CA2422891A1 - Novel proteins and nucleic acids encoding same - Google Patents

Abstract

Disclosed herein are nucleic acid sequences that encode novel polypeptides.
Also disclosed are polypeptides encoded by these nucleic acid sequences, and antiboides, which immunospecifically-bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. The invention further discloses therapeutic, diagnostic and research methods for diagnosis, treatment, and prevention of disorders involving any one of these novel human nucleic acids and proteins.

Description

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NOVEL PROTEINS AND NUCLEIC ACIDS ENCODING SAME
FIELD OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded thereby.
BACKGROUND OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded therefrom.
More specifically, the invention relates to nucleic acids encoding cytoplasmic, nuclear, membrane bound, and secreted polypeptides, as well as vectoxs, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides. The novel nucleic acids arid polypeptides are xeferred to herein as NOVX, or NOV 1, NOV2, NOV3, NOV4, NOVS, NOV6, NOV7, and NOV8 nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "NOVX"
nucleic acid or polypeptide sequences.
In one aspect, the invention provides an isolated NOVX nucleic acid molecule encoding a NOVX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26. In some embodiments, the NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80%
identical to a polypeptide comprising the amino acid sequences of SEQ ID
NOS:2, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, and 27. The nucleic acid can be, for example, a genomic DNA
fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID
NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.
Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NOVX nucleic acid (e.g., SEQ
ID NOS:l, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26) or a complement of said oligonucleotide.

Also included in the invention are substantially purified NOVX polypeptides (SEQ ID
NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27). In certain embodiments, the NOVX
polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide.
The invention also features antibodies that immunoselectively bind to NOVX
polypeptides, or fragments, homologs, analogs or derivatives thereof.
In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable carrier. The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX
polypeptide, or an antibody specific for a NOVX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression of the NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.
In another aspect, the invention includes a method of detecting the presence of a NOVX polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.
The invention also includes methods to identify specific cell or tissue types based on their expression of a NOVX.
Also included in the invention is a method of detecting the presence of a NOVX
nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX
polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.

2 Also within the scope of the invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., Cancer, Leukodystrophies, Breast cancer, Ovarian cancer, Prostate cancer, Uterine cancer, Hodgkin disease, Adenocarcinoma, Adrenoleukodystrophy,Cystitis, incontinence, Von Hippel-Lindau (VHL) syndrome, hypercalceimia, Endometriosis, Hirschsprung's disease, Crohn's Disease, Appendicitis, Cirrhosis, Liver failure, Wolfram Syndrome, Smith-Lemli-Opitz syndrome, Retinitis pigmentosa, Leigh syndrome; Congenital Adrenal Hyperplasia, Xerostomia; tooth decay and other dental problems; Inflammatory bowel disease, Diverticular disease, fertility, Infertility, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis , atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis , subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, Hemophilia, Hypercoagulation, Idiopathic thrombocytopenic purpura, obesity, Diabetes Insipidus and Mellitus with Optic Atrophy and Deafness, Pancreatitis, Metabolic Dysregulation, transplantation recovery, Autoimmune disease, Systemic lupus erythematosus, asthma, arthritis, psoriasis, Emphysema, Scleroderma, allergy, ARDS, Immunodeficiencies, Graft vesus host, Alzheimer's disease, Stroke, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Multiple sclerosis,Ataxia-telangiectasia, Behavioral disorders, Addiction, Anxiety, Pain, Neurodegeneration, Muscular dystrophy,Lesch-Nyhan syndrome,Myasthenia gravis, schizophrenia, and other dopamine-dysfunctional states, levodopa-induced dyskinesias, alcoholism, pileptic seizures and other neurological disorders, mental depression, Cerebellar ataxia, pure; Episodic ataxia, type 2;
Hemiplegic migraine, Spinocerebellar ataxia-6, Tuberous sclerosis, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Renal tubular acidosis, IgA nephropathy, and/or other pathologies and disorders of the like.
The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or a NOVX-specific antibody, or biologically-active derivatives or fragments thereof.
For example, the compositions of the pxesent invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above andlor other pathologies and disorders of the like. The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding NOVX may be useful in gene therapy, and NOVX may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
The invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. The method includes contacting a test compound with a NOVX
polypeptide and determining if the test compound binds to said NOVX
polypeptide. Binding of the test compound to the NOVX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly-expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of NOVX polypeptide in both the test animal and the control animal is compared. A change in the activity of NOVX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome.
In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX
polypeptide, a NOVX
nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount of the NOVX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the NOVX polypeptide present in a control sample. An alteration in the level of the NOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. Also, the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
In a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subject (e.g., a human subj ect), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
In yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded thereby.
Included in the invention are the novel nucleic acid sequences and their encoded polypeptides.
The sequences are collectively referred to herein as "NOVX nucleic acids" or "NOVX
polynucleotides" and the corresponding encoded polypeptides are referred to as "NOVX
polypeptides" or "NOVX proteins." Unless indicated otherwise, "NOVX" is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary of the NOVX
nucleic acids and their encoded polypeptides.
TABLE A. Sequences and Corresponding SEQ ID Numbers SEQ
ID

NOVX Internal IdentificationNO SEQ ID Homology NO

Assignment (nucleic(polypeptide) acid la 83420733 EXT Z 2 Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like 1b AL356413.6 3 2 Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like lc CG52997-02 4 5 Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like 2 101599929 EXT1 6 7 Attractin like 3a 124217931 EXT 8 9 Kinase-like 3b 124217931 10 11 RHO/RAC-interacting citron kinase-like 4 105827550 EXT 12 13 Plexin-like GMAC027612 A Z4 15 Dopamine receptor-like 6 GM523 a l A 16 17 Metabotropic Glutamate R eceptor 7a sggc draft_ba560a15_18 19 PV-1-like 2000723 dal 7b 2847264Ø32 20 21 PV-1-like 7c CG51878-03 22 23 PV-1-like 8a SC134914330_A 24 25 Papin-like 8b CG57026-04 26 27 Papiri-like NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
Additionally, NOVX
nucleic acids and polypeptides can also be used to identify proteiils that are members of the family to which the NOVX polypeptides belong.
NOV 1 is homologous to a Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like family of proteins. Thus, the NOV 1 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; cancer, cystitis, incontinence, fertility, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis , atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation recovery, and/or other pathologiesldisorders.
NOV2 is homologous to the Attractin-like family of proteins. Thus NOV2 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example;
Von Hippel-Lindau (VIIL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neurodegenexation, Diabetes, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Diabetes, Pancreatitis,Obesity, Endometriosis, Infertility, Hirschsprung's disease, Crohn's Disease, Appendicitis, Muscular dystrophy, Lesch-Nyhan syndrome, Myasthenia gravis, Cirrhosis, Liver failure, Breast cancer, Ovarian cancer, Prostate cancer, Uterine cancer and/or other pathologies/disorders.
NOV3 is homologous to a family of RHO/RAC-interacting citron kinase-like proteins.
Thus, the NOV3 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: asthma, arthritis, psoriasis, diabetes, and IBD, which require activated T cells, as well as diseases such as systemic lupus erythematosus that involve B cell activation, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Endocrine dysfunctions, Obesity, Growth and Reproductive disorders Hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, allergies, immunodeficiencies, transplantation, Lymphaedema, Hemophilia, Hypercoagulation,Idiopathic thrombocytopenic purpura , Immunodeficiencies, Graft vesus host, Hirschsprung's disease, Crohn's Disease, Appendicitis Inflammatory bowel disease, Diverticular disease and/or other pathologies/disorders.
NOV4 is homologous to the Plexin-like family ofproteins. Thus, NOV4 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neurodegeneration, Systemic lupus erythematosus, Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy, ARDS, Obesity, Metabolic Dysregulation, Infertility and/or other pathologies/disorders.
NOVS is homologous to the Dopamine receptor-like family of proteins. Thus NOVS
nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: schizophrenia, and other dopamine-dysfunctional states, Hypertension, Huntington's disease, levodopa-induced dyskinesias, alcoholism, Diabetes Insipidus and Mellitus with Optic Atrophy and Deafness, Wolfram Syndrome and/or other pathologies/disorders.

NOV6 is homologous to the Metabotropic Glutamate Receptor-like family of proteins.
Thus NOV6 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example:
pileptic seizuxes and other neurological disorders, Hodgkin disease, polycystic kidney disease, mental depression, Adenocarcinoma, Smith-Lemli-Opitz syndrome, Retinitis pigmentosa and/or other pathologies/disorders.
NOV7 is homologous to members of the PV-1-like family of proteins. Thus, the NOV7 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example;
Cerebellar ataxia, pure; Episodic ataxia, type 2; Hemiplegic migraine, familial; Leigh syndrome; Spinocerebellar ataxia-6; Psoriasis, susceptibility to; Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy, ARDS, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Muscular dystrophy, Myasthenia gravis, Hemophilia, Hypercoagulation, Idiopathic thrombocytopenic purpura, Immunodeficiencies, Graft vesus host, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Scleroderma, Obesity, Transplantation; fertility; cancer;
Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, Xerostomia;
tooth decay and other dental problems; Inflammatory bowel disease, Diverticular disease, Pancreatitis, and/or other pathologies/disorders.
NOV8 is homologous to the Papin-like family of proteins. Thus, NOV8 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; cancer, cystitis, incontinence, fertility, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation recovery and/or other pathologies/disorders.

The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., neurogenesis, cell differentiation, cell proliferation, hematopoiesis, wound healing and angiogenesis.
Additional utilities for the NOVX nucleic acids and polypeptides according to the invention are disclosed herein.
NOVl NOV 1 includes three novel Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit (IGFBP-ALS)-like proteins disclosed below. The disclosed sequences have been named NOV 1 a, NOV 1b, and NOV 1 c. The nucleotide sequences for NOV 1 a and b both code for the NOV 1 a protein sequence. The NOV 1 c nucleic acid sequence codes for the NOVlc protein sequence.
NOVla A disclosed NOV 1 a nucleic acid of 2838 nucleotides (also referred to as 83420733 EXT) encoding a novel Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like protein is shown in Table 1A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 184-186 and ending with a TAG codon at nucleotides 2707-2709. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 1A. The start and stop codons are in bold letters.
Table 1A. NOVla nucleotide sequence (SEQ ID NO:1).
AACTTTATGAAGCTATGGGACTTGACAAAAAGTGATATTTGAGAAGAAAGTACGCAGTGGTTGGTGTTTTCT
TTTTTTTAATAAAGGAATTGAATTACTTTGAACACCTCTTCCAGCTGTGCATTACAGATAACGTCAGGAAGA
GTCTCTGCTTTACAGAATCGGATTTCATCACATGACAACATGAAGCTGTGGATTCATCTCTTTTATTCATCT
CTCCTTGCCTGTATATCTTTACACTCCCAAACTCCAGTGCTCTCATCCAGAGGCTCTTGTGATTCTCTTTGC
AATTGTGAGGAAAAAGATGGCACAATGCTAATAAATTGTGAAGCAAAAGGTATCAAGATGGTATCTGAAATA
AGTGTGCCACCATCACGACCTTTCCAACTAAGCTTATTAAATAACGGCTTGACGATGCTTCACACAAATGAC
TTTTCTGGGCTTACCAATGCTATTTCAATACACCTTGGATTTAACAATATTGCAGATATTGAGATAGGTGCA
TTTAATGGCCTTGGCCTCCTGAAACAACTTCATATCAATCACAATTCTTTAGAAATTCTTAAAGAGGATACT
TTCCATGGACTGGAAAACCTGGAATTCCTGCAAGCAGATAACAATTTTATCACAGTGATTGAACCAAGTGCC
TTTAGCAAGCTCAACAGACTCAAAGTGTTAATTTTAAATGACAATGCTATTGAGAGTCTTCCTCCAAACATC
TTCCGATTTGTTCCTTTAACCCATCTAGATCTTCGTGGAAATCAATTACAAACATTGCCTTATGTTGGTTTT
CTCGAACACATTGGCCGAATATTGGATCTTCAGTTGGAGGACAACAAATGGGCCTGCAATTGTGACTTATTG
CAGTTAAAAACTTGGTTGGAGAACATGCCTCCACAGTCTATAATTGGTGATGTTGTCTGCAACAGCCCTCCA
TTTTTTAAAGGAAGTATACTCAGTAGACTAAAGAAGGAATCTATTTGCCCTACTCCACCAGTGTATGAAGAA
CATGAGGATCCTTCAGGATCATTACATCTGGCAGCAACATCTTCAATAAATGATAGTCGCATGTCAACTAAG
ACCACGTCCATTCTAAAACTACCCACCAAAGCACCAGGTTTGATACCTTATATTACAAAGCCATCCACTCAA
CTTCCAGGACCTTACTGCCCTATTCCTTGTAACTGCAAAGTCCTATCCCCATCAGGACTTCTAATACATTGT
CAGGAGCGCAACATTGAAAGCTTATCAGATCTGAGACCTCCTCCGCAAAATCCTAGAAAGCTCATTCTAGCG
GGAAATATTATTCACAGTTTAATGAAGTCTGATCTAGTGGAATATTTCACTTTGGAAATGCTTCACTTGGGA
AACAATCGTATTGAAGTTCTTGAAGAAGGATCGTTTATGAACCTAACGAGATTACAAAAACTCTATCTAAAT

GGTAACCACCTGACCAAATTAAGTAAAGGCATGTTCCTTGGTCTCCATAATCTTGAATACTTATATCTTGAA
TACAATGCCATTAAGGAAATACTGCCAGGAACCTTTAATCCAATGCCTAAACTTAAAGTCCTGTATTTAAAT
AACAACCTCCTCCAAGTTTTACCACCACATATTTTTTCAGGGGTTCCTCTAACTAAGGTAAATCTTAAAACA
AACCAGTTTACCCATCTACCTGTAAGTAATATTTTGGATGATCTTGATTTACTAACCCAGATTGACCTTGAG
GATAACCCCTGGGACTGCTCCTGTGACCTGGTTGGACTGCAGCAATGGATACAAAAGTTAAGCAAGAACACA
GTGACAGATGACATCCTCTGCACTTCCCCCGGGCATCTCGACAAAAAGGAATTGAAAGCCCTAAATAGTGAA
ATTCTCTGTCCAGGTTTAGTAAATAACCCATCCATGCCAACACAGACTAGTTACCTTATGGTCACCACTCCT
GCAACAACAACAAATACGGCTGATACTATTTTACGATCTCTTACGGACGCTGTGCCACTGTCTGTTCTAATA
TTGGGACTTCTGATTATGTTCATCACTATTGTTTTCTGTGCTGCAGGGATAGTGGTTCTTGTTCTTCACCGC
AGGAGAAGATACAAAAAGAAACAAGTAGATGAGCAAATGAGAGACAACAGTCCTGTGCATCTTCAGTACAGC
ATGTATGGCCATAAAACCACTCATCACACTACTGAAAGACCCTCTGCCTCACTCTATGAACAGCACATGGTG
AGCCCCATGGTTCATGTCTATAGAAGTCCATCCTTTGGTCCAAAGCATCTGGAAGAGGAAGAAGAGAGGAAT
GAGAAAGAAGGAAGTGATGCAAAACATCTCCAAAGAAGTCTTTTGGAACAGGAAAATCATTCACCACTCACA
GGGTCAAATATGAAATACAAAACCACGAACCAATCAACAGAATTTTTATCCTTCCAAGATGCCAGCTCATTG
TACAGAAACATTTTAGAAAAAGAAAGGGAACTTCAGCAACTGGGAATCACAGAATACCTAAGGAAAAACATT
GCTCAGCTCCAGCCTGATATGGAGGCACATTATCCTGGAGCCCACGAAGAGCTGAAGTTAATGGAAACATTA
ATGTACTCACGTCCAAGGAAGGTATTAGTGGAACAGACAAAAAATGAGTATTTTGAACTTAAAGCTAATTTA
CATGCTGAACCTGACTATTTAGAAGTCCTGGAGCAGCAAACATAGATGGAGAGTTTGAGGGCTTTCGCAGAA
ATGCTGTGATTCTGTTTTAAGTCCATACCTTGTAAATTAGTGCCTTACGTGAGTGTGTCATCCATCAGAACC
TAAGCACAGCAGSAAACTATGGAGAAAAAA
In a search of public sequence databases, the NOV 1 a nucleic acid sequence, located on chromsome 13 has 1173 of 1932 bases (61%) identical to a KIAA084~ mRNA from human (gb:GENBANK-ID:AB020655~acc:AB020655). Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
In all BLAST alignments herein, the "E-value" or "Expect" value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. For example, the probability that the subject ("Sbjct") retrieved from the NOVl BLAST analysis, e.g., thioredoxin mRNA from Ovis a~°ies, matched the Query NOV 1 sequence purely by chance is 7.4e 6g. The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences.
Essentially, the E value describes the random background noise that exists for matches between sequences.
The Expect value is used as a convenient way to create a significance threshold for reporting results. The default value used for blasting is typically set to 0.0001. In BLAST 2.0, the Expect value is also used instead of the P value (probability) to report the significance of matches. For example, an E value of one assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see one match with a similar score simply by chance. An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search. This is a result of automatic filtering of the query for low-IO

complexity sequence that is performed to prevent artifactual hits. The filter substitutes any low-complexity sequence that it finds with the letter "N" in nucleotide sequence (e.g., " ") or the letter "X" in protein sequences (e.g., "XXX~OOO~XX").
Low-complexity regions can result in high scores that reflect compositional bias rather than significant position-by-position alignment. (Wootton and Federhen, Methods Enzymol 266:554-571, 1996).
The disclosed NOV 1 a polypeptide (SEQ ID N0:2) encoded by SEQ ID NO:1 has 841 amino acid residues and is presented in Table 1B using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOVla has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0. 4600. In other embodiments, NOV 1 a may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (membrane) with a certainty of 0.1000, or extracellularly with a certainty of 0.1000. The most likely cleavage site for a NOV 1 a peptide is between amino acids and 21, at: LHS-QT.
IS SNP data for NOVla can be found below in Example 3. SAGE data can also be found below for NOV 1 a in Example 4.
Table 1B. Encoded NOVla protein sequence (SEA ID N0:2).
MKLWIHLFYSSLLACISLHSQTPVLSSRGSCDSLCNCEEKDGTMLINCEAKGIKMVSEISVPPS
RPFQLSLLNNGLTMLHTNDFSGLTNAISIHLGFNNIADIETGAFNGLGLLKQLHINHNSLEILK
EDTFHGLENLEFLQADNNFITVIEPSAFSKLNRLKVLILNDNAIESLPPNIFRFVPLTHLDLRG
NQLQTLPYVGFLEHIGRILDLQLEDNKWACNCDLLQLKTWLENMPPQSIIGDVVCNSPPFFKGS
ILSRLKKESICPTPPVYEEHEDPSGSLHLAATSSINDSRMSTKTTSILKLPTKAPGLTPYITKP
STQLPGPYCPIPCNCKVLSPSGLLIHCQERNIESLSDLRPPPQNPRKLILAGNIIHSLMKSDLV
EYFTLEMLHLGNNRIEVLEEGSFMNLTRLQKLYLNGNHLTKLSKGMFLGLHNLEYLYLEYNAIK
EILPGTFNPMPKLKVLYLNNNLLQVLPPHIFSGVPLTKVNLKTNQFTHLPVSNILDDLDLLTQI
DLEDNPWDCSCDLVGLQQWIQKLSKNTVTDDILCTSPGHLDKKELKALNSEILCPGLVNNPSMP
TQTSYLMVTTPATTTNTADTILRSLTDAVPLSVLILGLLIMFITIVFCAAGIVVLVLHRRRRYK
KKQVDEQMRDNSPVHLQYSMYGHKTTHHTTERPSASLYEQHMVSPMVHVYRSPSFGPKHLEEEE
ERNEKEGSDAKHLQRSLLEQENHSPLTGSNMKYKTTNQSTEFLSFQDASSLYRNILEKERELQQ
LGITEYLRKNIAQLQPDMEAHYPGAHEELKLMETLMYSRPRKVLVEQTKNEYFELKANLHAEPD
20 A search of sequence databases reveals that the NOVla amino acid sequence has 266 of 543 amino acid residues (49%) identical to, and 337 of 543 amino acid residues (62%) similar to the 977 amino acid residue KIAA0848 protein from human (SPTREMBL-ACC:094933) (E = 1.6e-16s), and 350 of 841 amino acid residues (41%) identical to, and 511 of 841 amino acid residues (60%) similar to the 845 amino acid residue Human gene 1 encoded secreted protein HMIAJ30 (patp:AAE01232) (E =1.6e-lss), public amino acid databases include the GenBank databases, SwissProt, PDB and PIR.
NOV 1 a is expressed in at least the following tissues: breast, heart, bladder, colon, prostate, brain, lung and uterus. TaqMan expression data for NOVla is shown below in Example 2.
NOVlb A disclosed NOVlb nucleic acid of 2526 nucleotides (also referred to as AL356413.6) encoding a novel Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like protein is shown in Table 1C. An open reading frame was identified beginning with an ATG initiation colon at nucleotides 1-3 and ending with a TAG colon at nucleotides 2524-2526. A putative untranslated region upstream from the initiation colon is underlined in Table 1C. The start and stop colons are in bold letters.
Table 1C. NOVIb nucleotide sequence (SEQ ID N0:3).
ATGAAGCTGTGGATTCATCTCTTTTATTCATCTCTCCTTGCCTGTATATCTTTACACTCCCAAAC
TCCAGTGCTCTCATCCAGAGGCTCTTGTGATTCTCTTTGCAATTGTGAGGAAAAAGATGGCACAA
TGCTAATAAATTGTGAAGCAAAAGGTATCAAGATGGTATCTGAAATAAGTGTGCCACCATCACGA
CCTTTCCAACTAAGCTTATTAAATAACGGCTTGACGATGCTTCACACAAATGACTTTTCTGGGCT
TACCAATGCTATTTCAATACACCTTGGATTTAACAATATTGCAGATATTGAGATAGGTGCATTTA
ATGGCCTTGGCCTCCTGAAACAACTTCATATCAATCACAATTCTTTAGAAATTCTTAAAGAGGAT
ACTTTCCATGGACTGGAAAACCTGGAATTCCTGCAAGCAGATAACAATTTTATCACAGTGATTGA
ACCAAGTGCCTTTAGCAAGCTCAACAGACTCAAAGTGTTAATTTTAAATGACAATGCTATTGAGA
GTCTTCCTCCAAACATCTTCCGATTTGTTCCTTTAACCCATCTAGATCTTCGTGGAAATCAATTA
CAAACATTGCCTTATGTTGGTTTTCTCGAACACATTGGCCGAATATTGGATCTTCAGTTGGAGGA
CAACAAATGGGCCTGCAATTGTGACTTATTGCAGTTAAAAACTTGGTTGGAGAACATGCCTCCAC
AGTCTATAATTGGTGATGTTGTCTGCAACAGCCCTCCATTTTTTAAAGGAAGTATACTCAGTAGA
CTAAAGAAGGAATCTATTTGCCCTACTCCACCAGTGTATGAAGAACATGAGGATCCTTCAGGATC
ATTACATCTGGCAGCAACATCTTCAATAAATGATAGTCGCATGTCAACTAAGACCACGTCCATTC
TAAAACTACCCACCAAAGCACCAGGTTTGATACCTTATATTACAAAGCCATCCACTCAACTTCCA
GGACCTTACTGCCCTATTCCTTGTAACTGCAAAGTCCTATCCCCATCAGGACTTCTAATACATTG
TCAGGAGCGCAACATTGAAAGCTTATCAGATCTGAGACCTCCTCCGCAAAATCCTAGAAAGCTCA
TTCTAGCGGGAAATATTATTCACAGTTTAATGAAGTCTGATCTAGTGGAATATTTCACTTTGGAA
ATGCTTCACTTGGGAAACAATCGTATTGAAGTTCTTGAAGAAGGATCGTTTATGAACCTAACGAG
ATTACAAAAACTCTATCTAAATGGTAACCACCTGACCAAATTAAGTAAAGGCATGTTCCTTGGTC
TCCATAATCTTGAATACTTATATCTTGAATACAATGCCATTAAGGAAATACTGCCAGGAACCTTT
AATCCAATGCCTAAACTTAAAGTCCTGTATTTAAATAACAACCTCCTCCAAGTTTTACCACCACA
TATTTTTTCAGGGGTTCCTCTAACTAAGGTAAATCTTAAAACAAACCAGTTTACCCATCTACCTG
TAAGTAATATTTTGGATGATCTTGATTTGCTAACCCAGATTGACCTTGAGGATAACCCCTGGGAC
TGCTCCTGTGACCTGGTTGGACTGCAGCAATGGATACAAAAGTTAAGCAAGAACACAGTGACAGA
TGACATCCTCTGCACTTCCCCCGGGCATCTCGACAAAAAGGAATTGAAAGCCCTAAATAGTGAAA
TTCTCTGTCCAGGTTTAGTAAATAACCCATCCATGCCAACACAGACTAGTTACCTTATGGTCACC
ACTCCTGCAACAACAACAAATACGGCTGATACTATTTTACGATCTCTTACGGACGCTGTGCCACT
GTCTGTTCTAATATTGGGACTTCTGATTATGTTCATCACTATTGTTTTCTGTGCTGCAGGGATAG
TGGTTCTTGTTCTTCACCGCAGGAGAAGATACAAAAAGAAACAAGTAGATGAGCAAATGAGAGAC
AACAGTCCTGTGCATCTTCAGTACAGCATGTATGGCCATAAAACCACTCATCACACTACTGAAAG
ACCCTCTGCCTCACTCTATGAACAGCACATGGTGAGCCCCATGGTTCATGTCTATAGAAGTCCAT
CCTTTGGTCCAAAGCATCTGGAAGAGGAAGAAGAGAGGAATGAGAAAGAAGGAAGTGATGCAAAA
CATCTCCAAAGAAGTCTTTTGGAACAGGAAAATCATTCACCACTCACAGGGTCAAATATGAAATA

CAAAACCACGAACCAATCAACAGAATTTTTATCCTTCCAAGATGCCAGCTCATTGTACAGAAACA
TTTTAGAAAAAGAA.AGGGAACTTCAGCAACTGGGAATCACAGAATACCTAAGGAAAAACATTGCT
CAGCTCCAGCCTGATATGGAGGCACATTATCCTGGAGCCCACGAAGAGCTGAAGTTAATGGAAAC
ATTAATGTACTCACGTCCAAGGAAGGTATTAGTGGAACAGACAAAAAATGAGTATTTTGAACTTA
AAGCTAATTTACATGCTGAACCTGACTATTTAGAAGTCCTGGAGCAGCAAACATAG
The disclosed NOV 1b nucleotide encodes the NOV 1 a protein sequence disclosed above in Table 1B.
NOVlc In the present invention, the target sequence identified previously, NOVlb, was subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on ira silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences or sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain -substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs.
Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported below, which is designated NOVlc (also referred to as Accession Number CG52997-02). This is a mature protein with 100% identity to the previously identified sequence NOVlb.
A disclosed NOVlc nucleic acid of 2531 nucleotides (also referred to as CG52997-02) encoding a novel Insulin Like Gxowth Factor Binding Protein Complex-Acid Labile Subunit-like pxotein is shown in Table ID. An open reading frame was identified beginning with a GAT initiation codon at nucleotides 2-4 and ending with a TAA codon at nucleotides 2513-2515. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 1D. The start and stop colons are in bold letters. Because the start colon is not a traditional ATG start colon, the reading frame shown below for NOV 1 c could be a partial reading frame that extends further in the 5' direction.
Table 1D. NOVlc nucleotide sequence (SEQ ID N0:4).
AGGCTCTTGTGATTCTCTTTGCAATTGTGAGGAAAAAGATGGCACAATGCTAATAAATTGTGAAGCAAAAGG
TATCAAGATGGTATCTGAAATAAGTGTGCTACCATCACGACCTTTCCAACTAAGCTTATTAAATAACGGCTT
GACGATGCTTCACACAAATGACTTTTCTGGGCTTACCAATGCTATTTCAATACACCTTGGATTTAACAATAT
TGCAGATATTGAGATAGGTGCATTTAATGGCCTTGGCCTCCTGAAACAACTTCATATCAATCACAATTCTTT
AGAAATTCTTAAAGAGGATACTTTCCATGGACTGGAAAACCTGGAATTCCTGCAAGCAGATAACAATTTTAT
CACAGTGATTGAACCAAGTGCCTTTAGCAAGCTCAACAGACTCAAAGTGTTAATTTTAAATGACAATGCTAT
TGAGAGTCTTCCTCCAAACATCTTCCGATTTGTTCCTTTAACCCATCTAGATCTTCGTGGAAATCAATTACA
AACATTGCCTTATGTTGGTTTTCTCGAACACATTGGCCGAATATTGGATCTTCAGTTGGAGGACAACAAATG
GGCCTGCAATTGTGACTTATTGCAGTTAAAAACTTGGTTGGAGAACATGCCTCCACAGTCTATAATTGGTGA
TGTTGTCTGCAACAGCCCTCCATTTTTTAAAGGAAGTATACTCAGTAGACTAAAGAAGGAATCTATTTGCCC
TACTCCACCAGTGTATGAAGAACATGAGGATCCTTCAGGATCATTACATCTGGCAGCAACATCTTCAATAAA
TGATAGTCGCATGTCAACTAAGACCACGTCCATTCTAAAACTACCCACCAAAGCACCAGGTTTGATACCTTA
TATTACAAAGCCATCCACTCAACTTCCAGGACCTTACTGCCCTATTCCTTGTAACTGCAAAGTCCTATCCCC
ATCAGGACTTCTAATACATTGTCAGGAGCGCAACATTGAAAGCTTATCAGATCTGAGACCTCCTCCGCAAAA
TCCTAGAAAGCTCATTCTAGCGGGAAATATTATTCACAGTTTAATGAAGTCTGATCTAGTGGAATATTTCAC
TTTGGAAATGCTTCACTTGGGAAACAATCGTATTGAAGTTCTTGAAGAAGGATCGTTTATGAACCTAACGAG
ATTACAAAAACTCTATCTAAATGGTAACCACCTGACCAAATTAAGTAAAGGCATGTTCCTTGGTCTCCATAA
TCTTGAATACTTATATCTTGAATACAATGCCATTAAGGAAATACTGCCAGGAACCTTTAATCCAATGCCTAA
ACTTAAAGTCCTGTATTTAAATAACAACCTCCTCCAAGTTTTACCACCACATATTTTTTCAGGGGTTCCTCT
AACTAAGGTAAATCTTAAAACAAACCAGTTTACCCATCTACCTGTAAGTAATATTTTGGATGATCTTGATTT
GCTAACCCAGATTGACCTTGAGGATAACCCCTGGGACTGCTCCTGTGACCTGGTTGGACTGCAGCAATGGAT
ACAAAAGTTAAGCAAGAACACAGTGACAGATGACATCCTCTGCACTTCCCCCGGGCATCTCGACAAAAAGGA
ATTGAAAGCCCTAAATAGTGAAATTCTCTGTCCAGGTTTAGTAAATAACCCATCCATGCCAACACAGACTAG
TTACCTTATGGTCACCACTCCTGCAACAACAACAAATACGGCTGATACTATTTTACGATCTCTTACGGACGC
TGTGCCACTGTCTGTTCTAATATTGGGACTTCTGATTATGTTCATCACTATTGTTTTCTGTGCTGCAGGGAT
AGTGGTTCTTGTTCTTCACCGCAGGAGAAGATACAAAAAGAAACAAGTAGATGAGCAAATGAGAGACAACAG
TCCTGTGCATCTTCAGTACAGCATGTATGGCCATAAAACCACTCATCACACTACTGAAAGACCCTCTGCCTC
ACTCTATGAACAGCACATGGTGAGCCCCATGGTTCATGTCTATAGAAGTCCATCCTTTGGTCCAAAGCATCT
GGAAGAGGAAGAAGAGAGGAATGAGAAAGAAGGAAGTGATGCAAAACATCTCCAAAGAAGTCTTTTGGAACA
GGAAAATCATTCACCACTCACAGGGTCAAATATGAAATACAAAACCACGAACCAATCAACAGAATTTTTATC
CTTCCAAGATGCCAGCTCATTGTACAGAAACATTTTAGAAAAAGAAAGGGAACTTCAGCAACTGGGAATCAC
AGAATACCTAAGGAAAAACATTGCTCAGCTCCAGCCTGATATGGAGGCACATTATCCTGGAGCCCACGAAGA
GCTGAAGTTAATGGAAACATTAATGTACTCACGTCCAAGGAAGGTATTAGTGGAACAGACAAAAAATGAGTA
TTTTGAACTTAAAGCTAATTTACATGCTGAACCTGACTATTTAGAAGTCCTGGAGCAGCAAACATAAGGGCG
AATTCTGCTGT
In a search of public sequence databases, the NOVlc nucleic acid sequence, located on chromsome 13 has 2471 of 2480 bases (99%) identical to a gb:GENBANK-ID:AK026427~acc:AK026427.1 mRNA from Homo Sapiens (Homo Sapiens cDNA: FLJ22774 frs, clone KAIA.1575) (E = 0.0). Public nucleotide databases include alI
GenBank databases and the GeneSeq patent database.
The disclosed NOVlc polypeptide (SEQ ID NO:S) encoded by SEQ ID N0:4 has 837 amino acid residues and is presented in Table 1E using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOVlc has no signal peptide and is likely to be localized in the plasma membrane with a certainty of 0. 4600. In other embodiments, NOVlc may also be localized to the endoplasmic reticulum (membrane) with a certainty of 0.1000, the endoplasmic reticulum (membrane) with a certainty of 0.1000, or extracellularly with a certainty of 0.1000.
Table 1E. Encoded NOVlc protein sequence (SEQ ID NO:S).
DSLFYSSLLACISLHSQTPVLSSRGSCDSLCNCEEKDGTMLINCEAKGIKMVSEISVLPSRPFQLSLLNNGL
TMLHTNDFSGLTNAISIHLGFNNIADIEIGAFNGLGLLKQLHINHNSLEILKEDTFHGLENLEFLQADNNFI
TVIEPSAFSKLNRLKVLILNDNAIESLPPNIFRFVPLTHLDLRGNQLQTLPYVGFLEHIGRILDLQLEDNKW
ACNCDLLQLKTWLENMPPQSIIGDWCNSPPFFKGSILSRLKKESICPTPPVYEEHEDPSGSLHLAATSSIN
DSRMSTKTTSILKLPTKAPGLIPYITKPSTQLPGPYCPIPCNCKVLSPSGLLIHCQERNIESLSDLRPPPQN
PRKLILAGNIIHSLMKSDLVEYFTLEMLHLGNNRIEVLEEGSFMNLTRLQKLYLNGNHLTKLSKGMFLGLHN
LEYLYLEYNAIKEILPGTFNPMPKLKVLYLNNNLLQVLPPHIFSGVPLTKVNLKTNQFTHLPVSNILDDLDL
LTQIDLEDNPWDCSCDLVGLQQWTQKLSKNTVTDDILCTSPGHLDKKELKALNSEILCPGLVNNPSMPTQTS
YLMVTTPATTTNTADTILRSLTDAVPLSVLILGLLIMFITIVFCAAGIWLVLHRRRRYKKKQVDEQMRDNS
PVHLQYSMYGHKTTHHTTERPSASLYEQHMVSPMVHVYRSPSFGPKHLEEEEERNEKEGSDAKHLQRSLLEQ
ENHSPLTGSNMKYKTTNQSTEFLSFQDASSLYRNILEKERELQQLGITEYLRKNIAQLQPDMEAHYPGAHEE
A search of public sequence databases reveals that the NOV 1 c amino acid sequence has 427 of 436 amino acid residues (97%) identical to, and 428 of 436 amino acid residues (98%) similar to, the 440 amino acid residue ptnr:SPTREMBL-ACC:Q9HSY7 protein from Honzo sapiezzs (Human) (CDNA: FLJ22774 FIS, CLONE KATA1575) (E = 5.7e?3o), public amino acid databases include the GenBank databases, SwissPxot, PDB and PIR.
NOV 1 c is expressed in at least the following tissues: Heart, Coronary Artery, Pancreas, Small Intestine, Peripheral Blood, Brain, Mammary gland/Breast, Uterus, Vulva, Prostate, Lung, Trachea, Skin, Colon. Expression information was derived from the tissue sources of the sequences that were included in the derivation of the sequence of NOVlc.
The proteins encoded by the NOVla, 1b and lc nucleotides are very closely homologous as is shown in the alignment in Table 1F. As shown, the sequences encoded by the NOVla and 1b nucleic acid sequences are 100% identical.
Table 1F Alignment of NOVla, 1b, and lc.

NOVla NOVlb NOVlc NOVla NOVlb NOVlc NOVla rrovlb 7o so so loo llo lzo ....I....I....I....I....I....I....l....l....l....l....l....~

NOVlc w ' .... ... .....:~. ... .... ... . .~... .... .. .....
NOVla . m.. ".il.6 a iu, ... ; ':~ ..i . .
'~ ~ . ~ '.
. ~

NOVlb r . .a ~ . a ~ . .., ' .

NOVlc ;gas ~:~~y~;~~s~t.~~,~~;r~~c aa~ ~~.~i~;~r *~r.~~ .5~t~.~~ ~l,r:~r.~~
tN.~n.i.5 .Yd~! !*a1y41:J:W1.7 u r ' i ' NOVla ci ' e~~;ii, ~~g~ ~'~ ~=
. "
"

NOVlb . ~ 'r " ~ ' NOV1C ! ~fJ~ai3;Zed'h*n~ai;~h ll~fcli Klas4 a:~~,h l~.W 7:J.Ytf.Y n.~rf-~Wi.'f .h*~s ly-"'f-~!1 a., I I ~
~

. . . _.. . . ... .. . .
NOVl ~. .Iii.... . ~ .. . ~a S~~ a .
. ' . . :.' ~ s. ,. ' . .. i ic r .p a NOVlb ' ' NOV1C Gig"fr~ i~:re #~*~/t*tW.~;~.'iiKvl.It:It !'?~C5t 3~*:let U:tJ.F.'l:l.Y tlrt~lr*. t~1(~ h~~h* ai..~U
-I ~
~

.. .. . .. . . . .. . .,..
NOVl wr.. ,,~ ~ . i . . . m"a. u. . . a a .. ,j r .. . r .o .
~.i~ ~TJr r NOVlb ' ~~ rr ' Novlo .

I ~

. ._ . . .
NOVla ' . ' : r . . .. 'i.
y,~i. H ~ . ' .. , i ii i s rr ~ ~
'I. .i.iir io'1.
_.

NOVlb I

NOV1C ~ H 9~1 II*-~G~ jllii*~ ~:IC JIY~Yb~:7Vi:3.i~r~ 'nltltlfl Y~lvlyll'II~[ IlIJ9.11*51.21 - -NOVla T "f'~ , ' iii . y . ~i p T~'V v'r~
s'i .. ..i " . ~y d.
I'.

NOVlb ~ . i , . . sa i.
.ri .u . ~
.

NOVle ' ' 40 ...~. . ...~ .I....~ .. .. ..~.. . .._.
;- .~ .,~. y .
: ..
' NOVla mii i .. " 'ai . , e .
s ' s ~ V r /
V ~. ' ~~
, NOVlb .. ~ . a ' '' .

NOVlc . '' I ~

.~.. .,I .. . .. .. .
NOVla n . . . r .
.. r~ . n. . i '' t . 's ' r s a ' a NOVlb ~ ' ' ~ ~
' NOVle . . i'a .- W i NOVla . ' '~ f i : ...
~ a ~ I ..
' 1i , ~

NOVlb 1 i ~

55 NOVlc r m ' ' ~ ~

NOVla a . . ..,1.. .. .. . ..
. ',.~~~;;. .:a. .~ '::. .,,.:.'i .. ...
a r :i. ~ .' ,. .v y w a -60 Novlb r . r NOVlc i a.s.' iwifi'*r4o't r~'faC V.7<~' f-~~If~l; , aylsJrtch~rw Aber;.l,rta iia, 'h*nai ~i;a~l 'r i 65 NOVl ~ ' '~ ~ ~" ' ~ 5'~ a . ~,I
I ~. ' I. ; . ' ~ . ~
. I ' ' i .ES : y ; !"~,.~, a ~ ~
~"3 ;
~ ;
-NOVlb . . ~ i . i~ , ~ ..
.

NOVlc [~.~~.~.. ~a~~ ~~~~w~ ~~i sxym, .w~r.~a~ x~;,. in.rwm.
~rrn~rnxr.~
- "

NOVla (SEQ ID N0:2) NOVlb ~ (SEQ ID N0:2) NOVlc (SEQ ID NO: S) Homologies to any of the above NOV 1 proteins will be shared by the other two proteins insofar as they are homologous to each other as shown above. Any reference to NOV 1 is assumed to refer to all three of the NOV 1 proteins in general, unless otherwise noted.
The disclosed NOV 1 a polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table 1 G.
Table 1G.
BLAST
results for NOVla Gene Protein/ OrganismLengthIdentityPo Eacpect Tndex/

Identifier (aa) (~) sitives ($) gi~14758126~ref~XP hypothetical 798 798/798 798/798 0.0 _ protein FLJ22774 (100%) (100%) 033182.1 [Homo sapiensl gi~14149932~ref~NP hypothetical 440 425/425 425/425 0.0 _ protein FLJ22774 (100%) (100%) 115605.1 [Homo sapiensl gi~6691962'emb~CAB6 bG256022.1 853 354/866 504/866 e-161 5788.1 (similar to (40%) (57%) IGFALS (insulin-like growth factor binding protein, acid labile subunit)) [Homo Sapiens]

gi~11360190~pirI~T4 hypothetical 314 314/314 314/314 e-160 6279 protein (100%) (100%) DKFZp56401278.1 -human (fragment) gi~14424224~sp~0949HYPOTHETICAL 966 356/915 534/915 e-158 91Y918 HUMAN P120TEIN KIAA0918 (38%) (57%) The homology between these and other sequences is shown graphically in the ClustalW analysis shown in Table 1H. In the ClustalW alignment of the NOV 1 protein, as well as all other ClustalW analyses herein, the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be altered to a much broader extent without altering protein structure or function.
Table 1H. ClustalW Analysis of NOV1 1) Novel NOVla (SEQ ID N0:2) 2) Novel NOVlb (SEQ ID N0:2) (Identical to NOVla)

3) Novel NOVlc (SEQ ID N0:5)

4) giI14758126Iref~XP'033182.1~ hypothetical protein FLJ22774 [Homo Sapiens]
(SEQ
ID N0:28)

5) gi~14149932~re~iNP 115605.1 hypothetical protein FLJ22774 [Homo Sapiens]
(SEQ
ID N0:29)

6) gi~6691962~emb~CAB65788.1~ bG256022.1 (similar to IGFALS (insulin-like growth factor binding protein, acid labile subunit)) [Homo Sapiens] (SEQ ID N0:30)

7) gi~11360190~pir~~T46279 hypothetical protein DKFZp56401278.1 - human (fragment (SEQ ID N0:31)

8) gi~14424224~sp~094991~Y918 HUMAN HYPOTHETICAL PROTEIN KIAA0918 (SEQ ID
N0:32) NOVla -------------------------MKLWIHLFY SLLACISLHSQTPVL
NOVlb -------------------------MKLWIHLFY~SLLAC1'SLHSQTPVL
IS NOVlc -----------------------------DSLFYSLLACTSLHSQTPVL
g1I147581261 -_________________________________________________ gii14149932~ __________________________________________________ gi~66919621 --------yFSLFRSIQLFADCK-KMFLWLFLIL----AISSTNADSD
gi~11360190~
ZO gi~14424224~ RRGAQGGKMHTCCPPVTLEQDLHRKMHSWMLQTL~;FAVTS~VLSCAETID

.. .I.. .~.. .~.. .~.. .~.. .~....
NOVla SSRGS SL ~ ~ EEK~T~G ~ v ZS NOVlb SSRGS SL EEK~G
NOVlc SSRGSSL EK~(GT L
gi~147581261 _______________._ gi~14149932~ _________________ gi ~ 6691962 ~ ISVEI~T-V~S~VSV~NU~i ~,. KVSS~YRP~T~,,KP ~ ~ F : FQ F
0 gi~11360190~ _____________ __ __ ___ _____ ________ gi1144242241 YYGEI~LkNA~P~EEK~GIT~SFSISP~RFPIL~SG~L

NOVla NOVlb I ~ ~ v NOVlc ~

gi~ 14758126~ ~ ~ v gi~ 14149932~ ~

40 66919621 ~ T LNF~._Ei I~Q L S ~ E
gi1 gii 11360190~_ __________ _._____________________ __________ gi 14424224~DTR~~PI~E~VVNYY~G~SI~,~eS~Q~T~RG~RR~,~I~

~ ~

4S y .. ..
.. .~.. .~..
.~ .~ .~..
.

NOVla i ~ ~ ~ ~ .

~
n NOVlb , , hn i m NOVlc v v~ i ~
v gi~ 147581261 ~ ~ ~
~

SO 14149932~ ~ ~m ~
gi~

gi~ 6691962~ ', a; KYR ~ LSF
~ ~mY~L
L
~

gi~ 11360190~_ _________________ _____ ____ __________ ___ gi~ 14424224~hQRA~L~
, ~
~Y~SG~LQLS~

NOVla ~ ~ v ~
~ v v ~

NOVlb ni v v ~ v~ v ~

NOVlc ~ v v ~ v giI 14758126~

gi~ 14149932~ ~ v v ~ v gi~ 6691962~ D S W RIB T ~~ PS

gi~ 11360190~_ ___________ _..
____-_ ____ __________ .__ gi~ 144242241~h ~
R~KI, ~eL~Q~MD~~P~S

NOVla ~ i PQST ~ ~ PFF S~ SRTt ~~5~ TPP~Y;E
NOVlb v ~PQST~.~PFFI~rS~BSRIaI~F'.rS2~TPP'(t'YE
1g NOVlc ~ ~T PQSI ~ S ~PFF S2 SRS ~~5~ ~TPPUYE~E
gi1147581261 ~ ~T'PQ~SI,S~pFFI~eST~SR~tF ~ESI~TPPVYE
gi1141499321 v-___________________________.______.___________ 81166919621 P ~YNTY~~AIQE'Ij'~'SDLY~R~~KETIV~yQE~LmMGTGS~7F
S gi~11360190~ _______ ____ _____ ________._ _________ _ g1 ~ 144242241 ~~,S~yD~DS~SYSALV~E~'IflFRLH~RD~DEt~S~QEbmRRL'I,SY

1O NOVla HED-i-~SGB... 1 uST~~IDSRMS.ICT.I~L..I. ~~____I_ I
NOVlb HED--- ~SGS H uG'ST~D,SRMS Kfi' IL ~ ~ ----------NOVlc HED---~SG~ ~STI~IDa~tMS KT 1L ~ ~G----------g7.1147581261 HED---~SG5 H S'ST~FD~T2MS KT SL ~ ~G----------g11141499321 ~. ~.:.~ _ IS 8i166919621 DVR-IL~PSQDENGY~IPNGH----~TQ~ZHR~yP~K-TTNPSK---gi111360190i _______ _____ _ __ ____________________ 8i 114424224 ~ EMRPQT~LS'~TGYLH~PA~V~S'VA~S~SA'S~Y~yPQL~yPQKGTRQPNKPRV

20 ....1.._.1....1.. .1 ~.. .1.. .
NOVla _______________ I. ~KpTQL~GPY ~I~' LS-PS L~~k NOVlb _______________ I~ ~KpTQL~GPY ~I~ LS-PS L3 NOVlc --------------- I~ ~KP~TQL~GPY ~I~ LS-PS L' gi114758126~ _______________ I xKPTQL~GPY ~I~ S-PS L.t 25 8i1141499321 ______________ __ _____________________ 8i 1 6691962 1 -ISGIVAGKALSNRN~SQISYQ'~RVP~LTP~F~yTAPSDL~ST~.~T
8i1113601901 ____________________ ~._____________________ _ 8i 114424224 1 RPTSRQPSKDLGYSNYG~S~AYQ"~KSPVPLEmTAQS~TT~QISDL

.1.. .1.. .1.. ..1.....1....1.,.-..1.. .,~,.. .~,.. .1 NOVla v ~ . ~P ~Q ~ I I HS~,M ~Lv.. F
. - . . -~ 1 NOVlb ~ ~ ~P~Q ~R I I HS ~L ~F E
NOVlc ~ ~ D ~p ~Q ~ I I HSZaMF ~Li, aFT E
3S 8i1147581261 ~ ~ D ~P~Q ~ I I HS~~L,, AFT E
8i1141499321 _______ ____ ____ ___ __ _ ___._____ 8i 16691962 1 ~I~Q~~I~IC~LS~KD~VDV~FTTJFEG~TL~~~
8i1113601901 __-____ ___ ____ ___ v -_____ 811144242241 ~K~TAEaQ~FC~Y~Y~TE~Y~AV;URR'.L~F~~ATGQLSL~

NOVla E~~'LE~G~S M. 1~~«'1.. .1."T,. '~G'.. L...1.. .1.. ,1 NOVlb ' E~~"G~ M ~"7 T KG1 L E.
4S NOVlc E'~~,GS M Q 3 T KG L
8i 1147581261 E~L~FiGM =-~TKG~L
8i1141499321 _ ._______ ________ __________ _________ 8i 1 6691962 1 ~Q~T~~ICGDVpHQy~E~YpEI~S~Q~L
SO 8i 1 14424224 1 SJ~IIQt? GD R"IE pE Y QS~Q~~'~Q~L
1 ~ ~ ..?~ ~ ~fl .1.. -1.. .I~.v'-1.. .1.. -1..'.-..1.. .,~._....1.. -L, NOVla . LP ~ ~P ~.T I~ SQ.
SS NOVlb LP ~ ~P ~ T~ ~Q
NOVlc LP ~- ~ ~PT~~~Q , 8i1147581261 LP ~ ~P T~
8i1141499321 -______ _____ ______ ___ _ _________ _____ ___ 8i166919621 ~SA~S~~h~KS~~e ~ ARI~~M
6O 8i1113601901 _______ ___ __________ ___ _____ _____.
8i 1 14424224 1 ~R~QSC '~~ QI,~F~SG~e jT~LRL~R~u~H~S

.1.,...1,. . . . ..1....1.. .1.. .1.. .1 . r ;J. r 6S NOVla ~D L..~.W n ~ ~ S ~ w NOVlb ~D L v ~ ~ ~ ~ S ~ w ~ ~ ~I~ w NOVlc ~D L v ~ ~ ~ ~ S ~ m v m 8i1147581261 ,._ ~D L ~ ~ ~ ~ ~ ~ v w v v m 8i1141499321 _ _______________________._____ T LkppQSIT
70 8i166919621 ~GVmQ~QS ~ ~ ~. ~ KL . GI~VIC~Ii gi,11360190~ - _____________________ gi ~ 14424224 ~ ~G~QOKS~I. . . ~ . ~I .MtC,L VET KVGV,T.~ ~EiVT

NOVla NOVlb NOVlc giI1475B126~
gi~14149932~ -DPSGSLHL~
gi16691962~ -PFTSP ~~I
gi~11360190~
gi114424224~ 'PRTSA ~ R

NOVla NOVlb NOVlc gi~147581261 gi1141499321 gi166919621 gi~113601901 gi1144242241 NOVla NOVlb NoVlc gi'14758126, gi1141499321 gi16691962~
gi~113601901 gi1144242241 .... ....~....~....~....~....I....~.... ....
NOVla ___, ~-~~.~ -:.~.~.. .y~ ______ ~v v ___ Novlb --- ~ ------ -NOVlc ___ ~ .~ ______ y~ __ gi~14758126~ ___ ~ _______ ___ gi1141499321 ____ S~j__________.p.pQ .R~I________~GNIIHS-____ gi~6691962~ -----T F~PQT~EQMS, H~'C L ES---------- TGFMFS~:7---gi~11360190~ ---- ~ ' -------- --gi I 14424224 ~ TPAGHVYEY'~PH . C~ - ~IjY'RS~EGNSVEDYKDL L(7TYSSNHHL

NOVla -----_-- ~~ ---NOVlb -------- ~~ ----NOVlc ------- ~ ---gi~147581261 ________ _____ gi114149932~ -----gi~66919621 ________ppG_____ gi~113601901 ________ _____ gi114424224~ QQQQQPPPPPQ~PQQQP

.~....~....~.... .~....~....~....~....~..
NOVla ..~ .. ---- --. .~.
NOVlb ..~ .. ____ __ NOVlc ..~ v ~ .. ____ __ gi~14758126~ ..~ .. ____ __ (5 gi~141499321 --- M1~LT, Q~L GN- T SKGMF G--------------LH
LI
gi~6691962~ LF-PS ~.8~7FI~F S;.-.. ~S~ ~G-___________________ gi~11360190~ ..~ ~ ~~ ' ----- -. . .
gi I 14424224 ~ ~TL SP .. ~ RF ~ PxJICHCSTTPAGNS Pls''YPKFPC,~P~ YTFS' ~;

NOVla NOVlb NOVlc S gi~14758126~
gi~14149932~
gi~6691962~
gi~11360190~
gi~14424224!

NOVla ~ -----NOVlb '~ -----1S NOVlc ~ -----giI147581261 ~ _____ gi~I4149932~ G-F ___________ -gi~66919621 n F,LNKI

gi~11360190~ ~ -gi~14424224~ ~ TFSQF

The presence of identifiable domains in NOV 1, as well as all other NOVX
proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks, 2S Pfam, ProDomain, and Prints, and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/
interpro).
DOMAIN results for NOV 1 as disclosed in Tables lI-IL, were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST analyses. This BLAST
analysis software samples domains found in the Smart and Pfam collections. For Table 1E
and all successive DOMAIN sequence alignments, fully conserved single residues are indicated by black shading or by the sign (~) and "strong" semi-conserved residues are indicated by grey shading or by the sign (+). The "strong" group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
3S Tables lI-1L lists the domain description from DOMAIN analysis results against NOV 1. This indicates that the NOV 1 sequence has properties similar to those of other proteins known to contain this domain.
Table 1I. Domain Analysis of NOVl qnl~Smart~smart00082, LRRCT, Leucine rich repeat C-terminal domain.
(SEQ ID N0:65) CD-Length = 51 residues, 100.0% aligned Score = 49.7 bits (117), Expect = 7e-07 G.O Query: 517 NPWDCSCDLVGLQQWIQKLSKNTVTDDILCTSPGHLDKKELKALNSEILCP 567 Sbjct: 1 NPFICDCELRWLLRWLQANRHLQDPVDLRCASPESLRGPLLLLLPSSFKCP 51 Table 1J. Domain Analysis of NOVl gnllSmartlsmartOD082, LRRCT, Leucine rich repeat C-terminal domain.
(SEQ ID N0:65) CD-Length = 51 residues, 100.0% aligned Score = 45.1 bits (105), Expect = 2e-05 Query: 218 NKWACNCDLLQLKTWLENMPPQSIIGDWCNSPPFFKGSILSRLKKESICP 268 I + I+~+I I II+ I+ I II +I +I I II
S Sbjct: 1 NPFICDCELRWLLRWLQANRHLQDPVDLRCASPESLRGPLLLLLPSSFKCP 51 Table 1K. Domain Analysis of NOV1 gnl~Pfam~pfam01463, LRRCT, Leucine rich repeat C-terminal domain.
Leucine Rich Repeats pfam00S60 are short sequence motifs present in a number of proteins with diverse functions and cellular locations.
Leucine Rich Repeats are often flanked by cysteine rich domains. This domain is often found at the C-terminus of tandem leucine rich repeats (SEQ ID N0:66) CD-Length = 51 residues, 100.0% aligned Score = 47.8 bits (112), Expect = 3e-06 Query: 517 NPWDCSCDLVGLQQWIQKLSKNTVTDDILCTSPGHLDKKELKALNSEILCP 567 II+ I I+I I +1+++ + +I+ I II I I+ I I+ II
Sbjct: 1 NPFICDCELRWLLRWLREPRRLEDPEDLRCASPESLRGPLLELLPSDFSCP 51 Table 1L. Domain Analysis of NOV1 gnl~Pfamlpfam01463, LRRCT, Leucine rich repeat C-terminal domain.
Leucine Rich Repeats pfam00560 are short sequence motifs present in a number of proteins with diverse functions and cellular locations.
Leucine Rich Repeats are often flanked by cysteine rich domains. This domain is often found at the C-terminus of tandem leucine rich repeats. (SEQ ID N0:66) CD-Length = 51 residues, 100.0% aligned Score = 46.2 bits (108), Expect = 7e-06 IS Query: 218 NKWACNCDLLQLKTWLENMPPQSIIGDWCNSPPFFKGSILSRLKKESICP 268 I + I+I+I I I~ I+ I II +I +I I + II
Sbjct: 1 NPFICDCELRWLLRWLREPRRLEDPEDLRCASPESLRGPLLELLPSDFSCP 51 Proteins belonging to the IGFBP-ALS family of proteins play an important role in regulating the levels of circulating hormones. The acid labile subunit of the complex plays an important role in regulating the stability of the complex and ensuring high levels of circulating hormones that are regulated by the IGFBP family of proteins. This protein also has a leucine rich repeat that is a common domain in many proteins that are important for the developing embryo. As a result this protein may play an important role in development and disease.

Insulin-like growth factors (IGFs) I and II are important regulators of cell proliferation and differentiation (Ueki I et al., Proc Natl Acad Sci U S A 2000 Jun 6;97(12):6868-73). After birth, plasma IGFs, representing mostly liver-derived IGFs, circulate in ternary complexes of I50 kDa consisting of one molecule each of IGF, IGF-binding protein (IGFBP) 3, and an acid labile subunit (ALS). Onset of ALS synthesis after birth is the primary factor driving the formation of ternary complexes. Capture of IGFs by ALS is thought to allow the development of a plasma reservoir without negative effects such as hypoglycemia and cell proliferation. To evaluate the importance of ALS and ternary complexes, we have created mice in which the ALS gene has been inactivated. The mutation was inherited in a Mendelian manner, without any effects on survival rates and birth weights. A growth deficit was observed in null mice after 3 weeks of life and reached 13% by IO weeks. This modest phenotype was observed despite reductions of 62 and 88% in the concentrations of plasma IGF-I and IGFBP-3, respectively. Increased turnover accounted for these reductions because indices of synthesis in liver and kidney were not decreased. Surprisingly, absence of ALS did not affect glucose and insulin homeostasis. Therefore, ALS is required for postnatal accumulation of IGF-I and IGFBP-3 but, consistent with findings supporting a predominant role for focally produced IGF-I, is not critical for growth. This model should be useful to determine whether presence of ALS is needed for other actions of liver-derived IGF-I and for maintenance of homeostasis in presence of high circulating levels of IGF-II.
In circulation, insulin-like growth factor-I (IGF-I) is bound in a trimeric complex of 150 kDa with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). (Moller S
et al., J Hepatol 2000 Mar;32(3):441-6). Whereas circulating IGF-I and IGFBP-3 are reported to be low in patients with chronic liver failure, the level of ALS has not been described in relation to hepatic dysfunction. The aim of the present study was therefore to measure circulating and hepatic venous concentrations of ALS in relation to hepatic function and the IGF axis.
The insulin-like growth factor (IGF) binding proteins (IGFBPs) were initially identified as tamer proteins for IGF-T and IGF-II in a variety of biologic fluids. (Rosenfeld RG et aL, Pediatrics 1999 Oct;104(4 Pt 2):1018-Z 1). Their presumed function was to protect IGF peptides from degradation and clearance, increase the half life of the IGFs, and deliver them to appropriate tissue receptors. The concept of IGFBPs as simple carrier proteins has been complicated, however, by a number of observations: 1) the six IGFBPs vary in their tissue expression and their regulation by other hormones and growth factors;
2) the IGFBPs are subjected to proteolytic degradation, thereby altering their affinities for the IGFs; 3) IGFBP-3 and IGFBP-5, in addition to binding IGFs, also can associate with an acid-labile subunit, thereby increasing further the half life of the IGFs; 4) in addition to modifying the access of IGF peptides to IGF and insulin receptors, several of the IGFBPs may be capable of increasing IGF action; 5) some of the IGFBPs may be capable of IGF-independent regulation of cell growth; 6) some of the IGFBPs are associated with cell membranes or possibly with membrane receptors; and 7) some of the IGFBPs have nuclear recognition sites and may be found within the nucleus. Additionally, a number of cDNAs identified xecently have been found to encode proteins that bind IGFs, but with substantially lower affinities than is the case with IGFBPs. The N-terminal regions of the predicted proteins are structurally homologous to the classic IGFBPs, with conservation of the cysteine-rich region. These observations suggest that these low-affinity binders are members of an IGFBP superfamily, capable of regulating cell growth by both IGF-dependent and IGF-independent mechanisms.insulin-like growth factor, insulin-like growth factor binding proteins.
Total IGF-I level in serum is a sensitive index during growth hormone (GH) replacement therapy of adults, since GH stimulates the hepatic expressions of both insulin-like growth factor (IGF-I) and acid-labile subunit (ALS) and the major part of IGF-I in the circulation is found in a ternary complex together with ALS and IGFBP-3. (Hall K et al., J
Endocrinol Invest 1999;22(5 Suppl):48-57) However, other regulators of the proteins constituting the ternary complex may influence IGF-I levels. In healthy subjects the serum IGF-I levels are low at birth, rise during childhood, with peak levels during puberty, and decline with increasing age. This pattern has been attributed to the age-dependent GH
production, but it is unknown whether the wide range of IGF-I levels within each age interval is due to GH production or GH sensitivity. In elderly twins approximately 60%
of IGF-I levels are genetically determined. The remaining environmental dependency of IGF-I is partly due to nutrition. Both caloric and protein content of the diet is of importance.
Thus, low IGF-I levels are found in GH deficient patients as well as in patients with GH resistance due to malnutrition or GH receptor defects. It is essential that IGF-I determination is performed by assays in which IGFBPs do not interfere, and that IGF-I concentration is evaluated in relation to age, i.e.
expressed in SD score, and the number of individuals constituting the reference intervals improves 'the sensitivity and specificity. Although determination of IGF-I is recommended in assessing GH deficiency in children, its diagnostic value in patients with adult onset of GH
deficiency is not agreed upon. In the age group above 40-80 years many patients with pituitarylhypothalamic disorders and GH peaks below 3 pg/1 during provocation tests have normal IGF-I levels. It is not clarified, whether the IGF-I levels within normal range for age is due to endogenous basal GH production being sufficient or other factors stimulating IGF-I, IGFBP-3 or ALS expressions.
Circulating insulin-like growth factors (IGFs) represent an important pool of potential hypoglycemic activity, which is largely inhibited by their sequestration in a heterotrimeric complex comprising growth factor, IGF-binding protein-3 (IGFBP-3), and acid-labile subunit (ALS). (Baxter RC Metabolism 1995 Oct;44(10 Suppl 4):12-7). Less than 1% of total IGFs circulate in the free form, yet even this amount might contribute significantly to circulating insulin-like activity. The ternary binding protein complex appears to inhibit insulin-like activity of bound IGFs by preventing their egress from the circulation.
Although the integrity of this complex might be affected by limited proteolysis of IGFBP-3 in pregnancy and catabolic conditions, the evidence that this increases IGF bioavailability, and thus hypoglycemic potential, is as yet unclear. However, in patients with IGF-II-secreting tumors, hypoglycemia may result from a failure of the ternary complex to adequately sequester the IGFs. Improvement in complex formation, by treatment with corticosteroids or growth hormone, alleviates the hypoglycemia, even if (as seen with growth hormone treatment) IGF-II hypersecretion persists. In these patients, blood glucose levels are inversely correlated with IGFBP-2 levels, suggesting that this protein might play a part in transporting IGFs to their target tissues. Conversely, ALS levels correlate positively with blood glucose, emphasizing the importance of the ternary complex in preventing hypoglycemia. Unlike the other IGF-binding proteins, IGFBP-1 is acutely regulated in the circulation, in a manner consistent with its acting as a glucose counterregulator. It might act in this way by inhibiting the activity of free IGFs in the circulation.
Leucine-rich repeats (LRRs) are relatively short motifs (22-28 residues in length) found in a variety of cytoplasmic, membrane and extracellular proteins (InterPro). Although these proteins are associated with widely different functions, a common property involves protein-protein interaction. Little is known about the 3D structure of LRRs, although it is believed that they can form amphipathic structures with hydrophobic surfaces capable of interacting with membranes. In vitro studies of a synthetic LRR from Drosophila Toll protein have indicated that the peptides form gels by adopting beta-sheet structures that form extended filaments. These results are consistent with the idea that LRRs mediate protein-protein interactions and cellular adhesion. Other functions of LRR-containing pxoteins include, for example, binding to enzymes and vascular repair. The 3D structure of ribonuclease inhibitor, a protein containing 15 LRRs, has been determined, revealing LRRs to be a new class of alpha/beta fold. LRRs form elongated non-globular structures and are often flanked by cysteine rich domains.
The disclosed NOV 1 nucleic acid of the invention encoding a Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit -like protein includes the nucleic acid whose sequence is provided in Table 1A, C and E or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 1A, C, or E while still encoding a protein that maintains its Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids ox nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
The disclosed NOV 1 protein of the invention includes the Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like protein whose sequence is provided in Table 1B or 1E. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 1B or 1E while still encoding a protein that maintains its Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 60% percent of the residues may be so changed.
The invention further encompasses antibodies and antibody fragments, such as Fab or (Fab)z, that bind immunospecifically to any of the proteins of the invention.
The above defined information for tlus invention suggests that this Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like protein (NOVl) may function as a member of a "Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit family". Therefore, the NOV 1 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to: protein tlierapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene deliverylgene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
The NOV 1 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in cancer including but not limited to various pathologies and disorders as indicated below. For example, a cDNA encoding the Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like protein (NOV 1) may be useful in gene therapy, and the Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit -like protein (NOV 1) may be useful when administered to a subject in need thereof.
By way of nonlirniting example, the compositions of the present invention will have efficacy for treatment of patients suffering from cancer, cystitis, incontinence, fertility, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis , atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation recovery. The NOVl nucleic acid encoding the Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
NOVl nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOVl substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies"
section below. The disclosed NOVl protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV 1 epitope is from about amino acids 10 to 50. In another embodiment, a NOV 1 epitope is from about amino acids 80 to 120. In additional embodiments, NOVl epitopes are from about amino acids 180 to 220, from about amino acids 230 to 300, from about amino acid 330 to 350, from about amino acid 370 to 400, from about amino acid 480 to 540, from about amino acid 550 to 560, and from about amino acids 620 to 840. These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.

A disclosed NOV2 nucleic acid of 3609 nucleotides (also referred to as 101599929 EXTl) encoding a novel Attractin like protein is shown in Table 2A.
An open reading frame was identified beginning with an ATG initiation colon at nucleotides 7-9 and ending with a TAA colon at nucleotides 3562-3564. A putative untranslated region upstream from the initiation colon and downstream from the termination colon is underlined in Table 2A, and the start and stop colons are in bold letters.
Table 2A. NOV2 nucleotide sequence (SEQ ID N0:6).
CGGCACAGGACCGGCTCCTGCTTCTCGGGCCGCTGTGTCAACTCCACCTGCCTCTGCGACCCGGGCTGGGTG
GGGGACCAGTGCCAGCACTGCCAGGGCAGGTTCAGGTTAACAGAACCTTCTGGATATTTAACAGATGGCCCA
ATTAACTATAAATATAAAACTAAATGTACTTGGCTCATTGAAGGCCCAAATGCAGTGTTAAGATTAAGATTC
AATCATTTTGCTACAGAATGTAGCTGGGATCATATGTATGTTTATGATGGAGATTCAATATATGCACCTTTA
ATAGCTTCTTTTAGTGGTTTGATAGTCCCTGAAATAAGGGGCAATGAAACTGTGCCTGAAGTTGTTACTACA
TCTGGCTATGCACTGTTACATTTTTTTAGTGATGCTGCGTATAATCTAACTGGTTTCAACATTTTCTATTCG
ATCAATTCTTGTCCTAACAATTGCTCTGGTCATGGGAAGTGTACAACTAGTGTCTCTGTTCCAAGTCAAGTA
TATTGTGAATGTGATAAATACTGGAAGGGTGAAGCTTGTGATATTCCTTACTGTAAAGCCAATTGCGGCAGT
CCAGATCACGGTTACTGTGACCTGACTGGAGAAAAATTATGTGTCTGCAATGATAGTTGGCAAGGTATAGGT
CCTGATTGTTCTTTGAATGTTCCCTCTACTGAGTCTTACTGGATTCTGCCAAACGTTAAACCCTTCAGTCCT
TCTGTAGGTCGGGCTTCACATAAAGCAGTTTTACACGGGAAATTTATGTGGGTGATTGGTGGATATACTTTT
AACTACAGTTCTTTTCAAATGGTCCTAAGTTACAATTTAGAAAGCAGTATATGGAATGTAGGAACTCCATCA
AGGGGACCTCTCCAGAGATATGGACACTCTCTTGCTTTATATCAGGAAAACATCTTTATGTATGGAGGCAGA
ATTGAAACAAATGATGGCAATGTCACAGATGAATTATGGGTTTTTAACATACATAGTCAGTCATGGAGTACA
AAAACTCCTACTGTTCTTGGACATGGTCAGCAGTATGCTGTGGAGGGACATTCAGCACATATTATGGAGTTG
GATAGTAGAGATGTTGTCATGATCATAATATTTGGATATTCTGCAATATATGGTTATACAAGCAGCATACAG
GAATACCATATCTGTTCAAACACTTGGCTTGTTCCAGAAACTAAAGGAGCTATTGTACAAGGTGGATATGGC
CATACTAGTGTGTATGATGAAATAACAAAGTCCATTTATGTTCATGGAGGGTATAAAGCATTGCCAGGGAAC
AAATATGGATTGGTTGATGATCTTTATAAATATGAAGTTAACACTAAGACTTGGACTATTTTGAAAGAAAGT
GGGTTTGCCAGATACCTTCATTCAGCTGTTCTTATCAATGGAGCTATGCTTATTTTTGGAGGAAATACCCAT
AATGACACTTCCTTGAGTAACGGTGCAAAATGTTTTTCTGCCGATTTCCTGGCATATGACATATGCCCAGGC
TGGAGTGCAGTGGCACGATCTCAGCTCACTGCCACCTCCACCTCCCACGTTCAAGCGATTCTCAATAGGTCC
ATGTATATATTTGGGGGATTTTCTAGTGTACTCCTTAATGATATCCTTGTATACAAGCCTCCAAATTGCAAG
GCTTTCAGAGATGAAGAACTTTGTAAAAATGCTGGTCCAGGGATAAAATGTGTTTGGAATAAAAATCACTGT
GAATCTTGGGAATCTGGGAATACTAATAATATTCTTAGAGCAAAGTGCTTTTCTAAAAGAAATCTCTGCAGT
GACAGATGTTACAGATATGCAGATTGTGCCAGCTGTACTGCCAATACAAATGGGTGCCAATGGTGTGATGAC
AAGAAATGCATTTCGGCAAATAGTAACTGCAGTATGGTTAGTATTTTTGGGTATATAACCTTGCCTTCACAG
TTCCCATTCTATTATTGCTACAGATATGCAGATTGTGCCAGCTGTACTGCCAATACAAATGGGTGCCAATGG
TGTGATGACAAGAAATGCATTGCTTTACCAGCTCATCTTTGTGGAGAAGGATGGAGTCATATTGGGGATGCT
TGTCTTAGAGTCAATTCCAGTAGAGAAAACTATGACAATGCAAAACTTTATTGCTATAATCTTAGTGGAAAT
CTTGCTTCATTAACAACCTCAAAAGAAGTAGAATTTGTTCTGGATGAAATACAGAAGTATACACAACAGAAA
GTATCACCTTGGGTAGGCTTGCGCAAGATCAATATATCCTATTGGGGATGGGAAGACATGTCTCCTTTTACA
AACACAACACTACAGTGGCTTCCTGGCGAACCCAATGATTCTGGGTTTTGTGCATATCTGGAAAGGGCTGCA
GTGGCAGGCTTAAAAGCTAATCCTTGTACATCTATGGCAAATGGCCTTGTCTGTGAAAAACCTGTTAATCAA
AATGCGAGGCCGTGCAAAAAGCCATGCTCTCTGAGGACATCATGTTCCAACTGTACAAGCAATGGCATGGAG
TGTATGTGGTGCAGCAGTACGAAACGATGTGTTGACTCTAATGCCTATATCATCTCTTTTCCATATGGACAA
TGTCTAGAGTGGCAAACTGCCACCTGCTCCCGTGCTCAAAATTGTTCTGGATTGAGAACCTGTGGACAGTGT
TTGGAACAGCCTGAATGTGGCTGGTGCAATGATCCTAGTAATACAGGAAGAGGACATTGCATTGAAGGTTCT
TCACGGGGACCAATGAAGCTTATTGGAATGCACCACAGTGAGATGGTTCTTGACACCAATCTTTGCCCCAAA
GAAAAGAACTATGAGTGGTCCTTTATCCAGTGTCCAGCTTGCCAGTGTAATGGACATAGCACTTGCATCAAT
AATAATGTGTGCGAACAGTGTAAAAATCTCACCACAGGAAAGCAGTGTCAAGATTGTATGCCAGGTTATTAT
GGAGATCCAACCAATGGTGGACAGTGCACAGCTTGTACATGCAGTGGCCATGCAAATATCTGTCATCTGCAC
ACAGGAAAATGTTTCTGCACAACTAAAGGAATAAAAGGTGACCAATGCCAATTGTGTGACTCTGAAAATCGC
TATGTTGGTAATCCACTTAGAGGAACATGTTATTGTAAGTATAGCCTTTTGATTGATTATCAATTTACCTTC
AGCTTATTACAGGAAGATGATCGCCACCATACTGCCATAAACTTTATAGCAAACCCAGAACAGGTGAGGAAA
AATCTGGATATATCAATTAATGCATCAAACAACTTTAATCTCAACATTACGTGGTCTGTCGGTTCAGCTGGA
ACAATATCTGGGGAAGAGACTTCTATAGTTTCCAAGAATAATATAAAGGAATACAGAGATAGTTTTTCCTAT
GAAAAATTTAACTTTAGAAGCAATCCTAACATTACATTCTATGTGTACGTCAGCAACTTTTCCTGGCCTATT
AAAATACAGGTAAGTGTTAAGAGTATTTACTTCTAATGACCATAATATCATTAAGAAAAGAATGGTGCTTTT
GTCCAAAGT

The disclosed NOV2 nucleic acid sequence, localized to chromsome 10, has 494 of 694 bases (71 %) identical to an Attractin protein mRNA from mouse (GENBANK-ID:
AF119821) (E = 2.9e 204).
A NOV2 polypeptide (SEQ ID N0:7) encoded by SEQ ID N0:6 has 1185 amino acid residues and is presented using the one-letter code in Table 2B. Signal P, Psort andlor Hydropathy results predict that NOV2 does not contain a signal peptide and is likely to be localized in the mitochondrial membrane space with a certainty of 0.3600. In other embodiments, NOV2 may also be localized to the microbody (peroxisome) with a certainty of 0.3000, or the lysosome (lumen) with a certainty of 0.1000.
Table 2B. Encoded NOV2 protein sequence (SEQ ID N0:7).
MQKADSSTETMKEKRTAAAITVRHRTGSCFSGRCVNSTCLCDPGWVGDQCQHCQGRFRLTEPSGYLTDGPIN
YKYKTKCTWLIEGPNAVLRLRFNHFATECSWDHMYWDGDSIYAPLIASFSGLIVPEIRGNETVPEWTTSG
YALLHFFSDAAYNLTGFNIFYSINSCPNNCSGHGKCTTSVSVPSQVYCECDKYWKGEACDIPYCKANCGSPD
HGYCDLTGEKLCVCNDSWQGIGPDCSLNVPSTESYWILPNVKPFSPSVGRASHKAVLHGKFMWVIGGYTFNY
SSFQMVLSYNLESSTWNVGTPSRGPLQRYGHSLALYQENIFMYGGRIETNDGNVTDELWVFNIHSQSWSTKT
PTVLGHGQQYAVEGHSAHIMELDSRDVVMIIIFGYSATYGYTSSIQEYHICSNTWLVPETKGAIVQGGYGHT
SVYDEITKSIYVHGGYKALPGNKYGLVDDLYKYEVNTKTWTILKESGFARYLHSAVLINGAMLIFGGNTHND
TSLSNGAKCFSADFLAYDICPGWSAVARSQLTATSTSHVQAILNRSMYIFGGFSSVLLNDILVYKPPNCKAF
RDEELCKNAGPGIKCVWNKNHCESWESGNTNNILRAKCFSKRNLCSDRCYRYADCASCTANTNGCQWCDDKK
CTSANSNCSWSIFGYITLPSQFPFYYCYRYADCASCTANTNGCQWCDDKKCIALPAHLCGEGWSHIGDACL
RVNSSRENYDNAKLYCYNLSGNLASLTTSKEVEFVLDEIQKYTQQKVSPWVGLRKINISYWGWEDMSPFTNT
TLQWLPGEPNDSGFCAYLERAAVAGLKANPCTSMANGLVCEKPVNQNARPCKKPCSLRTSCSNCTSNGMECM
WCSSTKRCVDSNAYIISFPYGQCLEWQTATCSRAQNCSGLRTCGQCLEQPECGWCNDPSNTGRGHCIEGSSR
GPMKLIGMHHSEMVLDTNLCPKEKNYEWSFIQCPACQCNGHSTCINNNVCEQCKNLTTGKQCQDCMPGYYGD
PTNGGQCTACTCSGHANICHLHTGKCFCTTKGIKGDQCQLCDSENRYVGNPLRGTCYCKYSLLIDYQFTFSL
LQEDDRHHTAINFIANPEQVRKNLDISINASNNFNLNITWSVGSAGTISGEETSIVSKNNIKEYRDSFSYEK
FNFRSNPNITFYVYVSNFSWPIKTQVSVKSIYF
The disclosed NOV2 amino acid sequence has 703 of 1197 amino acid residues (58%) identical to, and 895 of 1197 amino acid residues (74%) similar to, the 1198 amino acid residue Attractin protein from human (SPTREMBL-ID:075882) (E = 0.0), and 703 of 1197 amino acid residues (58%) identical to, and 895 of 1197 amino acid residues (74%) similar to, the 1198 amino acid residue human soluble Attractin-1 protein (patp:AAV70689) (E = 0.0).
NOV2 is expressed in at least the following tissues: Brain, Kidney, Muscle, Pancreas, Prostate, Uterus, Breast, Colon, Ovary, and Liver. In addition, the sequence is predicted to be expressed in the following tissues because of the expression pattern of (GENBANK-ID:
AFl 19821) a closely related attractin homolog in mouse : Brain, Heart, Kidney, Liver, Lung, Skin, Spinal cord, and Pituitary.
NOV2 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 2C.

Table 2C. BLAST
results for Gene Index/ Protein/ Length TdentityPositivesExpect Identifier Organism (aa) (%) (%) gi1131600511embICACdJ741H3.1.11011 566/1015739/10150.0 32456,1 (attractin (55%) (72%) (with dipeptidylpe ptidase IV

activity) secreted isoform) [Homo sapiensl gi145853071gbIAAD25attractin 1428 677/1174865/11740.0 372.11AF119821[Mus (57%) (73%) musculus]

gi1134313131spIQ9WUATTRACTIN 1428 679/1174871/11740.0 601ATRN_MOUSE PRECURSOR (57s) (73%) (MAHOGANY

PROTEIN) gi~6912258~ref~NPattractin;1198 703/1210894/12100.0 _ attractin (58%) (73%) 36202.1 (with dipeptidylpe ptidase IV

activity);

mahogany protein [Homo sapiens]

gi~12275312~dbj~BABattractin 1275 680/1177870/11770.0 21018.11 [Rattus (57%) (73%) norvegicus]

The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 2D.
Table 2D. ClustalW Analysis of NOV2 1) NOV2 (SEQ ID N0:7) 2) gi113160D511embICAC32456.11 dJ741H3.1.1 (attractin (with dipeptidylpeptidase IV
activity) secreted isoform) [Homo Sapiens] (SEQ ID N0:32) 2) gi145853071gb1AAD25372.11AF119S21 1 attractin [Mus musculus] (SEQ ID N0:33) 3) gi1134313131sp1Q9WU601ATRN_MOUSE ATTRACTIN PRECURSOR (MAHOGANY PROTEIN) (SEQ ID
N0:34) 4) gi169122581ref1NP 036202.11 attractin; attractin (with dipeptidylpeptidase IV
activity); mahogany protein [Homo Sapiens] (SEQ ID N0:35) 5) gi1122753121dbj1BAB21018.11 attractin [Rattus norvegicus] (SEQ ID N0;36) . ..1 ..~....I. ' ..1 ...1....1....1....1....1 NOV2 ___MQ~DSS~TMKEKR~A~AITVRH~T~____________________ gi1131600511 __________________________________________________ gi1458S3071 -MVA ' ~ 8 'GS ~~P' :RQHRPCTATGAWRP ~RARL
gi1134313131 -MVA ~ 'GS ~~P~ RQHRPCTATGAWRP ~RARL
gi169122581 __ ~R ..L. S ___________,_ ___ gi1122753121 MV ~E'' 'G T' ~~P' KERQHRPCAATGAWRP 'RAGL
60 70 80 90 lOD
....1....~....~....1....1....1....1....1....1....1 NOV2 __________________________________________________ gi'231600511 __________________________________________________ gi145853071 CLPRVLSRALPPP---PLLPLLFSLLLLPLPREAEAAAVAAAVSGSAAAE
gi1134313131 CLPRVLSRALPPP---PLLPLLFSLLLLPLPREAEAAAVAAAVSGSAAAE
gi169122581 -____________________________________g____________ gi1122753121 CLPRVLSRALSPPPLLPLLPLLFSLLLLPLPREAEP.AAVAAAVSGSAAAE

.1....1....1...,1....1.. ~. . .1....1....1....1 NOV2 -------SCFS~VNS~--~L~.DP .. ~ ~ Q~EP~YL~.
gi1131600511 1~ giI45853071 AKECDRP _-_________ ___ T_____ _ ________________ gi1134313131 AKECDRP . 'T
gi169122581 ______ ~ . ~ .
gi1122753121 AKECDRP . 'T
160 170 lao 190 200 NoV2 m1 '_ ~~. y , .
I _____ gi113160051 gi145853071 _________________ __ .v gi1134313131 Q' RTM' gi169122581 Q' RZ v gi I 12275312 I Q' ICxM' .1....I.... .1....I.. .1....I.. .1....I
NOV2 ~S ~'~' IRVT ~ " F~~I
gi1131600511 __________________________________________________ r y , v gi145853071 t T
gi1134313131 '~ T
gi169122581 '. ~ " S
gi1122753121 '. T ~ "

t,,. .1....I ..I... .I . .I.. .,I.. .I.. .I,.. .I.. .I
3S NOV2 ~TS. T~' . P Q, . 'Y S'DH
~~~ ._ gi1131600511 ______________I " "-DT'y ' gi~4585307~ ~ ~S
gi1134313131 . ~S
gi169122581 ~ ~I -DT
gi~122753121 ~ ~S -~T

~,1.~... . .1. . . I . . . . I . . . . . ~,.w. . ., . . . . I . . . . I . .
n N~ Y
NOV2 . ; L~GE,ECL S .GI 'D . Li- . 'STS ILPNVKPFSPSV . ' 45 gi1131600511 S ~ ' SD ~--gi145853071 ~ ~ P~ ~.7-- I' . __~j gi1134313131 ~T' P-- ' T' gi169122581 S ~ ' SD~.--gi1122753121 ~T' P .-- x' ~ ,", .I.. .I.. .1....1_....I.. . ..I.. .1....I..
NOV2 ' ~..yl' . KF . ~ T :f. S~Q -S - . E SI . -~7GTPSRGP?:iQ' .rr, . a gi1131600511 SS gi145853071 ~ S ~ T ' S
gi1134313131 ~ ~ ~ T ' S
gi169122581 gi1122753121 E . S ~ S 5 .I... ..
NOV2 Q. F . ETND SQ. ST.. TVLGH
Y EN
gi1131600511 ~ ~ -gi145853071 ~ ~ -gi1134313131 ~ ~ - ' gi169122581 ~ ~ - _ gi1122753121 " . ~ -....~....I....I....I....I....I....I....I....I....I

gi~13160051~
giI4585307~
gi~13431313~
gi~69122581 gi~122753121 57.0 S20 530 540 550 1 O NOV2 ~7PE ' I ~ . EI , tSI ,~ ' LP n G , m I
gi~13160051~ ~ ~ ~ ~ ~ ~ ' giI4585307~ ~ ' ~ .D~ ~m gi~13431313~ ~ ~ ~ .D~ ~m gi~6912258~ v ~ v ~ ~ ~m IS gi~1227537.2~ Q . ~ . . ~ ~.. ~ H

NOV2 G ~ L .J
n ~. r r ~ r v IGT~'..I S' Lr~N .

20 13160051~. . o ~~ .
gi~ v gi~ 4585307~. . . .

gi~ 13431313~~ ~ m gi1 6912258~

gi~ 12275312~

Lr~ n V v 1f. ~k NOV2 . . ~' . CPG- , V ~ Sid TATST--S QA~2L RS , T , $
tv gi~13160051~ ~ ~ ~ m 30 gi~4585307~ . . ~ m .
x~
gi~13431313~ ~ ~ ~ m m gi~6912258~ ~ ~ ' m ~~'STMMSTDLAIP~
gi~12275312~ ~ ~ ~ ~~ . ~ ..

i ~r~
NOV2 .~. 1"KPP IC~F~ EL KN--H E, ,SGNTNN--I
r~ v gi~13160051~ .I . .' Q I .E
n gi~4585307~ . . .~ Q T .
40 g1~13431313~ . ~ ~~ Q T ~ ~ ~
gi~6912258~ ~I . .~ ~ ~ I .E
gi~12275312~ ~x.. ~ ~~ ~ .< Q T ~ ~ v 4S .~....~....~....~....~.... ..
v. v NOV2 R&ST' CS. Yix'..Y' A Q .K I"S S S--gi~13160051~ ~ m n~~ ~ ~ ~- S
gi~4585307~ ~ ~~ m ~ ~ r- T
gi~13431313~
SO gi~6912258~
gi~12275312~ ~ m m ~ ~-v SS NOV2 ,F ,ITL~SQFIF,,.YYADI. ,TANT " I~,CDI~~-.vy I
~~v ~,~.
~v v gi~13160051~ . F~.. ~ ~
gi~4585307~ . S ~w ~ m - v ~ v gi~13431313~ . ~ ' . ~ .. - . ~ .
gi~6912258~ 1 FR ~ ' .. - . ~ .
60 gipzz7salz~ . ~ . ~ .. - . ~ .

~_y.v.~~ . . ~....~.. ~....'..
NOV2 ~r VN,~S . ' . 5 , te ~ z LAG E
E SHIT v 1 , s v 6S 131600511 I ~ 'F 'L
gi~

gi~ 4585307~ ~ 'S 'F S. L
' gi~ 134313131 . 'S 'F ~.
' gi~ 69122581 I . F ' 'L .
' gi~ 12275312~ I ~ 'S 'F S
~

7 0 , .. ...
NOV2 D~I!______vKY~QQKsJS. --I.
~ a ~ w gi~131600511 ~ ~T ~ v b ~ n v I~
gi~4585307~ , gi113431313~
gi~6912258~ ~ 'I v v ~ v gi~12275312~ ~ ' ~ ~ T
l0 910 920 930 940 950 NO V2 ~SA Y RAA;V SMA KiIQIM~ItPS
E ~mr v TCK
gi 13160051 ~ ' n ~ n ~ ~ y v P L
T ~
I ui ~~ I,I
I

gi~ 4585307~ ~ v v ~ . .
v ~i v gi~ 13431313~

gi1 6912258~

gi~ 12275312~

20 . . .. . . .~
.~. .~.. . ~.. . ..
. I . -. .

NOV2 S S ~y ~T~l v Y v.L Q 4'CSRA v.
m x n II
. .
G

gi n " ~'i v . .
~ v ~ , ~ .. v 13160051 ,~
~

gi~4585307~ E S v v S
v gi~13431313~ 9 ~ ~ S
~

gi ~ ~ T' ~ ~

~

gi w, ~ ~ .~' ~ ~ ' ~

. . . .

3O NOV2 LR GQ ~ ~': 1:2 S'~'. M MH
E~ '!'~-----I

gi~13160051~ ~' ~ i ~PT F
~

gi~4585307~ ~ ~' ' ~S

gi~13431313~ ~ ~' ' ~S

gi~6912258~ ~ ~' ~PT F

35 gi~122753121 ~' ~ iST

...
NOV2 ~IS~NL~ TNL P~t~I~I E nQ . ' T v r . . NV ~,y ~

4~ gi~13160051~' ~ ~ n v v giI4585307~

gi~13431313~ ~ ~ ' giI6912258~ v ~ v v gi~12275312~' ~ ~ ' ~ ~

1110 1120 1130 1140 llso ~
a . . . . . . .
NOV2 .Q .S~D NIP ~' .Q . S.- ~....~. ~. . ,.. ..
Y ~ . - L'F . ~Q
T . .
.T I~I I
~

gi~13160051~ ~ m v s $0 gi~4585307~ ~' gi1134313131 ~' gi~6912258~ ~' gi~12275312~ ~' ~' E

NOV2 .~. 5 C ~ ~ L~ .T~
v.. v, .S v ~m'vlI3 gi~13160051~

gi~4585307~ ~ ~ ~ ~
.v gi~13431313~ t ' t gi~6912258~ ~ ' ~ ~ ~ m ~

gi~12275312~ ~ ' ~ ~ ~ m ~

65 .~. ... ...~ ....~ ....~... .
. L....
.~.
.

NOV2 ~ ~5 SVG-S .
VR fCN Ii5 r siVn 'EQ TST
' gi~13160051~ '~ ~ '~ S 1 ~

giI4585307 ~ 1 ~F1 1 P
,, gi113431313~ ~ ~ '~ T P
~

7o giI6912258~ ~ ~ ~ S
~

gi ~ 12275312 I ~ r r ~ r r v ~ T~P~T~iI~

. I
.. .
. .
..' .
~ .
~

S NOV 2 ~ ~~ S., S ____' ~: ' SVKSIYF-__ r giI131600511 r r QTEv-____-______ gi~4585307~ r r r.rAFSrHSNFMDLVQFFV

gi~13431313~ r r rTAFSrHSNFMDLVQFFV

giI6912258~ r r v QTEr_-_ _________ g1~122753121 r r r RVTS--NOV2 _-________________________________________________ 1S gi~13160051~-___________________-___________________-_________ giI4585307~TFFSCFLSLLLVAAVVWKIKQSCWASRRREQLLREMQQMASRPFASVNVA

giI134313131TFFSCFLSLLLVAAVVWKIKQSCWASRRREQLLREMQQMASRPFASVNVA

gi~6912258~-____________,____________________________________ gi~122753121__________________________________________________ NOV2 __________________________________________________ gi~131600511 ZS giI4585307~LETDEEPPDLIGGSIKTVPKPIALEPCFGNKAAVLSVFVRLPRGLGGIPP

gi~134313131LETDEEPPDLIGGSIKTVPKPIALEPCFGNKAAVLSVFVRLPRGLGGIPP

gi~6912258~_______________________-__________________________ gi 112275312~_____________________-____________________________ NOV2 _____________-_______________________________ gi ~131600511____________________________________,________ giI45853071PGQSGLAVASALVDISQQMPIVYKEKSGAVRNRKQQPPAQPGTCI

3S gi 113431313~PGQSGLAVASALVDISQQMPIVYKEKSGAVRNRKQQPPAQPGTCI

gi ~6912258~_______________________________________-_____ gi ~12275312~_____________________________-______-________ 40 Tables 2E-I list the domain description from DOMAIN analysis results against NOV2.
This indicates that the NOV2 sequence has properties similar to those of other proteins known to contain this domain.
Table 2E Domain Analysis of NOV2 gnl~Smart~smart00034, CLECT, C-type lectin (CTL) or carbohydrate-recognition domain (CRD); Many of these domains function as calcium-dependent carbohydrate binding modules. (SEQ ID N0:67) CD-Length -- 124 residues, 100.0% aligned Score = 70.1 bits (170), Expect = 7e-13 4S Query: 708 CGEGW-SHIGDACLRVNSSRENYDNAKLYCYNhSGNLASLTTSKEVEFVLDEIQKYTQQK 766 Sbjct: 1 CPSGWVSYPGGKCYKFSTEKKTWADAQAFCQSLGAHLASIHSEEENDFI,LSLLKNSNSDY 60 Query: 767 VSPWVGLRKINI-SYWGWEDMSPFTNTTLQWLPGEPNDSGFCAYLERAAVAGLKANPCTS 825 S0 ~+~~ + + ~ ~ ~ ~ + + ~ ~~~~ ~~ ~ ~ + III
Sbjct: 61 Y--WIGLSRPDSNGSWQWSDGSGPVDYS-NWAPGEPGGSGNCWLSTSGGGKWNDVSCTS 117 Query: 826 MANGLVCE 833 +~~
SS Sbjct: 118 -KLPFICE 124 Table 2F Domain Analysis of NOV2 qnllSmartlsmartOD042, CUB, Domain first found in Clr, Cls, uEGF, and bone morphogenetic protein.; This domain is found mostly among developmentally-regulated proteins. Spermadhesins contain only this domain.. (SEQ ID N0:68) CD-Length = 114 residues, 96.5% aligned Score = 66.2 bits (160), Expect = 1e-11 Query: 58 RLTEPSGYLT--DGPINYKYKTKCTWLIEGPNA-VLRLRFNHFATE----CSWDHMYVYD 110 II II +I + I +I I 1 I I + I+I I I I++I++ +1I
S Sbjct: 4 TLTASSGTITSPNYPNSYPNNLNCVWTISAPPGYRIELKFTDFDLESSDNCTYDYVEIYD 63 Query: 111 GDSIYAPLIASFSGLIVPEIRGNETVPEWTTSGYALLHFFSDAAXNLTGFNIFYSI 167 I I +1I+ I I +I I + ++I + I II++ I~+ II
Sbjct: 64 GPSTSSPLLGRFCGSELPP-------PIISSSSNSMTVTFVSDSSVQKRGFSARYSA 113 Table 2G Domain Analysis of NOV2 gnllSmartlsmart00042, CUB, Domain first found in Clr, Cls, uEGF, and bone morphogenetic protein.; This domain is found mostly among developmentally-regulated proteins. Spermadhesins contain only this domain.. (SEQ ID N0:68) CD-Length = 114 residues, 96.5% aligned Score = 66.2 bits (160), Expect = 1e-11 Query: 58 RLTEPSGYLT--DGPINYKYKTKCTWLIEGPNA-VLRLRFNHFATE----CSWDHMYVYD 110 IS II II +I + I +I I I I I -~ I+I I I I++I++ +1I
Sbjct: 4 TLTASSGTITSPNYPNSYPNNLNCWTISAPPGYRIELKFTDFDLESSDNCTYDYVEIYD 63 Query: 111 GDSIYAPLIASFSGLIVPEIRGNETVPEWTTSGYALLHFFSDAAYNLTGFNIFYSI 167 I I +1I+ I I +I I + ++I + I II++ II+ II
2O Sbjct: 64 GPSTSSPLLGRFCGSELPP-------PIISSSSNSMTVTFVSDSSVQKRGFSARYSA 113 Table 2H Domain Analysis of NOV2 gnllPfamlpfam00431, CUB, CUB domain. (SEQ ID N0:69) CD-Length = 110 residues, 97.3% aligned Score = 63.9 bits (154), Expect = 5e-11 Query: 58 RLTEPSGYLT--DGPINYKYKTKCTWLIEGPNAV-LRLRFNHFATE----CSWDHMYVYD 110 25 III II ++ + I +I +I I I I + I I I I I +I++ + I
Sbjct: 4 VLTESSGSISSPNYPNDYPPNKECWTIRAPPGYRVELTFQDFDLEDHTGCRYDYVEIRD 63 Query: 111 GDSIYAPLIASFSGLIVPEIRGNETVPEWTTSGYALLHFFSDAAYNLTGFNIFY 165 II +1I+ I ~ I ++I++I + I III+ + II I
3O Sbjct: 64 GDGSSSPLLGKFCGSGPP--------EDIVSSSNRMTIKFVSDASVSKRGFKATY 110 Table 2I Domain Analysis of NOV2 c~nl Pfam~pfam00D59, lectin_c, Lectin C-type domain. This family includes both long and short form C-type (SEQ ID N0:70) CD-Length = 107 residues, 100.0 aligned Score = 49.3 bits (116), Expect = 1e-06 Query: 725 SRENYDNAKLYCYNLSGNLASLTTSKEVEFVLDEIQKYTQQKVSPWVGLRKINIS-YWGW 783 Sbjct: 1 ESKTWAEAQAACQKLGGGLVSIQSAEEQDFLTSLTKAS---NSYAWIGLTDTNTEGTWVW 57 Query: 784 EDMSPFTNTTLQWLPGEPND---SGFCAYLERAAVAGLKANPCTSMANGLVCEK 834 +
Sbjct: 58 TDGSPVNYTN--WAPGEPNNRGNKEDCVEIYTDG-NKWNDEPCGSK-LPYVCEF 107 The protein of invention is highly homologous to the protein attractin, which is a membrane-associated or secreted molecule (depending upon the splice variant) in activated T
cells. It has a protease activity and is thought to modify the N-terminals of cytokines and chernokines, enabling the cells to interact and form clusters. The mouse ortholog of soluble attractin has been demonstrated to play a role in obesity and metabolic regulation. The protein of invention shows characteristic domains involved in protein-protein interactions, such as the CUB domain and the ketch motif. It also shows the presence of 4 plexin repeats, a lectin C-type domain and two laminin EGF-like domains, thus sharing its domain structure with attractin. Although attractin is predicted to be localized in the mitochondria) matrix, like the protein of invention, it is either secreted or located as a transmembrane protein at the plasma membrane. The protein of invention may therefore have diverse physiological roles in the tissues that it is expressed.
Attractin is a rapidly upregulated membrane-associated molecule on activated T
calls (Duke-Cohan JS, et al. Adv Exp Med Biol 2000;477:173-85). It is a member of the CUB
family of extracellular guidance and development proteins, sharing with them a protease activity similar to that of Dipeptidyl peptidase IV (DPPIV/CD26). Most remarkably, and in sharp contrast to CD26, it is released from the T cell and is presumed to be a major source of a soluble serum-circulating attractin. Genomic sequencing reveals that the soluble form is not a proteolytic product of the membrane form, but is in fact the result of alternative splicing.
Recent results prove that the loss of murine membrane attractin results in the mahogany mutation with severe repercussions upon skin pigmentation and control of energy metabolism.
In each of these latter instances, there is a strong likelihood that attractin is moderating the interaction of cytokines with their respective receptors. Attractin is likely performing a similar function in the immune system through capture and pxoteolytic modification of the N-terminals of several cytokines and chemokines. This regulatory activity allows cells to interact and form immunoregulatory clusters and subsequently aids in downregulating chemokine/cytokine activity once a response has been initiated. These two properties are likely to be affected by the balance of membrane-expressed to soluble attractin.
Attractin was initially identified as a soluble human plasma protein with dipeptidyl peptidase IV activity that is expressed and released by activated T
lymphocytes. It has also been identified as the product of the murine mahogany gene with connections to control of pigmentation and energy metabolism. (Tang W et al., Proc Natl Acad Sci U S A
2000 May 23;97(11):6025-30). The mahogany product, however, is a transmembrane protein, raising the possibility of a human membrane attractin in addition to the secreted form.
The genomic structure of human attractin reveals that soluble attractin arises from transcription of 25 sequential exons on human chromosome 20p13, where the 3' ternlinal exon contains sequence from a long interspersed nuclear element-1 (LINE-1) retrotransposon element that includes a stop codon and a polyadenylation signal. The mRNA isoform for membrane attractin splices over the LINE-1 exon and includes five exons encoding transmembrane and cytoplasmic domains with organization and coding potential almost identical to that of the mouse gene.
The relative abundance of soluble and transmembrane isoforms measured by reverse transcription-PCR is differentially regulated in lymphoid tissues. Because activation of peripheral blood leukocytes with phytohemagglutinin induces strong expression of cell surface attractin followed by release of soluble attractin, these results suggest that a genomic event unique to mammals, LINE-1 insertion, has provided an evolutionary mechanism for regulating cell interactions during an inflammatory reaction.
The MahoganylAttractin gene (Atrn) has been proposed as a downstream mediator of Agouti signaling because yellow hair color and obesity in lethal yellow (A(y)) mice are suppressed by the mahogany (Atrn(mg)) mutation. (Lu Xy et al., FEBS Lett 1999 Nov 26;462(1-2):101-7). The present study examined the distribution of Atrn mRNA
in the brain and spinal cord by iya situ hybridization. Atrn mRNA was found widely distributed throughout the central nervous system, with high levels in regions of the olfactory system, some limbic structures, regions of the brainstem, cerebellum and spinal cord. In the hypothalamus, Atrn mRNA was found in specific nuclei including the suprachiasmatic nucleus, the supraoptic nucleus, the medial preoptic nucleus, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the arcuate nucleus. These results suggest a broad spectrum of physiological functions for the Atrn gene product.
Completely different lines of experimentation have identified attractin, a protein that seems to have multiple roles in regulating physiological processes. (Jackson IJ Trends Genet 1999 Nov;lS(11):429-31). It affects the balance between agonist and antagonist at receptors on melanocytes, modifies behaviour and basal metabolic rate, and mediates an interaction between activated T cells and macrophages. It may well be a target fox development of drugs to treat obesity.
Agouti protein and agouti-related protein are homologous paracrine signalling molecules that normally regulate hair colour and body weight, respectively, by antagonizing signalling through melanocortin receptors. (Gunn TM, et al., Nature 1999 Mar 11;398(6723):152-6). Expression of Agouti is normally limited to the skin, but rare alleles from which Agouti is expressed ubiquitously, such as lethal yellow, have pleiotropic effects that include a yellow coat, obesity, increased linear growth, and immune defects. The mahogany (mg) mutation suppresses the effects of lethal yellow on pigmentation and body weight, and results of our previous genetic studies place mg downstream of transcription of Agouti but upstream of melanocortin receptors. Here positional cloning was used to identify a candidate gene for mahogany, Mgca. The predicted protein encoded by Mgca is a 1,428-amino-acid, single-transmembrane-domain protein that is expressed in many tissues, including pigment cells and the hypothalamus. The extracellular domain of the Mgca protein is the orthologue of human attractin, a circulating molecule produced by activated T
cells that has been implicated in immune-cell interactions. These observations provide new insight into the regulation of energy metabolism and indicate a molecular basis for crosstalk between melanocorEin-receptor signalling and immune function.
Attractin is a normal human serum glycoprotein of 175 kDa that is rapidly expressed on activated T cells and released extracellularly after 48-72 hr. (Duke-Cohan JS et al., Proc Natl Acad Sci U S A 1998 Sep 15;95(19):11336-41). Attractin has been cloned and, in its natural serum form, it mediates the spreading of monocytes that become the focus for the clustering of nonproliferating T lymphocytes. There are two mRNA species with hematopoietic tissue-specific expression that code for a 134-kDa protein with a putative serine protease catalytic serine, four EGF-like motifs, a CUB domain, a C type lectin domain, and a domain homologous with the ligand-binding region of the common gamma cytokine chain.
Except for the latter two domains, the overall structure shares high homology with the Cae~aoJ~Izabditis elegans F33C8.1 protein, suggesting that attractin has evolved new domains and functions in parallel with the development of cell-mediated immunity.
The disclosed NOV2 nucleic acid of the invention encoding a Attractin-like protein includes the nucleic acid whose sequence is provided in Table 2A or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 2A while still encoding a protein that maintains its Attractin -like activities and physiological functions, or a fragment of such a nucleic acid.
The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In the mutant or variant nucleic acids, and their complements, up to about 29%
percent of the bases may be so changed.
The disclosed NOV2 protein of the invention includes the Attractin -like protein whose sequence is provided in Table 2B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 2B while still encoding a protein that maintains its Attractin -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 43% percent of the residues may be so changed.
The NOV2 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Multiple sclerosis,Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neurodegeneration, Diabetes, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA
nephropathy, Hypercalceimia, Diabetes, Pancreatitis,Obesity, Endometriosis, Infertility, Hirschsprung's disease , Crohn's Disease, Appendicitis, Muscular dystrophy,Lesch-Nyhan syndrome, Myasthenia gravis, Cirrhosis, Liver failure, Breast cancer, Ovarian cancer, Prostate cancer, Uterine cancer andlor other pathologies/disorders. The NOV2 nucleic acid encoding Afitractin-like protein, and the Attractin-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
NOV2 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in thexapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies"
section below. The disclosed NOV2 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV2 epitope is from about amino acids 1 to 20. In anothex embodiment, a NOV2 epitope is from about amino acids 50 to 130. In additional embodiments, NOV2 epitopes are from about amino acids 140 to 150, from about 180 to 380, from about amino acids 400 to 500, from about amino acids 530 to 550, from about amino acids 580 to 680, from about amino acids 700 to 740, from about amino acids 760 to 780, from about amino acids 820 to 900, and from about amino acids 950 to 1200. These novel proteins can be used in assay systems for functional analysis of various human disorders, which are useful in understanding of pathology of the disease and development of new drug targets for various disorders.

NOV3 includes three novel Insulin Like Growth Factor Binding Protein Complex-Acid Labile Subunit (IGFBP-ALS)-like proteins disclosed below. The disclosed sequences have been named NOV3a and NOV3b.
NOV3a A disclosed NOV3a nucleic acid of 6201 nucleotides (also referred to as 124217931 EXT) encoding a novel Kinase-like protein is shown in Table 3a. An open reading frame was identified beginning with a ATG initiation codon at nucleotides 57-59 and ending with a TGA codon at nucleotides 6199-6201. The start and stop codons are in bold letters.
Table 3A. NOV3a Nucleotide Sequence (SEQ ID N0:8) ATGTTGAAGTTCAAATATGGAGCGCGGAATCCTTTGGATGCTGGTGCTGCTGAACCCATTGCCAGCCGGGC
CTCCAGGCTGAATCTGTTCTTCCAGGGGAAACCACCCTTTATGACTCAACAGCAGATGTCTCCTCTTTCCC
GAGAAGGGATATTAGATGCCCTCTTTGTTCTCTTTGAAGAATGCAGTCAGCCTGCTCTGATGAAGATTAAG
CACGTGAGCAACTTTGTCCGGAAGTGTTCCGACACCATAGCTGAGTTACAGGAGCTCCAGCCTTCGGCAAA
GGACTTCGAAGTCAGAAGTCTTGTAGGTTGTGGTCACTTTGCTGAAGTGCAGGTGGTAAGAGAGAAAGCAA
CCGGGGACATCTATGCTATGAAAGTGATGAAGAAGAAGGCTTTATTGGCCCAGGAGCAGGTTTCATTTTTT
GAGGAAGAGCGGAACATATTATCTCGAAGCACAAGCCCGTGGATCCCCCAATTACAGTATGCCTTTCAGGA
CAAAAATCACCTTTATCTGGTGATGGAATATCAGCCTGGAGGGGACTTGCTGTCACTTTTGAATAGATATG
AGGACCAGTTAGATGAAAACCTGATACAGTTTTACCTAGCTGAGCTGATTTTGGCTGTTCACAGCGTTCAT
CTGATGGGATACGTGCATCGGGACATCAAGCCTGAGAACATTCTCGTTGACCGCACAGGACACATCAAGCT
GGTGGATTTTGGATCTGCCGCGAAAATGAATTCAAACAAGGTGAATGCCAAACTCCCGATTGGGACCCCAG
ATTACATGGCTCCTGAAGTGCTGACTGTGATGAACGGGGATGGAAAAGGCACCTACGGCCTGGACTGTGAC
TGGTGGTCAGTGGGCGTGATTGCCTATGAGATGATTTATGGGAGATCCCCCTTCGCAGAGGGAACCTCTGC
CAGAACCTTCAATAACATTATGAATTTCCAGCGGTTTTTGAAATTTCCAGATGACCCCAAAGTGAGCAGTG
ACTTTCTTGATCTGATTCAAAGCTTGTTGTGCGGCCAGAAAGAGAGACTGAAGTTTGAAGGTCTTTGCTGC
CATCCTTTCTTCTCTAAAATTGACTGGAACAACATTCGTAACGCTCCTCCCCCCTTCGTTCCCACCCTCAA

GTCTGACGATGACACCTCCAATTTTGATGAACCAGAGAAGAATTCGTGGGTTTCATCCTCTCCGTGCCAGC
TGAGCCCCTCAGGCTTCTCGGGTGAAGAACTGCCGTTTGTGGGGTTTTCGTACAGCAAGGCACTGGGGATT
CTTGGTAGATCTGAGTCTGTTGTGTCGGGTCTGGACTCCCCTGCCAAGACTAGCTCCATGGAAAAGAAACT
TCTCATCAAAAGCAAAGAGCTACAAGACTCTCAGGACAAGTGTCACAAGATGGAGCAGGAAATGACCCGGT
TACATCGGAGAGTGTCAGAGGTGGAGGCTGTGCTTAGTCAGAAGGAGGTGGAGCTGAAGGCCTCTGAGACT
CAGAGATCCCTCCTGGAGCAGGACCTTGCTACCTACATCACAGAATGCAGTAGCTTAAAGCGAAGTTTGGA
GCAAGCACGGATGGAGGTGTCCCAGGAGGATGACAAAGCACTGCAGCTTCTCCATGATATCAGAGAGCAGA
GCCGGAAGCTCCAAGAAATCAAAGAGCAGGAGTACCAGGCTCAAGTGGAAGAAATGAGGTTGATGATGAAT
CAGTTGGAAGAGGATCTTGTCTCAGCAAGAAGACGGAGTGATCTCTACGAATCTGAGCTGAGAGAGTCTCG
GCTTGCTGCTGAAGAATTCAAGCGGAAAGCGACAGAATGTCAGCATAAACTGTTGAAGGCTAAGGATCAGG
GGAAGCCTGAAGTGGGAGAATATGCGAAACTGGAGAAGATCAATGCTGAGCAGCAGCTCAAAATTCAGGAG
CTCCAAGAGAAACTGGAGAAGGCTGTAAAAGCCAGCACGGAGGCCACCGAGCTGCTGCAGAATATCCGCCA
GGCAAAGGAGCGAGCCGAGAGGGAGCTGGAGAAGCTGCAGAACCGAGAGGATTCTTCTGAAGGCATCAGAA
AGAAGCTGGTGGAAGCTGAGGAACGCCGCCATTCTCTGGAGAACAAGGTAAAGAGACTAGAGACCATGGAG
CGTAGAGAAAACAGACTGAAGGATGACATCCAGACAAAATCCCAACAGATCCAGCAGATGGCTGATAAAAT
TCTGGAGCTCGAAGAGAAACATCGGGAGGCCCAAGTCTCAGCCCAGCACCTAGAAGTGCACCTGAAACAGA
AAGAGCAGCACTATGAGGAAAAGATTAAAGTATTGGACAATCAGATAAAGAAAGACCTGGCTGACAAGGAG
ACACTGGAGAACATGATGCAGAGACACGAGGAGGAGGCCCATGAGAAGGGCAAAATTCTCAGCGAACAGAA
GGCGATGATCAATGCTATGGATTCCAAGATCAGATCCCTGGAACAGAGGATTGTGGAACTGTCTGAAGCCA
ATAAACTTGCAGCAAATAGCAGTCTTTTTACCCAAAGGAACATGAAGGCCCAAGAAGAGATGATTTCTGAA
CTCAGGCAACAGAAATTTTACCTGGAGACACAGGCTGGGAAGTTGGAGGCCCAGAACCGAAAACTGGAGGA
GCAGCTGGAGAAGATCAGCCACCAAGACCACAGTGACAAGAATCGGCTGCTGGAACTGGAGACAAGATTGC
GGGAGGTGAGTCTAGAGCACGAGGAGCAGAAACTGGAGCTCAAGCGCCAGCTCACAGAGCTACAGCTCTCC
CTGCAGGAGCGCGAGTCACAGTTGACAGCCCTGCAGGCTGCACGGGCGGCCCTGGAGAGCCAGCTTCGCCA
GGCGAAGACAGAGCTGGAAGAGACCACAGCAGAAGCTGAAGAGGAGATCCAGGCACTCACGGCACATAGAG
ATGAAATCCAGCGCAAATTTGATGCTCTTCGTAACAGCTGTACTGTGATCACAGACCTGGAGGAGCAGCTA
AACCAGCTGACCGAGGACAACGCTGAACTCAACAACCAAAACTTCTACTTGTCCAAACAACTCGATGAGGC
TTCTGGCGCCAACGACGAGATTGTACAACTGCGAAGTGAAGTGGACCATCTCCGCCGGGAGATCACGGAAC
GAGAGATGCAGCTTACCAGCCAGAAGCAAACGATGGAGGCTCTGAAGACCACGTGCACCATGCTGGAGGAA
CAGGTCATGGATTTGGAGGCCCTAAACGATGAGCTGCTAGAAAAAGAGCGGCAGTGGGAGGCCTGGAGGAG
CGTCCTGGGTGATGAGAAATCCCAGTTTGAGTGTCGGGTTCGAGAGCTGCAGAGGATGCTGGACACCGAGA
AACAGAGCAGGGCGAGAGCCGATCAGCGGATCACCGAGTCTCGCCAGGTGGTGGAGCTGGCAGTGAAGGAG
CACAAGGCTGAGATTCTCGCTCTGCAGCAGGCTCTCAAAGAGCAGAAGCTGAAGGCCGAGAGCCTCTCTGA
CAAGCTCAATGACCTGGAGAAGAAGCATGCTATGCTTGAAATGAATGCCCGAAGCTTACAGCAGAAGCTGG
AGACTGAACGAGAGCTCAAACAGAGGCTTCTGGAAGAGCAAGCCAAATTACAGCAGCAGATGGACCTGCAG
AAAAATCACATTTTCCGTCTGACTCAAGGACTGCAAGAAGCTCTAGATCGGGCTGATCTACTGAAGACAGA
AAGAAGTGACTTGGAGTATCAGCTGGAAAACATTCAGGTGCTCTATTCTCATGAAAAGGTGAAAATGGAAG
GCACTATTTCTCAACAAACCAAACTCATTGATTTTCTGCAAGCCAAAATGGACCAACCTGCTAAAAAGAAA
AAGGTGCCTCTGCAGTACAATGAGCTGAAGCTGGCCCTGGAGAAGGAGAAAGCTCGCTGTGCAGAGCTAGA
GGAAGCCCTTCAGAAGACCCGCATCGAGCTCCGGTCCGCCCGGGAGGAAGCTGCCCACCGCAAAGCAACGG
ACCACCCACACCCATCCACGCCAGCCACCGCGAGGCAGCAGATCGCCATGTCTGCCATCGTGCGGTCGCCA
GAGCACCAGCCCAGTGCCATGAGCCTGCTGGCCCCGCCATCCAGCCGCAGAAAGGAGTCTTCAACTCCAGA
GGAATTTAGTCGGCGTCTTAAGGAACGCATGCACCACAATATTCCTCACCGATTCAACGTAGGACTGAACA
TGCGAGCCACAAAGTGTGCTGTGTGTCTGGATACCGTGCACTTTGGACGCCAGGCATCCAAATGTCTAGAA
TGTCAGGTGATGTGTCACCCCAAGTGCTCCACGTGCTTGCCAGCCACCTGCGGCTTGCCTGCTGAATATGC
CACACACTTCACCGAGGCCTTCTGCCGTGACAAAATGAACTCCCCAGGTCTCCAGACCAAGGAGCCCAGCA
GCAGCTTGCACCTGGAAGGGTGGATGAAGGTGCCCAGGAATAACAAACGAGGACAGCAAGGCTGGGACAGG
AAGTACATTGTCCTGGAGGGATCAAAAGTCCTCATTTATGACAATGAAGCCAGAGAAGCTGGACAGAGGCC
GGTGGAAGAATTTGAGCTGTGCCTTCCCGACGGGGATGTATCTATTCATGGTGCCGTTGGTGCTTCCGAAC
TCGCAAATACAGCCAAAGCAGATGTCCCATACATACTGAAGATGGAATCTCACCCGCACACCACCTGCTGG
CCCGGGAGAACCCTCTACTTGCTAGCTCCCAGCTTCCCTGACAAACAGCGCTGGGTCACCGCCTTAGAATC
AGTTGTCGCAGGTGGGAGAGTTTCTAGGGAAAAAGCAGAAGCTGATGCTAAACTGCTTGGAAACTCCCTGC
TGAAACTGGAAGGTGATGACCGTCTAGACATGAACTGCACGCTGCCCTTCAGTGACCAGGTAGTGTTGGTG
GGCACCGAGGAAGGGCTCTACGCCCTGAATGTCTTGAAAAACTCCCTAACCCATGTCCCAGGAATTGGAGC
AGTCTTCCAAATTTATATTATCAAGGACCTGGAGAAGCTACTCATGATAGCAGGTGAAGAGCGGGCACTGT
GTCTTGTGGACGTGAAGAAAGTGAAACAGTCCCTGGCCCAGTCCCACCTGCCTGCCCAGCCCGACATCTCA
CCCAACATTTTTGAAGCTGTCAAGGGCTGCCACTTGTTTGGGGCAGGCAAGATTGAGAACGGGCTCTGCAT
CTGTGCAGCCATGCCCAGCAAAGTCGTCATTCTCCGCTACAACGAAAACCTCAGCAAATACTGCATCCGGA
AAGAGATAGAGACCTCAGAGCCCTGCAGCTGTATCCACTTCACCAATTACAGTATCCTCATTGGAACCAAT
AAATTCTACGAAATCGACATGAAGCAGTACACGCTCGAGGAATTCCTGGATAAGAATGACCATTCCTTGGC
ACCTGCTGTGTTTGCCGCCTCTTCCAACAGCTTCCCTGTCTCAATCGTGCAGGTGAACAGCGCAGGGCAGC
GAGAGGAGTACTTGCTGTGTTTCCACGAATTTGGAGTGTTCGTGGATTCTTACGGAAGACGTAGCCGCACA
GACGATCTCAAGTGGAGTCGCTTACCTTTGGCCTTTGCCTACAGAGAACCCTATCTGTTTGTGACCCACTT
CAACTCACTCGAAGTAATTGAGATCCAGGCACGCTCCTCAGCAGGGACCCCTGCCCGAGCGTACCTGGACA
TCCCGAACCCGCGCTACCTGGGCCCTGCCATTTCCTCAGGAGCGATTTACTTGGCGTCCTCATACCAGGAT
AAATTAAGGGTCATTTGCTGCAAGGGAAACCTCGTGAAGGAGTCCGGCACTGAACACCACCGGGGCCCGTC
CACCTCCCGCAGCAGCCCCAACAAGCGAGGCCCACCCACGTACAACGAGCACATCACCAAGCGCGTGGCCT
CCAGCCCAGCGCCGCCCGAAGGCCCCAGCCACCCGCGAGAGCCAAGCACACCCCACCGCTACCGCGAGGGG
CGGACCGAGCTGCGCAGGGACAAGTCTCCTGGCCGCCCCCTGGAGCGAGAGAAGTCCCCCGGCCGGATGCT

CCGTGAGGACCCCGCTGTCCCAGGTGAACAAGGTGAGGCAGCATTCCGAGGCCTGTGTGTCTGTTGCGGAG
GCCAGGAGTGACTTGGGGAACTGA
The disclosed NOV3a nucleic acid sequence maps to chromosome 13 and has 5518 of 6158 bases (89%) identical to rho/rac-interacting citron kinase (Crik) m)RNA
from Mus musculus (GENBANK-ID:AF086824) (E = 0.0).
A disclosed NOV3a protein (SEQ ID N0:9) encoded by SEQ ID N0:8 has 2066 amino acid residues, and is presented using the one-letter code in Table 3B.
Signal P, Psort and/or Hydropathy results predict that NOV3a does not have a signal peptide, and is likely to be localized to the nucleus with a certainty of 0.9800. In other embodiments NOV3a is also likely to be localized to microbody (peroxisome) with a certainty of 0.3000, to the mitochondria) membrane space with a certainty of 0.1000, or to the lysosome (lumen) with a certainty of 0.1000.
Table 3B. Encoded NOV3a protein sequence (SEQ ID N0:9).
MLKFKYGARNPLDAGAAEPTASRASRLNLFFQGKPPFMTQQQMSPLSREGILDALFVLFEECSQPALMKIK
HVSNFVRKCSDTIAELQELQPSAKDFEVRSLVGCGHFAEVQWREItATGDIYAMKVMKKKALLAQEQVSFF
EEERNILSRSTSPWTPQLQYAFQDKNHLYLVMEYQPGGDLLSLLNRYEDQLDENLIQFYLAELILAVHSVH
LMGYVHRDIKPENILVDRTGHIKLVDFGSAAKMI~1SNKVNAKLPTGTPDYMAPEVLTVMNGDGKGTYGLDCD
WWSVGVIAYEMIYGRSPFAEGTSARTFNNIMNFQRFLKFPDDPKVSSDFLDLIQSLLCGQKERLKFEGLCC
HPFFSKIDWNNIRNAPPPFVPTLKSDDDTSNFDEPEKNSWVSSSPCQLSPSGFSGEELPFVGFSYSKALGI
LGRSESWSGLDSPAKTSSMEKKLLIKSKELQDSQDKCHKMEQEMTRLHRRVSEVEAVLSQKEVELKASET
QRSLLEQDLATYITECSSLKRSLEQARMEVSQEDDKALQLLHDIREQSRKLQEIKEQEYQAQVEEMRLMMN
QLEEDLVSARRRSDLYESELRESRLAAEEFKRKATECQHKLLKAKDQGKPEVGEYAKLEKINAEQQLKIQE
LQEKLEKAVKASTEATELLQNIRQAKERAERELEKLQNREDSSEGIRKKLVEAEERRHSLENKVKRLETME
RRENRLKDDTQTKSQQIQQMADKILELEEKHREAQVSAQHLEVHLKQKEQHYEEKIKVLDNQIKKDLADKE
TLENMMQRHEEEAHEKGKILSEQKAMINAMDSKIRSLEQRIVELSEANKLAANSSLFTQRNMKAQEEMISE
LRQQKFYLETQAGKLEAQNRKLEEQLEKISHQDHSDKNRLLELETRLREVSLEHEEQKLELKRQLTELQLS
LQERESQLTALQAARAALESQLRQAKTELEETTAEAEEEIQALTAHRDEIQRKFDALRNSCTVTTDLEEQL
NQLTEDNAELNNQNFYLSKQLDEASGANDEIVQLRSEVDHLRREITEREMQLTSQKQTMEALKTTCTMLEE
QVMDLEALNDELLEKERQWEAWRSVLGDEKSQFECRVRELQRMLDTEKQSRARADQRITESRQWELAVKE
HKAEILALQQALKEQKLKAESLSDKLNDLEKKHAMLEMNARSLQQKLETERELKQRLLEEQAKLQQQMDLQ
KNHIFRLTQGLQEALDRADLLKTERSDLEYQLENIQVLYSHEKVKMEGTISQQTKLIDFLQAKMDQPAKKK
KVPLQYNELKLALEKEKARCAELEEALQKTRIELRSAREEAAHRKATDHPHPSTPATARQQTAMSAIVRSP
EHQPSAMSLLAPPSSRRKESSTPEEFSRRLKERMHHNIPHRFNVGLNMRATKCAVCLDTVHFGRQASKCLE
CQVMCHPKCSTCLPATCGLPAEYATHFTEAFCRDKMNSPGLQTKEPSSSLHLEGWMKVPRNNKRGQQGWDR
KYIVLEGSKVLIYDNEAREAGQRPVEEFELCLPDGDVSIHGAVGASELANTAKADVPYILKMESHPHTTCW
PGRTLYLLAPSFPDKQRWVTALESWAGGRVSREKAEADAKLLGNSLLKLEGDDRLDMNCTLPFSDQWLV
GTEEGLYALNVLKNSLTHVPGIGAVFQIYTIKDLEKLLMIAGEERALCLVDVKKVKQSLAQSHLPAQPDIS
PNIFEAVKGCHLFGAGKIENGLCICAAMPSKWILRYNENLSKYCIRKEIETSEPCSCIHFTNYSILIGTN
KFYEIDMKQYTLEEFLDKNDHSLAPAVFAASSNSFPVSIVQVNSAGQREEYLLCFHEFGVFVDSYGRRSRT
DDLKWSRLPLAFAYREPYLFVTHFNSLEVTEIQARSSAGTPARAYLDTPNPRYLGPAISSGAIYLASSYQD
KLRVICCKGNLVKESGTEHHRGPSTSRSSPNKRGPPTYNEHITKRVASSPAPPEGPSHPREPSTPHRYREG
RTELRRDKSPGRPLEREKSPGRMLSTRRERSPGRLFEDSSRGRLPAGAVRTPLSQVNKVRQHSEACVSVAE
ARSDLGN
The disclosed NOV3a amino acid has 1969 of 2053 amino acid residues (95%) identical to, and 2009 of 2053 amino acid residues (97%) similar to, the 2055 amino acid residue rho/rac-interacting citron kinase (Crik) protein from Mus nausculus (SPTREMBL-ACC:088938) (E= 0.0).

TaqMan expression data for NOV3a is found below is Example 2.
NOV3b A disclosed NOV3b nucleic acid of 6189 nucleotides (designated CuraGen Acc.
No.
CG106764-O1) encoding a novel RHO/RAC-interacting citron kinase-like is shown in Table 3C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TAA codon at nucleotides 6160-6162. A
putative untranslated region downstream from the termination codon is underlined in Table 3C, and the start and stop codons are in bold letters.
Table 3C. NOV3b Nucleotide Sequence (SEQ m N0:10) ATGTTGAAGTTCAAATATGGAGCGCGGAATCCTTTGGATGCTGGTGCTGCTGAACCCATTGCCAGCCGGGCCTCC
AGGCTGAATCTGTTCTTCCAGGGGAAACCACCCTTTATGACTCAACAGCAGATGTCTCCTCTTTCCCGAGAAGGG
ATATTAGATGCCCTCTTTGTTCTCTTTGAAGAATGCAGTCAGCCTGCTCTGATGAAGATTAAGCACGTGAGCAAC
TTTGTCCGGAAGTGTTCCGACACCATAGCTGAGTTACAGGAGCTCCAGCCTTCGGCAAAGGACTTCGAAGTCAGA
AGTCTTGTAGGTTGTGGTCACTTTGCTGAAGTGCAGGTGGTAAGAGAGAAAGCAACCGGGGACATCTATGCTATG
AAAGTGATGAAGAAGAAGGCTTTATTGGCCCAGGAGCAGGTTTCATTTTTTGAGGAAGAGCGGAACATATTATCT
CGAAGCACAAGCCCGTGGATCCCCCAATTACAGTATGCCTTTCAGGACAAAAATCACCTTTATCTGGTGATGGAA
TATCAGCCTGGAGGGGACTTGCTGTCACTTTTGAATAGATATGAGGACCAGTTAGATGAAAACCTGATACAGTTT
TACCTAGCTGAGCTGATTTTGGCTGTTCACAGCGTTCATCTGATGGGATACGTGCATCGGGACATCAAGCCTGAG
AACATTCTCGTTGACCGCACAGGACACATCAAGCTGGTGGATTTTGGATCTGCCGCGAAAATGAATTCAAACAAG
GTGAATGCCAAACTCCCGATTGGGACCCCAGATTACATGGCTCCTGAAGTGCTGACTGTGATGAACGGGGATGGA
AAAGGCACCTACGGCCTGGACTGTGACTGGTGGTCAGTGGGCGTGATTGCCTATGAGATGATTTATGGGAGATCC
CCCTTCGCAGAGGGAACCTCTGCCAGAACCTTCAATAACATTATGAATTTCCAGCGGTTTTTGAAATTTCCAGAT
GACCCCAAAGTGAGCAGTGACTTTCTTGATCTGATTCAAAGCTTGTTGTGCGGCCAGAAAGAGAGACTGAAGTTT
GAAGGTCTTTGCTGCCATCCTTTCTTCTCTAAAATTGACTGGAACAACATTCGTAACGCTCCTCCCCCCTTCGTT
CCCACCCTCAAGTCTGACGATGACACCTCCAATTTTGATGAACCAGAGAAGAATTCGTGGGTTTCATCCTCTCCG
TGCCAGCTGAGCCCCTCAGGCTTCTCGGGTGAAGAACTGCCGTTTGTGGGGTTTTCGTACAGCAAGGCACTGGGG
ATTCTTGGTAGATCTGAGTCTGTTGTGTCGGGTCTGGACTCCCCTGCCAAGACTAGCTCCATGGAAAAGAAACTT
CTCATCAAAAGCAAAGAGCTACAAGACTCTCAGGACAAGTGTCACAAGATGGAGCAGGAAATGACCCGGTTACAT
CGGAGAGTGTCAGAGGTGGAGGCTGTGCTTAGTCAGAAGGAGGTGGAGCTGAAGGCCTCTGAGACTCAGAGATCC
CTCCTGGAGCAGGACCTTGCTACCTACATCACAGAATGCAGTAGCTTAAAGCGAAGTTTGGAGCAAGCACGGATG
GAGGTGTCCCAGGAGGATGACAAAGCACTGCAGCTTCTCCATGATATCAGAGAGCAGAGCCGGAAGCTCCAAGAA
ATCAAAGAGCAGGAGTACCAGGCTCAAGTGGAAGAAATGAGGTTGATGATGAATCAGTTGGAAGAGGATCTTGTC
TCAGCAAGAAGACGGAGTGATCTCTACGAATCTGAGCTGAGAGAGTCTCGGCTTGCTGCTGAAGAATTCAAGCGG
AAAGCGACAGAATGTCAGCATAAACTGTTGAAGGCTAAGGATCAGGGGAAGCCTGAAGTGGGAGAATATGCGAAA
CTGGAGAAGATCAATGCTGAGCAGCAGCTCAAAATTCAGGAGCTCCAAGAGAAACTGGAGAAGGCTGTAAAAGCC
AGCACGGAGGCCACCGAGCTGCTGCAGAATATCCGCCAGGCAAAGGAGCGAGCCGAGAGGGAGCTGGAGAAGCTG
CAGAACCGAGAGGATTCTTCTGAAGGCATCAGAAAGAAGCTGGTGGAAGCTGAGGAACGCCGCCATTCTCTGGAG
AACAAGGTAAAGAGACTAGAGACCATGGAGCGTAGAGAAAACAGACTGAAGGATGACATCCAGACAAAATCCCAA
CAGATCCAGCAGATGGCTGATAAAATTCTGGAGCTCGAAGAGAAACATCGGGAGGCCCAAGTCTCAGCCCAGCAC
CTAGAAGTGCACCTGAAACAGAAAGAGCAGCACTATGAGGAAAAGATTAAAGTATTGGACAATCAGATAAAGAAA
GACCTGGCTGACAAGGAGACACTGGAGAACATGATGCAGAGACACGAGGAGGAGGCCCATGAGAAGGGCAAAATT
CTCAGCGAACAGAAGGCGATGATCAATGCTATGGATTCCAAGATCAGATCCCTGGAACAGAGGATTGTGGAACTG
TCTGAAGCCAATAAACTTGCAGCAAATAGCAGTCTTTTTACCCAAAGGAACATGAAGGCCCAAGAAGAGATGATT
TCTGAACTCAGGCAACAGAAATTTTACCTGGAGACACAGGCTGGGAAGTTGGAGGCCCAGAACCGAAAACTGGAG
GAGCAGCTGGAGAAGATCAGCCACCAAGACCACAGTGACAAGAATCGGCTGCTGGAACTGGAGACAAGATTGCGG
GAGGTGAGTCTAGAGCACGAGGAGCAGAAACTGGAGCTCAAGCGCCAGCTCACAGAGCTACAGCTCTCCCTGCAG
GAGCGCGAGTCACAGTTGACAGCCCTGCAGGCTGCACGGGCGGCCCTGGAGAGCCAGCTTCGCCAGGCGAAGACA
GAGCTGGAAGAGACCACAGCAGAAGCTGAAGAGGAGATCCAGGCACTCACGGCACATAGAGATGAAATCCAGCGC
AAATTTGATGCTCTTCGTAACAGCTGTACTGTGATCACAGACCTGGAGGAGCAGCTAAACCAGCTGACCGAGGAC
AACGCTGAACTCAACAACCAAAACTTCTACTTGTCCAAACAACTCGATGAGGCTTCTGGCGCCAACGACGAGATT
GTACAACTGCGAAGTGAAGTGGACCATCTCCGCCGGGAGATCACGGAACGAGAGATGCAGCTTACCAGCCAGAAG
CAAACGATGGAGGCTCTGAAGACCACGTGCACCATGCTGGAGGAACAGGTCATGGATTTGGAGGCCCTAAACGAT
GAGCTGCTAGAAAAAGAGCGGCAGTGGGAGGCCTGGAGGAGCGTCCTGGGTGATGAGAAATCCCAGTTTGAGTGT
CGGGTTCGAGAGCTGCAGAGGATGCTGGACACCGAGAAACAGAGCAGGGCGAGAGCCGATCAGCGGATCACCGAG
TCTCGCCAGGTGGTGGAGCTGGCAGTGAAGGAGCACAAGGCTGAGATTCTCGCTCTGCAGCAGGCTCTCAAAGAG

CAGAAGCTGAAGGCCGAGAGCCTCTCTGACAAGCTCAATGACCTGGAGAAGAAGCATGCTATGCTTGAAATGAAT
GCCCGAAGCTTACAGCAGAAGCTGGAGACTGAACGAGAGCTCAAACAGAGGCTTCTGGAAGAGCAAGCCAAATTA
CAGCAGCAGATGGACCTGCAGAAAAATCACATTTTCCGTCTGACTCAAGGACTGCAAGAAGCTCTAGATCGGGCT
GATCTACTGAAGACAGAAAGAAGTGACTTGGAGTATCAGCTGGAAAACATTCAGGTGCTCTATTCTCATGAAAAG
GTGAAAATGGAAGGCACTATTTCTCAACAAACCAAACTCATTGATTTTCTGCAAGCCAAAATGGACCAACCTGCT
AAAAAGAAAAAGGTGCCTCTGCAGTACAATGAGCTGAAGCTGGCCCTGGAGAAGGAGAAAGCTCGCTGTGCAGAG
CTAGAGGAAGCCCTTCAGAAGACCCGCATCGAGCTCCGGTCCGCCCGGGAGGAAGCTGCCCACCGCAAAGCAACG
GACCACCCACACCCATCCACGCCAGCCACCGCGAGGCAGCAGATCGCCATGTCTGCCATCGTGCGGTCGCCAGAG
CACCAGCCCAGTGCCATGAGCCTGCTGGCCCCGCCATCCAGCCGCAGAAAGGAGTCTTCAACTCCAGAGGAATTT
AGTCGGCGTCTTAAGGAACGCATGCACCACAATATTCCTCACCGATTCAACGTAGGACTGAACATGCGAGCCACA
AAGTGTGCTGTGTGTCTGGATACCGTGCACTTTGGACGCCAGGCATCCAAATGTCTAGAATGTCAGGTGATGTGT
CACCCCAAGTGCTCCACGTGCTTGCCAGCCACCTGCGGCTTGCCTGCTGAATATGCCACACACTTCACCGAGGCC
TTCTGCCGTGACAAAATGAACTCCCCAGGTCTCCAGACCAAGGAGCCCAGCAGCAGCTTGCACCTGGAAGGGTGG
ATGAAGGTGCCCAGGAATAACAAACGAGGACAGCAAGGCTGGGACAGGAAGTACATTGTCCTGGAGGGATCAAAA
GTCCTCATTTATGACAATGAAGCCAGAGAAGCTGGACAGAGGCCGGTGGAAGAATTTGAGCTGTGCCTTCCCGAC
GGGGATGTATCTATTCATGGTGCCGTTGGTGCTTCCGAACTCGCAAATACAGCCAAAGCAGATGTCCCATACATA
CTGAAGATGGAATCTCACCCGCACACCACCTGCTGGCCCGGGAGAACCCTCTACTTGCTAGCTCCCAGCTTCCCT
GACAAACAGCGCTGGGTCACCGCCTTAGAATCAGTTGTCGCAGGTGGGAGAGTTTCTAGGGAAAAAGCAGAAGCT
GATGCTAAACTGCTTGGAAACTCCCTGCTGAAACTGGAAGGTGATGACCGTCTAGACATGAACTGCACGCTGCCC
TTCAGTGACCAGGTAGTGTTGGTGGGCACCGAGGAAGGGCTCTACGCCCTGAATGTCTTGAAAAACTCCCTAACC
CATGTCCCAGGAATTGGAGCAGTCTTCCAAATTTATATTATCAAGGACCTGGAGAAGCTACTCATGATAGCAGGT
GAAGAGCGGGCACTGTGTCTTGTGGACGTGAAGAAAGTGAAACAGTCCCTGGCCCAGTCCCACCTGCCTGCCCAG
CCCGACATCTCACCCAACATTTTTGAAGCTGTCAAGGGCTGCCACTTGTTTGGGGCAGGCAAGATTGAGAACGGG
CTCTGCATCTGTGCAGCCATGCCCAGCAAAGTCGTCATTCTCCGCTACAACGAAAACCTCAGCAAATACTGCATC
CGGAAAGAGATAGAGACCTCAGAGCCCTGCAGCTGTATCCACTTCACCAATTACAGTATCCTCATTGGAACCAAT
AAATTCTACGAAATCGACATGAAGCAGTACACGCTCGAGGAATTCCTGGATAAGAATGACCATTCCTTGGCACCT
GCTGTGTTTGCCGCCTCTTCCAACAGCTTCCCTGTCTCAATCGTGCAGGTGAACAGCGCAGGGCAGCGAGAGGAG
TACTTGCTGTGTTTCCACGAATTTGGAGTGTTCGTGGATTCTTACGGAAGACGTAGCCGCACAGACGATCTCAAG
TGGAGTCGCTTACCTTTGGCCTTTGCCTACAGAGAACCCTATCTGTTTGTGACCCACTTCAACTCACTCGAAGTA
ATTGAGATCCAGGCACGCTCCTCAGCAGGGACCCCTGCCCGAGCGTACCTGGACATCCCGAACCCGCGCTACCTG
GGCCCTGCCATTTCCTCAGGAGCGATTTACTTGGCGTCCTCATACCAGGATAAATTAAGGGTCATTTGCTGCAAG
GGAAACCTCGTGAAGGAGTCCGGCACTGAACACCACCGGGGCCCGTCCACCTCCCGCAGCAGCCCCAACAAGCGA
GGCCCACCCACGTACAACGAGCACATCACCAAGCGCGTGGCCTCCAGCCCAGCGCCGCCCGAAGGCCCCAGCCAC
CCGCGAGAGCCAAGCACACCCCACCGCTACCGCGAGGGGCGGACCGAGCTGCGCAGGGACAAGTCTCCTGGCCGC
CCCCTGGAGCGAGAGAAGTCCCCCGGCCGGATGCTCAGCACGCGGAGAGAGCGGTCCCCCGGGAGGCTGTTTGAA
GACAGCAGCAGGGGCCGGCTGCCTGCGGGAGCCGTGAGGACCCCGCTGTCCCAGGTGAACAAGGTGTGGGACCAG
TCTTCAGTATAAATCTCAGCCAGAAAAACCAACTCCTCA
The disclosed NOV3b nucleic acid sequence of this invention has 2894 of 2908 bases (99%) identity with KIAA1531 mRNA from Hom~ Sapiens (GENBANK-ID: AB040964) (E
=
0.0).
A NOV3b polypeptide (SEQ ID NO:11) encoded by SEQ ID NO:10 is 2053 amino acid residues and is presented using the one letter code in Table 3D. The SignalP, Psort and Hydropathy, Psort, and/or SignalP data suggest that the NOV3b protein has no signal peptide and may be localized to nucleus with a certainty of 0.9800. In other embodiments, NOV3b may also be localized to the microbody (peroxisome) with a certainty of 0.300, the mitochondria) matrix space with a certainty of 0.100 or the lysosome (lumen) with a certainty of 0.100.
Table 3D. Encoded NOV3b protein sequence (SEQ ID NO:11) MLKFKYGARNPLDAGAAEPIASRASRLNLFFQGKPPFMTQQQMSPLSREGILDALFVLFEECSQPALMKIKHV
SNFVRKCSDTIAELQELQPSAKDFEVRSLVGCGHFAEVQVVREKATGDIYAMKVMKKKALLAQEQVSFFEEER
NILSRSTSPWIPQLQYAFQDKNHLYLVMEYQPGGDLLSLLNRYEDQLDENLIQFYLAELILAVHSVHLMGYVH
RDIKPENILVDRTGHIKLVDFGSAAKMNSNKVNAKLPIGTPDYMAPEVLTVMNGDGKGTYGLDCDWWSVGVIA

YEMIYGRSPFAEGTSARTFNNIMNFQRFLKFPDDPKVSSDFLDLIQSLLCGQKERLKFEGLCCHPFFSKIDWN
NIRNAPPPFVPTLKSDDDTSNFDEPEKNSWVSSSPCQLSPSGFSGEELPFVGFSYSKALGILGRSESWSGLD
SPAKTSSMEKKLLIKSKELQDSQDKCHKMEQEMTRLHRRVSEVEAVLSQKEVELKASETQRSLLEQDLATYIT
ECSSLKRSLEQARMEVSQEDDKALQLLHDIREQSRKLQEIKEQEYQAQVEEMRLMMNQLEEDLVSARRRSDLY
ESELRESRLAAEEFKRKATECQHKLLKAKDQGKPEVGEYAKLEKINAEQQLKIQELQEKLEKAVKASTEATEL
LQNIRQAKERAERELEKLQNREDSSEGIRKKLVEAEERRHSLENKVKRLETMERRENRI,KDDIQTKSQQIQQM
ADKILELEEKHREAQVSAQHLEVHLKQKEQHYEEKIKVLDNQTKKDLADKETLENMMQRHEEEAHEKGKILSE
QKAMINAMDSKIRSLEQRIVELSEANKLAANSSLFTQRNMKAQEEMISELRQQKFYLETQAGKLEAQNRKLEE
QLEKISHQDHSDKNRLLELETRLREVSLEHEEQKLELKRQLTELQLSLQERESQLTALQAARAALESQLRQAK
TELEETTAEAEEEIQALTAHRDEIQRKFDALRNSCWITDLEEQLNQLTEDNAELNNQNFYLSKQLDEASGAN
DEIVQLRSEVDHLRREITEREMQLTSQKQTMEALKTTCTMLEEQVMDLEALNDELLEKERQWEAWRSVLGDEK
SQFECRVRELQRMLDTEKQSRARADQRITESRQWELAVKEHKAEILALQQALKEQKLKAESLSDKLNDLEKK

IQVLYSHEKVKMEGTISQQTKLIDFLQAKMDQPAKKKKVPLQWELKLALEKEKARCAELEEALQKTRIELRS
AREEAAHRKATDHPHPSTPATARQQIAMSAIVRSPEHQPSAMSLLAPPSSRRKESSTPEEFSRRLKERMHHNT
PHRFNVGLNMRATKCAVCLDTVHFGRQASKCLECQVMCHPKCSTCLPATCGLPAEYATHFTEAFCRDKMNSPG
LQTKEPSSSLHLEGWMKVPRNNKRGQQGWDRKYIVLEGSKVLTYDNEAREAGQRPVEEFELCLPDGDVSIHGA

NSLLKLEGDDRLDMNCTLPFSDQWLVGTEEGLYALNVLKNSI,THVPGIGAVFQIYIIKDLEKLLMIAGEERA
LCLVDVKKVKQSLAQSHLPAQPDISPNIFEAVKGCHLFGAGKTENGLCICAAMPSKWILRYNENLSKYCIRK
EIETSEPCSCIHFTNYSILIGTNKFYEIDMKQYTLEEFLDKNDHSLAPAVFAASSNSFPVSIVQWSAGQREE
YLLCFHEFGVFVDSYGRRSRTDDLKWSRLPLAFAYREPYLFVTHFNSLEVIEIQARSSAGTPARAYLDIPNPR
YLGPAISSGAIYLASSYQDKLRVICCKGNLVKESGTEHHRGPSTSRSSPNKRGPPTYNEHITKRVASSPAPPE
GPSHPREPSTPHRYREGRTELRRDKSPGRPLEREKSPGRMLSTRRERSPGRLFEDSSRGRLPAGAVRTPLSQV
NKWDQSSV
The disclosed NOV3b amino acid sequence has 638 of 647 amino acid residues (98%) identical to, and 643 of 647 amino acid residues (99%) similar to, the KIAA1531 PROTEIN of 1060 amino acid residue prekallikrein-like protein from Homo sapiens (BAA96055) (E = 0.0).
NOV3b is expressed primarily in normal brain but not in other normal tissues.
Lower expression is seen in several tumor types.
NOV3b also has homology to the amino acid sequences shown in the BLASTP data listed in Table 3E.
Table 3E. BLAST
results for NOV3b Gene Index/ Protein/ Length IdentityPositivesExpect Tdentifier Organism (aa) (%) (%) gi1147680101refIXPcitron 883 849/883 849/883 0.0 (rho-_ interacting, (96%) (96%) 045786.1 serine/threo nine kinase 21 ) [Homo sapiens]

gi162252171sp101457CITRON 1286 1165/12861165/12860.0 81CTRO HUMAN PROTEIN (90%) (90%) gi145895421dbjIBAA7KIAA0949 940 887/940 887/940 0.0 6793.11 protein (94%) (94%) [Homo Sapiens]

gi133605141gb1AAC27Citron-K 1641 1476/16831490/16830.0 933.11 kinase (87%) (87%) [Mus musculus]

gi113458601spIP4902CITRON 1597 1427/15891442/15890.0 SICTRO MOUSE PROTEIN (89%) (89%) I

The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 3F.
Table 3F. ClustalW Analysis of NOV3 1) NOV3a (SEQ ID N0:9) 2) NOV3b (SEQ ID N0:11) 3) gi~14768010~ref~XP_045786.1~ citron (rho-interacting, serine/threonine kinase 21) [Homo sapiens] (SEQ ID N0:37) 4) gi~6225217~sp~01457B~CTRO_HUMAN CITRON PROTEIN (SEQ ID N0:38) 1~ 5) gi~4589542~dbj~BAA76793.1~] KIAA0949 protein [Homo Sapiens] (SEQ ID
N0:39) 6) gi~3360514~gbIAAC27933.1~ Citron-K kinase [Mus musculus] (SEQ ID N0:40) 7) giI1345860~spIP49025ICTR0 MOUSE CITRON PROTEIN (SEQ ID N0:41) ....
NOV3a MLKFKYGARNPLDAGAAEPIASRASRLNLFFQGKPPFMTQQQMSPLSREGILDALFVLFE60 NOV3b MLKFKYGARNPLDAGAAEPIASRASRLNLFFQGKPPFMTQQQMSPLSREGILDALFVLFE60 gi~14768010~ref ____________________________________________________________1 gi~6225217~sp~0 ____________________________________________________________1 gy 4589542~db31 ____________________________________________________________ gi~3360514~gb~A __________________,_________________________________________1 gi~1345860~sp~P ____________________________________________________________1 ....
NOV3a ECSQPALMKIKHVSNFVRKCSDTIAELQELQPSAKDFEVRSLVGCGHFAEVQWREKATG120 NOV3b ECSQPALMKIKHVSNFVRKCSDTIAELQELQPSAKDFEVRSLVGCGHFAEVQWREKATG120 giI14768010~ref ____________________________________________________________1 gi~6225217Isp~0 ____________________________________________________________1 giI4589542~dbj~ ___________________________________________________________ giI3360514IgbI A
____________________________________________________________1 gi11345860~sp~ P
_______________________________,____________________________1 ....
NOV3a DIYAMKVMKKKALLAQEQVSFFEEERNILSRSTSPWIPQLQYAFQDKNHLYLVMEYQPGG180 NOV3b DIYAMKVMKKKALLAQEQVSFFEEERNILSRSTSPWIPQLQYAFQDKNHLYLVMEYQPGG180 giI14768010~ref ____________________________________________________________1 4~ gi~6225217~sp~0 ____________________________________________________________1 gi14589542~dbjl ____________________________________________________________1 gi~3360514~gb~ A
____________________________________________________________1 gi~1345860~sp~ P
___________________________________________________________ 4.5 190 200 210 220 230 240 ....
NOV3a DLLSLLNRYEDQLDENLIQFYLAELILAVHSVHLMGYVHRDIKPENILVDRTGHIKLVDF240 NOV3b DLLSLLNRYEDQLDENLIQFYLAELILAVHSVHLMGYVHRDIKPENILVDRTGHIKLVDF~240 gi~14768010~ref ____________________________________________________________1 gi~6225217~sp~0 ____________________________________________________________1 gi~4589542~dbjl ___________________________________________________________ giI3360514IgbIA ____________________________________________________________1 gi~1345860~sp'P____________________________________________________________1 ....
NOV3a GSAAKMNSNKVNAKLPIGTPDYMAPEVLTVMNGDGKGTYGLDCDWWSVGVIAYEMIYGRS300 NOV3b GSAAKMNSNKVNAKLPIGTPDYMAPEVLTVMNGDGKGTYGLDCDWWSVGVIAYEMIYGRS300 gi I14768010~ref ________________________,___________________________________1 gi 16225217~sp~0 ____________________________________________________________1 gi ~4589542~dbjl ____________________________________________________________1 gi ~3360514~gb~A___________________________________________________________ gi ~1345860~sp~P___________________________________________________________ (5 310 320 330 340 350 360 ....

NOV3a PFAEGTSARTFNNIMNFQRFLKFPDDPKVSSDFLDLIQSLLCGQKERLKFEGLCCHPFFS360 NOV3b PFAEGTSARTFNNIMNFQRFLKFPDDPKVSSDFLDLIQSLLCGQKERLKFEGLCCHPFFS360 gi ~14768010~ref ___________________________________________________________ gi~6225217~sp~0 ___________________________________________________________1 $ gi~4589542~dbjl ___________________________________________________________1 gi~3360514~gb~A ___________________________________________________________1 -gi~1345860~sp~P -___________________________________________________________1 ....

NOV3a KIDWNNIRNAPPPFVPTLKSDDDTSNFDEPEKNSWSSSPCQLSPSGFSGEELPFVGFSY420 NOV3b KIDWNNIRNAPPPFVPTLKSDDDTSNFDEPEKNSWSSSPCQLSPSGFSGEELPFVGFSY420 gi~14768010~ref -__________________________________________________________ gi~6225217~sP~0 -__________________________________________________________1 -gi~4589542~dbjl ____________________________________________________________1 gi~3360514~gbIA ------------PFVPTLKSDDDTSNFDEPEKNSWSSSVCQLSPSGFSGEELPFVGFSY48 gi~1345860~sp~P ____________________________________________________________1 ....

NOV3a SICALGILGRSESWSGLDSPAKTSSMEKKLLIKSKELQDSQDKCHKMEQEMTRLHRRVSE480 NOV3b SKALGILGRSESWSGLDSPAKTSSMEKKJ,LIKSKELQDSQDKCHKMEQEMTRLHRRVSE480 gi~14768010~ref -__________________________________________________________ gi~6225217~sp~0 ____________________________________________________________1 2$ gi~4589542~dbjl ____________________________________________________________1 gi~3360514~gb~A

gi~1345860Isp~P -----------------------------MLLGEEAM---------MEQEMTRLHRRVSE22 ....

NOV3a VEAVLSQKEVELKASETQRSLLEQDLATYITECSSLKRSLEQARMEVSQEDDKALQLLHD540 NOV3b VEAVLSQKEVELKASETQRSLLEQDLATYITECSSLKRSLEQARMEVSQEDDKALQLLHD540 gi~14768010~ref _______________________________________,___,_______________ gi~6225217~ sp~0 _______________________________________,___,________________1 3$ gi~4589542~ dbjl ___________________________________,________________________1 giI3360514~ gbIA

giI1345860I spIP

....

NOV3a IREQSRKLQEIKEQEYQAQVEEMRLMMNQLEEDLVSARRRSDLYESELRESRLAAEEFKR600 NOV3b IREQSRKI,QEIKEQEYQAQVEEMRLMMNQLEEDLVSARRRSDLYESELRESRLAAEEFKR600 giI14768010~ref ____________________________________________________________1 gi~6225217~sp10 ____________________________________________________________1 4$ gi~4589542~dbjl ____________________________________________________________1 giI3360514Igb~A
IREQSRKLQEIKEQEYQAQVEEMRLMI~TQLEEDLVSARRRSDLYESELRESRLAAEEFKR228 gi~1345860~spIP

....

NOV3a KATECQHKLLKAKDQGKPEVGEYAKLEKINAEQQLKIQELQEKLEKAVICASTEATELLQN660 NOV3b KATECQHKLLKAKDQGKPEVGEYAKLEKINAEQQLKIQELQEKLEKAVKASTEATELLQN660 giI14768010~ref ____________________________________________________________1 $$ gi~4589542~dbj~ ___-_______________________________________________________ giI3360514~gbIA

gi~1345860~spIP

....

NOV3a IRQAKERAERELEKLQNREDSSEGIRKKLVEAEERRHSLENKVKRLETMERRENRLKDDI720 NOV3b IRQAKERAERELEKLQNREDSSEGIRKKLVEAEERRHSLENKVKRLETMERRENRLKDDI720 gi~14768010~ref ____________________________________________________________ gi~6225217~sp10 ___________________________________________________________ 6$ gi~4589542~dbj~
___________________________________________________________1 -gi~3360514~gbIA IRQAKERAERELEKLHNREDSSEGIKKKLVEAEE--------------------------giI1345860~splp IRQAKERAERELEKI~HNREDSSEGIKKKI,VEAEERRHSLENKVKRLETMERRENRLKDDI262 ....~....~....~....~....~....~....~....~....~....~....~....) NOV3a QTKSQQIQQMADKILELEEKHREAQVSAQHLEVHLKQKEQHYEEKI ~ v ~ ~~ 780 NOV3b QTKSQQIQQMADKILELEEKHREAQVSAQHLEVHLKQKEQHYEEKI ~ ~ ~ ~~ 780 gi~14768010~ref -_____,_____________________________________________________ 1 gi~6225217~sp~'O -_____________________________________________ ~ v ~ ~v 13 gi~4589542~dbjl ____________________________________________________________ 1 gi~3360514~gbIA ----------------LEEKHREAQVSAQHLEVHLKQKEQHYEEKI ~ ~ ~ ~~ 366 giI1345860IspIP QTKSEQIQQMADKILELEEKHREAQVSAQHLEVHLKQKEQHYEEKI ~ ~ ~ ~~ 322 . .
NOV3a v~ v ~ ~ w ~ ~~~ v 840 NOV3b t' ~ ~ ~ ~' ~ 840 gi~14768010~ref _-_________________________-_________,_____________________ gi~6225217~sp~0 Q: w ~ ~ ~ w ~ 73 gi~4589542~dbjl ____________________________________________________________ 1 g1~3360514~gb~A $ w ~ ~ ~ w ~ 426 gi~1345860~sp~P ~$ ~~ ~ ~ ~ w ~ 382 ..
NOV3a ' i ~v ~ ~w ~~ ~~ ~ , m ~ 900 NOV3b I ~~ ~~~ ~~ ~~ ~ ~ m ~ 900 gi~14768010~ref -__________________________________________________________ gi~6225217~sp~0 ~~ ~~~ ~~ ~~ ' ~ m ~ 133 gi~4589542~dbj~ _________________________________________________.__________ 1 gi~3360514~gb~A ~va ~w 3 W ~y ~ ~ w ~ $486 gi~1345860~sp~P ~~ w~ ~~ ~m ' ~ ~~ S 442 NOV3a ~ ~ ~ ~ ~~ ~ ~ 960 ~' NOV3b ~ ~ ~ ~ ~ ~ ~ 960 ~' giI147680101ref -_____________________ _____ ______________________________ ___ gi~6225217~ sp~0 v v v v v v.... ~ i93 ~

gi~4589542~ dbj~ ____________ _____ _____ _____ ______ __________ _____ _________ -__ gi~3360514~ gbIA ~ ~ ~ t ~ ~ ~ 546 ' ~' gi~1345860~ sp~P ~ '~ ~ ~ ~ ~ ~ 502 ~' ....~....~._ .. .y ..~.. ..~... _~.... . .
.. .. ... ...

NOV3a ~ ~ w ~ w ~ m ~ v ~ 1020 ~

NOV3b v v ~ ~ v ~ m m . ~~ ~r 1020 ~

gi~14768010~ref _________________________________ __________ _____ _________ ___ gi~62252171sp10 v v' m ~ ~ v v 253 ~ v ~ ~

gi14589542~dbjl ____________ _____ _____ _____ ______ __________ ____- _________ ___ giI3360514~gb~A ~ ~ ~ ~ ~~ ~ ~ ~ ~ 606 gi~1345860~sp~P v ~ v w ~v v v v v ~ 562 . ..~.. ..~.. .~.. .~.. .~..
.
.

NOV3a ~ ~ ~~ ~~ ~ W ~ 1080 ~

NOV3b ~ ' ~ ~ ~~ ~ ~ ~ 1080 ~

giI14768010~ref _________________ __________ ________________ _____ _________ ___ gi~6225217~sp~0 ~ ~ ~ ~ ~ ~ 313 ~ ~ ~
' giI4589S42~dbj~ -___________ _____ _____ _____ ______ __________ ____. _________ ___ gi~3360514~gb~A ~ a ~ ~ v ~ n 666 v v ~

giI1345860IspIP im v m ~ o a ew ~ may y 622 a v ~

....
NOV3a v ~W v ~ ,~ ~ 1140 v ~
~

NOV3b v i m v 1140 v v ~
i v gi~14768010~ref -__________________________________ __________ ____________ ___1 gi~6225217~sp10 w ~ .i~ . ., v v 3 ~ v 73 4589542~dbjl -________________________________ ,.m ~

gi~

gi~ 3360514~gbIA w ~m ~ 726 ~ ~
~ ~

giI 1345860~spIP

....

NOV3a 1200 NOV3b 1200 gi~14768010~ref 30 $ gi~4589542~dbjl 87 gi~3360514~gb~A

gi~1345860~sp~P 742 ....
. .

NOV3a ~~~ ~ ~ ~ ~ ~iv v a w 1260 ~ ~ v m ~

NOV3b ~~~ ~~ ~ ' ~ ~ ~ w 1260 ~ ~ m n gi~14768010~ref ~~~ ~ ~ m ~ ~ ~ ~ ~
~ ~ ~ 90 gi~6225217~sp~0 ~~~ ~~ ~ ~'m ~ ~ ~ w 493 ~ ~

~.$gi~4589542~dbj~ w w v ~~ ~ v v w 147 ~ v gi~3360514~gb~A ~~~ ~~ ~ m ~ ~ ~ ~ ~
~ ~ ~ 846 gi~1345860~sp~P w v v v~ ~ v v w 802 ~ ~

NOV3a 1320 NOV3b 1320 gi~14768010~ref gi~62252171sp~0 2$ giI4589542~dbj~

gi~3360514~gb~A

gi~1345860~spIP 862 NOV3a 1380 NOV3b 1380 giI14768010Iref 210 3$ giI4589542~dbj~ 267 gi~3360514~gb~A 966 giI1345860~sp~P 922 NOV3a 1440 NOV3b 1440 giI14768010~ref 270 gi~6225217 1sp10 giI4589542~ dbjl gi~3360514I gbIA 1026 gi~1345860 ~spIP

$0 NOV3a 1500 NOV3b 1500 giI14768010~ref 330 gi~62252171sp~0 $$ gi~4589542~dbj~ 387 gi~3360514~gb~A

gi~1345860~spIP

NOV3a 1560 NOV3b 1560 gi~14768010~ref 390 gi~62252171sp~0 6$ gi~4589542~dbj~ 447 gi~3360514Igb~A

gi~1345860~sp~P 1102 ....

NOV3a r r 2038 NOV3b r r 2038 gi1 147680101ref r r 868 gi1 62252171sp10 r r 1271 gi1 4589542~dbjl - r r 925 gi1 33605141gbIA D r r 1626 gi1 13458601spIP D r r 1582 l0 .1....1....1....1....1...
NOV3a ~ RQH EACVSVAEARSDLGN 2066 r_ NOV3b ~ r~ ------------ 2053 gi1147680101ref v m ____________ g83 gi162252171sp10 ~ rr ____________ 1286 15 ___________ giI45895421dbjl ~ r~ - 940 gi133605141gbIA ~ rr ------------ 1641 gi~13458601sPIP v m ____________ 1597 20 Tables 3G-K list the domain description from DOMAIN analysis results against NOV3b. This indicates that the NOV3 sequence has properties similar to those of other proteins known to contain this domain.
Table 3G Domain Analysis of NOV3b gnllPfamlpfam00780, CNH, CNH domain. Domain found in NIICl-like kinase, mouse citron and yeast ROM1, ROM2. Unpublished observations (SEQ ID
N0:71) CD-Length = 304 residues, 99.7 aligned Score = 238 bits (607), Expect = 2e-63 2S Query: 1619 DMNCTLPFSDQ--VVLVGTEEGLYALNVLKN--SLTHVPGIGAVFQIYIIKDLEKLLMIA

I ~ + +p p p p +I + +I 1 +++++ p 1l+

Sbjct:2 TAKCNHPTTCDAKNLLVGTEEGLYVLNRSDQGGTLEKIISRRSVTQIWVLEENNVLLMIS61 Query:1675GE---ERALCLVDVKKVKQSLAQSHLPAQPDISPNIFEAVKGCHLFGAGKIENGLCICAA1731 30 ~+ I ~ +++ ~ +~ + ~ + ++ I 1111111 + l +11I

Sbjct:62 GKKPYLYAHPLSGLRE-KDALGSARLVIRKNVWVK-IEDVKGCHLFAVVNGKRILFLCAA119 Query:1732MPSKWIL-RYNENLSKYCIR-----KEIETSEPCSCIHFTNY---SILIGTNKFYEIDM1782 +~I I +~ II + I ~ + ++ I I +~ I+

3S Sbjct:120 LPSSVQLLQWYNPLKKFKLFKSKFLKKLIVPVPLFVLLTSSSFELPKICIGVDK-NGFDV178 Query:1783KQYTLEEFLDKNDHSLAPAVFAASSNSFPVSIVQVNSAGQREEYLLCFHEFGVFVDSYG-1841 ~+ +~ I II ~ II I + +I loll ~~~~~1+ I

Sbjct:179 VQFHQTSLVSKEDLSLPNLNEETSKKPIPVIQVPQSD----DELLLCFDEFGVFVNLQGM234 Query:1842RRSRTDDLKWSRLPLAFAYREPYLFVTHFNSLEVIEIQARSSAGTPARAYLDIPNPRYLG1901 I I +~ II~ ~~~~ I I +I ~+ I + I II

Sbjct:235 RRSRKPILTWEFMPEYFAYHEPYLLAFHSNGIEIRSIETGELLQELADR--EARKIRVLG292 45 Query:1902PAISSGAIYLASSY 1915 1 I +1I

Sbjct:293 S--SDRKILVSSSP 304 Table 3H Domain Analysis of NOV3b gnllSmartlsmart00220, S_TKC, Serine/Threonine protein kinases, catalytic domain; Phosphotransferases. Serine or threonine-specific kinase subfamily. (SEQ TD N0:72) CD-Length = 256 residues, 100.0 aligned Score = 230 bits (587), Expect = 5e-61 Query: 97 FEVRSLVGCGHFAEVQWREKATGDIYAMFWKKKALLAQEQVSFFEEERNILSRSTSPW 156 +I+ ++I I I +I + I+I II I+II+II+ I +++
I II + I

Sbjct:1 YELLEVLGKGAFGKVYLARDKKTGKLVAIKVIKKEKLK-KKKRERILREIKILKKLDHPN59 Query:157IPQLQYAFQDKNHLYLVMEYQPGGDLLSLLNRYEDQLDENLIQFYLAELILAVHSVHLMG216 I +I I+I + 1111111 IIII II + +I I+ +1I
+++ I+ +I I

Sbjct:60 IVKLYDVFEDDDKLYLVMEYCEGGDLFDLLKKR-GRLSEDEARFYARQILSALEYLHSQG118 IO Query:217YVHRDIKPENILVDRTGHIKLVDFGSAAKMNSNKVNAKLPIGTPDYMAPEVLTVMNGDGK276 +ill+Illlil+I II+II III I +++I +111+Ilillll Sbjct:119IIHRDLKPENILLDSDGHVKLADFGLAKQLDSGGTLLTTFVGTPEYMAPEVLL------G172 Query:277GTYGLDCDWWSVGVIAYEMIYGRSPFAEGTSARTFNNIMNFQRFLKFPDDPKVSSDFLDL336 II 1 II+III II+ I+ II + I + +I + II

Sbjct:173KGYGKAVDIWSLGVILYELLTGKPPFPGDDQLLALFKKIGKPPPPFPPPEWKISPEAKDL232 Query:337IQSLLC-GQKERLKFEGLCCHPFF 359 2O Sbjct:233IKKLLVKDPEKRLTAEEALEHPFF 256 Table 3I Domain Analysis of NOV3b gnllSmartlsmart00036, CNH, Domain found in NIKl-like kinases, mouse citron and yeast ROM1, ROM2; Unpublished observations. (SEQ ID
N0:73) CD-Length = 301 residues, 99.7& aligned Score = 226 bits (577), Expect = 8e-60 Query: 1619 DMNCTLPFSDQ--WLVGTEEGLYALNVLKN--SLTHVPGIGAVFQIYIIKDLEKLLMIA 1674 I + ++IIIIIIIII II+ +I + I I II+++++ IIII+

Sbjct:2 TAKWNHPTTCDAKILLVGTEEGLWLNISDQHGTLEKLIGRRSVTQIWVLEENNVLLMIS61 Query:1675GEERALC---LVDVKKVKQSLAQSHLPAQPDISPNIFEAVKGCHLFGAGKIENGLCTCAA1731 I++ I I + + I +I + I + ++ 111111 + I +I
I

3O Sbjct:62 GKKPQLYSHPLSALTE-KDALGSARLVIRKNVLTK-IPDVKGCHLCAWNGKRILFLCHA119 Query:1732MPSKWIL-RYNENLSKYCIR-----KEIETSEPCSCIHFTNY---SILIGTNKFYEIDM1782 + 1 II+I II + I + + I I++I 1+

Sbjct:120 LQS5VVLLQWYNPLKKFKLFKSKFLFPLISPVPVFVELVSSSFELPGICIGSDK-NGGDV178 Query:1783KQYTLEEFLDKNDHSLAPAVFAASSNSFPVSTVQVNSAGQREEYLLCFHEFGVFVDSYG-1841 I+ + I 1 1l II I I +I III+ 111111+ I

Sbjct:179 VQFH-QSLVSKEDLSLPFLSEETSSKPISWQVP------ADELLLCYDEFGVFVNLYGM231 4Q Query:1842RRSRTDDLKWSRLPLAFAYREPYLFVTHFNSLEVIEIQARSSAGTPARAYLDIPNPRYLG1901 IIII I I +I +11I III I I +I+ +I I i II

Sbjct:232 RRSRNPTLHWEFMPESFAYHSPYLLAFHDNGTEIRSTKTGELLQELADR--KTRKIRLLG289 Query:1902PAISSGAIYLASSY 1915 I I I+II

Sbjct:290 S--SDRKILLSSSP 301 Table 3J Domain Analysis of NOV3b gnllPfamlpfam00069, pkinase, Protein kinase domain. (SEQ ID N0:74) CD-Length = 256 residues, 100.0& aligned Score = 189 bits (481), Expect = 1e-48 Query: 97 FEVRSLVGCGHFAEVQWREKATGDIYAMKVMKKKALLAQEQVSFFEEERNILSRSTSPW 156 +I+ +I I I +I + I II+I I+I++II+I I I I II
I + I

Sbjct:1 YELGEKLGSGAFGKVYKGKHKDTGEIVAIKILKKRSL--SEKKKRFLREIQILRRLSHPN58 Query:157IPQLQYAFQDKNHLYLVMEYQPGGDLLSLLNRYEDQLDENLIQFYLAELILAVHSVHLMG216 I +I I++ +IIIIIIII IIII I I I I + +++ I
I

Sbjct:59 IVRLLGVFEEDDHLYLVMEYMEGGDLFDYLRRNGLLLSEKEAKKIALQILRGLEYLHSRG118 1O Query:217YVHRDIKPENILVDRTGHIKLVDFGSAAKMNS-NKVNAKLPIGTPDYNIAPEVLTVMNGDG275 IIII+111111+I I I III I I I ++ +11I+1111111 I I

Sbjct:119TVHRDLKPENTLLDENGTVKIADFGLARKLESSSYEKLTTFVGTPEYMAPEVL---EGRG175 Query:276KGTYGLDCDWWSVGVIAYEMIYGRSPF-AEGTSARTFNNIMNFQRFLKFPDDPKVSSDFL334 I I Il+III II++ I+ II I + I I I I++

Sbjct:176---YSSKVDWSLGVILYELLTGKLPFPGIDPLEELFRIKERPRLRLPLP--PNCSEELK230 Query:335DLIQSLLCGQ-KERLKFEGLCCHPFF 359 + ++ +

Sbjct:231DLIKKCLNKDPEKRPTAKEILNHPWF 256 Table 3K Domain Analysis of NOV3b gnllSmartlsmart00219, TyrKC, Tyrosine kinase, catalytic domain;
Phosphotransferases. Tyrosine-specific kinase subfamily. (SEQ ID
N0:75) CD-Length = 258 residues, 95.0% aligned Score = 91.7 bits (226), Expect = 4e-19 Query: 104 GCGHFAEVQVVREKATGDIYAMKUMKKKA.LLAQE-QVSFFEEERNILSRSTSPWIPQLQY 162 I I I I I I I + +I I I I+ I I ++ + I I +I

Sbjct:8 GEGAFGEVYKGTLKGKGGVEVEVAVKTLKEDASEQQIEEFLREARLMRKLDHPNIVKLLG67 Query:163AFQDKNHLYLVMEYQPGGDLLSLLNRYEDQLDENLIQFYLAELI-LAVHSVHLMGYVHRD221 ++ I +1111 V III I++ + I I + + +1111 3O Sbjct:68 VCTEEEPLMIVMEYMEGGDLLDYLRKNRPKELSLSDLLSFALQIARGMEYLESKNFVHRD127 Query:222IKPENILVDRTGHIKLVDFGSAAKMNSNKVNAKLPIGTPD--YMAPEVLTVMNGDGKGTY279 I II +I+ III I + + I +1111 I I +

Sbjct:128LAARNCLVGENKTVKIADFGLARDLYDDDYYRKKKSPRLPIRWMAPESLK------DGKF181 Query:280GLDCDWWSVGVIAYEMI-YGRSPFAEGTSARTFNNIMNFQRFLKFPDDPKVSSDFLDLIQ338 I II II+ +I I II+ ++ I + I+ I + II+

Sbjct:182TSKSDWSFGVLLWEIFTLGESPYPGMSNEEVLEYLKKGYRLPQPPNCP---DEIYDLML238 Query:339SLLCGQ---KERLKFE 351 I ++I I

Sbjct:239Q--CWAEDPEDRPTFS 252 Recent data shows the identification of a novel serine/threonine kinase belonging to the myotoluc dystrophy kinase family (DiCunto et al. Eur J Immunol 2000 Dec;30(12):3403-10.).
The kinase can be produced in at least two different isoforms: a approximately 240-kDa protein (Citron Rho-interacting kinase, CRIK), in which the kinase domain is followed by the sequence of Citron, a previously identified Rho/Rac binding protein; a approximately 54-kDa protein (CRIK-short kinase (SK)), which consists mostly of the kinase domain.
CRIK and CRIK-SK proteins are capable of phosphorylating exogenous substrates as well as of autophosphorylation, when tested by iTa vitro kinase assays after expression into COS7 cells.
CRIK kinase activity is increased several fold by coexpression of costitutively active Rho, while active Rac has more limited effects. Kinase activity of endogenous CRIK
is indicated by iya vitYO kinase assays after immunoprecipitation with antibodies recognizing the Citron moiety of the protein. When expressed in keratinocytes, full-length CRIK, but not CRIK-SK, localizes into corpuscular cytoplasmic structures and elicits recruitment of actin into these structures. The previously reported Rho-associated kinases ROCK I and II are ubiquitously expressed. In contrast, CRIK exhibits a restricted pattern of expression, suggesting that this kinase may fulfill a more specialized function in specific cell types.
T cell receptor (TCR) engagement increases integrin-mediated adhesion to APC, resulting in the stabilization of the T cell. APC interaction and the close apposition of the two cell membranes. Engagement of either the TCR or CD3 chimeras with immobilized antibodies causes the rapid spreading of T cells in an integrin-independent fashion (Borroto et al. Eur J
Immunol 1999 Nov;29(11):3609-20). This effect concurs with the polymerization of the actin cytoskeleton and is dependent on the integrity of the immunoreceptor tyrosine-based activation motifs of the CD3 subunits. Expression of a dominant negative mutant of RhoA, as well as the Rho-specific inhibitor C3 toxin, abolished TCR-induced spreading.
In contrast, constitutively active or dominant negative forms of Rac and Cdc42 did not affect cell .
spreading. Signals emanating from the TCR can directly induce T cell spreading, independently of integrins, and via a Rho-dependent reorganization of the actin cytoskeleton.
Motile lymphocytes adopt a polarized morphology with different adhesion molecules (ICAM, CD43 and CD44) and ERM actin-binding proteins concentrated on the uropod, a slender posterior appendage with important functions in cell-cell interactions and lymphocyte recruitment. The role of Rho family of GTPases (Rho, Rac and Cdc42) in the control of lymphocyte polarity and migration has been studied by analyzing the effects of exogenously introduced Rho GTPase mutants. Transfection of T cell lines that constitutively display a polarized motile morphology with activated mutants of RhoA, Rac 1 and Cdc42 impaired cell polarization. A guanosine nucleotide exchange factor for Rac, Tiam-1, induced the same effect as activated Racl . Conversely, dominant negative forms of the three GTP-binding proteins induced a polarized phenotype in constitutively round-shaped T cells with redistribution of ICAM-3 and moesin to the uropod in an integrin-dependent manner. On the other hand, overexpression of dominant negative Cdc42 and activated mutants of all three Rho GTPases significantly inhibited SDF-lalpha-induced T cell chemotaxis.
Together, these data demonstrate that Rho GTPases regulate lymphocyte polarization and chemokine-induced migration, and underscore the key role of Cdc42 in lymphocyte directional migration.
Activated Rho GTPases trigger distinctive kinase cascades. In particular, ROCK
binds to Rho, and its kinase activity is moderately stimulated by this association.
The citron molecule (Madaule et al., 1995), a specific interactor of Rho and Rac, shares a significant degree of structural homology with ROCK; however, its lack of a kinase domain raised the question of its biologic function. By PCR of a mouse primary keratinocyte cDNA
library, Di Cunto et al. (1998) identified a novel serine/threonine kinase, CRIK (citron Rho-interacting kinase), belonging to the myotonic dystrophy kinase family. CRIK can be expressed as at least 2 isoforms, one of which encompasses the previously reported form of citron in almost its entirety. The long form of CRIK is a 240-kD protein in which the kinase domain is followed by the sequence of citron. The short form, CRIK-SK (short kinase), is an approximately 54 kD protein that consists mostly of the kinase domain. CRIK and CRIK-SK
proteins are capable of phosphorylating exogenous substrates as well as of autophosphorylation, when tested by in vitro kinase assays after expression into COS-7 cells. CRIK
kinase activity is increased several-fold by coexpression of constitutively active Rho, while active Rac has more limited effects. Kinase activity of the endogenous CRIK is indicated by ifa vitro kinase assays after immunoprecipitation with antibodies recognizing the citron moiety of the protein. When expressed in keratinocytes, full-length CRIK, but not CRIK-SK, localizes into corpuscular cytoplasmic structures and elicits recruitment of actin into these structures.
The previously reported Rho-associated kinases ROCKl and ROCK2 are ubiquitously expressed.
Northern blot analysis of mouse tissues revealed a restricted pattern of expression limited to keratinocytes, brain, spleen, lung, kidney, and an especially strong signal in testis. No expression was detectable in heart, liver, or skeletal muscle. The CRIK
protein contains a kinase domain, a coiled-coil domain, a leucine-rich domain, a Rho-Rac binding domain, a zinc finger region, a pleckstrin homology domain, and a putative SH3-binding domain. Di Cunto et al. (1998) reported cloning the human homolog of the CRIK kinase domain. They stated that the human homolog of citron is contained within a PAC clone (GenBank GENBANK
AC002563) mapping to chromosome 12q. By screening size-fractionated human brain cDNA
libraries for cDNAs encoding proteins larger than 50 kD, Nagase et al. ( 1999) identified CRIK

as cDNA KIAA0949 (GenBank GENBANK AB023166). Di Cunto et al. (1998) mapped the human CRTK gene to chromosome 12q24.1-q24.3.
Di Cunto et al. (2000) generated mice deficient in citron kinase by targeted disruption.
Citron-K -/- mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with dramatic depletion of microneurons in the olfactory bulb, hippocampus, and cerebellum.
These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Di Cunto et al. (2000) concluded that citron-K is essential for cytokinesis in vivo, in specific neuronal precursors only. Moreover, they suggested a novel molecular mechanism for a subset of human malfornlation syndromes of the central nervous system.
The disclosed NOV3 nucleic acid of the invention encoding a RHO/R.AC-interacting citron kinase -like protein includes the nucleic acid whose sequence is provided in Table 3A or 3C or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 3A or 3C
while still encoding a protein that maintains its RHO/RAC-interacting citron kinase -like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
The disclosed NOV3 protein of the invention includes the RHO/RAC-interacting citron kinase -like protein whose sequence is provided in Table 3B or 3D. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 3B or 3D while still encoding a protein that maintains its RHO/RAC-interacting citron kinase -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 13%
percent of the residues may be so changed.

The protein, similarity information, expression pattern, and map location for the RHOfRAC-interacting citron kinase-like protein and nucleic acid (NOV3) disclosed herein suggest that NOV3 may have important structural and/or physiological functions characteristic of the citron kinase-like family. Therefore, the NOV3 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications.
These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
The NOV3 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from asthma, arthritis, psoriasis, diabetes, and IBD, which require activated T cells, as well as diseases such as systemic lupus erythematosus that involve B cell activation, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Endocrine dysfunctions, Obesity, Growth and Reproductive disorders Hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, allergies, immunodeficiencies, transplantation, Lymphaedema, Hemophilia, Hypercoagulation, Idiopathic thrombocytopenic purpura , Irnmunodeficiencies, Graft vesus host, Hirschsprung's disease, Crohn's Disease, Appendicitis Inflammatory bowel disease, Diverticular disease, and/or other pathologies. The NOV3 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
NOV3 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies"
section below. For example the disclosed NOV3 protein have multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated epitope is from about amino acids 1 to 20. In another embodiment, a NOV3 epitope is from about amino acids 40 to 45. In additional embodiments, NOV3 epitopes are from about amino acids 110 to 150, from about amino acids 2I0 to 300, from about amino acids 410 to 900, from about amino acids 950 to 1200, from about amino acids 1250 to 1300, from about amino acids 1310 to 1450, from about amino acids 1490 to 1520, from about amino acids 1650 to 1680, from about amino acids 1800 to 1820, from about amino acids 1900 to 1920 and from about amino acids I 980 to 2053. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.

A disclosed NOV4 nucleic acid of 5691 nucleotides (designated CuraGen Acc. No.
105827550 EXT) encoding a novel Plexin-like protein is shown in Table 4A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 5683-5685. A putative untranslated region downstream from the termination codon is underlined in Table 4A, and the start and stop codons are in bold letters.
Table 4A. NOV4 Nucleotide Sequence (SEQ ID N0:12) ATGAAAGCCATGCCCTGGAACTGGACCTGCCTTCTCTCCCACCTCCTCATGGTGGGCATGGGCTCCTCCA
CTTTGCTCACCCGGCAGCCAGCCCCGCTGTCCCAGAAGCAGCGGTCATTTGTCACATTCCGAGGAGAGCC
CGCCGAGGGTTTCAATCACCTGGTGGTGGATGAGAGGACAGGACACATTTACTTGGGGGCCGTCAATCGG
ATTTACAAGCTCTCCAGCGACCTGAAGGTCTTGGTGACGCATGAGACAGGGCCGGACGAGGACAACCCCA
AGTGTTACCCACCCCGCATCGTCCAGACCTGCAATGAGCCCCTGACCACCACCAACAATGTCAACAAGAT
GCTCCTCATAGACTACAAGGAGAACAGGCTGATTGCCTGTGGGAGCCTGTACCAAGGCATCTGCAAGCTG
CTGAGGCTGGAGGACCTCTTCAAGCTGGGGGAGCCTTATCATAAGAAGGAGCACTATCTGTCAGGTGTCA
ACGAGAGCGGCTCAGTCTTTGGAGTGATCGTCTCCTACAGCAACCTGGATGACAAGCTGTTCATTGCCAC
GGCAGTGGATGGGAAGCCCGAGTATTTTCCCACCATCTCCAGCCGGAAACTGACCAAGAACTCTGAGGCG
GATGGCATGTTCGCGTACGTCTTCCATGATGAGTTCGTGGCCTCGATGATTAAGATCCCTTCGGACACCT
TCACCATCATCCCTGACTTTGATATCTACTATGTCTATGGTTTTAGCAGTGGCAACTTTGTCTACTTTTT
GACCCTCCAACCTGAGATGGTGTCTCCACCAGGCTCCACCACCAAGGAGCAGGTGTATACATCCAAGCTC
GTGAGGCTTTGCAAGGAGGACACAGCCTTCAACTCCTATGTAGAGGTGCCCATTGGCTGTGAGCGCAGTG
GGGTGGAGTACCGCCTGCTGCAGGCTGCCTACCTGTCCAAAGCGGGGGCCGTGCTTGGCAGGACCCTTGG
AGTCCATCCAGATGATGACCTGCTCTTCACCGTCTTCTCCAAGGGCCAGAAGCGGAAAATGAAATCCCTG
GATGAGTCGGCCCTGTGCATCTTCATCTTGAAGCAGATAAATGACCGCATTAAGGAGCGGCTGCAGTCTT
GTTACCGGGGCGAGGGCACGCTGGACCTGGCCTGGCTCAAGGTGAAGGACATCCCCTGCAGCAGTGCGCT
CTTAACCATTGACGATAACTTCTGTGGCCTGGACATGAATGCTCCCCTGGGAGTGTCCGACATGGTGCGT
GGAATTCCCGTCTTCACGGAGGACAGGGACCGCATGACGTCTGTCATCGCATATGTCTACAAGAACCACT
CTCTGGCCTTTGTGGGCACCAAAAGTGGCAAGCTGAAGAAGATCCGGGTGGATGGACCCAGGGGCAACGC
CCTCCAGTATGAGACGGTGCAGGTGGTGGACCCCGGCCCAGTCCTCCGGGATATGGCCTTCTCCAAGGAC
CACGAGCAACTCTACATCATGTCAGAGAGGCAGCTCACCAGAGTCCCTGTGGAGTCCTGTGGTCAGTATC
AGAGCTGCGGCGAGTGCCTTGGCTCAGGCGACCCCCACTGTGGCTGGTGTGTGCTGCACAACACGTGCAC
CCGGAAGGAGCGGTGTGAGCGGTCCAAGGAGCCCCGCAGGTTTGCCTCGGAGATGAAGCAGTGTGTCCGG
CTGACGGTCCATCCCAACAATATCTCCGTCTCTCAGTACAACGTGCTGCTGGTCCTGGAGACGTACAATG
TCCCGGAGCTGTCAGCTGGCGTCAACTGCACCTTTGAGGACCTGTCAGAGATGGATGGGCTGGTCGTGGG

CAATCAGATCCAGTGCTACTCCCCTGCAGCCAAGGAGGTGCCCCGGATCATCACAGAGAATGGGGACCAC
CATGTCGTACAGCTTCAGCTCAAATCAAAGGAGACCGGCATGACCTTCGCCAGCACCAGCTTTGTCTTCT
ACAATTGCAGCGTCCACAATTCGTGCCTGTCCTGCGTGGAGAGTCCATACCGCTGCCACTGGTGTAAATA
CCGGCATGTCTGCACCCATGACCCCAAGACCTGCTCCTTCCAGGAAGGCCGAGTGAAGCTGCCCGAGGAC
TGCCCCCAGCTGCTGCGAGTGGACAAGATCCTGGTGCCCGTGGAGGTGATCAAGCCTATCACGCTGAAGG
CCAAGAACCTCCCCCAGCCCCAGTCTGGGCAGCGTGGCTACGAATGCATCCTCAACATTCAGGGCAGCGA
GCAGCGAGTGCCCGCCCTGCGCTTCAACAGCTCCAGCGTACAGTGCCAGAACACCTCTTATTCCTATGAA
GGGATGGAGATCAACAACCTGCCCGTGGAGTTGACAGTCGTGTGGAATGGGCACTTCAACATTGACAACC
CAGCTCAGAATAAAGTTCACCTCTACAAGTGTGGAGCCATGCGTGAGAGCTGCGGGCTGTGCCTCAAGGC
TGACCCAGACTTCGCATGTGGCTGGTGCCAGGGCCCAGGCCAGTGCACCCTGCGCCAGCACTGCCCTGCC
CAGGAGAGCCAGTGGCTGGAGCTGTCTGGTGCCAAAAGCAAGTGCACAAACCCCCGCATCACAGAGATAA
TCCCGGTGACAGGCCCCCGGGAAGGGGGCACCAAGGTCACTATCCGAGGGGAGAACCTGGGCCTGGAATT
TCGCGACATCGCCTCCCATGTCAAGGTTGCTGGCGTGGAGTGCAGCCCTTTAGTGGATGGTTACATCCCT
GCAGAACAGATCGTGTGTGAGATGGGGGAGGCCAAGCCCAGCCAGCATGCAGGCTTCGTGGAGATCTGCG
TGGCTGTGTGTCGGCCTGAATTCATGGCCCGGTCCTCACAGCTCTATTACTTCATGACACTGACTCTCTC
AGATCTGAAGCCCAGCCGGGGGCCCATGTCCGGAGGGACCCAAGTGACCATCACAGGCACCAACCTGAAT
GCCGGAAGCAACGTGGTGGTGATGTTTGGAAAGCAGCCCTGTCTCTTCCACAGGCGATCTCCATCCTACA
TTGTCTGCAACACCACATCCTCAGATGAGGTGCTAGAGATGAAGGTGTCGGTGCAGGTGGACAGGGCCAA
GATCCACCAGGACCTGGTCTTTCAGTATGTGGAAGACCCCACCATCGTGCGGATTGAGCCAGAATGGAGC
ATTGTCAGTGGAAACACACCCATCGCCGTATGGGGGACCCACCTGGACCTCATACAGAACCCCCAGATCC
GTGCCAAGCATGGAGGGAAGGAGCACATCAATATCTGTGAGGTTCTGAACGCTACTGAGATGACCTGTCA
GGCGCCCGCCCTCGCTCTGGGTCCTGACCACCAGTCAGACCTGACCGAGAGGCCCGAGGAGTTTGGCTTC
ATCCTGGACAACGTCCAGTCCCTGCTCATCCTCAACAAGACCAACTTCACCTACTATCCCAACCCGGTGT
TTGAGGCCTTTGGTCCCTCAGGAATCCTGGAGCTCAAGCCTGGCACGCCCATCATCCTAAAGGGCAAGAA
CCTGATCCCGCCTGTGGCTGGGGGCAACGTGAAGCTGAACTACACTGTGCTGGTTGGGGAGAAGCCGTGC
ACCGTGACCGTGTCAGATGTCCAGCTGCTCTGCGAGTCCCCCAACCTCATCGGCAGGCACAAAGTGATGG
CCCGTGTCGGTGGCATGGAGTACTCCCCGGGGATGGTGTACATTGCCCCGGACAGCCCGCTCAGCCTGCC
CGCCATCGTCAGCATCGCAGTGGCTGGCGGCCTCCTCATCATTTTCATCGTGGCCGTGCTCATTGCCTAT
AAACGCAAGTCCCGCGAAAGTGACCTCACGCTGAAGCGGCTGCAGATGCAGATGGACAACCTGGAGTCCC
GTGTGGCCCTGGAGTGCAAGGAAGCCTTTGCCGAGCTGCAGACGGACATCCATGAGCTGACCAGTGACCT
GGATGGAGCCGGGATTCCGTTCCTGGACTATAGAACTTACACCATGCGGGTGCTGTTCCCAGGAATTGAA
GACCACCCTGTCCTCCGGGACCTTGAGGTCCCGGGCTACCGGCAGGAGCGTGTGGAGAAAGGCCTGAAGC
TCTTCGCCCAGCTCATCAACAACAAGGTGTTCCTGCTGTCCTTCATCCGCACGCTTGAGTCCCAGCGTAG
CTTCTCCATGCGCGACCGTGGCAACGTGGCCTCACTCATCATGACCGTGCTGCAGAGCAAGCTGGAGTAC
GCCACTGATGTGCTGAAGCAGCTGCTGGCCGACCTCATTGACAAGAACCTGGAGAGCAAGAACCACCCTA
AGCTGCTGCTCAGGAGGACTGAGTCAGTGGCTGAGAAGATGCTGACCAATTGGTTTACTTTCCTCCTCTA
CAAGTTCCTCAAGGAGTGTGCTGGGGAGCCCCTCTTCTCCCTGTTCTGTGCCATCAAGCAGCAGATGGAG
AAGGGCCCCATTGACGCCATCACGGGCGAGGCCCGCTACTCCTTGAGCGAGGACAAGCTCATCCGCCAGC
AGATTGACTACAAAACCCTGGTCCTGAGCTGTGTCAGCCCAGACAATGCCAACAGCCCCGAGGTCCCAGT
AAAGATCCTCAACTGTGACACCATCACTCAGGTCAAGGAGAAGATTCTGGATGCCATCTTCAAGAATGTG
CCTTGCTCCCACCGGCCCAAAGCTGCAGATATGGATCTGGAGTGGCGACAAGGAAGTGGGGCAAGGATGA
TCTTGCAGGATGAAGACATCACCACCAAGATTGAGAATGATTGGAAGCGACTGAACACACTGGCCCACTA
CCAGGTGCCAGATGGTTCCGTGGTGGCATTAGTGTCCAAGCAGGTGACAGCCTATAACGCAGTGAACAAC
TCCACCGTCTCCAGGACCTCAGCAAGTAAATATGAAAACATGATCCGGTACACGGGCAGCCCCGACAGCC
TCCGCTCACGGACACCTATGATCACTCCTGACCTGGAGAGTGGAGTCAAGATGTGGCACCTAGTGAAGAA
CCACGAGCACGGAGACCAGAAGGAGGGGGACCGGGGGAGCAAGATGGTGTCTGAAATCTACCTGACCCGA
CTCCTGGCCACTAAGGGCACACTGCAGAAGTTTGTGGATGACCTCTTTGAGACCATCTTCAGCACGGCAC
ACCGTGGCTCTGCCCTGCCCCTGGCCATCAAGTACATGTTTGACTTCCTGGATGAGCAGGCTGATAAACA
TGGCATTCATGACCCGCACGTCCGCCATACCTGGAAGAGCAATTGCCTGCCCCTGAGGTTTTGGGTCAAC
ATGATCAAGAACCCGCAGTTTGTGTTTGACATCCATAAGAACAGCATCACAGACGCCTGCCTCTCTGTGG
TGGCTCAGACCTTCATGGACTCTTGCTCCACGTCAGAGCACCGGCTGGGCAAGGACTCGCCCTCCAACAA
GCTGCTGTATGCCAAGGACATCCCCAGCTACAAGAATTGGGTGGAGAGGTATTACTCAGACATAGGGAAG
ATGCCAGCCATCAGCGACCAAGACATGAACGCATACCTGGCTGAGCAGTCCCGGATGCACATGAATGAGT
TCAACACCATGAGTGCACTCTCAGAGATCTTCTCCTATGTGGGCAAATACAGCGAGGAGATCCTTGGACC
TCTGGACCACGATGACCAGTGTGGGAAGCAGAAACTGGCCTACAAACTAGAACAAGTCATAACCCTCATG
AGCTTAGACAGCTGAAATAAA
The nucleic acid sequence of NOV4, localized on chromosome 7, has 4004 of 5567 bases (71%) identical to a plexin-2 mRNA from mouse (GENBANK-ID: D86949) (E =
0.0).
A NOV4 polypeptide (SEQ ID N0:13) encoded by SEQ ID N0:12 is 1896 amino acid residues and is presented using the one letter code in Table 4B. Signal P, Psort andlor Hydropathy results predict that NOV4 has no signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.46. In other embodiments, NOV4 may also be localized to the endoplasmic reticulum (membxane) with a certainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or the outside with a certainty of 0.1000.
Table 4B. NOV4 protein sequence (SEQ ID N0:13) MKAMPWNWTCLLSHLLMVGMGSSTLLTRQPAPLSQKQRSFVTFRGEPAEGFNHLVVDERTGHIYLGAVNRIYKL
SSDLKVLVTHETGPDEDNPKCYPPRIVQTCNEPLTTTNNVNKMLLIDYKENRLIACGSLYQGICKLLRLEDLFK
LGEPYHKKEHYLSGVNESGSVFGVIVSYSNLDDKLFIATAVDGKPEYFPTISSRKLTKNSEADGMFAYVFHDEF
VASMIKIPSDTFTIIPDFDIYYVYGFSSGNFVYFLTLQPEMVSPPGSTTKEQVYTSKLVRLCKEDTAFNSYVEV
PIGCERSGVEYRLLQAAYLSKAGAVLGRTLGVHPDDDLLFTVFSKGQKRKMKSLDESALCIFILKQINDRIKER
LQSCYRGEGTLDLAWLKVKDIPCSSALLTIDDNFCGLDMNAPLGVSDMVRGIPVFTEDRDRMTSVIAYVYKNHS
LAFVGTKSGKLKKIRVDGPRGNALQYETVQVVDPGPVLRDMAFSKDHEQLYIMSERQLTRVPVESCGQYQSCGE
CLGSGDPHCGWCVLHNTCTRKERCERSKEPRRFASEMKQCVRLTVHPNNISVSQYNVLLVLETYNVPELSAGVN
CTFEDLSEMDGLVVGNQIQCYSPAAKEVPRIITENGDHHVVQLQLKSKETGMTFASTSFVFYNCSVHNSCLSCV
ESPYRCHWCKYRHVCTHDPKTCSFQEGRVKLPEDCPQLLRVDKILVPVEVTKPITLKAKNLPQPQSGQRGYECI
LNIQGSEQRVPALRFNSSSVQCQNTSYSYEGMEINNLPVELTVVWNGHFNIDNPAQNKVHLYKCGAMRESCGLC
LKADPDFACGWCQGPGQCTLRQHCPAQESQWLELSGAKSKCTNPRITEIIPVTGPREGGTKVTIRGENLGLEFR
DIASHVKVAGVECSPLVDGYIPAEQIVCEMGEAKPSQHAGFVEICVAVCRPEFMARSSQLYYFMTLTLSDLKPS
RGPMSGGTQVTITGTNLNAGSNVWMFGKQPCLFHRRSPSYIVCNTTSSDEVLEMKVSVQVDRAKIHQDLVFQY
VEDPTIVRIEPEWSIVSGNTPIAVWGTHLDLIQNPQIRAKHGGKEHINICEVLNATEMTCQAPALALGPDHQSD
LTERPEEFGFILDNVQSLLILNKTNFTYYPNPVFEAFGPSGILELKPGTPIILKGKNLIPPVAGGNVKLNYTVL
VGEKPCTVTVSDVQLLCESPNLIGRHKVMARVGGMEYSPGMVYIAPDSPLSLPAIVSIAVAGGLLIIFIVAVLI
AYKRKSRESDLTLKRLQMQMDNLESRVALECKEAFAELQTDIHELTSDLDGAGIPFLDYRTYTMRVLFPGIEDH
PVLRDLEVPGYRQERVEKGLKLFAQLINNKVFLLSFIRTLESQRSFSMRDRGNVASLIMTVLQSKLEYATDVLK
QLLADLIDKNLESKNHPKLLLRRTESVAEKMLTNWFTFLLYKFLKECAGEPLFSLFCAIKQQMEKGPIDATTGE
ARYSLSEDKLIRQQIDYKTLVLSCVSPDNANSPEVPVKILNCDTITQVKEKILDAIFKNVPCSHRPKAADMDLE
WRQGSGARMILQDEDITTKIENDWKRLNTLAHYQVPDGSWALVSKQVTAYNAVNNSTVSRTSASKYENMTRYT
GSPDSLRSRTPMITPDLESGVKMWHLVKNHEHGDQKEGDRGSKMVSEIYLTRLLATKGTLQKFVDDLFETTFST
AHRGSALPLAIKYMFDFLDEQADKHGIHDPHVRHTWKSNCLPLRFWVNMIKNPQFVFDIHKNSITDACLSVVAQ
TFMDSCSTSEHRLGKDSPSNKLLYAKDIPSYKNWVERYYSDIGKMPAISDQDMNAYLAEQSRMHMNEFNTMSAL
SEIFSYVGKYSEEILGPLDHDDQCGKQKLAYKLEQVITLMSLDSNK
The full amino acid sequence of the protein of the invention was found to have 1263 of 1857 amino acid residues (68%) identical to, and 1501 of 1857 amino acid residues (80%) similar to, the 1884 amino acid residue plexin-2 protein from mouse (SPTREMBL-P70207) (E =0.0), and 364 of 801 amino acid residues (45%) identical to, and 520 of 801 amino acid residues (64%) similar to, the 2135 amino acid residue Human Plexin protein (patp:AAU00019) (E =2.6-283).
The disclosed NOV4 protein is expressed in at least the following tissues:
fibroblast like synoviocytes (normal), fetal brain, adipose, microvascular endothelial cells-lung, thalamus, fetal cerebral cortex, temporal lobe, parietal lobe, fetal cerebellum, and testis.
TaqMan expression data for NOV4 is shown below in Example 2.
NOV4 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 4C.

Table 4C. BLAST
results for Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (%) (%) gi~13649119~reflXp_SEX gene [Homo1871 1121/1841413/18460.0 010150.21 Sapiens] 6 (60%)(75%) gi~3413888~dbj~BAA3KIAA0463 protein1963 1270/1871508/18700.0 2308.1 [Homo Sapiens] 0 (67%)(79%) gi~2134135~pir~~T51Plexin - African1905 1220/1911468/19150.0 553 clawed frog 5 (63%)(75%) gi~14424639~gb~AAHOUnkown(protein813 641/810717/810 0.0 for 9343.1IAAH09343IMAGE:4130636) (79%) (88%) [Homo Sapiens]

gi~10047165~dbj~BABKIAA1550 protein593 513/513513/513 0.0 13376.1 [Homo Sapiens] (100%) (100%) The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 4D.
Table 4D ClustalW Analysis of NOV4 J 1) NOV4 (SEQ ID N0:13) 2) giI13649119IrefIXP 010150.21 SEX gene [Homo Sapiens] (SEQ ID N0:42) 3) gi13413888IdbjIBAA32308.1~ KIAA0463 protein [Homo Sapiens] (SEQ ID N0:43) 4) gi12134135IpirIII51553 Plexin - African clawed frog (SEQ ID N0:44) 5) gi114424639~gb~AAH09343.11AAH09343 Unkown(protein for IMAGE:4130636) [Homo Sapiens] (SEQ ID N0:45) 6) gi110047165Idbj~BAB13376.11 KIAA155D protein [Homo Sapiens] (SEQ ID N0:46) ....I....I....I....I....I....I....I....I....I....I
NOV4 __________________________________________________ gi~136491191 __________________________________________________ gi13413888~ GFGRLPDSELRAGRGASRRPQQPAAAEVDRAGTEGQTDVAELESCEGQPG
gi~2134I35~ __________________________________________________ gi114424639~ ______________________________-___________________ giI10D47165~ __________________________________________________ ....I....I....I....I....I....I....I...."I,....I....I
NOV4 -----------------------MKAMPWNWTCLL~HL,~VGMGSSTLLT
gi~136491191 -------------------------MP--------SIC~.~(LLLFLAVG-G
gi~3413888~ KVEQMSTHRSRLLTAAPLSMEQRRPWPRALEVDSR-~aWtiSVVWVLLAPP
giI2134135~ -----------------MLLHAERPLP----FHLW'FL~II,iLGSWIATGDG
gi1144246391 ______________________________________..__________ gi~100471651 __________________________________________________ ....I....I....I....~....L.....~....I~....I....I....I
NOV4 RQPAPLSQKQRSFVTFRGEPAEGFFILVVDERT Ei'YLGAVNRIYKLSSD
gi~136491191 ALGNRPFRAFW--TDTTLTHLAV~TRVTGEVF ~'NRVFKLAPNLTELR
3S gi~34138881 AAGMPQFSTFHSENRDWTFNHLTVQGTGAV RVYKLTGNLTIQV
gi~21341351 SP--KDFRTFTG--SDWSLTHLWTKTGEV RIYKLSNNLTLLR
gi~144246391 __________________________________________________ gi~100471651 __________________________________________________ ....I....I....I....~....I....I....~....I....I....I
NOV4 LKVLVTH TGPDED 'KCYPPRIVQTCNEPhTTTNN~NKMLLIDYKENRL
gi~136491191 AHVTGP NARCYP'PSMRVCAHRLAPVDNINKLLIDYAARRLVACGS
gi~34138881 AHKTGPE NKSCYP'LIVQPCSEVLTLTNNVNKLL[f;IDYSENRLLACGS
4S gi~21341351 THVTGP NEKCYP'PSVQSCPHGLITTNNVNKLLIDYSDNRLIACGS
gi~144246391 __________________________________________________ gi~1D047165~ __________________________________________________ NOV4 IAC SLYQGICKL RLEDLFKLG~PKKEHYLSGVNESGS~~FGVIVSYS
gi ~ 13649119' IW ICQFLRLDD FKLGEPHHRTtE~t33'LSGAQEPDSMAGVIS~EQGQGPSK
gi ~ 3413888 ~ LYQ CKLLRLDD FILVEPSHICVKE~L3YLSSVNKTGTMYGVISTRSEGEDGK
gi~2134135~ ASQ ICQFLRLDD FKLGEPHHRItE~tiYLSSUNESGTMSGVIEVPNGQNK
gi~144246391 _______________________.__________________________ gi~10047165~ -_________________________________________________ gi~136491191 LFVGTAVDGKSEYFPTLSSRIQ.~ISDEDSADMFSLVYQDEFVSSQTKIPSD

gi13413888~ LFIGTAVDGKQDYFPTLSSRICtiPRDPESSAMLDYELHSDFVSSLIKIPSD

gi~2134135~ LFVGTPIDGKSEYFPTLSSRIICtaLGNEENAEMFGFVYQDEFVSSQLKIPSD

IS gi~144246391 ___________________,_.____________________________ gi~100471651 __________________________________________________ 2O NOV4 K'I:PSDTFTI SSGNF~YFLL~PEMVSPP STTKEQVYT
PDFDIYYW

gi.13649119 T~LtSLYPAFD FLT~'iQLD-QQTLLDT EKFFTSKIV
~ I YYIYGFVSAS

gi 3413888 ALVSHFD FLT~tQPEP~.'GVAINS DLFYTSRIV
~ I FYIYGP'AS
T
Ii gi12134135~ ~ YYLTT~QLD-QLTSPDS EQFFTSKIV
~
TSKFPTFD
YYVYS~SSEQ

2S giI100471651 __________________________________________________ NOV4 SKLVRLCISEDTAFNSYVEVPIGCERSG'~EYRLLQ~~!AYLSKAGAVLGRTLG

3O gi113649119~ RMCAGDSFXYVEFPIGCSWRGVEYR
VQSAHL~KPGLLLAQALGVPAD
~

gi~34138881 LQAAYL~KPGDSLAQAFNITSQ
RLCKDDPFHYVSLPFGCTRAGVEYR

giI21341351 RLCVDDPT~FYYVEFPIGCMKDGVEYRIQDAYLKPGKRLAKELGISER

k gi~144246391 -_______________ _______.__._______________________ gi~100471651 ___________________,______________________________ NOV4 VHPDDDLFTVFSKGQKRKMK~'',LDESALCIF LKQ 'IT~ERLQSCYRG

gi~136491191 EDVLFTFSQGQKNRASPPRQ';~ILCLFTLS R 'I(TSCYGEGTL

O gi~3413888~ DDVLFA~FSKGQKQYHHPPDDALCAFPI LQ K ' QSCYt~GEGNL

gi~2134135~ EDILFFSQGQKNRTKPPKEVLCLFTL KD KE'xf?SCYGDGKL

gi~144246391 _________,___________-_________________ gi~10047165~ __________________________________________________ NOV4 EGTLI..WLKVKDIPCSSALLTIDDNFCGLDMNAPLG'iTSDMVRGIPVFTE

gi1136491191 ALPWLKELPCINTPMQINGNFCGLVLNQP~aGGLHU~EGLPLLADSTDG

gi134138881 ELNWLGKDVQCTKAPVPIDDNFCGLDINQPTiGGSTP~EGLTLYTTSRDR

SO gi121341351 SLPWLKELGCINSPLQIDDNFCGQDFNQP~iGGTVT;'~EGTPLFLDKEDG

gi~144246391 _______________________________.__________________ giI100471651 __________________________________________________ SS

gi~13649119~ MASVAAYTYRQHSWFIGTRS SLKKVRVDGFQDAH----LYETVPVVDG

giI34138881 MTSVASWYNGYSWFVGTKS KLKKIRADGPPHGG--VQYEMVSVLKDG

gi~21341351 MTSVAAYDYRGHTWFAGTRS RVKKILVDLSASSSHLVQQYENVWHEG

O gi~1442463 91__________________________________________________ giI100471651 __________________________________________________ 6S NOV4 PGPj'ULRDMAFSKDHEQLMSE;R'QLTRVPVESCGQYQSCGECLGSGDPHC

gi~136491191 SPI~iRDLLFSPDHRHIYL~SEICQUSQLPVETCEQYQSCAACLGSGDPHCG

gi ~34138881 SPITiRDMAFSIDQRYLYVi~SER~UTRVPVESCEQYTTCGECLSSGDPHCG

gi ~2134135~ NAIL'sRDLVLSPDRQYIYAL~TEK~UTRVPVESCEQYESCDTCLGSRDPHCG

gi ~144246391 __________________________________________________ 70 gi ~100471651 __________________________________________________ .I....I....I....I....I....I....I....I....I.. " I
NOV4 GWC(fiLI3NTCTRKERCERSKEPRRFASEMI~QCVRLTVHPh~NISVSQYNV~L
S giI136491191 WCV~RI~RCCREGACLGASAPHGFAEELS~CVQVRVRPN~1VSVTSPGVQ~,T
gi I 3413888 I WCA'tiHL~MCSRRDKCQQAWEPNRFAASISQCVSLAVHPS'-~,sISVSEHSRL'~tS
gi I 2134135 I WCV~iHCSRKDKCERADELHRFTSDQRQCVQLTVHPKI~TISVTVSEVp~r~V
giI144246391 __________________________________________________ giI10047165~ __,_______________________________________________ l~

I,,...I....I....I. .I....I.. .I _,.I....I
NOV4 "ULETYNVPELSAGVNCTFEDLSEMDGLWGN,~~~Q Y ~ . PRL';ITEN
gi I 136491191 j'O7TLHNVPDLSFiGVSCAFEAAAENEAVT.rLPSGIfL P ~SLQET~RATRGH
IS giI34138881 ~WSDAPDLSAGIACAFGNLTEVEGQVS-GSQ'VI I ~GP~CDVPV',IPLDQ
giI21341351 QAWNVPDLSAGVNCSFEDFTEMEGRIZ-DG~C~j T ~SA~IP~TRGH
giI14424639~ ._________._____________________~_________________ gi~10047165~ ______________________,___________________________ .I.... .I....I....I.. .I....I.. .I....I
NOV4 GDHHV,~7Q Q . MT STS HNS ESP~R
gi I 13649119 I GATRT~V,,R Q L GAD LQS GSPY"P
2S gi I 2134135 I GDKRVVE QY KI SVD QS ,~ GP
giI144246391 _____._____________________________.______________ giI100471651 __________________________________________________ 30 ..I....I.. .I.. .I.. .I. .I... L.~...I....I.. .I
NOV4 PKT . ~ P . ' LLiRVDK3;T. ' EVE
gi I 136491191 ' .-T SRPHE ~ '~ SP ~~~i~PSGD~L~ GVMQ~L;
giI34138881 '-L DPTT ~ VNIS QLVPTEEIL~' GE ~I ' giI21341351 AAD ~ "j S ~QZI~PSSQ~YI' GW 'x T
3S giI144246391 _________________~_____________________ ._._______ gi~100471651 __________________________________________________ .I.._.I....I.....I.. .I.._ . .I....I.. .I....I
NOV4 '~~1 ~1'~ ILN~Q SEQ' P~L~ . 8..1 t T S . MEINT~L
g1 I 13 649119 I ~ ~ ~ ~ ~ RS~Q RQQ' P' ~ u~ - ~ ~ S BFIu'HGT~T
g1I34138881 ~~~1 1R, LN~tTQ I ' P~ S 1 1 S Q M1.~IS~L
giI21341351 ~v~v ~ ; I~FH~P SVT~ T~L' ~ I~ ~ T E IS..,L
g1I144246391 _______________.__________________________________ 4S gilloo4716sj __________________________________________________ NOV4 P~L,-~-~. .. . .QNf......I... I . .I.- .~PD.I'. .~
r r n r wrv S0 giI136491191 EL70F~r~ P ~ 'PSFRAL ~,'Q~P w P I
giI21341351 L~ I ~ ~QNI..~ S~~~ 5~R E
giI144246391 _______________________________________.__________ gi I 100471651 -~~7LH~AQNGiIIPD~Q
SS

.I.. I.. .~ . .I .I.
NOV4 GPGQ ~ ~ ~ Q~'Q . ItEL GAK a~ . E T
gi I 13649119 I SE Q T ' ~Pi~CT L QKG~ S ' ()0 gi I 3413888 GE ~ TPS~'P IiD S ~'' ~Q E ~S 'P
giI21341351 SE '~ 'TL~f~P TAN'~R~TD~ICmItF~E
giI144246391 _ ~____ ____ _ _ ____ .___ _ gi I 10047165 I GPG(~~~ ' Q~"'~QQIsELpGAKS.~TE~I~STT~R~F~

NOV4 ~I . E~R~~ASH~..I.-E.S~UDI..P .. 'G.~~KPSQH
gi I 13 649119 I LSR'Et7G -~F:~R STPAE S ~ E'~'.~~iLVPSPP
giI34138881 S~ ~Q P T~ FGE I~ ~ G ~-LVGTT
'JD gi I 2134135 I T E'2 R~Ei1 RFGI3VM SE S ~ ~ TNGRTRVH

gi1144246391 __________________________________________________ gi I 10047165 I ~R~.E~E~R~AS~E~S~VDG~P ~~GE~-KPSQH

.1....1....1....1.....1....1.. .~.. .1Y_,..
NOV4 '~.'~~. F . T' . RPM."~'~7 MA S ~~L~ TL'TLSD'ii ' . Q .
gi I 13649119 I PLP L GD SA~RTQ E~S TPpFDQCt~S ~ RL S
gi~21341351 E1l~AJrrQ RL GE SQDlCY~Y'RAI P~~C6kAlT LP~FNRSIT~I' ~L IS E
;... ~
l~ gi~144246391 -____._ ____'_=_____ __- ___ _______________ gi1100471651 A~F~T~AV~RPF'..~MAR~S~L~YQMTLTLSDLK~~'~'M~Q~T~

I S NOV4 T . . . I . MF~t KIP . L H BPS ~~TS DE-VLMKI7S'~Q~ . ~ ' gi1136491191 SS =TVRDSE Q DA I~PL~TLGPSQAP~TiiAIm gi13413888~ HY C~ S ~YL~~T E Y ~MSE APP SNGLGPVP'V~~V'S m gi ~ 2134135 I N D SVAI ~P SW~'~AKEK;1'PQGPS-TG~CAETQ~LT ' gi1144246391 -gi ~ 10047165 ~ TNV~MF~eK(~P~L~IRQ~PSY~.;TS~DE-VL~'ME~V~UQ't~

..I,.. .I.. .~.. .I.. .I.. ..1,....I
NOV4 ~ K~!H-QD Q .'f ~ ' T ' T DL ~ ' Q ' gi113649119~ ~ 1SSP IYT Tn ~ ~T'L ' I S T S T L : vE~R ' gi134138881 D S QFE I ~~R'VQ~ isTS~ F ~E~
gi ~ 2134135 I ' TNrTSE," ~T~Q,,. T~I~T~eM~ KE
gi1144246391 _ ____ _____ ________________ __________._ ___ g11100471651 ~K~Fi-QD~Vk'Q1V' '~T~I~DL~Q~y .1._..1._..I.. .I .1.._.1....1....1.. .1....I
NOV4 HIT ~LE E Q~' LGPDHQS~LT ' ~ 13:n Y ' L
gi113649119~ ~t R I TT T Q 1 D L ~~G1FLGRPQPRAQ ~~ L~
gi134138881 FN~e S T TL L~~,S~TTDYRPGL~ ~~ F
gi121341351 iY'GD KE ~ T1i D L~~9U,DNPLRSPP~NGTJ~~~ I In gi1144246391 ------GTR , T Ti, L~~S TTDYRPGL~ ~~~ ~F
gi1100471651 G~eK~HI~E~Q~~: LGPDHQS~LT '~ Zy .1....I... . .-,I,.. .L... _I..
NOV4 . ~ ~ T . . T T
gi 13649119 ~TARSR~B T ~S P
gi'34138881 ~ Y~ r I ~T L S~ Q ~ ~ C~~~' gi121341351 T 5 L ~ ~ P T ~S ~ T ~p gi1144246391 ~ Y I ~ ~T L S~ Q ' ' ~ C~~~S
gi I 10 04 716 5 I ~ .,.,,~ T . . F ~ I ~ T ~ ~ ~ ~ ..

50 .I.... . .~....~.... ....I.. .,~.. .I.. y . .
NOV4 G.~. ~ T Y ~ I
gi113649119~ -5 GQ~ SL v ~~.Q l~Qp ~..L L -, L
gi134138881 -G T~ E ~ p~
giI2134135~ -~TF; ;T' L E ~ TF
gi1144246391 -T' ~ P' gi110047165~ G ~ T ~ I

NOV4 M; .IPn ply ,'1w . ~.. .II F I... .I.. .~.I.. .I
r gi1136491191 T~~S~~ ~~MN(GL~ . '.TQ
gi I 3413888 I ~S~~~VSttIy L ~ S IVIiI
gi I 213413 5 I ~14Q~~'~ ~ L ~ G ~ I~~I i gi114424639~ ~S~SVIS~ L S IV~I
gi1100471651 M~/Y~Ap~ P S ~ I FaU ' S

.i.. .I.. .I.... .1....I. I~~ I~ I
NOV4 w ~ v ~ ~ ~
70 gi1136491191 ~_,~ ~ ~ ~ ~EVQ

gi~34138881 . . . ~ y ~R~
gi~2134135~ . . . . . . .GA ~F E ' gi1144246391 . . . . . S. .R~ .
gi~lOD471651 . . . G__________ ___________________ .~....I....~....~....I....I....l.~~ ~I....~....I
~ ~ wu~!v NOV4 . ' PGYRQER . I S S
gi~136491191 ,TPP~~,,,,,,- -- . LEIS
IQ gi~34138881 . ~ Q~-.~~~,IGQQH ~ T L
gi ~ 2134135 I . ' Q---- 5 . ;T aH
gi~144246391 . ' QG~~~''GQQH , I-. L
gi~100471651 E.~~G ~, -____________________.______________, 1$ 1460 1470 1480 1490 1500 .... .... .... .... ..... .... ....~~.....L.....I....
NOV4 .~ ~.~ .S~ . i Sa v ' v giI13649119) .S '.~ T ~ .Silt ' G~ . . E 8a 2O gy 34138881 .~ ~.~ .GR . Sv D a gi~2134135 .' '.~ .GE . S. E S
gi~14424639~ .' '.' .G~2 . $. a giI1D0471651 ______________________.____________.______________ 25 .... ....I.... .... ..:.-I-...I_...~....I.... ....
NOV4 T Y S F ~ ..
g11136491191 T L ' ..
v giI34138881 gi~21341351 T Y ~ ..
30 g11144246391 Y ' ..
giI100471651 ____________________ VC2 HICSXCUCI F . GWAA

..I....I._..~....I..._I...
3$ NOV4 .~ . ~.. ~ _________________ g11136491191 ." . ~.. __________________T
gi~34138881 .~ . ~.. ~ __________________I ::
v giI21341351 .' . '.. PCADDVGLSDESCCRSPQT s gi~144246391 .' ~~ . ~.. ~. __________________I '.
gi11D0471651 GSA RCVC C -_______________,_________________ NOV4 z~~..v,rl.. ~..~.~..~ .. . . .- ~~IF 'C H~~ -~' -.
4$ g11136491191 C~Ei QY~ ~S v W ~T G~~ .~. .E, r N v g1134138881 ~ ~~ . . I .~ ,~~ . .
giI21341351 ~'n ~ T' . . a ~. .~~ -_ g11144246391 ~, T~ . . I .~ ,.. , , g11100471651 C .____ __ ~ T~yC_____________~ YTS~QAGQQ-__ $0 .... .... .... ....I.... .... .... .... .... ....I
I l I
r ~ ' V ~ ~ V
NOV4 ~. S_'~,T .. .I . . P. S pT
g11136491191 ~. ~ T~TT w . C. . '.
$$ gi13413888~ ~. ~~~~ .. .I . . S~ ~ .T S
gi~21341351 .. .'. x w . . . . S ~ ~ .
g11144246391 '~ 'I "_.. m ~T ~ v ~~ _. ~ vT
g11100471651 ______________________________________.___________ ... .~.. .~.. .I..
NOV4 ..VN~TS'~'V~""~S_ E~IMI'YT. '~. ~T~ ~~
g11136491191 F;'PF'~-~ ' E LL'TAS ~~ ~.Q T~
g1134138881 I:P ~I~'2SI ' SF'YT ~~ ~.
65 g1 I 2134135 I T,SN'ST!F'~FC~u ~L ' 8 ML ~TAS ~~. T L
g11144246391 ~P IG~'~5~ ' SF~YT ~ ~.
gi110047165~ __________________________________________________ 70 ....I....I....~....~....I....I....~....~....I....I
6$

v. -i NOV4 E~ m m v m I
gi1136491191 ~ ~ ~HR m v m v gi134138881 gi121341351 ~ mL~ R m ~ m I
gi1144246391 v ~ y ~~ ~ v m L
gi1100471651 . I.. .I. .. .~....I .... I....I. .. .I.. I.... I.. ..I
1O NOV4 ~ ~ ~ y . ' ~M ~
v r n w ' r KPH
' gi1136491191 ~ ~ ~ yRQ S~P~
rr w gi134138881 ~ ~ ~ W ~
S

gi121341351 ~ ~ ~m Q ~
y gi114424639 ~ ~ ~ S ~

15 gi110047165~ ____ ____________________ ___________ _______________ NOV4 -... . .: ... .. . . . .

gi1136491191 1 ~ ~ ' ~

gi134138881 ~ ~ ~

gi121341351 ~ ~ 1 ~

gi1144246391 1 ~ ~ ~

gi1100471651 ____ ____________________ ___________ _______________ ?5 : ....I....~ .... ... .
-- : ....I 'I~ ,..
.-.I-..- J '~.

NOV4 ... . S . m .. ~ . , . S.
. ~ . _ ' v E' S -_ T
.,~

giI136491191 m ~ . S S~ ~
v r F

O gi134138881 ~ L, m . , gi121341351 ~ m ~ LS

gi1144246391 ~ _ L~ m v ~ ~
~

gi1100471651 ____ ____________________ . ______.________ .__________ I ..I.
.
I..
1._..I
...I.
.I_..
.
.

NOV4 S~ hGP ~CG~L ~L TL L
I ~ ~ K

gi1136491191 T I T S KH RQtL SL S~
n ~T --gi134138881 S Sx 2 Ev vR ~L L
la _ EQ~

4O gi 121341351 IT ~ iT E~ ~ RS~L DT 'QS
II Em ~ --~ ~

gi 1144246391 S S ~G ~ ~ ~~ __ EQ~ I

gi1100471651 ____ _ _ ______ , ___________ .
~
_____~__________,____ 45 Tables 4E-K list the domain description from DOMAIN analysis results against NOV4. This indicates that the NOV4 sequence has properties similar to those of other proteins known to contain this domain.
Table 4E. Domain Analysis of NOV4 gnl~Smart~smart00630, Sema, semaphorin domain (SEQ ID N0:76) CD-Length = 430 residues, 100.0 aligned Score = 226 bits (575), Expect = 1e-59 SO Query: 51 FNHLVVDERTGHIYLGAVNRIYKLSSDLKVLVTHETGPDEDNPKCYPPRIVQTCNEPLTT 110 +I++II I +I+Il II+I II +I +11l +I I
Sbjct: 1 LQNLLLDEDNGTLYVGARNRLYVLSLNLISEAEVKTGPVLSSPDCEECV----SKGKDPP 56 Query: 111 TNNVNKM-LLIDYKENRLIACGS-LYQGICKLLRLEDLFKLGEPYHKKEHYLSGVNESGS 168 55 I+ 1l + II+II + I+ II+ +I +I+I+ I +I +I I + + +
Sbjct: 57 TDCVNFIRLLLDYNADHLLVCGTNAFQPVCRLINLGNLDRL-EVGRESGRGRCPFDPQHN 115 Query:169 VFGVIVSYSNLDDKLFIATAVD--GKPEYFPTISSRKLTKNSEADGMFAYVFHDEFVASM226 I+I I +I++ I I 1 I + I + I

Sbjct:116 STAVLV-----DGELYVGTVADFSGSDPAIYRSLSVRRLKGT-------SGPSLRTVLYD163 S Query:227 IKIPSDTFTIIPDFDIYYVYGFSSGNFVYFLTLQPEMVSPPGSTTKEQVYTSKLVRLCKE286 + + + +1I I II+1111 + + + I++ I+II

Sbjct:164 SRWLN---------EPNFVYAFESGDFVYFFFRETAVEDENC----GKAWSRVARVCKN210 Query:287 DTA--------FNSYVEVpIGC---ERSGVEYRLLQAAYLSKAGAVLGRTLGVHPDDDLL335 1O I + I+++ + I +
IIII+I II+ II+I

SbjCt:211 DVGGPRSLSKKWTSFLKARLECSVPGEFPFYFNELQAAFLLPAGS---------ESDDVL261 Query:336 FTVFSKGQKRKMKSLDESALCIFILKQINDRIKERLQSCYRGEGTLDL----AWLKVKDI391 + III + II+I I I II I + I I +

IS SbjCt:262 YGVFSTSS----NPIPGSAVCAFSLSDINAVFNEPFKECETGNSQWLPYPRGLVPFPRPG317 Query:392 PC-----SSALLTIDDNFCGLDMNAPLGVSDMVRGIPVF--TEDRDRMTSV----IAYVY440 I II I I I + I I+I I+ +1I+ +

Sbjet:318 TCPNTPLSSKDLPDDVLNFIKTHPLMDEWQPLTGRPLFVKTDSNYLLTSIAVDRVRTDG377 Query:441 KNHSLAFVGTKSGKLKKTRVDGPRGN--ALQYETVQVVDPGPVLRDMAFSKDH

I+++ I+II I++ I+ + + ++ ~ + I III + I+
I

Sbjct:378 GNYTVLFLGTSDGRILKVVLSRSSSSSESWLEEISVFDPGSPVSDLVLSPKK

Table 4F. Domain Analysis of NOV4 gnllPfam~pfamD1403, Sema, Sema domain. The Sema domain occurs in semaphorins, which are a large family of secreted and transmembrane proteins, some of which function as repellent signals during axon guidance. Sema domains also occur in the hepatocyte growth factor receptor. (SEQ ID N0:77) CD-Length = 433 residues, 100.0~s aligned Score = 186 bits (471), Expect = 1e-47 Query: 51 FNHLVVDERTGHIYLGAVNRIYKLS----SDLKVLVTHETGPDEDNPKCYPPRIVQTCNE 106 1 I++II I +I+ll II+I I+ I++ I I I I +I
Sbjct: 1 FVTLLLDEDRGRLYVGARNRVYVLNLEDLSEVLNLKTGWPGSCETCEECNMKGKS----- 55 3O Query: 107 PLTTTNNVNKMLLTDYKENRLIACGS-LYQGICKLLRLEDLFKLGEPYHKKEHYLSGVNE 165 III I + +I I + I 1l+ +I +I I+ I III I + +
Sbjct: 56 PLTECTNFIR-VLQAYNDTHLYVCGTNAFQPVCTLINLGDLFSLDVDNEEDGCGDCPYDP 114 Query: 166 SGSVFGVIVSYSNLDDKLFIATAVDGKPEYFPTISSRKLTKNSEADGMFAYVFHDEFVAS 225 3S I+ I+I +I+ I +I + + + + III +
Sbjct: 1l5 LGNTTSVLVQ----GGELYSGTVID------FSGRDPSIRRLLGSHDGLRTEFHD---SK 161 Query: 226 MIKIPSDTFTIIPDFDIYYVYGFSSGNFVYFLTLQPEMVSPPGSTTKEQVYTSKLVRLCK 285 + +I+ + + I+II+ I I + III + + I I++ I+II
4O Sbjct: 162 WLNLPNFVDS----YPIHYVHSF-SDDKVYFFFRETAVEDSNCKT-----IHSRVARVCK 211 Query: 286 EDTAFNSYVEVPIG---------CERSGVE----YRLLQAAYLSKAGAVLGRTLGVHPDD 332 I II+I+ I I + IIII++ II I
Sbjct: 212 NDPGGRSYLELNKWTTFLKARLNCSIPGEGTPFYFNELQAAFVLPTGA---------DTD 262 Query: 333 DLLFTVFSKGQKRKMKSLDESALCIFILKQIND--RIKERLQSCYRGEGTLDLAWLKVKD 390 +I+ II+ I It+I I + Il + II + +
Sbjct: 263 PVLYGVFTTSS----NSSAGSAVCAFSMSDINQVFEGPFKHQSPNSKWLPYRGKVPQPRP 318 SO Query: 391 IPCSSA-LLTIDDNFCGLDMNAPLG--VSDMVRGIPVFTEDRDR-------MTSVIAYVY 440 I +I I + I+ II I + +I+I + I I
Sbjct: 319 GQCPNASGLNLPDDTLNFIRCHPLMDEWPPLHNVPLFVGQSGNYRLTSIAVDRVRAGDG 378 Query: 441 KNHSLAFVGTKSGKLKKIRVDGPRGNA-----LQYETVQW-DPGPVLRDMAFSKD 490 SS + +++ I+1 I++ I +I I ++ + I I I p I + 1 +
Sbjct: 379 QIYTVLFLGTDDGRVLK-QVVLSRSSSASYLVVVLEESLVFPDGEPVQRMVISSKN 433 Table 4G. Domain Analysis of NOV4 gnl~Pfam~pfam01833, TIG, IPT/TIG domain. This family consists o~ a domain that has an immunoglobulin like fold. These domains are found in cell surface receptors such as Met and Ron as well as in intracellular transcription factors where it is involved in DNA
binding. CAUTION: This family does not currently recognise a significant number of members. (SEQ ID N0:78) CD-Length = 85 residues, 98.8% aligned Score = 69.7 bits (169), Expect = le-l2 Query: 955 TLSDLKPSRGPMSGGTQVTITGTNLNAGSNWVMFGKQPCLFHRRSPSYIVCNTTSSDEV 1014 ++ + II II+I~II++1111+II +I ++ I II I + I III I
S Sbjct: 2 VITSISPSSGPLSGGTEITITGSNLGSGEDIKVTFGGTECDWSQEASQIVCKTPPYANG 61 Query: 1015 LEMKVSVQVDRA-KIHQDLVFQW 1037 I+I +I + I II
Sbjct: 62 GPQPVTVSLDGGGLSSSPVTFTYV 85 Table 4H. Domain Analysis of NOV4 gnl~Pfam~pfam01833, TIG, IPT/TIG domain. This family consists of a domain that has an immunoglobulin like fold. These domains are found in cell surface receptors such as Met and Ron as well as in intracellular transcription factors where it is involved in DNA
binding. CAUTION: This family does not currently recognise a significant number of members. (SEQ ID N0:79) CD-Length = 85 residues, 91.8% aligned Score = 54.3 bits (129), Expect = 6e-08 Query: 858 PRITEIIPVTGPREGGTKVTIRGENLGLEFRDIASHVKVAGVECSPLVDGYIPAEQIVCE 917 I II I I +1I III++II I III I I II + IIII+
IS Sbjct: 1 PVITSISPSSGPLSGGTEITITGSNLGSGED---IKVTFGGTECDWSQEA---SQIVCK 54 Query: 918 MGE-AKPSQHAGFVEICVAVCRPE 940 I I +
Sbjct: 55 TPPYANGGPQPVTVSLDGGGLSSS 78 Table 4I. Domain Analysis of NOV4 gnllPfam~pfam01833, TIG, IPT/TIG domain. This family consists of a domain that has an immunoglobulin like fold. These domains are found in cell surface receptors such as Met and Ron as well as in intracellular transcription factors where it is involved in DNA
binding. CAUTION: This family does not currently recognise a significant number of members. (SEQ ID NO:80) CD-Length = 85 residues, 100.0% aligned Score = 45.8 bits (107), Expect = 2e-O5 Query: 1040 PTIVRIEPEWSIVSGNTPIAWGTHLDLIQNPQIRAKHGGKEHINICEVLN--ATEMTCQ 1097 I ! I I +I! I I + I++I ++ ~+ II I I+I++ I+++ I+
2S Sbjct: 1 PVITSISPSSGPLSGGTEITITGSNLGSGED--IKVTFGGTE----CDWSQEASQIVCK 54 Query: 1098 APALALGPDHQSDLTERPEEFGFILDNVQSLLILNKTNFTYY 1139 I I I+ II I + III
Sbjct: 55 TPPYA---------NGGPQPVTVSLDGGG--LSSSPVTFTYV 85 Table 4J Domain Analysis of NOV4 gnl~Smart~smart00423, PSI, domain found in Plexins, Semaphorins and Integrins (SEQ TD N0:81) CD-Length = 47 residues, 100.0% aligned Score = 46.6 bits (1D9), Expect = le-OS
Query: 655 NCSVHNSCLSCVESPYR-CHWCKYRHVCTHDPKTCSFQEGRVKLPEDCP 702 II + II I+ + I II + II I II
1O Sbjct: 1 RCSAYTSCSECLLARDPYCAWCSSQGRCTSGE-RCDSLRQNW-SSGQCP 47 Table 4K. Domain Analysis of NOV4 gnl~Smartlsmart00429, IPT, ig-like, plexins, transcription factors (SEQ ID N0:82) CD-Length = 93 residues, 100.0% aligned Score = 57.8 bits (138), Expect = 6e-09 Query. 1039 DPTIVRIEPEWSIVSGNTPIAVWGTHLDLIQNPQIRAKHGGKEHINICEVLNATEMTCQA 1098 II I II I +1I I I + I +1I I + I + ++ I + I+
Sbjct: 1 DPVITRISPNSGPLSGGTRITLCGKNLDSISWFVEVGVGEVPCTFLPSDVSQTAIVCKT 60 Query: 1099 PALALGPDHQSDLTERPEEFGFILDNVQSLLILNKTNFTYY 1139 I I I I 1 + III
2O Sbjct: 61 PPYHNIP----GSVPVRVEVGLRNGGVPGE----PSPFTYV 93 Semaphorins are a large family of secreted or cell-bound signals, known to guide axons in developing nervous tissue. They are expressed in a variety of adult and embryonic tissues and are thought to have a broader spectrum of functions. Recent evidence suggests that semaphorins and their receptors play a key role in the control of cellular interactions, most likely in cell-cell repulsion (Tamagnone and Comoglio. Trends Cell Biol 2000 Sep;10(9):377-83.). A subset of semaphorins interacts with neuropilins - cell-surface molecules lacking a signalling-competent cytoplasmic domain. Another large family of transmembrane molecules, namely plexins, bind specifically to semaphorins. Thus plexins, alone, or in association with neuropilins, behave as fully functional semaphorin receptors. The intracellular responses elicited by plexins are unknown, but their large cytoplasmic moiety, containing the strikingly conserved sex-plexin (SP) domain, is likely to trigger novel signal-transduction pathways.

Chemorepulsive signals such as the semaphorins play an essential role in navigating axons over large distances in the developing nervous system. The effects of one of these repulsive cues, semaphorin 3A (Sema3A), are mediated by the membrane protein neuropilin-1 (Npn-1). Recent work has shown that neuropilin-1 is essential but not sufficient to form functional Sema3A receptors and indicates that additional components are required to transduce signals from the cell surface to the cytoskeleton (Rohm et al. Mech Dev 2000 May;93(1-2):95-104). It has been shown that members of the plexin family interact with the neuropilins and act as co-receptors for Sema3A. Neuropilin/plexin interaction restricts the binding specificity of neuropilill-1 and allows the receptor complex to discriminate between two different semaphorins. Deletion of the highly conserved cytoplasmic domain of Plexin-A1 or -A2 creates a dominant negative Sema3A receptor that renders sensory axons resistant to the repulsive effects of Sema3A when expressed in sensoxy ganglia. These data suggest that functional semaphorin receptors contain plexins as signal-transducing and neuropilins as ligand-binding subunits.
Class 1 and 3 semaphorins repulse axons but bind to different cell surface proteins.
Two known semaphorin-binding proteins, plexin 1 (Plex 1) and neuropilin-1 (NP-1), form a stable complex (Strittmatter. Cell 1999 Oct 1;99(1):59-69.). Plex 1 alone does not bind semaphorin-3A (Sema3A), but the NP-1/Plex 1 complex has a higher affinity for Sema3A
than does NP-1 alone. While Sema3A binding to NP-1 does not alter nonneuronal cell morphology, Sema3A interaction with NP-1/Plex 1 complexes induces adherent cells to round up. Expression of a dominant-negative Plex 1 in sensory neurons blocks Sema3A-induced growth cone collapse. Sema3A treatment leads to the redistribution of growth cone NP-1 and plexin into clusters. Thus, physiologic Sema3A receptors consist of NP-1/plexin complexes.
As mentioned previously, the semaphorin family of molecules contains members known to deliver guidance cues to migrating axons during development.
Semaphorins also have been identified on the surface of hematopoietic cells and, interestingly, in the genomes of certain Iytic viruses. Recent studies indicate that semaphorins bind with high affinity to at least two different receptor families and are biologically active on immune cells as well as neuronal cells (Spriggs. Curr Opin Immunol 1999 Aug;l1(4):387-91.).
The mammalian olfactory system is capable of discriminating among a large variety of odor molecules and is therefore essential for the identification of food, enemies and mating partners. The assembly and maintenance of olfactory connectivity have been shown to depend on the combinatorial actions of a variety of molecular signals, including extracellular matrix, cell adhesion and odorant receptor molecules (Pasterkamp et al. Cell Mol Biol Sep;45(6):763-79). Recent studies have identified semaphorins and their receptors as putative molecular cues involved in olfactory pathfmding, plasticity and regeneration.
Neuropilins were shown to serve as receptors for secreted class 3 semaphorins, whereas members of the plexin family are receptors for class 1 and V (viral) semaphorins.
In Drosophila, plexin A is a functional receptor for semaphorin-la. The human plexin gene family comprises at least nine members in four subfamilies (Goodman et al. Cell 1999 Oct 1;99(1):71-80.). Plexin-B1 is a receptor for the transmembrane semaphorin Sema4D
(CD100), and plexin-C1 is a receptor for the GPI-anchored semaphorin Sema7A
(Sema-Kl).
Secreted (class 3) semaphorins do not bind directly to plexins, but rather plexins associate with neuropilins, coreceptors for these semaphorins. Plexins are widely expressed:
in neurons, the expression of a truncated plexin-A1 protein blocks axon repulsion by Sema3A.
The cytoplasmic domain of plexins associates with a tyrosine kinase activity.
Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. Plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and trigger a novel signal transduction pathway controlling cell repulsion.
Plexin is a type I membrane protein which was identified in XeTaopus nervous system by hybridoma technique. Molecular cloning studies demonstrated that the extracellular segment of the plexin protein possesses three internal repeats of cysteine cluster which are homologous to the cysteine-rich domain of the c-met proto-oncogene protein product. A cell aggregation test revealed that the plexin protein mediated cell adhesion via a homophilic binding mechanism, in the presence of calcium ions (Fujisawa et al. Dev Neurosci 1997;19(1):101-5.). Plexin was expressed in the neuronal elements composing particular neuron circuits in Xefaopus CNS and PNS. These findings indicate that plexin is a new member of the Ca(2+)-dependent cell adhesion molecules, and suggest that the molecule plays an important role in neuronal cell contact and neuron network formation.
Plexin (previously referred to as B2) is a neuronal cell surface molecule that has been identified in Xenopus. cDNA cloning reveals that plexin has no homology to known neuronal cell surface molecules but possesses, in its extracellular segment, three internal repeats of cysteine clusters that are homologous to the cysteine-rich domain of the c-met proto-oncogene protein product. The exogenous plexin proteins expressed on the surfaces of L
cells by cDNA
transfection mediate cell adhesion via a homophilic binding mechanism, under the presence of calcium ions (Fujisawa. Neuron 1995 Jun;l4(6):l 189-99.). Plexin is expressed in the receptors and neurons of particular sensory systems. These findings indicate that plexin is a novel calcium-dependent cell adhesion molecule and suggest its involvement in specific neuronal cell interaction and/or contact.
The disclosed NOV4 nucleic acid of the invention encoding a Plexin -like protein includes the nucleic acid whose sequence is provided in Table 4A or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 4A while still encoding a protein that maintains its Plexin -like activities and physiological functions, or a fragment of such a nucleic acid.
The invention further includes nucleic acids whose sequences a.re complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids ox nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the rnodifred nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In the mutant or variant nucleic acids, and their complements, up to about 29%
percent of the bases may be so changed.
The disclosed NOV4 protein of the invention includes the Plexin -like protein whose sequence is provided in Table 4B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 4B while still encoding a protein that maintains its Plexin -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 40% percent of the residues may be so changed.
The protein similarity information, expression pattern, and map location for the Plexin-like protein and nucleic acid (NOV4) disclosed herein suggest that this NOV4 protein may have important structural and/or physiological functions characteristic of the Plexin family.
Therefore, the NOV4 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleie acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene deliverylgene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.

The NOV4 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neurodegeneration, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderrna, allergy, ARDS, Obesity, Metabolic Dysregulation, Infertility, andlor other pathologies. The NOV4 nucleic acids, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
NOV4 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies"
section below. For example, the disclosed NOV4 protein has multiple hydrophilic regions, each of which can be used as an imrnunogen. In one embodiment, a contemplated epitope is from about amino acids 20 to 30. In another embodiment, a NOV4 epitope is from about amino acids 180 to 190. In additional embodiments, NOV4 epitopes are from about amino acids 180 to 270, from about amino acids 310 to 320" from about amino acids 380 to 390, from about amino acids 400 to 405, from about amino acids 420 to 550, from about amino acids 620 to 630, from about amino acids 650 to 700, from about amino acids 790 to 900, from about amino acids 1040 to 1050, from about amino acids 1100 to 1120, from about amino acids 1220 to 1240, from about amino acids 1410 to 1420, from about amino acids 1450 to 1500, from about amino acids 1580 to 1600, from about amino acids 1620 to 1650, from about amino acids 1720 to 1730 and from about amino acids 1800 to 1900.
These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOVS
A disclosed NOVS nucleic acid of 1535 nucleotides (also referred to as GMAC027612 A) encoding a novel dopamine receptor-like protein is shown in Table SA. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 7-9 and ending with a TAA codon at nucleotides 1447-1449. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table SA, and the start and stop codons are in bold letters.
Table SA. NOVS Nucleotide Sequence (SEQ ID N0:14) CCCGAAATGCTGCCGCCAAGGAGCAACGACACCGCGTACCCGGGGCAGTTAGCGCTATACCA
GCAGCTGGCGCAGGGGAATGCCGTGGGGGGCTCGGCGGGGGCACCGCCACTGGGGCCCGTGC
AGGTGGTCACCGCCTGCCTGCTGACCCTACTCGTCATCTGGACCTTGCTGGGCAACGTGCTG
GTGTCCGCAGCCATCGTGTGGAGCCGCCACCTGCGCGCCAAGATGACCAACGTCTTCATCGT
GTCTCTACCTGTGTCAGACCTCTTCGTGGCGCTGCTGGTCATGTCCTGGAAGGCAGTCGCCG
AGGTGGCCGGTTACTGGCCCTTTGAAGCGTTCTGCGACGTCTGGGTGGCCTTCGACATCATG
TGCTCCACCGCCTCCATCCTGAACCTGTGCGTCATCAGCGTGGCCCGCTACTGGGCCATCTC
CAGGCCCTTCCGCTACGAGCGCAAGATGACCCAGCGCATGGCCTTGGTCATGGTCCGCCCGG
CCTGGACCTTGTCCAGCCTCATCTCCTTCATTCCGGTCCAGCTCAACTGGCACAGGGACCAG
GCGGTCTCTTGGGGTGGGCTGGACCTGCCAAACAACCTGGCCAACTGGACGCCCTGGGAGGA
GGCCGTTTGGGAGCCCGACGTGAGGGCAGAGAACTGTGACTCCAGCCTGAATCGAACCTACG
CCATCCCTTCCTCGCTCATCAGCTTCTACATCCCCATGGCCATCATGATCGTGACCTACACG
CGCATCTACCGCATCGCCCAGGTGCAGATCCGCAGGATTTCCTCCCTGGAGAGGGCCGCAGA
GCACGTGCAGAGCTGCCGGAGCAGCGCAGGCTGCACGCCCGACACCAGCCTGCGGTTTTCCA
TCAAGAAGGAGACCGAGGTTCTCAAGACCCTGTCGGTGATCATGGGGGTCTTCGTGTGTTGC
TGGCTGCCCTTCTTCATCCTTAACTGCATGGTTCCTTTCTGCAGTGGACACCCCAAAGGCCC
TCCGGCCGGCTTCCCCTGCGTCAGTGAGACCACATTCGATGTCTTCATCTGGTTCTGCTGGG
CCAACTCCTCACTCAACCCAGTCCCCAGTCACTATGCCTTCAACGCCGACTTCCGGAAGGTG
TTTGCCCAGCTGCTGGGGTGCAGCCACGTCTGCTCCCGCACGCCGGTGGAGACGGTGAACAT
CAGCAATGAGCTCATCTCCTACAACCAAGACACGGTCTTCCACAAGGAAATCGCAGCTGCCT
ACATCCACATGATGCCCAACGCCATTCCCCCCGGGGACCGGGAGGTGGACAACGATGAGGAG
GAGGAGAGTCCTTTCGATCGCATGTCCCAGATCTATCAGACATCCCCAGATGGTGACCATGT
TGCAGAGTCTGTCTGGGAGCTGGACTGCGAGGGGGAGATTTCTTTAGACAAAATAACACCTT
TCACCCCAAATGGATTCCATTAAACTGCATTAAGAAACCCCCTCATGGATCTGCATAACCAC
ACAGACATTGACAAGCATGCACACACAAGCAAATACATGGCTTTCCA
The NOVS nucleic acid was identified on chromosome 4 and has 1494 of 1536 bases (97%) identical to a Human dopamine receptor (DS) transcribed pseudogene mRNA
from Homo sapieras (GENBANK-ID: M75867) (E = 0.0) A disclosed NOVS polypeptide (SEQ ID NO:15) encoded by SEQ ID N0:14 is 480 amino acid residues and is presented using the one-letter code in Table SB.
Signal P, Psort andlor Hydropathy results predict that NOVS has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6400. In other embodiments, NOVS
may also be localized to the Golgi body with acertainty of 0.4600, the endoplasmic reticulum (membrane) with a certainty of 0.3700, or the endoplasmic reticulum (lumen) with a certainty of 0.1000.
The most likely cleavage site for a NOVS peptide is between amino acids 63 and 64, at: VSA-AI.
Table SB. Encoded NOVS protein sequence (SEQ m NO:15) MLPPRSNDTAYPGQLALYQQLAQGNAVGGSAGAPPLGPVQVVTACLLTLLVIWTLLGNVLVSAAIVWSRHLR
AKMTNVFIVSLPVSDLFVALLVMSWKAVAEVAGYWPFEAFCDVWVAFDIMCSTASILNLCVISVARYWAISR
PFRYERKMTQRMALVMVRPAWTLSSLISFIPVOLNWHRDQAVSWGGLDLPNNLANWTPWEEAVWEPDVRAEN

CDSSLNRTYAIPSSLISFYIPMAIMIVTYTRIYRIAQVQIRRISSLERAAEHVQSCRSSAGCTPDTSLRFSI
KKETEVLKTLSVIMGVFVCCWLPFFILNCMVPFCSGHPKGPPAGFPCVSETTFDVFIWFCWANSSLNPVPSH
YAFNADFRKVFAQLLGCSHVCSRTPVETVNISNELISYNQDTVFHKEIAAAYIHMMPNAIPPGDREVDNDEE
EESPFDRMSQIYQTSPDGDHVAESVWELDCEGEISLDKITPFTPNGFH
The disclosed NOVS amino acid sequence has 437 of 480 amino acid residues (91 %) identical to, and 446 of 480 amino acid residues (92%) similar to, the 477 amino acid residue DOPAMINE RECEPTOR (D(5) DOPAMINE RECEPTOR) (D1BETA DOPAMINE
RECEPTOR) protein from Homo sapieras (Human) (P21918) (E = 3.3e-237).
NOVS is expressed in at least the following tissues: fetal heart, pooled human melanocyte, fetal heart, and pregnant uterus. TaqMan data for NOVS is shown below in Example 2.
NOVS also has homology to the amino acid sequences shown in the BLASTP data listed in Table 5C.
Table 5C. BLAST
results for NOVS

Gene Index/ Protein/ LengthIdentityPositivesExpect Organism Identifier (aa) (%) (%) 0 dopamine 477 437/480446/480 0.0 gi~4503391Iref~NPreceptor - D5; Dopamine (91%) (92%) 00789.11 receptor [Homo sapiens]

0 D(1B) DOPAMINE475 376/480403/480 O.D
gi16978781~ref~NP

- RECEPTOR (78%) (83%) 36900.11 (D(5) DOPAMINE

RECEPTOR) gi~1169230Isp~P4229D(1B) DOPAMINE457 299/445341/445 e-158 O1DBDR XENLA RECEPTOR (67%) (76%) (D(5) DOPAMINE

RECEPTOR) gi~1362719~pir~~H55dopamine 486 321/483353/483 e-157 receptor 886 D1B - chicken (66~) (72%) gi~1518040~gb~AAC60dopamine 458 297/446346/446 e-154 070.11 receptor (66%) (76%) [Anguilla anguilla]

The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 5D.
Table SD Clustal W Sequence Alignment 1) NOV5 (SEQ TD NO:15) 2) giI45033911ref1NP 000789.11 dopamine receptor D5; Dopamine receptor D1B
[Homo sapiens] (SEQ ID N0:47) 3) gi169787811ref1NP 036900.11 D(1B) DOPAMINE RECEPTOR (D(5) DOPAMINE
RECEPTOR) (SEQ ID N0:48) 4) gi111692301sp1P422901DBDR_XENLA D(1B) DOPAMINE RECEPTOR (D(5) DOPAMINE
RECEPTOR) (SEQ ID N0:49) 5) gi113627191pir11B55886 dopamine receptor D1B - chicken (SEQ ID NO:50) 6) gi~15180401gb1AAC60070.11 dopamine D1B receptor [Anguilla anguilla] (SEQ TD
N0:51) I I....I,.. .1....p ...1....I.. .I... ..I
NOVS .LP RS~DTAYP-GQL .YQQLAQ V ~'AGAPPL.--P ~. ..C
gi145033911 LP GSIyG~AYP-GQF~YQQLAQG~AV~'~AGAPPL~--PST C
gi169787811 LP GR~R~AQP-ARL QRQLAQVI7AP SA--TPL --P
gi111692301 YQ~FQHLD~--DQVASWQSPEMLf~KSVSRESQRRICELVA ~I GS
gi113627191 ------MLRG---GRSP~PPP---AGPPGe~'iRGQAGA~-- ~ GS
gi115180401 ~GS~AKYL~UHETQSVPFFIGEIMWI~TSESA~EKTDGKKELIVRT GC

v....1-. .I.. .I.. .I._..I....I....I....I....I....I
1~ ~ I~ ~ v t NOVS Z S~~ S~ \ P ~ S
gi145033911 ~ I ~S~ V
gi169787811 ~ .. .S~ , IS gi111692301 L F F I T~ '~F~ ~ R ~ L
gi113627191 L F ~Y~ ~ L ~
gi~15180401 5 L I S~VLTCF~ ~T ~

.1....1....1....1....1.... .... ....I.. .1....I
y.W ~n i V
NOV5 ~ E~
gi145033911 ~ ~ w gi169787811 T W ~ I ~~
gi111692301 y ~ ~~ S~
gi113627191 ~ Q ~ ~~ S~
gi115180401 P ~ ~ ~ T ~~ S~

.I.. .."I.. .I.. .I.. ....~......I....I
y n r NOVS ~~ . ;RP~ . S.. ~ ~ . ~D3~5AVWGG----L-gi14503391) v~ ,~G~~ T ~ v DQAA;~WGG--__L__ gi169787811 v~ 'GL~ 2 ~ WCAGQG________ V~ ~.
gi11169230i ~~ v~ IST~ ~ v ~ KS~-__ _________ gi113627191 w ~ ~G ~ ~ GGDA~TAAAAGDIED
giI1518D40I ~' ;,F IS'~~ ~ BAS------------....I....I....I....1....1....1....1....1....1....1 NOVS ____________________D__________LP PEE~~'I',,'VWEPD
40 gi145033911 ____________________D__________LP P&~E~FWEPD
gi169787811 _______________________________Q GL S~G~PEil~~G-WELE
gi111692301 _______________________________T LNHSTG-______ gi113627191 GFDTGWEAAGAFTTWAEDMSTTWVALAAMTPS GTSGNNTVPGP----gi115180401 _______________________________D EVP7I~G~S~'G~K-____ ..,,.,.I.... .... .... .... ..... .... ....I.... ....I
~
NOV5 V ' ~ P ~ v v gi145033911 ~ ~ ~v v gi169787811 GRT ~
gi111692301 ___ , ~~~, T T
gi113627191 ==~ S ~
gi115180401 ~ E ~ ~~T

I I .... ....1....I
.... .... ....1....1....I.... ....I..
V y ~ v NOV5 ~ ' Fa'AGCT----PD F E
gi145033911 ~~ SAACA----PD
gi~69787811 ~~ ' RGAYE----PDP F
gi~11692301 ~~ RVDSCSRHHQ ~T T
gi113627191 ~~ ~CI3HVDC---HHH EMS 12 I
gi I 15180401 ~ ~ '~'~RLEC---QHHN~' I~T I F

.I... ~, NOV5 S=GK.~P~IF~. n..1 .C.~
gi145033911 S G~E ~P~ F~
gi169787811 SSGDAE ~K F~ ~I
gi111692301 -RS G ~Q~ L~ ~I
gi113627191 E-SP~SD~ ~ L~ I

gi I 1518040 I ~T~D-RPOTDHTF~~eL~DD~STi~

S NOV5 tops ~ ~ ~ ~ . Q ~ . . 5 . . . . I . ,.
v v gi145033911 -_ ., .Q S ..I' ~r r gi169787811 ~~ ~ ~Q S F~ ~ Q m v giI11692301 ~~ ~ SS m L
gi113627191 m ~ S GQ S ~ m -gi115180401 m ~ ~S _ m L

NOVS ~'a.:.. I PPGDRF-,STD E-EES~n ~.I~'YQISP~ DHVA
1S gi145033911 I '~TPGNR~VDE ~- ~~ Ft YQ SiP~Gi~PVA
gi169787811 _SSGDR"P-yVG~E E- ~ ~ ~ P P~GI~L
gi111692301 ~CIv ~ ~ v Q ~ L
I 7~CEEN ~ ~~ ~ P- -~P TD
gi11362719 gillslso4ol 11- It~cl,n --T .~z ~F H- -~~IT

.I.. .I.. .~.- ,~
NOV5 =W .~.- D F~-=-r gi145033911 W ~ - D ~ F~- -gi169787811 ~W ~ - E -' S~L ' CFDKTA
gi111692301 MC ~S- ~SMS I,~~y ---gi113627191 ~uC ~ - L~-=-gi 11518040 1 ~C~ ADC C D~. Ll~Af- -Tables SE list the domain description from DOMAIN analysis results against NOVS.
This indicates that the NOVS sequence has properties similar to those of other proteins known to contain this domain.
Table SE. Domain Analysis of NOVS
gnllPfam~pfam00001, 7tm 1, 7 transmembrane receptor (rhodopsin family). (SEQ ID N0:83) CD-Length = 254 residues, 99.2% aligned Score = 188 bits (478), Expect = 5e-49 3S Query: 57 GNVLVSAAIVWSRHLRAKMTNVFIVSLPVSDLFVALLVMSWKAVAEVAGYWPF-EAFCDV 115 II+II I+ ++ II II+I+++I I+II I + I I I I I +I I +
Sbjct: 1 GNLLVILVILRTKKLR-TPTNIFLLNLAVADLLFLLTLPPWALYYLVGGDWVFGDALCKL 59 Query: 116 WVAFDIMCSTASILNLCVISVARYWAISRPFRYERKMTQRMALVMVRPAWTLSSLISFIP 175 I ++ IIII I II+ II II I II I I I I 1++ I I+ I+I I
Sbjct: 60 VGALFVVNGYASILLLTAISIDRYLAIVHPLRYRRIRTPRRAKVLILLVWVLALLLSLPP 119 Query: 176 VQLNWHRDQAVSWGGLDLPNNLANWTPWEEAVWEPDVRAENCDSSLNRTYATPSSLISFY 235 + +I I I + + I+ I+I + I+I+ I
4S Sbjct: 120 LLFSWLR------------TVEEGNTTVCLTDFPEE--------SVKRSYVLLSTLVGFV 159 SO
Query: 236 IPMAIMIVTYTRIYRIAQVQIRRISSLERAAEHVQSCRSSAGCTPDTSLRFSIKKETEVL 295 +I+ +++I IIII I + + I II+ I +
Sbjct: 160 LPLLVILVCYTRILRTLRKRARSQ----------------------RSLKRRSSSERKAA 197 Query: 296 KTLSVIMGVFVCCWLPFFILNCMVPFCSGHPKGPPAGFPCVSETTFDVFIWFCWANSSLN 355 I I I++ III IIII+ I+ + I I I + +I + II II
Sbjct: 198 KMLLVVWVFVLCWLPYHIVLLLDSLC-------LLSIWRVLPTALLITLWLAYVNSCLN 250 SS Query: 356 PV 357 I+

Sbjct: 251 PI 252 NOVS also has homology to proteins found in the pate patent database as shown in Table SE.
Table SE. BLAST
results for NOVS for patp Database Gene Index/ Protein/ LengthIdentityPositivesExpect Identifier Organism (aa) (%) (%) patp:AAR22546 Truncated 479 453/480460/480 1.7e-Dopamine (94%) (95%) 244 receptor encoded by pseudogene clone GL-39 Homo Sapiens patp:AAR'79381 Dopamine 477 436/48045/480 2.Oe-receptor D5 - Homo (90%) (92%) 236 Sapiens The rhodopsin-like GPCRs themselves represent a widespread protein family that includes hormone, neurotransmitter and light receptors, all of which transduce extracellular signals through interaction with guanine nucleotide-binding (G) proteins.
Although their activating ligands vary widely in structure and character, the amino acid sequences of the receptors are very similar and are believed to adopt a common structural framework comprising 7 transmembrane (TM) helices.
G-protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions (including various autocrine, paracrine and endocrine processes). They show considerable diversity at the sequence level, on the basis of which they can be separated into distinct groups. The term clan is used to describe the GPCRs, as they embrace a group of families for which there are indications of evolutionary relationship, but between which there is no statistically significant similarity in sequence.
The currently known clan members include the rhodopsin-like GPCRs, the secretin-like GPCRs, the cAMP
receptors, the fungal mating pheromone receptors, and the metabotropic glutamate receptor family.
The diverse physiologic actions of dopamine are mediated by its interaction with 2 types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Three groups reported the cloning of the D1 dopamine receptor gene (Dearry et al., 1990 ; Zhou et al., 1990 ; Sunahara et al., 1990 ). The gene encodes a pxotein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Northern blot analysis and ifa situ hybridization showed that the mRNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart (Dearry et al., 1990 ). Sunahara et al. (1990) reported that the DRD1 gene is intronless and, by Southern blot hybridization to DNAs from a hybrid cell panel, they mapped the gene to chromosome 5. Family linkage studies confirmed this assignment and suggested that it is in the same general region as the gene for glucocorticoid receptor and DSS22, a marker about 12 cM from GRL. This places it in the Sq31-q34 region near the structurally homologous genes for beta-2-adrenergic receptor and alpha-1-adrenergic receptor. Using pulsed Eeld gel electrophoresis and a range of different restriction enzyme digests, Boultwood et al. (1991) established that GRL and DRD1 are on the same 300-kb genomic DNA
fragment.
Grandy et al. (1990) used the recently cloned DRD1 gene to map the locus to chromosome 5 in rodent-human somatic cell hybrids. Fluorescence in situ hybridization refined the localization to Sq35.1. A 2-allele EcoRI RFLP associated with DRD1 allowed confirmation of the localization by linkage analysis in CEPH families. The homologous gene in the mouse is located on chromosome 13.
The distal end of Sq, Sq31.1-qter, contains the genes for 2 adrenergic receptors, ADRB2 and ADRA1B and the dopamine receptor type 1A gene. Krushkal et al.
(1998) used an efficient discordant sib-pair ascertainment scheme to investigate the impact of this region of the genome on variation in systolic blood pressure in young Caucasians. They measured 8 highly polymorphic markers spanning this positional candidate gene-rich region in 427 individuals from 55 3-generation pedigrees containing 69 discordant sib pairs, and calculated multipoint identity by descent probabilities. The results of genetic linkage and association tests indicated that the region between markers DSS2093 and DSS462 was significantly linked to 1 or more polymorphic genes influencing interindividual variation in systolic blood pressure levels. Since the ADRA1B and DRD1A genes axe located close to these markers, the data suggested that genetic variation in 1 or both of these G protein-coupled receptors, which participate in the control of vascular tone, plays an important role in influencing interindividual variation in systolic blood pressure levels The disclosed NOVS nucleic acid of the invention encoding a Dopamine receptor -like protein includes the nucleic acid whose sequence is provided in Table SA or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table SA while still encoding a protein that maintains its Dopamine receptor -like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlirniting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications axe carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
The disclosed NOVS protein of the invention includes the Dopamine receptor -like protein whose sequence is provided in Table SB. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table SB while still encoding a protein that maintains its Dopamine receptor -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 34% percent of the residues may be so changed.
The NOVS nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in schizophrenia, and other dopamine-dysfunctional states, Hypertension, Huntington's disease, levodopa-induced dyskinesias, alcoholism, Diabetes Insipidus and Mellitus with Optic Atrophy and Deafness, Wolfram Syndrome and/or other pathologies and disorders. For example, a cDNA encoding the dopamine receptor-like protein may be useful in gene therapy, and the dopamine receptox-like protein may be useful when administered to a subj ect in need thereof. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from schizophrenia, and other dopamine-dysfunctional states, Hypertension, Huntington's disease, levodopa-induced dyskinesias, alcoholism, Diabetes Insipidus and Mellitus with Optic Atrophy and Deafliess, Wolfram Syndrome, as well as other diseases, disorders and conditions. The NOVS nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
NOVS nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies"
section below. For example the disclosed NOVS protein have multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated NOVS

epitope is from about amino acids 1 to 10. In other embodiments, NOVS epitope is from about amino acids 125 to 150, from about amino acids 175 to 230, from about amino acids 250 to 300, from about amino acids 320 to 330, from about amino acids 350 to 370, from about amino acids 380 to 410, or from about amino acids 420 to 460. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.

A disclosed NOV6 nucleic acid of 2657 nucleotides (also referred to as GM523 e-1 A) encoding a novel Metabotropic Glutamate Receptor-like protein is shown in Table 6A. An open xeading frame was identified beginning with an ATG
initiation codon at nucleotides 6-8 and ending with a TGA codon at nucleotides 2643-2645. A
putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 6A, and the start and stop codons are in bold letters.
Table 6A. NOV6 Nucleotide Sequence (SEQ ID N0:16) GATTCATGAAGATGTTGACAAGACTACAAGTTCT'1'ATGT'1'AGCT'1"1'GT'L"1"1'CAAAGCUCUA'1"1"1"
1"1'AU'1'C:'1'C
TTTAGGAGATCACAACTTTATGAGGAGAGAAATTAAAATAGAAGGAGACCTTGTTTTAGGGGGCTTATTT
CCTATTAATGAAAAAGGCACTGGAACTGAAGAGTGTGGACGAATCAATGAAGACAGAGGTATCCAACGCC
TGGAGGCCATGTTGTTTGCCATTGATGAAATCAACAAAGACAATTACTTGCTTCCAGGAGTGAAGCTGGG
GGTTCACATTTTGGATACATGTTCAAGAGACACCTATGCATTAGAGCAGTCACTGGAGTTTGTCAGAGCA
TCGTTGACTAAAGTGGATGAAGCTGAATATATGTGTCCTGATGGATCATATGCTATTCAAGAAAACATCC
CACTACTCATTGCAGGAGTCATTGGCGGTTCGTACAGCAGTGTTTCCATACAGGTAGCAAACCTGCTGAG
GCTCTTCCAGATCCCTCAGATAAGCTACGCCTCCACCAGTGCCAAACTCAGCGACAAATCGCGCTATGAT
TATTTTGCCAGGACCGTGCCCCCTGACTTCTACCAGGCCAAAGCCATGGCCGAGATCTTGCGCTACTTTA
ACTGGACCTATGTGTCCACTGTTGCCTCTGAAGGTGACTATGGGGAGACAGGGATTGAGGCCTTCGAGCA
GGAAGCAAGGCTACGCAACATCTGCATCGCCACTGCTGAAAAGGTGGGGCGCTCCAACATCCGCAAGTCC
TACGACAGCGTGATCCGTGAGCTCCTGCAGAAACCTAACGCGCGAGTTGTGGTCCTGTTCATGCGCAGTG
ATGACTCACGAGAGTTGATCGCTGCAGCCAGCCGCGTGAATGCTTCCTTCACCTGGGTGGCCAGCGATGG
CTGGGGTGCACAGGAGAGCATTGTCAAGGGCAGTGAGCACGTCGCCTATGGAGCCATCACCCTGGAGCTG
GCGTCCCACCCTGTTCGTCAGTTTGATCGCTACTTCCAGAGCCTCAACCCCTACAACAATCATCGTAACC
CCTGGTTCCGAGACTTCTGGGAGCAGAAGTTCCAGTGCAGCCTCCAGAACAAGAGAAACCACAGACAGAT
TTGTGACAAGCACCTGGCCATTGACAGCAGCAACTATGAACAAGAATCCAAGATCATGTTTGTGGTGAAT
GCAGTGTATGCCATGGCGCATGCGCTGCACAAAATGCAACGCACCCTCTGTCCCAACACCACCAAGCTCT
GTGATGCAATGAAGATCCTGGATGGAAAGAAGTTGTACAAAGATTATTTGCTGAAAATCAACTTCCTTGC
TCCATTCAACCCAAATAAAGGAGCAGACAGCATTGTGAAGTTTGACACTTACGGAGACGGGATGGGAAGA
TACAACGTGTTCAACTTCCAGCATATAGGTGGAAAGTATTCCTACTTAAAAGTTGGCCACTGGGCAGAAA
CTTTATATCTAGATGTGGACTCTATTCATTGGTCCCGGAACTCAGTCCCCACTTCCCAGTGCAGTGATCC
CTGTGCCCCCAATGAAATGAAAAACATGCAGCCAGGAGATGTTTGCTGCTGGATCTGCATCCCATGTGAG
CCCTATGAATACCTGGTTGATGAGTTCACCTGCATGGATTGTGGCCCTGGCCAGTGGCCCACTGCAGACC
TATCTGGATGCTACAACCTTCCAGAGGATTACATCAGGTGGGAAGATGCCTGGGCAATAGGCCCAGTCAC
TATTGCCTGCCTGGGTTTTATGTGTACATGCATAGTCATAACTGTTTTTATCAAGCACAACAACACACCC
TTGGTCAAAGCATCAGGCCGAGAACTCTGCTACATCTTGTTATTTGGAGTTAGCCTGTCCTATTGCATGA
CATTCTTCTTCATTGCTAAGCCATCGCCTGTCATCTGTGCATTGCGCCGACTTGGGCTTGGGACCTCCTT
TGCCATCTGTTATTCAGCTCTCCTGACCAAGACAAACTGCATCGCTCGCATCTTTGATGGGGTCAAGAAT
GGCGCTCAGAGGCCAAAATTCATCAGCCCCAGTTCTCAGGTTTTTATCTGCCTGGGTTTGATACTGGTGC
AAATTGTGATGGTGTCTGTGTGGCTTATCTTGGAGACTCCAGGTACTAGAAGATACACCCTGCCAGAGAA
GCGGGAAACAGTCATCCTAAAATGCAATGTCAAAGATTCCAGCATGTTGATCTCTCTGACCTATGACGTG
GTTCTGGTGATTCTATGCACTGTGTATGCCTTCAAAACAAGGAAGTGTCCTGAAAACTTCAATGAAGCCA
AGTTCATAGGCTTCACCATGTACACCACCTGCATCATCTGGTTGGCATTCCTCCCTATATTTTATGTGAC
8l ATCAAGTGACTACAGAGTACAGACGACAACAATGTGCATCTCCGTTAGCTTGAGTGGTTTCGTGGTCTTG
GGCTGTTTGTTTGCCCCCAAGGTGCACATTGTCCTGTTCCAACCCCAGAAGAATGTGGTCACACACAGAC
TTCACCTCAACAGGTTCAGTGTCAGTGGAACTGCGACCACATATTCTCAGGCCTCTGCAAGCACGTATGT
GCCAACGGTGTGCAATGGGCGGGAAGTCCTCGACTCCACCACCTCATCTCTGTGATTGTGAATTGCA
The disclosed NOV6 nucleic acid sequence has 2522 of 2658 bases (94%) identical to a Rat metabotropic glutamate receptor 3 mRNA from Rattus norvegicus (GENBANI~-ID:
M92076) (E = 0.0).
A disclosed NOV6 polypeptide (SEQ ID N0:17) encoded by SEQ ID N0:16 is 879 amino acid residues and is presented using the one-letter amino acid code in Table 6B. Signal P, Psort andlor Hydropathy results predict that NOV6 contains a signal peptide and is likely to be localized in the endoplasmic reticulum (membrane) with a certainty of 0.6850. In other embodiments, NOV6 is also likely to be localized to the plasma membrane with a certainty of 0.6400, to the Golgi body with a certainty of 0.4600, or to the endoplasmic reticulum (lumen) with a certainty of 0.1000. The most likely cleavage site for a NOV6 peptide is between amino acids 24 and 25, at: SLG-DH.
Table 6B. Encoded NOV6 protein sequence (SEQ ID N0:17).
MKMLTRLQVLMLALFSKGFLVSLGDHNFMRREIKIEGDLVLGGLFPINEKGTGTEECGRINEDRGIQRLEAMLFA
IDEINKDNYLLPGVKI~GVHILDTCSRDTYALEQSLEFVRASLTKVDEAEYMCPDGSYAIQENIPLLIAGVIGGSY
SSVSIQVANLLRLFQIPQISYASTSAKLSDKSRYDYFARTVPPDFYQAKAMAEILRYFNWTYVSTVASEGDYGET
GIEAFEQEARLRNICIATAEKVGRSNIRKSYDSVIRELLQKPNARVVVLFMRSDDSRELIAAASRVNASFTWVAS
DGWGAQESIVKGSEHVAYGAITLELASHPVRQFDRYFQSLNPYNNHRNPWFRDFWEQKFQCSLQNKRNHRQICDK
HLATDSSNYEQESKIMFVVNAVYAMAHALHKMQRTLCPNTTKLCDAMKILDGKKLYKDYLLKINFLAPFNPNKGA
DSIVKFDTYGDGMGRYNVFNFQHIGGKYSYLKVGHWAETLYLDVDSIHWSRNSVPTSQCSDPCAPNEMKNMQPGD
VCCWICIPCEPYEYLVDEFTCMDCGPGQWPTADLSGCYNLPEDYIRWEDAWAIGPVTIACLGFMCTCIVITVFIK
HNNTPLVKASGRELCYILLFGVSLSYCMTFFFIAKPSPVICALRRLGLGTSFAICYSALLTKTNCIARIFDGVKN
GAQRPKFISPSSQVFICLGLILVQIVMVSVWLILETPGTRRYTLPEKRETVILKCNVKDSSMLISLTYDWLVIL
CTVYAFKTRKCPENFNEAKFIGFTMYTTCIIWLAFLPIFYVTSSDYRVQTTTMCISVSLSGFVVLGCLFAPKVHI
I5 The disclosed NOV6 amino acid sequence has 877 of 879 amino acid residues (99%) identical to, and 878 of 879 amino acid residues (99%) similar to, the 879 amino acid residue METABOTROPIC GLUTAMATE RECEPTOR 3 PROTEIN protein from Mus musculus (Mouse (Q9QYS2) (E = 0.0).
NOV6 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 6C.
Table 6C. BLAST results for NOV6 Gene Index/ Protein/ Organism Length Identity Positives Expect Identifier (aa) (%) - I (%) gi~400254~sp~P31422METABOTROPIC 879 864/879874/879 0.0 ~MGR3 RAT GLUTAMATE (98%) (99%) PRECURSOR

gii6288800~gb~AAF06metabotropic 879 877/879878/879 D.0 741.1~AF1707D1glutamate (99%) (99%) receptor 3 protein [MUs musculus]

gi~11279202~pir~~JCmetabotropic 879 875/879876/879 0.0 7160 glutamate (99%) (99%) receptor subtype 3 precursor -mouse The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 6D.
Table 6D Information for the ClustalW proteins S Z) NOV6 (SEQ ID N0:17) 2) gi~400254~sp~P31422~MGR3 RAT METABOTROPIC GLUTAMATE RECEPTOR 3 PRECURSOR
(SEQ ID
N0:52) 3) giI6288800~gb~AAF06741.1~AF170701_1 metabotropic glutamate receptor 3 protein [Mus musculus] (SEQ ID N0:53) 4) gi~11279202~pir~~JC7160 metabotropic glutamate receptor subtype 3 precursor -mouse (SEQ ID N0:54) _I____I_._I.._1____1____1..._1....1__..1....1 . . .
NOV6 t v r v gi~ 4002541 .I L . .' i gi~ 62888001 '. . .

gi~ 11279202~ '. .t .

....~....~.... .... ....~.... .... .... .... ....
NOV6 . ' ~ .' .~ . . ~~ .
gi~400254~ .' .' . . 'I' .
gi~62888001 .~ .~ . . .
gi~11279202~ R .' .~ . . ~ .

I___I_._I..__1____1____1..__1_...1__..1....1 NOV6 'W v v o ~ v 30 4002541 '. . . ' . .
gi~

giI 6288800~'. . . . .

gi~ 11279202~'. . . . .

35 .... .... .... .... .... .... .... .... .... ....
NOV6 W ~v v ~v ~ ~ t~~~ v~w gi~400254~ . ~ ~ ~. . . ~~. .~
gi~62888001 . ~ ~ ~~ . . ~~. .~
gi~11279202~ . ~ ~ ~~ . . ~~. .~

....~.... .... ....~.... .... ....~....~....~....
NOV6 ~~' ~
gi~400254~ 'F ~ ~ ~
gi~6z8saoo~ ~ ~ ~
gi~11279202~ ~ ~

..,.
NOV6 ~ ~ ~ .. ' gi14D02541 gi162888001 gi111279202!

.,. .1..

v~~v g114D0254!
gi162888001 v~w .... ....1.... .... .... ....

1S 8i1400254!
gi162888DO1 8i111279202! v Table 6E-F lists the domain description from DOMAIN analysis results against NOV6.
This indicates that the NOV6 sequence has properties similar to those of other proteins known to contain this domain.
Table 6E. Domain Analysis of NOV6 gnl~Pfam~pfam01094, ANF_receptor, Receptor family ligand binding region. This family includes extracellular ligand binding domains of a wide range of receptors. This family also includes the bacterial amino acid binding proteins of known structure. (SEQ ID N0:84) CD-Length = 402 residues, 98.5% aligned Score = 323 bits (827), Expect = 3e-89 2S Query: 62 EDRGIQRLEAMLFAIDEINKDNYLLPGVKLGVHILDTCSRDTYALEQSLEFVRASLTKVD 121 III 111111 I I II 1 Illl+ II+ III I Illll II I I I
Sbjct: 6 AVRGITRLEAMLGAFDRINADPALLPGLALGLAILDINSLRNVALEQSFTFVYGLLIKCD 65 Query: 122 EAEYMCPDGSYAIQENIPLLIAGVIGGSYSSVSIQUANLLRLFQIPQISYASTSAKLSDK 181 + I I I+ + +I I 1 III I II III II+ +1111 Sbjct: 66 CSSVRCAGGDLALTHGVAGVIGPSCSSSAIQV----ANLASLLNIPMISYGSTAPELSDK 121 Query: 182 SRYDYFARTVPPDFYQAKAMAEILRYFNWTYVSTVASEGDYGETGIEAFEQEARLRNICI 241 +11 I+II+I I +I II +I ++III III I I+I III I 111 + I I II
3S Sbjct: 122 TRYPTFSRTIPSDAFQGLAMVDIFKHFNWNYVSWYSDGTYGEEGCEAFIEALRARGGCI 181 Query: 242 ATAEKVGR--SNIRKSYDSVIRELLQKPNARVWLFMRSDDSRELIAAASRVN--ASFTW 297 ! + !+! + +I ++!!! + 11111+ + III+ Il I+
Sbjct: 182 ALSVKIGEFDRGDEEDFDKLLRELKR--RARVWMCGHGETLRELLEAALRLGLTGEDYV 239 Query: 298 VASDGWGAQESIVKGSEHVAYGAITLELASHPVRQFDRYFQSLNPYNNHRNPWFRDFWEQ 357 II + I I 111 111+ + +! !! + II IIIII +11++
Sbjct: 240 FISDDLFNKSLPA---EPGAPGAI--ELANASMLRFAYYFVLVLTLNNPRNPWFLEFWKE 294 4S Query: 358 KFQCSLQNKRNHRQICDKHLAIDSSNYEQESKIMFVVNAVYAMAHALHKMQRTLCPNTT- 416 I I+Il+ IIII 1 II +11l IIIII 1 +
Sbjct: 295 NFICALQDFLT------------LEPYEQEGKAGFVYDAVYLYAHALHNTTLALGGSWVD 342 Query: 417 --KLCDAMKILDGKKLYKDYLLKINFLAPFNPNKGADSIVKFDTYGDGMGRYNVFNFQHI 474 SO II + + I I I II II I I + + I+
Sbjct: 343 GEKLVQHL-------------RNLTFEGVTGP-------VTFDENGDRDGDYVLLDTQNT 382 Query: 475 GGK-----YSYLKVGHWAE 488 +I II I I
Sbjct: 383 ETGQLKWGTYDGVGKWTE 401 Table 6F. Domain Analysis of NOV6 gnllPfamlpfam00003, 7tm 3, 7 transmembrane receptor (metabotropic E
family). (SEQ ID N0:85) CD-Length = 256 residues, 100.0 aligned Score = 323 bits (827), Expect = 3e-89 S Query: 576 WAIGPVTIACLGFMCTCIVITVFIKHNNTPLVKASGRELCYILLFGVSLSYCMTFFFIAK 635 I I +I II + I I+ II+II +1I+1111 III I+II I+ I I +I II I
Sbjct: 1 LGTVLVALAVLGIVLTLFVLWFVKHRDTPIVKASNRELSYLLLIGLILCYLCSFLFIGK 60 Query: 636 PSPVTCALRRLGLGTSFAICYSALLTKTNCIARIFDGVKNGAQRPKFISPSSQVFICLGL 695 to II I III+ I I +111111 III + III I I+ +111111 +1I I I I
Sbjct: 61 PSETSCILRRILFGLGFTLCYSALLAKTNRVLRIFRAKKPGSGKPKFISPWAQVLIVLIL 120 Query: 696 TLVQIVMVSWLILETPGTRRYTLPEKRETVILKCNVKDS-SMLISLTYDVVLVILCTW 754 +I+I+++ +11++I I II + +II+II + ++ I II +I +11I
IS Sbjct: 121 VLIQVIICVIWLWEPPRPTIDIYSEKEK-TILECNKGSMVAFVVVLGYDGLLAVLCTFL 179 Query: 755 AFKTRKCPENFNEAKFIGFTMYTTCIIWLAFLPIFYVTSSDYRVQTTTMCISVSLSGFW 814 II II llllllllllll+I I II+I+II+II+ I+ +1I I+ 1 I+
Sbjct: 180 AFLTRNLPENFNEAKFIGFSMLTFCIVWVAFIPIYL--STPGKVQVAVEIFSILASSTVL 237 Query: 815 LGCLFAPKVHIVLFQPQKN 833 IIIII II +I+II+I+II
Sbjct: 238 LGCLFVPKCYIILFRPEKN 256 2S G-protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions (including various autocrine, paracrine and endocrine processes). They show considerable diversity at the sequence level, on the basis of which they can be separated into distinct groups. The term clan is used to describe the GPCRs, as they embrace a group of families for which there are indications of evolutionary relationship, but between which there is no statistically significant similarity in sequence.
The currently known clan members include the rhodopsin-like GPCRs, the secretin-like GPCRs, the cAMP
receptors, the fungal mating pheromone receptors, and the metabotropic glutamate receptor family.
The metabotropic glutamate receptors are functionally and pharmacologically distinct 3 S from the ionotropic glutamate receptors. They are coupled to G-proteins and stimulate the inositol phosphate/Ca2+ intracellular signalling pathway. The amino acid sequences of the receptors contain high proportions of hydrophobic residues grouped into 7 domains, in a manner reminiscent of the rhodopsins and other receptors believed to interact with G-proteins.
However, while a similar 3D framework has been proposed to account for this, there is no significant sequence identity between these and receptors of the rhodopsin-type family: the metabotropic glutamate receptors thus bear their own distinctive'7TM' signature. This 7TM
signature is also shared by the calcium-sensing receptors, and GABA (gamma-amino-butyric acid) type B (GABA(B)) receptors.
At least eight sub-types of metabotropic receptor (MGRl-8) have been identified in cloning studies. The sub-types differ in their agonist pharmacology and signal transduction pathways.
The mGluR3 gene consists of six exons and spans over 95 kb. Exon 1 and its preceding putative promoter are located distantly from the following protein-coding region. In the mGluR family, mGluR3 and mGluRS are both expressed in neuronal and glial cells and are upregulated during the early postnatal period. They are, however, coupled to two distinct signaling cascades and have been shown to exert opposite influences on some functions of cultured astrocytes. In cultured astrocytes, mGluR3 and mGluRS mRNA levels were significantly increased by exposure to epidermal growth factor (EGF), basic hbroblast growth factor (bFGF), or transforming growth factor-alpha; and EGF was more efficacious than bFGF
in producing this increase. Hence, mGluR3 and mGluRS mRNAs are concertedly upregulated in cultured astrocytes by specific growth factors. This fording suggests that the two mGluR
subtypes may play an important role in maintaining the proper balance of astrocyte functions via two distinct signal transduction mechanisms.
Glutamate receptors are divided into 2 distinct classes: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). The iGluRs consist of N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors. Non-NMDA receptors are further subdivided into 2 groups: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and kainate receptors. The mGluRs are divided into 3 groups according to agonist selectivity, coupling to different effector systems, and sequence homology.
Group I includes mGluRl and mGIuRS, which are coupled to inositol phospholipid metabolism.
Group II, which includes mGluR2 and mGluR3, and group III, which includes mGluR4, mGluR6, mGluR7, and mGluRB, are negatively coupled to adenylate cyclase activity. Each mGluR
possesses a large extracellular domain. Okamoto et al. (1998) expressed mGlurl-alpha (mGluRlA) in insect cells on a baculovirus system. They isolated a soluble mGluR that encodes only the extracellular domain and retains a ligand binding characteristic similar to that of the full-length receptor. Their observations demonstrated that a ligand binding event in mGluRs can be dissociated from the membrane domain.

Smitt et al. (2000) demonstrated that autoantibody against mGluRlA was responsible for severe paraneoplastic cerebellar ataxia in 2 patients. The disorder developed in both patients while they were in remission from Hodgkin disease. One, a teenager, had been in remission for 2 years when truncal ataxia, intention tremor, and gait ataxia developed. This patient improved clinically with loss of cells in the cerebrospinal fluid when treated with plasma exchanges, oral prednisone, and 2 courses of intravenous immune globulin. The second patient reported by Smitt et al. (2000) was in her late forties and, in addition to successfully treated Hodgkin disease, had polycystic kidney disease requiring hemodialysis for many years. Therapy was less successful in this patient, possibly because of delay in initiation.
The disclosed NOV6 nucleic acid of the invention encoding a Metabotropic Glutamate Receptor -like protein includes the nucleic acid whose sequence is provided in Table 6A or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 6A while still encoding a protein that maintains its Metabotropic Glutamate Receptor -like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications axe carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
The disclosed NOV6 protein of the invention includes the Metabotropic Glutamate Receptor -like protein whose sequence is provided in Table 6B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 6B while still encoding a protein that maintains its Metabotropic Glutamate Receptor -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 10% percent of the residues may be so changed.
The above defined information for this invention suggests that these Metabotropic Glutamate Receptor -like proteins (NOV6) may function as a member of a "Metabotropic Glutamate Receptor family". Therefore, the NOV6 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration iya vivo and iJ2 vit3°o of all tissues and cell types composing (but not limited to) those defined here.
The nucleic acids and proteins of NOV6 are useful in potential therapeutic applications implicated in immune disorders and airway pathologies such as epileptic seizures and other neurological disorders, Hodgkin disease, polycystic kidney disease, mental depression, Adenocarcinoma, Smith-Lemli-Opitz syndrome, Retinitis pigmentosa, and/or other pathologies and disorders For example, a cDNA encoding NOV6 may be useful in gene therapy, and NOV6 may be useful when administered to a subject in need thereof. By way of nonlimiting example, NOV6 will have efficacy for treatment of patients suffering from epileptic seizures and other neurological disorders, Hodgkin disease, polycystic kidney disease, mental depression, Adenocarcinoma, Smith-Lemli-Opitz syndrome, Retinitis pigmentosa. The novel NOV6 nucleic acid encoding NOV6 protein" or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
NOV6 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies"
section below. For example the disclosed NOV6 protein have multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated epitope is from about amino acids 20 to 30. In other embodiments, NOV6 epitope is from about amino acids SO to 70, from about amino acids 100 to 140, from about amino acids 180 to 200, from about amino acids 210 to 280, from about amino acids 310 to 400, from about amino acids 450 to 510, from about amino acids 520 to 560, from about amino acids 600 to 610, from about amino acids 660 to 680, from about amino acids 700 to 720, from about amino acids 750 to 770, or from about amino acids 800 to 850. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets fox various disorders.

NOV7 includes three novel PV-1-like proteins disclosed below. The disclosed proteins have been named NOV7a, NOV7b, and NOV7c.
NOV7a A disclosed NOV7a nucleic acid of 1366 nucleotides (also referred to sggc draft ba560a15 20000723 dal) encoding a novel PV-1-like receptor protein is shown in Table 7A. An open reading frame was identified beginning with an ATG
initiation codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 1327-1329. In Table 7A, the 3' untranslated region is underlined and the start and stop codons are in bold letters.
Table 7A. NOV7a Nucleotide Sequence (SEQ ID N0:18) CCTGCGCTACTTCTTCCTCTTCGTCTCCCTCATCCAATTCCTCATCATCCTGGGGCTCGTGCTCTTCATGG
TCTATGGCAACGTGCACGTGAGCACAGAGTCCAACCTGCAGGCCACCGAGCGCCGAGCCGAGGGCCTATAC
AGTCAGCTCCTAGGGCTCACGGCCTCCCAGTCCAACTTGACCAAGGAGCTCAACTTCACCACCCGCGCCAA
GGATGCCATCATGCAGATGTGGCTGAATGCTCGCCGCGACCTGGACCGCATCAATGCCAGCTTCCGCCAGT
GCCAGGGTGACCGGGTAATCTACACGAACAATCAGAGGTACATGGCTGCCATCATCTTGAGTGAGAAGCAA
TGCAGAGATCAATTCAAGGACATGAACAAGAGCTGCGATGCCTTGCTCTTCATGCTGAATCAGAAGGTGAA
GACGCTGGAGGTGGAGATAGCCAAGGAGAAGACCATTTGCACTAAGGATAAGGAAAGCGTGCTGCTGAACA
AACGCGTGGCGGAGGAACAGCTGGTTGAATGCGTGAAAACCCGGGAGCTGCAGCACCAAGAGCGCCAGCTG
GCCAAGGAGCAACTGCAAAAGGTGCAAGCCCTCTGCCTGCCCCTGGACAAGGACAAGTTTGAGATGGACCT
TCGTAACCTGTGGAGGGACTCCATTATCCCACGCAGCCTGGACAACCTGGGTTACAACCTCTACCATCCCC
TGGGCTCGGAATTGGCCTCCATCCGCAGAGCCTGCGACCACATGCCCAGCCTCATGAGCTCCAAGGTGGAA
GGTCAGTGCCGGAGCCTCCGGGCGGATATCGAACGCGTGGCCCGCGAGAACTCAGACCTCCAACGCCAGAA
GCTGGAAGCCCAGCAGGGCCTGCGGGCCAGTCAGGAGGCGAAACAGAAGGTGGAGAAGGAGGCTCAGGCCC
GGGAGGCCAAGCTCCAAGCTGAATGCTCCCGGCAGACCCAGCTAGCGCTGGAGGAGAAGGCGGTGCTGCGG
AAGGAACGAGACAACCTGGCCAAGGAGCTGGAAGAGAAGAAGAGGGAGGCGGAGCAGCTCAGGATGGAGCT
GGCCATCAGAAACTCAGCCCTGGACACCTGCATCAAGACCAAGTCGCAGCCGATGATGCCAGTGTCAAGGC
CCATGGGCCCTGTCCCCAACCCCCAGCCCATCGACCCAGCTAGCCTGGAGGAGTTCAAGAGGAAGATCCTG
GAGTCCCAGAGGCCCCCTGCAGGCATCCCTGTAGCCCCATCCAGTGGCTGAGGAGGCTCCGGCACTGACCT
AAGGGCGAATCCCAGCA
The disclosed NOV7a nucleic acid sequence, localized to chromosome 19, has 945 of 1345 bases (70%) identical to a 1968 by PV-1 mRNA from Rattus norvegicus (GENBANI~-ID: AF154831 ~acc:AF154831) (E = 1.1e lzi), A disclosed NOV7a polypeptide (SEQ ID N0:19) encoded by SEQ ID N0:18 is 442 amino acid residues and is pxesented using the one-letter amino acid code in Table 7B. Signal P, Psort and/or Hydropathy results predict that NOV7a has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.7900. In other embodiments, NOV7a is also likely to be localized to the nucleus with a certainty of 0.6000, to the microbody (peroxisome) with a certainty of 0.3000, or the Golgi body with a certainty of 0.3000. The most likely cleavage site for a NOV7a peptide is between amino acids 50 and 51, at: YVG-NV.
SNP data for NOV7a can be found below in Example 3.
Table 7B. Encoded NOV7a protein sequence (SEQ m N0:19).
MGLAMEHGGSYARAGGSSRGCWYYLRYFFLFVSLIQFLIILGLVLFMVYGNVHVSTESNLQATE
RRAEGLYSQLLGLTASQSNLTKELNFTTRAKDAIMQMWLNARRDLDRINASFRQCQGDRVIYTN
NQRYMAAIILSEKQCRDQFKDMNKSCDALLFMLNQKVKTLEVEIAKEKTICTKDKESVLLNKRV
AEEQLVECVKTRELQHQERQLAKEQLQKVQALCLPLDKDKFEMDLRNLWRDSIIPRSLDNLGYN
LYHPLGSELASIRRACDHMPSLMSSKVEGQCRSLRADIERVARENSDLQRQKLEAQQGLRASQE
AKQKVEKEAQAREAKLQAECSRQTQLALEEKAVLRKERDNLAKELEEKKREAEQLRMELAIRNS
ALDTCIKTKSQPMMPVSRPMGPVPNPQPIDPASLEEFKRKILESQRPPAGIPVAPSSG
The disclosed NOV7a amino acid sequence has 266 of 442 amino acid residues (60%) identical to, and 347 of 442 amino acid residues (78%) similar to, the 438 amino acid residue PV-1 protein from Rattus norvegicus (SPTREMBL-ACC:Q9WV78) ( 1.6e 14z), and 439 of 442 amino acid residues (99%) identical to, and 439 of 442 amino acid residues (99%) similar to, the 479 amino acid residue Human ORFX ORF1918 polypeptide sequence (patp:AAB42154 ) (3.2e 229) TaqMan data for NOV7a can be found below in Example 2.
NOV7b A disclosed NOV7b nucleic acid of 1421 nucleotides (also referred to 2847264Ø32) encoding a novel PV-1-like receptor protein is shown in Table 7C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 55-57 and ending with a TGA codon at nucleotides 1381-1383. In Table 7C, the 5' and 3' untranslated regions are underlines and the start and stop codons are in bold letters.
Table 7C. NOV7b Nucleotide Sequence (SEQ m N0:20) GTACGGAGGGTCCTACGCTCGGGCGGGGGGCAGCTCTCGGGGCTGCTGGTATTACCTGCGCTACTTCTTCC
TCTTCGTCTCCCTCATCCAATTCCTCATCATCCTGGGGCTCGTGCTCTTCATGGTCTATGGCGACGTGCAC
GTGAGCACAGAGTCCAACCTGCAGGCCACCGAGCGCCGAGCCGAGGGCCTATACAGTCAGCTCCTAGGGCT
CACGGCCTCCCAGTCCAACTTGACCAAGGAGCTCAACTTCACCACCCGCGCCAAGGATGCCATCATGCAGA
TGTGGCTGAATGCTCGCCGCGACCTGGACCGCATCAATGCCAGCTTCCGCCAGTGCCAGGGTGACCGGGTC
ATCTACACGAACAATCAGAGGTACATGGCTGCCATCATCTTGAGTGAGAAGCAATGCAGAGATCAATTCAA
GGACATGAACAAGAGCTGCGATGCCTTGCTCTTCATGCTGAATCAGAAGGTGAAGACGCTGGAGGTGGAGA
TAGCCAAGGAGAAGACCATTTGCACTAAGGATAAGGAAAGCGTGCTGCTGAACAAACGCGTGGCGGAGGAA
CAGCTGGTTGAATGCGTGAAAACCCGGGAGCTGCAGCACCAAGAGCGCCAGCTGGCCAAGGAGCAACTGCA
AAAGGTGCAAGCCCTCTGCCTGCCCCTGGACAAGGACAAGTTTGAGATGGACCTTCGTAACCTGTGGAGGG
ACTCCATTATCCCACGCAGCCTGGACAACCTGGGTTACAACCTCTACCATCCCCTGGGCTCGGAATTGGCC
TCCATCCGCAGAGCCTGCGACCACATGCCCAGCCTCATGAGCTCCAAGGTGGAGGAGCTGGCCCGGAGCCT
CCGGGCGGATATCGAACGCGTGGCCCGCGAGAACTCAGACCTCCAACGCCAGAAGCTGGAAGCCCAGCAGG
GCCTGCGGGCCAGTCAGGAGGCGAAACAGAAGGTGGAGAAGGAGGCTCAGGCCCGGGAGGCCAAGCTCCAA
GCTGAATGCTCCCGGCAGACCCAGCTAGCGCTGGAGGAGAAGGCGGTGCTGCGGAAGGAACGAGACAACCT
GGCCAAGGAGCTGGAAGAGAAGAAGAGGGAGGCGGAGCAGCTCAGGATGGAGCTGGCCATCAGAAACTCAG
CCCTGGACACCTGCATCAAGACCAAGTCGCAGCCGATGATGCCAGTGTCAAGGCCCATGGGCCCTGTCCCC

AACCCCCAGCCCATCGACCCAGCTAGCCTGGAGGAGTTCAAGAGGAAGATCCTGGAGTCCCAGAGGCCCCC
TGCAGGCATCCCTGTAGCCCCATCCAGTGGCTGAGGAGGCTCCAGGCCTGAGGACCAAGGGATGGCCCGAC
T
The disclosed NOV7b nucleic acid sequence, localized to chromosome 19, has 969 of 1383 bases (70%) identical to a PV-1 mRNA from Rattus norvegicus (GENBANK-ID:
AFI54831 ) (E = 2.5e lzs), A disclosed NOV7b polypeptide (SEQ ID N0:21) encoded by SEQ ID N0:20 is 442 amino acid residues and is presented using the one-letter amino acid code in Table 7D. Signal P, Psort and/or Hydropathy results predict that NOV7b has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.7900. In other embodiments, NOV7b is also likely to be localized to the nucleus with a certainty of 0.6000, to the microbody (peroxisome) with a certainty of 0.3000, or the Golgi body with a certainty of 0.3000. The most likely cleavage site for a NOV7b peptide is between amino acids 50 and 51, at: YVG-NV.
Table 7D. Encoded NOV7b protein sequence (SEQ ID N0:21).
SQLLGLTASQSNLTKELNFTTRAKDAIMQMWLNARRDLDRINASFRQCQGDRVIYTNNQRYMAAIILSEKQ
CRDQFKDMNKSCDALLFMLNQKVKTLEVEIAKEKTICTKDKESVLLNKRVAEEQLVECVKTRELQHQERQL
AKEQLQKVQALCLPLDKDKFEMDLRNLWRDSTIPRSLDNLGYNLYHPLGSELASIRRACDHMPSLMSSKVE
ELARSLRADIERVARENSDLQRQKLEAQQGLRASQEAKQKVEKEAQAREAKLQAECSRQTQLALEEKAVLR
KERDNLAKELEEKKREAEQLRMELAIRNSALDTCIKTKSQPMMPVSRPMGPVPNPQPIDPASLEEFKRKIL
The disclosed NOV7b amino acid sequence has 268 of 442 amino acid residues (60%) identical to, and 350 of 442 amino acid residues (79%) similar to the 438 amino acid residue PV-1 protein from Rattus norvegicus (SPTREMBL-ACC:Q9WV78), and 454of 457 amino acid residues (99%) identical to, and 457 of 457 amino acid residues (100%) similar to the 479 amino acid residue Human ORFX ORF 1918 polypeptide sequence (patp:AAB42154) (E
=
1. 3 e-23~).
NOV7b is expressed in at least the following tissues: lymph node, bone marrow, spleen, mammary gland, thyroid, stomach, fetal kidney, heart, fetal liver. In addition, the sequence is predicted to be expressed in lung because of the expression pattern of (GENBANI~-ID: Q9WV78) a closely related PV-1 hornolog in species Rattus norvegicus. It has also been reported to be expressed in muscle and brain (J Cell Biol 1999 Jun 14;145(6):1189-98). Endothelium of the fenestrated peritubular capillaries of the kidney and those of the intestinal villi, pancreas, and adrenals have also been shown to express PV-1 (Proc Natl Acad Sci U S A 1999 Nov 9;96(23):13203-7) TaqMan data for NOV7b can be found below in Example 2.
NOV7c A disclosed NOV7c nucleic acid of 2024 nucleotides (also referred to CG51878-03) encoding a novel PV-1-like receptor protein is shown in Table 7E. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TGA
codon at nucleotides 1327-1329. In Table 7E, the 3' untranslated region is underlined and the start and stop codons are in bold letters.
Table 7A. NOV7c Nucleotide Sequence (SEQ ID N0:22) ATGGGTCTGGCCATGGAGCACGGAGGGTCCTACGCTCGGGCGGGGGGCAGCTCTCGGGGCTGCTGGTATTA
CCTGCGCTACTTCTTCCTCTTCGTCTCCCTCATCCAATTCCTCATCATCCTGGGGCTCGTGCTCTTCATGG
TCTATGGCAACGTGCACGTGAGCACAGAGTCCAACCTGCAGGCCACCGAGCGCCGAGCCGAGGGCCTATAC
AGTCAGCTCCTAGGGCTCACGGCCTCCCAGTCCAACTTGACCAAGGAGCTCAACTTCACCACCCGCGCCAA
GGATGCCATCATGCAGATGTGGCTGAATGCTCGTCGCGACCTGGACCGCATCAATGCCAGCTTCCGCCAGT
GCCAGGGTGACCGGGTCATCTACACGAACAATCAGAGGTACATGGCTGCCATCATCTTGAGTGAGAAGCAA
TGCAGAGATCAATTCAAGGACATGAACAAGAGCTGCGATGCCTTGCTCTTCATGCTGAATCAGAAGGTGAA
GACGCTGGAGGTGGAGATAGCCAAGGAGAAGACCATTTGCACTAAGGATAAGGAAAGCGTGCTGCTGAACA
AACGCGTGGCGGAGGAACAGCTGGTTGAATGCGTGAAAACCCGGGAGCTGCAGCACCAAGAGCGCCAGCTG
GCCAAGGAGCAACTGCAAAGGGTGCAAGCCCTCTGCCTGCCCCTGGACAAGGACAAGTTTGAGATGGACCT
TCGTAACCTGTGGAGGGACTCCATTATCCCACGCAGCCTGGACAACCTGGGTTACAACCTCTACCATCCCC
TGGGCTCGGAATTGGCCTCCATCCGCAGAGCCTGCGACCACATGCCCAGCCTCGTGAGCTCCAAGGTGGAG
GAGCTGGCCCGGAGCCTCCGGGCGGATATCGAACGCGTGGCCCGCGAGAACTCAGACCTCCAACGCCAGAA
GCTGGAAGCCCAGCAGGGCCTGCGGGCCAGTCAGGAGGCGAAACAGAAGGTGGAGAAGGAGGCTCAGGCCC
GGGAGGCCAAGCTCCAAGCTGAATGCTCCCGGCAGACCCAGCTAGCGCTGGAGGAGAAGGCGGTGCTGCGG
AAGGAACGAGACAACCTGGCCAAGGAGCTGGAAGAGAAGAAGAGGGAGGCGGAGCAGCTCAGGATGGAGCT
GGCCATCAGAAACTCAGCCCTGGACACCTGCATCAAGACCAAGTCGCAGCCGATGATGCCAGTGTCAAGGC
CCATGGGCCCTGTCCCCAACCCCCAGCCCATCGACCCAGCTAGCCTGGAGGAGTTCAAGAGGAAGATCCTG
GAGTCCCAGAGGCCCCCTGCAGGCATCCCTGTAGCCCCATCCAGTGGCTGAGGAGGCTCCAGGCCTGAGGA
CCAAGGGATGGCCCGACTCGGCGGTTTGCGGAGGATGCAGGGATATGCTCACAGCGCCCGACACAACCCCC
TCCCGCCGCCCCCAACCACCCAGGGCCACCATCAGACAACTCCCTGCATGCAAACCCCTAGTACCCTCTCA
CACCCGCACCCGCGCCTCATGATCCCTCACCCAGAGCACACGGCCGCGGAGATGACGTCACGCAAGCAACG
GCGCTGACGTCACATATCACCGTGGTGATGGCGTCACGTGGCCATGTAGACGTCACGAAGAGATATAGCGA
TGGCGTCGTGCAGATGCAGCACGTCGCACACAGACATGGGGAACTTGGCATGACGTCACACCGAGATGCAG
CAACGACGTCACGGGCCATGTCGACGTCACACATATTAATGTCACACAGACGCGGCGATGGCATCACACAG
ACGGTGATGATGTCACACACAGACACAGTGACAACACACACCATGACAACGACACCTATAGATATGGCACC
AACATCACATGCACGCATGCCCTTTCACACACACTTTCTACCCAATTCTCACCTAGTGTCACGTTCCCCCG
ACCCTGGCACACGGGCCAAGGTACCCACAGGATCCCATCCCCTCCCGCACAGCCCTGGGCCCCAGCACCTC
CCCTCCTCCAGCCTCCTGGCCTCCCGGTAGTACACG
The disclosed NOV7c nucleic acid sequence, localized to chromosome 19p13, has 2009 of 2015 bases (99%) identical to a gb:GENBANI~-ID:AF326591 ~acc:AF326591.1 mRNA from Homo sapie~as (Homo Sapiens fenestrated-endothelial linked structure protein (EELS) mRNA, complete cds) (E =0.0).
A disclosed NOV7c polypeptide (SEQ ID N0:23) encoded by SEQ ID N0:22 is 442 amino acid residues and is presented using the one-letter amino acid code in Table 7F. Signal P, Psort and/or Hydropathy results predict that NOV7c has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.7900. In other embodiments, NOV7c is also likely to be localized to the nucleus with a certainty of 0.6000, to the microbody (peroxisome) with a certainty of 0.3000, or the Golgi body with a certainty of 0.3000. The most likely cleavage site for a NOV7c peptide is between amino acids 50 and 51, at: YVG-NV.
Table 7F. Encoded NOV7c protein sequence (SEQ ID N0:23).
MGLAMEHGGSYARAGGSSRGCWYYLRYFFLFVSLIQFLIILGLVLFMVYGNVHVSTESNLQATERRAEGLY
SQLLGLTASQSNLTKELNFTTRAKDAIMQMWLNARRDLDRINASFRQCQGDRVIYTNNQRYMAAIILSEKQ
CRDQFKDMNKSCDALLFMLNQKVKTLEVEIAKEKTICTKDKESVLLNKRVAEEQLVECVKTRELQHQERQL
AKEQLQRVQALCLPLDKDKFEMDLRNLWRDSIIPRSLDNLGYNLYHPLGSELASIRRACDHMPSLVSSKVE
ELARSLRADIERVARENSDLQRQKLEAQQGLRASQEAKQKVEKEAQAREAKLQAECSRQTQLALEEKAVLR
KERDNLAKELEEKKREAEQLRMELAIRNSALDTCIKTKSQPMMPVSRPMGPVPNPQPIDPASLEEFKRKIL
ESQRPPAGIPVAPSSG
The disclosed NOV7c amino acid sequence has 440 of 442 amino acid residues (99%) identical to, and 442 of 442 amino acid residues (100%) similar to, the 442 amino acid residue ptnr:SPTREMBL-ACC:Q9BX97 protein from Homo sapiefas (Human) (PVl PROTEIl~ (E =
6.9e 2st) NOV7c is expressed in at least the following tissues: Heart, Adrenal Gland/Suprarenal gland, Thyroid, Salivary Glands, Liver, Bone Marrow, Spleen, Lymph Node, Mammary gland/Breast, Placenta, Prostate, Lung, Kidney, Pancreas, Bone Marrow, and Small Intestine.
Expression information was derived from the tissue sources of the sequences that were included in the derivation of the sequence of CuraGen Acc. No. CG51878-03. The sequence is predicted to be expressed in the following tissues because of the expression pattern of (GENBANK-ID: gb:GENBANK-ID:AF326591~acc:AF326591.1) a closely related Homo sapiens fenestrated-endothelial linked structure protein (FELS) mRNA.
The NOV7a, 7c and 7c are very closely homologous as is shown in the alignment in Table 7G.
Table 7G Alignment of NOV7a, 7b, and 7c.
NOV7a 60 NOV7b 60 NOV7c 60 7o so 90 loo 1l0 120 NOV7a 120 NOV7b 120 NOV7c 120 NOV7a 180 NOV7b 180 NOV7c 180 l90 NOV7a 240 NOV7b 240 NOV7c 240 NOV7a 300 NOV7b 300 NOV7c 300 NOV7a 360 NOV7b 360 NOV7c 360 NOV7a 420 NOV7b 420 NOV7c 420 .1 ....1....1 ....1..
NOV7a ~' 442 (SEQ
ID
N0:19) 5 NOV7b ~' 442 (SEQ
ID
N0:21) NOV7c ~' 442 (SEQ
ID
N0:23) Homologies to any of the above proteins will be shared by the other two proteins insofar as they are homologous to each other as shown above.
Any reference to is assumed to refer to all three of the proteins in general, unless otherwise noted.

NOV7a also has homology to the amino acid sequence shown in the BLASTP
data listed in Table 7H.

Table 7H. BLAST
results for NOV7a Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (%) (%) gi~12963353~gb~AAKl1fenestrated- 442 438/442438/442 0.0 226.1 endothelial (99%) (99%) linked structure protein [Homo sapiens]

gi~13775238~ref~NPfenestrated- 442 439/442439/442 0.0 _ endothelial (99%) (99%) 12600.11 linked structure protein; PV-1 protein [Homo sapiens]) gi199105201refINPPV-1 [Rattus 438 266/442347/442 e-131 4471.1 norvegicus] (60%) (78%) gi~141613941gb1AAK54MECA32 [Mus 438 270/442348/442 e-125 730.1 AF369900 musculus] (61%) (78%) gi~14161698~ref~NPplasmalemma 438 269/442346/442 e-125 15774. 1 vesicle ( 60%) (77%) associated protein [Mus musculus]

The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 7I.
Table 7I. Information for the ClustalW proteins J 1) NOV7 (SEQ ID N0:19) 2) giI12963353~gb~AAK11226.1~ fenestrated-endothelial linked structure protein [Homo Sapiens] (SEQ ID N0:55) 3) giI13775238~refINP_112600.11 fenestrated-endothelial linked structure protein;
PV-1 protein [Homo Sapiens]) (SEQ ID N0:56) 4) gi~9910520~ref~NP_064471.1~ PV-1 [Rattus norvegicus] (SEQ ID N0:57) 5) gi~14161394~gb~AAK54730.1~AF369900_1 MECA32 [Mus musculus] (SEQ ID N0:58) 6) giI14161698~refINP_115774.1~ plasmalemma vesicle associated protein [Mus musculus] (SEQ ID N0:59) .... ....I........~....I.
....~....~....
....I...

NOV7 ~''HGGS .
n v r GSS

giI129633531HGGS GSS

gi~13775238~
r n V r F,HGGS GSS

gi~99105201S RS-P ~ RD

gi1141613941RS-P T DQ ' ~ I
T~ D

gi~141616981SRS-P Q ~ I

~.1.... .~.. .p.. .~.. -I..-...~....~
NOV 7 ~ ~ Lei ~ . . ~ . . ~ F~ ~ ~ v w~V ~r v,~..,, ~ ~,a, ~ a v v .. .
gi~12963353~ ~ R~~ ~L~ T' ~S FjT ~ 'I
V v a gi~13775238~ S R~~ vLL ~' ~S, T FT v ' v giI99105201 S ' I.. ~ S.-~ ~ I~ L ~T, ~
0 gi~141613941 1 S ~ I~~DS ~VV S' ~ ~, . ILL ET v gi ~ 14161698 I ~S ;: ~ I ~ ~ .. S~9 ' ~ ., S If.~. i'LV~aE~T

NOV7 M~.~I..ZYTa'~~ I.~~.InR"UI.T..Q'.M~~ ~'..v....yF~..,M
v ~ v X ~ tC W
~ ~ ' YM ~~~ K L?
gi ~ 12963353 I M ' ~., ' ' ~ ~ ~1~~I ~W -~ ~ ~F a I
gi~1377523B~ M ~ 7.i ' w v WI~T Q~ :pig.. v yFv gi~9910520~ QL TT~~EVE' ~ w ~ ALT I FI~~ ~ QE~La~G
gi~141613941 QL TTw EM ' ~ w ~ ~L3:T I FI~~ ~ ~LaE
0 gi' 14161698 ~ QL~TT'~'EM_,_' ~ ' ~ ~ ~L1T I FT ~ ~ ~ Q~ aE~1 ~ ~ u~~~ ~ r ~ V w ~ ~w ~ a w yr v NOV7 4S 3Q ~ T~ vT~ :: ~ , L v E
.V.
gi ~ 12963353 I Q T~ ~Ts~ ~ L ~ E
gi~13775238~ S Q T~ yT~ ~ L t E
gi~9910520~ T E~ GE ~ S ~ 'Q~ ~Q
gi~141613941 GE ~, S ~ QT
gi~14161698) ~ G~ S ~ 'Q~
. _ -.1... . .L..~....~.., .~....p..
NOV7 ' Lw w ~ ~ ~Kp &y ' ~ T
gi~12963353~ T' L~ ~ ~ t ~ ~ ~KIj E ~ '~ I P
gi~13775238~ a ~ Lv v v,i' v v yCD E ~' w T~P~
gi~99105201 v v v T~ ~ v ~ E ~ L ~v L
gi~14161698~ v v ,~.~T~ ~S ~' ~._~ . -L~... :; L~Y:..

~r r m NOV7 n. .L~EfP GS L~ S" -'H 'S~ SS GQC'S '~D.
gi ~ 12963353 ~ ~ ;YF~P GS L~ r" ~ ~SLISS ~'S ' ~D
gi ~ 13775238 ~ ~ LYktP GS L' I" H ~S,L SS "S ~'D
gi I 9910520 ~ ~ Z -~FT~IS P FS "T ~5~ 'GI T " ~ T~
gi I 14161394 ~ ~7 P --~T'~Q MP Y ~ "T ~S~'GI T I " ~ T' g1I14161698~ ~' P --~II'~Q MP Y'~L~"T~E~S~'GT PP I "G,:' ~ ~ T

.J..
NOV7 -S ~v (;~Q~G,LR~Sv L~Q~VE . S'~ ~ ~
gi~12963353~ SD '~ QQ;G~, v QVE S'~ ~ ~
gi~13775238~ SI) '~ C~'GLw T~Q~VE S'v v ~
g1 I 9910520 ~ G~ ' ~ LERA,Tf,~GEt ~ T~tAGT ' ~ ' T,Q T w ~ ~
giI14161394~ '~ L~FnAA' ,._~ .', ' GT w' T.Q ' '~ ~
gi~14161698~ __. __'v L~'!~ _.~ , __~ GT '~~~ T;Q _~

.~.. .~.. .p . ~..I-. -NOV7 ~~ -. - ~ ~ 1 T' Q~M.,'~~~", gi ~ 12963353 I ' ~ EF. ~ ~ 'T~ ~ y T Q:M
gi l 137752381 KE' ~ E v ~ ~ I 'T Q ~~l:11~''.M
V 1' gi~9910520~ T~'~ E~2- ' L v ~T ,'I ~ ~ L QP
gii14161698~ .. ~ E. ~ L ~ 'T I ~ ~ L~ ~P

..
~ _..
~

S .p Q ~.. ,~ ~.. GI ..S
NOV7 V V I r gi~ 12963353~S 'P Q' ~ ~1~~ GI S

gi~ 137752381S 'PM Q ,.. ~~ ~...GI...S

gi~ 9910520~- LPP P ~. ~...L .p gi~ 14161394~- ' 'PP ~ ~'L QP
S ~

3$ 14161698~ ' P P ~ ~L QP
gi~ S

PV-1 is a novel endothelial protein shown by immunocytochemical tests to be specifically associated with the stomatal diaphragms of caveolae in lung endothelium (Stan RV, et.al.; Proc Natl Acad Sci U S A 1999 Nov 9;96(23):13203-7). Although the highest expression levels of both mRNA and protein are in the lung, PV-1 also has been found to be expressed in other organs. Using a specific antibody to the extracellular domain of PV-1, the survey on the presence of this protein at light and electron microscope level has been extended in several rat organs. It has been shown by immunofluorescence the antibody recognizes with high specificity the endothelium of the fenestrated peritubular capillaries of the kidney and those of the intestinal villi, pancreas, and adrenals. By imrnunolocalization at electron microscope level, the antibody recognizes specifically the diaphragms of the fenestrae and the stomatal diaphragms of caveolae and transendothelial chanxiels in the endothelia of these vascular beds. No signal was detected in the continuous endothelium of the heart, skeletal muscle, intestinal muscularis, or brain capillaries or the nondiaphragmed fenestrated endothelium of kidneyglomeruli. Taken together, the findings define the only antigen to be localized thus far in fenestral diaphragms. They also show that the stornatal diaphragms of caveolae and transendothelial channels and the fenestral diaphragms might be biochemically related, in addition to being morphologically similar structures.
By using an immunoisolation procedure (Stan, R.-V., W.G. Roberts, K. Ihida, D.
Predescu, L., Saucan, L. Ghitescu, and G.E. Palade. 1997. Mol. Biol. Cell.
8:595-605) developed in our laboratory, a caveolar subfraction from rat lung endothelium has been isolated and the proteins of this subfraction have been partially characterized which include an apparently caveolae-specific glycoprotein is proposed to be called PV-1 (formerly known as gp68). The isolation and partial sequencing of PV-1, combined with the cloning of the full length PV-1 cDNA led to the following conclusions: (a) PV-1 is a novel single span type II
integral membrane protein (438 amino acids long) which forms homodimers in situ; (b) the transmembrane domain of PV-1 is near the NH2 terminus defining a short cytoplasmic endodomain and a large COOH-terminal ectodomain exposed to the blood plasma;
(c) PV-1 is N-glycosylated and its glycan antennae bear terminal nonreducing galactosyl residues in alphal-3 linkage. PV-1 is expressed mostly in the lung but both the messenger RNA and the protein can be detected at lower levels also in kidney, spleen, liver, heart, muscle, and brain.
No signal could be detected in testis and two lower molecular weight forms were detected in brain. Immunocytochemical studies carned out by immunodiffusion on rat lung with an anti-PV-1 polyclonal antibody directed against a COOH-terminal epitope reveal a specific localization of PV-1 to the stomatal diaphragms of rat lung endothelial caveolae and confirm the extracellular orientation of the PV-1 COOH terminus (Stan, R.V. J. Cell.
Biol. 1999, Jun.
14; 145(6): 189-98).
Immunohistochemistry revealed initial expression of the stage-specific glycoprotein, GP68, in various mesenchymal tissue substructures of mouse embryos (Morita T, et.al.;
Okajimas Folia Anat Jpn 1998 Oct;75(4):185-95). During the 11-15th days of gestation, GP68 was localized in the primitive meninges, chondroblasts and perichondrium of pre-cartilaginous vertebral bodies and ribs, connective tissue cells of the dermis, the epicardium and endocardium of the heart, the epimysium and perimysium of skeleton musclature, and the basement membranes of splanchnic organs. Double staining for laminin expression indicated coincidental expression in identical tissue substructures. However, laminin was expressed in days 10-18 embryos and the neonate. Therefore, GP68 is coincidentally expressed with laminin in mesenchymal tissues between the 11th and 15th day of gestation, and may play a role as a laminin-associated protein. In the light of these results, a hypothesis concerning the relationship between these two proteins and the mechanisms of non-integrin laminin-associated proteins during normal embryogenesis is discussed further.

The microvascular endothelium is organized as a highly differentiated squamous epithelium whose main function is to mediate the exchanges of water, macromolecules, and small solutes between the blood plasma and the interstitial fluid. The endothelial structures implicated so far in the transendothelial transport are the caveolae, transendothelial channels, intercellular junctions, and the fenestrae. Caveolae are flask-shaped or spherical plasma membrane invaginations and associated vesicles of 70-nm average outer diameter that can occur singly or in chains or clusters . In invaginated form, their membranes is in continuity layer by layer with the plasmalemma proper, and, in some microvascular beds (e.g., the continuous endothelium of the lung and the fenestrated and sinusoidal endothelia), their introits or necks are provided with a stomatal diaphragm.
The transendothelial channels axe channels of 60-70-nm diameter that run across the endothelial cell. They seem to be formed by the fusion of either one caveola with both luminal and abluminal aspects of the plasmalemma or by chains of usually two to four caveolae. These channels are provided with two diaphragms (one luminal and one abluminal) only in fenestrated endothelia and not in their continuous counterparts.
The diaphragmed fenestrae are characteristic structural elements of all fenestrated endothelia (e.g., kidney peritubular capillaries and ascending vasa recta, capillaries of intestinal villi, pancreas, adrenal cortex, endocrine glands, and choriocapillaries of the brain and eye). They are round openings or windows cutting through the endothelial cell, have a constant diameter of 63-68 nm, and occur only in the attenuated parts of the cell, in clusters referred to as "sieve plates". In en face electron microscopic images, the fenestrae appear circular, but several studies have shown that they have an 8-fold symmetry.
The rim of the fenestra (where the abluminal plasmalemma is continuing the luminal plasmalemma) is the anchoring line for the fenestral diaphragm . In normal sections, the diaphragm appears as a very thin (5-6 nm) single-layer barrier provided with a central density or knob. Deep-etch rapid-freeze techniques have revealed the structure of the diaphragm to be composed of radial fibrils (7-nm diameter) starting at the rim and interweaving in a central mesh (the equivalent of the central knob in orthogonal sections).
Although the chemical composition of endothelial caveolae started to yield some insights, the molecular components of transendothelial channels and fenestrae remained elusive. The chemistry of these endothelial microdomains has been investigated with nonspecific "general" probes (charged molecules and lectins alone or in combination with various degrading enzymes), which yielded some information on the surface charge, type of molecules conferring the charge, and type of glycan antennae found on the glycoproteins and glycolipids. No specific component of the fenestral or transendothelial channels diaphragms has been identified so far.
Proteins reported to be contained within caveolae include G protein-coupled receptors (GPCR) (Ostrom RS , et al., J Pharmacol Exp Ther 2000 Aug;294(2):407-12), scavenger receptor class B type I (SR-BI) (Krieger M Annu Rev Biochem 1999;68:523-58), Monocarboxylate transporters (Bonen A, Med Sci Sports Exerc 2000 Apr;32(4):778-89), endothelial NOS (eNOS) (Done BC Acta Physiol Scand 2000 Jan;168(1):27-31). IP3 receptor-like protein, Ca2+ ATPase, several PI~C isoforrns. (Isshiki M, et al., Cell Calcium 1999 Nov;26(5):201-8). and GPI-anchored molecules (Martins VR, Braz J Med Biol Res Ju1;32(7):853-9).
The disclosed NOV7 nucleic acid of the invention encoding a PV-1 -like protein includes the nucleic acid whose sequence is provided in Table 7A, 7C, or 7E or a fragment thereof. The invention also includes a mutant ox variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 7A, 7C, or 7E while still encoding a protein that maintains its PV-1 -like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 30% percent of the bases may be so changed.
The disclosed NOV7 protein of the invention includes the PV-1 -like protein whose sequence is provided in Table 7B, 7D, or 7F. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 7B, 7D, or 7F while still encoding a pxotein that maintains its PV-1 -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 40% percent of the residues may be so changed.
The protein similarity information, expression pattern, and map location for the PV-1-like protein and nucleic acid (NOV7) disclosed herein suggest that NOV7 may have important structural and/or physiological functions characteristic of the PV-1-like family. Therefore, the NOV7 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targetinglcytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
The NOV7 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below andlor other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from Cerebellar ataxia, pure;
Episodic ataxia, type 2; Hemiplegic migraine, familial; Leigh syndrome; Spinocerebellar ataxia-6;
Psoriasis, susceptibility to; Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy, ARDS, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Muscular dystrophy, Myasthenia gravis, Hemophilia, Hypercoagulation, Idiopathic thrombocytopenic purpura, Immunodeficiencies, Graft vesus host, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis ,Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Scleroderma, Obesity, Transplantation; fertility; cancer; Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, Xerostomia; tooth decay and other dental problems;
Inflammatory bowel disease, Diverticular disease, Pancreatitis, and/or other pathologiesldisorders. The NOV7 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
NOV7 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies"
section below. For example the disclosed NOV7 protein have multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated epitope is from about amino acids 5 to 25. In other embodiments, NOV7 epitope is from about amino acids 50 to 75, from about amino acids 80 to 160, from about amino acids 175 to 275, from about amino acids 280 to 380, or from about amino acids 385 to 430.
This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.

NOVS includes two novel Papin-like proteins disclosed below. The disclosed proteins have been named NOVBa, and NOVBb.
NOVBa A disclosed NOVBa nucleic acid of 8640 nucleotides (also referred to as SC134914330 A) encoding a novel papin-like protein is shown in Table 8A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 101-103 and ending with a TGA codon at nucleotides 8543-8545. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 8A. The start and stop codons are in bold letters.
Table 8A. NOV8a nucleotide sequence (SEQ ID NO:24).
AGCTGATGATGGCCAGGGACCCCAGGGGACGTGGGGCCCTGTGGGGTCTGGCCCCCAGGAGCAAGACCTCTG
ATGATGCTGGTGTCTGGGAGTGAGCACCATGCCCATCACCCAGGACAATGCCGTGCTGCACCTGCCCCTCCT
CTACCAGTGGCTGCAGAACAGCCTGCAGGAAGGTGGGGATGGGCCGGAGCAGCGGCTCTGCCAGGCGGCCAT
CCAGAAGCTGCAGGAGTACATCCAGCTGAACTTTGCTGTGGATGAGAGTACGGTCCCACCTGATCACAGCCC
CCCCGAAATGGAGATCTGTACTGTGTACCTCACCAAGGAGCTGGGGGACACAGAGACTGTGGGCCTGAGTTT
TGGGAACATCCCTGTTTTCGGGGACTATGGTGAAAAGCGCAGGGGGGGCAAGAAGAGGAAAACCCACCAGGG
TCCTGTGCTGGATGTGGGCTGCATCTGGGTGACAGAGCTGAGGAAGAACAGCCCAGCAGGGAAGAGTGGGAA
GGTCCGACTGCGGGATGAGATCCTCTCACTGAATGGGCAGCTGATGGTTGGAGTTGATGTCAGTGGGGCCAG
TTACCTGGCTGAGCAGTGCTGGAATGGCGGCTTTATCTACCTGATCATGCTGCGTCGCTTTAAGCACAAAGC
CCACTCCACTTATAATGGCAACAGTAGCAACAGCTCTGAACCAGGAGAAACACCTACCTTGGAGCTGGGTGA
CCGAACTGCGAAAAAGGGGAAACGAACCAGAAAGTTTGGGGTCATCTCCAGGCCTCCTGCCAACAAGGCCCC
TGAAGAATCCAAGGGCAGCGCTGGCTGTGAGGTGTCCAGTGACCCCAGCACTGAGCTGGAGAACGGCCTGGA
CCCTGAACTTGGAAACGGCCATGTCTTTCAGCTAGAAAATGGCCCAGATTCTCTCAAGGAGGTGGCTGGACC
CCATCTAGAGAGGTCAGAAGTGGACAGAGGGACAGAGCATAGAATTCCAAAGACAGATGCTCCTCTGACCAC
AAGCAATGACAAACGCCGCTTCTCAAAAGGTGGGAAGACGGACTTCCAATCGAGTGACTGCCTGGCACGGTC
CAAGGAGGAAGTTGGCCGAATATGGAAGATGGAGCTGCTCAAAGAATCGGATGGGCTGGGAATTCAGGTTAG
TGGAGGCCGAGGATCAAAGCGCTCACCTCACGCTATCGTTGTCACTCAAGTGAAGGAAGGAGGTGCCGCTCA
CAGGCTCAGGGATGGCAGGCTGTCCTTAGGAGATGAGCTGCTGGTAATCAATGGTCATTTACTGGTCGGGCT
CTCCCACGAGGAAGCAGTGGCCATTCTTCGCTCCGCCACGGGAATGGTGCAGCTTGTGGTGGCCAGCAAGGT
AGGTGTGCTTTCTGCATTTCAGATGCCTGGGACAGATGAACCCCAAGATGTGTGCGGTGCTGAGGAATCCAA
GGGGAACTTGGAAAGTCCCAAACAGGGCAGCAATAAAATCAAGCTCAAGAGTCGCCTTTCAGGTAGGTGGGG
GCTCTACCTGATGCAGCCTGTCGGGGGTGTACACCGCCTTGAGTCAGTTGAAGAATATAACGAGCTGATGGT

GCGGAATGGGGACCCCCGGATCCGGATGTTGGAGGTCTCCCGAGATGGCCGGAAACACTCCCTCCCGCAGCT
GCTGGACTCTTCCAGTGCCTCACAGGAATACCACATTGTGAAGAAGTCTACCCGCTCCTTAAGCACGACTCA
GGTGGAATCTCCTTGGAGGCTCATTCGGCCATCCGTCATCTCGATCATTGGGTTGTACAAAGAAAAAGGCAA
GGGCCTTGGCTTTAGTATTGCTGGAGGTCGAGACTGCATTCGTGGACAGATGGGGATTTTTGTCAAGACCAT
CTTCCCAAATGGATCAGCTGCAGAGGACGGAAGACTTAAAGAAGGTGATGAAATCCTAGATGTAAATGGAAT
ACCAATAAAGGGCTTGACATTTCAAGAAGCCATTCATACCTTTAAGCAAATCCGGAGTGGATTATTTGTTTT
AACGGTACGCACAAAGTTGGTGAGCCCCAGCCTCACACCCTGCTCGACACCCACACACATGAGCAGATCCGC
CTCCCCGAACTTCAATACCAGTGGGGGAGCCTCGGCGGGAGGTTCCGATGAAGGCAGTTCTTCATCCCTGGG
TCGGAAGACCCCTGGGCCCAAGGACAGGATCGTCATGGAAGTAACACTCAACAAAGAGCCAAGAGTTGGATT
AGGCATTGGTGCCTGCTGCTTGGCTCTGGAAAACAGTCCTCCTGGCATCTACATTCACAGCCTTGCTCCAGG
ATCAGTGGCCAAGATGGAGAGCAACCTGTCGCGGGGATCAATCCTGGAAGTGAACTCCGTCAACGTCCGCCA
TGCTGCTTTAAGCAAAGTCCACGCCATCTTGAGTAAATGCCCTCCAGGACCCGTTCGCCTTGTCATCGGCCG
GCACCCTAATCCAAAGGTGAATCAGGTTTCCGAGCAGGAAATGGATGAAGTCATAGCACGCAGCACTTATCA
GGAGAGCAAAGAGGCCAATTCCTCTCCTGGCTTAGGTACTGTAATCTCAATCGGATGTTTTCTTCTTCAACA
GGACTCCCTTATTTCTGAATCTGAACTCTCCCAGTACTTTGCCCACGATGTCCCTGGCCCCTTGTCAGACTT
CATGGTGGCCGGTTCTGAGGACGAGGATCACCCGGGAAGTGGCTGCAGCACGTCGGAGGAGGGCAGCCTGCC
TCCCAGCACCTCCACTCACAAGGAGCCTGGAAAACCCAGAGCCAACAGCCTCGTGACTCTTGGGAGCCATCG
GGCTTCTGGGCTCTTCCACAAGCAGGTGACAGTTGCCAGACAAGCCAGTCTCCCCGGAAGCCCACAGGCCCT
CCGAAACCCTCTCCTCCGCCAGAGGAAGGTAGGCTGCTACGATGCCAACGATGCCAGTGATGAGGAAGAGTT
TGACAGAGAAGGGGACTGCATTTCACTCCCAGGGGCCCTCCCGGGTCCCATCAGGCCTCTGTCAGAGGATGA
CCCGAGGCGTGTCTCAATTTCCTCTTCCAAGGGCATGGACGTCCACAACCAAGAGGAACGACCCCGGAAAAC
ACTGGTGAGCAAGGCCATCTCGGCACCTCTTCTTGGTAGCTCAGTGGACTTAGAGGAGAGTATCCCAGAGGG
CATGGTGGATGCTGCGTCCTATGCAGCCAACCTCACGGACTCTGCAGAGGCCCCCAAGGGGAGCCCTGGAAG
CTGGTGGAAGAAGGAACTGTCAGGATCAAGTAGCGCACCCAAATTGGAATACACAGTCCGTACAGACACCCA
GAGTCCGACAAACACTGGGAGCCCCAGTTCCCCCCAGCA.~AAAAGTGAAGGCCTGGGCTCCAGGCACAGACC
AGTGGCCAGGGTAAGCCCCCACTGCAAGAGATCCGAGGCTGAGGCCAAGCCCAGTGGCTCACAGACAGTGAA
CCTGACTGGCAGAGCCAATGATCCATGCGATCTGGACTCGAGAGTCCAGGCCACTTCTGTCAAAGTGACTGT
CGCTGGCTTTCAGCCAGGTGGAGCTGTGGAGAAGGAATCTCTGGGAAAGCTGACCACTGGAGATGCTTGTGT
CTCTACCAGCTGTGAACTAGCCAGTGCTCTGTCCCATCTGGATGCCAGCCACCTCACAGAGAACCTGCCCAA
AGCTGCATCAGAGCTGGGGCAACAACCCATGACTGAACTGGACAGCTCCTCGGACCTCATCTCTTCCCCAGG
GAAGAAGGGGGCCGCTCATCCTGACCCCAGCAAGACCTCTGTAGACACAGGGAAAGTCAGTCGGCCAGAGAA
TCCCAGCCAGCCTGCATCGCCCAGGGTCGCCAAGTGCAAGGCCAGGTCTCCAGTCAGGCTCCCCCATGAGGG
CAGCCCCTCCCCAGGGGAGAAAGCAGCGGCTCCCCCTGACTACAGCAAGACTCGATCAGCATCGGAAACCAG
CACACCCCACAATACCAGGAGGGTGGCTGCCCTCAGGGGAGCGGGACCTGGAGCAGAGGGAATGACACCAGC
TGGTGCTGTCCTGCCAGGAGACCCCCTCACATCCCAGGAGCAGAGACAGGGAGCTCCAGGTAACCACAGTAA
GGCTCTGGAAATGACAGGAATCCATGCACCTGAAAGCTCCCAGGAGCCTTCCCTGCTGGAGGGAGCAGATTC
TGTGTCCTCAAGGGCACCGCAGGCCAGCCTCTCCATGCTGCCATCCACTGACAACACCAAAGAAGCATGTGG
CCATGTCTCGGGGCACTGCTGCCCGGGGGGGAGTAGAGAGAGCCCTGTGACGGACATTGACAGCTTCATCAA
GGAGCTGGATGCTTCTGCAGCAAGGTCTCCGTCTTCCCAGACGGGGGACAGTGGCTCTCAGGAGGGCAGTGC
TCAGGGCCACCCACCAGCCGGGGCTGGAGGTGGGAGCTCCTGCCGTGCCGAACCAGTCCCGGGGGGCCAGAC
CTCCTCCCCGAGGAGGGCCTGGGCTGCTGGTGCCCCCGCCTACCCACAATGGGCCTCCCAGCCTTCGGTTTT
AGATTCAATTAATCCCGACAAACATTTTACTGTGAACAAAAACTTTCTGAGCAACTACTCTAGAAATTTTAG
CAGTTTTCATGAAGACAGCACCTCCCTATCAGGCCTGGGTGACAGCACGGAGCCGTCTCTGTCATCCATGTA
TGGCGATGCTGAGGATTCTTCTTCTGACCCTGAGTCACTCACTGAAGCCCCACGAGCTTCTGCCAGGGACGG
CTGGTCCCCTCCTCGTTCCCGTGTGTCTTTGCACAAGGAAGATCCTTCGGAGTCAGAAGAGGAACAGATTGA
GATTTGTTCCACACGTGGCTGCCCCAATCCACCCTCGAGTCCTGCTCATCTTCCCACCCAGGCTGCCATCTG
TCCTGCCTCAGCCAAAGTTCTGTCATTAAAATACAGCACTCCGAGAGAGTCGGTGGCCAGTCCCCGTGAGAA
GGTCGCCTGCTTGCCAGGCTCATACACTTCAGGCCCAGACTCTTCCCAGCCATCATCACTCTTGGAGATGAG
CTCTCAGGAGCATGAAACTCATGCGGACATAAGCACTTCACAGAACCACAGGCCCTCGTGTGCAGAAGAAAC
CACAGAAGTCACCAGCGCTAGCTCAGCCATGGAAAACAGTCCGCTGTCTAAAGTAGCCAGGCATTTTCACAG
TCCGCCCATCATTCTCAGCTCCCCCAACATGGTAAATGGCTTGGAACATGACCTGCTAGATGACGAAACCCT
GAATCAATACGAAACAAGCATTAATGCAGCTGCCAGTCTGTCCTCCTTCAGTGTGGATGTCCCTAAGAATGG
AGAATCTGTTTTGGAAAACCTCCACATCTCTGAAAGTCAAGACCTGGATGACTTGCTACAGAAACCAAAAAT
GATCGCTAGGAGGCCCATCATGGCCTGGTTTAAAGAAATAAATAAACATAACCAAGGCACACATTTGAGGAG
CAAAACCGAGAAGGAACAACCTCTAATGCCTGCCAGAAGTCCCGACTCCAAGATTCAGATGGTGAGTTCAAG
CCAAAAAAAGGGCGTTACTGTGCCTCATAGCCCTCCTCAGCCGAAAACAAACCTGGAAAATAAGGACCTGTC
TAAGAAGAGTCCGGCAGAAATGCTTCTGACTAATGGTCAGAAGGCAAAGTGTGGTCCGAAGCTGAAGAGGCT
CAGCCTCAAGGGCAAGGCCAAAGTCAACTCTGAGGCCCCTGCTGCGAATGCTGTGAAGGCTGGGGGGACGGA
CCACAGGAAACCCTTGATCTCACCCCAGACCTCCCACAAAACACTTTCTAAGGCAGTGTCACAGCGGCTCCA
TGTAGCCGACCACGAGGACCCTGACAGAAACACCACAGCTGCCCCCAGGTCCCCCCAGTGTGTGCTGGAAAG
CAAGCCACCTCTTGCCACCTCTGGGCCACTGAAACCCTCAGTGTCTGACACGAGCATCAGGACATTTGTCTC
GCCCCTGACCTCTCCCAAGCCTGTTCCTGAGCAAGGCATGTGGAGCAGGTTCCACATGGCTGTCCTCTCTGA
ACCCGACAGAGGTTGCCCAACCACCCCTAAATCTCCTAAGTGTAGAGCAGAGGGCAGGGCGCCCCGTGCTGA
CTCCGGGCCGGTGAGTCCGGCAGCGTCTAGGAACGGCATGTCCGTGGCAGGGAACAGACAGAGTGAGCCGCG
CCTGGCCAGCCATGTGGCAGCAGACACAGCCCAACCCAGGCCGACTGGCGAAAAAGGAGGCAACATAATGGC
CAGCGATCGCCTCGAAAGAACAAACCAGCTGAAAATCGTGGAGATTTCTGCTGAAGCAGTGTCAGAGACTGT
ATGTGGTAACAAGCCAGCTGAAAGCGACAGACGGGGAGGGTGCTTGGCCCAGGGCAACTGTCAGGAGAAGAG
TGAAATCAGGCTCTATCGCCAGGTCGCAGAATCATCCACAAGTCATCCATCCTCACTCCCATCTCATGCCTC
CCAGGCAGAGCAGGAAATGTCACGATCATTCAGCATGGCAAAACTGGCGTCCTCCTCCTCCTCCCTTCAAAC

AGCCATTAGAAAGGCAGAATACTCCCAGGGAAAATCAAGCCTGATGTCAGACTCCCGAGGGGTGCCCAGAAA
CAGCATTCCAGGGGGCCCCTCGGGGGAGGACCATCTCTACTTCACCCCAAGGCCAGCGACCAGGACCTACTC
CATGCCAGCCCAGTTCTCAAGCCATTTTGGACGGGAGGGTCACCCCCCACACAGCCTGGGTCGCTCTCGGGA
CAGCCAGGTCCCTGTGACAAGCAGTGTTGTCCCCGAGGCAAAGGCATCCAGAGGTGGTCTTCCCAGCCTGGC
TAATGGACAGGGCATATATAGTGTAAAGCCGCTGCTGGACACATCGAGGAATCTTCCAGCCACAGATGAAGG
GGATATCATTTCAGTCCAGGAGACGAGCTGCCTAGTCACAGACAAAATCAAAGTCACCAGACGACACTACTG
CTATGAGCAGAACTGGCCCCATGAATCTACCTCATTTTTCTCTGTGAAGCAGCGGATCAAGTCTTTTGAGAA
CCTGGCCAATGCTGACCGGCCTGTAGCCAAGTCCGGGGCTTCCCCATTTTTGTCGGTGAGCTCCAAGCCTCC
CATTGGGAGGCGGTCTTCCGGCAGCATTGTTTCCGGGAGCCTGGGCCACCCAGGTGACGCAGCAGCAAGGTT
GTTGAGACGCAGCTTGAGTTCCTGCAGCGAAAACCAAAGCGAAGCCGGCACCCTCCTGCCCCAGATGGCCAA
GTCTCCCTCAATCATGACACTGACCATCTCTCGGCAGAACCCACCAGAGACCAGTAGCAAGGGCTCTGATTC
GGAACTAAAGAAATCACTTGGTCCTTTGGGAATTCCCACCCCAACGATGACCCTGGCTTCTCCTGTTAAGAG
GAACAAGTCCTCGGTACGCCACACGCAGCCCTCGCCCGTGTCCCGCTCCAAGCTCCAGGAGCTGAGAGCCTT
GAGCATGCCTGACCTTGACAAGCTCTGCAGCGAGGATTACTCAGCAGGGCCGAGCGCCGTGCTCTTCAAAAC
TGAGCTGGAGATCACCCCCAGGAGGTCACCTGGCCCTCCTGCTGGAGGCGTTTCGTGTCCCGAGAAGGGCGG
GAACAGGGCCTGTCCAGGAGGAAGTGGCCCTAAAACCAGTGCTGCTGAGACACCCAGTTCAGCCAGTGATAC
GGGTGAAGCTGCCCAGGATCTGCCTTTTAGAAGAAGCTGGTCAGTTAATTTGGATCAACTTCTAGTCTCAGC
GGGGGACCAGCAAAGATTACAGTCTGTTTTATCGTCAGTGGGATCGAAATCTACCATCCTAACTCTCATTCA
GGAAGCGAAAGCACAATCAGAGAATGAAGAAGATGTTTGCTTCATAGTCTTGAATAGAAAAGAAGGCTCAGG
TCTGGGATTCAGTGTGGCAGGAGGGACAGATGTGGAGCCAAAATCAATCACGGTCCACAGGGTGTTTTCTCA
GGGGGCGGCTTCTCAGGAAGGGACTATGAACCGAGGGGATTTCCTTCTGTCAGTCAACGGCGCCTCACTGGC
TGGCTTAGCCCACGGGAATGTCCTGAAGGTTCTGCACCAGGCACAGCTGCACAAAGATGCCCTCGTGGTCAT
CAAGAAAGGGATGGATCAGCCCAGGCCCTCTGCCCGGCAGGAGCCTCCCACAGCCAATGGGAAGGGTTTGCT
GTCCAGAAAGACCATCCCCCTGGAGCCTGGCATTGGGAGAAGTGTGGCTGTACACGATGCTCTGTGTGTTGA
AGTGCTGAAGACCTCGGCTGGGCTGGGACTGAGTCTGGATGGGGGAAAATCATCGGTGACGGGAGATGGGCC
CTTGGTCATTAAAAGAGTGTACAAAGGTGGTGCGGCTGAACAAGCTGGAATAATAGAAGCTGGAGATGAAAT
TCTTGCTATTAATGGGAAACCTCTGGTTGGGCTCATGCACTTTGATGCCTGGAATATTATGAAGTCTGTCCC
AGAAGGACCTGTGCAGTTATTAATTAGAAAGCATAGGAATTCTTCATGAATTTTAACAAGAATCATTTTCTC
AGTTCTCTTCTTTCTTTAGCAAATCAGAGTGACTTCTTTAAACCACAGGTTGTTGAAATGGCCAACACTGGT
In a search of public sequence databases, the NOVBa nucleic acid sequence, located on chromsome 5 has 997 of 1128 bases (88%) identical to a Papin mIRNA from Rattus noyvegicus (GENBANK-ID: AF1694I 1). Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
SNP data for NOV 1 a can be found below in Example 3.
The disclosed NOV8a polypeptide (SEQ ID N0:25) encoded by SEQ ID N0:24 has 2814 amino acid residues and is presented in Table 8B using the one-letter amino acid code.
Signal P, Psort and/or Hydropathy results predict that NOVBa has no signal peptide and is likely to be localized in the nucleus with a certainty of 0.7000. In other embodiments, NOV8a may also be localized to the microbody (peroxisome) with a certainty of 0.3000, the mitochondrial matrix space with a certainty of 0.1000, or the lysosome (lumen) with a certainty of 0.1000.
Table 8B. Encoded NOVBa protein sequence (SEQ m N0:25).
MPITQDNAVLHLPLLYQWLQNSLQEGGDGPEQRLCQAAIQKLQEYIQLNFAVDESTVPPDHSPPEMEICTVY
LTKELGDTETVGLSFGNIPVFGDYGEKRRGGKKRKTHQGPVLDVGCIWVTELRKNSPAGKSGKVRLRDEILS
LNGQLMVGVDVSGASYLAEQCWNGGFIYLIMLRRFKHKAHSTYNGNSSNSSEPGETPTI,ELGDRTAKKGKRT
RKFGVISRPPANKAPEESKGSAGCEVSSDPSTELENGLDPELGNGHVFQLENGPDSLKEVAGPHLERSEVDR
GTEHRIPKTDAPLTTSNDKRRFSKGGKTDFQSSDCLARSKEEVGRIWKMELLKESDGLGIQVSGGRGSKRSP
HAIWTQVKEGGAAHRLRDGRLSLGDELLVINGHLLVGLSHEEAVAILRSATGMVQLWASKVGVLSAFQMP
GTDEPQDVCGAEESKGNLESPKQGSNKIKLKSRLSGRWGLYLMQPVGGVHRLESVEEYNELMVRNGDPRIRM
LEVSRDGRKHSLPQLLDSSSASQEYHIVKKSTRSLSTTQVESPWRLIRPSVISIIGLYKEKGKGLGFSIAGG
104 .

RDCIRGQMGIFVKTIFPNGSAAEDGRLKEGDEILDVNGIPIKGLTFQEAIHTFKQIRSGLFVLTVRTKLVSP
SLTPCSTPTHMSRSASPNFNTSGGASAGGSDEGSSSSLGRKTPGPKDRIVMEVTLNKEPRVGLGIGACCLAL
ENSPPGIYIHSLAPGSVAKMESNLSRGSILEVNSVNVRHAALSKVHAILSKCPPGPVRLVIGRHPNPKVNQV
SEQEMDEVIARSTYQESKEANSSPGLGTVISIGCFLLQQDSLISESELSQYFAHDVPGPLSDFMVAGSEDED
HPGSGCSTSEEGSLPPSTSTHKEPGKPRANSLVTLGSHRASGLFHKQVTVARQASLPGSPQALRNPLLRQRK
VGCYDANDASDEEEFDREGDCISLPGALPGPIRPLSEDDPRRVSISSSKGMDVHNQEERPRKTLVSKAISAP
LLGSSVDLEESIPEGMVDAASYAANLTDSAEAPKGSPGSWWKKELSGSSSAPKLEYTVRTDTQSPTNTGSPS
SPQQKSEGLGSRHRPVARVSPHCKRSEAEAKPSGSQTVNLTGRANDPCDLDSRVQATSVKVTVAGFQPGGAV
EKESLGKLTTGDACVSTSCELASALSHLDASHLTENLPKAASELGQQPMTELDSSSDLISSPGKKGAAHPDP
SKTSVDTGKVSRPENPSQPASPRVAKCKARSPVRLPHEGSPSPGEKAAAPPDYSKTRSASETSTPHNTRRVA
ALRGAGPGAEGMTPAGAVLPGDPLTSQEQRQGAPGNHSKALEMTGIHAPESSQEPSLLEGADSVSSRAPQAS
LSMLPSTDNTKEACGHVSGHCCPGGSRESPVTDIDSFIKELDASAARSPSSQTGDSGSQEGSAQGHPPAGAG
GGSSCRAEPVPGGQTSSPRRAWAAGAPAYPQWASQPSVLDSINPDKHFTVNKNFLSNYSRNFSSFHEDSTSL
SGLGDSTEPSLSSMYGDAEDSSSDPESLTEAPRASARDGWSPPRSRVSLHKEDPSESEEEQIEICSTRGCPN
PPSSPAHLPTQAAICPASAKVLSLKYSTPRESVASPREKVACLPGSYTSGPDSSQPSSLLEMSSQEHETHAD
ISTSQNHRPSCAEETTEVTSASSAMENSPLSKVARHFHSPPIILSSPNMVNGLEHDLLDDETLNQYETSINA
AASLSSFSVDVPKNGESVLENLHISESQDLDDLLQKPKMIARRPIMAWFKEINKHNQGTHLRSKTEKEQPLM
PARSPDSKIQMVSSSQKKGVTVPHSPPQPKTNLENKDLSKKSPAEMLLTNGQKAKCGPKLKRLSLKGKAKVN
SEAPAANAVKAGGTDHRKPLISPQTSHKTLSKAVSQRLHVADHEDPDRNTTAAPRSPQCVLESKPPLATSGP
LKPSVSDTSIRTFVSPLTSPKPVPEQGMWSRFHMAVLSEPDRGCPTTPKSPKCRAEGRAPRADSGPVSPAAS
RNGMSVAGNRQSEPRLASHVAADTAQPRPTGEKGGNIMASDRLERTNQLKIVEISAEAVSETVCGNKPAESD
RRGGCLAQGNCQEKSEIRLYRQVAESSTSHPSSLPSHASQAEQEMSRSFSMAKLASSSSSLQTAIRKAEYSQ
GKSSLMSDSRGVPRNSIPGGPSGEDHLYFTPRPATRTYSMPAQFSSHFGREGHPPHSLGRSRDSQVPVTSSV
VPEAKASRGGLPSLANGQGIYSVKPLLDTSRNLPATDEGDIISVQETSCLVTDKIKVTRRHYCYEQNWPHES
TSFFSVKQRIKSFENLANADRPVAKSGASPFLSVSSKPPIGRRSSGSIVSGSLGHPGDAAARLLRRSLSSCS
ENQSEAGTLLPQMAKSPSTMTLTISRQNPPETSSKGSDSELKKSLGPLGIPTPTMTLASPVKRNKSSVRHTQ
PSPVSRSKLQELRALSMPDLDKLCSEDYSAGPSAVLFKTELEITPRRSPGPPAGGVSCPEKGGNRACPGGSG
PKTSAAETPSSASDTGEAAQDLPFRRSWSVNLDQLLVSAGDQQRLQSVLSSVGSKSTILTLIQEAKAQSENE
EDVCFIVLNRKEGSGLGFSVAGGTDVEPKSITVHRVFSQGAASQEGTMNRGDFLLSVNGASLAGLAHGNVLK
VLHQAQLHKDALWIKKGMDQPRPSARQEPPTANGKGLLSRKTIPLEPGIGRSVAVHDALCVEVLKTSAGLG
LSLDGGKSSVTGDGPLVIKRVYKGGAAEQAGIIEAGDEILAINGKPLVGLMHFDAWNIMKSVPEGPVQLLIR
KHRNSS
A search of sequence databases reveals that the NOVBa amino acid sequence has of 1741 amino acid residues (53%) identical to, and 1133 of 1741 amino acid residues (65%) similar to, the 2766 amino acid residue Papin protein from Rattus ho~vegicus (Q9QZR8) (E =
0.0), and 122 of 304 amino acid residues (40%) identical to, and 176 of 304 amino acid residues (57%) similar to, the 334 amino acid residue Human interleukin-16 monomer (patp:AAW19209 ) (E =1.Oe~6). Amino acid databases include the GenBank databases, SwissProt, PDB, PATP, and PIR. The global sequence homology (as defined by FASTA
alignment with the full length sequence of this protein) is 72.943% amino acid homology and 69.689 % amino acid identity. In addition, this protein contains the following protein domains (as defined by Interpro) at the indicated nucleotide positions: PDZ dofnains (IPR001478) at amino acid positions 336 to 422, 558 to 644, 700 to 784, 2597 to 2681, 2725 to 2810.
NOVBa is expressed in at least the following tissues: Nervous System. Brain.
Prosencephalon/Forebrain. Diencephalon. Pituitary Gland; Hematopoietic and Lymphatic System. Hematopoietic Tissues. Lymphoid tissue. Lymph node; Whole Organism. In addition, the sequence is predicted to be expressed in the following tissues because of the expression pattern of (GENBANK-ID: AF169411) a closely related Papin homolog in species Rattus faovwegicus: brain. TaqMan data for NOV8 can be found below in Example 2.

NOVBb A disclosed NOVBb nucleic acid of 8640 nucleotides (also referred to as CG57026-04) encoding a novel papin-like protein is shown in Table 8C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 101-103 and ending with a TGA codon at nucleotides 8534-8536. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 8C. The start and stop codons are in bold letters.
Table 8C. NOVBb nucleotide sequence (SEQ ID N0:26).
CTACCAGTGGCTGCAGAACAGCCTGCAGGAAGGTGGGGATGGGCCGGAGCAGCGGCTCTGCCAGGCGGCCAT
CCAGAAGCTGCAGGAGTACATCCAGCTGAACTTTGCTGTGGATGAGAGTACGGTCCCACCTGATCACAGCCC
CCCCGAAATGGAGATCTGTACTGTGTACCTCACCAAGGAGCTGGGGGACACAGAGACTGTGGGCCTGAGTTT
TGGGAACATCCCTGTTTTCGGGGACTATGGTGAAAAGCGCAGGGGGGGCAAGAAGAGGAAAACCCACCAGGG
TCCTGTGCTGGATGTGGGCTGCATCTGGGTGACAGAGCTGAGGAAGAACAGCCCAGCAGGGAAGAGTGGGAA
GGTCCGACTGCGGGATGAGATCCTCTCACTGAATGGGCAGCTGATGGTTGGAGTTGATGTCAGTGGGGCCAG
TTACCTGGCTGAGCAGTGCTGGAATGGCGGCTTTATCTACCTGATCATGCTGCGTCGCTTTAAGCACAAAGC
CCACTCCACTTATAATGGCAACAGTAGCAACAGCTCTGAACCAGGAGAAACACCTACCTTGGAGCTGGGTGA
CCGAACTGCGAAAAAGGGGAAACGAACCAGAAAGTTTGGGGTCATCTCCAGGCCTCCTGCCAACAAGGCCCC
TGAAGAATCCAAGGGCAGCGCTGGCTGTGAGGTGTCCAGTGACCCCAGCACTGAGCTGGAGAACGGCCTGGA
CCCTGAACTTGGAAACGGCCATGTCTTTCAGCTAGAAAATGGCCCAGATTCTCTCAAGGAGGTGGCTGGACC
CCATCTAGAGAGGTCAGAAGTGGACAGAGGGACAGAGCATAGAATTCCAAAGACAGATGCTCCTCTGACCAC
AAGCAATGACAAACGCCGCTTCTCAAAAGGTGGGAAGACGGACTTCCAATCGAGTGACTGCCTGGCACGGTC
CAAGGAGGAAGTTGGCCGAATATGGAAGATGGAGCTGCTCAAAGAATCGGATGGGCTGGGAATTCAGGTTAG
TGGAGGCCGAGGATCAAAGCGCTCACCTCACGCTATCGTTGTCACTCAAGTGAAGGAAGGAGGTGCCGCTCA
CAGGCTCAGGGATGGCAGGCTGTCCTTAGGAGATGAGCTGCTGGTAATCAATGGTCATTTACTGGTCGGGCT
CTCCCACGAGGAAGCAGTGGCCATTCTTCGCTCCGCCACGGGAATGGTGCAGCTTGTGGTGGCCAGCAAGGT
AGGTGTGCTTTCTGCATTTCAGATGCCTGGGACAGATGAACCCCAAGATGTGTGCGGTGCTGAGGAATCCAA
GGGGAACTTGGAAAGTCCCAAACAGGGCAGCAATAAAATCAAGCTCAAGAGTCGCCTTTCAGGTAGGTGGGG
GCTCTACCTGATGCAGCCTGTCGGGGGTGTACACCGCCTTGAGTCAGTTGAAGAATATAACGAGCTGATGGT
GCGGAATGGGGACCCCCGGATCCGGATGTTGGAGGTCTCCCGAGATGGCCGGAAACACTCCCTCCCGCAGCT
GCTGGACTCTTCCAGTGCCTCACAGGAATACCACATTGTGAAGAAGTCTACCCGCTCCTTAAGCACGACTCA
GGTGGAATCTCCTCGGAGGCTCATTCGGCCATCCGTCATCTCGATCATTGGGTTGTACAAAGAAAAAGGCAA
GGGCCTTGGCTTTAGTATTGCTGGAGGTCGAGACTGCATTCGTGGACAGATGGGGATTTTTGTCAAGACCAT
CTTCCCAAATGGATCAGCTGCAGAGGACGGAAGACTTAAAGAAGGGGATGAAATCCTAGATGTAAATGGAAT
ACCAATAAAGGGCTTGACATTTCAAGAAGCCATTCATACCTTTAAGCAAATCCGGAGTGGATTATTTGTTTT
AACGGTACGCACAAAGTTGGTGAGCCCCAGCCTCACACCCTGCTCGACACCCACACACATGAGCAGATCCGC
CTCCCCGAACTTCAATACCAGTGGGGGAGCCTCGGCGGGAGGTTCCGATGAAGGCAGTTCTTCATCCCTGGG
TCGGAAGACCCCTGGGCCCAAGGACAGGATCGTCATGGAAGTAACACTCAACAAAGAGCCAAGAGTTGGATT
AGGCATTGGTGCCTGCTGCTTGGCTCTGGAAAACAGTCCTCCTGGCATCTACATTCACAGCCTTGCTCCAGG
ATCAGTGGCCAAGATGGAGAGCAACCTGAGCCGCGGGGATCAAATCCTGGAAGTGAACTCCGTCAACGTCCG
CCATGCTGCTTTAAGCAAAGTCCACGCCATCTTGAGTAAATGCCCTCCAGGACCCGTTCGCCTTGTCATCGG
CCGGCACCCTAATCCAAAGGTTTCCGAGCAGGAAATGGATGAAGTCATAGCACGCAGCACTTATCAGGAGAG
CAAAGAGGCCAATTCCTCTCCTGGCTTAGGTACCCCCTTGAAGAGTCCCTCTCTTGCAAAAAAGGACTCCCT
TATTTCTGAATCTGAACTCTCCCAGTACTTTGCCCACGATGTCCCTGGCCCCTTGTCAGACTTCATGGTGGT
CGGTTCTGAGGACGAGGATCACCCGGGAAGTGGCTGCAGCACGTCGGAGGAGGGCAGCCTGCCTCCCAGCAC
CTCCACTCACAAGGAGCCTGGAAAACCCAGAGCCAACAGCCTCGTGACTCTTGGGAGCCATCGGGCTTCTGG
GCTCTTCCACAAGCAGGTGACAGTTGCCAGACAAGCCAGTCTCCCCGGAAGCCCACAGGCCCTCCGAAACCC
TCTCCTCCGCCAGAGGAAGGTAGGCTGCTACGATGCCAACGATGCCAGTGATGAGGAAGAGTTTGACAGAGA
AGGGGACTGCATTTCACTCCCAGGGGCCCTCCCGGGTCCCATCAGGCCTCTGTCAGAGGATGACCCGAGGCG
TGTCTCAATTTCCTCTTCCAAGGGCATGGACGTCCACAACCAAGAGGAACGACCCCGGAAAACACTGGTGAG
CAAGGCCATCTCGGCACCTCTTCTTGGTAGCTCAGTGGACTTAGAGGAGAGTATCCCAGAGGGCATGGTGGA
TGCTGCGTCCTATGCAGCCAACCTCACGGACTCTGCAGAGGCCCCCAAGGGGAGCCCTGGAAGCTGGTGGAA
GAAGGAACTGTCAGGATCAAGTAGCGCACCCAAATTGGAATACACAGTCCGTACAGACACCCAGAGTCCGAC

GGTAAGCCCCCACTGCAAGAGATCCGAGGCTGAGGCCAAGCCCAGTGGCTCACAGACAGTGAACCTGACTGG
CAGAGCCAATGATCCATGCGATCTGGACTCGAGAGTCCAGGCCACTTCTGTCAAAGTGACTGTCGCTGGCTT

TCAGCCAGGTGGAGCTGTGGAGAAGGAATCTCTGGGAAAGCTGACCACTGGAGATGCTTGTGTCTCTACCAG
CTGTGAACTAGCCAGTGCTCTGTCCCATCTGGATGCCAGCCACCTCACAGAGAACCTGCCCAAAGCTGCATC
AGAGCTGGGGCAACAACCCATGACTGAACTGGACAGCTCCTCGGACCTCATCTCTTCCCCAGGGAAGAAGGG
GGCCGCTCATCCTGACCCCAGCAAGACCTCTGTAGACACAGGGAAAGTCAGTCGGCCAGAGAATCCCAGCCA
GCCTGCATCGCCCAGGGTCGCCAAGTGCAAGGCCAGGTCTCCAGTCAGGCTCCCCCATGAGGGCAGCCCCTC
CCCAGGGGAGAAAGCAGCGGCTCCCCCTGACTACAGCAAGACTCGATCAGCATCGGAAACCAGCACACCCCA
CAATACCAGGAGGGTGGCTGCCCTCAGGGGAGCGGGACCTGGAGCAGAGGGAATGACACCAGCTGGTGCTGT
CCTGCCAGGAGACCCCCTCACATCCCAGGAGCAGAGACAGGGAGCTCCAGGTAACCACAGTAAGGCTCTGGA
AATGACAGGAATCCATGCACCTGAAAGCTCCCAGGAGCCTTCCCTGCTGGAGGGAGCAGATTCTGTGTCCTC
AAGGGCACCGCAGGCCAGCCTCTCCATGCTGCCATCCACTGACAACACCAAAGAAGCATGTGGCCATGTCTC
GGGGCACTGCTGCCCGGGGGGGAGTAGAGAGAGCCCTGTGACGGACATTGACAGCTTCATCAAGGAGCTGGA
TGCTTCTGCAGCAAGGTCTCCGTCTTCCCAGACGGGGGACAGTGGCTCTCAGGAGGGCAGTGCTCAGGGCCA
CCCACCAGCCGGGGCTGGAGGTGGGAGCTCCTGCCGTGCCGAACCAGTCCCGGGGGGCCAGACCTCCTCCCC
GAGGAGGGCCTGGGCTGCTGGTGCCCCCGCCTACCCACAATGGGCCTCCCAGCCTTCGGTTTTAGATTCAAT
TAATCCCGACAAACATTTTACTGTGAACAAAAACTTTCTGAGCAACTACTCTAGAAATTTTAGCAGTTTTCA
TGAAGACAGCACCTCCCTATCAGGCCTGGGTGACAGCACGGAGCCGTCTCTGTCATCCATGTATGGCGATGC
TGAGGATTCTTCTTCTGACCCTGAGTCACTCACTGAAGCCCCACGAGCTTCTGCCAGGGACGGCTGGTCCCC
TCCTCGTTCCCGTGTGTCTTTGCACAAGGAAGATCCTTCGGAGTCAGAAGAGGAACAGATTGAGATTTGTTC
CACACGTGGCTGCCCCAATCCACCCTCGAGTCCTGCTCATCTTCCCACCCAGGCTGCCATCTGTCCTGCCTC
AGCCAAAGTTCTGTCATTAAAATACAGCACTCCGAGAGAGTCGGTGGCCAGTCCCCGTGAGAAGGTCGCCTG
CTTGCCAGGCTCATACACTTCAGGCCCAGACTCTTCCCAGCCATCATCACTCTTGGAGATGAGCTCTCAGGA
GCATGAAACTCATGCGGACATAAGCACTTCACAGAACCACAGGCCCTCGTGTGCAGAAGAAACCACAGAAGT
CACCAGCGCTAGCTCAGCCATGGAAAACAGTCCGCTGTCTAAAGTAGCCAGGCATTTTCACAGTCCGCCCAT
CATTCTCAGCTCCCCCAACATGGTAAATGGCTTGGAACATGACCTGCTAGATGACGAAACCCTGAATCAATA
CGAAACAAGCATTAATGCAGCTGCCAGTCTGTCCTCCTTCAGTGTGGATGTCCCTAAGAATGGAGAATCTGT
TTTGGAAAACCTCCACATCTCTGAAAGTCAAGACCTGGATGACTTGCTACAGAAACCAAA1~ATGATCGCTAG
GAGGCCCATCATGGCCTGGTTTAAAGAAATAAATAAACATAACCAAGGCACACATTTGAGGAGCAAAACCGA
GAAGGAACAACCTCTAATGCCTGCCAGAAGTCCCGACTCCAAGATTCAGATGGTGAGTTCAAGCCAAAAAAA
GGGCGTTACTGTGCCTCATAGCCCTCCTCAGCCGAAAACAAACCTGGAAAATAAGGACCTGTCTAAGAAGAG
TCCGGCAGAAATGCTTCTGACTAATGGTCAGAAGGCAAAGTGTGGTCCGAAGCTGAAGAGGCTCAGCCTCAA
GGGCAAGGCCAAAGTCAACTCTGAGGCCCCTGCTGCGAATGCTGTGAAGGCTGGGGGGACGGACCACAGGAA
ACCCTTGATCTCACCCCAGACCTCCCACAAAACACTTTCTAAGGCAGTGTCACAGCGGCTCCATGTAGCCGA
CCACGAGGACCCTGACAGAAACACCACAGCTGCCCCCAGGTCCCCCCAGTGTGTGCTGGAAAGCAAGCCACC
TCTTGCCACCTCTGGGCCACTGAAACCCTCAGTGTCTGACACGAGCATCAGGACATTTGTCTCGCCCCTGAC
CTCTCCCAAGCCTGTTCCTGAGCAAGGCATGTGGAGCAGGTTCCACATGGCTGTCCTCTCTGAACCCGACAG
AGGTTGCCCAACCACCCCTAAATCTCCTAAGTGTAGAGCAGAGGGCAGGGCGCCCCGTGCTGACTCCGGGCC
GGTGAGTCCGGCAGCGTCTAGGAACGGCATGTCCGTGGCAGGGAACAGACAGAGTGAGCCGCGCCTGGCCAG
CCATGTGGCAGCAGACACAGCCCAACCCAGGCCGACTGGCGAAAAAGGAGGCAACATAATGGCCAGCGATCG
CCTCGAAAGAACAAACCAGCTGAAAATCGTGGAGATTTCTGCTGAAGCAGTGTCAGAGACTGTATGTGGTAA
CAAGCCAGCTGAAAGCGACAGACGGGGAGGGTGCTTGGCCCAGGGCAACTGTCAGGAGAAGAGTGAAATCAG
GCTCTATCGCCAGGTCGCAGAATCATCCACAAGTCATCCATCCTCACTCCCATCTCATGCCTCCCAGGCAGA
GCAGGAAATGTCACGATCATTCAGCATGGCAAAACTGGCGTCCTCCTCCTCCTCCCTTCAAACAGCCATTAG
AAAGGCAGAATACTCCCAGGGAAAATCAAGCCTGATGTCAGACTCCCGAGGGGTGCCCAGAAACAGCATTCC
AGGGGGCCCCTCGGGGGAGGACCATCTCTACTTCACCCCAAGGCCAGCGACCAGGACCTACTCCATGCCAGC
CCAGTTCTCAAGCCATTTTGGACGGGAGGGTCACCCCCCACACAGCCTGGGTCGCTCTCGGGACAGCCAGGT
CCCTGTGACAAGCAGTGTTGTCCCCGAGGCAAAGGCATCCAGAGGTGGTCTTCCCAGCCTGGCTAATGGACA
GGGCATATATAGTGTAAAGCCGCTGCTGGACACATCGAGGAATCTTCCAGCCACAGATGAAGGGGATATCAT
TTCAGTCCAGGAGACGAGCTGCCTAGTCACAGACAAAATCAAAGTCACCAGACGACACTACTGCTATGAGCA
GAACTGGCCCCATGAATCTACCTCATTTTTCTCTGTGAAGCAGCGGATCAAGTCTTTTGAGAACCTGGCCAA
TGCTGACCGGCCTGTAGCCAAGTCCGGGGCTTCCCCATTTTTGTCGGTGAGCTCCAAGCCTCCCATTGGGAG
GCGGTCTTCCGGCAGCATTGTTTCCGGGAGCCTGGGCCACCCAGGTGACGCAGCAGCAAGGTTGTTGAGACG
CAGCTTGAGTTCCTGCAGCGAAAACCAAAGCGAAGCCGGCACCCTCCTGCCCCAGATGGCCAAGTCTCCCTC
AATCATGACACTGACCATCTCTCGGCAGAACCCACCAGAGACCAGTAGCAAGGGCTCTGATTCGGAACTAAA
GAAATCACTTGGTCCTTTGGGAATTCCCACCCCAACGATGACCCTGGCTTCTCCTGTTAAGAGGAACAAGTC
CTCGGTACGCCACACGCAGCCCTCGCCCGTGTCCCGCTCCAAGCTCCAGGAGCTGAGAGCCTTGAGCATGCC
TGACCTTGACAAGCTCTGCAGCGAGGATTACTCAGCAGGGCCGAGCGCCGTGCTCTTCAAAACTGAGCTGGA
GATCACCCCCAGGAGGTCACCTGGCCCTCCTGCTGGAGGCGTTTCGTGTCCCGAGAAGGGCGGGAACAGGGC
CTGTCCAGGAGGAAGTGGCCCTAAAACCAGTGCTGCTGAGACACCCAGTTCAGCCAGTGATACGGGTGAAGC
TGCCCAGGATCTGCCTTTTAGAAGAAGCTGGTCAGTTAATTTGGATCAACTTCTAGTCTCAGCGGGGGACCA
GCAAAGATTACAGTCTGTTTTATCGTCAGTGGGATCGAAATCTACCATCCTAACTCTCATTCAGGAAGCGAA
AGCACAATCAGAGAATGAAGAAGATGTTTGCTTCATAGTCTTGAATAGAAAAGAAGGCTCAGGTCTGGGATT
CAGTGTGGCAGGAGGGACAGATGTGGAGCCAAAATCAATCACGGTCCACAGGGTGTTTTCTCAGGGGGCGGC
TTCTCAGGAAGGGACTATGAACCGAGGGGATTTCCTTCTGTCAGTCAACGGCGCCTCACTGGCTGGCTTAGC
CCACGGGAATGTCCTGAAGGTTCTGCACCAGGCACAGCTGCACAAAGATGCCCTCGTGGTCATCAAGAAAGG
GATGGATCAGCCCAGGCCCTCTGCCCGGCAGGAGCCTCCCACAGCCAATGGGAAGGGTTTGCTGTCCAGAAA
GACCATCCCCCTGGAGCCTGGCATTGGGAGAAGTGTGGCTGTACACGATGCTCTGTGTGTTGAAGTGCTGAA
GACCTCGGCTGGGCTGGGACTGAGTCTGGATGGGGGAAAATCATCGGTGACGGGAGATGGGCCCTTGGTCAT
TAAAAGAGTGTACAAAGGTGGTGCGGCTGAACAAGCTGGAATAATAGAAGCTGGAGATGAAATTCTTGCTAT
TAATGGGAAACCTCTGGTTGGGCTCATGCACTTTGATGCCTGGAATATTATGAAGTCTGTCCCAGAAGGACC

TGTGCAGTTATTAATTAGAAAGCATAGGAATTCTTCATGAATTTTAACAAGAATCATTTTCTCAGTTCTCTT
CTTTCTTTAGCAAATCAGAGTGACTTCTTTAAACCACAGGTTGTTGAAATGGCCAACACTGGTACAGACACG
In a search of public sequence databases, the NOVBa nucleic acid sequence, located on chromsome 5 has 5828 of 5941 bases (98%) identical to a gb:GENBANK-ID:AF338650~acc:AF338650.1 mRNA from Homo Sapiens (Homo sapiezzs PDZ domain-s containing protein AIPC (AIPC) mRNA, complete cds) (E = 0.0). Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
The disclosed NOVBb polypeptide (SEQ ID N0:27) encoded by SEQ ID N0:26 has 2811 amino acid residues and is presented in Table 8D using the one-letter amino acid code.
Signal P, Psort and/or Hydropathy results predict that NOV8b has no signal peptide and is likely to be localized in the nucleus with a certainty of 0.7000. In other embodiments, NOV8b is also likely to be localized to the microbody (peroxisome) with a certainty of 0.3000, the mitochondria) matrix space with a certainty of 0.1000, and to the lysosome (lumen) with a certainty of 0.1000.
Table 8D. Encoded NOVBb protein sequence (SEQ ID N0:27).
MPITQDNAVLHLPLLYQWLQNSLQEGGDGPEQRLCQAAIQKLQEYIQLNFAVDESTVPPDHSPPEMEICTVY
LTKELGDTETVGLSFGNIPVFGDYGEKRRGGKKRKTHQGPVLDVGCIWVTELRKNSPAGKSGKVRLRDEILS
LNGQLMVGVDVSGASYLAEQCWNGGFIYLIMLRRFKHKAHSTYNGNSSNSSEPGETPTLELGDRTAKKGKRT
RKFGVISRPPANKAPEESKGSAGCEVSSDPSTELENGLDPELGNGHVFQLENGPDSLKEVAGPHLERSEVDR
GTEHRIPKTDAPLTTSNDKRRFSKGGKTDFQSSDCLARSKEEVGRIWKMELLKESDGLGIQVSGGRGSKRSP
HAIWTQVKEGGAAHRLRDGRLSLGDELLVINGHLLVGLSHEEAVATLRSATGMVQLWASKVGVLSAFQMP
GTDEPQDVCGAEESKGNLESPKQGSNKIKLKSRLSGRWGLYLMQPVGGVHRLESVEEYNELMVRNGDPRIRM
LEVSRDGRKHSLPQLLDSSSASQEYHTVKKSTRSLSTTQVESPRRLTRPSVISIIGLYKEKGKGLGFSIAGG
RDCIRGQMGIFVKTIFPNGSAAEDGRLKEGDEILDVNGIPIKGLTFQEAIHTFKQIRSGLFVLTVRTKLVSP
SLTPCSTPTHMSRSASPNFNTSGGASAGGSDEGSSSSLGRKTPGPKDRIVMEVTLNKEPRVGLGIGACCLAL
ENSPPGIYIHSLAPGSVAKMESNLSRGDQILEVNSVNVRHAALSKVHAILSKCPPGPVRLVIGRHPNPKVSE
QEMDEVIARSTYQESKEANSSPGLGTPLKSPSLAKKDSLISESELSQYFAHDVPGPLSDFMWGSEDEDHPG
SGCSTSEEGSLPPSTSTHKEPGKPRANSLVTLGSHRASGLFHKQVTVARQASLPGSPQALRNPLLRQRKVGC
YDANDASDEEEFDREGDCISLPGALPGPIRPLSEDDPRRVSISSSKGMDVHNQEERPRKTLVSKAISAPLLG
SSVDLEESIPEGMVDAASYAANLTDSAEAPKGSPGSWWKKELSGSSSAPKLEYTVRTDTQSPTNTGSPSSPQ
QKSEGLGSRHRPVARVSPHCKRSEAEAKPSGSQTVNLTGRANDPCDLDSRVQATSVKVTVAGFQPGGAVEKE
SLGKLTTGDACVSTSCELASALSHLDASHLTENLPKAASELGQQPMTELDSSSDLISSPGKKGAAHPDPSKT
SVDTGKVSRPENPSQPASPRVAKCKARSPVRLPHEGSPSPGEKAAAPPDYSKTRSASETSTPHNTRRVAALR
GAGPGAEGMTPAGAVLPGDPLTSQEQRQGAPGNHSKALEMTGIHAPESSQEPSLLEGADSVSSRAPQASLSM
LPSTDNTKEACGHVSGHCCPGGSRESPVTDIDSFIKELDASAARSPSSQTGDSGSQEGSAQGHPPAGAGGGS
SCRAEPVPGGQTSSPRRAWAAGAPAYPQWASQPSVLDSINPDKHFTVNKNFLSNYSRNFSSFHEDSTSLSGL
GDSTEPSLSSMYGDAEDSSSDPESLTEAPRASARDGWSPPRSRVSLHKEDPSESEEEQIEICSTRGCPNPPS
SPAHLPTQAAICPASAKVLSLKYSTPRESVASPREKVACLPGSYTSGPDSSQPSSLLEMSSQEHETHADIST
SQNHRPSCAEETTEVTSASSAMENSPLSKVARHFHSPPIILSSPNMVNGLEHDLLDDETLNQYETSINAAAS
LSSFSVDVPKNGESVLENLHISESQDLDDLLQKPKMIARRPIMAWFKEINKHNQGTHLRSKTEKEQPLMPAR
SPDSKIQMVSSSQKKGVTVPHSPPQPKTNLENKDLSKKSPAEMLLTNGQKAKCGPKLKRLSLKGKAKVNSEA
PAANAVKAGGTDHRKPLISPQTSHKTLSKAVSQRLHVADHEDPDRNTTAAPRSPQCVLESKPPLATSGPLKP
SVSDTSIRTFVSPLTSPKPVPEQGMWSRFHMAVLSEPDRGCPTTPKSPKCRAEGRAPRADSGPVSPAASRNG
MSVAGNRQSEPRLASHVAADTAQPRPTGEKGGNIMASDRLERTNQLKIVEISAEAVSETVCGNKPAESDRRG
GCLAQGNCQEKSEIRLYRQVAESSTSHPSSLPSHASQAEQEMSRSFSMAKLASSSSSLQTAIRKAEYSQGKS
SLMSDSRGVPRNSIPGGPSGEDHLYFTPRPATRTYSMPAQFSSHFGREGHPPHSLGRSRDSQVPVTSSWPE
AKASRGGLPSLANGQGIYSVKPLLDTSRNLPATDEGDIISVQETSCLVTDKIKVTRRHYCYEQNWPHESTSF
FSVKQRIKSFENLANADRPVAKSGASPFLSVSSKPPIGRRSSGSIVSGSLGHPGDAAARLLRRSLSSCSENQ
SEAGTLLPQMAKSPSIMTLTISRQNPPETSSKGSDSELKKSLGPLGTPTPTMTLASPVKRNKSSVRHTQpSP
VSRSKLQELRALSMPDLDKLCSEDYSAGPSAVLFKTELEITPRRSPGPPAGGVSCPEKGGNRACPGGSGPKT

SAAETPSSASDTGEAAQDLPFRRSWSVNLDQLLVSAGDQQRLQSVLSSVGSKSTILTLIQEAKAQSENEEDV
CFIVLNRKEGSGLGFSVAGGTDVEPKSITVHRVFSQGAASQEGTMNRGDFLLSVNGASLAGLAHGNVLKVLH
QAQLHKDALWIKKGMDQPRPSARQEPPTANGKGLLSRKTIPLEPGIGRSVAVHDALCVEVLKTSAGLGLSL
DGGKSSVTGDGPLVTKRVYKGGAAEQAGIIEAGDEILAINGKPLVGLMHFDAWNIMKSVPEGPVQLLIRKHR
NSS
A search of sequence databases reveals that the NOVBa amino acid sequence has of 2045 amino acid residues (98%) identical to, and 2022 of 2045 amino acid residues (98%) similar to, the 2641 amino acid residue ptnr:TREMBLNEW-ACC:AAK07661 protein from Homo Sapiens (Human) (PDZ DOMAIN-CONTAINING PROTEIN AIPC) (E = 0.0). Public amino acid databases include the GenBank databases, SwissProt, PDB and PIR.
NOVBb is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain -thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. Expression information was derived from the tissue sources of the sequences that were included in the derivation of the sequence of CuraGen Acc. No. CG57026-04.The sequence is predicted to be expressed in the following tissues because of the expression pattern of (GENBANK-ID: gb:GENBANK-ID:AF338650~acc:AF338650.1) a closely related Homo sapieras PDZ domain-containing protein AIPC (AIPC) mRNA, complete cds homolog in species Homo sapiefzs : prostate. TaqMan data for NOVBb can be found below in Example 2.
The NOVBa, and 8b proteins are very closely homologous as as shown in the alignment in Table 8E.
Table 8E Alignment of NOVBa, and 8b.

NOVBa Novsb NOVBa NOVBb NOV8a NOV8b NOV8a NOV8b NOV8a ~r~.~io ~'~ylr~ai~ .~i.r'..~ . .~.
I I ~ I
NOVBb rt.~ is ti r~ a ~r i r~ r~~

NOVBa r~~i~°.~ .tr~yv rm < <~ii i1 ~~ s it i r~i rcv i NOVBb ~~~ It it v r ~~r ~ i~ a r t v er~

NOVBa r a ~ v ~ '~'r c ~ iC i r d il' ~ ~I e'1' ~ ii' i .~~~.~~' .,_;,~ ~
NOVBb r ~ i ~ r ~ . i r ii i i a r NOVBa ~~~i~~uT~~'~r~~~ia~eZSi~Wa~is vl~~'~'~~~~j~~~~i~i,~T,~~~~~~c~ ;.~ a NOVBb -r ~r r ~vr ~~vr m i w .I.. . ..~..
NOVBa m~. r~wi~ .r y n~~r r : - ..
NOVBb \~.yra wI: w~77~~viui~l~.'i;iWNei'.~.~."fi~:Lr dA11r1aFcf.~iG~CLVaI~.wiil,l:l.'Y4l;i~.~iill..'1 .~....~.. .I.. .~.... ..
NOVBa ~~~.~'~~,~ ..I ~unyl I ~ y w~~y r tlui~-~ r NOV8b ~ a ~R~ t ~~ ~ra r ~ r NOVBa '';': lir ~. ..~ ~r ~~~r Z. ~ : ~ ' ~~.~.~' 'r NOVBb ' ~I~ ~ I II
r r ~ v ~ ~ i .... .
~e!t .a ~' .rJ~;~a~.,:;~"iT
NOVBa '. ~~~~ ' ' NOV8b r' ,r." ~, ~

.i. .i....~~.~...i....~....~;~....i NOV 8 a ~~'r~' ~ ~ ~ S -~liE~~ w' ;S'a~~;~=I~ ~ ~ I i3'1'~~, ail NOVBb Q
79o BDO Blo a2o s3o 840 ...I...~I.~_ ~ ~SIGCF L~~,. ....
NOV8a KVN ~ ~r r NOV8b ~ nr r PKS-PST r 1 .i... .i.. .;,.~...i NOV8a ~W~y ~ .-~~A r.r~~ ~~ ~i~i i NOV8b ~ a ~ r r r ~ i .. ..I.. .I.. .~..
NOVBa ~i~E~~it e1~:~ ,~!.a~~ .,. ~i~i.~~-~~t r~ll~ r a.r~W i NOVBb n ~~ ri ~v~ .r. .' .~. r. r. r r~ er NOVBa ~t~~~~ ~~iI''a~i.:!;:~"~ ~,~~u~y ~~..,~~ ~,."t.~ ''~,~~.
NOVBb ~~ ~ ~ I ii r r ~ ~ ur ~ ~ i r NOVBa . rIW~ ~r~~~~~TSr~~~~~_~'~,~-~I~i~,3~~~r~~~~x~i~i :~ v ~ ; ~ .
NOVBb r i.~ r ~ r ~

CA

' NOVBa :" ' . . ~ . . . .
. . . . .
.

NOVBb . a . r . . .
.~.

NovB ' ~' . ', rj'; ~, i.. ~~ .: ~.~i .,. ~ ~'~..
~ii I I ~. ' ~i .~ : ~~
~ ~ a ;., ~~~~!

NOV8b ~ ~ c. , .., ~~ n~ ..
.
' ~ ' ' .
' ' ' NOVBa , ~.~, ~ c.%~,~:~j! . i. ~I"i"a ,r,;, l ',r ~

NOVBb ~ ~ efii . . c . !-'~

NOVBa . I ', ~ ~'~ ~~~'~ ia~~~:~~'~ ~~Zli~; ~:r'c~~ ~~~3:~ ~

v a ~~ e..
c a. ; ~
~~~~ .

v NOVBb ' ' NOV8a ~ ~ T~#~~'r'e ~.~ u~'~?i i~l~,Ta~ ~iPaTe~_Ti~.~~i ~ ~~

~>' s1 .~.5~-~S .

NOVBb ' ' ' I I

. ..,. .~.... ... . .. ... . .... .... ... ... .
NOVBa .. c ai .. . .... ~. . . . ....
v . i.r. . i.
i~ ;, r.

NOV8b v ~ a . . ,. II. ' . .
i~ ., .
..

I I I I I
I I I
I

NOVBa . I.. .I....I.. . _.... . .. .. . .. ... .
. i~'li'efm .. ~';~;:.~ .. ,. . . .
..~ . ',.'i'i . r : . '!~
.~ .
,. .

NOVBb a~ . . ~.. ., ,. , . r , ~ I
I

:. .. .I.. ,~ .. . .. .. .. ...
NOV8a ~!' :~~~: . '~ ~~i~aT: ... xe;. a"'~'~s. a i~ii~'~ t: i~"'~c;~
~' .
I: ~v~i~. i ~'~~.'~~~u - ~ .fi c ~
~ ; .~

NOVBb '. ~ . c. ~. , ia.. . , a ....
. r oV ~ ' ~ s . x'~i~
8 ' '~i~3~~es's~
a ~"-~i;~' NOV8b ~
~ ~~ , , v ,.."., ~

NOV8a . ~.1, ~. r~~~~~ ~:. .. . .. -~', .Ts' . .:'.

NOV8b . . a~~ . . . a NOV8a a w .' ~~~~'~'~'~JT~~I,.~ ~'~ ~''i~ ~ ~it~'~2'G~~ I ~ ~' . a ~'a ~~~~
:

NOV8b i r a m .. . up . , ~ n .

NOVB . . i i =: ,, ~. .. ~
.. . ii ~rr .~~~.~, r 1 a .y '"

NOVBb . . n. . , .i , .
.. , . , ~ ~

. ... . . . . .. . .I... . . ...
NOV8 il "i~'ii.,.. .. .. , . 'ii;t . r' . . .
n a y II ,.; ~ io r . ii.~ . . , ~.a .
NOVBb i NOV8a i, . ' =i . ~ . . .,: : a w ; . . . . . .
1~.~. , .
.

NOVBb a.. r , v . .
,.~ a.

NOVBa NOV8b ~' ~ ~. ~~. _,. .m .~I~ .~,. ~. , (SEQ ID N0:25) (SEQ ID N0:27) Homologies to either of the above NOVB proteins will be shared by the other NOVB
protein insofar as they are homologous to each other as shown above. Any reference to NOV8 is assumed to refer to both of the NOV8 proteins in general, unless otherwise noted.
The disclosed NOV8 polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table 8F.
Table 8F. BLAST
results for Gene Tndex/ Protein/ OrganismLengthIdentityPositivesExpect Identifier (aa) (%) (%) giI15295903IrefIXPsimilar to 1788 1712/17411716/17410.0 _ NONE RETURNED (98%) (98%) 043060.21 (R.

norvegicus) [Homo Sapiens]

gi~2224541~dbj~BAA2KIAA0300 [Homo1608 1608/16081608/16080.0 0760.11P Sapiens] (100%) (100%) gi1126211061ref1NPPAPIN [Rattus 2766 1906/28432147/28430.0 - norvegicus] (67%) (75%) 075229.11 gi1127514521gb1AAK0PDZ domain- 2641 2256/23432275/23430.0 7661.11AF338650containing (96%) (96%) protein AIPC

[Homo sa iens]

gi~12861607~dbjIBABputative [Mus 364 314/387 337/387 e-157 32241.1 musculus] (81%) (86%) The homology between these and other sequences is shown graphically in the ClustalW analysis shown in Table 8G. In the ClustalW aligmnent of the NOV8 protein, as well as all other ClustalW analyses herein, the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be altered to a much broader extent without altering protein structure or function.
Table 8G. ClustalW Analysis of NOV8 1) Novel NOVBa (SEQ ID N0:25) 2) Novel NOV8b (SEQ ID N0:27) 3) gi1152959031ref1XP 043060.21 similar to NONE RETURNED (R. norvegicus) [Homo Sapiens] (SEQ ID N0:60) 4) gi122245411dbj1BAA20760.11P KIAA0300 [Homo Sapiens] (SEQ ID N0:61) 5) gi112621106IreflNP 075229.11 PAPIN [Rattus norvegicus] (SEQ ID N0:62) 6) gi1127514521gb1AAK07661.11AF338650 1 PDZ domain-containing protein AIPC
[Homo Sapiens] (SEQ ID N0:63) 3~ 7) gi1128616071dbj1BAB32241.11 putative [MllS musculus] (SEQ ID N0:64) ...
NOV8a MPITQDNAVLHLPLLYQWLQNSLQEGGDGPEQRLCQAAIQKLQEYIQLNF

S NOVBb MPITQDNAVLHLPLLYQWLQNSLQEGGDGPEQRLCQAAIQKLQEYIQLNF

gi~15295903~ __________________________________________________ gi~2224541~ __________________________________________________ gi~12621106~ __________________________________________________ g1~12751452~ MPITQDNAVLHLPLLYQWLQNSLQEGGDGPEQRLCQAAIQKLQEYIQLNF

1O gi~128616071 ...
NOVBa AVDESTVPPDHSPPEMEICTVYLTKELGDTETVGLSFGNIPVFGDYGEKR

IS NOVBb AVDESTVPPDHSPPEMEICTVYLTKELGDTETVGLSFGNIPVFGDYGEKR

gi~152959031__________________________________________________ gi~2224541~__________________________________________________ giI126211061__________________________________________________ gi112751452~AVDESTVPPDHSPPEMEICTVYLTKELGDTETVGLSFGNIPVFGDYGEKR

gi~12861607~-_________________________________________________ ...
NOVBa RGGKKRKTHQGPVLDVGCIWVTELRKNSPAGKSGKVRLRDEILSLNGQLM

Z.SNOVBb RGGKKRKTHQGPVLDVGCIWVTELRKNSPAGKSGKVRLRDEILSLNGQLM

gi~15295903~__________________________________________________ gi 2224541__________________________________________________ ~

gi 12621106~__________________________________________________ giI127514521RGGKKRKTHQGPVLDVGCIWVTELRKNSPAGKSGKVRLRDEILSLNGQLM

gi~12861607~__________________________________________________ ...
NOV8a VGVDVSGASYLAEQCWNGGFIYLIMLRRFKHKAHSTYNGNSSNSSEPGET

3S NOVBb VGVDVSGASYLAEQCWNGGFIYLIMLFRFKHKAHSTYNGNSSNSSEPGET

gi~15295903~__________________________________________________ giI22245411__________________________________________________ gi~126211061__________________________________________________ gi~12751452~VGVDVSGASYLAEQCWNGGFIYLIMLRRFKHKAHSTYNGNSSNSSEPGET

40 gi~12861607~__________________________________________________ ...
NOVBa PTLELGDRTAKKGKRTRKFGVISRPPANKAPEESKGSAGCEVSSDPSTEL

4S NOVBb PTLELGDRTAKKGKRTRKFGVISRPPANKAPEESKGSAGCEVSSDPSTEL

giI15295903~__________________________________________________ gi~22245411__________________________________________________ giI126211061_______________________________________________..__ g1112751452~PTLELGDRTAKKGKRTRKFGVISRPPANKAPEESKGSAGCEVSSDPSTEL

S0 gi~12861607~__________________________________________________ ...
NOVBa ENGLDPELGNGHVFQLENGPDSLKEVAGPHLERSEVDRGTEHRIPKTDAP

SS NOVBb ENGLDPELGNGHVFQLENGPDSLKEVAGPHLERSEVDRGTEHRIPKTDAP

giI15295903~__________________________________________________ gi~22245411_______________________________________________,__ gi~126211061__________________________________________________ gi ~12751452~ENGADPELGNGHVFQLENGPDSLItEVAGPHLERSEVDRGTEHRIPKTDAP

giI12861607~__________________________________________________ ..
NOVBa LTTSNDKRRFSKGGKTDFQSSDCLARSKEEVGRIWKMELLKESDGLGIQV

GS NOVBb LTTSNDKRRFSKGGKTDFQSSDCLARSKEEVGRIWKMELLKESDGLGIQV

giI15295903~__________________________________________________ gi12224541~__________________________________________________ gi~12621106~__________________________________________________ gi~127514521LTTSNDKRRFSKGGKTDFQSSDCLA-RQEEVGRIWKMELLKESDGLGIQV

70 giI12861607~__________________________________________________ ..
NOVBa SGGRGSKRSPHAIWTQVKEGGAAHRLRDGRLSLGDELLVINGHLLVGLS

7S NOV8b SGGRGSKRSPHAIVVTQVKEGGAAHRLRDGRLSLGDELLVINGHLLVGLS

giI1529590_________________,________________-_______________ gy ii -_________________________________________________ gy 12621106_________________,________________________________ giI12751452~SGGRGSKRSPHAIVVTQVKEGGAAH-------------------------giI12861607~__________________________________________________ ...

NOVBa HEEAVAILRSATGMVQLWASKVGVLSAFQMPGTDEPQDVCGAEESKGNL

NOVBb HEEAVAILRSATGMVQLWASKVGVLSAFQMPGTDEPQDVCGAEESKGNL

giI15295903~__________________________________________________ g1 22245411---______________________________________________ 1~ i 12621106=
gi 1 -________________________________________________ gi112751452~__________________________________________________ giI128616071_________________________________________________-...

NOVBa ESPKQGSNKIKLKSRLSGRWGLYLMQPVGGVHRLESVEEYNELMVRNGDP

NOVBb ESPKQGSNKIKLKSRLSGRWGLYLMQPVGGVHRLESVEEYNELMVRNGDP

giI15295903~__________________________________________________ gi~2224541~-________________________________________________ gi~1 =
12621106--_-_____________________________________________ gi1127514521__________________________________________________ giI12861607~__________________________________________________ ...

NOV8a RIRMLEVSRDGRKHSLPQLLDSSSASQEYHIVKKSTRSLSTTQVESPWRL

NOVBb RIRMLEVSRDGRKHSLPQLLDSSSASQEYHIVKKSTRSLSTTQVESPRRL

gi~152959031__________________________________________________ g1122245411____________________________________________ gy 12621106_'__-1 _____________________________________________ gi1127514521--------------------------REYHIVKKSTRSLSTTQVESPWRL

giI12861607~__________________________________________________ ...

NOVBa IRPSVISIIGLYKEKGKGLGFSIAGGRDCIRGQMGIFVKTIFPNGSAAED

NOV8b IRPSVISIIGLYKEKGKGLGFSIAGGRDCIRGQMGIFVKTIFPNGSAAED

gi~15295903~__________________________________________________ gi 2224541~__________________________________________________ 4~ i ~ -___________________________-_____________________ gi 12621106 giI12751452~IRPSVISIIGLYKEKGKGLGFSIAGGRDCIRGQMGIFVKTIFPNGSAAED

gi~12861607~__________________________________________________ . .

NOVBa GRLKEGDEILDVNGIPIKGLTFQEAIHTFKQIRSGLFVLTVRTKLVSPSL

NOVBb GRLKEGDEILDVNGIPIKGLTFQEAIHTFKQIRSGLFVLTVRTKLVSPSL

gi I15295903~-_________________________________________________ gi 22245411__________________________________________________ i 12621106__________________________________________________ g1 1 gi1127514521GRLKEGDEILDVNGIPIKGLTFQEAIHTFKQIRSGLFVLTVRTKLVSPSL

gi~12861607~_______________.__________________________________ ..

NOV8a TPCSTPTHMSRSASPNFNTSGGASAGGSDEGSSSSLGRKTPGPKDRIVME

NOVSb TPCSTPTHMSRSASPNFNTSGGASAGGSDEGSSSSLGRKTPGPKDRIVME

giI15295903~ __________________________________________________ giI2224541~ ____________________-_________________________ I =' gi __-________________--___________________________ gi112751452~ TPCSTPTHMSRSASPNFNTSGGASAGGSDEGSSSSLGRKTPGPKDRIVME

gi~12861607~ ______________________________-___________________ . .

NOV8a VTLNKEPRVGLGIGACCLALENSPPGIYIHSLAPGSVAKMESNLSRGS-I

NOV8b VTLNKEPRVGLGIGACCLALENSPPGIYIHSLAPGSVAKMESNLSRGDQI

giI152959031__________________-____________________-__________ gi122245411__________________________________________________ gi 1 __________________________________________________ gi I12751452~VTLNKEPRVGLGIGACCLALENSPPGIYIHSLAPGSVAKMESNLSRGDQI

gi ~12861607~_____________________________________..____________ . .

NOV8a LEVNSVNVRHAALSKVHAILSKCPPGPVRLVIGRHPNPKVNQVSEQEMDE

NOVBb LEVNSVNVRHAALSKVHAILSKCPPGPVRLVI---GRHPNPKVSEQEMDE

giI15295903~ _______________________________-__________________ gi122245411 __________________________________________________ giI126211061 ___________________________-_ , ______________-___ gi1127514521 LEVNSVNVRHAALSKVHATLSKCP------------------VSEQEMDE
gi~12861607~ __________________________________________________ I

NOVBa ....
....1....1....1....1....
....
....1....1....
VIARSTYQESKEANSSPGLGTVISIGCFLLQQDSLISESELSQYFAHDVP

NOVBb VIARSTYQESKEANSSPGLGTPLKS-PSLAKKDSLISESELSQYFAHDVP

1O gi~15295903f gi122245411_________________________________-________________ gi~12621106__________________________________________________ ~

gi~12751452VIARSTYQESKfiANSSPGLGTPLKS-PSLAKKDSLISESELSQYFAHDVP

gi~12861607~__________________________________________________ ..
NOV8a GPLSDFMVAGSEDEDHPGSGCSTSEEGSLPPSTSTHKEPGKPRANSLWL

NOVSb GPLSDFMWGSEDEDHPGSGCSTSEEGSLPPSTSTHKEPGKPRANSLVTL

2O gi1152959031__________________________________________________ giI2224541~__________________________________________________ gi~12621106~____-_____________________________________________ gi~127514521GPLSDFMVAGSEDEDHPGSGCSTSEEGSLPP--STSSEPGKPRANSLVTL

gi~12861607~__________________________________________________ ~S

..
NOV8a GSHRASGLFHKQVTVARQASLPGSPQALRNPLLRQRKVGCYDANDASDEE

NOV8b GSHRASGLFHKQVTVARQASLPGSPQALRNPLLRQRKVGCYDANDASDEE

3 gi~15295903~
O

gi~22245411__________________________________________________ giI126211061__________________________________________________ gi~12751452~GSHRASGLFHKQVTVARQASLPGSPQALRNPLLRQRKVGCYDANDASDEE

giI12861607~__________________________________________________ NOV8a EFDREGDCISLPGALPG~iRPLEDDPRRVSISSSKGMDVHNQEERPRKT

NOV8b EFDREGDCISLPGALPG
TRPL EDDPRRVSISSSKGMDVHNQEERPRKT
~

giI152959031______________MLR
QpT~-__________-__________--___ gi122245411 gi~12621106~~TQD_____________________~,LHLP
________________ ~
~

g.i1127514521~
~I-.RPL'~EDDPRRVSISSSKGMDVHNQEERPRKT
EFDREGDCISLPGALP
G

g11128616071__________________.._____________________-_________ S

'1_,...I....1..
NOVSa .I....I....1....1....1....1....1 NOV8b LVSKA~TSAPLLGSSVDL~SIPEGMVDAASYAANLTD~AEAPKGSPGSWW
ESIPEGMVDAASYAANLTDAEAPKGSPGSWW
LVSKA~SAPLLGSSVDL

SO gi~15295903~~
---ATQRPPRGA--L GGRRNCQDQVAHPNWNTQ~~,'VQTPR-------gi 22245411__________________________________________________ giI126211061LLYEW QNSLREGGDSP~QRLCQAAIQKLQEYIQLNL~7DESTVPPDHSP

gi112751452~LVSKI~SAPLLGSSVDL~ESIPEGMVDAASYAANLTD~1EAPKGSPGSWW

gi1128616071_____.____________--______________________________ loso lo7a loso 1090 lloo ....1....~....1....x....1....1....1....1....1....1 NOVBa KKELSGSSSAPKyEYTVRTDTQSPTNTGSPSSPQ
EGLGSRHRPVA

~
NOVBb ~
llIA
KKELSGSSSAPKLEYTVRTDTQSPTNTGSPSSPQ
SEGLGSRHRPV
~~!1 6O gi1152959031 ~
----------------------VRRTLGAPVPPS
KAWAPGTDQWPG

gi122245411 __________________________________-_______________ gi1126211061 PEMEICTVYLTKQLGDTETVGLSFGNIPVFGDYGRGGKKRKTHQG~

gi~12751452~ KKELSGSSSAPKLEYTVRTDTQSPTNTGSPSSPQ~SEGLGSRHRPV
~,',1~1A

gi112861607~ __________________________________________________ '1130 1140 NOVBa SPHCKRSE.
~CP;~GSQTVN,LTG~~v'~' ,'.,".,~y.PCDLD
RVQATS
G~!QPG

NOV8b SPHCKRSE
~CPSGSQTV~TLTGPCDLDRVQATS
G~'.,QPG

70 gi I15295903~SPHCKRSE
P~GSQTV1~LTG~~PCDLD~SRVQATS
G:k'QPG

gi 22245411__________ ____ ____________ ______________ ___ gi 12621106LDVGCIWVT
1 LItKttSPAG
gi ~ 127514521G L ~EILSL~TGQLMV
D G~S'Y,...LAE

SPHCKRSEA~AXPSGSQTV~~TLTG' ~PCDLD~RVQATS~K~G~",QPG

gi ~12861607~_____________.__________________,_____________.___ ~
~
I~

NOV8a ~GK~TTGDACVST
GAVEK---E
CELA1~AL~HLDASHL
EN~pKAA~
L

NOVBb GAVEK---ES G TTGDACVSTSCE LDASH E P L
gi 152959031 GAVEKLCQES~GFC~TTGDACVSTCELA ALBHLDASHL~EN~PK~L
gi 22245411 ___________________.______________________________ gi1126211061 QCWNG-GFIY~~RRFKQKAHV~YNG~SEPGETP~L~GDQT ILK
811127514521 GAVEKLCQESlLJT.~..~~!lTTGDACVST;,,~',CE.~.,~G~,~,~L
HLDASHLLCC~~~17E~,,.~,l~~P L
8i1128616071 ---------- M RRFKQKAHL'I'YNG G SEPGETP LE GDQT KK

NOVBa ~y,.Qf7PMTELDS..,' .1 . .1:;:.1~..:1..,.. ~ : :::.1 NOVBb ~QQPMTELDS . '.' . ' .~~

8i1152959031 QQPMT--- . ~.~ . ~ .~~
8i122245411 _________ , .,. , K . ,..
8i1126211061 ~KItTRK-----FGV ~SISKTPEDS S~GC. DPNS LE~TG----gi1127514521 ~Q~PMT-__ , . .,. , .. . ,..
8i1128616071 1L~1KI?,,TRK-----FGA' II ~PEDS ~uGC. ~DPSS LEG----Novsa ~~~ ~ ~ ' NOVBb w .~ ~~i.
8i1152959031 ' T~w ' ~ ~ . ~ o . .., : ~ ~
8i122245411 .., 87.1126211061 --ADPLG'~,~GHAFEL ~HSLICD~' --HLE~ E,'AT7~EVELRVP~CTE
8i 1 127514521 1'y~T " ~
8i1128616071 --TDSLGGHAFEL ~,.SL2ZD~' --HLE~ EADREAELRVP;TE

NOVBa ' .' . .'.
~. ,- a an s NOVSb ~ .~ . .~.
.,~ .
8i1152959031 ~e~~~ ~~ ~ .~.~~ . .,. .' ,,., , . yy a ~,.. ,,. ,~ ,.,,., 8i122245411 ~ ~ I . .I ,. , y.v .. n 8i1126211061 ~PLSDSNDKRRFS:KT KTDFQSSDCLAR~EVRIWKMELLI SD 'GIQV
3 5 gillz7s14s21 . . . . .... . ,. , ,., ..
8i1128616071 ~PLSDSNDKRRF~..KT~KTNFQSSD;~LAR~SE RIWEMELLI SD~~GIQV

y : .
" .. ..
:; .
:. ..

40 ~ _i Novea NOVBb . ~ .

8i1 152959031 .a ~~.. v. .
.

8i1 22245411 . '~ .

8i 126211061 GRG KRSPHAI LGbELLVI LLL H
, QVK~GGAAHRDGRLS

8i 127514521 ~ .
. , 8i1 128616071 GRG KRSPHAI LLL H
,_,.~VKGGA~iHRDGRL'~LG~,'ELLU;~=

NOVBa " ~ .. .. . . .. .. . .
NOVBb . . .~ . .
giI152959031 . . .~ . . . ~.
gi~22245411 . . .~ . . ~ ~, 8i 1262110fi~ E~ I~ TGh>SX,,QL ~~SKMPG E;:iS.Q~ S E KGN-LE~PKQ CK
8i 12751452 . . .~ ~ . ~ ~.
8i1128616071 E~ ITGMVQL ~~SKMLG E~;SQ S E KGNtNLEPKQ K

60 Novea .. .. ; . ;.. .. . .. . .
NOVBb ~ . ~ .~ . ~.
8i1152959031 ..~. . , . ., . ,.; ,. ., v 8i122245411 ~ . .' . '~
giI126211061 T~~LSRLS VHRLESVEEYNELMVRNGDP~IRM bSRDG~KHSLP~LL
65 gi1127514s21 ~.
8i1128616071 M~~LSRLS VHRLESVEEYNELMVRNGDP~I ~VSRDG~KH~PI~LL

..1.. ...1....1 70 NOVBa .. .. '~ ~ ___ _________ NOVBb -. . --- --------gi1152959031 . , ___ _________ 8i122245411 . , _-_ _____-___ giI126211061 D TGT QE~HIVH3(S~P TTHVE$PWRLI ~ ,I I.,I LYKEKGKGLG
75 giI127514521 . . ___ _________ 8i ~ 128616071 D TGT ~E1HIVK,KS,~'.. TTHVE,~v~,~.PWRLI ~ ,I ICI ----------...
NOVBa __________________________________________________ NOVBb __________________________________________________ gi~1 __________________________________________________ g1~i __________________________________________________ giI12621106~FSIAGGRDCIRGQMGIFVKTIFPNGSAAEDGRLKEGDEILDVNGIPIKGL

gi~12751452~__________________________________________________ gy 12861607'__________________________________________________ 1~ 1610 1620 1630 1640 1650 ..
NOV8a __________________________________________________ NOVBb __________________________________________________ ~ __________________________________________________ D

gi~11 __________________________________________________ giI12621106~TFQEAIHTFKQIRSGLFVLTVRTKLLSPSLTPCSTPTHMSRSSSPSFNTN

giI127514521__________________________________________________ gi~12861607~__________________________________________________ ...
NOVBa ___________.______________________________________ NOVBb ________________________________,._________________ giI15295903~ __________________________________________________ gi~22245411 -_________________________________________________ gi'12621106~ SGGTPAGGGQEEGGSSSLGRKAPGPICDRIVMEVTLNKEPRVGLGIGACCL

gi~127514521 __________________________________________________ gi~12861607~ __________________________________________________ ..
NOVBa __________________________________________________ NOVBb __________________________________________________ 1 __________________________________________________ i 3 g __________________________________________________ 5 i j i giI126211061 ALENSPPGIYIHSLAPGSVAKMESNLSRGDQILEVNSVNVRHAALSKVHA

giI127514521 __________________________________________________ gi~12861607~ __________________________________________________ y y ~
~

NOVBa ....~....
...
...
....~....~_...~....~....p _..
___________________________________________..______ NOV8b __________________________________________________ 1 __________________________________________________ gi 45 i _______________,.__________________________________ i g1 gi~126211061 ILSKCPPGPVRLVIGRHPNPKVSEQEMDEVIARSTYQESREANSSPGLGT

gi~12751452~ __________________________________________________ gi~12861607~ __________________________________________________ ..
NOVBa _______________,.__________________________________ NOVBb __________________________________________________ gi 1529590-_________________________________________________ ~

55 gi i __________________________________________________ i g7.~12621106~PLKSPSLAKKDSLLSESELSQYFVHDGQGSLSDFWAGSEDEDHPGSGXE

gi ~12751452~_____________________________________,.____________ gi ~12861607~__________________________________________________ ...
NOV8a __________________________.________________________ NOVBb __________________________________________________ gi~1529590 __________________________________________________ ~

i __________________________________________________ g1~2224541 gi~12621106~ TSEDGSLLPVPSAHKARANSLVTLGSQRTSGLLHKQVTVARQASLPGSPQ

gi~12751452~ __________________________________________________ gi~12861607~ __________________________________________________ 1910 1920 1930 1940 7.950 NOVBa __________________________________________________ NOVBb __________________________________________________ gii1529590i~ __________________________________________________ 75 gi 2224541 __________________________________________________ gi~126211061 VLRNPLLRQRRVRCYDSNGGSDDEDFDGEGDCISLPGVLPGPGKPLVEDD
giI12751452~ __________________________________________________ gi~12861607~ __________________________________________________ 11g NOVBa ....I....
....1....I....I....I...,I....I....1....
__________________________________________________ NOVBb -_________________________________________________ gi1152959031 __________________________________________________ gi~2224541~ __________________________________________________ gi1126211061 TRPALTTSSKSIDVNKQEERLQKPLVSKACSVPLLGSSLDSEHSILNGAG

gi~12751452~ __________________,_______________________________ 1~ gi~12861607~ -_________________________________________________ ...
NOVBa ________________________________________,_________ 1~JNOVBb __________________________________________________ gi1152959031__________________________________________________ gi~22245411__________________________________________________ gi1126211061GTPPKVASLPGSGETPKNGPRGSGRKEMSGSRSSPKLEYRVPTDTQSPRS

gi~12751452~__________________________________________________ gi~12861607~_--_______________________________________________ ..
NOVBa ___,____________________________________,._________ ~S NOVBb __________________________________________________ gi1152959031 --________________________________________________ giI22245411 __________________________________________________ giI126211061 PENHTSPPQKSENLVSRHKPVARISPHYKRSDAEEAPGGTANGPCAQDLK

gi~127514521 __________________________________________________ giI12861607~ __________________________________________________ ...
NOVBa __________________________________________________ 35 NOVBb __________________________________________________ gi1152959031_________________________.,________________________ gi~22245411__________________________________________________ giI126211061VQASPVKDPVTSRQPGGTAEKELRGNPTPGDSSVPTNCGPASTPCHPNIG

gi1127514521__________________________________________________ 4~ gi~128616071__________________________________________________ ..
NOVBa __________________________________________________ 45 Noveb __________________________________________________ giI15295903~_-.-______________________________________________ gi~2224541~_________________________,________________________ giI12621106~LPTENPQGAAPECGPHPGTGWDGSSEHLCSPGKSREVHPDSSETPTVAEQ

gi1127514521_______________________________________________,__ gi1128616071__________________________________________________ ...
NOVBa __________________________________________________ SS NOVBb __________________________________________________ qy 31 ______________________________________________ q' 1 ___ 2224541_______________________________________________ gi1126211061VHQPESLSQPVSPRTSEPESQGISKMKPPSQRCVSPREKASTPPDSSRAW

gi1127514521_________________________________________________ gi112861607~__________________________________________________ ~

~
~

NOVBa ....
....
....
....
....
....
....
....~....
....
__________________________________________________ 65 NOVBb __________________________________________________ gi1152959031__________________________________________________ gi122245411__________________________________________________ gi1126211061AAPGDSSPSTRRIAVPMSTGAAPATAIPQASLVSQERSRGLSGPSKGLGT

gi112751452________________________________,._________________ gi 1 __________________________________________________ I
I
I
I
I
I

....
NOVBa ....
....
....
....
....I
....I....I....1....
__________________________________________________ 7S NOVBb __________________________________________________ gi115295903~__________________________________________________ gi I22245411__________________________________________________ gi I126211061KELCIPKSLKDGALLEDTAPASGKMSHASSPSGPVATERTLSGSPENPVT

gi~12751452~ __________________________________________________ giI128616071 __________________________________________________ ~~ '2360~ 2370~ .2380~ .2390. 2400 ~. .y ~ y ~~
NOVBa __________________________________________________ NOVBb __________________________________________________ giI15295903~ __________________________________________________ gi122245411 1O giI12621106~ DIDNFIEEASEARLSQSPQKADCRAHGDTFESQPPGGAGSSSSHHAQMVR
giI127514521 ____________________'._____________________________ gi~12861607~ __________________________________________________ ...
NOVSa __________________________________________________ NOVBb __________________________________________________ gi~15295903~ __________________________________________________ gi122245411 2O gi~126211061 SDQTSSPRKTGGTGSPPPQQWALQPSVLDSIHPDKHLAVNKTFLNNYSRN
giI12751452~ __________________________________________________ gi~12861607~ __________________________________________________ ....~....~....~....~....~.... .... .... .... ....
NOVBa ___________________________,. , ,. ..i. . , i.
NOVSb __________________________ ~ , r~ ..,-gi~152959031 ___________________________,. , .~, 81.122245411 ___________________________,~ , . . . .., 3O giI12621106~ FSNFHEDSISLSGPGGSSEPSPSSMY ~ ~ ~' D~G~
gi~12751452~ ___________________________,. , ,~ .., 8i 12861607 __________________________________________________ .... .... ....
.... .... .... ....
....
....
....

NOVBa ~ t ~ v ~

NOVBb ~ ~ ~

gi~ 15295903~ m a awa c ~
a gi~ 2224541~ .. ,. , .. . ,..

126211061 'LPE SPGS DGT- ----~,TAPPPTQ
gi~

8i1 12751452~ ~ v .
,..

gi~ 12861607~__________________________________________________ NOVBa m v NOVSb ~~ v~ v r.
gi~15295903~ ~~ v~ v gi~22245411 ~ ' ~ " ' - w 8i I 12621106 ~ LCP Pt7QQRAVCI~P GDIC F~ACF. ~G I ~ ~ ~ ~F F 1~ E
gi~12751452 gi~12861607~ __________________________________________________ NOVBa t NOVBb gi~ 15295903) giI 22245411 12621106~P W ~ T GIM, QSQ~T F
giI S T CRI~

gi~ 12751452~ r gi~ 12861607~__________________________________________________ y ....~...y ..
NOVBa ~ m ~ ~ ~
NOVBb v " isr~~i~-giI152959031 i~ a ~ ma m a . ~~~ . ~ ~ ~ n.
8i122245411 ~ m ~ ~~
gi~12621106~ G ' D G ~ WGAPK'~GAAp'AJVM' ;FALGA~, gi~12751452~
gi~128616071 __________________________________________________ .... .... .... .... .... .... .... .... .... ....
NOVBa w m v ~ "~mv v~
NOV8b giI15295903~ n. " " w~. ..i . ,m w ,~

gi122245411 m m ~ ' ~ ~ a~
gi1126211061 A RGI E~ ~ ~~T Sw ~ LT DSO S ~ TS ASS
gi1127514521 gi~12861607~ __________________________________________________ NOVBa . . . .. .
.. ..

NGVBb 1~ gi1152959031 ~' ~ ~

gi122245411 w ~ ~ w gi1126211061LLG TGH SR~K$ ~ T
PP GN-gi~12751452~

811128616071________ _________ __________________ _______________ NOVBa ~ m NOVBb ~ ~

2~ 811152959031v v a ~
." . v 8i12224541)~ ~

giI126211061C~P -S SP-- ~ T
S I~T P
GS

8i1127514521 ~

811128616071________ _________ __________________ _______________ .. 2880 2900 NOV8a ~ ~w NOV8b ~ ., ,.,. ., .- ~,- .
, 8i1152959031 8i122245411 .~. ~ o~~ :w a~,.
~ , ' 8i1126211061 H' I~QS T
MF KNTAGDT'~P P Q

8i1127514521 gi~12861607~ -_____________________________________________ --_-NOVBa ..
NOVBb ~ ' ~ 'm 8i1152959031 ~
8i122245411 ~
8i1126211061 ~ T ~ ~ L~,~ ' YL S~ S ~ S ~ SGP
8i1127514521 ~
gi~12861607~ __________________________________________________ ....~....1....1....1....1....1....1....1.... ....1 NOV8b w w ~~ '~~' 8i 152959031 ~~~ '~ ~ ~~~' gi~22245411 .., ,,..
8i1126211061 K~ H~ S ~P~ ~ S~,AL~ Iw QFT~G ~DL~VT ~QGIC
8i1127514521 .~, .~ , .,..
8i1128616071 _-________________________________________________ ....1....x....1....1....1....1.... ....1.... ....1 NOVBa m NoVBb m ~ m 8i1152959031 m ~ ~~ w ~ a 8i122245411 ~~ v 8i1126211061 EKK~ ~PP.y ~ S PP~N ~P ~' F T~
8i1127514521 ~ ~ m 8i1128616071 __________________________________________________ NOVBa ~~ . . . . .
NOV8b 8i1152959031 0 0 ~ ~v ~o . ~ v~ova 8i 22245411 w ~
8i 126211061 ~ ' R-ASE PE ~PFP~ Q' ~ TRW ' P~T
8i 127514521 8i 128616071 __________________________________________________ ....1....x....1....1....1....1....1...
NOVBa ~-NOVBb - . ' .

giI15295903~ - ' giI2224541~ - ' gi~12621106~ SLPA2CLPaSF.~.QPs Gt7IL ss~SP

gi~12751452~ -.

gi~12861607~______________ ___________________ _________________ NOVBa ' NOVBb ' gi~152959031 s s c.... r ~ 'es gi12224541~ . w gi~12621106~ . PS' PS PQDPQVPAMGGKLSE
T' ---jV

15 g11127514521'. ' gi~12861607~ _________________________________________________ _ 2o NOVBa ~ ~ ~
s . i. i~ .o :.
NOVBb . . W ~ . . i~ - t . t gi~152959031 . m, ,I~.. , ..,i :,~ ~ ~ -,I~F' w ~
m .. r1' a ~ , .. ,, gi~2224541~ ~ 's ~ ~,I~.. ~ ." . s. ~~I,. y~ .,"I, gi~12621106~ T a ~ ~~ E ~ SP . .~SA~DP ~ I'~P.' - 'Q C
25 gy 127s14sz~ , - .I.- . -, giI12861607~ __________________________________________________ 30 NOVSa ,- . i~
:i;

NOV8b . ' gi~15295903~ . a ~ . .~, ..
.
.

gi~2224541~ . .' gi~12621106~ 9 ' ' ' ' ' S T
CAT

35 giI127514521 . ' gi~12861607~ __________________________________________________ 40 Novsa ~ ~~~ ~ ~~ ~
r NOVBb ~ ~~~ ~ '~ ~
giI15295903~ .,,.. .~..~om:a~ ~.
gi~z2245411 ' ~~' ~ '~
gi1126211061 P --- S~S. T$'S ' S~ ~S','$' ~~ T S
45 giI12751452~ ~ ~~~ . '~ ~
giI12861607~ __________________________________________________ 5~ NOV8a '~ ~ ~
NOV8b '. ~ .
gi~15295903~ ~~~ " a ~
gi~2224541~ '~ ' ~
55 g1 12621106 ~ ~ PSP~P ~ S SP~ S
gi 127514521 '~ ~ ~
gi112861607~ __________________________________________________ 3410 3420 3430 3440 ~ 3450 NDVBa ~.~ ~ ~~ ~
NOV8b .~ ~
gi~15295903~ .' ~ '. . - .
gi122z45411 .' ~ ~~ ~ - ~
gi112621106~ . 'T ~. . G .D ~S~ Q
65 giI1z751452~ . ~. . - .
gi~12861607~ __________________________________________________ 7O NOVBa _______________ NOVBb _______________ :y.-.
gi115295903~ _______________ gi122245411 .. _________________ . ..
gi1126211061 ~ SQ~TSPAGSPARGHADFNGSTF1~ T~~YT S~P EPAI
75 gi~12751452~ _______________ gi~12861607~ --________________________________________________ NOVBa 1 w w w ~

NOV8b r ..". w " r , gi152959031 1 m ~~ m ~ r g12224541) 1 ~m ' - ' ' n1 gi1126211061At,~'GSR~',E9,VWATSG S r ~~~~~GI.L~cc p gi1127514521~ .. w r w , r "

gi~12861607~________ ____________ _________ _______ ______________ ...
NOVBa wiw ~ ~i 1 -NOVBb v~ 1 1 152959031 ~~ 1 g1 ~

gi 2224541~ m~ .~ 1 ~ v gi1 126211061 ~w T~ IT

gi1 127514521 ~~ ~ r giI 128616071__________________________________________________ NOVBa -,...,- , :i~n . -v nr y iu .~. ; ~i r/

NOVBb : ~I11 , '~I I .~ I ,.,".
~ 1 1 I I / '1 1 ' I

g1 r v~ 1 : ~ i v ~ ~I~1 a I 1r w 152959031 1 r ''~ ~
~ I

gi122245411 ~ vII- 1 ~ c1 , ,a r~Il1 gi ~ ~S 1 T SET ~ ET!

gi ~ ~ 1 ~ ~~

gi1128616071 _______________ _______ ________________ _____ _______ NOVB a ... ;'~ .. ..
~
~a NOVBb r~ w .1v g11152959031 m -, ~.. . ~.,~.. ,, - ." , gi122245411 w w ' -gi1126211061 r~ ~ S PFP w G ~

gi1127514521 1 gi1128616071 ______________ ________ ________________ _____ _______ NOV8a .1.... ....1... . ..1.. . . ...
~ .. .... ... ... .
1 ~w t NOVBb ~ . . , 1 .~. r.
v ., gi1152959031 . m ~ ,. . .a,. . , gi122245411 ~ r gi1126211061 ~ S w F. T
E

gi1127514521 gi1128616071 ______________ ________ ________________ _____ _______ NOVBa ~ ~ t I~I~ ~~In h;,~/rm ~~~ ~w ~n 1 . ,~- .
NOVBb 11 ~~ryn Ira r e~ y ,I r giI152959031 y1 r v-gi 22245411 gi1126211061 r~ ~ ~ 'D -gi1127514521 ~~ ' gi~128616071 --_-________________________________ Table 8H-J lists the domain description from DOMAIN analysis results against NOVB.
This indicates that the NOV8 sequence has properties similar to those of other proteins known to contain this domain.

Table 8H. Domain Analysis of NOV8 gnllSmart~smart00228, PDZ, Domain present in PSD-95, Dlg, and ZO-1/2.;
Also called DHR (Dlg homologous region) or GLGF (relatively well conserved tetrapeptide in these domains). Some PDZs have been shown to bind C-terminal polypeptides; others appear to bind internal (non-C-terminal) polypeptides. Different PDZs possess different binding specificities. (SEQ ID NO:86) CD-Length = 86 residues, 95.3% aligned Score = 69.3 bits (168), Expect = 3e-12 Query: 333 RIWKMELLKESDGLGIQVSGGRGSKRSPHAIWTQVKEGGAAHRLRDGRLSLGDELLVIN 392 +1I I III + p + I +1I+ I I I + I ~ II +I +I
Sbjct: 1 EPRLVELEKGGGGLGFSLVGGKDSGDGG--WVSSWPGSPAAK--AG-LKPGDVILEVN 55 S
Query: 393 GHLLVGLSHEEAVAILRSATGMVQLW 419 I + II+I III +I+ I I I I I
Sbjct: 56 GTSVEGLTHLEAVDLLKEAGGKVTLTV 82 Table 8I. Domain Analysis of NOV8 gnllSmart~smart00228, PDZ, Domain present in PSD-95, Dlg, and ZO-1/2.;
Also called DHR (Dlg homologous region) or GLGF (relatively well conserved tetrapeptide in these domains). Some PDZs have been shown to bind C-terminal polypeptides; others appear to bind internal (non-C-terminal) polypeptides. Different PDZs possess different binding specificities. (SEQ ID N0:86) CD-Length = 86 residues, 96.5% aligned Score = 66.2 bits (160), Expect = 2e-11 Query: 556 ISIIGLYKEKGKGLGFSIAGGRDCIRGQMGIFVKTIFPNGSAAEDGRLKEGDEILDVNGI 615 ++ I I I IIIII+ II+1 I I+ I ++ I II+ I II II II+III
IS Sbjct: 2 PRLVELEKGGG-GLGFSLVGGKD--SGDGGWVSSWPGSPAAKAG-LKPGDVILEVNGT 57 Query: 616 PIKGLTFQEAIHTFKQIRSGLFVLTVR 642 ++III II+ I+ + + +I
Sbjct: 58 SVEGLTHLEAVDLLKEAGGKVTLTVLR 84 Table 8J. Domain Analysis of NOVB
gnl~Smart~smart00228, PDZ, Domain present in PSD-95, Dlg, and ZO-1/2.;
Also called DHR (Dlg homologous region) or GLGF (relatively well conserved tetrapeptide in these domains). Some PDZs have been shown to bind C-terminal polypeptides; others appear to bind internal (non-C-terminal) polypeptides. Different PDZs possess different binding specificities. (SEQ ID N0:86) CD-Length = 86 residues, 97.7% aligned Score = 60.1 bits (144), Expect = 2e-09 Query: 2597 FIVLNRKEGSGLGFSVAGGTDVEPKSITVHRVFSQGAASQEGTMNRGDFLLSVNGASLAG 2656 +I I I IIIII+ II I + I I I++ I + II +I III 1+ I
2S Sbjct: 3 RLVELEKGGGGLGFSLVGGKDSGDGGWVSSWPGSPAAKAG-LKPGDVILEVNGTSVEG 61 Query: 2657 LAHGNVLKVLHQAQLHKDALWIKKG 2682 + +~ +~ ~ ~ ~++
Sbjot: 62 LTHLEAVDLLKEAG-GKVTLTVLRGG 86 Proteins belonging to the IGFBP-ALS family of proteins play an important role in regulating the levels of circulating horniones. The acid labile subunit of the complex plays an important role in regulating the stability of the complex and ensuring high levels of circulating hormones that are regulated by the IGFBP family of proteins. This protein also has a leucine rich repeat that is a common domain in many proteins that are important for the developing embryo. As a result this protein may play an important role in development and disease.
Insulin-like growth factors (IGFs) I and II are important regulators of cell proliferation and differentiation (IJeki I et al., Proc Natl Acad Sci U S A 2000 Jun 6;97(12):6868-73). After birth, plasma IGFs, representing mostlyliver-derived IGFs, circulate in ternary complexes of 150 kDa consisting of onemolecule each of IGF, IGF-binding protein (IGFBP) 3, and an acid labile subunit (ALS). Onset of ALS synthesis after birth is the primary factor driving the formation of ternary complexes. Capture of IGFs by ALS is thought to allow the development of a plasma reservoir without negative effects such as hypoglycemia and cell proliferation. To evaluate the importance of ALS and ternary complexes, mice have been created in which the ALS gene has been inactivated. The mutation was inherited in a Mendelian manner, without any effects on survival rates and birth weights. A growth deftcit was observed in null mice after 3 weeks of life and reached 13% by 10 weeks. This modest phenotype was observed despite reductions of 62 and 88% in the concentrations of plasma IGF-I and IGFBP-3, respectively. Increased turnover accounted for these reductions because indices of synthesis in liver and kidney were not decreased. Surprisingly, absence of ALS did not affect glucose and insulin homeostasis. Therefore, ALS is required for postnatal accumulation of IGF-I and IGFBP-3 but, consistent with findings supporting a predominant role for locally produced IGF-I, is not critical for growth. This model should be useful to determine whether presence of ALS is needed for other actions of liver-derived IGF-I and for maintenance of homeostasis in presence of high circulating levels of IGF-II.
In the circulation, insulin-Iike growth factor-I (IGF-I) is bound in a trimeric complex of 150 kDa with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). (Moller S
et al., J Hepatol 2000 Mar;32(3):441-6). Whereas circulating IGF-I and IGFBP-3 are reported to be low in patients with chronic liver failure, the level of ALS has not been described in relation to hepatic dysfunction. The aim of the present study was therefore to measure circulating and hepatic venous concentrations of ALS in relation to hepatic function and the IGF axis.
The insulin-like growth factor (IGF) binding proteins (IGFBPs) were initially identified as carrier proteins for IGF-I and IGF-II in a variety of biologic fluids (Rosenfeld RG
et al., Pediatrics 1999 Oct;104(4 Pt 2):1018-21). Their presumed function was to protect IGF
peptides from degradation and clearance, increase the half life of the IGFs, and deliver them to appropriate tissue receptors. The concept of IGFBPs as simple carrier proteins has been complicated, however, by a number of observations: 1) the six IGFBPs vary in their tissue expression and their regulation by other hormones and growth factors; 2) the IGFBPs are subjected to proteolytic degradation, thereby altering their affinities for the IGFs; 3) IGFBP-3 and IGFBP-5, in addition to binding IGFs, also can associate with an acid-labile subunit, thereby increasing further the half life of the IGFs; 4) in addition to modifying the access of IGF peptides to IGF and insulin receptors, several of the IGFBPs may be capable of increasing IGF action; 5) some of the IGFBPs may be capable of IGF-independent regulation of cell growth; 6) some of the IGFBPs are associated with cell membranes or possibly with membrane receptors; and 7) some of the IGFBPs have nuclear recognition sites and may be found within the nucleus. Additionally, a number of cDNAs identified recently have been found to encode proteins that bind IGFs, but with substantially lower affinities than is the case with IGFBPs. The N-terminal regions of the predicted proteins are structurally homologous to the classic IGFBPs, with conservation of the cysteine-rich region. These observations suggest that these low-affinity binders are members of an IGFBP superfamily, capable of regulating cell growth by both IGF-dependent and IGF-independent mechanisms.insulin-like growth factor, insulin-like growth factor binding proteins.
Total IGF-I level in serum is a sensitive index during growth hormone (GH) replacement therapy of adults, since GH stimulates the hepatic expressions of both insulin-like growth factor (IGF-I) and acid-labile subunit (ALS) and the major part of IGF-I in the circulation is found in a ternary complex together with ALS and IGFBP-3 (Hall K et al., J
Endocrinol Invest 1999;22(5 Suppl):48-57). However, other regulators of the proteins constituting the ternary complex may influence IGF-I levels. In healthy subjects the serum IGF-I levels are low at birth, rise during childhood, with peak levels during puberty, and decline with increasing age. This pattern has been attributed to the age-dependent GH
production, but it is unknown whether the wide range of IGF-I levels within each age interval is due to GH production or GH sensitivity. In elderly twins approximately 60%
of IGF-I levels axe genetically determined. The remaining environmental dependency of IGF-I is partly due to nutrition. Both caloric and protein content of the diet is of importance.
Thus, low IGF-I levels are found in GH deficient patients as well as in patients with GH resistance due to malnutrition or GH receptor defects. It is essential that IGF-I determination is performed by assays in which IGFBPs do not interfere, and that IGF-I concentration is evaluated in relation to age, i.e.
expressed in SD score, and the number of individuals constituting the reference intervals improves the sensitivity and specificity. Although determination of IGF-I is recommended in assessing GH deficiency in children, its diagnostic value in patients with adult onset of GH
deficiency is not agreed upon. In the age group above 40-80 years many patients with pituitaryll~ypothalamic disorders and GH peaks below 3 microgll during provocation tests have normal IGF-I levels. It is not clarified, whether the IGF-I levels within normal range for age is due to endogenous basal GH production being sufficient or other factors stimulating IGF-I, IGFBP-3 or ALS expressions.
Circulating insulin-like growth factors (IGFs) represent an important pool of potential hypoglycemic activity, which is largely inhibited by their sequestration in a heterotrimeric complex comprising growth factor, IGF-binding protein-3 (IGFBP-3), and acid-labile subunit (ALS) (Baxter RC Metabolism 1995 Oct;44(10 Suppl 4):12-7). Less than 1% of total IGFs circulate in the free form, yet even this amount might contribute significantly to circulating insulin-like activity. The ternary binding protein complex appears to inhibit insulin-like activity of bound IGFs by preventing their egress from the circulation.
Although the integrity of this complex might be affected by limited proteolysis of IGFBP-3 in pregnancy and catabolic conditions, the evidence that this increases IGF bioavailability, and thus hypoglycemic potential, is as yet unclear. However, in patients with IGF-II-secreting tumors, hypoglycemia may result from a failure of the ternary complex to adequately sequester the IGFs. Improvement in complex formation, by treatment with corticosteroids or growth hormone, alleviates the hypoglycemia, even if (as seen with growth hormone treatment) IGF-II hypersecretion persists. In these patients, blood glucose levels are inversely correlated with IGFBP-2 levels, suggesting that this protein might play a part in transporting IGFs to their target tissues. Conversely, ALS levels correlate positively with blood glucose, emphasizing the importance of the ternary complex in preventing hypoglycemia. Unlike the other IGF-binding proteins, IGFBP-1 is acutely regulated in the circulation, in a manner consistent with its acting as a glucose counterregulator. It might act in this way by inhibiting the activity of free IGFs in the circulation.
Leucine-rich repeats (LRRs) are relatively short motifs (22-28 residues in length) found in a variety of cytoplasmic, membrane and extracellular proteins (InterPro). Although these proteins are associated with widely different functions, a conunon property involves protein-protein interaction. Little is known about the 3D structure of LRRs, although it is believed that they can form amphipathic structures with hydrophobic surfaces capable of interacting with membranes. h2 vitro studies of a synthetic LRR from Drosophila Toll protein have indicated that the peptides form gels by adopting beta-sheet structures that form extended filaments. These results are consistent with the idea that LRRs mediate protein-protein interactions and cellular adhesion. Other functions of LRR-containing proteins include, for example, binding to enzymes and vascular repair . The 3-D structure of ribonuclease inhibitor, a protein containing 15 LRRs, has been determined, revealing LRRs to be a new class of alphalbeta fold. LRRs form elongated non-globular structures and are often flanked by cysteine rich domains.
The disclosed NOV8 nucleic acid of the invention encoding a papin-like protein includes the nucleic acid whose sequence is provided in Table 8A and C, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 8A and C while still encoding a pxotein that maintains its papin-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are tamed out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 12% percent of the bases may be so changed.
The disclosed NOV8 protein of the invention includes the papin-like protein whose sequence is provided in Table 8B and D. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 2 while still encoding a protein that maintains its papin-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 43%
percent of the residues may be so changed.
The invention further encompasses antibodies and antibody fragments, such as Fab or ~ab~2, that bind immunospecifically to any of the proteins of the invention.

The above defined information for this invention suggests that this papin-like protein (NOVB) may function as a member of a "papin family". Therefore, the NOV8 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to:
protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and ifa vitro of all tissues and cell types composing (but not limited to) those defined here.
The NOV8 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in cancer including but not limited to hitlamation, Autoimmune disorders, Aging and Cancer. For example, a cDNA encoding the papin-like protein (NOV8) may be useful in gene therapy, and the papin-like protein (NOVB) may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from cancer, cystitis, incontinence, fertility, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis , atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis , subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation recovery. The NOV8 nucleic acid encoding papin-like protein, and the papin-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
NOV 8 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOVB substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies"
section below. The disclosed NOV8 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV8 epitope is from about amino acids 10 to 50. In another embodiment, a NOV8 epitope is from about amino acids 80 to 120. In additional embodiments, NOVS epitopes are from about amino acids 180 to 220, from about amino acids 230 to 300, from about amino acid 330 to 350, from about amino acid 370 to 400, from about amino acid 480 to 540, from about amino acid 550 to 560, and from about amino acids 620 to 840. These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOVX Nucleic Acids and Polypeptides One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-stranded DNA.
An NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event.
Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences.
Longex length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one, which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA
of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information pxovided herein.
Using all or a portion of the nucleic acid sequence of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.), CURRENT
PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.) A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID
NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an NOVX polypeptide). A nucleic' acid molecule that is complementary to the nucleotide sequence shown SEQ ID NOS: l, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 is one that is sufficiently complementary to the nucleotide sequence shown SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID
NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.

Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95%
identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. S'ee e.g.
Ausubel, et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an NOVX
polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX
protein.
Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS: l, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, as well as a polypeptide possessing NOVX biological activity. Various biological activities of the NOVX proteins are described below.
An NOVX polypeptide is encoded by the open reading frame ("ORF") of an NOVX
nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG
"start" codon and terminates with one of the three "stop" codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bozza fide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX
homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26; or an anti-sense strand nucleotide sequence of SEQ ID
NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26; or of a naturally occurring mutant of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.
Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g.
the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express an NOVX protein, such as by measuring a level of an NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of an NOVX polypeptide"
refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically-active portion of NOVX" can be prepared by isolating a portion SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, that encodes a polypeptide having an NOVX
biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression i~a vitro) and assessing the activity of the encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 due to degeneracy of the genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ
m NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27.
In addition to the human NOVX nucleotide sequences shown in SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, if will be appreciated by those skilled in the art that DNA sequence polyrnorphisms that lead to changes in the amino acid sequences of the NOVX
polypeptides may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (OIRF) encoding an NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX
polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from the human SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 are intended to be within the scope of the invention.
Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX
cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (i. e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA
at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequences SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, corresponds to a naturally-occurnng nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, SX Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in 1X SSC, 0.1 % SDS at 37°C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT
PROTOCOLS IN
MOLECULAR BIOLOGY, john Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER
AND
EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, SX SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02%
PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH
7.4), 5 mM EDTA, and 0.1% SDS at SO°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Tohn Wiley &
Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY
MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Pf~oc Natl Acad Sci USA 78: 6789-6792.

Conservative Mutations In addition to naturally-occurnng allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, thereby leading to changes in the amino acid sequences of the encoded NOVX
proteins, without altering the functional ability of said NOVX proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the NOVX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding NOVX
proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences SEQ ID NOS:2, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, and 27.
Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID
NOS:2, S, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27; more preferably at least about 70%
homologous SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27; still more preferably at least about 80% homologous to SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27;
even more preferably at least about 90% homologous to SEQ ID NOS:2, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, or 27; and most preferably at least about 95% homologous to SEQ ID
NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27.
An isolated nucleic acid molecule encoding an NOVX protein homologous to the protein of SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

Mutations can be introduced into SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY, wherein the letters within each group represent the single letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to form protein:protein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and an NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).

Antisense Nucleic Acids Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an NOVX protein of SEQ ID
NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, or antisense nucleic acids complementary to an NOVX nucleic acid sequence of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an NOVX
protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the NOVX protein. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA.
For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, S-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-metlrylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid rnethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i. e., RNA
transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated irZ situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (3-units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl.
Acids Res. 15:
6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-rnethylribonucleotide (See, e.g., moue, et al. 1987. Nucl. Acids Res. 15:
6131-6148) or a chimeric RNA-DNA analogue (See, e.g., moue, et al., 1987, FEBSLett. Z15: 327-330.
Ribozymes and PNA Moieties Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX
mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for an NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of an NOVX cDNA disclosed herein (i.e., SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26). For example, a derivative of a Tet~alaynaeraa L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an NOVX-encoding mRNA. See, e.g., U.S.
Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. NOVX mRNA
can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel et al., (1993) Scieface 261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NOVX nucleic acid (e.g., the NOVX
promoter and/or enhancers) to form triple helical structures that prevent transcription of the NOVX gene in target cells. See, e.g., Helene, 1991. A~aticancer Dfzsg Des. 6:
569-84; Helene, et al. 1992. Ah.n. N. Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.

In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to impxove, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996.
Bioorg Med C72em 4: 5-23. As used herein, the teens "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996. supra;
Perry-O'Keefe, et al., 1996. Proc. Natl. Acid. Sci. USA 93: 14670-14675.
PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (See, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996. supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes (e.g., RNase H and DNA polymerises) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA
chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996, sups°a).
The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al., 1996.
supra and Fimi, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA
chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al., 1989. Niacl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment.
See, e.g., Finn, et al., 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5' DNA
segment and a 3' PNA segment. See, e.g., Petersen, et al., 1975. Bioorg. Med.
Claern. Lett. 5:
1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors iya vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci.
U.S.A. 86:
6553-6556; Lemaitre, et al., 1987. P~oc. Natl. Acad Sci. 84: 648-652; PCT
Publication No.
W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO
89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al., 1988. BioTechyaiyues 6:958-976) or intercalating agents (see, e.g., Zon, 1988.
Plaarm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
NOVX Polypeptides A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID
NOS:2, 5, 7,

9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID
NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
In general, an NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated NOVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX
antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NOVX proteins are produced by recombinant DNA technidues.
Alternative to recombinant expression, an NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material"
includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX
proteins, and most preferably less than about 5% of non-NOVX proteins. When the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the NOVX protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence shown in SEQ ID NOS:2, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, or 27) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of an NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A
biologically-1.45 active portion of an NOVX protein can be a polypeptide which is, for example,

10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein.
In an embodiment, the NOVX protein has an amino acid sequence shown SEQ ID
NOS:2, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, or 27. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, and retains the functional activity of the protein of SEQ ID NOS:2, 5, 7, 9,

11, 13, 15, 17, 19, 21, 23, 25, or 27, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX
protein is a protein that comprises an amino acid sequence at least about 45%
homologous to the amino acid sequence SEQ ID NOS:2, 5, 7, 9, 1 l, 13, 1 S, 17, 19, 21, 23, 25, or 27, and retains the functional activity of the NOVX proteins of SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27.
Determining Homology Between Two or More Sequences To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i. e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMoI Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP
extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.
The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a 1 S reference sequence over a comparison region.
Chimeric and Fusion Proteins The invention also provides NOVX chirneric or fusion proteins. As used herein, an NOVX "chimeric protein" or "fusion protein" comprises an NOVX polypeptide operatively-linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an NOVX protein SEQ ID NOS:2, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, or 27, whereas a "non-NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within an NOVX fusion protein the NOVX
polypeptide can correspond to all or a portion of an NOVX protein. In one embodiment, an NOVX fusion protein comprises at least one biologically-active portion of an NOVX protein.
In another embodiment, an NOVX fusion protein comprises at least two biologically-active portions of an NOVX protein. In yet another embodiment, an NOVX fusion protein comprises at least three biologically-active portions of an NOVX protein.
Within the fusion protein, the term "operatively-linked" is intended to indicate that the NOVX
polypeptide and the non-NOVX polypeptide are fused in-frame with one another. The non-NOVX
polypeptide can be fused to the N-terminus or C-terminus of the NOVX polypeptide.

In one embodiment, the fusion protein is a GST-NOVX fusion protein in which the NOVX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX
polypeptides.
hi another embodiment, the fusion protein is an NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is an NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member of the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an NOVX ligand and an NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of an NOVX cognate ligand.
Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with an NOVX ligand.
An NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX Agonists and Antagonists The invention also pertains to variants of the NOVX proteins that function as either NOVX agonists (i. e., mimetics) or as NOVX antagonists. Variants of the NOVX
protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the NOVX protein).
An agonist of the NOVX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the NOVX protein. An antagonist of the NOVX protein can inhibit one or more of the activities of the naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the NOVX proteins.
Variants of the NOVX proteins that function as either NOVX agonists (i. e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. S'ee, e.g., Narang, 1983.
Tetrahedron 39: 3;
Itakura, et al., 1984. Aianu. Rev. Biochem. 53: 323; Itakura, et al., 1984.
Science 198: 1056;
Ike, et al., 1983. Nucl. Acids Res. 11: 477.

Polypeptide Libraries In addition, libraries of fragments of the NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of an NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the NOVX
proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NOVX proteins.
The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants.
, See, e.g., Arkin and Yourvan, 1992. Pf°oc. Natl. Acad. Sci. USA 89:
7811-7815; Delgrave, et al., 1993. Protein Engifteerisag 6:327-331.
Anti-NOVX Antibodies Also included in the invention are antibodies to NOVX proteins, or fragments of NOVX proteins. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab~ and F(ab~~2 fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
Certain classes have subclasses as well, such as IgGI, IgGz, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
An isolated NOVX-related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX-related protein that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human NOVX-related protein sequence will indicate which regions of a NOVX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci.
USA 78:
3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.

Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a proteiiz of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
Examples of such irnmunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
Additional examples of adjuvants which can be employed include MPL-TDM
adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

Monoclonal Antibodies The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.
MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by I~ohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to fornl a hybridoma cell (Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Aeademic Press, (1986) pp.
59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are rnurine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Mantissas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLICATIONS, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in LT.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be 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 of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S.
Patent No.
4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
Humanization can be performed following the method of Winter and co-workers (Jones et al., Natu~~e, 321:522-525 (1986); Riechmann et al., Natuf°e, 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. (See also U.S. Patent No.
5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can 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 framew~rk 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., 1986; Riechmann et al., 1988; and Presta, Cu~~. Op.
Strz~ct. Biol., 2:593-596 (1992)).
Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein.
Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV
hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In:
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. PatentNos. 5,545,807; 5,545,806;
5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al. (BiolTech.nology 10, 779-783 (1992));
Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Bioteclaraolagy 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (hater°n. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT
publication W094/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO
96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins.
The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT
publication WO 99/53049.
Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F~ab~>z fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F~ab~>a fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F~ fragments.

Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding 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 organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
According to another approach described in WO 96!2701 l, 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 CH3 region of an antibody constant 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 chains) 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 can be prepared as full length antibodies or antibody fragments (e.g. F(ab')Z bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')Z 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'-ThlB 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.
Additionally, Fab' fragments can be directly recovered from E. coli and 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. Iznzzzurzol.
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 heavy-chain variable domain (VH) connected to a light-chain variable domain (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. Iznzzzunol.
152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., .I. Immufiol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII
(CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTIJBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
Heteroconjugate Antibodies 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. Patent No. 4,676,980), and for treatment of HIV infection (WO 91100360;
WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
For example, i~nmunotoxins can 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.
Patent No.
4,676,980.
Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residues) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability andlor increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can 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 that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, 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).
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 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 trieothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies.
Examples include zizBi, i3ih 131In, 9oY, and lg6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) 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 W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting 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) that is in turn conjugated to a cytotoxic agent.
In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an NOVX
protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX
protein possessing such a domain. Thus, antibodies that are specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Anti-NOVX antibodies rnay be used in methods known within the art relating to the localization and/or quantitation of an NOVX protein (e.g., for use in measuring levels of the NOVX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for NOVX
proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter "Therapeutics").
An anti-NOVX antibody (e.g., monoclonal antibody) can be used to isolate an NOVX
polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation.
An anti-NOVX antibody can facilitate the purification of natural NOVX
polypeptide from cells and of recombinantly-produced NOVX polypeptide expressed in host cells.
Moreover, an anti-NOVX antibody can be used to detect NOVX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the NOVX
protein. Anti-NOVX antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i. e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, (3-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include lzsh 131I' 35S ~r 3H.
NOVX Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein additional DNA
segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences) in a manner that allows for expression of the nucleotide sequence (e.g., in an iYz vitYO transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN

ENZYMOLOGY 185, Academic Press, San Diego, Cali~ (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX
proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Cali~ (1990). Alternatively, the recombinant expression vector can be transcribed and translated ih vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Esche>"ichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gezze 67: 31-40), pMAL
(New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N,J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gerze 69:301-315) and pET l 1d (Studier et al., GENE
EXPRESSION

TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Cali~ (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY
185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E.
coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the NOVX expression vector is a yeast expression vector.
Examples of vectors for expression in yeast Saccharonzyces cerivisae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30:
933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Cozp, San Diego, Calif.).
Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
20, In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufinan, et al., 1987. EMBO
J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987.
Genes Dev. 1:
268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immuzzol.
43:

235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740;
Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. LISA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA
molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA
molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA
as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any.prokaryotic or eukaryotic cell. For example, NOVX
protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vector. DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (I.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertihized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene, Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 can be introduced as a transgene into the genome of a non-human animal.
Alternatively, a non-human homologue of the human NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequences) can be operably-linked to the NOVX transgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos.
4,736,866;
4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX mRNA
in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared which contains at least a portion of an NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26), but more preferably, is a non-human homologue of a human NOVX gene.
For example, a mouse homologue of human NOVX gene of SEQ ID NOS:1, 3, 4, 6, 8, 10,

12, 14, 16, 18, 20, 22, 24, and 26 can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX
gene is functionally disrupted (i.e., no longer encodes a functional protein;
also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein). In the homologous recombination vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic acid of the NOVX
gene to allow for homologous recombination to occur between the exogenous NOVX
gene carned by the vector and an endogenous NOVX gene in an embryonic stem cell.
The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the S'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al., 1987. Cell 51:
503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX
gene has homologously-recombined with the endogenous NOVX gene are selected.
See, e.g., Li, et al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AI~m EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152.
A chirneric embryo can then be implanted into a suitable pseudopregmant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. CuYr. Opin. Biotechnol. 2: 823-829; PCT
International Publication Nos.: WO 90111354; WO 91101140; WO 9210968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P 1. For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Pz-oc. Natl.
Acad. Sci. USA 89:
6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharozzzyces cerevisiae. See, O'Gorman, et al., 1991. Sciezzce 251:1351-1355. If a cre/loxP
recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al., 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable Garners are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such Garners or diluents include, but are not limited to, water, saline, forger's solutions, dextrose solution, and 5%
human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components:
a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;
chelating agents such 1 S as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible Garner. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with Garners that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S.
Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated;
each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. P~oc. Natl. Acad. Sci.
USA 91: 3054-3057).
The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods The isolated nucleic acid molecules of the invention can be used to express NOVX
protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX rnRNA (e.g., in a biological sample) or a genetic lesion in an NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
Screening Assays The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i. e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX
protein activity.
The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of an NOVX
protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997. Afaticaracer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. PYOC. Natl. Acad. Sci. U.S.A. 90: 6909;
Erb, et al., 1994.
Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994..1. Med.
CJaem. 37: 2678;
Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Aragew. Chern.
Int. Ed. Engl. 33:
2059; Carell, et al., 1994. Ahgew. Chern. 132t. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J.
Med. Clzem. 37: 1233.
Libraries of compounds tnay be presented in solution (e.g., Houghten, 1992.
Biotechfziques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991.
J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to an NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell.
Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with lzsi~ 3sS~ lace or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX
protein, wherein determining the ability of the test compound to interact with an NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX
protein or a biologically-active portion thereof as compared to the known compound. .
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule. As used herein, a "target molecule" is a molecule with which an NOVX
protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. An NOVX target molecule can be a non-NOVX molecule or an NOVX protein or polypeptide of the invention. In one embodiment, an NOVX
target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.
Determining the ability of the NOVX protein to bind to or interact with an NOVX
target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i. e.
intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising an NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting an NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the NOVX protein or biologically-active portion thereof. Binding of the test compound to the NOVX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX
protein, wherein deterniining the ability of the test compound to interact with an NOVX
protein comprises determining the ability of the test compound to preferentially bind to NOVX
or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting NOVX
protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability of the NOVX protein to bind to an NOVX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability of the NOVX protein further modulate an NOVX target molecule. For example, the catalyticlenzymatic activity of the target molecule on an appropriate substrate can be determined as described, supYa.
In yet another embodiment, the cell-free assay comprises contacting the NOVX
protein or biologically-active portion thereof with a known compound which binds NOVX
protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX protein, wherein determining the ability of the test compound to interact with an NOVX protein comprises determining the ability of the NOVX protein to preferentially bind to or modulate the activity of an NOVX
target molecule.

The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution.
Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylinaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton° X-100, Triton° X-114, Thesit°, Isotridecypoly(ethylene glycol ether)", N-dodecyl--N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, fox example, as described, supra.
Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NOVX
protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies xeactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX
protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX
mRNA or protein in the cell is determined. The level of expression of NOVX
mRNA or protein in the presence of the candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i. e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression. Alternatively, when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression. The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect of the invention, the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317;
Zervos, et al., 1993. Cell72: 223-232; Madura, et al., 1993. .I. Biol. Chem.
268: 12046-12054;
Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.
Otacogene 8:
1693-1696; and Brent WO 94!10300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-by") and modulate NOVX activity. Such NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements of the NOVX
pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX
is fused to a gene encoding the DNA binding domain of a !mown transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey"
proteins are able to interact, in vivo, forming an NOVX-dependent complex, the DNA-binding and activation S domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.
The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way 1 S of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.
Chromosome Mapping Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the NOVX sequences, SEQ ID
2S NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or fragments or derivatives thereof, can be used to map the location of the NOVX genes, respectively, on a chromosome. The mapping of the NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers (preferably 1 S-2S by in length) from the NOVX sequences. Computer analysis of the NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the NOVX sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes; allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al., 1983. Scie~zce 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes.
Fluorescence iTa situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al., HUMAN
CHROMOSOMES: A
MANUAL of BASIC TECHrnQu~s (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single chxomosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites andlor multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in Mcl~usick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al., 1987.
Natuf°e, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease.
Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing The NOVX sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA
markers for RFLP
("restriction fragment length polymorphisms," described in U.S. Patent No.
5,272,057).
Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (ItFLPs).
Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS;1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
Predictive Medicine The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX
protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX
expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in an NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.

Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").
Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.) Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assays An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA.
The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA
or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX
protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample ira vitro as well as i~r vivo.
For example, ih vitro techniques for detection of NOVX mRNA include Northern hybridizations and in. situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. Ifa vits~o techniques for detection of NOVX genomic DNA
include Southern hybridizations. Furthermore, ifa vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mIZNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid.
Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX
expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity.

Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX
expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder.
Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant NOVX
expression or ~,0 activity).
The methods of the invention can also be used to detect genetic lesions in an NOVX
gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an NOVX-protein, or the misexpression of the NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from an NOVX gene; (ia) an addition of one or more nucleotides to an NOVX gene; (iii) a substitution of one or more nucleotides of an NOVX gene, (iv) a chromosomal rearrangement of an NOVX gene; (v) an alteration in the level of a messenger RNA transcript of an NOVX gene, (vi) aberrant modification of an NOVX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a xion-wild-type splicing pattern of a messenger RNA transcript of an NOVX gene, (viii) a non-wild-type level of an NOVX
protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate post-translational 1~6 modirication of an NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in an NOVX
gene. A
preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al., 1988. Scie~zce 241: 1077-1080; and Nakazawa, et al., 1994.
Proc. Natl.
Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23:
675-682).
This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an NOVY gene under conditions such that hybridization and amplification of the NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. P~oc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al., 1989. PYOG. Natl. Acad. Sci. USA 86: 1173-1177);
Q(3 Replicase (see, Lizardi, et al, 1988. BioTeclafzology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared.
Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence speciric ribozymes (see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example, genetic mutations in NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes.
This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence of the sample NOVX with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl.
Acad. Sci. USA
74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al., 1995.
Biotechzziques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Clzroznatograplzy 36:
127-162; and Griffin, et al., 1993. Appl. Bioclzerrz Biotechzaol. 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al., 1988. Proc. Natl.
Acad. Sci. USA 85:
4397; Saleeba, et al., 1992, llfethods Erazyrnol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutt enzyme of E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T
at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15: 1657-1662.
According to an exemplary embodiment, a probe based on an NOVX sequence, e.g., a wild-type NOVX
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86:
2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79.
Single-stranded DNA fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al., 1991.
Trends Genet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495.
When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 by of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and.
sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Bioph,ys. Chern. 265: 12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al., 1986. Nature 324: 163;
Saiki, et al., 1989. Proc. Natl. Acad. Sci, USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on selective PCR
amplification may be used in conjunction with the instant invention.
Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtecla. 11: 238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection. See, e.g., Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3'-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an NOVX
gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which NOVX is expressed may be utilized in the prognostic assays described herein.
However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.

Pharmacogenomics Agents, or modulators that have a stimulatory or inhibitory effect on NOVX
activity (e.g., NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX
protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual. can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Cliyz. Exp. Pha~nae~col. Physiol., 23: 983-985;
Linder, 1997. Cli~a.
Chena., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzyrnopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C 19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM
show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX (e.g., the ability to modulate aberrant cell proliferation andlor differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity.
In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation ox immune disorder can be used as a "read out" or markers of the immune xesponsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified.
Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can he quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an NOVX protein, mRNA, or genomic DNA in the preadministration sample;
(iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NOVX
protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX
protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of NOVX
to higher levels than detected, i.e., to increase the effectiveness of the agent.
Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness of the agent.

Methods of Treatment The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NOVX expression or activity. The disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions of the like.
These methods of treatment will be discussed more fully, below.
Disease and Disorders Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ai) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Scieyace 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disoxder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be, readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitYO for RNA or peptide levels, structure andlor activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, iu.
situ hybridization, and the like).
Prophylactic Methods In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity.
Subjects at risk for a disease that is caused or contributed to by aberrant NOVX
expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX
aberrancy, for example, an NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the invention are further discussed in the following subsections.
Therapeutic Methods Another aspect of the invention pertains to methods of modulating NOVX
expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurnng cognate ligand of an NOVX protein, a peptide, an NOVX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity.
Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX

that has been introduced into the cell. In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed ira vitf-o (e.g., by culturing the cell with the agent) or, alternatively, ira vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an NOVX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering an NOVX
protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX
expression or activity.
Stimulation of NOVX activity is desirable iy~ situations in which NOVX is abnormally downregulated and/or in which increased NOVX activity is likely to have a beneficial effect.
One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia).
Determination of the Biological Effect of the Therapeutic In various embodiments of the invention, suitable ih vitJ-o or ih vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.
In various specific embodiments, in vitro assays may be performed with representative cells of the types) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for ih vivo testing, any of the animal model system known in the art may be used prior to administration to human subjects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention The NOVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
As an example, a cDNA encoding the NOVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof.
By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from; metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias.
Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. A
further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Examples Example 1. Identification of NOVX clones The novel NOVX target sequences identified in the present invention were subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. Table 1 1A shows the sequences of the PCR primers used for obtaining different clones. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences from other species. These primers were then employed in PCR
amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain -amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma -Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The PCR
product derived from exon linking was cloned into the pCR2.1 vector from Invitrogen.
The resulting bacterial clone has an insert covering the entire open reading frame cloned into the pCR2. l vector. Table 17B shows a list of these bacterial clones. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported herein.
Table 11A. PCR Primers for Exon Linking NOVX Primer 1 (5' - 3') SEQ Primer 2 (5' - 3') SEQ

Clone ID ID

NO NO

NOVlc TCATCACATGACAACATGAAGCTGT87 GAAAGCCCTCAAACTCTCCATCTATG 88 NOV7a CCAATCTCTGATGCCCTGCGAT89 AGGTCAGTGCCGGAGCCTCC 90 Physical clone: Exons were predicted by homology and the intronlexon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.
Table 11B. Physical Clones for PCR products NOVX Clone Bacterial Clone NOV1 Physical 128940::83420733.698715.E24 clone:

NOV2 Physical ALf57059, AL3555530, AL356100, clone: AL022344, AC016042 NOV4 Physical AC009785 clone:

NOVS Genomic GMChromosome4 clone:

NOV7a Genomic gb AC010319 sapiens~chromosome 19 file: HTG Homo CTD-NOVB Physical AC008803, AC026718 clone: AC010449, Example 2. Quantitative expression analysis of clones in various cells and tissues The quantitative expression of various clones was assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed on a Perkin-Elmer Biosystems ABI PRISM~ 7700 Sequence Detection System. Various collections of samples are assembled on the plates, and referred to as Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing samples derived from tissues from normal and cancer sources), Panel 3 (containing cancer cell lines), Panel 4 (containing cells and cell lines from normal tissues and cells related to inflammatory conditions), AI
comprehensive~anel (containing normal tissue and samples from autoinflammatory diseases), Panel CNSD.01 (containing samples from normal and diseased brains) and CNS
neurodegeneration_panel (containing samples from normal and diseased brains).
First, the RNA samples were normalized to reference nucleic acids such as constitutively expressed genes (for example, [3-actin and GAPDH). Normalized RNA (5 u1) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (PE Biosystems; Catalog No. 4309169) and gene-speciftc primers according to the manufacturer's instructions. Probes and primers were designed for each assay according to Perkin Elmer Biosystem's Ps°ifneY Express Software package (version I
for Apple Computer's Macintosh Power PC) or a similar algorithm using the target sequence as input.
Default settings were used for reaction conditions and the following parameters were set before selecting primers: primer concentration = 250 nM, primer melting temperature (Tm) range =
58°-60° C, primer optimal Tm = 59° C, maximum primer difference = 2° C, probe does not have 5' G, probe Tm must be 10° C greater than primer Tm, amplicon size 75 by to 100 bp.
The probes and primers selected (see below) were synthesized by Synthegen (Houston, TX, USA). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3' ends of the probe, respectively. Their final concentrations were: forward and reverse primers, 900 nM
each, and probe, 200nM.
PCR conditions: Normalized RNA from each tissue and each cell line was spotted in each well of a 96 well PCR plate (Perkin Elmer Biosystems). PCR cocktails including two probes (a probe specific for the target clone and another gene-specific probe multiplexed with the target probe) were set up using 1X TaqManTM PCR Master Mix for the PE
Biosystems 7700, with 5 mM MgCl2, dNTPs (dA, G, C, U at 1:1:1:2 ratios), 0.25 Ulml AmpliTaq GoldTM
(PE Biosystems), and 0.4 U/~1 RNase inhibitor, and 0.25 U/wl reverse transcriptase. Reverse transcription was performed at 48° C for 30 minutes followed by amplification/PCR cycles as follows: 95° C 10 min, then 40 cycles of 95° C for 15 seconds, 60° C for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA
difference and multiplying by 100.
Panel 1 In the results for Panel l, the following abbreviations are used:
ca. = carcinoma, * = established from metastasis, met = metastasis, s cell var = small cell variant, non-s = non-sm = non-small, squam = squamous, p1. eff = p1 effusion = pleural effusion, glio = glioma, astro = astrocytoma, and neuro = neuroblastoma.
Panel 2 The plates for Panel 2 generally include 2 control wells and 94 test samples composed of RNA or cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI). The tissues are derived from human malignancies and in cases where indicated many malignant tissues have "matched margins"
obtained from noncancerous tissue just adjacent to the tumor. These are termed normal adjacent tissues and are denoted "NAT" in the results below. The tumor tissue and the "matched margins" are evaluated by two independent pathologists (the surgical pathologists and again by a pathologists at NDRI or CHTN). This analysis provides a gross histopathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage of the patient. These matched margins are taken from the tissue surrounding (i.e.
immediately proximal) to the zone of surgery (designated "NAT", for normal adjacent tissue, in Table RR).
In addition, RNA and cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death victims (accidents, etc.). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics, and Invitrogen.
RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA
contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
Panel 3D
The plates of Panel 3D are comprised of 94 cDNA samples and two control samples.
Specifically, 92 of these samples are derived from cultured human cancer cell lines, 2 samples of human primary cerebellar tissue and 2 controls. The human cell lines are generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: Squamous cell carcinoma of the tongue, breast cancer, prostate cancer, melanoma, epidermoid carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas, ovarianluterine/cervical, gastric, colon, lung and CNS cancer cell lines. In addition, there are two independent samples of cerebellum. These cells are all cultured under standard recommended conditions and RNA
extracted using the standard procedures. The cell lines in panel 3D and 1.3D
are of the most common cell lines used in the scientific literature.
RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA
contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
Panel 4 Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4r) or cDNA (Panel 4d) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene ,La Jolla, CA) and thymus and kidney (Clontech) were employed.
Total RNA from liver tissue from cirrhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, Inc., Hayward, CA). Intestinal tissue for RNA
preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA).
Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, rnicrovascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated. The following cytokines were used; IL-1 beta at approximately 1-5 ng/ml, TNF
alpha at approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml, IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml, IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1% serum.
Mononuclear cells were prepared from blood of employees at CuraGen Corporation, using Ficoll. LAK cells were prepared from these cells by culture in DMEM 5%
FCS
(Hyclone), 100 p.M non essential amino acids (Gibco/Life Technologies, Rockville, MD), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10-5 M (Gibco), and 10 mM
Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20 ng/ml PMA and 1-2 pg/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml and IL-18 at 5-10 ng/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100 pM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol S.5 x 10-5 M (Gibco), and 10 mM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5 wg/ml.
Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1:1 at a final concentration of approximately 2x106 cells/ml in DMEM 5% FCS (Hyclone), 100 p,M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol (5.5 x 10-5 M) (Gibco), and 10 mM
Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1- 7 days for RNA preparation.
Monocytes were isolated from mononuclear cells using CD14 Miltenyi Beads, +ve VS
selection columns and a Vario Magnet according to the manufacturer's instructions.
Monocytes were differentiated into dendritic cells by culture in DMEM 5% fetal calf serum (FCS) (Hyclone, Logan, UT), 100 pM non essential amino acids (Gibco), 1 mM
sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10-5 M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml GMCSF and 5 ng/ml IL-4 for 5-7 days. Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100 pM non essential amino acids (Gibco), 1 mM
sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10-5 M (Gibco), 10 mM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml. Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at 100 ng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 p,g/ml for 6 and 12-14 hours.
CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS
selection columns and a Vario Magnet according to the manufacturer's instructions. CD45RA and lymphocytes were isolated by depleting mononuclear cells of CDB, CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi beads and positive selection.
Then CD45R0 beads were used to isolate the CD45R0 CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45R0 CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), 100 ~M non essential amino acids (Gibco), 1 mM
sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10-5 M (Gibco), and 10 mM Hepes (Gibco) and plated at 106 cellslml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5 p,g/ml anti-CD28 (Pharmingen) and 3 ug/ml anti-CD3 (OI~T3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation. To prepare chronically activated CD8 lymphocytes, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5% FCS (Hyclone), 100 ~M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10-5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2.
The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days of the second expansion culture. The isolated NK
cells were cultured in DMEM 5% FCS (Hyclone), 100 pM non essential amino acids (Gibco), 1 mM

sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10-5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.
To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 106 cells/ml in DMEM S% FCS (Hyclone), 100 p.M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10-5 M (Gibco), and 10 mM
Hepes (Gibco). To activate the cells, we used PWM at 5 ~g/ml or anti-CD40 (Pharmingen) at approximately 10 pg/ml and IL-4 at 5-10 ng/ml. Cells were harvested for RNA
preparation at 24,48 and 72 hours.
To prepare the primary and secondary Thl/Th2 and Trl cells, six-well Falcon plates were coated overnight with 10 ~g/ml anti-CD28 (Pharmingen) and 2 ~g/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems, German Town, MD) were cultured at 10 -10 cells/ml in DMEM 5% FCS (Hyclone), 100 wM
non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10-5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (4 nglml). IL-12 (5 ng/ml) and anti-IL4 (1 ~g/ml) were used to direct to Thl, while IL-4 (5 ng/ml) and anti-IFN gamma (1 ~glml) were used to direct to Th2 and IL-10 at 5 ng/ml was used to direct to Trl . After 4-S days, the activated Thl, Th2 and Trl lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100 pM non essential amino acids (Gibco), 1 mM
sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10-5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (1 ng/ml). Following this, the activated Thl, Th2 and Trl lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti-CD95L (1 ~g/ml) to prevent apoptosis. After 4-5 days, the Thl, Th2 and Trl lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this way for a maximum of three cycles. RNA was prepared from primary and secondary Thl, Th2 and Trl after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.
The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in 0.1 mM dbcAMP at 5 x105 cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5 x 105 cells/ml. For the culture of these cells, we used DMEM or RPMI (as recommended by the ATCC), with the addition of 5% FCS (Hyclone), 100 p.M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 105 M (Gibco), 10 mM

Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at 10 ng/ml and ionomycin at 1 pg/ml for 6 and 14 hours. Keratinocyte line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in DMEM 5% FCS (Hyclone), 100 p.M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10-S M (Gibco), and 10 mM Hepes (Gibco).
CCD1106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF
alpha and 1 ng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and 25 ng/ml IFN gamma.
For these cell lines and blood cells, RNA was prepared by lysing approximately cells/ml using Trizol (Gibco BRL). Briefly, 1/10 volume of bromochloropropane (Molecular Research Corporation) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15 ml Falcon Tube. An equal volume of isopropanol was added and left at -20 degrees C overnight. The precipitated RNA was spun down at 9,000 rpm for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol. The pellet was redissolved in 300 p.1 of RNAse-free water and 35 ~l buffer (Promega) 5 p1 DTT, 7 p,1 RNAsin and 8 ~1 DNAse were added. The tube was incubated at 37 degrees C for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down and placed in RNAse free water. RNA was stored at -80 degrees C.
Panel CNSD.Ol The plates for Panel CNSD.O1 include two control wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor.
All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.
Disease diagnoses are taken from patient records. The panel contains two brains from each of the following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supernuclear Palsy, Depression, and "Normal controls".
Within each of these brains, the following regions are represented: cingulate gyrus, temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17 (occipital cortex). Not all brain regions are represented in all cases; e.g., Huntington's disease is characterized in part by neurodegeneration in the globus palladus, thus this region is impossible to obtain from confirmed Huntington's cases. Likewise Parkinson's disease is characterized by degeneration of the substantia nigra making this region more difficult to obtain. Normal control brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration.
RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA
contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
In the labels employed to identify tissues in the CNS panel, the following abbreviations are used:
PSP = Progressive supranuclear palsy Sub Nigra = Substantia nigra Glob Palladus= Globus palladus Temp Pole = Temporal pole Cing Gyr = Cingulate gyros BA 4 = Brodman Area 4 Panel CNS Neurodegeneration V1.0 The plates for Panel CNS Neurodegeneration V1.0 include two control wells and test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System).
Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.
Disease diagnoses are taken from patient records. The panel contains six brains from Alzheimer's disease (AD) pateins, and eight brains from "Normal controls" who showed no evidence of dementia prior to death. The eight normal control brains are divided into two categories: Controls with no dementia and no Alzheimer's like pathology (Controls) and controls with no dementia but evidence of severe Alzheimer's like pathology, (speciFically senile plaque load rated as level 3 on a scale of 0-3; 0 = no evidence of plaques, 3 = severe AD
senile plaque load). Within each of these brains, the following regions are represented:
Hippocampus, Temporal cortex (Broddmann Area 21), Somatosensory cortex (Broddmann area 7), and Occipital cortex (Brodmann area 17). These regions were chosen to encompass all levels of neurodegeneration in AD. The hippocampus is a region of early and severe neuronal loss in AD; the temporal cortex is known to show neurodegeneration in AD after the hippocampus; the somatosensory cortex shows moderate neuronal death in the late stages of the disease; the occipital cortex is spared in AD and therefore acts as a "control" region within AD patients. Not all brain regions are represented in all cases.
RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA
contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
In the labels employed to identify tissues in the CNS Neurodegeneration V 1.0 panel, the following abbreviations are used:
AD = Alzheimer's disease brain; patient was demented and showed AD-like pathology upon autopsy Control = Control brains; patient not demented, showing no neuropathology Control (Path) = Control brains; pateint not demented but showing sever AD-like pathology SupTemporal Ctx = Superior Temporal Cortex Inf Temporal Ctx = Inferior Temporal Cortex NOVla Expression of gene NOVla was assessed using the primer-probe sets Ag273b and Ag1094, described in Tables 12 and 13. Results from RTQ-PCR runs are shown in Tables 14, 15, 16, 17, and 18.
Table 12. Probe Name Ag273b PrimersSequences TM LengthStart SEQ ID

PositionNO:

Forward5'-CGGCTTGACGATGCTTCAC-3' 19 13 91 FAM-5'- 92 Probe TGACTTTTCTGGGCTTACCAATGCTATTTCAA- 32 37 3'-TAMRA

Reverse5'-GCACCTATCTCAATATCTGCAATATTG-3' 27 85 93 Table 13. Probe Name Ag1094 PrimersSequences TM Lengthstart SEQ
ID

PositionNO:

Forward5'-ATGGACTGGAAAACCTGGAA-3'59.4 20 192 94 FAM-5'- 95 Probe TCCTGCAAGCAGATAACAATTTTATCACA-66.5 29 213 3'-TAMRA

Reverse5'-TGCTAAAGGCACTTGGTTCA-3'59.5 20 247 96 Table 14. Panel 1 Relative Relative Ex cession Ex cession %

tm566f_ tm566f_ Tissue Name a 273b Tissue Name a 273b Endothelial cells 0.0 Renal ca. 786-0 0.0 Endothelial cells 0.0 Renal ca. A498 0.0 treated Pancreas 0.0 Renal ca. RXF 393 0.0 Pancreatic ca. 0.0 Renal ca. ACHN 0.0 Adrenal land 0.0 Renal ca. U0-31 0.0 Th oid 0.0 Renal ca. TK-10 0.0 Salav land 12.9 Liver 0.0 Pituit land 0.0 Liver fetal 0.0 Brain fetal 0.0 Liver ca. (he atoblast0.0 He G2 Brain whole 0.2 Lun 0.5 Brain am dala 0.0 Lun fetal 2.2 Brain (cerebellum)1.6 Lung ca. (small 0.0 cell) LX-1 Brain (hi ocam 0.0 Lun ca. small cell2.7 us) NCI-H69 Brain substantia 0.0 Lun ca. s.cell 44.1 ni a vac. SHP-77 Brain (thalamus 2.9 Lung ca. (large 0.0 cell)NCI-H460 Brain othalamus 0.0 Lun ca. non-sm. 0.0 cell) A549 S final cord 0.0 Lung ca. (non-s.cell)14.7 CNS ca. lio/astro 0.0 Lun ca non-s.cell 12.2 CNS ca. ( lio/astro)0.0 Lung ca. (non-s.cl)0.2 CNS ca. astro SW17830.0 Lun ca. s uam. 11.9 CNS ca. * (neuro;
met ) SI~-N- 6.6 Lun ca. s uam. 2.5 CNS ca. (astro) 0.0 Mammary land 4.8 Breast ca.* (p1.
CNS ca, astro SNB-7510.2 effusion) MCF- 0.4 Breast ca.* (pl.ef) CNS ca. ( lio) 24.3 MDA-MB- 0.0 CNS ca. lio U251 4.2 Breast ca.* 1. 7.2 effusion T47D

CNS ca. lio SF-29537.6 Breast ca. BT-549 0.0 Heart 1.5 Breast ca. MDA-N 0.0 Skeletal muscle 0.0 Ov 0.0 Bone marrow 0.0 Ovarian ca. OVCAR-30.0 Th us 0.4 Ovarian ca. OVCAR-40.0 S Teen 0.0 Ovarian ca. OVCAR-56.2 L h node 0.0 Ovarian ca. OVCAR-80.0 Colon ascendin 9.9 Ovarian ca. IGROV-10,0 ) Stomach 0.4 Ovarian ca.* ascites0.0 Small intestine 4.2 Uterus 0.0 Colon ca. SW480 0.0 Placenta 0.8 Colon ca.* SW480 0.0 Prostate 3.6 met SW620 Colon ca. HT29 34.4 Prostate ca.* one 100.0 met PC-3 Colon ca. HCT-116 0.0 Testis 0.0 Colon ca. CaCo-2 0.0 Melanoma Hs688 0.0 A .T

Colon ca. HCT-15 0.0 Melanoma* (met) 0.0 Hs688(B).T

Colon ca. HCC-29980.0 Melanoma UACC-62 0.3 Gastric ca. * (liver met) NCI- 1.3 Melanoma M14 0.0 Bladder 0.1 Melanoma LOX IMVI 0.0 Trachea 8.9 Melanoma* met SK-MEL-50.0 Kidney 0.2 Melanoma SK-MEL-280.2 Kidne fetal 1.3 Table 15. Panel 1.3D
Relative pression(%) Ex l.3Dtm2741fl.3Dtm2838f Tissue Name a 1094 a 1094 Liver adenocarcinoma 10.0 9.1 Pancreas 0.2 0.1 Pancreatic ca. CAPAN 2 0.0 0.0 Adrenal land 0.0 0.0 Thyroid 0.2 0.2 Saliv land 8.9 4.3 Pituit land 0.0 0.2 Brain (fetal) 0.1 0.0 Brain whole 0.8 0.6 Brain (amy data 0.2 0.1 Brain cerebellum 0.5 0.7 Brain (hi ocam us) 0.4 0.2 Brain substantia ni a 0.0 0.0 Brain thalamus 1.1 1.0 Cerebral Cortex 0.2 0.1 S final cord 0.2 0.0 CNS ca. liolastro) U87-MG 0.0 0.2 CNS ca. lio/astro U-118-MG 1.0 0.8 CNS ca. astro SW1783 1.1 0.9 CNS ca.* (neuro; met SK N-AS 26.4 26.8 CNS ca. astro SF-539 0.0 0.0 CNS ca. astro SNB-75 15.1 12.9 CNS ca. lio SNB-19 38.2 21.0 CNS ca. (glio) U251 3.3 3.7 CNS ca, lio SF-295 38.4 36.9 Heart fetal 0.2 0.5 Heart 0.6 0.3 Fetal Skeletal 2.9 2.2 Skeletal muscle 0.0 0.0 Bone marrow 0.0 0.2 Th us 0.4 0.1 S Teen 0.0 0.0 L h node 0.0 0.0 Colorectal 1.6 0.6 Stomach 1.6 1.6 Small intestine 4.2 3.7 Colon ca. SW480 0.0 0.0 Colon ca.* SW480 met SW620 0.4 0.1 Colon ca. HT29 21.0 25.5 Colon ca. HCT-116 0.0 0.0 Colon ca. CaCo-2 0.0 0.0 83219 CC Well to Mod Diff OD03866 0.0 0.0 Colon ca. HCC-2998 0.0 0.0 Gastric ca.* (liver met) NCI-N87 21.3 20.7 Bladder 0.1 0.0 Txachea 12.5 12.9 Kidne 0.0 0.0 Kidney (fetal) 0.9 0.6 Renal ca. 786-0 0.0 0.0 Renal ca. A498 2.0 1.5 Renal ca. RXF 393 0.0 0.0 Renal ca. ACHN 0.0 0.0 Renal ca. U0-31 0.0 0.0 Renal ca. TK-10 0.0 0.0 Liver 0.4 0.6 Liver (fetal) I 1.~ 1.0 Liver ca. (hepatoblast) HepG2 0.0 0.0 Lun ~ 1.8 1.4 Lun fetal 11.7 7.5 Lun ca. (small cell LX-1 0.2 0.0 Lun ca. small cell NCI-H69 2.4 3.1 Lung ca. s.cell var. SHP-77 100.0 100.0 Lun ca, lar a cell NCI-H460 0.0 0.0 Lun ca. non-sm. cell A549 0.3 0.8 Lun ca. non-s.cell NCI-H23 12.8 12.8 Lun ca non-s.cell HOP-62 3.9 3.7 Lun ca. non-s.cl) NCI-H522 0.1 0.0 Lun ca. s uam. SW 900 4.8 6.4 Lung ca. (s uam. NCI-H596 1.3 0.9 Marmn land 3.4 3.3 Breast ca. * 1. effusion MCF-7 1.2 0.7 Breast ca.* 1.e MDA-MB-231 0.0 0.0 Breast ca.* 1. effusion T47D 3.2 3.0 Breast ca. BT-549 2.1 1.7 Breast ca. MDA-N 0.0 0.0 Ov 0.7 0.3 Ovarian ca. OVCAR-3 0.4 0.3 Ovarian ca. OVCAR-4 0.1 0.0 Ovarian ca. OVCAR-5 8.5 6.2 Ovarian ca. OVCAR-8 0.0 0.0 Ovarian ca. IGROV-1 0.0 0.0 Ovarian ca.* ascites) SK-OV-3 0.3 0.2 Uterus 0.4 0.2 Placenta 1.0 1.3 Prostate 1.1 1.1 Prostate ca.* (bone met)PC-3 13.2 13.9 Testis 0.3 0.4 Melanoma Hs688 A .T 0.0 0.0 Melanoma* met Hs688 .T 0.0 0.0 Melanoma UACC-62 0.0 0.0 Melanoma M14 0.0 0.0 Melanoma LOX IMVI 0.0 0.0 Melanoma* (met) SK-MEL-5 0.1 0.5 (Adipose 0.5 0.4 Table 16. Panel 2D
Relative Expression(%) 2Dtm2837f ~ 2dtm2940f Tissue Name a~1094 ag1094 Normal Colon GENPAK 061003 12.4 11.4 83219 CC Well to Mod Diff OD03866 0.0 0.0 83220 CC NAT OD03866 1.0 1.5 83221 CC Gr.2 rectosigmoid (0D03868)0.3 0.0 83222 CC NAT OD03868 0.4 0.2 83235 CC Mod Diff OD03920 0.0 0.0 83236 CC NAT OD03920 0.8 0.8 83237 CC Gr.2 ascend colon OD03921 2.4 2.2 83238 CC NAT OD03921) 2.0 1.9 83241 CC from Partial He atectom 0.0 0.0 83242 Liver NAT (0D04309 0.2 0.3 874_72 Colon mets to lun OD04451-Ol 0.0 0.0 87473 Lun NAT (0D04451-02) 0.9 0.6 Normal Prostate Clontech A+ 6546-1 2.7 3.0 84140 Prostate Cancer OD04410 1.5 1.4 84141 Prostate NAT OD04410 6.5 8.0 87073 Prostate Cancer OD04720-O1 5.9 6.1 87074 Prostate NAT (0D04720-02 14.1 12.6 ormal Lun GENPAK 061010 3.3 3.5 83239 Lun Met to Muscle OD042~6 0.2 0.2 83240 Muscle NAT ODO4286) 0.0 0.0 84136 Lun Mali ant Cancer OD03126 7.9 6.0 84137 Lun NAT OD0312 1.8 2.5 84871 Lun Cancer OD04404 24.8 21.6 84872 Lung NAT (0D04404 1.8 1.7 84875 Lun Cancer OD04565 0.7 1,2 84876 Lun NAT OD04565) 0.5 0.7 85950 Lung Cancer (0D04237-01 13.5 12.5 85970 Lun NAT OD04237-02 1.4 1.0 83255 Ocular Mel Met to Liver (0D04310)0.0 0.0 83256 Liver NAT OD04310 0.2 0.4 84139 Melanoma Mets to Lun OD04321) 0.4 0.2 84138 Lung NAT (0D04321) 2.5 1.2 ormal Kidne GENPAK 061008 0.2 0.0 83786 Kidney Ca, Nuclear ade 2 OD043380.0 0.0 83787 Kidne NAT OD04338 0.2 0.2 83788 Kidney Ca Nuclear bade 1/2 0.0 0.0 (0D04339 83789 Kidne NAT (0D04339) 0.0 0.0 83790 Kidney Ca, Clear cell type 0.1 0.2 (0D04340) 183791 Kidney NAT (0D04340) 0.0 0.2 83792 Kidne Ca, Nuclear ade 3 OD043480.0 0.0 83793 Kidney NAT (0D04348) 0.0 0.1 87474 Kidne Cancer OD04622-O1 0.4 0.4 87475 Kidne NAT OD04622-03 0.0 0.0 85973 Kidney Cancer (0D04450-01) 0.0 0.0 85974 Kidney NAT (0D04450-03) 0.0 0.0 Kidne Cancer Clontech 8120607 0.4 0.3 Kidne NAT Clontech 8120608 0.0 0.0 Kidney Cancer Clontech 8120613 0.0 0.0 Kidne NAT Clontech 8120614 0.0 0.0 Kicliie Cancer Clontech 9010320 0.0 0.0 Kidne NAT Clontech 9010321 0.0 0.0 Normal Uterus GENPAK 061018 0.1 0.0 Uterus Cancer GENPAK 064011 0.5 0.6 Normal Th oid Clontech A+ 6570-1 0.5 0.4 Thyroid Cancer GENPAK 064010 0.0 0.0 Th oid Cmcer INVITROGEN A302152 0.0 0.0 Th oid NAT INVITROGEN A302153 0.2 0.1 Nornzal Breast GENPAK 061019 5.5 5.6 84877 Breast Cancer OD04566 0.5 0.7 85975 Breast Cancer OD04590-O1 3.0 3.9 85976 Breast Cancer Mets OD04590-03 1.4 1.7 87070 Breast Cancer Metastasis (0D04655-05100.0 100.0 GENPAK Breast Cancer 064006 1.7 2.0 Breast Cancer Res. Gen. 1024 0.8 0.8 Breast Cancer Clontech 9100266 3.5 4.1 Breast NAT Clontech 9100265 3.5 4.2 Breast Cancer INVITROGEN A209073 0.7 0.7 Breast NAT 1NVITROGEN A2090734 1.1 1.3 Normal Liver GENPAK 061009 2.0 1.7 Liver Cancer GENPAK 064003 0.0 0.0 Liver Cancer Research Genetics RNA 0.3 0.3 Liver Cancer Research Genetics RNA 0.0 0.0 Paired Liver Cancer Tissue Research 0.2 0.1 Genetics RNA 6004-T

Paired Liver Tissue Research Genetics0.0 0.1 Paired Liver Cancer Tissue Research 0.0 0.0 Genetics RNA 6005-T

Paired Liver Tissue Research Genetics0.1 0.1 Normal Bladder GENPAK 061001 0.2 0.1 Bladder Cancer Research Genetics 3.0 3.1 Bladder Cancer INVITROGEN A302173 1.0 0.8 87071 Bladder Cancer OD04718-O1 0.0 0.0 187072 Bladder Normal Ad'acent OD04718-03)4.1 3.4 Normal Ov Res. Gen. 0.0 0.0 Ovarian Cancer GENPAK 064008 1.6 1.4 87492 Ovary Cancer (0D04768-07 0.0 0.0 87493 Ovary NAT (0D04768-08) 0.0 0.0 Normal Stomach GENPAK 061017 1.0 1.7 Gastric Cancer Clontech 9060358 0.2 0.2 NAT Stomach Clontech 9060359 0.1 0.2 Gastric Cancer Clontech 9060395 0.4 0.7 AT Stomach Clontech 9060394 0.4 0,4 Gastric Cancer Clontech 9060397 0.1 0.3 NAT Stomach Clontech 9060396 0.1 0.2 Gastric Cancer GENPAK 064005 1.0 1.3 Table 17. Panel 3D
Relative Relative Ex ression Ex ression %

3dtm5226f 3dtm5226f Tissue Name a 1094 Tissue Name a 1094 ~

94954 Ca Ski_Cervical 94905 Daoy Medulloblastoma/ epidermoid carcinoma Cerebellum sscDNA 0.0 (metastasis sscDNA0.1 94906 TE671 Medulloblastom 94955 ES-2 Ovarian clear cell /Cerebellum sscDNA0.2 carcinoma sscDNA 0.0 94907 D283 94957 Ramos/6h stim -Medu11ob1astomalCerebell Stimulated with Med _ 0.2 PMA/ionomycin 6h 0.1 um sscDNA sscDNA

PFSI~-1 Primitive 94958 Ramos/14h 94908 stim_ _ Stimulated with NeuroectodermallCerebellum_s scDNA 1.6 PMAlionomycin 14h 0.0 sscDNA

94962 MEG-O1_Chronic myelogenous leukemia 94909 XF-498 CNS 30.4 (megokaryoblast 1.7 sscDNA sscDNA

94910 SNB- 94963 Raji_Burkitt's 78 CNS/glioma sscDNA0.7 1 homa sscDNA 0.0 268 CNS/glioblastoma 94964 Daudi Burkitt's sscDN

A 0.0 1 homa sscDNA 0.0 94965 U266 B-cell 94912 T98G Glioblastoma_ssc plasmacytoma/myeloma sscDN

DNA ' 3.3 A 0.2 Neuroblastoma 94968 CA46 Burkitt's SH

_ 22.4 1 homa sscDNA 0.0 metastasis) sscDNA

295 CNS/glioblastoma 94970 RL non-Hodgkin's sscDN B-A 27.2 cell lymphoma sscDNA0.1 94972 JMl~re-B-cell 94914 Cerebellum A 6.7 lymphomafleukemia 0.0 sscDN sscDNA

94973 Jurkat_T
cell 96777 Cerebellum 0.0 leukemia sscDNA 0.0 sscDNA

H292 Mucoepidermoid 94974 TF-lung carcinoma sscDN A 21.9 1 E oleukemia sscDNA2.1 94917 DMS-114 Small 94975 HUT 78 T-cell cell lung cancer sscDNA2.4 1 homa sscDNA 0.0 94918 DMS-79 Small cell lung 94977 U937_Histiocytic cancer/neuroendoerine0.0 lymphoma sscDNA 0.0 sscDNA

94919 NCI-H146_Small cell lung 94980 KU-812 Myelogenous caiicer/neuroendocrine100.0 leukemia sscDNA 8.2 sscDNA

94920 NCI-H526_Small cell lung 94981_769-P
Clear cell renal cancer/neuroendocrine0.0 _ 0.0 sscDNA carcinoma sscDNA

94921_NCI-N417_Small cell lung 94983 Caki-2 Clear cell renal cancerlneuroendocrine0.0 carcinoma sscDNA 0.3 sscDNA

94923 NCI-H82_Small cell lung 94984 SW 839 Clear cell renal cancer/neuroendocrine0.3 carcinoma sscDNA 0.0 sscDNA

Squamous cell lung cancer 94986 6401 Wilins' metastasis sscDNA 0.0 tumor sscDNA 0.0 94925 NCI-H1155_Large 94987 Hs766T Pancreatic cell leg carcinoma (LN

cancer/neuroendocrine65.1 metastasis sscDNA 4.9 sscDNA

94926 NCI-H1299_Large 94988 CAPAN-1 Pancreatic cell lung adenocarcinoma (liver cancer/neuroendocrine0.0 metastasis) sscDNA0.3 sscDNA

94989 SU86.86 Pancreatic 94927 NCI-H727_Lung carcinoma (liver carcinoid sscDNA 13.8 metastasis sscDNA 1.5 94928 NCI-UMC-11 94990 BxPC-3 Pancreatic Lung carcinoid sscDNA 28.7 adenocarcinoma 23.7 sscDNA

94929 LX-1 Small 94991 HPAC
cell lung Pancreatic cancer sscDNA 0.7 _ 76.8 adenocarcinoma sscDNA

94930 Colo-205 94992 MIA PaCa-2 Colon Pancreatic cancer sscDNA 0.0 carcinoma sscDNA 0.5 94993 CFPAC-1 Pancreatic 94931 KM12_Colon ductal cancer sscDNA 0.1 adenocarcinoma 0.5 sscDNA

94994 PANC-1 Pancreatic 94932 KM20L2 Colon epithelioid ductal cancer sscDNA 7.3 carcinoma sscDNA 6.9 94933 NCI-H716_Colon 94996 T24 Bladder carcinma cancer sscDNA 80.1 transitional cell 4.1 sscDNA

94935 SW-48 Colon 94997 5637 Bladder adenocarcinoma 0.3 carcinoma sscDNA 1.0 sscDNA

94936 SW1116_Colon 94998 HT-1197 Bladder adenocarcinoma 0.0 carcinoma sscDNA 2.4 sscDNA

94999 UM-UC-3 Bladder 94937 LS 174T Colon carcinma (transitional adenocarcinoma 0.0 cell sscDNA 0.0 sscDNA

94938_SW-948 Colon 95000 A204 Rhabdomyosarco adenocarcinoma 0.6 ma sscDNA 0.3 sscDNA

94939 SW-480 Colon 95001 HT-adenocarcinoma 0.0 1080 Fibrosarcoma 0.2 sscDNA sscDNA

94940 NCI-SNU-5 95002 MG-63 0steosarcoma Gastric carcinoma sscDNA 0.0 ( bone) sscDNA 0.2 94941 KATOIII_Gastric 1 Leiomyosarcoma carcinoma sscDNA 0.0 ( vulva) sscDNA 0.0 95004 SJRH30 Rhabdomyosar 94943 NCI-SNU-16 coma (met to bone Gastric carcinoma sscDNA 1.7 marrow) sscDNA 0.2 94944 NCI-SNU-1 95005 A431 Epidermoid Gastric carcinoma sscDNA 0.0 carcinoma sscDNA 0.0 94946 RF-1 Gastric 95007 WM266-adenocarcinoma 0.0 4 Melanoma sscDNA 0.4 sscDNA

95010 DU 145 Prostate 94947 RF'-48 Gastric carcinoma (brain adenocarcinoma 0.0 metastasis sscDNA 0.0 sscDNA

96778_MKN-45_Gastric 95012_MDA-MB-4.68_Breast carcinoma sscDNA 0.1 adenocarciiloma 1.3 sscDNA

94949 95013 SCG4_Squamous NCI-N87_Gastric cell _ 2.4 carcinoma of ton 0.2 carcinoma sscDNA a sscDNA

94951 OVCAR-S Ovarian 95014 SCC-9 Squamous cell carcinoma sscDNA 0.0 carcinoma of ton 0.0 a sscDNA

94952 1ZL95-2_Uterine 95015 SCC-15 Squamous cell carcinoma sscDNA 2.8 carcinoma of ton 0.3 a sscDNA

94953 HelaS3 Cervical 95017 CAL 27 Squamous cell ~ 0.2 carcinoma of tongue1.6 adenocarcinoma 1 sscDNA
sscDNA

Table 18. Panel 4D
Relative Relative Expression Expression 4Dtm2495f 4Dtm2495f Tissue Name a 1094 Tissue Name a 1094 93768 Secondary 93100 HUVEC
Thl anti-CD28/anti-CD3 0.0 Endothelial) IL-1b0.0 93769 Secondary 93779 HUVEC
Th2_anti-CD28/anti-CD3 0.1 Endothelial) IFN 0.0 gamma 93770 Secondary (Endothelial) TNF
Trl anti- alpha + IFN

CD28lanti-CD3 0.0 gamma 0.0 93573 Secondary 93101 HUVEC
Thl_resting da 4-6 in IL-2 0.0 Endothelial) TNF 0.3 alpha + IL4 93572 Secondary 93781 HLTVEC
Th2_resting da 4-6 in IL-2 0.0 Endothelial IL-11 0.0 93571 Secondary 93583 Lung Microvascular Trl_resting day 4-6 in IL-2 0.0 Endothelial Cells 0.0 none 93584 Liuig Microvascular 93568-primary Thl Endothelial Cells_TNFa anti- (4 CD28/anti-CD3 0.0 n ml and ILlb 1 0.0 n ml 93569_primary Th2_anti- 92662 Microvascular Dermal CD28/anti-CD3 0.0 endothelium none 0.0 92663 Microsvasular Dermal 93570~rimary Trl endothelium TNFa anti- (4 ng/ml) y CD28/anti-CD3 0.0 and ILlb 1 n ml 0.0 93773 Bronchial 93565~rimary Thl_resting epithelium TNFa dy (4 ng/ml) and 4-6 in IL-2 0.0 ILlb 1 n ml ** 3.7 93566-primary Th2_resting 93347 Small Airway dy 4-6 in IL-2 0.0 E ithelium none 4.5 93348 Small Airway 93567-primary Trl_resting Epithelium_TNFa dy (4 ng/ml) 4-6 in IL-2 0.0 and ILlb 1 n ml 1.7 lymphocyte anti-CD28/anti- 92668 Coronery Artery CD3 0.0 SMC restin 0.0 93352 CD45R0 CD4 92669 Coronery Artery lymphocyte anti-CD28/anti- SMC TNFa (4 ng/ml) and ILlb CD3 0.0 1 ng/ml 0.0 93251 CD8 Lymphocytes_anti-CD28/anti-CD3 0.0 93107 astrocytes 8.5 resting 93353 chronic CD8 Lymphocytes try 93108 astrocytes resting dy 4- TNFa (4 6 in IL-2 0.0 n ml and ILlb 0.2 l1 n ml 93574 chronic CD8 Lymphocytes try 92666 KU-812 activated CD3/CD28 0.3 aso hil restin 8.3 93354 CD4 none 0.0 (Baso hil) PMA/ionoycui100.0 93252 Secondary 93579 CCD1106 Thl/Th2lTrl anti-CD950.0 (Keratinocytes 70.2 CHll none (Keratinocytes) TNFa and -cells restin 0.0 IFN ** 3.0 93788 LAK cells 0.0 93791 Liver Cirrhosis1.3 93787 LAK cells 4.2 93792 Lu us Kidney0.0 IL-2+IL-12 93789 LAK cells_IL-2+IFN

aroma 0.0 93577 NCI-H292 16.6 93790 LAK cells 0.0 93358 NCI-H292 10.4 IL-2+ IL-18 IL-4 cells PMA/ionomycin and IL-18 0.0 93360 NCI-H292 20.2 93578 NK Cells 0.0 93359 NCI-H292 6.3 IL-2 restin IL-13 93109 Mixed Lymphocyte Reaction Two Wa 0.0 93357 NCI-H292 8.5 MLR IFN aroma 93110 Mixed Lymphocyte Reaction Two Way 0.0 93777 HPAEC - 0.0 MLR

93111 Mixed Lymphocyte 93778 HPAEC IL-1 betal'TNA

Reaction Two Way 0.0 alpha 0.0 MLR

93112 Mononuclear 93254 Normal Human Cells Lung (PBMCs) resting 0.0 Fibroblast none 36.3 93253 Normal Human Lung 93113 Mononuclear Fibroblast_TNFa Cells (4 ng/ml) and PBMCs PWM 0.0 IL-lb 1 ng/ml) 18.6 93114 Mononuclear 93257 Normal Human Cells Lung (PBMCs) PHA-L 0.0 Fibroblast IL-4 16.0 93256 Normal Human Lung 93249 Ramos cell 0.0 Fibroblast IL,-9 4.4 none 93250 Ramos (B 93255 Normal Human Lung cell ionom cin 0.3 Fibroblast IL-13 11.4 93349 B 1 hoc es 0.0 93258 Normal HumanI 36.9 PWM Lung Fibroblast IFN
gamma 93350 B lymphoytes 93106 Dermal Fibroblasts and IL-4 0.0 CCD1070 restin 0.1 (Eosinophil) dbcAMP' 93361 Dermal Fibroblasts differentiated 0.0 CD1070 TNF al ha 0.0 C 4 n ml (Eosinophil) dbcAMP/PMAion 93105 Dermal Fibroblasts omycin 0.0 CCD1070 IL-1 beta 0.0 1 n ml 93772 dermal fibroblast_IFN

93356 Dendritic 0.0 aroma 0.3 Cells none 93355 Dendritic Cells_LPS

100 n ml 0.0 93771 dermal fibroblast0.0 IL,-4 93775 Dendritic Cells_anti-CD40 0.0 93260 IBD Colitis 0.3 93774 Monoc es 0.0 93261 IBD Crohns 0.3 restin 93776 Monocytes --n /ml 0.0 735010 Colon normal11.4 93581 Macro ha 41.8 735019 Lun none 5.3 es resting 93582 Macrophages - -n ml 0.1 64028-1 Th us none1.2 93098 IiLTVEC

(Endothelial none 1.7 64030-1 Kidne none2.0 93099 HiJVEC

(Endothelial) starved0.0 Panel 1 Summary: A~73b Expression of the NOVIa gene is highest in a metastatic prostate cancer cell line PC-3 (CT = 26.8). There is also substantial expression of this gene in a number of lung cancer cell lines and brain cancer cell lines. Thus, expression of the NOV 1 a gene could be used to distinguish lung, prostate or brain cancer cell lines from other samples.
In addition, therapeutic inhibition of this gene product, through the use of small molecule drugs or antibodies, might have benefit in the treatment of lung, prostate or brain cancer.
Among other normal tissues this gene is also low to moderately expressed in heart, colon, small intestine, trachea, salivary gland, fetal liver, and mammary gland.
The NOV la gene encodes a novel insulin-like growth factor binding protein acid labile subunit. Among CNS tissues, this gene is expressed at moderate levels in cerebellum and thalamus. Insulin-like growth factor (IGF) has been shown to have neuroprotective effects, as is currently under investigation as a biopharmaceutical for the treatment of amyotropic lateral sclerosis. In serum, IGF is bound to both IGF-binding protein (IGFBP) and the acid labile subunit (IGFBP-ALS). In the brain, glia produce IGFBP; however the IGFBP-ALS
has not been detected in the CNS. Therefore, the NOVla gene may represent the CNS
equivalent of IGFBP-ALS. Because of the neuroprotective effects of IGF, therapeutic modulation of this gene or its protein product may be useful in treating diseases in which neuronal death/degeneration occur such as amyotropic lateral sclerosis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, or CNS injury such as stroke, head or spinal cord trauma.
Panel 1.3D Summary: A~1094 Results from two experiments using the same probe/primer set are in excellent agreement and are consistent with what is observed in Panel 1. Expression of the NOV 1 a gene is highest in a lung cancer cell line (CT =
28). There is also substantial expression of this gene in a number of additional lung cancer cell lines and brain cancer cell lines as well as in a metastatic prostate cancer cell line. Thus, NOVla gene expression could be used to distinguish prostate, lung or brain cancer cell lines from other samples. In addition, therapeutic inhibition of this gene product, through the use of small molecule drugs or antibodies, might have benefit in the treatment of prostate, lung or brain cancer.
Among CNS tissues, there is low but significant expression in thalamus and cerebellum. Please see Panel 1 summary for description of potential utility of this gene in the CNS.
Panel 2D Summary: A- 1g 094 Results from two experiments using the same probe/primer set are in good agreement. Expression of the NOVla gene is highest in a metastatic breast cancer sample (CT = 26-27). In addition, several other breast cancer and lung cancer samples show increased expression when compaxed to their normal adjacent margin samples. This observation is consistent with the results in Panel 1.3D
that show higher NOV 1 a gene expression in lung cancer cell lines. Thus, expression of this gene might be used to distinguish breast cancer or lung cancer tissue from their normal counterparts and might be of diagnostic value. Moreover, therapeutic modulation of the NOV 1 a gene or its gene product, through the use of small molecule drugs or antibodies, may be of benefit for treatment of breast or lung cancer.
Panel 3D Summary: A- 1g-094 Expression of the NOVla gene is highest in a small cell lung cancer cell line (CT = 28.5). In addition, there is significant expression of this gene in other lung cancer cell lines as well as in several brain cancer and pancreatic cancer cell lines.
These results are consistent with what is observed in the other panels. Thus, the expression of the NOV 1 a gene may be used to distinguish lung, breast or pancreas cancer cell line samples from other tissues. Moreover, therapeutic modulation of this gene or gene product, through the use of small molecule drugs or antibodies, may be of benefit for treatment of lung, breast or pancreatic cancer.
Panel 4D Summary: A-81094 The NOV 1 a gene is expressed at high levels in basophils (CT 28.3) as well as in keratinocytes and normal lung fibroblasts (independently of their activation status). In addition, this gene is expressed at a lower level in a muco-epidermoid cell line (H292). Expression of the NOV 1a gene is also found in normal lung which is consistent with the data from Panel 1.3D. The protein encoded by this transcript is a homolog of insulin-like growth factor binding protein acid labile subunit, a component of the systemic insulin-like growth factor-binding protein (IGFBP) complex. Therefore, this gene may play an important role in the biology of circulating IGFs. IGFs are involved in a wide array of cellular processes such as proliferation, prevention of apoptosis, and differentiation. Thus, the NOVIa gene may be a suitable target for protein therapeutic to modulate locally the mitogenic effect of IGF and could be useful in the treatment of emphysema, COPD, or skin related disease.
References:
1. Mewar R., McMorns F.A. (1997) Expression of insulin-like growth factor-binding protein messenger RNAs in developing rat oligodendrocytes and astrocytes. J.
Neurosci. Res 50:721-728.
Insulin-like growth factors, IGF-I and IGF-II, are potent regulators of oligodendrocyte development. Most of the IGF present in vivo is bound to members of a family of six high-affinity IGF-binding proteins (IGFBPs), which can either potentiate or inhibit IGF action, depending on other conditions. Additionally, serum contains a structurally unrelated protein, acid-labile sub-unit (ALS), which forms a ternary complex with IGF and IGFBP3.
In this study, reverse-transcriptase polymerase chain reaction (RT-PCR) was used to examine the expression of mRNAs for IGFBP 1-6 and ALS in purified populations of oligodendroglial cells and astrocytes. Astrocytes express all six IGFBPs. A2B5+/p4-oligodendrocyte precursors, 04+/O1- intermediate precursors, and O1+ oligodendrocytes express IGFBP3, 5, and 6, while IGFBP4 is expressed in oligodendrocyte precursors but not at more mature stages. They were unable to detect ALS mRNA in whole brain or in cultured oligodendroglial cells. The presence of differentially expressed IGFBPs in developing oligodendrocytes and astrocytes could significantly affect the biological activity of IGF-I and IGF-II in the central nervous system and the IGF-responsiveness of the IGFBP-expressing cells.

PMID: 9418960 2. Arnold P.M., Ma J.Y., Citron B.A., Zoubine M.N., Festoff B.W. (2000) Selective developmental regulation of gene expression for insulin-like growth factor-binding proteins in mouse spinal cord. Spine 25:1765-1770.
STUDY DESIGN: Prospective, randomized experimental study in mice. STUDY
OBJECTIVE: To determine whether insulin-like growth factor binding proteins (IGFBPs) are present in mouse spinal cord and, if so, what role they play in its development. SUMMARY
OF BACKGROUND DATA: Insulin-like growth factors are well recognized hormonal effectors of growth hormone and are expressed in the mammalian spinal cord.
The IGFBPs are a group of six genetically distinct proteins that bind IGFs and modulate their bioactivity. They appear in the brain during development, localize to the neuromuscular junction, and promote motor neuron survival. The benefit of IGF-I in amyotrophic lateral sclerosis ALS and its potential use in preventing motor neuron apoptosis in spinal cord injury dictates that studies of the presence and response of IGFBPs in that tissue be performed. METHODS: The IGFBPs in mouse spinal cord were analyzed by Western ligand blot, Western immunoblot, and reverse transcription-polymerase chain reaction at various time points from embryonic day 14 to postnatal day 30. RESULTS: Three IGFBPs with molecular masses of 24, 28, and 32 kDa were found, the latter two being the most prominent. The data indicate that these are IGFBP-4, -5, and -2. CONCLUSION: Both IGFBP-2 and BP-5 are developmentally regulated in mouse spinal cord, with higher levels of those at early embryonic stages indicating their potential role in development of the mouse spinal cord.
PMID:10888943 3. Corse A.M., Bilak M.M., Bilak S.R., Lehar M., Rothstein J.D., Kuncl R.W.
(1999) Preclinical testing of neuroprotective neurotrophic factors in a model of chronic motor neuron degeneration. Neurobiol. Dis. 6:335-346.
Many neurotrophic factors have been shown to enhance survival of embryonic motor neurons or affect their response to injury. Few studies have investigated the potential effects of neurotrophic factors on more mature motor neuxons that might be relevant for neurodegenerative diseases. Using organotypic spinal cord cultures from postnatal rats, researchers have demonstrated that insulin-like growth factor-I (IGF-I) and glial-derived neuroirophic factor (GDNF) significantly increase choline acetyltransferase (ChAT) activity, but brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) do not. Surprisingly, ciliary neurotrophic factor (CNTF) actually reduces ChAT
activity compared to age-matched control cultures. Neurotrophic factors have also been shown to alter the sensitivity of some neurons to glutamate neurotoxicity, a postulated mechanism of injury in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS).
Incubation of organotypic spinal cord cultures in the presence of the glutamate transport inhibitor threo-hydroxyaspartate (THA) reproducibly causes death of motor neurons which is glutamate-mediated. In this model of motor neuron degeneration, IGF-I, GDNF, and NT-4/5 are potently neuroprotective, but BDNF, CNTF, and NT-3 are not. The organotypic glutamate toxicity model appears to be the best preclinical predictor to date of success in human clinical trials in ALS.
NOV3a Expression of gene NOV3a was assessed using the primer-probe set Ag2100, described in Table 19. Results from RTQ-PCR runs are shown in Tables 20, 21, 22, 23, and 24.
Table 19. Probe Name Ag2100 Start SEQ
ID

PrimersSequences TM Length PositionNO:

Forward5'-AGATCCCTGGAACAGAGGATT-3'59 21 2446 97 TET-5'- 98 Probe TGTCTGAAGCCAATAAACTTGCAGCA-67.926 2474 3'-TAMRA

Reverse~ 5'-CCTTCATGTTCCTTTGGGTAA-3'~ 21 2513 99 I
58.9~ ~

Table 20. Panel 1.3D
Relative Relative Ex ression Ex ression %

l.3dtm3300t_ l.3dtm3300t_ Tissue Name a 2100 Tissue Name a 2100 Liver adenocarcinoma11.7 Kidne fetal 1.8 Pancreas 0.0 Renal ca. 786-0 7.1 .

Pancreatic ca. 3.2 Renal ca. A498 3.7 Adrenal land 1.4 Renal ca. RXF 393 3.1 Thyroid 0.1 Renal ca. ACHN 4.4 Saliv land 0.1 Renal ca. U0-31 6.3 (Pituitary gland 2.1 Renal ca. TK-10 3.2 Brain (fetal) 2.1 Liver 0.0 Brain whole 24.7 Liver fetal 3.8 Brain am dala 11.2 Liver ca. a atoblast3.2 He G2 Brain (cerebellum)2.7 Lun 0.3 Brain i ocam us 36.3 Lun fetal 0.9 Brain (substantia 1.5 Lun ca. small cell6.6 nigra LX-1 Brain thalamus 30.4 Lun ca. small cell8.5 Cerebral Cortex 100.0 Lun ca. s.cell 7.5 var. SHP-77 S iiial cord 2.5 Lun ca. lax a cell0.0 CNS ca. lio/astro 6.4 Lun ca. non-sm. 0.2 U87-MG cell A549 CNS ca. lio/astro 33.7 Lun ca. (non-s.cell10.4 CNS ca. astro SW17835.9 Lun ca (non-s.cell1.4 CNS ca.* (neuro;
met) SK N- 14.5 Lun ca. non-s.cl 5.3 CNS ca. astro) 7.4 Lun ca. s uam. 3.2 CNS ca. astro SNB-755.8 Lun ca. (s uam. 7.2 CNS ca. (glio SNB-191.0 Mammary gland 0.2 Breast ca.* (p1.
CNS ca. lio U251 2.4 effusion) MCF- 5.6 Breast ca.* (pl.ef) CNS ca. lio SF-2950.9 MDA-MB- 14.5 Heart (fetal 0.4 Breast ca.* ( 1. 2.4 effusion) T47D

Heart 0.1 Breast ca. BT-549 6.8 Fetal Skeletal 3.4 Breast ca. MDA 14.0 N

Skeletal muscle 0.0 Ovary 2.2 Bone marrow 5.4 Ovarian ca. OVCAR-32.5 Thymus 2.1 Ovarian ca. OVCAR-40.8 S Teen 0.6 Ovarian ca. OVCAR-52.7 L h node 0.4 Ovarian ca. OVCAR-83.2 Colorectal 1.8 Ovarian ca. IGROV-12.0 Stomach 1.0 Ovarian ca. * ascites7.4 Small intestine 1.6 Uterus 0.0 Colon ca. SW480 13.1 Placenta 0.2 Colon ca.* (SW480 4.5 Prostate 0.2 met SW620 Colon ca. HT29 4.1 Prostate ca.* (bone2.0 met)PC-3 Colon ca. HCT-116 5.0 Testis 4.0 Colon ca. CaCo-2 5.9 Melanoma Hs688 0.7 A .T

83219 CC Well to Mod Diff 2.8 Melanoma* met Hs68~0.3 (0D03866) B .T

Colon ca. HCC-29983.7 Melanoma UACC-62 0.5 Gastric ca. * (liver met) NCI- 2.3 Melanoma M14 7.2 Bladder 0.9 Melanoma LOX IMVI 2.8 Trachea 0.7 Melanoma* met SK-MEL-55.8 Kidne 0.7 Adi ose 0.2 Table 21. Panel 2.2 Relative Relative Ex ression Ex ression %

2.2x4tm6379t 2.2x4tm6379t Tissue Name a 2100 Tissue Name a 2100 b2 b2 Normal Colon GENPAK

061003 6.2 83793 Kidne NAT 30.3 98938 Kidney malignant cancer 97759 Colon cancer13.4 OD06204B 3.6 97760 Colon cancer 98939 Kidney normal NAT adjacent OD06064) 9.0 tissue OD06204E 10.5 85973 Kidney Cancer 97778 Colon cancerOD061594.5 OD04450-O1 2.4 97779 Colon cancer 85974 Kidney NAT
NAT (OD04450-OD06159 5.9 03 13.3 98861 Colon cancer Kidney Cancer (OD06297- Clontech 04 3.8 8120613 6.6 98862 Colon cancer NAT

(0D06297-015) 10.0 Kidney NAT Clontech1.2 83237 CC Gr.2 ascend Kidney Cancer colon Clontech (0D03921) 4.3 9010320 1.6 83238 CC NAT OD039212.8 Kidne NAT Clontech4.5 97766 Colon cancer Kidney Cancer metastasis Clontech OD06104) 1.7 8120607 0.5 97767 Lun NAT OD061043.1 Kidne NAT Clontech1.7 87472 Colon mets ormal Uterus GENPAK
to lung (0D04451-01 9.6 061018 1.1 87473 Lung NAT Uterus Cancer (OD04451- GENPAK

02 3.2 064011 1.5 Normal Prostate onnal Thyroid Clontech A+ Clontech A+

6546-1 (8090438) 1.2 6570-1 (7080817 0.0 84140 Prostate Thyroid Cancer Cancer GENPAK

(0D04410) 0.0 064010 0.6 84141 Prostate . Thyroid Cancer NAT INVITROGEN

OD04410 0.7 A302152 5.3 Thyroid NAT INVITROGEN

onnal Ov Res. Gen.2.8 A302153 0.0 98863 Ovarian cancer Normal Breast GENPAK

(0D06283-03) 11.7 061019 3.0 98865 Ovarian cancer NATlfallopian tube 84877 Breast Cancer (0D06283-07) 3.0 OD04566 8.1 Ovarian Cancer GENPAK

064008 1.1 Breast Cancer.Res.2.9 Gen. 1024 97773 Ovarian cancer 85975 Breast Cancer OD06145 0.9 ( OD04590-O1) 14.7 97775 Ovarian cancer 85976 Breast Cancer NAT Mets (0D06145) 0.0 ( OD04590-03) 3.2 98853 Ovarian cancer 87070 Breast Cancer Metastasis (0D06455-03 15.8 ( OD04655-05) 5.4 '98854 Ovarian GENPAK Breast NAT Cancer ~,(OD06455-07) 1.8 064006 3.1 Fallo ian tube Breast Cancer Clontech Normal Lun GENPAK 1.2 9100266 2.6 92337 Invasive poor diff. lung adeno (0D04945-01 8.4 Breast NAT Clontech2.3 92338 Lung NAT Breast Cancer INVITROGEN
(OD04945-03 1.2 A209073 1.8 84136 Lung Malignant Breast NAT INVITROGEN
Cancer OD03126 5.0 A2090734 2.5 97763 Breast cancer 84137 Lun NAT OD031260.6 OD06083 17.1 90372 Lung Cancer 97764 Breast cancer node (OD05014A) 10.1 metastasis (0D06083)14.6 Normal Liver GENPAK

90373 Lun NAT OD05014B9.0 061009 0.4 Liver Cancer Research Genetics 97761 Lun cancer 10.1 RNA 1026 0.0 97762 Lung cmcer Liver Cancer Research NAT Genetics OD0608I) 4.0 RNA 1025 1.8 Paired Liver Cancer Tissue 85950 Lung Cancer Research Genetics (OD04237- RNA 6004-01 4.1 T 1.1 85970 Lung NAT Paired Liver Tissue (OD04237- Research 02) 2.0 Genetics RNA 6004 2.5 N

Paired Liver Cancer Tissue 83255 Ocular Mel Research Genetics Met to Liver RNA 6005-(OD04310 0.9 T 1.6 Paired Liver Tissue Research 83256 Liver NAT 0.4 Genetics RNA 6005-N0.0 84139 Melanoma Mets to Lung (0D04321) 10.4 Liver Cancer GENPAK0.7 ormal Bladder GENPAK.

84138 Lun NAT (0D04321)2.0 061001 2.9 Normal Kidney GENPAK Bladder Cancer Research 061008 5.0 Genetics RNA 1023 1.5 83786 Kidney Ca, Bladder Cancer Nuclear INVITROGEN

ade 2 (0D04338) 15.3 A302173 17.8 ormal Stomach GENPAK

83787 Kidne NAT 5.1 061017 10.4 OD04338) 83788 Kidney Ca Gastric Cancer Nuclear grade Clontech 112 (0D04339 100.0 9060397 1.1 AT Stomach Clontech 83789 Kidney NAT 9.3 9060396 0.7 83790 Kidney Ca, Gastric Cancer Clear cell Clontech a (0D04340) 14.0 9060395 2.8 AT Stomach Clontech 83791 Kidne NAT 11.2 9060394 2.8 83792 Kidney Ca, Gastric Cancer Nuclear GENPAK

grade 3 (0D04348) 9.0 064005 6.0 Table 22. Panel 3D
Relative Relative Ex ression Ex ression %

3dx4tm5110t 3dx4tm5110t Tissue Name a 2100 Tissue Name a 2100 a2 a2 94954 Ca Ski_Cervical 94905 Daoy_Medulloblastoma/ epidermoid carcinoma Cerebellum sscDNA 7.3 (metastasis sscDNA21.0 94906 TE671 Medulloblastom 94955 ES-2 Ovarian clear cell !Cerebellum sscDNA3.8 carcinoma sscDNA 11.7 94907 D283 94957 Ramos/6h stun_ Med_MedulloblastomalCerebell Stimulated with lun sscDNA 15.7 PMA/ionomycin 6h 10.8 sscDNA

94908 PFSK-1 Primitive 94958 Ramos/14h stim_ NeuroectodermallCerebellum_s Stimulated with scDNA 11.2 PMA/ionomycin 14h 6.2 sscDNA

94962 MEG-O1 Chronic myelogenous leukemia 94909 XF-498 CNS 21.2 (megokaryoblast) 5.8 sscDNA sscDNA

94910 SNB- 94963 Raji Burkitt's 78 CNS/glioma sscDNA11.3 lym home sscDNA 6.8 268 CNS/glioblastoma_sscDN 94964 Daudi_Burkitt's A 7.6 1 n home sscDNA 14.7 94965 U266 B-cell 94912 T98G_Glioblastoma_ssc plasmacytoma/myeloma sscDN

DNA 12.0 A 5.1 96776_SK-N-SH_Neuroblastoma 94968 CA46 Burkitt's metastasis) sscDNA5.6 1 home sscDNA 5.0 295 CNS/glioblastoma 94970 RL non-Hodgkin's sscDN B-A 12.4 cell l home sscDNA3.8 94972_JMl~re-B-cell 94914 Cerebellum 16.1 lymphomalleukemia 11.5 sscDNA sscDNA

94973 Jurkat T
cell 96777 Cerebellum 3.6 leukemia sscDNA 12.5 sscDNA

H292_Mucoepidermoid 94974 TF-lung carcinoma sscDNA 14.0 1 Erythroleukemia 9.9 sscDNA

94917 DMS-114 Small 94975 HUT 78_T-cell cell lung cancer_sscDNA10.3 lym home sscDNA 14.7 94918 DMS-79 Small cell lung 94977 U937 Histiocytic cancer/neuroendocrine100.0 lym home sscDNA 8.1 sscDNA

94919 NCI-H146_Small cell lung 94980 KU-812 Myelogenous cancer/neuroendocrine14.2 leukemia sscDNA 17.7 sscDNA

94920 NCI-H526_Small cell lung 94981 769-P_Clear cell renal cancer/neuroendocrine19.8 carcinoma sscDNA 6.3 sscDNA

94921_NCI-N417_Small 94983_Caki-2_Clear cell cell renal lun 5.7 carcinoma sscDNA 9.5 cancer/neuroendocrine_sscDNA

94923 NCI-H82_Small cell lung 94984 SW 839 Clear cell renal cancer/neuroendocrine10.1 carcinoma sscDNA 5.2 sscDNA

94924 NCI-H157_Squamous cell lung cancer 94986 6401 Wilms' metastasis sscDN A 13.8 tumor sscDNA 6.3 94925 NCI-H1155_Large 94987 Hs766T Pancreatic cell lung carcinoma (LN

cancer/neuroendocrine36.0 metastasis sscDNA 15.7 sscDNA

94926 NCI-H1299_Large 94988 CAPAN-1 Pancreatic cell lung adenocarcinoma (liver cancer/neuroendocrine22.7 metastasis sscDNA 8.6 sscDNA

94989 SU86.86 Pancreatic 94927 NCI-H727_Lung carcinoma (liver carcinoid sscDNA 14.3 metastasis) sscDNA14.0 94928 NCI-UMC-11 94990 BxPC-3 Pancreatic Lung carcinoid sscDNA 25.8 adenocarcinoma 9.4 sscDNA

94929 LX-1 Small 94991 HPAC Pancreatic cell lung cancer sscDNA 11.0 adenocarcinoma 14.4 sscDNA

94930 Colo-205 94992 MIA PaCa-2 Colon Pancreatic cancer sscDNA 12.7 carciizoma sscDNA 2.6 94993 CFPAC-1 Pancreatic 94931 KM12 Colon ductal cancer sscDNA 17.1 adenocarcinoma 38.5 sscDNA

94994 PANG-1 Pancreatic 94932 KM20L2 Colon epithelioid ductal cancer sscDNA 7.0 carcinoma sscDNA 19.5 94933 NCI-H716_Colon 94996 T24_Bladder carcinma cancer sscDNA 19.4 (transitional cell9.0 sscDNA

94935 SW-48 Colon 94997 5637 Bladder adenocarcinoma 10.6 carcinoma sscDNA 10.5 sscDNA

94936 SW1116 Colon 94998 HT-1197 Bladder adenocarcinoma 7.7 carcinoma sscDNA 4.8 sscDNA

94999 UM-UC-3_Bladder 94937 LS 174T Colon carcinma (transitional adenocarcinoma 9.8 cell) sscDNA 13.3 sscDNA

94938 SW-948 Colon 95000 A204 Rhabdomyosarco adenocarcinoma 1.4 ma sscDNA 15.2 sscDNA

94939 SW-480 Colon 95001 HT-adenocarcinoma 7.6 1080 Fibrosarcoma 11.9 sscDNA sscDNA

94940 NCI-SNU-5 95002 MG-63 0steosarcoma Gastric carcinoma sscDNA 14.8 (bone sscDNA 7.3 94941 KATOIII_Gastric 1 Leiomyosarcoma carcinoma sscDNA 18.8 vulva sscDNA 47.8 95004 SJRH30 Rhabdomyosar 94943 NCI-SNU-16 coma (met to bone Gastric carcinoma sscDNA 12.5 marrow) sscDNA 10.2 94944 NCI-SNU-1 95005 A431 Epidermoid Gastric carcinoma sscDNA 12.3 carcinoma sscDNA 12.1 94946 RF-1 Gastric 95007 WM266-adenocarciiloma 5.3 4 Melanoma sscDNA 21.8 sscDNA

95010 DU 145 Prostate 94947 1ZF-48 Gastric carcinoma (brain adenocarciiioma 7.7 metastasis) sscDNA0.2 sscDNA

96778_MKN-45_Gastric 95012_MDA-MB-4.68_Breast carcinoma sscDNA 11.7 adenocarcinoma 5.6 sscDNA

94949_NCI-N87_Gastric 95013 SCC-4_Squamous cell carcinoma sscDNA 9.3 carcinoma of tongue0.3 sscDNA

94951 OVCAR-5 Ovarian 95014 SGC-9 Squamous cell carcinoma sscDNA 3.0 carcinoma of ton 0.3 a sscDNA

94952 RL95-2'Uteriiie 95015 SCC-15 Squamous cell carcinoma sscDNA 4.5 carcinoma of ton 0.2 a sscDNA

94953 HelaS3~Cervical 95017 CAL 27 Squamous cell adenocarcinoma 9.0 carcinoma of tongue19.8 sscDNA sscDNA

Table 23. Panel 4D
Relative Relative Ex ression Ex ression %

4dtm3359t 4dtm3359t Tissue Name a 2100 Tissue Name a 2100 93768 Secondary 93100 HUVEC
Thl anti-CD28/anti-CD3 15.4 Endothelial IL-lb12.2 93769 Secondary 93779 HUVEC
Th2_anti-CD28/anti-CD3 11.9 (Endothelial) 16.6 IFN gamma 93770 Secondary (Endothelial)~TNF
Trl anti- alpha + IFN

CD28/anti-CD3 15.6 anima 11.8 93573 Secondary 93101 HUVEC
Thl_resting da 4-6 in IL-2 4.9 (Endothelial TNF 11.4 al ha + IL4 93572 Secondary 93781 HUVEC
Th2_resting da 4-6 in IL-2 3.3 ndothelial IL-11 8.2 93571 Secondary 93583 Lung Microvascular Trl_resting day 4-6 in IL-2 6.0 Endothelial Cells7.3 none 93584 Lung Microvascular 93568~rimary Thl Endothelial Cells_TNFa anti- (4 CD28/anti-CD3 13.6 n ml and lLlb 6.2 1 n ml 93569-primary Th2_anti- 92662 Microvascular Dermal CD28/anti-CD3 12.0 endothelium none 23.3 92663 Microsvasular Dermal 93570~rimary Trl endothelium TNFa anti- (4 ng/ml) CD28/anti-CD3 22.2 and ILlb 1 n /ml 10.5 93773 Bronchial 93565~rimary Thl epithelium TNFa resting dy (4 ng/ml) and 4-6 in IL-2 100.0 ILlb 1 ng/ml ** 0.6 93566~rimary Th2 93347 Small Airway resting dy 4-6 in IL-2 37.9 E ithelium none 1.6 93348 Small Airway 93567_primary Trl_resting Epithelium TNFa dy (4 ng/ml) 4-6 in 1L-2 29.3 and ILlb (1 n 7.4 ml) lymphocyte anti-CD28/anti- 92668 Coronery Artery CD3 13.6 SMC restin 4.4 93352 CD45R0 CD4 92669 Coronery Artery lymphocyte anti-CD28/anti- SMC_TNFa (4 ng/ml) and ILlb CD3 15.4 1 ng/ml) 2.0 93251 CD8 Lymphocytes_anti-CD28/anti-CD3 10.6 93107 astroc es 1.3 restin 93353 chronic CD8 Lymphocytes 2ry_resting 93108 astrocytes_TNFa dy 4- (4 6 in IL-2 7.9 ng/ml) and ILlb 0.5 (1 n ml) 93574 chronic CD8 Lymphocytes 2ry_activated 92666 KU-812 CD3/CD28 17.3 Baso hil restin 22.4 92667 KiJ-812 93354 CD4 none 0.5 (Baso hil PMA/ionoycin28.5 93252_Secondary 93579 CCD1106 Thl/Th2/Trl anti-CD9517.1 (Keratinocytes) 14.3 CH11 none (Keratinocytes) TNFa and ~
-93103 LAK 3.6 IFN ** 18.4 cells restin 93788 LAK cells 16.8 93791 Liver Cirrhosis0.5 93787 LAIC cells 8.4 93792 Lu us Kidne 3.3 IL-2+IL-12 93789 LAK cells_IL-2+IFN

anima 16.4 93577 NCI-H292 29.5 93790 LAK cells 16.8 93358 NCI-H292 27.7 IL-2+ IL-18 IIr4 cells PMA/ionomycin and IL-18 0.6 93360 NCI-H292 32.3 93578 NK Cells 15.3 93359 NCI-H292 13.4 IL-2 restin Ih-13 93109 Mixed Lymphocyte Reaction Two Wa 1.8 93357 NCI-H292 11.0 MLR IFN a 93110 Mixed Lymphocyte Reaction Two Wa 6.1 93777 HPAEC - 8.5 MLR

93111 Mixed Lymphocyte 93778 HPAEC IL-1 beta/TNA

Reaction Two Way R 10.1 alpha 7.7 ML

93112 Mononuclear 93254 Normal Human Cells Lung (PBMCs restin 0.1 Fibroblast none 6.3 93253 Normal Human Lung 93113 Mononuclear Fibroblast TNFa Cells (4 ng/ml) and (PBMCs PWM 25.5 IL-lb 1 n ml 9.0 93114 Mononuclear 93257 Normal Human Cells Lung (PBMCs) PHA-L 24.0 Fibroblast IL-4 3.7 93256 Normal Human Lung 93249 Ramos (B 17.7 Fibroblast IL-9 5.0 cell none 93250 Ramos (B 93255 Normal Human Lung cell) ionomycin 92.0 Fibroblast IL-13 1.7 93258 Normal Human Lung 93349 B 1 hoc es 48.6 Fibroblast IFN 3.4 PWM anvna 93350 B lymphoytes_CD40L 93106 Dermal Fibroblasts and IL-4 16.4 CCD1070 restin 57.4 (Eosinophil) dbcAMP 93361 Dermal Fibroblasts differentiated 10.5 CD 1070 TNF al 79.0 C ha 4 n ml 93248 EOL-1 93105 Dermal Fibroblasts (Eosino hil) dbcAMP/PMAion7.0 CCD1070 IL-1 beta 21.8 1 n ml omycin 93772 dermal fibroblast_IFN

93356 Dendritic 0.5 gamma 22.2 Cells none 93355 Dendritic Cells_LPS

100 n ml 0.0 93771 dermal fibroblast45.7 93775 Dendritic Cells_anti-CD40 0.0 93260 IBD Colitis 0.9 93774 Monocytes 0.2 93261 IBD Crohns 1.0 restin 93776 Monocytes ng/ml 0.0 735010 Colon normal3.7 93581 Macro ha 4.4 735019 Lun none 1.5 es restin 93582 Macrophages ng/ml 0.6 64028-1 Thymus 13.0 none Endothelial none 24.7 64030-1 Kidne none31.2 (Endothelial starved43.5 Table 24. AI comprehensive_panel v1.0 Relativepression(%) Ex tm7130t_tm7159t_ Tissue Name a 2100 a 2100 a2 b1 110967 COPD-F 0.5 0.8 110980 COPD-F 1.5 1.2 110968 COPD-M 0.4 0.6 110977 COPD-M 1.5 1.9 110989 Emphysema-F 4.2 6.0 110992 Em h sema-F 2.8 2.9 110993 Em h sema-F 0.9 0.8 110994 Em h sema-F 0.7 0.4 110995 Em h sema-F 2.0 5.4 110996 Emphysema-F 2.2 2.4 110997 Asthma-M 1.9 3.1 111001 Asthma-F 1.4 2.7 111002 Astluna-F 1.0 1.0 111003 Ato is Asthma-F 4.0 2.2 111004 Ato is Asthma-F 16.6 17.0 111005 Ato is Asthma-F 7.2 5.5 111006 Ato is Asthma-F 0.9 0.7 111417 Aller -M 1.9 2.4 112347 Aller -M 0.0 0.0 112349 Normal Lun -F 0.0 0.0 112357 Normal Lun -F 6.1 6.0 112354 Normal Lun -M 1.5 2.3 112374 Crohns-F 2.9 5.2 112389 Match Control Crohns-F 9.0 6.8 112375 Crohns-F 2.5 3.8 112732 Match Control Crohns-F 3.8 5.4 112725 Crohns-M 0.1 0.7 112387 Match Control Crohns-M 1.0 1.4 112378 Crohns-M 0.0 0.0 112390 Match Control Crohns-M Z.5 1.8 112726 Crohns-M 3.8 5.9 112731 Match Control Crohns-M 3.6 6.7 112380 Ulcer Col-F 4.9 4.9 112734 Match Control Ulcer Col-F 12.6 12.0 112384 Ulcer Col-F 6.6 10.2 112737 Match Control Ulcer Col-F 4.1 6.1 112386 Ulcer Col-F 0.5 1.2 112738 Match Control Ulcer Col-F 7.5 7.9 112381 Ulcer Col-M 0.1 0.0 112735 Match Control Ulcer Col-M 2.9 2.3 112382 Ulcer Col-M 6.8 8.4 112394 Match Control Ulcer Col-M 0.5 0.5 112383 Ulcer Col-M 12.1 14.6 112736 Match Control Ulcer Col-M 3.5 5.3 112423 Psoriasis-F 1.4 1.1 112427 Match Control Psoriasis-F 2.9 1.8 112418 Psoriasis-M 0.8 0.8 112723 Match Control Psoriasis-M 6.1 7.4 112419 Psoriasis-M 1.0 1.3 112424 Match Control Psoriasis-M 0.4 1.2 112420 Psoriasis-M 1.8 2.4 112425 Match Control Psoriasis-M 2.2 2.7 104689 (MF) OA Bone-Backus 12.1 13.2 104690 MF Ad' "Normal" Bone-Backus 5.4 4.2 104691 MF OA S ovium-Backus 43.3 35.7 104692 (BA) OA Cartilage-Backus 0.9 0.4 104694 BA OA Bone-Backus 16.8 16.7 104695 (BA) Ad' "Normal" Bone-Backus6.5 6.1 104696 A OA S iovium-Backus 24.0 24.2 104700 (SS) OA Bone-Backus 12.3 35.1 104701 SS) Ad' "Normal" Bone-Backus7.9 9.5 104702 SS OA S ovium-Backus 8.3 7.9 117093 OA Cartila a Re 7 2.0 2.3 112672 OA Bones 1.9 0.8 112673 OA Synovium5 0.3 1.2 112674 OA S ovial Fluid cells5 0.5 0.4 117100 OA Cartila a Re 14 0.4 0.3 112756 OA Bone9 I 100.0 100.0 112757 OA Synovium9 0.5 0.2 112758 OA S ovial Fluid Cells9 0.8 1.5 117125 RA Cartila a Re 2 1.0 0.6 113492 Bone2 RA 2.8 3.6 113493 S novium2 RA 1.7 0.7 113494 S Fluid Cells RA 0.9 2.1 I 13499 Cartila e4 RA Z.I 1.8 113500 Bone4 RA 1.8 2.5 113501 S ovium4 RA 2.1 2.3 113502 S Fluid Cells4 RA 1.0 0.8 113495 Cartilage3 RA 2.5 2.6 113496 Bone3 RA 2.0 2.1 113497 Synovium3 RA 1.3 1.4 113498 S Fluid Cells3 RA 2.9 3.2 117106 Normal Cartila a Re 20 0.1 0.7 113663 Bone3 Normal 0.3 0.1 113664 S ovium3 Normal 0.0 0.0 113665 Syn Fluid Cells3 Normal 0.1 0.2 117107 Normal Cartila a Re 22 0.9 0.3 113667 Bone4 Normal 0.4 0.7 113668 S ovium4 Normal 1.0 1.1 113669 Syn Fluid Cells4 Normal 1.0 I 0.7 Panel 1.3D Summary: A-_ 2a 100 Expression of the NOV3a gene is highest in cerebral cortex (CT = 26.3). This gene is expressed at more moderate levels in other parts of the CNS
including amygdala, cerebellum, hippocampus, substantia nigra, thalamus, spinal cord, and fetal brain. Expression of the NOV3a gene in other normal tissues was lower than that in brain, suggesting a specific function for this protein in the CNS. Thus, this gene may be useful as a marker to distinguish brain from other tissues. The NOV3a gene encodes a protein with homology to citron-kinase. Citron-kinase (Citron-K) has been proposed by in vitro studies to be a crucial effector of Rho in regulation of cytokinesis. Citron-K is essential for cytokinesis in vivo in specific neuronal precursors and may play a fundamental role in specific human malfonnative syndromes of the CNS. General inhibitors of the RHOIRAC-INTERACTING
CITRON KINASE family disrupt endothelial tight junctions, suggesting that specific modulators of this brain-preferential family member could be useful in delivery of therapeutics across the blood brain barrier. These general inhibitors also influence intracellular calcium flux, which is a central component of many important neuronal processes, such as apoptosis, neurotransmitter release and signal transduction. Thus, modulators of NOV3a protein function may prove useful in the treatment of neurodegenerative disorders involving apoptosis, such as spinal muscular atrophy, Alzheimer's disease, Huntington's disease, Parkinson's disease, and others. Diseases involving neurotransmitters or signal transduction, such as schizophrenia, mania, stroke, epilepsy and depression may also benefit from agents that modulate the function of the NOV3a gene product.
The NOV3a gene also shows low expression in several metabolic tissues including adrenal gland (CT = 32), pituitary gland (CT = 32) and fetal heart (CT = 34).
Interestingly, this gene is expressed at higher levels in adult skeletal muscle (CT = 37) compared to fetal skeletal muscle (CT = 31) as well as in adult liver (CT = 40) compared to fetal liver (CT = 31). Thus, the NOV3A gene may be used to differentiate between the fetal and adult skeletal muscle and liver. Moreover, the therapeutic modulation of this gene, specifically its use in replacement type therapy through the administration of purified protein, might be beneficial in the treatment of diseases involving the degeneration of liver or skeletal muscle, such as muscular dystrophy.
Panel 2.2 Summary: A-82100 Expression of the NOV3a gene is highest in a kidney cancer sample (CT = 28). In addition, there are a number of normal tissue/cancer tissue pairs in which this gene is expressed at higher levels in the tumor than the normal matched tissue.
Thus, expression of the NOV3a gene could be used to distinguish between cancerous tissue and normal tissue. In addition, therapeutic modulation of this gene product, through the use of small molecule drugs or antibodies, might be of benefit in the treatment of cancer.
Panel 3D Summary: A-82100 Expression of the NOV3a gene is highest in a lung cancer cell line (CT = 26). However, low to moderate expression is also seen in the majority of cancer cell lines on this panel, suggesting that this gene may play an important role in many cell types.
Panel 4D Summary: A- 2g 100 The NOV3a gene is highly induced in Ramos B cells treated with PMA and ionomycin, in non-transformed B cells treated with PWM
and in PBMC
treated with PWM. All three of these observations are consistent with this transcript being induced in B cells after activation. Upon activation, T cells also produce this transcript, PBMC treated with PHA (T cell mitogen) express the transcript as well as primary activated Thl cells. Primary Trl and Th2 express the transcript to a lesser extent.
Fibroblost and endothelial cell lines on this panel also express NOV3a gene, although at lower levels as compared to the activated lymphocytes.

Role in inflammation: The NOV3a gene product has homology to the RHO/RAC-interacting citron kinase. The citron kinase may play an important role in T
cell activation, by regulating TCR-mediated T cell spreading, chemotaxis and other chernokine responses and in apoptosis. Since the protein encoded for by the NOV3a gene has high homology to this kinase, it too could contribute to T cell motility, activation and apoptosis.
Likewise, this putative kinase may also be important in B cell motility, antigen receptor mediated activation and apoptosis.
Therapeutic function: Small molecule therapeutics designed against the protein encoded for by the NOV3a gene could reduce or inhibit inflammation. Anti-sense therapeutics that would block the translation of the transcript and protein production could also inhibit inflammatory processes. These types of therapeutics could be important in the treatment of diseases such as osteoarthritis. Likewise, these therapeutics could be important in the treatment of asthma, psoriasis, diabetes, and IBD, which require activated T cells, as well as diseases that involve B cell activation such as systemic lupus erythematosus.
AI comprehensive-panel v1.0 Summary: A~2100 The NOV3A gene is highly expressed in bone isolated from 5 different osteoarthritic (OA) patients, synovium in 3 out of 5 OA patients, but not in cartilege from OA patients nor in any tissues from rheumatoid arthritis (RA) patients or in control samples. Thus, small molecule therapeutics designed against the protein encoded for by the NOV3a gene could reduce or inhibit inflammation.
Anti-sense therapeutics that would block the translation of the transcript and protein production could also inhibit inflammatory processes. These types. of therapeutics could be important in the treatment of diseases such as osteoarthritis References:
1. Di Cunto F., Imarisio S., Hirsch E., Broccoli V., Bulfone A., Migheli A., Atzori C., Turco E., Triolo R., Dotto G.P., Silengo L., Altruda F. (2000) Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis. Neuron 28:115-127.
Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K-/-mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures.
Their brains display defective neurogenesis, with depletion of specific neuronal populations.
These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. The results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS.
PMID: 11086988 2. Jezior J.R., Brady J.D., Rosenstein D.L, McCammon K.A., Miner A.S., Ratz P.H.
(2001) Dependency of detrusor contractions on calcium sensitization and calcium entry through LOE-908-sensitive channels. Br. J. Pharmacol. 134:78-87.
The subcellular mechanisms regulating stimulus-contraction coupling in detrusor remain to be determined. Ca(2+)-free solutions, Ca(2+) channel blockers, cyclopiazonic acid (CPA), and RhoA kinase (ROK) inhibitors were used to test the hypothesis that Ca(2+) influx and Ca(2+) sensitization play primary roles. In rabbit detrusor, peak bethanechol (BE)-induced force was inhibited 90% by incubation for 3 min in a Ca(2+)-free solution. By comparison, a min incubation of rabbit femoral artery in a Ca(2+)-free solution reduced receptor-induced force by only 5%. In detrusor, inhibition of sarcoplasmic reticular (SR) Ca(2+) release by 2APB, or depletion of SR Ca(2+) by CPA, inhibited BE-induced force by only 27%. The 20 CPA-insensitive force was abolished by LaCI(3). By comparison, 2APB
inhibited receptor-induced force in rabbit femoral artery by 71 %. In the presence of the non-selective cation channel (NSCC) inhibitor, LOE-908, BE did not produce an increase in [Ca(2+)](i) but did produce weak increases in myosin phosphorylation and force. Inhibitors of ROK-induced Ca(2+) sensitization, HA-1077 and Y-27632, inhibited BE-induced force by approximately 50%, and in combination with LOE-908, nearly abolished force. These data suggest that two principal muscarinic receptor-stimulated detrusor contractile mechanisms include NSCC
activation, that elevates [Ca(2+)](i) and ROK activation, that sensitizes cross bridges to Ca(2+).
PMID:11522599 Walsh S.V., Hopkins A.M., Chen J., Narumiya S., Parkos C.A., Nusrat A. (2001) Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia. Gastroenterology 121:566-579.

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Claims (49)

1. WHAT IS CLAIMED IS:
   An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
   (a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27;
   (b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;
   (c) an amino acid sequence selected from the group consisting SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27; and (d) a variant of an amino acid sequence selected from the group consisting of SEQ ID
   NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence.
2. 2 The polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence selected from the group consisting SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27.
3. The polypeptide of claim 2, wherein said allelic variant comprises an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID
   NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.
4. 4. The polypeptide of claim 1, wherein the amino acid sequence of said variant comprises a conservative amino acid substitution.
5. 5. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:
   (a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 1S, 17, 19, 21, 23, 25, and 27;
   (b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, S, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;
   (c) an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27;
   (d) a variant of an amino acid sequence selected from the group consisting SEQ
   ID
   NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence;
   (e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising an amino acid sequence chosen from the group consisting of SEQ ID NOS:2, S, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, or a variant of said polypeptide, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence; and (f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or (e).
6. 6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally-occurring allelic nucleic acid variant.
7. 7. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally-occurring polypeptide variant.
8. 8. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ
   ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.
9. 9. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
   (a) a nucleotide sequence selected from the group consisting of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26;
   (b) a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, provided that no more than 20% of the nucleotides differ from said nucleotide sequence;
   (c) a nucleic acid fragment of (a); and (d) a nucleic acid fragment of (b).
10. 10. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule hybridizes under stringent conditions to a nucleotide sequence chosen from the group consisting SEQ ID NOS:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or a complement of said nucleotide sequence.
11. 11. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
    (a) a first nucleotide sequence comprising a coding sequence differing by one or more nucleotide sequences from a coding sequence encoding said amino acid sequence, provided that no more than 20% of the nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence;
    (b) an isolated second polynucleotide that is a complement of the first polynucleotide;
    and (c) a nucleic acid fragment of (a) or (b).
12. 12. A vector comprising the nucleic acid molecule of claim 11.
13. 13. The vector of claim 12, further comprising a promoter operably-linked to said nucleic acid molecule.
14. 14. A cell comprising the vector of claim 12.
15. 15. An antibody that binds immunospecifically to the polypeptide of claim 1.
16. 16. The antibody of claim 15, wherein said antibody is a monoclonal antibody.
17. 17. The antibody of claim 15, wherein the antibody is a humanized antibody.
18. 18. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising:
    (a) providing the sample;
    (b) contacting the sample with an antibody that binds immunospecifically to the polypeptide; and (c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.
19. 19. A method for determining the presence or amount of the nucleic acid molecule of claim 5 in a sample, the method comprising:
    (a) providing the sample;
    (b) contacting the sample with a probe that binds to said nucleic acid molecule; and (c) determining the presence or amount of the probe bound to said nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in said sample.
20. 20. The method of claim 19 wherein presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type.
21. 21. The method of claim 20 wherein the cell or tissue type is cancerous.
22. 22. A method of identifying an agent that binds to a polypeptide of claim 1, the method comprising:
    (a) contacting said polypeptide with said agent; and (b) determining whether said agent binds to said polypeptide.
23. 23. The method of claim 22 wherein the agent is a cellular receptor or a downstream effector.
24. 24. A method for identifying an agent that modulates the expression or activity of the polypeptide of claim 1, the method comprising:
    (a) providing a cell expressing said polypeptide;
    (b) contacting the cell with said agent, and (c) determining whether the agent modulates expression or activity of said polypeptide, whereby an alteration in expression or activity of said peptide indicates said agent modulates expression or activity of said polypeptide.
25. 25. A method for modulating the activity of the polypeptide of claim 1, the method comprising contacting a cell sample expressing the polypeptide of said claim with a compound that binds to said polypeptide in an amount sufficient to modulate the activity of the polypeptide.
26. 26. A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the polypeptide of claim 1 in an amount sufficient to treat or prevent said NOVX-associated disorder in said subject.
27. 27. The method of claim 26 wherein the disorder is selected from the group consisting of cardiomyopathy and atherosclerosis.
28. 28. The method of claim 26 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
29. 29. The method of claim 26, wherein said subject is a human.
30. 30. A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the nucleic acid of claim 5 in an amount sufficient to treat or prevent said NOVX-associated disorder in said subject.
31. 31. The method of claim 30 wherein the disorder is selected from the group consisting of cardiomyopathy and atherosclerosis.
32. 32. The method of claim 30 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
33. 33. The method of claim 30, wherein said subject is a human.
34. 34. A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the antibody of claim 15 in an amount sufficient to treat or prevent said NOVX-associated disorder in said subject.
35. 35. The method of claim 34 wherein the disorder is diabetes.
36. 36. The method of claim 34 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
37. 37. The method of claim 34, wherein the subject is a human.
38. 38. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically-acceptable carrier.
39. 39. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically-acceptable carrier.
40. 40. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically-acceptable carrier.
41. 41. A kit comprising in one or more containers, the pharmaceutical composition of claim 38.
42. 42. A kit comprising in one or more containers, the pharmaceutical composition of claim 39.
43. 43. A kit comprising in one or more containers, the pharmaceutical composition of claim 40.
44. 44. A method for determining the presence of or predisposition to a disease associated with altered levels of the polypeptide of claim 1 in a first mammalian subject, the method comprising:
    (a) measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and (b) comparing the amount of said polypeptide in the sample of step (a) to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease;
    wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to said disease.
45. 45. The method of claim 44 wherein the predisposition is to a cancer.
46. 46. A method for determining the presence of or predisposition to a disease associated with altered levels of the nucleic acid molecule of claim 5 in a first mammalian subject, the method comprising:
    (a) measuring the amount of the nucleic acid in a sample from the first mammalian subject; and (b) comparing the amount of said nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease;
    wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
47. 47. The method of claim 46 wherein the predisposition is to a cancer.
48. 48. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising an amino acid sequence of at least one of SEQ ID NOS:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, or a biologically active fragment thereof.
49. 49. A method of treating a pathological state in a mammal, the method comprising administering to the mammal the antibody of claim 15 in an amount sufficient to alleviate the pathological state.