu Ser
320 325 330
Leu Leu Ala Cys Phe Phe Ala Pro Ile Ser Val Ile Pro Thr Tyr
335 340 345
Val Tyr Pro Leu Ala Leu Tyr Gly Phe Met Val Phe Phe Leu Ile
350 355 360
Asn Pro Thr Lys Thr Phe Tyr Tyr Lys Ser Arg Phe Trp Leu Leu
365 370 375
Lys Leu Leu Phe Arg Val Phe Thr Ala Pro Phe His Lys Val Gly
380 385 390
Phe Ala Asp Phe Trp Leu Ala Asp Gln Leu Asn Ser Leu Ser Val
395 400 405
Ile Leu Met Asp Leu Glu Tyr Met Ile Cys Phe Tyr Ser Leu Glu
410 415 420
Leu Lys Trp Asp Glu Ser Lys Gly Leu Leu Pro Asn Asn Ser Glu
425 430 435
Glu Ser Gly Ile Cys His Lys Tyr Thr Tyr Gly Val Arg Ala Ile
440 445 450
Val Gln Cys Ile Pro Ala Trp Leu Arg Phe Ile Gln Cys Leu Arg
455 460 465
Arg Tyr Arg Asp Thr Lys Arg Ala Phe Pro His Leu Val Asn Ala
470 475 480
Gly Lys Tyr Ser Thr Thr Phe Phe Met Val Thr Phe Ala Ala Leu
485 490 495
Tyr Ser Thr His Lys Glu Arg Gly His Ser Asp Thr Met Val Phe
500 505 510
Phe Tyr Leu Trp Ile Val Phe Tyr Ile Ile Ser Ser Cys Tyr Thr
515 520 525
Leu Ile Trp Asp Leu Lys Met Asp Trp Gly Leu Phe Asp Lys Asn
530 535 540
Ala Gly Glu Asn Thr Phe Leu Arg Glu Glu Ile Val Tyr Pro Gln
545 550 555
Lys Ala Tyr Tyr Tyr Cys Ala Ile Ile Glu Asp Val Ile Leu Arg
560 565 570
Phe Ala Trp Thr Ile Gln Ile Ser Ile Thr Ser Thr Thr Leu Leu
575 580 585
Pro His Ser Gly Asp Ile Ile Ala Thr Val Phe Ala Pro Leu Glu
590 595 600
Val Phe Arg Arg Phe Val Trp Asn Phe Phe Arg Leu Glu Asn Glu
605 610 615
His Leu Asn Asn Cys Gly Glu Phe Arg Ala Val Arg Asp Ile Ser
620 625 630
Val Ala Pro Leu Asn Ala Asp Asp Gln Thr Leu Leu Glu Gln Met
635 640 645
Met Asp Gln Asp Asp Gly Val Arg Asn Arg Gln Lys Asn Arg Ser
650 655 660
Trp Lys Tyr Asn Gln Ser Ile Ser Leu Arg Arg Pro Arg Leu Ala
665 670 675
Ser Gln Ser Lys Ala Arg Asp Thr Lys Val Leu Ile Glu Asp Thr
680 685 690
Asp Asp Glu Ala Asn Thr
695
85
635
PRT
Homo Sapien
85
Met Ser Val Gly Val Ser Thr Ser Ala Pro Leu Ser Pro Thr Ser
1 5 10 15
Gly Thr Ser Val Gly Met Ser Thr Phe Ser Ile Met Asp Tyr Val
20 25 30
Val Phe Val Leu Leu Leu Val Leu Ser Leu Ala Ile Gly Leu Tyr
35 40 45
His Ala Cys Arg Gly Trp Gly Arg His Thr Val Gly Glu Leu Leu
50 55 60
Met Ala Asp Arg Lys Met Gly Cys Leu Pro Val Ala Leu Ser Leu
65 70 75
Leu Ala Thr Phe Gln Ser Ala Val Ala Ile Leu Gly Val Pro Ser
80 85 90
Glu Ile Tyr Arg Phe Gly Thr Gln Tyr Trp Phe Leu Gly Cys Cys
95 100 105
Tyr Phe Leu Gly Leu Leu Ile Pro Ala His Ile Phe Ile Pro Val
110 115 120
Phe Tyr Arg Leu His Leu Thr Ser Ala Tyr Glu Tyr Leu Glu Leu
125 130 135
Arg Phe Asn Lys Thr Val Arg Val Cys Gly Thr Val Thr Phe Ile
140 145 150
Phe Gln Met Val Ile Tyr Met Gly Val Val Leu Tyr Ala Pro Ser
155 160 165
Leu Ala Leu Asn Ala Val Thr Gly Phe Asp Leu Trp Leu Ser Val
170 175 180
Leu Ala Leu Gly Ile Val Cys Thr Val Tyr Thr Ala Leu Gly Gly
185 190 195
Leu Lys Ala Val Ile Trp Thr Asp Val Phe Gln Thr Leu Val Met
200 205 210
Phe Leu Gly Gln Leu Ala Val Ile Ile Val Gly Ser Ala Lys Val
215 220 225
Gly Gly Leu Gly Arg Val Trp Ala Val Ala Ser Gln His Gly Arg
230 235 240
Ile Ser Gly Phe Glu Leu Asp Pro Asp Pro Phe Val Arg His Thr
245 250 255
Phe Trp Thr Leu Ala Phe Gly Gly Val Phe Met Met Leu Ser Leu
260 265 270
Tyr Gly Val Asn Gln Ala Gln Val Gln Arg Tyr Leu Ser Ser Arg
275 280 285
Thr Glu Lys Ala Ala Val Leu Ser Cys Tyr Ala Val Phe Pro Phe
290 295 300
Gln Gln Val Ser Leu Cys Val Gly Cys Leu Ile Gly Leu Val Met
305 310 315
Phe Ala Tyr Tyr Gln Glu Tyr Pro Met Ser Ile Gln Gln Ala Gln
320 325 330
Ala Ala Pro Asp Gln Phe Val Leu Tyr Phe Val Met Asp Leu Leu
335 340 345
Lys Gly Leu Pro Gly Leu Pro Gly Leu Phe Ile Ala Cys Leu Phe
350 355 360
Ser Gly Ser Leu Ser Thr Ile Ser Ser Ala Phe Asn Ser Leu Ala
365 370 375
Thr Val Thr Met Glu Asp Leu Ile Arg Pro Trp Phe Pro Glu Phe
380 385 390
Ser Glu Ala Arg Ala Ile Met Leu Ser Arg Gly Leu Ala Phe Gly
395 400 405
Tyr Gly Leu Leu Cys Leu Gly Met Ala Tyr Ile Ser Ser Gln Met
410 415 420
Gly Pro Val Leu Gln Ala Ala Ile Ser Ile Phe Gly Met Val Gly
425 430 435
Gly Pro Leu Leu Gly Leu Phe Cys Leu Gly Met Phe Phe Pro Cys
440 445 450
Ala Asn Pro Pro Gly Ala Val Val Gly Leu Leu Ala Gly Leu Val
455 460 465
Met Ala Phe Trp Ile Gly Ile Gly Ser Ile Val Thr Ser Met Gly
470 475 480
Phe Ser Met Pro Pro Ser Pro Ser Asn Gly Ser Ser Phe Ser Leu
485 490 495
Pro Thr Asn Leu Thr Val Ala Thr Val Thr Thr Leu Met Pro Leu
500 505 510
Thr Thr Phe Ser Lys Pro Thr Gly Leu Gln Arg Phe Tyr Ser Leu
515 520 525
Ser Tyr Leu Trp Tyr Ser Ala His Asn Ser Thr Thr Val Ile Val
530 535 540
Val Gly Leu Ile Val Ser Leu Leu Thr Gly Arg Met Arg Gly Arg
545 550 555
Ser Leu Asn Pro Ala Thr Ile Tyr Pro Val Leu Pro Lys Leu Leu
560 565 570
Ser Leu Leu Pro Leu Ser Cys Gln Lys Arg Leu His Cys Arg Ser
575 580 585
Tyr Gly Gln Asp His Leu Asp Thr Gly Leu Phe Pro Glu Lys Pro
590 595 600
Arg Asn Gly Val Leu Gly Asp Ser Arg Asp Lys Glu Ala Met Ala
605 610 615
Leu Asp Gly Thr Ala Tyr Gln Gly Ser Ser Ser Thr Cys Ile Leu
620 625 630
Gln Glu Thr Ser Leu
635
86
351
PRT
Homo Sapien
86
Met Ala Leu Thr Gly Ala Ser Asp Pro Ser Ala Glu Ala Glu Ala
1 5 10 15
Asn Gly Glu Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu
20 25 30
Val Val Ser Leu Tyr Trp Val Thr Ser Ile Ser Met Val Phe Leu
35 40 45
Asn Lys Tyr Leu Leu Asp Ser Pro Ser Leu Arg Leu Asp Thr Pro
50 55 60
Ile Phe Val Thr Phe Tyr Gln Cys Leu Val Thr Thr Leu Leu Cys
65 70 75
Lys Gly Leu Ser Ala Leu Ala Ala Cys Cys Pro Gly Ala Val Asp
80 85 90
Phe Pro Ser Leu Arg Leu Asp Leu Arg Val Ala Arg Ser Val Leu
95 100 105
Pro Leu Ser Val Val Phe Ile Gly Met Ile Thr Phe Asn Asn Leu
110 115 120
Cys Leu Lys Tyr Val Gly Val Ala Phe Tyr Asn Val Gly Arg Ser
125 130 135
Leu Thr Thr Val Phe Asn Val Leu Leu Ser Tyr Leu Leu Leu Lys
140 145 150
Gln Thr Thr Ser Phe Tyr Ala Leu Leu Thr Cys Gly Ile Ile Ile
155 160 165
Gly Gly Phe Trp Leu Gly Val Asp Gln Glu Gly Ala Glu Gly Thr
170 175 180
Leu Ser Trp Leu Gly Thr Val Phe Gly Val Leu Ala Ser Leu Cys
185 190 195
Val Ser Leu Asn Ala Ile Tyr Thr Thr Lys Val Leu Pro Ala Val
200 205 210
Asp Gly Ser Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala
215 220 225
Cys Ile Leu Phe Leu Pro Leu Leu Leu Leu Leu Gly Glu Leu Gln
230 235 240
Ala Leu Arg Asp Leu Ala Gln Leu Gly Ser Ala His Phe Trp Gly
245 250 255
Met Met Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val
260 265 270
Thr Gly Leu Gln Ile Lys Phe Thr Ser Pro Leu Thr His Asn Val
275 280 285
Ser Gly Thr Ala Lys Ala Cys Ala Gln Thr Val Leu Ala Val Leu
290 295 300
Tyr Tyr Glu Glu Thr Lys Ser Phe Leu Trp Trp Thr Ser Asn Met
305 310 315
Met Val Leu Gly Gly Ser Ser Ala Tyr Thr Trp Val Arg Gly Trp
320 325 330
Glu Met Lys Lys Thr Pro Glu Glu Pro Ser Pro Lys Asp Ser Glu
335 340 345
Lys Ser Ala Met Gly Val
350
87
351
PRT
Homo Sapien
87
Met Ala Leu Thr Gly Ala Ser Asp Pro Ser Ala Glu Ala Glu Ala
1 5 10 15
Asn Gly Glu Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu
20 25 30
Val Val Ser Leu Tyr Trp Val Thr Ser Ile Ser Met Val Phe Leu
35 40 45
Asn Lys Tyr Leu Leu Asp Ser Pro Ser Leu Arg Leu Asp Thr Pro
50 55 60
Ile Phe Val Thr Phe Tyr Gln Cys Leu Val Thr Thr Leu Leu Cys
65 70 75
Lys Gly Leu Ser Ala Leu Ala Ala Cys Cys Pro Gly Ala Val Asp
80 85 90
Phe Pro Ser Leu Arg Leu Asp Leu Arg Val Ala Arg Ser Val Leu
95 100 105
Pro Leu Ser Val Val Phe Ile Gly Met Ile Thr Phe Asn Asn Leu
110 115 120
Cys Leu Lys Tyr Val Gly Val Ala Phe Tyr Asn Val Gly Arg Ser
125 130 135
Leu Thr Thr Val Phe Asn Val Leu Leu Ser Tyr Leu Leu Leu Lys
140 145 150
Gln Thr Thr Ser Phe Tyr Ala Leu Leu Thr Cys Gly Ile Ile Ile
155 160 165
Gly Gly Phe Trp Leu Gly Val Asp Gln Glu Gly Ala Glu Gly Thr
170 175 180
Leu Ser Trp Leu Gly Thr Val Phe Gly Val Leu Ala Ser Leu Cys
185 190 195
Val Ser Leu Asn Ala Ile Tyr Thr Thr Lys Val Leu Pro Ala Val
200 205 210
Asp Gly Ser Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala
215 220 225
Cys Ile Leu Phe Leu Pro Leu Leu Leu Leu Leu Gly Glu Leu Gln
230 235 240
Ala Leu Arg Asp Phe Ala Gln Leu Gly Ser Ala His Phe Trp Gly
245 250 255
Met Met Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val
260 265 270
Thr Gly Leu Gln Ile Lys Phe Thr Ser Pro Leu Thr His Asn Val
275 280 285
Ser Gly Thr Ala Lys Ala Cys Ala Gln Thr Val Leu Ala Val Leu
290 295 300
Tyr Tyr Glu Glu Thr Lys Ser Phe Leu Trp Trp Thr Ser Asn Met
305 310 315
Met Val Leu Gly Gly Ser Ser Ala Tyr Thr Trp Val Arg Gly Trp
320 325 330
Glu Met Lys Lys Thr Pro Glu Glu Pro Ser Pro Lys Asp Ser Glu
335 340 345
Lys Ser Ala Met Gly Val
350
88
208
PRT
Homo Sapien
88
Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala
1 5 10 15
Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu
20 25 30
Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe Val His Ile Ile
35 40 45
Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg
50 55 60
Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp
65 70 75
Asn Val Phe Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu
80 85 90
Gln Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser
95 100 105
Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro
110 115 120
Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly
125 130 135
Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser
140 145 150
Gly Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys
155 160 165
Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Phe Asp Phe
170 175 180
Ile Gly Gly Phe Asp Pro Phe Pro Leu Tyr His Val Asn Glu Lys
185 190 195
Pro Ser Ser Leu Leu Ser Lys Gln Val Tyr Leu Pro Ala
200 205
89
208
PRT
Homo Sapien
89
Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala
1 5 10 15
Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu
20 25 30
Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe Val His Ile Ile
35 40 45
Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg
50 55 60
Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp
65 70 75
Asn Val Phe Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu
80 85 90
Lys Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser
95 100 105
Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro
110 115 120
Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly
125 130 135
Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser
140 145 150
Cys Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys
155 160 165
Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Phe Asp Phe
170 175 180
Ile Gly Gly Phe Asp Pro Phe Pro Leu Tyr His Val Asn Glu Lys
185 190 195
Pro Ser Ser Leu Leu Ser Lys Gln Val Tyr Leu Pro Ala
200 205
90
181
PRT
Homo Sapien
90
Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala
1 5 10 15
Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu
20 25 30
Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe Val His Ile Ile
35 40 45
Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg
50 55 60
Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp
65 70 75
Asn Val Phe Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu
80 85 90
Lys Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser
95 100 105
Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro
110 115 120
Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly
125 130 135
Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser
140 145 150
Cys Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys
155 160 165
Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Cys Leu Arg
170 175 180
Lys
91
181
PRT
Homo Sapien
91
Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala
1 5 10 15
Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu
20 25 30
Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe Val His Ile Ile
35 40 45
Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg
50 55 60
Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp
65 70 75
Asn Val Phe Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu
80 85 90
Gln Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser
95 100 105
Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro
110 115 120
Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly
125 130 135
Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser
140 145 150
Gly Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys
155 160 165
Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Cys Leu Arg
170 175 180
Lys
92
382
PRT
Homo Sapien
92
Met Ala Val Leu Phe Leu Leu Leu Phe Leu Cys Gly Thr Pro Gln
1 5 10 15
Ala Ala Asp Asn Met Gln Ala Ile Tyr Val Ala Leu Gly Glu Ala
20 25 30
Val Glu Leu Pro Cys Pro Ser Pro Pro Thr Leu His Gly Asp Glu
35 40 45
His Leu Ser Trp Phe Cys Ser Pro Ala Ala Gly Ser Phe Thr Thr
50 55 60
Leu Val Ala Gln Val Gln Val Gly Arg Pro Ala Pro Asp Pro Gly
65 70 75
Lys Pro Gly Arg Glu Ser Arg Leu Arg Leu Leu Gly Asn Tyr Ser
80 85 90
Leu Trp Leu Glu Gly Ser Lys Glu Glu Asp Ala Gly Arg Tyr Trp
95 100 105
Cys Ala Val Leu Gly Gln His His Asn Tyr Gln Asn Trp Arg Val
110 115 120
Tyr Asp Val Leu Val Leu Lys Gly Ser Gln Leu Ser Ala Arg Ala
125 130 135
Ala Asp Gly Ser Pro Cys Asn Val Leu Leu Cys Ser Val Val Pro
140 145 150
Ser Arg Arg Met Asp Ser Val Thr Trp Gln Glu Gly Lys Gly Pro
155 160 165
Val Arg Gly Arg Val Gln Ser Phe Trp Gly Ser Glu Ala Ala Leu
170 175 180
Leu Leu Val Cys Pro Gly Glu Gly Leu Ser Glu Pro Arg Ser Arg
185 190 195
Arg Pro Arg Ile Ile Arg Cys Leu Met Thr His Asn Lys Gly Val
200 205 210
Ser Phe Ser Leu Ala Ala Ser Ile Asp Ala Ser Pro Ala Leu Cys
215 220 225
Ala Pro Ser Thr Gly Trp Asp Met Pro Trp Ile Leu Met Leu Leu
230 235 240
Leu Thr Met Gly Gln Gly Val Val Ile Leu Ala Leu Ser Ile Val
245 250 255
Leu Trp Arg Gln Arg Val Arg Gly Ala Pro Gly Arg Gly Asn Arg
260 265 270
Met Arg Cys Tyr Asn Cys Gly Gly Ser Pro Ser Ser Ser Cys Lys
275 280 285
Glu Ala Val Thr Thr Cys Gly Glu Gly Arg Pro Gln Pro Gly Leu
290 295 300
Glu Gln Ile Lys Leu Pro Gly Asn Pro Pro Val Thr Leu Ile His
305 310 315
Gln His Pro Ala Cys Val Ala Ala His His Cys Asn Gln Val Glu
320 325 330
Thr Glu Ser Val Gly Asp Val Thr Tyr Pro Ala His Arg Asp Cys
335 340 345
Tyr Leu Gly Asp Leu Cys Asn Ser Ala Val Ala Ser His Val Ala
350 355 360
Pro Ala Gly Ile Leu Ala Ala Ala Ala Thr Ala Leu Thr Cys Leu
365 370 375
Leu Pro Gly Leu Trp Ser Gly
380
93
783
PRT
Homo Sapien
93
Met Ser Gly Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp
1 5 10 15
Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu
20 25 30
Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys
35 40 45
Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys
50 55 60
Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile
65 70 75
Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu
80 85 90
Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser
95 100 105
Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro Arg Pro Cys Leu
110 115 120
Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser
125 130 135
Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln
140 145 150
Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp
155 160 165
Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln
170 175 180
Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro
185 190 195
Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe
200 205 210
Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg Pro Arg
215 220 225
His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile
230 235 240
Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala
245 250 255
Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro
260 265 270
Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu
275 280 285
Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val Asp
290 295 300
Pro Trp Leu His Gln His Arg Thr Cys Pro Leu Cys Val Phe Asn
305 310 315
Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg
320 325 330
Ser Tyr Gln Glu Pro Gly Arg Arg Leu His Leu Ile Arg Gln His
335 340 345
Pro Gly His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly
350 355 360
Pro Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro Gly Pro Phe
365 370 375
Leu Pro Ser Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe
380 385 390
Pro Arg Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu
395 400 405
Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Met Ser His
410 415 420
Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu
425 430 435
Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr
440 445 450
Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp
455 460 465
Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val
470 475 480
Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser
485 490 495
Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr
500 505 510
Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser
515 520 525
Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly
530 535 540
Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg His
545 550 555
His His Tyr Lys Lys Arg Phe Gln Trp His Gly Arg Lys Pro Gly
560 565 570
Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr
575 580 585
Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln Gln Val Thr Gly
590 595 600
Ser Asn Ser Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys
605 610 615
Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser
620 625 630
Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser
635 640 645
Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro
650 655 660
Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His
665 670 675
Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr
680 685 690
Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly
695 700 705
Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser
710 715 720
Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu
725 730 735
Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp
740 745 750
Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu
755 760 765
Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu
770 775 780
Gln Ala Val
94
510
PRT
Homo Sapien
94
Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp
1 5 10 15
Leu Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro
20 25 30
Ala Gly Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly
35 40 45
Gln Asp Ala Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu
50 55 60
Gln Val Gly Gln Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly
65 70 75
Ala Gln Glu Leu Ala Leu Leu His Ser Lys Tyr Gly Leu His Val
80 85 90
Ser Pro Ala Tyr Glu Gly Arg Val Glu Gln Pro Pro Pro Pro Arg
95 100 105
Asn Pro Leu Asp Gly Ser Val Leu Leu Arg Asn Ala Val Gln Ala
110 115 120
Asp Glu Gly Glu Tyr Glu Cys Arg Val Ser Thr Phe Pro Ala Gly
125 130 135
Ser Phe Gln Ala Arg Leu Arg Leu Arg Val Leu Val Pro Pro Leu
140 145 150
Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu Glu Gly Gln Gly Leu
155 160 165
Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser Pro Ala Pro Ser
170 175 180
Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr Ser Ser Arg Ser
185 190 195
Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser Glu Phe His Leu
200 205 210
Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr Cys Val Val
215 220 225
Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His Ile Leu
230 235 240
His Val Ser Phe Leu Ala Glu Ala Ser Val Arg Gly Leu Glu Asp
245 250 255
Gln Asn Leu Trp His Ile Gly Arg Glu Gly Ala Met Leu Lys Cys
260 265 270
Leu Ser Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu
275 280 285
Asp Gly Pro Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu
290 295 300
Gly Phe Pro Pro Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys
305 310 315
His Val Ser Asn Glu Phe Ser Ser Arg Asp Ser Gln Val Thr Val
320 325 330
Asp Val Leu Asp Pro Gln Glu Asp Ser Gly Lys Gln Val Asp Leu
335 340 345
Val Ser Ala Ser Val Val Val Val Gly Val Ile Ala Ala Leu Leu
350 355 360
Phe Cys Leu Leu Val Val Val Val Val Leu Met Ser Arg Tyr His
365 370 375
Arg Arg Lys Ala Gln Gln Met Thr Gln Lys Tyr Glu Glu Glu Leu
380 385 390
Thr Leu Thr Arg Glu Asn Ser Ile Arg Arg Leu His Ser His His
395 400 405
Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val Gly Leu Arg Ala
410 415 420
Glu Gly His Pro Asp Ser Leu Lys Asp Asn Ser Ser Cys Ser Val
425 430 435
Met Ser Glu Glu Pro Glu Gly Arg Ser Tyr Ser Thr Leu Thr Thr
440 445 450
Val Arg Glu Ile Glu Thr Gln Thr Glu Leu Leu Ser Pro Gly Ser
455 460 465
Gly Arg Ala Glu Glu Glu Glu Asp Gln Asp Glu Gly Ile Lys Gln
470 475 480
Ala Met Asn His Phe Val Gln Glu Asn Gly Thr Leu Arg Ala Lys
485 490 495
Pro Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg Gly His Leu Val
500 505 510
95
523
PRT
Homo Sapien
95
Met Thr Gln Asn Lys Leu Lys Leu Cys Ser Lys Ala Asn Val Tyr
1 5 10 15
Thr Glu Val Pro Asp Gly Gly Trp Gly Trp Ala Val Ala Val Ser
20 25 30
Phe Phe Phe Val Glu Val Phe Thr Tyr Gly Ile Ile Lys Thr Phe
35 40 45
Gly Val Phe Phe Asn Asp Leu Met Asp Ser Phe Asn Glu Ser Asn
50 55 60
Ser Arg Ile Ser Trp Ile Ile Ser Ile Cys Val Phe Val Leu Thr
65 70 75
Phe Ser Ala Pro Leu Ala Thr Val Leu Ser Asn Arg Phe Gly His
80 85 90
Arg Leu Val Val Met Leu Gly Gly Leu Leu Val Ser Thr Gly Met
95 100 105
Val Ala Ala Ser Phe Ser Gln Glu Val Ser His Met Tyr Val Ala
110 115 120
Ile Gly Ile Ile Ser Gly Leu Gly Tyr Cys Phe Ser Phe Leu Pro
125 130 135
Thr Val Thr Ile Leu Ser Gln Tyr Phe Gly Lys Arg Arg Ser Ile
140 145 150
Val Thr Ala Val Ala Ser Thr Gly Glu Cys Phe Ala Val Phe Ala
155 160 165
Phe Ala Pro Ala Ile Met Ala Leu Lys Glu Arg Ile Gly Trp Arg
170 175 180
Tyr Ser Leu Leu Phe Val Gly Leu Leu Gln Leu Asn Ile Val Ile
185 190 195
Phe Gly Ala Leu Leu Arg Pro Ile Ile Ile Arg Gly Pro Ala Ser
200 205 210
Pro Lys Ile Val Ile Gln Glu Asn Arg Lys Glu Ala Gln Tyr Met
215 220 225
Leu Glu Asn Glu Lys Thr Arg Thr Ser Ile Asp Ser Ile Asp Ser
230 235 240
Gly Val Glu Leu Thr Thr Ser Pro Lys Asn Val Pro Thr His Thr
245 250 255
Asn Leu Glu Leu Glu Pro Lys Ala Asp Met Gln Gln Val Leu Val
260 265 270
Lys Thr Ser Pro Arg Pro Ser Glu Lys Lys Ala Pro Leu Leu Asp
275 280 285
Phe Ser Ile Leu Lys Glu Lys Ser Phe Ile Cys Tyr Ala Leu Phe
290 295 300
Gly Leu Phe Ala Thr Leu Gly Phe Phe Ala Pro Ser Leu Tyr Ile
305 310 315
Ile Pro Leu Gly Ile Ser Leu Gly Ile Asp Gln Asp Arg Ala Ala
320 325 330
Phe Leu Leu Ser Thr Met Ala Ile Ala Glu Val Phe Gly Arg Ile
335 340 345
Gly Ala Gly Phe Val Leu Asn Arg Glu Pro Ile Arg Lys Ile Tyr
350 355 360
Ile Glu Leu Ile Cys Val Ile Leu Leu Thr Val Ser Leu Phe Ala
365 370 375
Phe Thr Phe Ala Thr Glu Phe Trp Gly Leu Met Ser Cys Ser Ile
380 385 390
Phe Phe Gly Phe Met Val Gly Thr Ile Gly Gly Leu Thr Phe His
395 400 405
Cys Leu Leu Lys Met Met Ser Trp Ala Leu Gln Lys Met Ser Ser
410 415 420
Ala Ala Gly Val Tyr Ile Phe Ile Gln Ser Ile Ala Gly Leu Ala
425 430 435
Gly Pro Pro Leu Ala Gly Leu Leu Val Asp Gln Ser Lys Ile Tyr
440 445 450
Ser Arg Ala Phe Tyr Ser Cys Ala Ala Gly Met Ala Leu Ala Ala
455 460 465
Val Cys Leu Ala Leu Val Arg Pro Cys Lys Met Gly Leu Cys Gln
470 475 480
Arg His His Ser Gly Glu Thr Lys Val Val Ser His Arg Gly Lys
485 490 495
Thr Leu Gln Asp Ile Pro Glu Asp Phe Leu Glu Met Asp Leu Ala
500 505 510
Lys Asn Glu His Arg Val His Val Gln Met Glu Pro Val
515 520
96
124
PRT
Homo Sapien
96
Met Leu Leu Trp Val Ile Leu Leu Val Leu Ala Pro Val Ser Gly
1 5 10 15
Gln Phe Ala Arg Thr Pro Arg Pro Ile Ile Phe Leu Gln Pro Pro
20 25 30
Trp Thr Thr Val Phe Gln Gly Glu Arg Val Thr Leu Thr Cys Lys
35 40 45
Gly Phe Arg Phe Tyr Ser Pro Gln Lys Thr Lys Trp Tyr His Arg
50 55 60
Tyr Leu Gly Lys Glu Ile Leu Arg Glu Thr Pro Asp Asn Ile Leu
65 70 75
Glu Val Gln Glu Ser Gly Glu Tyr Arg Cys Gln Ala Gln Gly Ser
80 85 90
Pro Leu Ser Ser Pro Val His Leu Asp Phe Ser Ser Glu Met Gly
95 100 105
Phe Pro His Ala Ala Gln Ala Asn Val Glu Leu Leu Gly Ser Ser
110 115 120
Asp Leu Leu Thr
97
977
PRT
Homo Sapien
97
Met Leu Leu Trp Val Ile Leu Leu Val Leu Ala Pro Val Ser Gly
1 5 10 15
Gln Phe Ala Arg Thr Pro Arg Pro Ile Ile Phe Leu Gln Pro Pro
20 25 30
Trp Thr Thr Val Phe Gln Gly Glu Arg Val Thr Leu Thr Cys Lys
35 40 45
Gly Phe Arg Phe Tyr Ser Pro Gln Lys Thr Lys Trp Tyr His Arg
50 55 60
Tyr Leu Gly Lys Glu Ile Leu Arg Glu Thr Pro Asp Asn Ile Leu
65 70 75
Glu Val Gln Glu Ser Gly Glu Tyr Arg Cys Gln Ala Gln Gly Ser
80 85 90
Pro Leu Ser Ser Pro Val His Leu Asp Phe Ser Ser Ala Ser Leu
95 100 105
Ile Leu Gln Ala Pro Leu Ser Val Phe Glu Gly Asp Ser Val Val
110 115 120
Leu Arg Cys Arg Ala Lys Ala Glu Val Thr Leu Asn Asn Thr Ile
125 130 135
Tyr Lys Asn Asp Asn Val Leu Ala Phe Leu Asn Lys Arg Thr Asp
140 145 150
Phe His Ile Pro His Ala Cys Leu Lys Asp Asn Gly Ala Tyr Arg
155 160 165
Cys Thr Gly Tyr Lys Glu Ser Cys Cys Pro Val Ser Ser Asn Thr
170 175 180
Val Lys Ile Gln Val Gln Glu Pro Phe Thr Arg Pro Val Leu Arg
185 190 195
Ala Ser Ser Phe Gln Pro Ile Ser Gly Asn Pro Val Thr Leu Thr
200 205 210
Cys Glu Thr Gln Leu Ser Leu Glu Arg Ser Asp Val Pro Leu Arg
215 220 225
Phe Arg Phe Phe Arg Asp Asp Gln Thr Leu Gly Leu Gly Trp Ser
230 235 240
Leu Ser Pro Asn Phe Gln Ile Thr Ala Met Trp Ser Lys Asp Ser
245 250 255
Gly Phe Tyr Trp Cys Lys Ala Ala Thr Met Pro His Ser Val Ile
260 265 270
Ser Asp Ser Pro Arg Ser Trp Ile Gln Val Gln Ile Pro Ala Ser
275 280 285
His Pro Val Leu Thr Leu Ser Pro Glu Lys Ala Leu Asn Phe Glu
290 295 300
Gly Thr Lys Val Thr Leu His Cys Glu Thr Gln Glu Asp Ser Leu
305 310 315
Arg Thr Leu Tyr Arg Phe Tyr His Glu Gly Val Pro Leu Arg His
320 325 330
Lys Ser Val Arg Cys Glu Arg Gly Ala Ser Ile Ser Phe Ser Leu
335 340 345
Thr Thr Glu Asn Ser Gly Asn Tyr Tyr Cys Thr Ala Asp Asn Gly
350 355 360
Leu Gly Ala Lys Pro Ser Lys Ala Val Ser Leu Ser Val Thr Val
365 370 375
Pro Val Ser His Pro Val Leu Asn Leu Ser Ser Pro Glu Asp Leu
380 385 390
Ile Phe Glu Gly Ala Lys Val Thr Leu His Cys Glu Ala Gln Arg
395 400 405
Gly Ser Leu Pro Ile Leu Tyr Gln Phe His His Glu Asp Ala Ala
410 415 420
Leu Glu Arg Arg Ser Ala Asn Ser Ala Gly Gly Val Ala Ile Ser
425 430 435
Phe Ser Leu Thr Ala Glu His Ser Gly Asn Tyr Tyr Cys Thr Ala
440 445 450
Asp Asn Gly Phe Gly Pro Gln Arg Ser Lys Ala Val Ser Leu Ser
455 460 465
Ile Thr Val Pro Val Ser His Pro Val Leu Thr Leu Ser Ser Ala
470 475 480
Glu Ala Leu Thr Phe Glu Gly Ala Thr Val Thr Leu His Cys Glu
485 490 495
Val Gln Arg Gly Ser Pro Gln Ile Leu Tyr Gln Phe Tyr His Glu
500 505 510
Asp Met Pro Leu Trp Ser Ser Ser Thr Pro Ser Val Gly Arg Val
515 520 525
Ser Phe Ser Phe Ser Leu Thr Glu Gly His Ser Gly Asn Tyr Tyr
530 535 540
Cys Thr Ala Asp Asn Gly Phe Gly Pro Gln Arg Ser Glu Val Val
545 550 555
Ser Leu Phe Val Thr Val Pro Val Ser Arg Pro Ile Leu Thr Leu
560 565 570
Arg Val Pro Arg Ala Gln Ala Val Val Gly Asp Leu Leu Glu Leu
575 580 585
His Cys Glu Ala Pro Arg Gly Ser Pro Pro Ile Leu Tyr Trp Phe
590 595 600
Tyr His Glu Asp Val Thr Leu Gly Ser Ser Ser Ala Pro Ser Gly
605 610 615
Gly Glu Ala Ser Phe Asn Leu Ser Leu Thr Ala Glu His Ser Gly
620 625 630
Asn Tyr Ser Cys Glu Ala Asn Asn Gly Leu Val Ala Gln His Ser
635 640 645
Asp Thr Ile Ser Leu Ser Val Ile Val Pro Val Ser Arg Pro Ile
650 655 660
Leu Thr Phe Arg Ala Pro Arg Ala Gln Ala Val Val Gly Asp Leu
665 670 675
Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Ser Pro Ile Leu
680 685 690
Tyr Trp Phe Tyr His Glu Asp Val Thr Leu Gly Lys Ile Ser Ala
695 700 705
Pro Ser Gly Gly Gly Ala Ser Phe Asn Leu Ser Leu Thr Thr Glu
710 715 720
His Ser Gly Ile Tyr Ser Cys Glu Ala Asp Asn Gly Pro Glu Ala
725 730 735
Gln Arg Ser Glu Met Val Thr Leu Lys Val Ala Val Pro Val Ser
740 745 750
Arg Pro Val Leu Thr Leu Arg Ala Pro Gly Thr His Ala Ala Val
755 760 765
Gly Asp Leu Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Pro
770 775 780
Leu Ile Leu Tyr Arg Phe Phe His Glu Asp Val Thr Leu Gly Asn
785 790 795
Arg Ser Ser Pro Ser Gly Gly Ala Ser Leu Asn Leu Ser Leu Thr
800 805 810
Ala Glu His Ser Gly Asn Tyr Ser Cys Glu Ala Asp Asn Gly Leu
815 820 825
Gly Ala Gln Arg Ser Glu Thr Val Thr Leu Tyr Ile Thr Gly Leu
830 835 840
Thr Ala Asn Arg Ser Gly Pro Phe Ala Thr Gly Val Ala Gly Gly
845 850 855
Leu Leu Ser Ile Ala Gly Leu Ala Ala Gly Ala Leu Leu Leu Tyr
860 865 870
Cys Trp Leu Ser Arg Lys Ala Gly Arg Lys Pro Ala Ser Asp Pro
875 880 885
Ala Arg Ser Pro Pro Asp Ser Asp Ser Gln Glu Pro Thr Tyr His
890 895 900
Asn Val Pro Ala Trp Glu Glu Leu Gln Pro Val Tyr Thr Asn Ala
905 910 915
Asn Pro Arg Gly Glu Asn Val Val Tyr Ser Glu Val Arg Ile Ile
920 925 930
Gln Glu Lys Lys Lys His Ala Val Ala Ser Asp Pro Arg His Leu
935 940 945
Arg Asn Lys Gly Ser Pro Ile Ile Tyr Ser Glu Val Lys Val Ala
950 955 960
Ser Thr Pro Val Ser Gly Ser Leu Phe Leu Ala Ser Ser Ala Pro
965 970 975
His Arg
98
146
PRT
Homo Sapien
98
Met Leu Leu Trp Cys Pro Pro Gln Cys Ala Cys Ser Leu Gly Val
1 5 10 15
Phe Pro Ser Ala Pro Ser Pro Val Trp Gly Thr Arg Arg Ser Cys
20 25 30
Glu Pro Ala Thr Arg Val Pro Glu Val Trp Ile Leu Ser Pro Leu
35 40 45
Leu Arg His Gly Gly His Thr Gln Thr Gln Asn His Thr Ala Ser
50 55 60
Pro Arg Ser Pro Val Met Glu Ser Pro Lys Lys Lys Asn Gln Gln
65 70 75
Leu Lys Val Gly Ile Leu His Leu Gly Ser Arg Gln Lys Lys Ile
80 85 90
Arg Ile Gln Leu Arg Ser Gln Cys Ala Thr Trp Lys Val Ile Cys
95 100 105
Lys Ser Cys Ile Ser Gln Thr Pro Gly Ile Asn Leu Asp Leu Gly
110 115 120
Ser Gly Val Lys Val Lys Ile Ile Pro Lys Glu Glu His Cys Lys
125 130 135
Met Pro Glu Ala Gly Glu Glu Gln Pro Gln Val
140 145
99
235
PRT
Homo Sapien
99
Met Arg Glu Leu Ala Ile Glu Ile Gly Val Arg Ala Leu Leu Phe
1 5 10 15
Gly Val Phe Val Phe Thr Glu Phe Leu Asp Pro Phe Gln Arg Val
20 25 30
Ile Gln Pro Glu Glu Ile Trp Leu Tyr Lys Asn Pro Leu Val Gln
35 40 45
Ser Asp Asn Ile Pro Thr Arg Leu Met Phe Ala Ile Ser Phe Leu
50 55 60
Thr Pro Leu Ala Val Ile Cys Val Val Lys Ile Ile Arg Arg Thr
65 70 75
Asp Lys Thr Glu Ile Lys Glu Ala Phe Leu Ala Val Ser Leu Ala
80 85 90
Leu Ala Leu Asn Gly Val Cys Thr Asn Thr Ile Lys Leu Ile Val
95 100 105
Gly Arg Pro Arg Ala Asp Phe Phe Tyr Arg Cys Phe Pro Asp Gly
110 115 120
Val Met Asn Ser Glu Met His Cys Thr Gly Asp Pro Asp Leu Val
125 130 135
Ser Glu Gly Arg Lys Ser Phe Pro Ser Ile His Ser Ser Phe Ala
140 145 150
Phe Ser Gly Leu Gly Phe Thr Thr Phe Tyr Leu Ala Gly Lys Leu
155 160 165
His Cys Phe Thr Glu Ser Gly Arg Gly Lys Ser Trp Arg Leu Cys
170 175 180
Ala Ala Ile Leu Pro Leu Tyr Cys Ala Met Met Ile Ala Leu Ser
185 190 195
Arg Met Cys Asp Tyr Lys His His Trp Gln Asp Ser Phe Val Gly
200 205 210
Gly Val Ile Ala Leu Ile Phe Ala Tyr Ile Cys Tyr Arg Gln His
215 220 225
Tyr Pro Pro Leu Gly Gln His Ser Leu Pro
230 235
100
252
PRT
Homo Sapien
100
Met Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile Val Val Val
1 5 10 15
Met Met Val Met Val Val Val Ile Thr Cys Leu Leu Ser His Tyr
20 25 30
Lys Leu Ser Ala Arg Ser Phe Ile Ser Arg His Ser Gln Gly Arg
35 40 45
Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly Cys Leu Trp Pro Ser
50 55 60
Glu Ser Thr Val Ser Gly Asn Gly Ile Pro Glu Pro Gln Val Tyr
65 70 75
Ala Pro Pro Arg Pro Thr Asp Arg Leu Ala Val Pro Pro Phe Ala
80 85 90
Gln Arg Glu Arg Phe His Arg Phe Gln Pro Thr Tyr Pro Tyr Leu
95 100 105
Gln His Glu Ile Asp Leu Pro Pro Thr Ile Ser Leu Ser Asp Gly
110 115 120
Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr Leu Gln Leu Arg
125 130 135
Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg Glu Ser Val Arg Ala
140 145 150
Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp Leu Met Asp Ser Ala
155 160 165
Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser
170 175 180
Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro Pro Pro
185 190 195
Thr Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln
200 205 210
His Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg
215 220 225
Leu His His Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp
230 235 240
Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro Leu
245 250
101
252
PRT
Homo Sapien
101
Met Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile Val Val Val
1 5 10 15
Met Met Val Met Val Val Val Ile Thr Cys Leu Leu Ser His Tyr
20 25 30
Lys Leu Ser Ala Arg Ser Phe Ile Ser Arg His Ser Gln Gly Arg
35 40 45
Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly Cys Leu Trp Pro Ser
50 55 60
Glu Ser Thr Val Ser Gly Asn Gly Ile Pro Glu Pro Gln Val Tyr
65 70 75
Ala Pro Pro Arg Pro Thr Asp Arg Leu Ala Val Pro Pro Phe Ala
80 85 90
Gln Arg Glu Arg Phe His Arg Phe Gln Pro Thr Tyr Pro Tyr Leu
95 100 105
Gln His Glu Ile Asp Leu Pro Pro Thr Ile Ser Leu Ser Asp Gly
110 115 120
Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr Leu Gln Leu Arg
125 130 135
Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg Glu Ser Val Arg Ala
140 145 150
Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp Leu Met Asp Ser Ala
155 160 165
Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser
170 175 180
Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro Pro Pro
185 190 195
Thr Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln
200 205 210
His Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg
215 220 225
Leu His His Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp
230 235 240
Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro Leu
245 250
102
465
PRT
Homo Sapien
102
Met Gly Gly Ala Val Val Asp Glu Gly Pro Thr Gly Val Lys Ala
1 5 10 15
Pro Asp Gly Gly Trp Gly Trp Ala Val Leu Phe Gly Cys Phe Val
20 25 30
Ile Thr Gly Phe Ser Tyr Ala Phe Pro Lys Ala Val Ser Val Phe
35 40 45
Phe Lys Glu Leu Ile Gln Glu Phe Gly Ile Gly Tyr Ser Asp Thr
50 55 60
Ala Trp Ile Ser Ser Ile Leu Leu Ala Met Leu Tyr Gly Thr Gly
65 70 75
Pro Leu Cys Ser Val Cys Val Asn Arg Phe Gly Cys Arg Pro Val
80 85 90
Met Leu Val Gly Gly Leu Phe Ala Ser Leu Gly Met Val Ala Ala
95 100 105
Ser Phe Cys Arg Ser Ile Ile Gln Val Tyr Leu Thr Thr Gly Val
110 115 120
Ile Thr Gly Leu Gly Leu Ala Leu Asn Phe Gln Pro Ser Leu Ile
125 130 135
Met Leu Asn Arg Tyr Phe Ser Lys Arg Arg Pro Met Ala Asn Gly
140 145 150
Leu Ala Ala Ala Gly Ser Pro Val Phe Leu Cys Ala Leu Ser Pro
155 160 165
Leu Gly Gln Leu Leu Gln Asp Arg Tyr Gly Trp Arg Gly Gly Phe
170 175 180
Leu Ile Leu Gly Gly Leu Leu Leu Asn Cys Cys Val Cys Ala Ala
185 190 195
Leu Met Arg Pro Leu Val Val Thr Ala Gln Pro Gly Ser Gly Pro
200 205 210
Pro Arg Pro Ser Arg Arg Leu Leu Asp Leu Ser Val Phe Arg Asp
215 220 225
Arg Gly Phe Val Leu Tyr Ala Val Ala Ala Ser Val Met Val Leu
230 235 240
Gly Leu Phe Val Pro Pro Val Phe Val Val Ser Tyr Ala Lys Asp
245 250 255
Leu Gly Val Pro Asp Thr Lys Ala Ala Phe Leu Leu Thr Ile Leu
260 265 270
Gly Phe Ile Asp Ile Phe Ala Arg Pro Ala Ala Gly Phe Val Ala
275 280 285
Gly Leu Gly Lys Val Arg Pro Tyr Ser Val Tyr Leu Phe Ser Phe
290 295 300
Ser Met Phe Phe Asn Gly Leu Ala Asp Leu Ala Gly Ser Thr Ala
305 310 315
Gly Asp Tyr Gly Gly Leu Val Val Phe Cys Ile Phe Phe Gly Ile
320 325 330
Ser Tyr Gly Met Val Gly Ala Leu Gln Phe Glu Val Leu Met Ala
335 340 345
Ile Val Gly Thr His Lys Phe Ser Ser Ala Ile Gly Leu Val Leu
350 355 360
Leu Met Glu Ala Val Ala Val Leu Val Gly Pro Pro Ser Gly Gly
365 370 375
Lys Leu Leu Asp Ala Thr His Val Tyr Met Tyr Val Phe Ile Leu
380 385 390
Ala Gly Ala Glu Val Leu Thr Ser Ser Leu Ile Leu Leu Leu Gly
395 400 405
Asn Phe Phe Cys Ile Arg Lys Lys Pro Lys Glu Pro Gln Pro Glu
410 415 420
Val Ala Ala Ala Glu Glu Glu Lys Leu His Lys Pro Pro Ala Asp
425 430 435
Ser Gly Val Asp Leu Arg Glu Val Glu His Phe Leu Lys Ala Glu
440 445 450
Pro Glu Lys Asn Gly Glu Val Val His Thr Pro Glu Thr Ser Val
455 460 465
103
445
PRT
Homo Sapien
103
Met Ala Ala Pro Thr Pro Ala Arg Pro Val Leu Thr His Leu Leu
1 5 10 15
Val Ala Leu Phe Gly Met Gly Ser Trp Ala Ala Val Asn Gly Ile
20 25 30
Trp Val Glu Leu Pro Val Val Val Lys Glu Leu Pro Glu Gly Trp
35 40 45
Ser Leu Pro Ser Tyr Val Ser Val Leu Val Ala Leu Gly Asn Leu
50 55 60
Gly Leu Leu Val Val Thr Leu Trp Arg Arg Leu Ala Pro Gly Lys
65 70 75
Asp Glu Gln Val Pro Ile Arg Val Val Gln Val Leu Gly Met Val
80 85 90
Gly Thr Ala Leu Leu Ala Ser Leu Trp His His Val Ala Pro Val
95 100 105
Ala Gly Gln Leu His Ser Val Ala Phe Leu Ala Leu Ala Phe Val
110 115 120
Leu Ala Leu Ala Cys Cys Ala Ser Asn Val Thr Phe Leu Pro Phe
125 130 135
Leu Ser His Leu Pro Pro Arg Phe Leu Arg Ser Phe Phe Leu Gly
140 145 150
Gln Gly Leu Ser Ala Leu Leu Pro Cys Val Leu Ala Leu Val Gln
155 160 165
Gly Val Gly Arg Leu Glu Cys Pro Pro Ala Pro Ile Asn Gly Thr
170 175 180
Pro Gly Pro Pro Leu Asp Phe Leu Glu Arg Phe Pro Ala Ser Thr
185 190 195
Phe Phe Trp Ala Leu Thr Ala Leu Leu Val Ala Ser Ala Ala Ala
200 205 210
Phe Gln Gly Leu Leu Leu Leu Leu Pro Pro Pro Pro Ser Val Pro
215 220 225
Thr Gly Glu Leu Gly Ser Gly Leu Gln Val Gly Ala Pro Gly Ala
230 235 240
Glu Glu Glu Val Glu Glu Ser Ser Pro Leu Gln Glu Pro Pro Ser
245 250 255
Gln Ala Ala Gly Thr Thr Pro Gly Pro Asp Pro Lys Ala Tyr Gln
260 265 270
Leu Leu Ser Ala Arg Ser Ala Cys Leu Leu Gly Leu Leu Ala Ala
275 280 285
Thr Asn Ala Leu Thr Asn Gly Val Leu Pro Ala Val Gln Ser Phe
290 295 300
Ser Cys Leu Pro Tyr Gly Arg Leu Ala Tyr His Leu Ala Val Val
305 310 315
Leu Gly Ser Ala Ala Asn Pro Leu Ala Cys Phe Leu Ala Met Gly
320 325 330
Val Leu Cys Arg Ser Leu Ala Gly Leu Gly Gly Leu Ser Leu Leu
335 340 345
Gly Val Phe Cys Gly Gly Tyr Leu Met Ala Leu Ala Val Leu Ser
350 355 360
Pro Cys Pro Pro Leu Val Gly Thr Ser Ala Gly Val Val Leu Val
365 370 375
Val Leu Ser Trp Val Leu Cys Leu Gly Val Phe Ser Tyr Val Lys
380 385 390
Val Ala Ala Ser Ser Leu Leu His Gly Gly Gly Arg Pro Ala Leu
395 400 405
Leu Ala Ala Gly Val Ala Ile Gln Val Gly Ser Leu Leu Gly Ala
410 415 420
Val Ala Met Phe Pro Pro Thr Ser Ile Tyr His Val Phe His Ser
425 430 435
Arg Lys Asp Cys Ala Asp Pro Cys Asp Ser
440 445
104
398
PRT
Homo Sapien
104
Met His Thr Val Ala Thr Ser Gly Pro Asn Ala Ser Trp Gly Ala
1 5 10 15
Pro Ala Asn Ala Ser Gly Cys Pro Gly Cys Gly Ala Asn Ala Ser
20 25 30
Asp Gly Pro Val Pro Ser Pro Arg Ala Val Asp Ala Trp Leu Val
35 40 45
Pro Leu Phe Phe Ala Ala Leu Met Leu Leu Gly Leu Val Gly Asn
50 55 60
Ser Leu Val Ile Tyr Val Ile Cys Arg His Lys Pro Met Arg Thr
65 70 75
Val Thr Asn Phe Tyr Ile Ala Asn Leu Ala Ala Thr Asp Val Thr
80 85 90
Phe Leu Leu Cys Cys Val Pro Phe Thr Ala Leu Leu Tyr Pro Leu
95 100 105
Pro Gly Trp Val Leu Gly Asp Phe Met Cys Lys Phe Val Asn Tyr
110 115 120
Ile Gln Gln Val Ser Val Gln Ala Thr Cys Ala Thr Leu Thr Ala
125 130 135
Met Ser Val Asp Arg Trp Tyr Val Thr Val Phe Pro Leu Arg Ala
140 145 150
Leu His Arg Arg Thr Pro Arg Leu Ala Leu Ala Val Ser Leu Ser
155 160 165
Ile Trp Val Gly Ser Ala Ala Val Ser Ala Pro Val Leu Ala Leu
170 175 180
His Arg Leu Ser Pro Gly Pro Arg Ala Tyr Cys Ser Glu Ala Phe
185 190 195
Pro Ser Arg Ala Leu Glu Arg Ala Phe Ala Leu Tyr Asn Leu Leu
200 205 210
Ala Leu Tyr Leu Leu Pro Leu Leu Ala Thr Cys Ala Cys Tyr Ala
215 220 225
Ala Met Leu Arg His Leu Gly Arg Val Ala Val Arg Pro Ala Pro
230 235 240
Ala Asp Ser Ala Leu Gln Gly Gln Val Leu Ala Glu Arg Ala Gly
245 250 255
Ala Val Arg Ala Lys Val Ser Arg Leu Val Ala Ala Val Val Leu
260 265 270
Leu Phe Ala Ala Cys Trp Gly Pro Ile Gln Leu Phe Leu Val Leu
275 280 285
Gln Ala Leu Gly Pro Ala Gly Ser Trp His Pro Arg Ser Tyr Ala
290 295 300
Ala Tyr Ala Leu Lys Thr Trp Ala His Cys Met Ser Tyr Ser Asn
305 310 315
Ser Ala Leu Asn Pro Leu Leu Tyr Ala Phe Leu Gly Ser His Phe
320 325 330
Arg Gln Ala Phe Arg Arg Val Cys Pro Cys Ala Pro Arg Arg Pro
335 340 345
Arg Arg Pro Arg Arg Pro Gly Pro Ser Asp Pro Ala Ala Pro His
350 355 360
Ala Glu Leu His Arg Leu Gly Ser His Pro Ala Pro Ala Arg Ala
365 370 375
Gln Lys Pro Gly Ser Ser Gly Leu Ala Ala Arg Gly Leu Cys Val
380 385 390
Leu Gly Glu Asp Asn Ala Pro Leu
395
105
359
PRT
Homo Sapien
105
Met Ser Met Asn Asn Ser Lys Gln Leu Val Ser Pro Ala Ala Ala
1 5 10 15
Leu Leu Ser Asn Thr Thr Cys Gln Thr Glu Asn Arg Leu Ser Val
20 25 30
Phe Phe Ser Val Ile Phe Met Thr Val Gly Ile Leu Ser Asn Ser
35 40 45
Leu Ala Ile Ala Ile Leu Met Lys Ala Tyr Gln Arg Phe Arg Gln
50 55 60
Lys Ser Lys Ala Ser Phe Leu Leu Leu Ala Ser Gly Leu Val Ile
65 70 75
Thr Asp Phe Phe Gly His Leu Ile Asn Gly Ala Ile Ala Val Phe
80 85 90
Val Tyr Ala Ser Asp Lys Glu Trp Ile Arg Phe Asp Gln Ser Asn
95 100 105
Val Leu Cys Ser Ile Phe Gly Ile Cys Met Val Phe Ser Gly Leu
110 115 120
Cys Pro Leu Leu Leu Gly Ser Val Met Ala Ile Glu Arg Cys Ile
125 130 135
Gly Val Thr Lys Pro Ile Phe His Ser Thr Lys Ile Thr Ser Lys
140 145 150
His Val Lys Met Met Leu Ser Gly Val Cys Leu Phe Ala Val Phe
155 160 165
Ile Ala Leu Leu Pro Ile Leu Gly His Arg Asp Tyr Lys Ile Gln
170 175 180
Ala Ser Arg Thr Trp Cys Phe Tyr Asn Thr Glu Asp Ile Lys Asp
185 190 195
Trp Glu Asp Arg Phe Tyr Leu Leu Leu Phe Ser Phe Leu Gly Leu
200 205 210
Leu Ala Leu Gly Val Ser Leu Leu Cys Asn Ala Ile Thr Gly Ile
215 220 225
Thr Leu Leu Arg Val Lys Phe Lys Ser Gln Gln His Arg Gln Gly
230 235 240
Arg Ser His His Leu Glu Met Val Ile Gln Leu Leu Ala Ile Met
245 250 255
Cys Val Ser Cys Ile Cys Trp Ser Pro Phe Leu Val Thr Met Ala
260 265 270
Asn Ile Gly Ile Asn Gly Asn His Ser Leu Glu Thr Cys Glu Thr
275 280 285
Thr Leu Phe Ala Leu Arg Met Ala Thr Trp Asn Gln Ile Leu Asp
290 295 300
Pro Trp Val Tyr Ile Leu Leu Arg Lys Ala Val Leu Lys Asn Leu
305 310 315
Tyr Lys Leu Ala Ser Gln Cys Cys Gly Val His Val Ile Ser Leu
320 325 330
His Ile Trp Glu Leu Ser Ser Ile Lys Asn Ser Leu Lys Val Ala
335 340 345
Ala Ile Ser Glu Ser Pro Val Ala Glu Lys Ser Ala Ser Thr
350 355
106
819
PRT
Homo Sapien
106
Met Ser Arg Met Ser Arg His Pro Asp Lys Asp Leu Ala Gln Gly
1 5 10 15
Pro Phe Asn Thr Cys Cys Gly Cys Thr Leu Met Ala Ser Pro Ala
20 25 30
Asn Leu Pro Pro Asn Thr Gln Ala Ala Ala Glu Arg Ala Leu Ser
35 40 45
Gln Ser Arg Trp Lys Arg Val Gln Val Pro Ala Pro Ala Ser Leu
50 55 60
Ser Pro Phe Pro Leu Ala Met Ala Ser Val Ala Phe Trp Ile Ser
65 70 75
Ile Leu Ile Gly Cys Glu Glu Gln Thr Leu Cys Arg Gly Trp Arg
80 85 90
Ser Pro Val Gly Asp Gly Cys Ala His Val Pro Pro Gln Glu Arg
95 100 105
Ala Thr Ala Glu Ala Asp Pro Pro Gly Arg Cys Ser Thr Ser Thr
110 115 120
Ala Ser Ser Thr Ile Cys Gly Leu Trp His Leu Ser Pro Arg Leu
125 130 135
Gln Leu Leu Pro Pro Leu His Ser Arg Gln Gly Glu Glu Ser Gly
140 145 150
Lys Thr Glu Lys Val Leu Leu Trp Gly Arg Glu Gly Leu His Val
155 160 165
Trp Lys Pro Gly Val Leu Gln Pro Asp Val His Gly Thr Ser Asn
170 175 180
Leu Gly Asn Cys Ser Phe Leu His Gly Leu Val Thr Ala Pro Ser
185 190 195
Cys Pro Arg Arg Ala Gly Ala Glu Leu Leu Asn Ser Leu Gly Ser
200 205 210
Gln Phe Ala Ile Ser Leu Phe Glu Val Gln Ser Gly Thr Glu Pro
215 220 225
Ser Ile Thr Gly Val Ala Thr Ser Gly Gln Cys Arg Ala Met Pro
230 235 240
Leu Lys His Tyr Leu Leu Leu Leu Val Gly Cys Gln Ala Trp Gly
245 250 255
Ala Gly Leu Ala Tyr His Gly Cys Pro Ser Glu Cys Thr Cys Ser
260 265 270
Arg Ala Ser Gln Val Glu Cys Thr Gly Ala Arg Ile Val Ala Val
275 280 285
Pro Thr Pro Leu Pro Trp Asn Ala Met Ser Leu Gln Ile Leu Asn
290 295 300
Thr His Ile Thr Glu Leu Asn Glu Ser Pro Phe Leu Asn Ile Ser
305 310 315
Ala Leu Ile Ala Leu Arg Ile Glu Lys Asn Glu Leu Ser Arg Ile
320 325 330
Thr Pro Gly Ala Phe Arg Asn Leu Gly Ser Leu Arg Tyr Leu Ser
335 340 345
Leu Ala Asn Asn Lys Leu Gln Val Leu Pro Ile Gly Leu Phe Gln
350 355 360
Gly Leu Asp Ser Leu Glu Ser Leu Leu Leu Ser Ser Asn Gln Leu
365 370 375
Leu Gln Ile Gln Pro Ala His Phe Ser Gln Cys Ser Asn Leu Lys
380 385 390
Glu Leu Gln Leu His Gly Asn His Leu Glu Tyr Ile Pro Asp Gly
395 400 405
Ala Phe Asp His Leu Val Gly Leu Thr Lys Leu Asn Leu Gly Lys
410 415 420
Asn Ser Leu Thr His Ile Ser Pro Arg Val Phe Gln His Leu Gly
425 430 435
Asn Leu Gln Val Leu Arg Leu Tyr Glu Asn Arg Leu Thr Asp Ile
440 445 450
Pro Met Gly Thr Phe Asp Gly Leu Val Asn Leu Gln Glu Leu Ala
455 460 465
Leu Gln Gln Asn Gln Ile Gly Leu Leu Ser Pro Gly Leu Phe His
470 475 480
Asn Asn His Asn Leu Gln Arg Leu Tyr Leu Ser Asn Asn His Ile
485 490 495
Ser Gln Leu Pro Pro Ser Ile Phe Met Gln Leu Pro Gln Leu Asn
500 505 510
Arg Leu Thr Leu Phe Gly Asn Ser Leu Lys Glu Leu Ser Leu Gly
515 520 525
Ile Phe Gly Pro Met Pro Asn Leu Arg Glu Leu Trp Leu Tyr Asp
530 535 540
Asn His Ile Ser Ser Leu Pro Asp Asn Val Phe Ser Asn Leu Arg
545 550 555
Gln Leu Gln Val Leu Ile Leu Ser Arg Asn Gln Ile Ser Phe Ile
560 565 570
Ser Pro Gly Ala Phe Asn Gly Leu Thr Glu Leu Arg Glu Leu Ser
575 580 585
Leu His Thr Asn Ala Leu Gln Asp Leu Asp Gly Asn Val Phe Arg
590 595 600
Met Leu Ala Asn Leu Gln Asn Ile Ser Leu Gln Asn Asn Arg Leu
605 610 615
Arg Gln Leu Pro Gly Asn Ile Phe Ala Asn Val Asn Gly Leu Met
620 625 630
Ala Ile Gln Leu Gln Asn Asn Gln Leu Glu Asn Leu Pro Leu Gly
635 640 645
Ile Phe Asp His Leu Gly Lys Leu Cys Glu Leu Arg Leu Tyr Asp
650 655 660
Asn Pro Trp Arg Cys Asp Ser Asp Ile Leu Pro Leu Arg Asn Trp
665 670 675
Leu Leu Leu Asn Gln Pro Arg Leu Gly Thr Asp Thr Val Pro Val
680 685 690
Cys Phe Ser Pro Ala Asn Val Arg Gly Gln Ser Leu Ile Ile Ile
695 700 705
Asn Val Asn Val Ala Val Pro Ser Val His Val Pro Glu Val Pro
710 715 720
Ser Tyr Pro Glu Thr Pro Trp Tyr Pro Asp Thr Pro Ser Tyr Pro
725 730 735
Asp Thr Thr Ser Val Ser Ser Thr Thr Glu Leu Thr Ser Pro Val
740 745 750
Glu Asp Tyr Thr Asp Leu Thr Thr Ile Gln Val Thr Asp Asp Arg
755 760 765
Ser Val Trp Gly Met Thr His Ala His Ser Gly Leu Ala Ile Ala
770 775 780
Ala Ile Val Ile Gly Ile Val Ala Leu Ala Cys Ser Leu Ala Ala
785 790 795
Cys Val Gly Cys Cys Cys Cys Lys Lys Arg Ser Gln Ala Val Leu
800 805 810
Met Gln Met Lys Ala Pro Asn Glu Cys
815
107
3014
PRT
Homo Sapien
107
Met Ala Pro Pro Pro Pro Pro Val Leu Pro Val Leu Leu Leu Leu
1 5 10 15
Ala Ala Ala Ala Ala Leu Pro Ala Met Gly Leu Arg Ala Ala Ala
20 25 30
Trp Glu Pro Arg Val Pro Gly Gly Thr Arg Ala Phe Ala Leu Arg
35 40 45
Pro Gly Cys Thr Tyr Ala Val Gly Ala Ala Cys Thr Pro Arg Ala
50 55 60
Pro Arg Glu Leu Leu Asp Val Gly Arg Asp Gly Arg Leu Ala Gly
65 70 75
Arg Arg Arg Val Ser Gly Ala Gly Arg Pro Leu Pro Leu Gln Val
80 85 90
Arg Leu Val Ala Arg Ser Ala Pro Thr Ala Leu Ser Arg Arg Leu
95 100 105
Arg Ala Arg Thr His Leu Pro Gly Cys Gly Ala Arg Ala Arg Leu
110 115 120
Cys Gly Thr Gly Ala Arg Leu Cys Gly Ala Leu Cys Phe Pro Val
125 130 135
Pro Gly Gly Cys Ala Ala Ala Gln His Ser Ala Leu Ala Ala Pro
140 145 150
Thr Thr Leu Pro Ala Cys Arg Cys Pro Pro Arg Pro Arg Pro Arg
155 160 165
Cys Pro Gly Arg Pro Ile Cys Leu Pro Pro Gly Gly Ser Val Arg
170 175 180
Leu Arg Leu Leu Cys Ala Leu Arg Arg Ala Ala Gly Ala Val Arg
185 190 195
Val Gly Leu Ala Leu Glu Ala Ala Thr Ala Gly Thr Pro Ser Ala
200 205 210
Ser Pro Ser Pro Ser Pro Pro Leu Pro Pro Asn Leu Pro Glu Ala
215 220 225
Arg Ala Gly Pro Ala Arg Arg Ala Arg Arg Gly Thr Ser Gly Arg
230 235 240
Gly Ser Leu Lys Phe Pro Met Pro Asn Tyr Gln Val Ala Leu Phe
245 250 255
Glu Asn Glu Pro Ala Gly Thr Leu Ile Leu Gln Leu His Ala His
260 265 270
Tyr Thr Ile Glu Gly Glu Glu Glu Arg Val Ser Tyr Tyr Met Glu
275 280 285
Gly Leu Phe Asp Glu Arg Ser Arg Gly Tyr Phe Arg Ile Asp Ser
290 295 300
Ala Thr Gly Ala Val Ser Thr Asp Ser Val Leu Asp Arg Glu Thr
305 310 315
Lys Glu Thr His Val Leu Arg Val Lys Ala Val Asp Tyr Ser Thr
320 325 330
Pro Pro Arg Ser Ala Thr Thr Tyr Ile Thr Val Leu Val Lys Asp
335 340 345
Thr Asn Asp His Ser Pro Val Phe Glu Gln Ser Glu Tyr Arg Glu
350 355 360
Arg Val Arg Glu Asn Leu Glu Val Gly Tyr Glu Val Leu Thr Ile
365 370 375
Arg Ala Ser Asp Arg Asp Ser Pro Ile Asn Ala Asn Leu Arg Tyr
380 385 390
Arg Val Leu Gly Gly Ala Trp Asp Val Phe Gln Leu Asn Glu Ser
395 400 405
Ser Gly Val Val Ser Thr Arg Ala Val Leu Asp Arg Glu Glu Ala
410 415 420
Ala Glu Tyr Gln Leu Leu Val Glu Ala Asn Asp Gln Gly Arg Asn
425 430 435
Pro Gly Pro Leu Ser Ala Thr Ala Thr Val Tyr Ile Glu Val Glu
440 445 450
Asp Glu Asn Asp Asn Tyr Pro Gln Phe Ser Glu Gln Asn Tyr Val
455 460 465
Val Gln Val Pro Glu Asp Val Gly Leu Asn Thr Ala Val Leu Arg
470 475 480
Val Gln Ala Thr Asp Arg Asp Gln Gly Gln Asn Ala Ala Ile His
485 490 495
Tyr Ser Ile Leu Ser Gly Asn Val Ala Gly Gln Phe Tyr Leu His
500 505 510
Ser Leu Ser Gly Ile Leu Asp Val Ile Asn Pro Leu Asp Phe Glu
515 520 525
Asp Val Gln Lys Tyr Ser Leu Ser Ile Lys Ala Gln Asp Gly Gly
530 535 540
Arg Pro Pro Leu Ile Asn Ser Ser Gly Val Val Ser Val Gln Val
545 550 555
Leu Asp Val Asn Asp Asn Glu Pro Ile Phe Val Ser Ser Pro Phe
560 565 570
Gln Ala Thr Val Leu Glu Asn Val Pro Leu Gly Tyr Pro Val Val
575 580 585
His Ile Gln Ala Val Asp Ala Asp Ser Gly Glu Asn Ala Arg Leu
590 595 600
His Tyr Arg Leu Val Asp Thr Ala Ser Thr Phe Leu Gly Gly Gly
605 610 615
Ser Ala Gly Pro Lys Asn Pro Ala Pro Thr Pro Asp Phe Pro Phe
620 625 630
Gln Ile His Asn Ser Ser Gly Trp Ile Thr Val Cys Ala Glu Leu
635 640 645
Asp Arg Glu Glu Val Glu His Tyr Ser Phe Gly Val Glu Ala Val
650 655 660
Asp His Gly Ser Pro Pro Met Ser Ser Ser Thr Ser Val Ser Ile
665 670 675
Thr Val Leu Asp Val Asn Asp Asn Asp Pro Val Phe Thr Gln Pro
680 685 690
Thr Tyr Glu Leu Arg Leu Asn Glu Asp Ala Ala Val Gly Ser Ser
695 700 705
Val Leu Thr Leu Gln Ala Arg Asp Arg Asp Ala Asn Ser Val Ile
710 715 720
Thr Tyr Gln Leu Thr Gly Gly Asn Thr Arg Asn Arg Phe Ala Leu
725 730 735
Ser Ser Gln Arg Gly Gly Gly Leu Ile Thr Leu Ala Leu Pro Leu
740 745 750
Asp Tyr Lys Gln Glu Gln Gln Tyr Val Leu Ala Val Thr Ala Ser
755 760 765
Asp Gly Thr Arg Ser His Thr Ala His Val Leu Ile Asn Val Thr
770 775 780
Asp Ala Asn Thr His Arg Pro Val Phe Gln Ser Ser His Tyr Thr
785 790 795
Val Ser Val Ser Glu Asp Arg Pro Val Gly Thr Ser Ile Ala Thr
800 805 810
Leu Ser Ala Asn Asp Glu Asp Thr Gly Glu Asn Ala Arg Ile Thr
815 820 825
Tyr Val Ile Gln Asp Pro Val Pro Gln Phe Arg Ile Asp Pro Asp
830 835 840
Ser Gly Thr Met Tyr Thr Met Met Glu Leu Asp Tyr Glu Asn Gln
845 850 855
Val Ala Tyr Thr Leu Thr Ile Met Ala Gln Asp Asn Gly Ile Pro
860 865 870
Gln Lys Ser Asp Thr Thr Thr Leu Glu Ile Leu Ile Leu Asp Ala
875 880 885
Asn Asp Asn Ala Pro Gln Phe Leu Trp Asp Phe Tyr Gln Gly Ser
890 895 900
Ile Phe Glu Asp Ala Pro Pro Ser Thr Ser Ile Leu Gln Val Ser
905 910 915
Ala Thr Asp Arg Asp Ser Gly Pro Asn Gly Arg Leu Leu Tyr Thr
920 925 930
Phe Gln Gly Gly Asp Asp Gly Asp Gly Asp Phe Tyr Ile Glu Pro
935 940 945
Thr Ser Gly Val Ile Arg Thr Gln Arg Arg Leu Asp Arg Glu Asn
950 955 960
Val Ala Val Tyr Asn Leu Trp Ala Leu Ala Val Asp Arg Gly Ser
965 970 975
Pro Thr Pro Leu Ser Ala Ser Val Glu Ile Gln Val Thr Ile Leu
980 985 990
Asp Ile Asn Asp Asn Ala Pro Met Phe Glu Lys Asp Glu Leu Glu
995 1000 1005
Leu Phe Val Glu Glu Asn Asn Pro Val Gly Ser Val Val Ala Lys
1010 1015 1020
Ile Arg Ala Asn Asp Pro Asp Glu Gly Pro Asn Ala Gln Ile Met
1025 1030 1035
Tyr Gln Ile Val Glu Gly Asp Met Arg His Phe Phe Gln Leu Asp
1040 1045 1050
Leu Leu Asn Gly Asp Leu Arg Ala Met Val Glu Leu Asp Phe Glu
1055 1060 1065
Val Arg Arg Glu Tyr Val Leu Val Val Gln Ala Thr Ser Ala Pro
1070 1075 1080
Leu Val Ser Arg Ala Thr Val His Ile Leu Leu Val Asp Gln Asn
1085 1090 1095
Asp Asn Pro Pro Val Leu Pro Asp Phe Gln Ile Leu Phe Asn Asn
1100 1105 1110
Tyr Val Thr Asn Lys Ser Asn Ser Phe Pro Thr Gly Val Ile Gly
1115 1120 1125
Cys Ile Pro Ala His Asp Pro Asp Val Ser Asp Ser Leu Asn Tyr
1130 1135 1140
Thr Phe Val Gln Gly Asn Glu Leu Arg Leu Leu Leu Leu Asp Pro
1145 1150 1155
Ala Thr Gly Glu Leu Gln Leu Ser Arg Asp Leu Asp Asn Asn Arg
1160 1165 1170
Pro Leu Glu Ala Leu Met Glu Val Ser Val Ser Asp Gly Ile His
1175 1180 1185
Ser Val Thr Ala Phe Cys Thr Leu Arg Val Thr Ile Ile Thr Asp
1190 1195 1200
Asp Met Leu Thr Asn Ser Ile Thr Val Arg Leu Glu Asn Met Ser
1205 1210 1215
Gln Glu Lys Phe Leu Ser Pro Leu Leu Ala Leu Phe Val Glu Gly
1220 1225 1230
Val Ala Ala Val Leu Ser Thr Thr Lys Asp Asp Val Phe Val Phe
1235 1240 1245
Asn Val Gln Asn Asp Thr Asp Val Ser Ser Asn Ile Leu Asn Val
1250 1255 1260
Thr Phe Ser Ala Leu Leu Pro Gly Gly Val Arg Gly Gln Phe Phe
1265 1270 1275
Pro Ser Glu Asp Leu Gln Glu Gln Ile Tyr Leu Asn Arg Thr Leu
1280 1285 1290
Leu Thr Thr Ile Ser Thr Gln Arg Val Leu Pro Phe Asp Asp Asn
1295 1300 1305
Ile Cys Leu Arg Glu Pro Cys Glu Asn Tyr Met Lys Cys Val Ser
1310 1315 1320
Val Leu Arg Phe Asp Ser Ser Ala Pro Phe Leu Ser Ser Thr Thr
1325 1330 1335
Val Leu Phe Arg Pro Ile His Pro Ile Asn Gly Leu Arg Cys Arg
1340 1345 1350
Cys Pro Pro Gly Phe Thr Gly Asp Tyr Cys Glu Thr Glu Ile Asp
1355 1360 1365
Leu Cys Tyr Ser Asp Pro Cys Gly Ala Asn Gly Arg Cys Arg Ser
1370 1375 1380
Arg Glu Gly Gly Tyr Thr Cys Glu Cys Phe Glu Asp Phe Thr Gly
1385 1390 1395
Glu His Cys Glu Val Asp Ala Arg Ser Gly Arg Cys Ala Asn Gly
1400 1405 1410
Val Cys Lys Asn Gly Gly Thr Cys Val Asn Leu Leu Ile Gly Gly
1415 1420 1425
Phe His Cys Val Cys Pro Pro Gly Glu Tyr Glu Arg Pro Tyr Cys
1430 1435 1440
Glu Val Thr Thr Arg Ser Phe Pro Pro Gln Ser Phe Val Thr Phe
1445 1450 1455
Arg Gly Leu Arg Gln Arg Phe His Phe Thr Ile Ser Leu Thr Phe
1460 1465 1470
Ala Thr Gln Glu Arg Asn Gly Leu Leu Leu Tyr Asn Gly Arg Phe
1475 1480 1485
Asn Glu Lys His Asp Phe Ile Ala Leu Glu Ile Val Asp Glu Gln
1490 1495 1500
Val Gln Leu Thr Phe Ser Ala Gly Glu Thr Thr Thr Thr Val Ala
1505 1510 1515
Pro Lys Val Pro Ser Gly Val Ser Asp Gly Arg Trp His Ser Val
1520 1525 1530
Gln Val Gln Tyr Tyr Asn Lys Pro Asn Ile Gly His Leu Gly Leu
1535 1540 1545
Pro His Gly Pro Ser Gly Glu Lys Met Ala Val Val Thr Val Asp
1550 1555 1560
Asp Cys Asp Thr Thr Met Ala Val Arg Phe Gly Lys Asp Ile Gly
1565 1570 1575
Asn Tyr Ser Cys Ala Ala Gln Gly Thr Gln Thr Gly Ser Lys Lys
1580 1585 1590
Ser Leu Asp Leu Thr Gly Pro Leu Leu Leu Gly Gly Val Pro Asn
1595 1600 1605
Leu Pro Glu Asp Phe Pro Val His Asn Arg Gln Phe Val Gly Cys
1610 1615 1620
Met Arg Asn Leu Ser Val Asp Gly Lys Asn Val Asp Met Ala Gly
1625 1630 1635
Phe Ile Ala Asn Asn Gly Thr Arg Glu Gly Cys Ala Ala Arg Arg
1640 1645 1650
Asn Phe Cys Asp Gly Arg Arg Cys Gln Asn Gly Gly Thr Cys Val
1655 1660 1665
Asn Arg Trp Asn Met Tyr Leu Cys Glu Cys Pro Leu Arg Phe Gly
1670 1675 1680
Gly Lys Asn Cys Glu Gln Ala Met Pro His Pro Gln Leu Phe Ser
1685 1690 1695
Gly Glu Ser Val Val Ser Trp Ser Asp Leu Asn Ile Ile Ile Ser
1700 1705 1710
Val Pro Trp Tyr Leu Gly Leu Met Phe Arg Thr Arg Lys Glu Asp
1715 1720 1725
Ser Val Leu Met Glu Ala Thr Ser Gly Gly Pro Thr Ser Phe Arg
1730 1735 1740
Leu Gln Ile Leu Asn Asn Tyr Leu Gln Phe Glu Val Ser His Gly
1745 1750 1755
Pro Ser Asp Val Glu Ser Val Met Leu Ser Gly Leu Arg Val Thr
1760 1765 1770
Asp Gly Glu Trp His His Leu Leu Ile Glu Leu Lys Asn Val Lys
1775 1780 1785
Glu Asp Ser Glu Met Lys His Leu Val Thr Met Thr Leu Asp Tyr
1790 1795 1800
Gly Met Asp Gln Asn Lys Ala Asp Ile Gly Gly Met Leu Pro Gly
1805 1810 1815
Leu Thr Val Arg Ser Val Val Val Gly Gly Ala Ser Glu Asp Lys
1820 1825 1830
Val Ser Val Arg Arg Gly Phe Arg Gly Cys Met Gln Gly Val Arg
1835 1840 1845
Met Gly Gly Thr Pro Thr Asn Val Ala Thr Leu Asn Met Asn Asn
1850 1855 1860
Ala Leu Lys Val Arg Val Lys Asp Gly Cys Asp Val Asp Asp Pro
1865 1870 1875
Cys Thr Ser Ser Pro Cys Pro Pro Asn Ser Arg Cys His Asp Ala
1880 1885 1890
Trp Glu Asp Tyr Ser Cys Val Cys Asp Lys Gly Tyr Leu Gly Ile
1895 1900 1905
Asn Cys Val Asp Ala Cys His Leu Asn Pro Cys Glu Asn Met Gly
1910 1915 1920
Ala Cys Val Arg Ser Pro Gly Ser Pro Gln Gly Tyr Val Cys Glu
1925 1930 1935
Cys Gly Pro Ser His Tyr Gly Pro Tyr Cys Glu Asn Lys Leu Asp
1940 1945 1950
Leu Pro Cys Pro Arg Gly Trp Trp Gly Asn Pro Val Cys Gly Pro
1955 1960 1965
Cys His Cys Ala Val Ser Lys Gly Phe Asp Pro Asp Cys Asn Lys
1970 1975 1980
Thr Asn Gly Gln Cys Gln Cys Lys Glu Asn Tyr Tyr Lys Leu Leu
1985 1990 1995
Ala Gln Asp Thr Cys Leu Pro Cys Asp Cys Phe Pro His Gly Ser
2000 2005 2010
His Ser Arg Thr Cys Asp Met Ala Thr Gly Gln Cys Ala Cys Lys
2015 2020 2025
Pro Gly Val Ile Gly Arg Gln Cys Asn Arg Cys Asp Asn Pro Phe
2030 2035 2040
Ala Glu Val Thr Thr Leu Gly Cys Glu Val Ile Tyr Asn Gly Cys
2045 2050 2055
Pro Lys Ala Phe Glu Ala Gly Ile Trp Trp Pro Gln Thr Lys Phe
2060 2065 2070
Gly Gln Pro Ala Ala Val Pro Cys Pro Lys Gly Ser Val Gly Asn
2075 2080 2085
Ala Val Arg His Cys Ser Gly Glu Lys Gly Trp Leu Pro Pro Glu
2090 2095 2100
Leu Phe Asn Cys Thr Thr Ile Ser Phe Val Asp Leu Arg Ala Met
2105 2110 2115
Asn Glu Lys Leu Ser Arg Asn Glu Thr Gln Val Asp Gly Ala Arg
2120 2125 2130
Ala Leu Gln Leu Val Arg Ala Leu Arg Ser Ala Thr Gln His Thr
2135 2140 2145
Gly Thr Leu Phe Gly Asn Asp Val Arg Thr Ala Tyr Gln Leu Leu
2150 2155 2160
Gly His Val Leu Gln His Glu Ser Trp Gln Gln Gly Phe Asp Leu
2165 2170 2175
Ala Ala Thr Gln Asp Ala Asp Phe His Glu Asp Val Ile His Ser
2180 2185 2190
Gly Ser Ala Leu Leu Ala Pro Ala Thr Arg Ala Ala Trp Glu Gln
2195 2200 2205
Ile Gln Arg Ser Glu Gly Gly Thr Ala Gln Leu Leu Arg Arg Leu
2210 2215 2220
Glu Gly Tyr Phe Ser Asn Val Ala Arg Asn Val Arg Arg Thr Tyr
2225 2230 2235
Leu Arg Pro Phe Val Ile Val Thr Ala Asn Met Ile Leu Ala Val
2240 2245 2250
Asp Ile Phe Asp Lys Phe Asn Phe Thr Gly Ala Arg Val Pro Arg
2255 2260 2265
Phe Asp Thr Ile His Glu Glu Phe Pro Arg Glu Leu Glu Ser Ser
2270 2275 2280
Val Ser Phe Pro Ala Asp Phe Phe Arg Pro Pro Glu Glu Lys Glu
2285 2290 2295
Gly Pro Leu Leu Arg Pro Ala Gly Arg Arg Thr Thr Pro Gln Thr
2300 2305 2310
Thr Arg Pro Gly Pro Gly Thr Glu Arg Glu Ala Pro Ile Ser Arg
2315 2320 2325
Arg Arg Arg His Pro Asp Asp Ala Gly Gln Phe Ala Val Ala Leu
2330 2335 2340
Val Ile Ile Tyr Arg Thr Leu Gly Gln Leu Leu Pro Glu Arg Tyr
2345 2350 2355
Asp Pro Asp Arg Arg Ser Leu Arg Leu Pro His Arg Pro Ile Ile
2360 2365 2370
Asn Thr Pro Met Val Ser Thr Leu Val Tyr Ser Glu Gly Ala Pro
2375 2380 2385
Leu Pro Arg Pro Leu Glu Arg Pro Val Leu Val Glu Phe Ala Leu
2390 2395 2400
Leu Glu Val Glu Glu Arg Thr Lys Pro Val Cys Val Phe Trp Asn
2405 2410 2415
His Ser Leu Ala Val Gly Gly Thr Gly Gly Trp Ser Ala Arg Gly
2420 2425 2430
Cys Glu Leu Leu Ser Arg Asn Arg Thr His Val Ala Cys Gln Cys
2435 2440 2445
Ser His Thr Ala Ser Phe Ala Val Leu Met Asp Ile Ser Arg Arg
2450 2455 2460
Glu Asn Gly Glu Val Leu Pro Leu Lys Ile Val Thr Tyr Ala Ala
2465 2470 2475
Val Ser Leu Ser Leu Ala Ala Leu Leu Val Ala Phe Val Leu Leu
2480 2485 2490
Ser Leu Val Arg Met Leu Arg Ser Asn Leu His Ser Ile His Lys
2495 2500 2505
His Leu Ala Val Ala Leu Phe Leu Ser Gln Leu Val Phe Val Ile
2510 2515 2520
Gly Ile Asn Gln Thr Glu Asn Pro Phe Leu Cys Thr Val Val Ala
2525 2530 2535
Ile Leu Leu His Tyr Ile Tyr Met Ser Thr Phe Ala Trp Thr Leu
2540 2545 2550
Val Glu Ser Leu His Val Tyr Arg Met Leu Thr Glu Val Arg Asn
2555 2560 2565
Ile Asp Thr Gly Pro Met Arg Phe Tyr Tyr Val Val Gly Trp Gly
2570 2575 2580
Ile Pro Ala Ile Val Thr Gly Leu Ala Val Gly Leu Asp Pro Gln
2585 2590 2595
Gly Tyr Gly Asn Pro Asp Phe Cys Trp Leu Ser Leu Gln Asp Thr
2600 2605 2610
Leu Ile Trp Ser Phe Ala Gly Pro Ile Gly Ala Val Ile Ile Ile
2615 2620 2625
Asn Thr Val Thr Ser Val Leu Ser Ala Lys Val Ser Cys Gln Arg
2630 2635 2640
Lys His His Tyr Tyr Gly Lys Lys Gly Ile Val Ser Leu Leu Arg
2645 2650 2655
Thr Ala Phe Leu Leu Leu Leu Leu Ile Ser Ala Thr Trp Leu Leu
2660 2665 2670
Gly Leu Leu Ala Val Asn Arg Asp Ala Leu Ser Phe His Tyr Leu
2675 2680 2685
Phe Ala Ile Phe Ser Gly Leu Gln Gly Pro Phe Val Leu Leu Phe
2690 2695 2700
His Cys Val Leu Asn Gln Glu Val Arg Lys His Leu Lys Gly Val
2705 2710 2715
Leu Gly Gly Arg Lys Leu His Leu Glu Asp Ser Ala Thr Thr Arg
2720 2725 2730
Ala Thr Leu Leu Thr Arg Ser Leu Asn Cys Asn Thr Thr Phe Gly
2735 2740 2745
Asp Gly Pro Asp Met Leu Arg Thr Asp Leu Gly Glu Ser Thr Ala
2750 2755 2760
Ser Leu Asp Ser Ile Val Arg Asp Glu Gly Ile Gln Lys Leu Gly
2765 2770 2775
Val Ser Ser Gly Leu Val Arg Gly Ser His Gly Glu Pro Asp Ala
2780 2785 2790
Ser Leu Met Pro Arg Ser Cys Lys Asp Pro Pro Gly His Asp Ser
2795 2800 2805
Asp Ser Asp Ser Glu Leu Ser Leu Asp Glu Gln Ser Ser Ser Tyr
2810 2815 2820
Ala Ser Ser His Ser Ser Asp Ser Glu Asp Asp Gly Val Gly Ala
2825 2830 2835
Glu Glu Lys Trp Asp Pro Ala Arg Gly Ala Val His Ser Thr Pro
2840 2845 2850
Lys Gly Asp Ala Val Ala Asn His Val Pro Ala Gly Trp Pro Asp
2855 2860 2865
Gln Ser Leu Ala Glu Ser Asp Ser Glu Asp Pro Ser Gly Lys Pro
2870 2875 2880
Arg Leu Lys Val Glu Thr Lys Val Ser Val Glu Leu His Arg Glu
2885 2890 2895
Glu Gln Gly Ser His Arg Gly Glu Tyr Pro Pro Asp Gln Glu Ser
2900 2905 2910
Gly Gly Ala Ala Arg Leu Ala Ser Ser Gln Pro Pro Glu Gln Arg
2915 2920 2925
Lys Gly Ile Leu Lys Asn Lys Val Thr Tyr Pro Pro Pro Leu Thr
2930 2935 2940
Leu Thr Glu Gln Thr Leu Lys Gly Arg Leu Arg Glu Lys Leu Ala
2945 2950 2955
Asp Cys Glu Gln Ser Pro Thr Ser Ser Arg Thr Ser Ser Leu Gly
2960 2965 2970
Ser Gly Gly Pro Asp Cys Ala Ile Thr Val Lys Ser Pro Gly Arg
2975 2980 2985
Glu Pro Gly Arg Asp His Leu Asn Gly Val Ala Met Asn Val Arg
2990 2995 3000
Thr Gly Ser Ala Gln Ala Asp Gly Ser Asp Ser Glu Lys Pro
3005 3010
108
181
PRT
Homo Sapien
108
Met Val Asp Val Lys Cys Leu Ser Asp Cys Lys Leu Gln Asn Gln
1 5 10 15
Leu Glu Lys Leu Gly Phe Ser Pro Gly Pro Ile Leu Pro Ser Thr
20 25 30
Arg Lys Leu Tyr Glu Lys Lys Leu Val Gln Leu Leu Val Ser Pro
35 40 45
Pro Cys Ala Pro Pro Val Met Asn Gly Pro Arg Glu Leu Asp Gly
50 55 60
Ala Gln Asp Ser Asp Asp Ser Glu Glu Leu Asn Ile Ile Leu Gln
65 70 75
Gly Asn Ile Ile Leu Ser Thr Glu Lys Ser Lys Lys Leu Lys Lys
80 85 90
Trp Pro Glu Ala Ser Thr Thr Lys Arg Lys Ala Val Asp Thr Tyr
95 100 105
Cys Leu Asp Tyr Lys Pro Ser Lys Gly Arg Arg Trp Ala Ala Arg
110 115 120
Ala Pro Ser Thr Arg Ile Thr Tyr Gly Thr Ile Thr Lys Glu Arg
125 130 135
Asp Tyr Cys Ala Glu Asp Gln Thr Ile Glu Ser Trp Arg Glu Glu
140 145 150
Gly Phe Pro Val Gly Leu Lys Leu Ala Val Leu Gly Ile Phe Ile
155 160 165
Ile Val Val Phe Val Tyr Leu Thr Val Glu Asn Lys Ser Leu Phe
170 175 180
Gly
109
620
PRT
Homo Sapien
109
Met Ser Lys Ser Lys Cys Ser Val Gly Leu Met Ser Ser Val Val
1 5 10 15
Ala Pro Ala Lys Glu Pro Asn Ala Val Gly Pro Lys Glu Val Glu
20 25 30
Leu Ile Leu Val Lys Glu Gln Asn Gly Val Gln Leu Thr Ser Ser
35 40 45
Thr Leu Thr Asn Pro Arg Gln Ser Pro Val Glu Ala Gln Asp Arg
50 55 60
Glu Thr Trp Gly Lys Lys Ile Asp Phe Leu Leu Ser Val Ile Gly
65 70 75
Phe Ala Val Asp Leu Ala Asn Val Trp Arg Phe Pro Tyr Leu Cys
80 85 90
Tyr Lys Asn Gly Gly Gly Ala Phe Leu Val Pro Tyr Leu Leu Phe
95 100 105
Met Val Ile Ala Gly Met Pro Leu Phe Tyr Met Glu Leu Ala Leu
110 115 120
Gly Gln Phe Asn Arg Glu Gly Ala Ala Gly Val Trp Lys Ile Cys
125 130 135
Pro Ile Leu Lys Gly Val Gly Phe Thr Val Ile Leu Ile Ser Leu
140 145 150
Tyr Val Gly Phe Phe Tyr Asn Val Ile Ile Ala Trp Ala Leu His
155 160 165
Tyr Leu Phe Ser Ser Phe Thr Thr Glu Leu Pro Trp Ile His Cys
170 175 180
Asn Asn Ser Trp Asn Ser Pro Asn Cys Ser Asp Ala His Pro Gly
185 190 195
Asp Ser Ser Gly Asp Ser Ser Gly Leu Asn Asp Thr Phe Gly Thr
200 205 210
Thr Pro Ala Ala Glu Tyr Phe Glu Arg Gly Val Leu His Leu His
215 220 225
Gln Ser His Gly Ile Asp Asp Leu Gly Pro Pro Arg Trp Gln Leu
230 235 240
Thr Ala Cys Leu Val Leu Val Ile Val Leu Leu Tyr Phe Ser Leu
245 250 255
Trp Lys Gly Val Lys Thr Ser Gly Lys Val Val Trp Ile Thr Ala
260 265 270
Thr Met Pro Tyr Val Val Leu Thr Ala Leu Leu Leu Arg Gly Val
275 280 285
Thr Leu Pro Gly Ala Ile Asp Gly Ile Arg Ala Tyr Leu Ser Val
290 295 300
Asp Phe Tyr Arg Leu Cys Glu Ala Ser Val Trp Ile Asp Ala Ala
305 310 315
Thr Gln Val Cys Phe Ser Leu Gly Val Gly Phe Gly Val Leu Ile
320 325 330
Ala Phe Ser Ser Tyr Asn Lys Phe Thr Asn Asn Cys Tyr Arg Asp
335 340 345
Ala Ile Val Thr Thr Ser Ile Asn Ser Leu Thr Ser Phe Ser Ser
350 355 360
Gly Phe Val Val Phe Ser Phe Leu Gly Tyr Met Ala Gln Lys His
365 370 375
Ser Val Pro Ile Gly Asp Val Ala Lys Asp Gly Pro Gly Leu Ile
380 385 390
Phe Ile Ile Tyr Pro Glu Ala Ile Ala Thr Leu Pro Leu Ser Ser
395 400 405
Ala Trp Ala Val Val Phe Phe Ile Met Leu Leu Thr Leu Gly Ile
410 415 420
Asp Ser Ala Met Gly Gly Met Glu Ser Val Ile Thr Gly Leu Ile
425 430 435
Asp Glu Phe Gln Leu Leu His Arg His Arg Glu Leu Phe Thr Leu
440 445 450
Phe Ile Val Leu Ala Thr Phe Leu Leu Ser Leu Phe Cys Val Thr
455 460 465
Asn Gly Gly Ile Tyr Val Phe Thr Leu Leu Asp His Phe Ala Ala
470 475 480
Gly Thr Ser Ile Leu Phe Gly Val Leu Ile Glu Ala Ile Gly Val
485 490 495
Ala Trp Phe Tyr Gly Val Gly Gln Phe Ser Asp Asp Ile Gln Gln
500 505 510
Met Thr Gly Gln Arg Pro Ser Leu Tyr Trp Arg Leu Cys Trp Lys
515 520 525
Leu Val Ser Pro Cys Phe Leu Leu Phe Val Val Val Val Ser Ile
530 535 540
Val Thr Phe Arg Pro Pro His Tyr Gly Ala Tyr Ile Phe Pro Asp
545 550 555
Trp Ala Asn Ala Leu Gly Trp Val Ile Ala Thr Ser Ser Met Ala
560 565 570
Met Val Pro Ile Tyr Ala Ala Tyr Lys Phe Cys Ser Leu Pro Gly
575 580 585
Ser Phe Arg Glu Lys Leu Ala Tyr Ala Ile Ala Pro Glu Lys Asp
590 595 600
Arg Glu Leu Val Asp Arg Gly Glu Val Arg Gln Phe Thr Leu Arg
605 610 615
His Trp Leu Lys Val
620
110
442
PRT
Homo Sapien
110
Met Gly Leu Ala Met Glu His Gly Gly Ser Tyr Ala Arg Ala Gly
1 5 10 15
Gly Ser Ser Arg Gly Cys Trp Tyr Tyr Leu Arg Tyr Phe Phe Leu
20 25 30
Phe Val Ser Leu Ile Gln Phe Leu Ile Ile Leu Gly Leu Val Leu
35 40 45
Phe Met Val Tyr Gly Asn Val His Val Ser Thr Glu Ser Asn Leu
50 55 60
Gln Ala Thr Glu Arg Arg Ala Glu Gly Leu Tyr Ser Gln Leu Leu
65 70 75
Gly Leu Thr Ala Ser Gln Ser Asn Leu Thr Lys Glu Leu Asn Phe
80 85 90
Thr Thr Arg Ala Lys Asp Ala Ile Met Gln Met Trp Leu Asn Ala
95 100 105
Arg Arg Asp Leu Asp Arg Ile Asn Ala Ser Phe Arg Gln Cys Gln
110 115 120
Gly Asp Arg Val Ile Tyr Thr Asn Asn Gln Arg Tyr Met Ala Ala
125 130 135
Ile Ile Leu Ser Glu Lys Gln Cys Arg Asp Gln Phe Lys Asp Met
140 145 150
Asn Lys Ser Cys Asp Ala Leu Leu Phe Met Leu Asn Gln Lys Val
155 160 165
Lys Thr Leu Glu Val Glu Ile Ala Lys Glu Lys Thr Ile Cys Thr
170 175 180
Lys Asp Lys Glu Ser Val Leu Leu Asn Lys Arg Val Ala Glu Glu
185 190 195
Gln Leu Val Glu Cys Val Lys Thr Arg Glu Leu Gln His Gln Glu
200 205 210
Arg Gln Leu Ala Lys Glu Gln Leu Gln Lys Val Gln Ala Leu Cys
215 220 225
Leu Pro Leu Asp Lys Asp Lys Phe Glu Met Asp Leu Arg Asn Leu
230 235 240
Trp Arg Asp Ser Ile Ile Pro Arg Ser Leu Asp Asn Leu Gly Tyr
245 250 255
Asn Leu Tyr His Pro Leu Gly Ser Glu Leu Ala Ser Ile Arg Arg
260 265 270
Ala Cys Asp His Met Pro Ser Leu Met Ser Ser Lys Val Glu Glu
275 280 285
Leu Ala Arg Ser Leu Arg Ala Asp Ile Glu Arg Val Ala Arg Glu
290 295 300
Asn Ser Asp Leu Gln Arg Gln Lys Leu Glu Ala Gln Gln Gly Leu
305 310 315
Arg Ala Ser Gln Glu Ala Lys Gln Lys Val Glu Lys Glu Ala Gln
320 325 330
Ala Arg Glu Ala Lys Leu Gln Ala Glu Cys Ser Arg Gln Thr Gln
335 340 345
Leu Ala Leu Glu Glu Lys Ala Val Leu Arg Lys Glu Arg Asp Asn
350 355 360
Leu Ala Lys Glu Leu Glu Glu Lys Lys Arg Glu Ala Glu Gln Leu
365 370 375
Arg Met Glu Leu Ala Ile Arg Asn Ser Ala Leu Asp Thr Cys Ile
380 385 390
Lys Thr Lys Ser Gln Pro Met Met Pro Val Ser Arg Pro Met Gly
395 400 405
Pro Val Pro Asn Pro Gln Pro Ile Asp Pro Ala Ser Leu Glu Glu
410 415 420
Phe Lys Arg Lys Ile Leu Glu Ser Gln Arg Pro Pro Ala Gly Ile
425 430 435
Pro Val Ala Pro Ser Ser Gly
440
111
170
PRT
Homo Sapien
111
Met Met Ala Gly Met Lys Ile Gln Leu Val Cys Met Leu Leu Leu
1 5 10 15
Ala Phe Ser Ser Trp Ser Leu Cys Ser Asp Ser Glu Glu Glu Met
20 25 30
Lys Ala Leu Glu Ala Asp Phe Leu Thr Asn Met His Thr Ser Lys
35 40 45
Ile Ser Lys Ala His Val Pro Ser Trp Lys Met Thr Leu Leu Asn
50 55 60
Val Cys Ser Leu Val Asn Asn Leu Asn Ser Pro Ala Glu Glu Thr
65 70 75
Gly Glu Val His Glu Glu Glu Leu Val Ala Arg Arg Lys Leu Pro
80 85 90
Thr Ala Leu Asp Gly Phe Ser Leu Glu Ala Met Leu Thr Ile Tyr
95 100 105
Gln Leu His Lys Ile Cys His Ser Arg Ala Phe Gln His Trp Glu
110 115 120
Leu Ile Gln Glu Asp Ile Leu Asp Thr Gly Asn Asp Lys Asn Gly
125 130 135
Lys Glu Glu Val Ile Lys Arg Lys Ile Pro Tyr Ile Leu Lys Arg
140 145 150
Gln Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu Lys Arg
155 160 165
Asp Ser Tyr Tyr Tyr
170
112
502
PRT
Homo Sapien
112
Met Leu Leu Arg Ser Ala Gly Lys Leu Asn Val Gly Thr Lys Lys
1 5 10 15
Glu Asp Gly Glu Ser Thr Ala Pro Thr Pro Arg Pro Lys Val Leu
20 25 30
Arg Cys Lys Cys His His His Cys Pro Glu Asp Ser Val Asn Asn
35 40 45
Ile Cys Ser Thr Asp Gly Tyr Cys Phe Thr Met Ile Glu Glu Asp
50 55 60
Asp Ser Gly Leu Pro Val Val Thr Ser Gly Cys Leu Gly Leu Glu
65 70 75
Gly Ser Asp Phe Gln Cys Arg Asp Thr Pro Ile Pro His Gln Arg
80 85 90
Arg Ser Ile Glu Cys Cys Thr Glu Arg Asn Glu Cys Asn Lys Asp
95 100 105
Leu His Pro Thr Leu Pro Pro Leu Lys Asn Arg Asp Phe Val Asp
110 115 120
Gly Pro Ile His His Arg Ala Leu Leu Ile Ser Val Thr Val Cys
125 130 135
Ser Leu Leu Leu Val Leu Ile Ile Leu Phe Cys Tyr Phe Arg Tyr
140 145 150
Lys Arg Gln Glu Thr Arg Pro Arg Tyr Ser Ile Gly Leu Glu Gln
155 160 165
Asp Glu Thr Tyr Ile Pro Pro Gly Glu Ser Leu Arg Asp Leu Ile
170 175 180
Glu Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu Pro Leu Leu
185 190 195
Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Lys Gln Ile
200 205 210
Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg Gly
215 220 225
Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser
230 235 240
Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His
245 250 255
Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly
260 265 270
Ser Trp Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly
275 280 285
Ser Leu Tyr Asp Tyr Leu Lys Ser Thr Thr Leu Asp Ala Lys Ser
290 295 300
Met Leu Lys Leu Ala Tyr Ser Ser Val Ser Gly Leu Cys His Leu
305 310 315
His Thr Glu Ile Phe Ser Thr Gln Gly Lys Pro Ala Ile Ala His
320 325 330
Arg Asp Leu Lys Ser Lys Asn Ile Leu Val Lys Lys Asn Gly Thr
335 340 345
Cys Cys Ile Ala Asp Leu Gly Leu Ala Val Lys Phe Ile Ser Asp
350 355 360
Thr Asn Glu Val Asp Ile Pro Pro Asn Thr Arg Val Gly Thr Lys
365 370 375
Arg Tyr Met Pro Pro Glu Val Leu Asp Glu Ser Leu Asn Arg Asn
380 385 390
His Phe Gln Ser Tyr Ile Met Ala Asp Met Tyr Ser Phe Gly Leu
395 400 405
Ile Leu Trp Glu Val Ala Arg Arg Cys Val Ser Gly Gly Ile Val
410 415 420
Glu Glu Tyr Gln Leu Pro Tyr His Asp Leu Val Pro Ser Asp Pro
425 430 435
Ser Tyr Glu Asp Met Arg Glu Ile Val Cys Ile Lys Lys Leu Arg
440 445 450
Pro Ser Phe Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg Gln
455 460 465
Met Gly Lys Leu Met Thr Glu Cys Trp Ala His Asn Pro Ala Ser
470 475 480
Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser
485 490 495
Glu Ser Gln Asp Ile Lys Leu
500
113
2403
DNA
Homo Sapien
113
ttgaagtgca ttgctgcagc tggtagcatg agtggtggcc accacctgca 50
gctggctgcc ctctggccct ggctgctgat ggctaccctg caggcaggct 100
ttggacgcac aggactggta ctggcagcag cggtggagtc tgaaagatca 150
gcagaacaga aagctgttat cagagtgatc cccttgaaaa tggaccccac 200
aggaaaactg aatctcactt tggaaggtgt gtttgctggt gttgctgaaa 250
taactccagc agaaggaaaa ttaatgcagt cccacccgct gtacctgtgc 300
aatgccagtg atgacgacaa tctggagcct ggattcatca gcatcgtcaa 350
gctggagagt cctcgacggg ccccccaccc ctgcctgtca ctggctagca 400
aggctcggat ggcgggtgag cgaggagcca gtgctgtcct ctttgacatc 450
actgaggatc gagctgctgc tgagcagctg cagcagccgc tggggctgac 500
ctggccagtg gtgttgatct ggggtaatga cgctgagaag ctgatggagt 550
ttgtgtacaa gaaccaaaag gcccatgtga ggattgagct gaaggagccc 600
ccggcctggc cagattatga tgtgtggatc ctaatgacag tggtgggcac 650
catctttgtg atcatcctgg cttcggtgct gcgcatccgg tgccgccccc 700
gccacagcag gccggatccg cttcagcaga gaacagcctg ggccatcagc 750
cagctggcca ccaggaggta ccaggccagc tgcaggcagg cccggggtga 800
gtggccagac tcagggagca gctgcagctc agcccctgtg tgtgccatct 850
gtctggagga gttctctgag gggcaggagc tacgggtcat ttcctgcctc 900
catgagttcc atcgtaactg tgtggacccc tggttacatc agcatcggac 950
ttgccccctc tgcatgttca acatcacaga gggagattca ttttcccagt 1000
ccctgggacc ctctcgatct taccaagaac caggtcgaag actccacctc 1050
attcgccagc atcccggcca tgcccactac cacctccctg ctgcctacct 1100
gttgggccct tcccggagtg cagtggctcg gcccccacga cctggtccct 1150
tcctgccatc ccaggagcca ggcatgggcc ctcggcatca ccgcttcccc 1200
agagctacac atccccgggc tccaggagag cagcagcgcc tggcaggagc 1250
ccagcacccc tatgcacaag gctggggact gagccacctc caatccacct 1300
cacagcaccc tgctgcttgc ccagtgcccc tacgccgggc caggccccct 1350
gacagcagtg gatctggaga aagctattgc acagaacgca gtgggtacct 1400
ggcagatggg ccagccagtg actccagctc agggccctgt catggctctt 1450
ccagtgactc tgtggtcaac tgcacggaca tcagcctaca gggggtccat 1500
ggcagcagtt ctactttctg cagctcccta agcagtgact ttgaccccct 1550
agtgtactgc agccctaaag gggatcccca gcgagtggac atgcagccta 1600
gtgtgacctc tcggcctcgt tccttggact cggtggtgcc cacaggggaa 1650
acccaggttt ccagccatgt ccactaccac cgccaccggc accaccacta 1700
caaaaagcgg ttccagtggc atggcaggaa gcctggccca gaaaccggag 1750
tcccccagtc caggcctcct attcctcgga cacagcccca gccagagcca 1800
ccttctcctg atcagcaagt caccagatcc aactcagcag ccccttcggg 1850
gcggctctct aacccacagt gccccagggc cctccctgag ccagcccctg 1900
gcccagttga cgcctccagc atctgcccca gtaccagcag tctgttcaac 1950
ttgcaaaaat ccagcctctc tgcccgacac ccacagagga aaaggcgggg 2000
gggtccctcc gagcccaccc ctggctctcg gccccaggat gcaactgtgc 2050
acccagcttg ccagattttt ccccattaca cccccagtgt ggcatatcct 2100
tggtccccag aggcacaccc cttgatctgt ggacctccag gcctggacaa 2150
gaggctgcta ccagaaaccc caggcccctg ttactcaaat tcacagccag 2200
tgtggttgtg cctgactcct cgccagcccc tggaaccaca tccacctggg 2250
gaggggcctt ctgaatggag ttctgacacc gcagagggca ggccatgccc 2300
ttatccgcac tgccaggtgc tgtcggccca gcctggctca gaggaggaac 2350
tcgaggagct gtgtgaacag gctgtgtgag atgttcaggc ctagctccaa 2400
cca 2403
114
783
PRT
Homo Sapien
114
Met Ser Gly Gly His His Leu Gln Leu Ala Ala Leu Trp Pro Trp
1 5 10 15
Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu
20 25 30
Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys
35 40 45
Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys
50 55 60
Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile
65 70 75
Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu
80 85 90
Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser
95 100 105
Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro His Pro Cys Leu
110 115 120
Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser
125 130 135
Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln
140 145 150
Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp
155 160 165
Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln
170 175 180
Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro
185 190 195
Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe
200 205 210
Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg Pro Arg
215 220 225
His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile
230 235 240
Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala
245 250 255
Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro
260 265 270
Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu
275 280 285
Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val Asp
290 295 300
Pro Trp Leu His Gln His Arg Thr Cys Pro Leu Cys Met Phe Asn
305 310 315
Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg
320 325 330
Ser Tyr Gln Glu Pro Gly Arg Arg Leu His Leu Ile Arg Gln His
335 340 345
Pro Gly His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly
350 355 360
Pro Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro Gly Pro Phe
365 370 375
Leu Pro Ser Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe
380 385 390
Pro Arg Ala Thr His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu
395 400 405
Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Leu Ser His
410 415 420
Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu
425 430 435
Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr
440 445 450
Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp
455 460 465
Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val
470 475 480
Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser
485 490 495
Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr
500 505 510
Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser
515 520 525
Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly
530 535 540
Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg His
545 550 555
His His Tyr Lys Lys Arg Phe Gln Trp His Gly Arg Lys Pro Gly
560 565 570
Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr
575 580 585
Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln Gln Val Thr Arg
590 595 600
Ser Asn Ser Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys
605 610 615
Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser
620 625 630
Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser
635 640 645
Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro
650 655 660
Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His
665 670 675
Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr
680 685 690
Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly
695 700 705
Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser
710 715 720
Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu
725 730 735
Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp
740 745 750
Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu
755 760 765
Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu
770 775 780
Gln Ala Val
115
2407
DNA
Homo Sapien
115
ccctttgaag tgcattgctg cagctggtag catgagtggt ggccaccagc 50
tgcagctggc tgccctctgg ccctggctgc tgatggctac cctgcaggca 100
ggctttggac gcacaggact ggtactggca gcagcggtgg agtctgaaag 150
atcagcagaa cagaaagctg ttatcagagt gatccccttg aaaatggacc 200
ccacaggaaa actgaatctc actttggaag gtgtgtttgc tggtgttgct 250
gaaataactc cagcagaagg aaaattaatg cagtcccacc cgctgtacct 300
gtgcaatgcc agtgatgacg acaatctgga gcctggattc atcagcatcg 350
tcaagctgga gagtcctcga cgggcccccc gcccctgcct gtcactggct 400
agcaaggctc ggatggcggg tgagcgagga gccagtgctg tcctctttga 450
catcactgag gatcgagctg ctgctgagca gctgcagcag ccgctggggc 500
tgacctggcc agtggtgttg atctggggta atgacgctga gaagctgatg 550
gagtttgtgt acaagaacca aaaggcccat gtgaggattg agctgaagga 600
gcccccggcc tggccagatt atgatgtgtg gatcctaatg acagtggtgg 650
gcaccatctt tgtgatcatc ctggcttcgg tgctgcgcat ccagtgccgc 700
ccccgccaca gcaggccgga tccgcttcag cagagaacag cctgggccat 750
cagccagctg gccaccagga ggtaccaggc cagctgcagg caggcccggg 800
gtgagtggcc agactcaggg agcagctgca gctcagcccc tgtgtgtgcc 850
atctgtctgg aggagttctc tgaggggcag gagctacggg tcatttcctg 900
cctccatgag ttccatcgta actgtgtgga cccctggtta catcagcatc 950
ggacttgccc cctctgcatg ttcaacatca cagagggaga ttcattttcc 1000
cagtccctgg gaccctctcg atcttaccaa gaaccaggtc gaagactcca 1050
cctcattcgc cagcatcccg gccatgccca ctaccacctc cctgctgcct 1100
acctgttggg cccttcccgg agtgcagtgg ctcggccccc acgacctggt 1150
cccttcctgc catcccagga gccaggcatg ggccctcggc atcaccgctt 1200
ccccagagct gcacatcccc gggctccagg agagcagcag cgcctggcag 1250
gagcccagca cccctatgca caaggctggg gactgagcca cctccaatcc 1300
acctcacagc accctgctgc ttgcccagtg cccctacgcc gggccaggcc 1350
ccctgacagc agtggatctg gagaaagcta ttgcacagaa cgcagtgggt 1400
acctggcaga tgggccagcc agtgactcca gctcagggcc ctgtcatggc 1450
tcttccagtg actctgtggt caactgcacg gacatcagcc tacagggggt 1500
ccatggcagc agttctactt tctgcagctc cctaagcagt gactttgacc 1550
ccctagtgta ctgcagccct aaaggggatc cccagcgagt ggacatgcag 1600
cctagtgtga cctctcggcc tcgttccttg gactcggtgg tgcccacagg 1650
ggaaacccag gtttccagcc atgtccacta ccaccgccac cggcaccacc 1700
actacaaaaa gcggttccag tggcatggca ggaagcctgg cccagaaacc 1750
ggagtccccc agtccaggcc tcctattcct cggacacagc cccagccaga 1800
gccaccttct cctgatcagc aagtcaccag atccaactca gcagcccctt 1850
cggggcggct ctctaaccca cagtgcccca gggccctccc tgagccagcc 1900
cctggcccag ttgacgcctc cagcatctgc cccagtacca gcagtctgtt 1950
caacttgcaa aaatccagcc tctctgcccg acacccacag aggaaaaggc 2000
gggggggtcc ctccgagccc acccctggct ctcggcccca ggatgcaact 2050
gtgcacccag cttgccagat ttttccccat tacaccccca gtgtggcata 2100
tccttggtcc ccagaggcac accccttgat ctgtggacct ccaggcctgg 2150
acaagaggct gctaccagaa accccaggcc cctgttactc aaattcacag 2200
ccagtgtggt tgtgcctgac tcctcgccag cccctggaac cacatccacc 2250
tggggagggg ccttctgaat ggagttctga caccgcagag ggcaggccat 2300
gcccttgtcc gcactgccag gtgctgtcgg cccagcctgg ctcagaggag 2350
gaactcgagg agctgtgtga acaggctgtg tgagatgttc aggcctagct 2400
ccaacca 2407
116
783
PRT
Homo Sapien
116
Met Ser Gly Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp
1 5 10 15
Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu
20 25 30
Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys
35 40 45
Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys
50 55 60
Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile
65 70 75
Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu
80 85 90
Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser
95 100 105
Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro Arg Pro Cys Leu
110 115 120
Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser
125 130 135
Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln
140 145 150
Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp
155 160 165
Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln
170 175 180
Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro
185 190 195
Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe
200 205 210
Val Ile Ile Leu Ala Ser Val Leu Arg Ile Gln Cys Arg Pro Arg
215 220 225
His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile
230 235 240
Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala
245 250 255
Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro
260 265 270
Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu
275 280 285
Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val Asp
290 295 300
Pro Trp Leu His Gln His Arg Thr Cys Pro Leu Cys Met Phe Asn
305 310 315
Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg
320 325 330
Ser Tyr Gln Glu Pro Gly Arg Arg Leu His Leu Ile Arg Gln His
335 340 345
Pro Gly His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly
350 355 360
Pro Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro Gly Pro Phe
365 370 375
Leu Pro Ser Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe
380 385 390
Pro Arg Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu
395 400 405
Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Leu Ser His
410 415 420
Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu
425 430 435
Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr
440 445 450
Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp
455 460 465
Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val
470 475 480
Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser
485 490 495
Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr
500 505 510
Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser
515 520 525
Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly
530 535 540
Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg His
545 550 555
His His Tyr Lys Lys Arg Phe Gln Trp His Gly Arg Lys Pro Gly
560 565 570
Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr
575 580 585
Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln Gln Val Thr Arg
590 595 600
Ser Asn Ser Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys
605 610 615
Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser
620 625 630
Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser
635 640 645
Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro
650 655 660
Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His
665 670 675
Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr
680 685 690
Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly
695 700 705
Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser
710 715 720
Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu
725 730 735
Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp
740 745 750
Thr Ala Glu Gly Arg Pro Cys Pro Cys Pro His Cys Gln Val Leu
755 760 765
Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu
770 775 780
Gln Ala Val
117
2403
DNA
Homo Sapien
117
ttgaagtgca ttgctgcagc tggtagcatg agtggtggcc accacctgca 50
gctggctgcc ctctggccct ggctgctgat ggctaccctg caggcaggct 100
ttggacgcac aggactggta ctggcagcag cggtggagtc tgaaagatca 150
gcagaacaga aagctgttat cagagtgatc cccttgaaaa tggaccccac 200
aggaaaactg aatctcactt tggaaggtgt gtttgctggt gttgctgaaa 250
taactccagc agaaggaaaa ttaatgcagt cccacccgct gtacctgtgc 300
aatgccagtg atgacgacaa tctggagcct ggattcatca gcatcgtcaa 350
gctggagagt cctcgacggg ccccccaccc ctgcctgtca ctggctagca 400
aggctcggat ggcgggtgag cgaggagcca gtgctgtcct ctttgacatc 450
actgaggatc gagctgctgc tgagcagctg cagcagccgc tggggctgac 500
ctggccagtg gtgttgatct ggggtaatga cgctgagaag ctgatggagt 550
ttgtgtacaa gaaccaaaag gcccatgtga ggattgagct gaaggagccc 600
ccggcctggc cagattatga tgtgtggatc ctaatgacag tggtgggcac 650
catctttgtg atcatcctgg cttcggtgct gcgcatccgg tgccgccccc 700
gccacagcag gccggatccg cttcagcaga gaacagcctg ggccatcagc 750
cagctggcca ccaggaggta ccaggccagc tgcaggcagg cccggggtga 800
gtggccagac tcagggagca gctgcagctc agcccctgtg tgtgccatct 850
gtctggagga gttctctgag gggcaggagc tacgggtcat ttcctgcctc 900
catgagttcc atcgtaactg tgtggacccc tggttacatc agcatcggac 950
ttgccccctc tgcatgttca acatcacaga gggagattca ttttcccagt 1000
ccctgggacc ctctcgatct taccaagaac caggtcgaag actccacctc 1050
attcgccagc atcccggcca tgcccactac cacctccctg ctgcctacct 1100
gttgggccct tcccggagtg cagtggctcg gcccccacga cctggtccct 1150
tcctgccatc ccaggagcca ggcatgggcc ctcggcatca ccgcttcccc 1200
agagctgcac atccccgggc tccaggagag cagcagcgcc tggcaggagc 1250
ccagcacccc tatgcacaag gctggggaat gagccacctc caatccacct 1300
cacagcaccc tgctgcttgc ccagtgcccc tacgccgggc caggccccct 1350
gacagcagtg gatctggaga aagctattgc acagaacgca gtgggtacct 1400
ggcagatggg ccagccagtg actccagctc agggccctgt catggctctt 1450
ccagtgactc tgtggtcaac tgcacggaca tcagcctaca gggggtccat 1500
ggcagcagtt ctactttctg cagctcccta agcagtgact ttgaccccct 1550
agtgtactgc agccctaaag gggatcccca gcgagtggac atgcagccta 1600
gtgtgacctc tcggcctcgt tccttggact cggtggtgcc cacaggggaa 1650
acccaggttt ccagccatgt ccactaccac cgccaccggc accaccacta 1700
caaaaagcgg ttccagtggc atggcaggaa gcctggccca gaaaccggag 1750
tcccccagtc caggcctcct attcctcgga cacagcccca gccagagcca 1800
ccttctcctg atcagcaagt caccagatcc aactcagcag ccccttcggg 1850
gcggctctct aacccacagt gccccagggc cctccctgag ccagcccctg 1900
gcccagttga cgcctccagc atctgcccca gtaccagcag tctgttcaac 1950
ttgcaaaaat ccagcctctc tgcccgacac ccacagagga aaaggcgggg 2000
gggtccctcc gagcccaccc ctggctctcg gccccaggat gcaactgtgc 2050
acccagcttg ccagattttt ccccattaca cccccagtgt ggcatatcct 2100
tggtccccag aggcacaccc cttgatctgt ggacctccag gcctggacaa 2150
gaggctgcta ccagaaaccc caggcccctg ttactcaaat tcacagccag 2200
tgtggttgtg cctgactcct cgccagcccc tggaaccaca tccacctggg 2250
gaggggcctt ctgaatggag ttctgacacc gcagagggca ggccatgccc 2300
ttatccgcac tgccaggtgc tgtcggccca gcctggctca gaggaggaac 2350
tcgaggagct gtgtgaacag gctgtgtgag atgttcaggc ctagctccaa 2400
cca 2403
118
783
PRT
Homo Sapien
118
Met Ser Gly Gly His His Leu Gln Leu Ala Ala Leu Trp Pro Trp
1 5 10 15
Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu
20 25 30
Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys
35 40 45
Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys
50 55 60
Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile
65 70 75
Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu
80 85 90
Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser
95 100 105
Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro His Pro Cys Leu
110 115 120
Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser
125 130 135
Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln
140 145 150
Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp
155 160 165
Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln
170 175 180
Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro
185 190 195
Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe
200 205 210
Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg Pro Arg
215 220 225
His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile
230 235 240
Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala
245 250 255
Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro
260 265 270
Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu
275 280 285
Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val Asp
290 295 300
Pro Trp Leu His Gln His Arg Thr Cys Pro Leu Cys Met Phe Asn
305 310 315
Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg
320 325 330
Ser Tyr Gln Glu Pro Gly Arg Arg Leu His Leu Ile Arg Gln His
335 340 345
Pro Gly His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly
350 355 360
Pro Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro Gly Pro Phe
365 370 375
Leu Pro Ser Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe
380 385 390
Pro Arg Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu
395 400 405
Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Met Ser His
410 415 420
Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu
425 430 435
Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr
440 445 450
Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp
455 460 465
Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val
470 475 480
Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser
485 490 495
Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr
500 505 510
Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser
515 520 525
Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly
530 535 540
Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg His
545 550 555
His His Tyr Lys Lys Arg Phe Gln Trp His Gly Arg Lys Pro Gly
560 565 570
Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr
575 580 585
Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln Gln Val Thr Arg
590 595 600
Ser Asn Ser Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys
605 610 615
Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser
620 625 630
Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser
635 640 645
Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro
650 655 660
Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His
665 670 675
Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr
680 685 690
Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly
695 700 705
Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser
710 715 720
Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu
725 730 735
Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp
740 745 750
Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu
755 760 765
Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu
770 775 780
Gln Ala Val
119
4839
DNA
Homo Sapien
119
ggaaagctag cggcagaggc tcagccccgg cggcagcgcg cgccccgctg 50
ccagcccatt ttccggacgc cacccgcggg cactgccgac gcccccgggg 100
ctgccgaggg gaggccgggg gggcgcagcg gagcgcggtc ccgcgcactg 150
agccccgcgg cgccccggga acttggcggc gacccgagcc cggcgagccg 200
gggcgcgcct cccccgccgc gcgcctcctg catgcggggc cccagctccg 250
ggcgccggcc ggagcccccc ccggccgccc ccgagccccc cgcgccccgc 300
gccgcgccgc cgcgccgtcc atgcaccgct tgatgggggt caacagcacc 350
gccgccgccg ccgccgggca gcccaatgtc tcctgcacgt gcaactgcaa 400
acgctctttg ttccagagca tggagatcac ggagctggag tttgttcaga 450
tcatcatcat cgtggtggtg atgatggtga tggtggtggt gatcacgtgc 500
ctgctgagcc actacaagct gtctgcacgg tccttcatca gccggcacag 550
ccaggggcgg aggagagaag atgccctgtc ctcagaagga tgcctgtggc 600
cctcggagag cacagtgtca ggcaacggaa tcccagagcc gcaggtctac 650
gccccgcctc ggcccaccga ccgcctggcc gtgccgccct tcgcccagcg 700
ggagcgcttc caccgcttcc agcccaccta tccgtacctg cagcacgaga 750
tcgacctgcc acccaccatc tcgctgtcag acggggagga gcccccaccc 800
taccagggcc cctgcaccct ccagcttcgg gaccccgagc agcagctgga 850
actgaaccgg gagtcggtgc gcgcaccccc aaacagaacc atcttcgaca 900
gtgacctgat ggatagtgcc aggctgggcg gcccctgccc ccccagcagt 950
aactcgggca tcagcgccac gtgctacggc agcggcgggc gcatggaggg 1000
gccgccgccc acctacagcg aggtcatcgg ccactacccg gggtcctcct 1050
tccagcacca gcagagcagt gggccgccct ccttgctgga ggggacccgg 1100
ctccaccaca cacacatcgc gcccctagag agcgcagcca tctggagcaa 1150
agagaaggat aaacagaaag gacaccctct ctagggtccc caggggggcc 1200
gggctggggc tgcgtaggtg aaaaggcaga acactccgcg cttcttagaa 1250
gaggagtgag aggaaggcgg ggggcgcagc aacgcatcgt gtggccctcc 1300
cctcccacct ccctgtgtat aaatatttac atgtgatgtc tggtctgaat 1350
gcacaagcta agagagcttg caaaaaaaaa aagaaaaaag aaaaaaaaaa 1400
accacgtttc tttgttgagc tgtgtcttga aggcaaaaga aaaaaaattt 1450
ctacagtagt ctttcttgtt tctagttgag ctgcgtgcgt gaatgcttat 1500
tttcttttgt ttatgataat ttcacttaac tttaaagaca tatttgcaca 1550
aaacctttgt ttaaagatct gcaatattat atatataaat atatataaga 1600
taagagaaac tgtatgtgcg agggcaggag tatttttgta ttagaagagg 1650
cctattaaaa aaaaaagttg ttttctgaac tagaagagga aaaaaatggc 1700
aatttttgag tgccaagtca gaaagtgtgt attaccttgt aaagaaaaaa 1750
attacaaagc aggggtttag agttatttat ataaatgttg agattttgca 1800
ctatttttta atataaatat gtcagtgctt gcttgatgga aacttctctt 1850
gtgtctgttg agactttaag ggagaaatgt cggaatttca gagtcgcctg 1900
acggcagagg gtgagccccc gtggagtctg cagagaggcc ttggccagga 1950
gcggcgggct ttcccgaggg gccactgtcc ctgcagagtg gatgcttctg 2000
cctagtgaca ggttatcacc acgttatata ttccctaccg aaggagacac 2050
cttttccccc ctgacccaga acagccttta aatcacaagc aaaataggaa 2100
agttaaccac ggaggcaccg agttccaggt agtggttttg cctttcccaa 2150
aaatgaaaat aaactgttac cgaaggaatt agtttttcct cttctttttt 2200
ccaactgtga aggtccccgt ggggtggagc atggtgcccc tcacaagccg 2250
cagcggctgg tgcccgggct accagggaca tgccagaggg ctcgatgact 2300
tgtctctgca gggcgctttg gtggttgttc agctggctaa aggttcaccg 2350
gtgaaggcag gtgcggtaac tgccgcactg gaccctagga agccccaggt 2400
attcgcaatc tgacctcctc ctgtctgttt cccttcacgg atcaattctc 2450
acttaagagg ccaataaaca acccaacatg aaaaggtgac aagcctgggt 2500
ttctcccagg ataggtgaaa gggttaaaat gagtaaagca gttgagcaaa 2550
caccaacccg agcttcgggc gcagaattct tcaccttctc ttcccctttc 2600
catctccttt ccccgcggaa acaacgcttc ccttctggtg tgtctgttga 2650
tctgtgtttt catttacatc tctcttagac tccgctcttg ttctccaggt 2700
tttcaccaga tagatttggg gttggcggga cctgctggtg acgtgcaggt 2750
gaaggacagg aaggggcatg tgagcgtaaa tagaggtgac cagaggagag 2800
catgaggggt ggggctttgg gacccaccgg ggccagtggc tggagcttga 2850
cgtctttcct ccccatgggg gtgggagggc ccccagctgg aagagcagac 2900
tcccagctgc taccccctcc cttcccatgg gagtggcttt ccattttggg 2950
cagaatgctg actagtagac taacataaaa gatataaaag gcaataacta 3000
ttgtttgtga gcaacttttt tataacttcc aaaacaaaaa cctgagcaca 3050
gttttgaagt tctagccact cgagctcatg catgtgaaac gtgtgcttta 3100
cgaaggtggc agctgacaga cgtgggctct gcatgccgcc agcctagtag 3150
aaagttctcg ttcattggca acagcagaac ctgcctctcc gtgaagtcgt 3200
cagcctaaaa tttgtttctc tcttgaagag gattctttga aaaggtcctg 3250
cagagaaatc agtacaggtt atcccgaaag gtacaaggac gcacttgtaa 3300
agatgattaa aacgtatctt tcctttatgt gacgcgtctc tagtgcctta 3350
ctgaagaagc agtgacactc ccgtcgctcg gtgaggacgt tcccggacag 3400
tgcctcactc acctgggact ggtatcccct cccagggtcc accaagggct 3450
cctgcttttc agacacccca tcatcctcgc gcgtcctcac cctgtctcta 3500
ccagggaggt gcctagcttg gtgaggttac tcctgctcct ccaacctttt 3550
tttgccaagg tttgtacacg actcccatct aggctgaaaa cctagaagtg 3600
gaccttgtgt gtgtgcatgg tgtcagccca aagccaggct gagacagtcc 3650
tcatatcctc ttgagccaaa ctgtttgggt ctcgttgctt catggtatgg 3700
tctggatttg tgggaatggc tttgcgtgag aaaggggagg agagtggttg 3750
ctgccctcag ccggcttgag gacagagcct gtccctctca tgacaactca 3800
gtgttgaagc ccagtgtcct cagcttcatg tccagtggat ggcagaagtt 3850
catggggtag tggcctctca aaggctgggc gcatcccaag acagccagca 3900
ggttgtctct ggaaacgacc agagttaagc tctcggcttc tctgctgagg 3950
gtgcaccctt tcctctagat ggtagttgtc acgttatctt tgaaaactct 4000
tggactgctc ctgaggaggc cctcttttcc agtaggaagt tagatggggg 4050
ttctcagaag tggctgattg gaaggggaca agcttcgttt caggggtctg 4100
ccgttccatc ctggttcaga gaaggccgag cgtggctttc tctagccttg 4150
tcactgtctc cctgcctgtc aatcaccacc tttcctccag aggaggaaaa 4200
ttatctcccc tgcaaagccc ggttctacac agatttcaca aattgtgcta 4250
agaaccgtcc gtgttctcag aaagcccagt gtttttgcaa agaatgaaaa 4300
gggaccccat atgtagcaaa aatcagggct gggggagagc cgggttcatt 4350
ccctgtcctc attggtcgtc cctatgaatt gtacgtttca gagaaatttt 4400
ttttcctatg tgcaacacga agcttccaga accataaaat atcccgtcga 4450
taaggaaaga aaatgtcgtt gttgttgttt ttctggaaac tgcttgaaat 4500
cttgctgtac tatagagctc agaaggacac agcccgtcct cccctgcctg 4550
cctgattcca tggctgttgt gctgattcca atgctttcac gttggttcct 4600
ggcgtgggaa ctgctctcct ttgcagcccc atttcccaag ctctgttcaa 4650
gttaaactta tgtaagcttt ccgtggcatg cggggcgcgc acccacgtcc 4700
ccgctgcgta agactctgta tttggatgcc aatccacagg cctgaagaaa 4750
ctgcttgttg tgtatcagta atcattagtg gcaatgatga cattctgaaa 4800
agctgcaata cttatacaat aaattttaca attctttgg 4839
120
287
PRT
Homo Sapien
120
Met His Arg Leu Met Gly Val Asn Ser Thr Ala Ala Ala Ala Ala
1 5 10 15
Gly Gln Pro Asn Val Ser Cys Thr Cys Asn Cys Lys Arg Ser Leu
20 25 30
Phe Gln Ser Met Glu Ile Thr Glu Leu Glu Phe Val Gln Ile Ile
35 40 45
Ile Ile Val Val Val Met Met Val Met Val Val Val Ile Thr Cys
50 55 60
Leu Leu Ser His Tyr Lys Leu Ser Ala Arg Ser Phe Ile Ser Arg
65 70 75
His Ser Gln Gly Arg Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly
80 85 90
Cys Leu Trp Pro Ser Glu Ser Thr Val Ser Gly Asn Gly Ile Pro
95 100 105
Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro Thr Asp Arg Leu Ala
110 115 120
Val Pro Pro Phe Ala Gln Arg Glu Arg Phe His Arg Phe Gln Pro
125 130 135
Thr Tyr Pro Tyr Leu Gln His Glu Ile Asp Leu Pro Pro Thr Ile
140 145 150
Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys
155 160 165
Thr Leu Gln Leu Arg Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg
170 175 180
Glu Ser Val Arg Ala Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp
185 190 195
Leu Met Asp Ser Ala Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser
200 205 210
Asn Ser Gly Ile Ser Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met
215 220 225
Glu Gly Pro Pro Pro Thr Tyr Ser Glu Val Ile Gly His Tyr Pro
230 235 240
Gly Ser Ser Phe Gln His Gln Gln Ser Ser Gly Pro Pro Ser Leu
245 250 255
Leu Glu Gly Thr Arg Leu His His Thr His Ile Ala Pro Leu Glu
260 265 270
Ser Ala Ala Ile Trp Ser Lys Glu Lys Asp Lys Gln Lys Gly His
275 280 285
Pro Leu