WO1998007838A1 - Higher animal telomerase protein and gene encoding the same - Google Patents

Higher animal telomerase protein and gene encoding the same Download PDF

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Publication number
WO1998007838A1
WO1998007838A1 PCT/JP1997/002904 JP9702904W WO9807838A1 WO 1998007838 A1 WO1998007838 A1 WO 1998007838A1 JP 9702904 W JP9702904 W JP 9702904W WO 9807838 A1 WO9807838 A1 WO 9807838A1
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polypeptide
sequence
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ser
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PCT/JP1997/002904
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French (fr)
Japanese (ja)
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Fuyuki Ishikawa
Hideo Nakamura
Kazuhiro Takahashi
Yasuhiro Fujino
Naozumi Harada
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Mitsubishi Chemical Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a gene encoding a telomerase protein of a higher animal cell and a gene product thereof.
  • telomeres Both ends of linear DNA in eukaryotic chromosomes, such as animal cells, are called telomeres and have a complex higher-order structure consisting of a special DNA sequence and proteins that bind to it.
  • Telomere DNA is composed of characteristic repetitive sequences rich in thymine (T) and guanine (G) (the opposite strands are adenine (A) and cytosine (C)), for example, telomere in vertebrate cell chromosomes.
  • TDNA is composed of 6 bases of TTAGGG (CCCTAA on the opposite strand). Southern blotting analysis using this sequence revealed that the average length of telomere repeats in human somatic cells was 7-10 kilobases.
  • telomere structure is thought to have an important function in chromosome stabilization. For example, morphology studies using yeast have revealed that telomeres are located at the periphery of the cell nucleus, and telomeres act as "anchors" that anchor chromosomes to specific locations in the nucleus, and are located in the cell nucleus. Suggests that it may control physical interactions between chromosomes. It has also been suggested that it has a function to prevent inactivation of chromosomal functions by shortening each replication of eukaryotic linear double-stranded DNA as follows.
  • telomeres and the region adjacent to them may adopt a hairpin structure or function as a buffer band for shortening.
  • telomeres have the function of preventing chromosome shortening.
  • the proliferative capacity decreases as the cells pass, and eventually becomes “senescent” cells that have lost proliferative capacity.
  • Immortalized cells that have acquired permanent growth ability may be obtained by introducing a certain oncogene into the cell.
  • RNA-dependent DNA polymerase which extends telomere repeats, has attracted attention as one of the mechanisms for controlling the average length of telomere repeats.
  • This enzyme is an enzyme that adds the same 6-base repeat sequence to the 3 'end of a synthetic oligonucleotide (TTGGGG) derived from the tetramer repeat sequence of Tetrahymena from the prokaryotic tetrahymena macronuclear extract.
  • TTGGGG synthetic oligonucleotide
  • telomere consists of an 80 kD subunit that binds to type I RNA and a 95 kD subunit that binds to the DNA end that serves as a primer, and may have a primary structure relatively similar to RNA virus RNA polymerase. It was revealed.
  • telomerase is understood to be an essential enzyme for cell growth in unicellular eukaryotes.
  • telomerase activity was not detected early in the passage after the introduction of the cancer gene and was detected in a cell population that had acquired infinite proliferative capacity. . It is also said that telomerase activity is detected in most of actual human cancer cells, but telomerase activity is not detected in many normal cells. From these findings, it is possible to speculate that cancer cells may escape from shortening of telomere DNA by expressing telomerase activity during the establishment process, and acquire permanent growth ability. Therefore, telomerase inhibitors are useful as highly selective anticancer agents, and it is expected that early diagnosis of cancer will be possible by measuring telomerase activity.
  • telomerase RNA subunits does not always correlate with telomerase activity (AviIon et al., Cancer Res., 56, 645, 1996).
  • telomerase itself has not yet been separated and purified, and its actual substance state remains unknown. Since it is necessary to use a complicated detection method using PCR, almost no enzymological research has been conducted on telomerase. Furthermore, since the expression of telomerase activity cannot be determined at the individual cell level using pathological sections or the like, it is difficult to analyze the exact relationship between telomerase and cancer malignancy.
  • the present inventors have conducted intensive studies to isolate and identify a higher animal telomerase protein, and succeeded for the first time in cloning a gene encoding a higher animal telomerase protein, and further obtained a gene from the gene.
  • the present invention has been completed based on these findings.
  • the full-length amino acid sequence of human 'telomerase protein was reported (Science, 275, pp. 973-977, February 14, 1997), but the nucleotide sequence and amino acid sequence of c-DNA were It differs in many respects from what we have elucidated.
  • the present invention provides a polypeptide specified by the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, wherein the polypeptide is a rat-derived telomerase protein. Further, according to the present invention, substitution, insertion, and / or deletion by one or more amino acid residues are present in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence is substantially present.
  • a polypeptide characterized by functioning as a higher animal telomerase protein including human is provided. According to a preferred embodiment, the above polypeptide capable of functioning as a telomerase protein in a human organism is provided.
  • polypeptide identified by the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing wherein the polypeptide is a partial polypeptide of a human-derived telomerase protein. It is characterized by being a peptide. Further, according to the present invention, substitution, insertion, and / or deletion by one or more amino acid residues is present in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and substantially contains human. There is provided a polypeptide characterized by functioning as a partial polypeptide of a higher animal telomerase protein.
  • the amino acid sequence described in 13 includes substitution, insertion, and / or deletion by one or more amino acid residues, and substantially functions as a higher telomerase protein containing humans.
  • the present invention provides a polypeptide characterized by the above-mentioned, and in a preferred embodiment thereof, the above-mentioned polypeptide which can function as a telomerase protein in a living body of a human.
  • nucleotide sequence encoding each of the above polypeptides.
  • nucleotide sequence include a DNA sequence and an RNA sequence.
  • nucleic acid numbers from 199 to nucleic acid numbers of the DNA sequence described in SEQ ID NO: 1 in the sequence listing are set to be from nucleic acid number (Excluding codons), or the DNA specified by nucleic acid No. 1 to nucleic acid No. 487 of the DNA sequence described in SEQ ID No. 2 in the sequence listing, or the DNA specified in SEQ ID No.
  • a recombinant vector containing the DNA sequence, a transformant into which the recombinant vector has been introduced, and a polypeptide which is a gene product of the DNA sequence from a culture obtained by culturing the transformant comprising a step of separating and collecting.
  • an antibody capable of specifically recognizing each of the above-mentioned polypeptides, a nucleic acid probe comprising a nucleotide sequence capable of binding complementarily to part or all of the above-mentioned nucleotide sequences
  • antibodies or nucleic acid probes are useful as reagents for detecting cancer cells, and a pharmaceutical composition for cancer diagnosis containing the above antibodies or nucleic acid probes is provided as one embodiment of the present invention.
  • the molecular weight by SDS (sodium sodium dodecyl sulfate) -polyacrylamide electrophoresis (PAGE) is about 240 kDa for the inactive form.
  • SDS sodium sodium dodecyl sulfate
  • PAGE polyacrylamide electrophoresis
  • Ma A method of screening a substance that acts on the expression of the enzymatic activity of a higher animal telomerase protein, which is a subunit of a higher animal telomerase protein contained in a cell or tissue that has been brought into contact with a test substance. Also provided is a method comprising the step of measuring the molecular weight of the polypeptide.
  • the step of contacting with the test substance is carried out by a culturing step in the presence of the test substance or a step of administering the test substance to an animal;
  • the molecular weight is measured by SDS-polyacrylamide.
  • the above method comprising the steps of measuring the abundance ratio of an inactive polypeptide of about 240 kDa and an active polypeptide of about 230 kDa.
  • the above method comprising the step of determining that the substance is a substance that inhibits the expression of the enzymatic activity of a higher animal telomerase protein; and comparing the abundance ratio of a 230 kDa polypeptide in the absence of the test substance And the polypeptide
  • the above method including the step of determining that the test substance is a substance that activates the expression of the enzyme activity of a higher animal telomerase protein, Provided.
  • FIG. 1 is a diagram showing a restriction enzyme cleavage map of a cDNA clone of a rat telomerase protein gene.
  • Figure 2 compares the homology of the DNA sequence of the cDNA fragment of the human telomerase protein gene amplified by PCR with the predicted amino acid sequence of the rat and Tetrahymena p80, respectively. It is a figure showing a result.
  • R indicates the rat gene
  • H indicates the human gene
  • p80 indicates the Tetrahymena p80 gene.
  • Figure 3 shows telomerase derived from human cancer cell (PA-1) or rat cancer cell (AH66F) extract using beads coated with a specific antibody against the recombinant rat telomerase protein fragment.
  • FIG. 4 shows the results of immunoprecipitation of activity. The results of studies using a method combining PCR and ELISA are shown.
  • FIG. 4 is a diagram showing a restriction enzyme cut map of a cDNA clone of a human telomerase protein gene.
  • a first aspect of the polypeptide of the present invention is specified by the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and corresponds to a polypeptide constituting a mouse-derived telomerase protein.
  • the polypeptide provided by the present invention is not limited to the specific polypeptide described in SEQ ID NO: 1, but may have one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.
  • Polypeptides in which substitutions, insertions, and / or deletions by residues are present and which can substantially function as a telomerase protein of a higher animal including human are also included in the scope of the present invention.
  • Higher animal telomerase protein containing such a polypeptide as a sub-unit is also included in the scope of the present invention.
  • a second embodiment of the polypeptide of the present invention is specified by the amino acid sequence of SEQ ID NO: 2 in the sequence listing, and corresponds to a partial polypeptide of the polypeptide constituting the human-derived telomerase protein. is there.
  • the polypeptide provided by the present invention is not limited to the specific polypeptide set forth in SEQ ID NO: 2, but may be one or more than one of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
  • a polypeptide which has a substitution, insertion and / or deletion by an amino acid residue and which can function as a partial polypeptide of a telomerase protein of a higher animal, preferably a human. Tides are also included in the scope of the present invention.
  • a third embodiment of the polypeptide of the present invention is specified by the amino acid sequence shown in SEQ ID NO: 13 in the sequence listing, and corresponds to a polypeptide constituting a human-derived telomerase protein.
  • the polypeptide provided by the present invention is represented by SEQ ID NO: 13. It is not limited to the specific polypeptides described, but may be substituted, inserted, and / or deleted by one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 13 in the sequence listing. Is included in the scope of the present invention, wherein the polypeptide can substantially function as a telomerase protein of a higher animal including human. Higher animal telomerase proteins containing such polypeptides as a sub-unit are also included in the scope of the present invention.
  • the polypeptide of the present invention also includes a polypeptide containing each of the above polypeptides as a partial sequence.
  • a polypeptide obtained by binding an appropriate amino acid sequence having a property of improving the expression efficiency to each of the above polypeptides a polypeptide obtained by binding a signal sequence to each of the above polypeptides
  • a fusion protein of a so-called tag sequence in which another protein is bound to the above polypeptide so that the reading frame does not change so as to confirm the expression of the above polypeptide is also included in the scope of the present invention. You.
  • any nucleotide sequence encoding any of the above polypeptides is encompassed by the nucleotide sequence of the present invention.
  • Gene encoding the telomerase protein of the present invention may be referred to as “telomerase protein gene” in the present specification, a nucleotide encoding the full length of the polypeptide constituting the telomerase protein or a part thereof)
  • sequence is used to mean a nucleotide sequence encoding a polypeptide included in the first, second, and third embodiments, and preferably a DNA sequence. Can be.
  • the term “higher animal” is used as a concept including mammals including humans. It is expected that polypeptides constituting the telomerase protein derived from such higher animals, preferably mammals, have high homology. Therefore, based on the method for cloning the mouse-derived telomerase protein gene disclosed in detail in the present specification and the information on the gene, those skilled in the art can understand the polymorphism of the telomerase protein derived from higher animals. It goes without saying that a gene encoding a peptide can be easily obtained and its gene product can be obtained.
  • the telomerase protein gene of the present invention can be obtained, for example, by the following method.
  • Examples of the DNA library containing the telomerase protein gene of the present invention include immortalized higher animal cell lines, preferably humans, monkeys, pomas, magpies, higgies, dogs, cats, puppies, rats, mice, and the like.
  • a plasmid cDNA library, a phage cDNA library, a phage genomic library, or the like can be used which is prepared by a known conventional method using RNA prepared from the above cell line.
  • a tissue such as a cancer or an immortalized higher animal cell line is ground in liquid nitrogen, homogenized in an aqueous solution of guanidine isothiocyanate, and then used in Chirgwin.
  • a tissue such as a cancer or an immortalized higher animal cell line is ground in liquid nitrogen, homogenized in an aqueous solution of guanidine isothiocyanate, and then used in Chirgwin.
  • RNA is separated as a precipitate by cesium chloride equilibrium density gradient centrifugation according to [Biochemistry 18, 5294-5299 (11979)].
  • a commercially available extraction reagent such as RNAzo1 (Te1Test) can be used. After isolating the RNA, the total RNA is purified by phenol extraction and ethanol precipitation, and purified by oligo (dT) cellulose column chromatography to obtain poly (A) containing the mRNA of the target telomerase protein. Contained mRNA
  • Primer DNA composed of synthetic DNA containing the 01 igo (dT) sequence as described in [Nature 329, 836–838 (1987)] is hybridized, and reverse transcriptase is used. Synthesize single-stranded cDNA. Similar sequences are also used in commercially available cDNA synthesis kits, and such sequences may be used. Thereafter, the PCR reaction may be performed using a synthetic DNA for the PCR reaction with a commercially available primer (usually the one itself attached to the kit). When a primer DNA such as that described in the above-mentioned literature [Nature 329, 836-838 (1987)] is used, a sequence complementary to that sequence is designed and used as a primer for PCR reaction. It is preferable to prepare them in advance.
  • E. coli DNA polymerase I E. coli DNA ligase
  • RNase H double-stranded cDNA is synthesized according to a conventional method.
  • a small fragment of DNA which is cut with a restriction enzyme, such as a so-called EcoRI adapter, is digested with T4 DNA ligase. Add to both ends of DNA chain.
  • a restriction enzyme cleavage point in cDNA is methylated with a DNA methylase such as EcoRI methylase (for example, in the case of EcoRI methylase, the EcoRI cleavage point is methylated).
  • a DNA methylase such as EcoRI methylase
  • the EcoRI cleavage point is methylated.
  • Protect the cDNA from cleavage of the EcoRI enzyme add a so-called EcoRI linker to the end of the cDNA using T4 DNA ligase, and then use the restriction enzyme EcoRI. The same result can be obtained by cutting only the linker-DNA portion with the above.
  • a cleavage site for another restriction enzyme such as BamHI as a vector cloning site
  • the above-described series of terminal treatment operations can be performed, for example, by binding one BamHI adapter or BamHI. Similar results can be obtained by processing with a combination of mHI methylase, BamHI linker, BamHI, and the like.
  • the cDNA chain end-treated as described above is commercially available; I phage vector, for example; a phage vector such as IZAP (Promega Biotech) or pGEM2 (Promega Biotech)
  • I phage vector for example; a phage vector such as IZAP (Promega Biotech) or pGEM2 (Promega Biotech)
  • the recombinant sphage DNA group or the recombinant plasmid DNA group can be produced by inserting the plasmid into the EcoRI cleavage site of the plasmid vector according to a conventional method.
  • the complementary (T) can be produced using a vector to which, for example, pCRII (In Vitrogen) or pT7 (ogen ⁇ age ⁇ ) is added.
  • a commercially available in vitro 'packaging' kit for example, Gigapack Gold (Promega, Inc.), is used for so-called in vitro 'Packaging can be performed to produce sphage particles having the recombinant sphage DNA.
  • packaging may be performed in accordance with the conditions in the instruction manual of a commercially available kit. Profit
  • the obtained spheroid particles are transduced into a host such as E. coli according to a conventional method, for example, the method of T. Maniatis et al. (“Molecular Cloning”, Card Spring Laboratories, 1998).
  • a phage cDNA library can be prepared by growing the obtained transformant.
  • a plasmid cDNA library can be obtained by transforming a host such as Escherichia coli, for example, and growing the resulting transformant according to a conventional method.
  • these phages or transformants such as Escherichia coli are propagated, and transferred onto a Nymouth membrane or a ditrocellulose membrane such as GenScreen Plus (Dupnt), for example.
  • GenScreen Plus GenScreen Plus
  • a [ 32 P] -labeled probe prepared from a partial fragment of the higher animal lipoprotein melase protein gene amplified by the method described below was used for the phage DNA or plasmid DNA.
  • the soybean is selected by the plaque hybridization method to obtain all or part of the cDNA clone encoding the higher animal telomerase protein gene of interest.
  • Probes used to select a cDNA clone encoding the higher animal telomerase protein gene from the phage cDNA library or the plasmid cDNA library can be selected by a conventional method, for example, a commercially available kit.
  • telomere sequence derived from a gene encoding a known telomerase protein (Col 1 ins et al., Ce 11, 81, 677-686, 1995), and homology to its amino acid sequence
  • a program such as TB LAS TN in a gene bank such as the National Center for Biotechnology Information (NCBI) to search for the DNA sequence of a gene of another organism capable of encoding an amino acid sequence having
  • NCBI National Center for Biotechnology Information
  • An amino acid sequence having a certain degree of homology can be used as a probe by synthesizing an oligonucleotide with reference to a DNA sequence capable of coding the amino acid sequence.
  • PCR primers are designed based on the DNA sequence of similar genes, and longer DNA is obtained by the so-called degenerative PCR method to obtain a professional DNA. It may be used as a bus.
  • the type II used in the PCR method includes a phage cDNA library derived from cells containing the target probe DNA, a plasmid cDNA library, or cDNA synthesized from extracted RNA according to a conventional method. Can be used.
  • PCR primers were designed as if the probe DNA had been designed, and a part of the telomerase protein gene of higher animals was so-called PCR. Can also be obtained.
  • the type ⁇ used in the PCR method in addition to the phage cDNA library and the plasmid cDNA library described above, and cDNA directly synthesized from RNA extracted from immortalized cells according to a conventional method, are used directly. be able to. After the PCR reaction, the reaction solution was analyzed by agarose-polyacrylamide gel electrophoresis, and fragments of the expected size were recovered from the DNA fragments amplified by the two primers.
  • PCR-II PCR-II-II
  • a new PCR primer was designed and synthesized based on the obtained partial sequence of the higher animal telomerase protein gene, and a PCR primer or cDNA was designed based on the sequence of the higher animal telomerase protein.
  • a gene encoding the full length of a higher animal telomerase protein can also be obtained by repeatedly amplifying DNA with the above-mentioned primer synthesized above.
  • the DNA fragment can be subjected to agarose or polyacrylamide gel electrophoresis and analyzed, recovered, and purified according to a conventional method.
  • the obtained purified DNA fragment is inserted into a vector into which a PCR fragment such as pCR-II can be directly incorporated, and E. coli is transformed with the obtained recombinant vector, and the DNA fragment is prepared according to a conventional method.
  • a conventional method Was prepared according to the dideoxy method of Sanger et al. [Proc. Natl. Acad. Sc USA, 74, 5463, 1977].
  • the base sequence of the target DNA fragment can be determined. Sequence determination can also be performed by an automated sequencer, such as ABI 373A (Applied 'Bio' Systems).
  • the length of a sequence that can be determined using an automatic sequencer is generally limited. It may be difficult to analyze all regions at once. In such a case, analysis can be facilitated by digesting the fragment with an appropriate restriction enzyme, separating and recovering the fragment by gel electrophoresis, and reinserting the recovered fragment into an appropriate vector. it can. In addition to such operations (sub-cloning), it is also possible to select an appropriate sequence from the base sequence determined by the automatic sequencer, design a new primer, and continue analysis from there. .
  • telomere sequence By joining the sequences of the DNA fragments thus determined so as to overlap each other, for example, a full-length polypeptide constituting the higher animal telomerase protein as described in SEQ ID NO: 1 or 13 in the sequence listing can be obtained.
  • the nucleotide sequence encoding the nucleotide sequence to be encoded or the partial polypeptide sequence constituting the higher animal telomerase protein as described in SEQ ID NO: 2 in the Sequence Listing can be determined.
  • the nucleotides of the present invention include DNA and RNA, and SEQ ID NOs: 1, 13, and 2 in the sequence listing show full-length polypeptides constituting rat and human derived telomerase proteins, respectively. And a DNA sequence encoding a partial polypeptide sequence constituting a human-derived telomerase protein have been described as preferred embodiments.
  • the nucleotides of the present invention include, in addition to the DNA sequences specified by SEQ ID NOs: 1, 13, and 2 above, one or more amino acids relative to the amino acid sequence of the polypeptide encoded by them.
  • Residue substitutions, insertions, and / or deletions have been introduced and include nucleotides that encode a polypeptide that functions substantially as a full-length or partial polypeptide of a higher animal telomerase protein. Modification of the amino acid sequence by such substitution, insertion, and deletion or deletion of amino acid residues can be performed, for example, by the methods described in Nucleic Acid Res., Vol. 10, 6487. — 6500 (1 982), Methodsin Enzymo l., Vo l. 2 1 7, 2 18-227 (1993), and Vo 2 1 7, 27 0-278 (1993) ) And the like, but the method is not limited to these methods, and any method available to those skilled in the art may be used.
  • telomere protein gene DNA obtained as described above as a hybridization probe or PCR primer
  • a higher animal telomerase protein gene of another species is isolated in a similar manner. be able to.
  • a PCR primer derived from the highest homology of the amino acid sequence of the Tetrahymena telomerase protein (p80) and the rat telomerase protein the corresponding portion of the human telomerase protein is used.
  • the amino acid sequence can be determined, and its full-length cDNA can also be obtained.
  • the higher animal telomerase protein gene DNA or its DNA fragment obtained as described above may be modified at both ends or at one end thereof, or by itself, in a known expression vector in a manner known per se.
  • the recombinant vector for gene expression thus produced is introduced into a known cell such as Escherichia coli, yeast, or an animal cell host by a method known per se to transform the gene.
  • a transformant can be manufactured.
  • telomerase protein of the present invention The method for producing a higher animal telomerase protein of the present invention will be described in detail.
  • a promoter is located at a position at which DNA encoding the higher animal telomerase protein obtained as described above can be transcribed. Is used.
  • telomere proteins are relatively large proteins, and their refolding is important for obtaining biological activity. Generally, animal cells should be used as hosts when refolding is considered. Is advantageous.
  • the higher animal telomerase may exist as a complex composed of several kinds of proteins and RNA subunits. When purified from a recombinant as a biologically active higher animal telomerase, the higher animal telomerase to be introduced is used. It is preferred that the species from which the protein is derived and the species from which the host cell is derived coincide. Needless to say, after producing a higher animal telomerase protein in Escherichia coli, it can be reconstituted with other components in vitro as an active complex.
  • animal cells examples include CHO cells (species: hamster), COS cells (species: monkey), NIH3T3 cells (species: mouse), Rat-1 (species: rat), VA-13 (organism: human) Cells and the like.
  • the expression plasmid using these cells as a host is preferably a promoter derived from the SV40 promoter or a viral gene. Into this downstream, a higher animal lipomelase protein gene is inserted from the 5 'side.
  • two or three higher animal telomerase protein genes may be inserted from the 5 'side, or each higher animal telomerase protein gene You may connect two or three units with a promoter such as SV40 inserted on the 'side. It is preferable to include a polyadenylation site downstream of the higher animal telomerase protein gene. For example, those derived from the SV40 DNA, / 3-globin gene or the metrotionein gene can be used.
  • Such an expression vector may have a selection marker when transformed into an animal cell such as a CH0 cell.
  • a selectable marker for example, a DHFR gene that provides methotrexate resistance, a neomycin derivative G—418 resistance gene, or the like can be used.
  • a promoter derived from SV40 is inserted on the 5 'side of each resistance gene, and a polyadenylation site is included on the 3' side of each resistance gene.
  • these resistance genes are inserted into the expression vector of the higher animal telomerase protein, they may be inserted downstream of the polyadenylation site of the higher animal telomerase protein gene. Further, the expression vector may not have the transformant selection marker.
  • the higher animal telomere Double transformation is preferably performed using a vector having a marker for transformant selection together with an expression vector for the CT / JP97 / 0204 protein, for example, pSV2neo, pSV2gpt, pMTV dhfr and the like.
  • the cells in which the expression of the higher animal telomerase protein has been confirmed may be repeatedly transformed by changing the selection ability.
  • the plasmid vector used in the expression vector include the SV40 early promoter, the splice sequence DNA derived from the ⁇ 3 / 3-globin gene of the egret, and the polyadenylation from the 99 / globin gene of the egret.
  • Expression vectors can be transferred to animal cells using calcium phosphate or cationic.
  • a transfection method using 1 ip id as a DNA carrier is common.
  • Culture of the transformed animal cells can be performed by suspension culture or adherent culture according to a conventional method. Use MEM, RPMI 1640, etc.
  • the culture can be performed in the presence of 10% serum or in the presence of an appropriate amount of insulin, dexamethasone, or transfusion, or in the absence of serum. Since it is considered that a large amount of the higher animal telomerase protein is present in the animal cells expressing the higher animal telomerase protein, the higher animal telomerase is used by using a protein extract obtained from a culture of the transformant. It is possible to separate and purify proteins.
  • the culture supernatant containing the produced higher animal telomerase protein can be purified by chromatography using various chromatographies, for example, heparin sepharose or blue sepharose.
  • the expression vector is a promoter, a ribosome binding (SD) sequence, a higher animal telomerase protein gene. It preferably contains a gene that controls a promoter, a transcription termination sequence, and a promoter.
  • the promoter include those derived from Escherichia coli and phage, such as tributophan synthase (trp), lactose operin (1 ac), sphage PL, PR, and the promoter of the early gene of T5 phage. ⁇ 26 promoters and the like. These may also be sequences that have been independently modified and designed, for example, the pac promoter [Agric. Biol. Chem. 52, 983-988, 1988].
  • the ribosome binding sequence may be derived from Escherichia coli, phage, etc., but a consensus sequence having 4 or more consecutive bases complementary to the 3′-terminal region of 16S ribosomal RNA prepared by DNA synthesis may be used. You may have it.
  • the transcription termination sequence is not necessarily required, but preferably has a / 0-independent sequence, such as a riboprotein terminator or a trp operon terminator.
  • the sequence of these factors required for expression on the expression plasmid is preferably, for example, in the order of 5 'upstream, promoter, SD sequence, higher animal telomerase protein gene, and transcription termination factor.
  • a method of increasing the copy number of a transcription unit on a vector by inserting a plurality of units of the SD sequence on the expression vector and the higher animal telomerase protein gene in the same direction Japanese Patent Laid-Open No. 1-95798) The method described in public announcements) can also be used.
  • telomerase protein or its partial polypeptide can be used to easily recover and purify the expressed higher animal telomerase protein or its partial polypeptide from a transformant such as Escherichia coli.
  • a transformant such as Escherichia coli.
  • an amino acid sequence in which six or more histidines are arranged downstream of a promoter is coded using the property of binding an amino acid sequence in which six or more histidines are arranged, that is, a protein having a histidine tag to a chelate column.
  • a telomerase protein gene containing a histidine tag or a partial polypeptide thereof can be expressed by arranging a DNA to be expressed and further binding a higher animal telomerase protein gene downstream thereof.
  • the expressed higher animal telomerase protein or its partial polypeptide can be easily purified using a chelate column. Furthermore, a polypeptide specifically cleaved by a protease such as thrombin, TEV protease, or factor X between the histidine tag and the polypeptide constituting the higher animal telomerase protein or a partial polypeptide thereof.
  • a protease such as thrombin, TEV protease, or factor X between the histidine tag and the polypeptide constituting the higher animal telomerase protein or a partial polypeptide thereof.
  • pUAI2 Japanese Patent Application Laid-Open No. 1-95798
  • commercially available pKK233-2 Pharmacia
  • pGEX series Pharmacia
  • PP ro EX-I Gibco BRL
  • Transformation of the host can be performed according to a conventional method.
  • insect cells for example, according to the manual of Max Knock (MAXBAC Tm , BACULOVIRUS EXPRE SSI ON SYSTEM MANUAL VERS I ON 1.4), which is a baculovirus expression kit from Invitrogen, Inc. This kit can be used. At this time, it is preferable to change the distance from the polyhedrin promoter to the initiation codon in order to increase the expression level.
  • Max Knock MAXBAC Tm , BACULOVIRUS EXPRE SSI ON SYSTEM MANUAL VERS I ON 1.4
  • Culturing of the transformant can be performed according to a conventional method available to those skilled in the art.
  • An appropriate cultivation temperature is 28 ° C to 42 ° C.
  • lactose operon (lac) promoter add IPTG so that the final concentration will be about 1 mM when the absorbance of the bacterial cell culture at 600 nm becomes about 0.5. It is necessary to induce expression.
  • telomerase protein or its partial polypeptide isolated and purified by the above method
  • mammals such as monkeys, sheep, rabbits, rats and mice
  • Polyclones that specifically recognize zeoproteins Null or monoclonal antibodies can be made.
  • a culture solution of a transformant or an extract of a gene product into which an expression vector containing a higher animal Tesla merase protein gene has been introduced can be used.
  • the higher animal telomerase complex can be concentrated and purified.
  • it expresses a fusion protein of a higher animal teliptic melanase protein and a so-called “tag sequence” such as glutathione-1S-transferase and polyhistidine in an eukaryotic immortalized cell line having telomerase activity.
  • the vector is introduced, and the resulting extract of the transformant is specifically bound to a “tag sequence” such as glutathione 'Sepharose (Pharmacia), Nigel NTA agarose (QIAGEN), etc.
  • a “tag sequence” such as glutathione 'Sepharose (Pharmacia), Nigel NTA agarose (QIAGEN), etc.
  • telomere ⁇ a gene encoding a protein that physically binds tightly to a higher animal telomerase protein is obtained using various transformants including yeast. Isolation ⁇ Can be identified. For such a purpose, for example, a “Mtch Maker Kit” of C1ontech can be used.
  • the expression level of the above gene can be monitored at the protein level by using the above specific antibody for the higher animal tepa-merase protein, and the expression level at the gene level can be monitored using a nucleic acid probe or a PCR primer. can do.
  • a nucleic acid probe or a PCR primer it is possible to detect cancer cells and diagnose a disease caused by a change in telomerase activity and a disease accompanied by a change in telomerase activity.
  • the ELI SA method using a specific antibody
  • the determination can be performed by the Western 'plot method, the Southern or Northern plot method using a nucleic acid probe, or the PCR method using an oligonucleotide primer.
  • an antibody capable of specifically recognizing the polypeptide of the present invention or a nucleic acid probe comprising a nucleotide sequence capable of binding complementarily to part or all of the nucleotide sequence of the present invention can be used as a reagent for detecting cancer cells, Or it is useful as an active ingredient of a pharmaceutical composition for cancer diagnosis.
  • rat-derived telomerase protein includes an inactive polypeptide having a molecular weight of about 240 kDa by SDS-polyacrylamide electrophoresis, The presence of an activated polypeptide with a molecular weight of about 230 kDa has been confirmed by SDS-polyacrylamide electrophoresis.
  • the existence of a mechanism whereby an inactive polypeptide of about 240 kDa is first expressed and converted to an active polypeptide of about 230 kDa has been proved. Therefore, similar inactive and active polypeptides are present in other higher animals, and a similar mechanism exists for converting inactive to active polypeptides. It is obvious to those skilled in the art. All of these molecular species (subunits) are included in the scope of the present invention.
  • the abundance ratio of the active polypeptide and the inactive polypeptide By measuring the abundance ratio of the active polypeptide and the inactive polypeptide, it is possible to screen for a substance that acts on the telomerase activation mechanism.
  • This screening method is typically carried out in tissues or cells of a higher animal after administration of a test substance, or tissues or cells of a higher animal cultured in the presence of the test substance in a culture system. Measuring the abundance ratio of active and inactive polypeptides and comparing the abundance ratio in the absence of the test substance. Generally, the molecular weight may be measured by SDS-polyacrylamide electrophoresis.
  • the molecular weight of a telomerase protein subunit contained in cells or tissues that have not been contacted with the test substance is measured by SDS-polyacrylamide electrophoresis, and a polypeptide of about 240 kDa and about 230 Check the abundance ratio of kDa to the polypeptide in advance.
  • administer the test substance or culture in the presence of the test substance was measured in the same manner, and the presence of about 240 kDa polypeptide and about 230 kDa polypeptide was observed. Measure the ratio.
  • the test substance will activate the mechanism of telomerase activation. It can be determined that it inhibits. On the other hand, if the abundance of the approximately 230 kDa protein is increased, it can be determined that the test substance promotes telomerase activation. It should be understood that substances confirmed to act on the telomerase activation mechanism in this way are also included in the scope of the present invention.
  • Example 1 Obtaining rat telomerase protein gene
  • RNA was prepared by the method of Ch omc zynski (An a 1. Biochem., 162, 156-159. 987). That is, 1 0 8 Z 1 9 cells, guaiacolsulfonate two gin isothiocyanate Xia Ne one Bok solution [4 M guaiacolsulfonate two gin isothiocyanate Xia sulfonate (Wako Pure Chemical), 25 mM Kuen Sanna preparative potassium (Wako Pure Chemical), 0 1 M 2-mercaptoethanol, 0.5% sodium sarcosinate (Wako Pure Chemical Industries, Ltd.)], and mixed with 0.1 volume of 2 M sodium acetate (pH 4.0).
  • the precipitate obtained was dissolved again in guanidine isothiocynate solution, an equal volume of isopropanol was added, and the mixture was cooled at 20 ° C. for 1 hour, and then centrifuged at 15,000 ⁇ g for 20 minutes to obtain total RNA. Was recovered.
  • RNA was purified as follows. That is, 0.2 mg of total RNA was dissolved in ImMEDTA and 20 mM Tris-HCl (pH 7.5), heat-treated at 70 ° C. for 5 minutes, and quenched on ice. To this solution was added a 5 M NaC1 solution to a final concentration of 0.5 M. The solution was applied to a 01 igo-dT cellulose column (type 7, 1 cmxlcm, Pharmacia), and 1 mM EDTA and After washing the column with 2 OmM Tris-HCl buffer (pH 7.5) containing 0.5 M NaC1, the bound fraction was eluted with sterile deionized water, and 4 g of po 1 y (A) + RNA was obtained.
  • the cDNA obtained as described above was subjected to the step (1) using the method of Riey et al. (Vectorette method, Nucleic Acid Res., 18, 2887-2890).
  • the unknown cDNA sequence located further 5 'upstream of the portion corresponding to the obtained cDNA sequence was analyzed.
  • 60 ng of cDNA was treated with T4 polymerase to blunt the ends, and then 10 units of the restriction enzyme Pvu II (manufactured by Toyobo, using the supplied buffer) and 37 I wrote for two hours.
  • the digested DNA was purified by phenol / cohol-form treatment and ethanol precipitation, and then the vector unit shown in Table 2 below (annealed vct A and vct B) 3 pmoles was ligated to DNA ligase.
  • Table 2 The vector unit shown in Table 2 below (annealed vct A and vct B) 3 pmoles was ligated to DNA ligase.
  • VctA 5 AAGGAGAGGACGCTG
  • VctB 5 CTCTCC CTTCTCGAA
  • the cDNA in which the Vectoretteunit is connected to the blunt end is type- ⁇ and hybridized to one strand of the Vectoretteunit shown in Table 3 below.
  • PCR using a vct G oligonucleotide primer and a Ra PC 5 'oligonucleotide primer that hybridizes to the cDNA sequence shown in SEQ ID NO: 3 to perform Ra PC 5' oligonucleotide primer CDNA containing an unknown portion 5 ′ upstream from the binding site of was amplified.
  • the amplification reaction was carried out according to a conventional method using a thermal cycler for PCR. A heat retention cycle of 1 minute at C, 1 minute at 65, and 2 minutes at 72 ° C was repeated 35 times.
  • VctG 5 'CGGTACCGAATCGTA
  • PCR product was purified by treatment with phenolic noroform and ethanol precipitation, a portion was ligated to pT7B1ueT vector (Pharmacia) using DNA ligase, and the transformed recombinant was used. Escherichia coli was selected with ampicillin to prepare a plasmid DNA.
  • the DNA sequence of the inserted PCR product was determined by the Sanger method using ABI373A Sequencer (Applied Biosystems). As a result, the nucleotide sequence described in SEQ ID NO: 4 in the sequence listing was found in cDNA inserted into the plasmid RaPC53.
  • nucleotide sequence of nucleic acid numbers 1 to 170 described in SEQ ID NO: 3 in the sequence listing predicted from the complementary strand DNA was found to be the same as that of the sequence listing in actual rat cells. It was confirmed that it corresponded to the nucleotide sequences of nucleic acid numbers 1 to 244 of SEQ ID NO: 4.
  • the base sequence (5 '— TCTC TCCTAG-3') of nucleic acid number 163 to 172 of SEQ ID NO: 3 in the sequence listing is splicingacceptor Since the consensus sequence at site corresponds to 5'-PyPyPyPyPyPyNCAG-3 ', this result is not due to artifacts, but to RNA editing by splicing. It was thought that the result was done. Therefore, the [T] of base number 170 in SEQ ID NO: 3 in the sequence listing is actually [ ⁇ ] in the sequence of SEQ ID NO: 4, and the stop codon TAG is lysine AAG. I was In addition, it was found that the open, reading, and flames were further increasing toward the 5 'upstream.
  • the amino acid sequence of the open reading frame is homologous to the amino acid sequence of Tetrahymena p80 predicted in step (1) (High S core: 94, Probability: 1.7). x 1 0 one 3) in comparison with the Amino acid sequence of rat Bok from showing a, which shows a higher homology (H igh S core: 1 2 5, P robability:. l 6 x 1 0- 18 ),
  • the [A] at position 312 in SEQ ID NO: 3 is [T], indicating that the corresponding amino acid is mutated from asparagine (AA C) to isoloisin (ATC). .
  • po1y (A) + RNA was obtained from the rat 3Y1-derived SV-3Y1-C66 cells transformed with the SV40 virus in the same manner as in step (1).
  • CDNA was prepared using a cDNA synthesis kit from STRATAGENE. Preparation of cDNA was performed according to the manual, but 1ststrand synthesis reaction was performed by adding both random hexamer 'oligonucleotide and oligo dT primer as primers at a final concentration of 2 / M each.
  • the obtained phage particles were infected with Escherichia coli C600hf1A strain according to a conventional method and amplified, and the phage particles were collected. Through a series of operations, about 5 million phage clones were obtained.
  • phage clones were infected with E. coli C600hf1A strain according to a conventional method, and cultured on a NZY agar medium on a plate. Two replicas of the phage particles copied on a nylon membrane were prepared, washed and treated with alkali, and then the RaPC53 obtained in step (2) was labeled with 32 P and used as a probe, and the probe was hybridized. Phage clones were screened. As a result, three positive signals were found, and phage particles were collected from them. After cloning the phage particles in the same manner, the plasmid containing the cDNA fragment inserted according to the Stratagene manual was used. (RET1, RET2, RET3) were recovered by the invivoexcision method.
  • the termination codon of the same frame was not found on the 5 'side further than the ATG on the 5' end, so that the 5 ' — Investigations were performed using the Rapid Amplification of cDNA Ends (RACE) method.
  • RACE Rapid Amplification of cDNA Ends
  • the 5'-RACE method was performed according to the manual using a 5'-RACE kit of C1onetech Inc.
  • step (3) 2 g of po1y (A) + RNA obtained from SV-3Y1-C66 cells is complementary to the nucleic acid number 1493-3151 of SEQ ID NO: 1 in the sequence listing Oligonucleotide primer NcEX 3′10 pmo1e having a unique DNA sequence was mixed, heated and quenched. Reverse transcriptase to reaction mixture
  • RNA ligase (GIB CO BRL Superscript), substrate nucleotides and buffers The solution was added and reacted at 42 ° C for 1 hour. After terminating the reaction by adding EDTA, the type II RNA was decomposed by an aliquot treatment, and single-stranded cDNA was isolated by isopropanol precipitation. In addition, an anchor primer for 5'-RACE [5'-P (+) ANC] 4 pmoles was ligated to half of the cDNA using RNA ligase. The reaction was performed at 37 ° C. for 3 hours in the presence of 25% PEG.
  • an oligonucleotide primer complementary to the anchor DNA is used as a type I single-stranded cDNA that is reverse-transcribed with NcEX 3 'and further has an anchor DNA sequence added to the 3' end.
  • RACE PCR amplification of DNA using PRM and the oligonucleotide primer Ra PC 5 'of the DNA sequence complementary to nucleic acid numbers 1039 to 106 of SEQ ID NO: 1 in the sequence listing. went.
  • a PCR was performed according to the attached manual using a 1/20 volume of single-stranded cDNA and a primer of 1 O pmo 1 e each, using TaQ polymerase of GIBCORBRL. However, to avoid non-specific DNA amplification, the reaction was started manually using a hot-start method, followed by 30 seconds at 94 ° C, 1 minute at 55 ° C, and 2 minutes at 72 ° C. This cycle was repeated 35 times.
  • the PCR product was incorporated into the pT7B1ueT vector, and the DNA sequence of the inserted DNA was analyzed. As a result, 10 clones among these clones had almost the same DNA sequence.
  • RACE 3 and RACE 5 which are representative clones, are located at the positions shown in FIG. 1, and nucleic acid numbers 199 to 201 of SEQ ID NO: 1 in the sequence listing.
  • HPET5 SEQ ID NO: 5 in the sequence listing
  • HP ET3 SEQ ID NO: 6 in the sequence listing
  • rat DN derived from step SV3Y1-C66 cells obtained in step (3) of Example 1
  • c DNA PCR was performed in the usual manner using cDNA derived from PA-11 cells derived from human ovarian teratoma obtained by the same method as described above, but PCR was performed using PA-1 cell-derived cDNA and SV as a positive control.
  • the target DNA was not amplified from cDNA derived from 3Y1-C66 cells.
  • HPET 5-2 (SEQ ID NO: 7 in Sequence Listing) corresponding to amino acid number 376 to 385 in SEQ ID NO: 1 in Sequence Listing or amino acid number 380 in SEQ ID NO: 1 in Sequence Listing is used.
  • HP ET 5-3 (SEQ ID NO: 8) corresponding to -388, HP ET 3-2 (SEQ ID NO: 5) corresponding to the amino acid number 532 to 540 of SEQ ID NO: 1 as an antisense primer Using SEQ ID NO: 9) or HP ET3-3 (SEQ ID NO: 10 in the sequence listing) corresponding to amino acid numbers 534 to 542 of SEQ ID NO: 1 in the sequence listing, cDNA derived from SV-3Y1-C66 cells For each type III of cDNA and PA-1 cell-derived cDNA, PCR was performed in the usual manner using four combinations of primers.
  • oligonucleotide primers capable of PCR-amplifying the human 'telomerase protein cDNA fragment were designed.
  • H TPC5 SEQ ID NO: 11 in the sequence listing
  • hTPC3 corresponding to 455
  • PCR was performed using hTPC5 and hTPC3 as primers with about 1/20 of these cDNAs as type III.
  • Amp 1 itaq Gold Perkin-Elmer
  • Amp 1 itaq Gold was used, and after heat treatment at 95 ° C for 10 minutes, 95 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C
  • the 30 second heat retention cycle was repeated 35 times.
  • the expected DNA fragment of approximately 390 bp was amplified when the human cancer cell-derived cDNA was type III, but the type III (1) negative control and human placenta total RNA-derived cNA were amplified.
  • DNA was type II, it was not detected.
  • oligonucleotides 'primers' corresponding to the 5 'and 3' sides of the cDNA insertion site of gt10 used as the vector of the cDNA library (5'A gt10 And 3′ ⁇ gt10) (Clontech) and hTPC5 and hTPC3 as primers to determine the unknown portion upstream of 5 ′ side of hTPC5 or downstream of 3′side of hTPC3.
  • One million phages of the cDNA library were type I, and four combinations of primers (hTPC 5 vs. 5's gt10 or 3's gtl O and hTPC 3 vs.
  • RNA zol solution (T e1 — Test) from about 100 million Raji cells and PA-1 cells, respectively. 62, 156-159, 1987) to obtain total RNA, and apply the obtained total RNA to a 0.1 igo-d T cellulose column (type 7, lcmxlcm, Pharmacia). As a result, about 100 / g of poly (A) + RNA was obtained.
  • cDNA synthesis 5 ⁇ g of poly (A) + RNA was used for type I.
  • double-stranded cDNA was synthesized using reverse transcriptase, ribonuclease 1 ⁇ and Escherichia coli DNA polymerase attached to cDNA synthesis module (Amersham) according to the attached instructions.
  • the T4 DNA polymerase attached to the cDNA synthesis module (Amersham) was used.
  • phenol / chloroform extraction was performed, and the aqueous layer of the supernatant was recovered.
  • T4 DNA ligase reaction solution “66 mM Tris-HCl buffer (pH 7.6), 6.6 mM MgC 10 (Wako Pure Chemical Industries, Ltd.), 1 OmM dithiolysate Toll (DTT, Wako Pure Chemical), 66 mM adenosine 5'-triphosphate
  • the reaction product was developed on a Sephacryl S-200 column (lcmx 4 cm) according to a conventional method, and a 10 mM Tris-HCl buffer solution (pH 7.0) containing ImM EDTA and 0.5 mM NaC1 was used. Using 5), the cDNA to which an EcoRI adapter was added at the end was eluted. The eluted cDNA was recovered by ethanol precipitation, and the precipitate was dried.
  • phage particles were infected and spread in E. coli C600hf1A strain according to a conventional method, and phage particles were collected. Through a series of operations, about 200,000 phage clones were obtained per 100 ng of cDNA. About 100,000 phage clones were infected with E. coli C600hf1A strain according to a conventional method, and cultured on a NZY agar medium on a plate.
  • the human telomerase protein cDNA fragment obtained in the step (1) of Example 2 was labeled with 32 P and used as a probe, and phage clones hybridizing to the probe were screened.
  • the phage particles were collected from the obtained positive signal, cloned by the same method, and the plasmid containing the inserted cDNA was purified by the in vivooexcision method according to the manual of Stratagene. Collected at
  • the mRNA obtained in the above step (2) was converted into type ⁇ , and cDNA was amplified by the RACE method using a Marathon Tm cDNA Amplification kit (Clontech). In the following reactions, synthetic DNA primers
  • cDNA was synthesized. Purified p o 1 y (A) + RNA lig and c D NA reverse transcription primer —, 5'-
  • the amplification reaction was carried out using a primer complementary to a part of the sequence of the human 'telomerase protein cDNA and a primer complementary to the adapter primer added to the 3' end (5'-C CATC CTAATACGACTCACTATAGGGC-3 '(27 nuclei Reotide)], and TaQDNA polymerase.
  • One-tenth volume of the reaction solution was analyzed by 5% PAGE.
  • 51 of the above reaction solution was diluted 50-fold, and a second amplification reaction was performed using 5/1.
  • the second amplification reaction was performed according to the first amplification reaction.
  • the diluted reaction mixture 51 was made into type III, a primer complementary to a part of the sequence of human 'telomerase protein cDNA and located inside the primer used for the first amplification reaction, and 5' — ACTCACTATAGGGCTCGAGCGGC-
  • An amplification reaction with Taq DNA polymerase was performed using 3 ′ (23 nucleotides). Bring the total volume of the reaction mixture to 50 ⁇ 1 and incubate at 94 ° C for 1 minute followed by 60 at 94 ° C for 30 seconds. The incubation was performed at 30 ° C. for 30 seconds and at 68 ° C. for 5 minutes for 30 cycles, and finally, the reaction was completed at 72 ° C. for 7 minutes. After completion of the reaction, one-tenth volume of the reaction solution was analyzed by 5% PAGE.
  • the cDNA fragment amplified from the gel fragment was recovered, purified, and inserted into the cloning site of plasmid vector pCRII (Invitrogen) using T4 DNA ligase to obtain the DNA fragment.
  • Escherichia coli JM109 strain was transformed with the obtained recombinant vector.
  • X—Ga1—I PTG—LB Aminified DNA from three transformants that appeared on the agar medium and were not colored by X—Ga1 according to the standard method Then, the analysis was performed. Furthermore, the nucleotide sequence of cDNA was determined using the prepared plasmid DNA. As a result, a cDNA fragment having the nucleotide sequence of the 3 ′ untranslated region was obtained.
  • the reaction of the 5'-RAC E method was performed according to the 3'-RAC E method.
  • Synthetic DNA primers were synthesized using an ABI 394 DNA synthesizer, except for the primers attached to the Rathon cDNA Amplification kit.
  • a buffer and dNTP attached to Marathon TM cDNA Amplification kit were used.
  • type III a cDNA to which an adapter-primer was added at both ends was used as in the reaction of the 3'-RACE method.
  • the first amplification reaction was performed during the reaction of the 3'-RACE method, which was complementary to a part of the sequence of the human 'telomerase protein cDNA and a primer complementary to an adapter primer added to the 3' end.
  • the primer used for the following step was 5'-C CATC CTAATACGACTCACTATAGGGC- 3 '(27 nucleotides).
  • the total amount of the reaction solution was adjusted to 50 1 and an amplification reaction was carried out using Taq DNA polymerase.
  • the reaction was performed at 94 ° C for 1 minute, followed by 30 cycles of incubation at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 5 minutes, and finally ⁇ 2
  • the reaction was completed at 7 ° C for 7 minutes.
  • one-tenth volume of the reaction solution was analyzed by 5% PAGE.
  • 5; u1 of the above reaction solution was diluted 50-fold, and the second amplification reaction was performed using 51 as type III.
  • the second amplification reaction was performed according to the first amplification reaction.
  • primers primers complementary to a part of the sequence of human telomerase protein cDNA and located inside the primer used in the first amplification reaction and 5 ' -Performed using AC TCACTATAGGGCTC GAGC GGC-3 '(23 nucleotides).
  • the reaction was incubated at 94 ° C for 1 minute followed by 60 minutes at 94 ° C for 30 seconds.
  • the incubation was performed for 30 cycles at 30 seconds at C and for 5 minutes at 68, followed by incubation at 72 for 7 minutes to complete the reaction.
  • one-tenth volume of the reaction solution was analyzed with 5% PAGE.
  • telomere protein A cDNA fragment having the sequence of the 5 'untranslated region was obtained.
  • Example 3 Obtaining human 'telomerase protein gene
  • a cDNA library was first prepared using PA-1 cells. This library was screened using the PCR product obtained by using the aforementioned hTPC5 (SEQ ID NO: 11 in the sequence listing) and the aforementioned hTPC3 (SEQ ID NO: 12 in the sequence listing) as a primer. The human ⁇ telomerase protein gene full-length cDNA was obtained.
  • po 1 y (A) T RNA was obtained from PA-1 cells. That is, 1 0 8 cells were homogenized in guaiacolsulfonate two gin isothiocyanate Xia sulphonate solution was added and mixed of 2 M sodium acetate 0.1 volume (pH 4. 0). An equal volume of water-saturated phenol and 0.2 volume of a mixed solution of formaldehyde / isoamyl alcohol were added to the homogenate, mixed vigorously, and the aqueous layer of the supernatant was recovered by centrifugation. An equal volume of isopropanol was mixed with the collected aqueous layer, cooled at 120 ° C for 1 hour, and centrifuged.
  • the obtained precipitate was again dissolved in a guanidine isothiocyanate solution, an equal volume of isopropanol was added, the mixture was cooled at 120 ° C. for 1 hour, and then total RNA was recovered by centrifugation.
  • RNA was dissolved in ImM EDTA and 20 mM Tris-HCl (pH 7.5), heat treated for 70 minutes, and quenched on ice. NaC1 solution was added to this solution to a final concentration of 0.5 M, and developed on a 01 igo-d T cellulose column (type 7, lcmxlcm, Pharmacia). After washing the column with 2 OmM Tris-HCl buffer (pH 7.5) containing EDTA and 0.5 M NaC1, the bound fraction is eluted with sterile demineralized water and Po1y (A) + RNA was obtained.
  • cDNA was prepared using a cDNA synthesis kit from Stratagene. 1ststrand synthesis was performed by adding both random hexamer oligonucleotide and oligonucleotide dt primer as primers to a final concentration of 2 / M each. Blunt cDNA ends using T4 DNA polymerase After the conversion, an Eco RI adapter was added to the end. The reaction product was developed on Sephacryl S-500 force column to remove unreacted EcoRI adapter and small size cDNA. cDNA was recovered by ethanol precipitation and inserted into ⁇ phage DN ⁇ .
  • the sZAP phage DNA bound to cDNA was packaged into phage particles using a GI GAPACK GOLD II kit from Stratagene. By a series of operations, about 10 million phage clones were obtained.
  • phage clones were infected with E. coli C600hf1A strain according to a conventional method, and cultured on NZY agar medium on a plate. Two replicas in which the phage particles were copied onto a nylon membrane were prepared, washed, and washed. A PCR product using hTPC5 and hTPC3 as primers was labeled with 32 P and used as a probe, and phage clones hybridizing to this probe were screened.
  • restriction maps were prepared for plasmids pHB01 and pHB04, cDNAs of 1.lkbp and 7.4 kbp were inserted, respectively, and overlapping positional relationships as shown in Fig. 4 It turned out to be.
  • a deletion mutant cDNA was prepared according to a conventional method, and the DNA sequences of pHB01 and pHB04 were decoded.
  • a large open reading frame covering a region of about 8.1 kbp, including a C-terminal stop 'codon, was included.
  • the amino acid sequence predicted from this open reading frame showed high homology of 70% or more identity with the amino acid sequence at the C-terminal side of rat telomerase protein. This sequence was determined to be that of the human telomerase protein.
  • step (1) Obtaining human 'telomerase protein cDNA—Obtaining upstream sequence (5'-RACE method)
  • the DNA sequence obtained in the step (1) was the sequence of the nucleic acid sequence from position 756 onwards of the DNA sequence shown in SEQ ID NO: 13 in the sequence listing, but it had the primary structure of rat 'telomerase protein. From the comparison, it was considered that the open 'leading' frame extended further toward the N-terminal. Therefore, 5'-Rapid Amp liricationofc DNA Ends
  • step (1) 2 g of po1y (A) + RNA obtained from PA-1 cells and a DN complementary to the nucleic acid number 1165 to 1187 of SEQ ID NO: 13 in the sequence listing Oligonucleotide primers of A sequence TLP CM3
  • RNA ligase (SUPER SCRIPTII from GIBCO BRL), a substrate nucleotide, and a buffer were added and reacted at 42 ° C. for 1 hour. After terminating the reaction by adding EDTA, type I RNA was decomposed by alkali treatment, and single-stranded cDNA was isolated by isopropanol precipitation. Further, half of this cDNA was ligated with 5′-RACE force primer [5′-P (+) ANC] 4 pmo 1 using RNA ligase.
  • a single-stranded cDNA that was reverse-transcribed with TL PCM3 and further added with an anchor DNA sequence at the 3 ′ end was type III, and the oligonucleotide primer RACE—PRM 2 complementary to the anchor DNA was used.
  • the DNA was amplified by PCR using an oligonucleotide primer TLPNE having a DNA sequence complementary to the nucleic acid number 1024-1046 of SEQ ID NO: 13 in the sequence listing.
  • PCR was performed using a 1/20 amount of single-stranded cDNA and each lOpmol primer, and using Taq polymerase from GICO BRL in accordance with the attached manual.
  • the reaction was started with a manual hot-start method and then cycled for 30 seconds at 94 ° C, 1 minute at 60, and 2 minutes at 72 ° C. Was repeated 35 times.
  • the PCR product was incorporated into the pT7B1ueT vector, and the amplified DNA was inserted.
  • three of these clones had almost the same DNA sequence.
  • a representative clone, RACE-L4 was located at the position shown in FIG.
  • An initiation codon was present at nucleic acid numbers 156 to 158 of SEQ ID NO: 13 in the sequence listing, and a termination codon of the same frame was present at nucleic acid numbers 144 to 146 upstream. Since the length of the amplified DNA is almost uniform up to 157 bp upstream of the start codon, it is highly likely that the cDNA corresponding to the 5 'end of the actual mRNA was obtained.
  • Example 4 Acquisition of recombinant rat telomerase protein and preparation of specific antibody Schistosoma japonicum glutathione-1 S-transferase and rat 'telomerase protein (amino acid number 2 17 to SEQ ID NO: 1 in the sequence listing)
  • a fusion protein (GST-p80hom) with the 345th partial polypeptide was expressed using Escherichia coli, and the purified gene product was used as an antigen to immunize egrets.
  • the same portion of the rat telomerase protein was expressed as a fusion protein with histidine-hexamer (6His-p80hom) using another expression vector, and purified gene product was used.
  • a polyclonal antibody that recognizes rat 'telomerase protein from egret antiserum (specific for the portion corresponding to amino acids 217 to 345 of SEQ ID NO: 1 in the sequence listing) Polyclonal antibody).
  • the expression plasmid vector pGEX2T (Pharmacia) is cleaved with the restriction enzyme SmaI, and an oligonucleotide linker having a HindIII cleavage site is inserted into the expression vector pGEXH. 12 were produced. After digestion of this vector with the restriction enzyme Ec0RI, the ends were blunted using T4 polymerase (Toyobo), and further digested with the restriction enzyme HindIII.
  • the plasmid RaPC53 containing the rat telomerase protein cDNA fragment was cleaved with the restriction enzyme BamHI, and the ends were blunt-ended using T4 polymerase (Toyobo). Then, it is further digested with the restriction enzyme HindIII, subjected to polyacrylamide gel electrophoresis, and a partial DNA fragment of rat 'telomerase protein cDNA (nucleic acid No. 648- About 390 bp of Hindi II-corresponding to 1034; BamHI-derived blunt-ended DNA fragment) was isolated.
  • the HindIII-blended pGEXH12 vector thus obtained is ligated to a rat DNA fragment derived from the telomerase protein cDNA using a DNA ligase kit (Takara Shuzo).
  • E. coli strain JM109 (Toyobo) was transformed using the obtained recombinant vector.
  • a restriction map of each plasmid was prepared, and pGEXp80hom / JM109 having the correct recombinant plasmid was selected.
  • pGEXp80hom / JMl09 was inoculated into a 50 ml LB medium containing ampicillin, and cultured with shaking at 37 overnight. The next day, dilute this 10-fold with the same medium, further culture at 37 ° C for 1 hour, add 1/6 to a final concentration of 0.3 mM, and use GDS with a molecular weight of about 44 kDa by SDS-PAGE. — P80hom was expressed. Recombinant E. coli expressing GST-p80hom contains a final concentration of 1.5% sodium sarcosylate according to the method of Frangoni (An al. Biochem., 210, 179, 1993).
  • glutathione 'Sepharose beads (Pharmacia) were added and suspended. After incubating at 4 ° C for 40 minutes, the beads were washed with a phosphate buffer (PBS) containing 1% Triton X-100 and packed in a column. The GST-p80hom bound to the beads was eluted with a Hepes buffer containing 25 mM reduced glutathione and 0.1% Triton X-100.
  • PBS phosphate buffer
  • 0.7 mg of GST-p80hom was obtained from a 100 ml culture of recombinant.
  • the fusion protein was transformed into a GST with an apparent molecular weight of about 29 kDa and a rat 'telomerase protein fragment of about 16 kDa in SDS-PAGE (sequence in Sequence Listing).
  • the portion of the rat's telomerase protein shown in No. 1 (corresponding to amino acids No. 217-345) was cut into two. The latter was immobilized on a PVDF membrane, and the amino acid sequence at the N-terminus was analyzed by the Edman method, and it was confirmed that the amino acid sequence was the same as the expected amino acid sequence.
  • Two Japanese native male herons (shake 1 and! ⁇ 2) weighing approximately 2.6 kg According to the method, antiserum was obtained by immunization with a mixture of 100 g of GST-p80hom and Freund's adjuvant at one time.
  • Plasmid RaPC53 was digested with restriction enzymes HindIII and BamHI, and the approximately 390 bp HindiII-BamHI DNA fragment of rat telomerase protein cDNA (sequence listing). (Corresponding to nucleic acid numbers 648 to 1034 in SEQ ID NO: 1), and this fragment was subcloned into HindIII-BamHI site of pBlueScript (Toyobo).
  • a DNA fragment corresponding to the nucleic acid number 648-1034 of rat telomerase protein cDNA was digested from this plasmid using the restriction enzymes XhoI and NotI.
  • the XhoI-NotI DNA fragment was isolated, and the expression plasmid vector pProEX-I (Gibco BRL) cut with the restriction enzymes Sa1I and NotI and DNA ligation kit (Takara Shuzo).
  • the resulting recombinant vector was used to transform E. coli strain JM109 (Toyobo).
  • a restriction map of each plasmid was prepared, and PProEXp80hom / JM109 having the correct recombinant plasmid was selected.
  • pProEXp80hom / JM109 was inoculated into a 50 ml LB medium containing ampicillin, and cultured with shaking at 37 ° C overnight. The next day, the culture was diluted 10-fold with the same medium, and further cultured at 37 ° C for 1 hour. IPTG was added to a final concentration of ImM, and 6H with a molecular weight of about 18 kDa was determined by SDS-PAGE. is—p80hom was expressed. Recombinant Escherichia coli expressing 6His-p80hom was dissolved in a binding buffer containing 6M guanidine hydrochloride according to the protocol of Qiagen, and the solution was dissolved in Ni-NTA-agarose (Qiagen).
  • telomerase activity in a rat or human cell extract was immunoprecipitated.
  • total IgG was purified from R1 pre-immune serum using protein A sepharose (Pharmacia) (PI-1), and obtained from the IgG and R1 hyperimmune serum.
  • an S100 extract was prepared according to the method of Counter et al. (EMB 0 J., 11, 1921, 1992). Was prepared.
  • Telomerase activity was measured by the ELISA method using digoxigenin-labeled dUPP and an anti-digoxigenin antibody according to the method of Tatema tsu ssu et al. (Oncgene, 13, 2265—2274, 1996).
  • an oligonucleotide bpTG 3 Bioti n y l a t e d 5 '
  • the reaction was carried out at 30 ° C. for 1 hour using 0.8 mM each of monodeoxynucleotides (TTP, dATP, dGTP) as substrates.
  • the enzymatic reaction was stopped by adding excess EDTA.
  • streptavidin (GIB CO BRL) was cross-linked to a 96-well polycarbonate microtiter plate (Takara) using EDC (Sigma), and a blocking agent (Belinger Mannheim Yamanouchi) Was blocked at 37 ° C for 2 hours.
  • the telomerase extension reaction product 251 diluted with TBS was added to each of the above-mentioned tubes, and the mixture was incubated at 37 ° C. for 30 minutes to bind to streptavidin on the plate. After discarding the sample solution, an excess amount of a biotin solution was added, and the mixture was incubated at 37 ° C for 30 minutes, and excess streptavidin was blocked.
  • Streptavidin prepared to 5 mg Zm 1 using OmM sodium carbonate buffer (pH 9.6) was dispensed into white polystyrene 96-well microtiter plates at a rate of 1001 Z Thereafter, the cells were kept warm at 37 for 1 hour and coated with streptavidin. After discarding the streptavidin solution, a blocking agent was dispensed at a ratio of 150 11 / ⁇ ⁇ and blocking was performed at 37 ° C for 2 hours. To this vial, add the PCR product diluted 20-fold with TBS in 100 ⁇ 1 Zells, and add 3 ⁇ l. C was incubated for 30 minutes to bind to the plate.
  • each well was washed 5 times with 1.51% Zell's 0.05% Tween 20ZTBS, and then diluted with TBS 5000-fold with an anti-digoxigenin-labeled anti-lipoxyphosphatase antibody (Beilinger Mannheim). (Yamanouchi) and kept at 37 ° C for 30 minutes. The plate was washed five times with 150 ⁇ 1 noel of 0.05% Tween 2 OZTB S, and then diluted CS-PD (D) 100-fold with 0.1 M diethanolamine buffer (pH 9.5).
  • Example 7 Expression of human 'telomerase protein mRNA in human cancer cells and normal tissues
  • RNA from human normal tissues such as spleen, thymus, ⁇ , testis, ovary, small intestine, large intestine, heart, placenta, lung, liver, skeletal muscle, and ⁇ is clear A new 10.7 kb band was detected.
  • po 1 y (A) + In the RNA block a short 8.6 kb species was observed in addition to the 10.7 kb band.
  • Example 8 Purification of rat telomerase protein and identification of molecular species
  • the fractions having telomerase activity were collected, applied to an anion exchange column (trade name: Resource Q, Pharmacia) saturated with a TMG buffer (containing no dithiothreitol) containing 5 OmM potassium chloride, and potassium chloride was used. Step elution was performed. Next, the fractions having telomerase activity were collected, and the metal (Zn 2+ ) chelate was saturated with a TMG buffer (containing no dithiothreitol) containing 0.5 M potassium chloride and ImM imidazole. The solution was applied to a T-column (trade name: High Trap Cleaning, Pharmacia), and stepwise elution was performed using imidazole.
  • the eluted fraction having telomerase activity was subjected to 15-40% glycerol concentration gradient centrifugation (Beckman SW28, 25,000 rpm, 2 ° C, 24 hours). As a result, a protein with a sedimentation coefficient of 44 S was found to be a protein correlated with telomerase activity. P97 / 0204 was obtained and its molecular weight was calculated to be approximately 1500 kDa.
  • the fractions generated by glycerol concentration gradient centrifugation were separated by 6% SDS-PAGE, and subjected to pestic blot with a specific antibody against the recombinant rat telomerase protein obtained in Example 4.
  • Three antibody-reactive bands (molecular weights on SDS-PAGE of about 240 kDa, 230 kDa and 55 kDa) were observed in the protein fraction showing activity.
  • the band of 55 kDa was confirmed to be a 240 kDa or 230 kDa protein degradation product by heat treatment experiments.
  • rat telomerase protein was composed of a 240 kDa protein (hereinafter sometimes referred to as “p240”) as a component, and a 23 OkDa protein (hereinafter “p240”). p230 ”) as a component.
  • p240 240 kDa protein
  • p230 23 OkDa protein
  • Rat hepatoma-derived cell line AH66F cells sown on a 10 cm plastic dish were incubated with 250 ⁇ CiZm1 [ ⁇ S] methionine (trade name Tran 35 S-1 abe 1, ICN) and 10% fetal calf Pulse label for 30 minutes in 1 ml DMEM medium (without methionine and cystine, Lifetech Oriental) containing serum (JRH Biosciences), then add a large excess of non-radioactive methionine to the medium did.
  • the cells were collected and immunoprecipitated in the same manner as in Example 4 using a specific antibody against the recombinant rat telomerase protein.
  • the obtained immunoprecipitate was subjected to 6% SDS-PAGE and then to autoradiography.
  • p240 was mainly immunoprecipitated immediately after the pulse labeling (0 hour).
  • p240 decreased and p230 increased.
  • the rat telomerase protein is first expressed as a protein having p240, and then modified to become a protein having p230.
  • the correlation between the abundance ratio of p240 / p230 in rat normal tissues and rat liver cancer cell line AH66F cells and telomerase activity was examined. First, an S100 extract was prepared from rat liver, kidney, testis, and AH66F cells according to the method of Counter et al.
  • telomere activity was higher in AH66F cells, testis and liver than in higher order, and was not detected in kidney.
  • the abdominal ratio of p230 was AH66F cells, testis, and liver in descending order. Almost no p230 was observed in the kidney.
  • telomerase protein derived from a higher animal which is essential for cell growth and has been suggested to be involved in cancer cell growth, and a gene encoding the same are provided.
  • the telomerase protein and the gene encoding the telomerase protein of the present invention are useful for elucidating, for example, biological control mechanisms such as cell growth and cell aging, and are expected to be particularly useful for the development of therapeutic drugs for cancer.
  • the antibody specifically recognizing the telomerase protein of the present invention is useful as a reagent for detecting cancer cells, and is expected to be useful as a clinical test agent for early detection of cancer. .
  • telomerase protein of the present invention differs in the molecular weight in the SDS-polyacrylamide electrophoresis between the active form and the inactive form
  • screening of a drug acting on the telomerase protein can be carried out. It is possible to do. 4 Sequence Listing SEQ ID NO: 1
  • GCC AAC CCG AGG TAC CTG TGC ACC CTG ACG CAG CGG CAG CTT CGG GCG 2100
  • GCA AAT GCC AAC AGA CTT TGT CCC AAG AGT CAC TTG CAA GGG CCT CCC 2340
  • Lys Gin lie lie Trp Gin His Val Asn Ser Lys Cys Leu Phe Val Ser
  • CAG CTG GCT TTC CAG TAC ACC CAT CCC AAG TCT CTA AAC TGC ATC ACC 5652
  • GGC AGG GAT CGG AAT CTC CTC TGC TGG GAC GTC AAG GTA GCC CAA GCC 6516 Gly Arg Asp Arg Asn Leu Leu Cys Trp Asp Val Lys Val Ala Gin Ala
  • AAG CAC CGG GCC AAG AGA CAC CCC CGC CGG CCA CCC CGC TCT CCA GGG 96 Lys His Arg Ala Lys Arg His Pro Arg Arg Pro Pro Arg Ser Pro Gly
  • GAG AAA AAG AAT CCT CCA AGG TTC ACC CTG AAG AAG CTG GTT CAG CGA 240 Glu Lys Lys Asn Pro Pro Pro Arg Phe Thr Leu Lys Lys Leu Val Gin Arg 65 70 75 80 CTG CAC ATC CAC AAG CCT GCC CAG CAC GTT CAA GCC CTG CTG GGT TAC 288 Leu His lie His Lys Pro Ala Gin His Val Gin Ala Leu Leu Gly Tyr
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • GCC AAT ATA GTG TTG GAA GTG GCT GCC CTC TTG CCA GCC TGC CGC CCC 336 Ala Asn lie Val Lys Ala Val Ala Ala Leu Leu Pro Ala Cys Arg Pro
  • Sequence type nucleic acid
  • R indicates A or G, Y or T.
  • Sequence type nucleic acid
  • R indicates A or G
  • N indicates A
  • G G
  • C indicates A
  • W indicates A or T.
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • ARRTTICKIA RCATIGCCAT RAAIGG 26 SEQ ID NO: 10
  • Sequence type nucleic acid
  • R indicates A or G
  • 1 indicates inosine
  • K indicates G or T.
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • TCT GAC CTG AAG ACC ATG GAG AAA CCA CAT GGA CAT GTT TCT GCC CAC 458 Ser Asp Leu Lys Thr Met Glu Lys Pro His Gly His Val Ser Ala His
  • VOV OVO OVO WO VOV 313 VIV 3VX 90V V33 III XOI VOV OVO
  • AAG ACT GCC ATC AAG CTC CAG GCT CAA GTC CAG GAG TTT GAT GAA AAT 2426 Lys Thr Ala He Lys Leu Gin Ala Gin Val Gin Glu Phe Asp Glu Asn
  • GGC CAA AGG GTT CCT GTG GAC AGG GTC ATC CTC CTT GGC CAA AGC ATG 2522 Gly Gin Arg Val Pro Val Asp Arg Val He Leu Leu Gly Gin Ser Met
  • GCA CAG CTG TTT GTG GGG ATT CTG GGC TCC CGT TAT GGA AAC ATT CCC 3098 Ala Gin Leu Phe Val Gly lie Leu Gly Ser Arg Tyr Gly Asn lie Pro
  • GGT TCC CAG GGG GCT CAG GGT CAG GCA CTG GAT GTG GCA GTG TCG GCC 6074
  • Leu Ala Trp lie Ser Pro Lys Val Leu Val Ser Gly Ala Glu Asp Gly

Abstract

A telomerase protein originating in higher animals involving human being. This protein and a gene encoding the same are useful in, for example, the clarification of biological control mechanisms such as cell growth and aging and expected to be applicable to, in particular, the development of remedies for cancer. A method for screening substances acting on the expression of the enzyme activity of the higher animal telomerase protein involves the step of measuring the molecular weight of the telomerase protein contained in cells or tissues in contact with a test substance by, for example, the SDS polyacrylamide electrophoresis method.

Description

明 細 書 高等動物テロメラ—ゼ蛋白質及びそれをコ一ドする遣伝子  Description Higher animal telomerase protein and gene encoding it
技術分野 Technical field
本発明は高等動物細胞のテロメラーゼの蛋白質をコードする遺伝子及びその遺 伝子産物に関するものである。 背景技術  The present invention relates to a gene encoding a telomerase protein of a higher animal cell and a gene product thereof. Background art
動物細胞などの真核細胞染色体の線状 DNAの両末端はテロメァと呼ばれ、 特殊な DNA配列とそれに結合する蛋白質からなる複雑な高次構造をとつている。 テロメ ァ DNA は、 チミ ン（T) 及びグァニン（G) (反対鎖はアデニン（A) 及びシ トシン（C) ) の豊富な特徴的繰り返し配列からなり、 例えば、 脊椎動物細胞染色体のテロメ Both ends of linear DNA in eukaryotic chromosomes, such as animal cells, are called telomeres and have a complex higher-order structure consisting of a special DNA sequence and proteins that bind to it. Telomere DNA is composed of characteristic repetitive sequences rich in thymine (T) and guanine (G) (the opposite strands are adenine (A) and cytosine (C)), for example, telomere in vertebrate cell chromosomes.
TDNA は TTAGGG (反対鎖は CCCTAA) の 6塩基の繰り返しで構成されている。 この配 列を利用したサザンプロッティ ング解析により、 ヒ ト体細胞のテロメァ繰り返し 配列の平均長は 7キロ〜 10キロベースであることが明らかにされた。 TDNA is composed of 6 bases of TTAGGG (CCCTAA on the opposite strand). Southern blotting analysis using this sequence revealed that the average length of telomere repeats in human somatic cells was 7-10 kilobases.
テロメァ構造は染色体の安定化に重要な機能を有すると考えられている。 例え ば、 テロメァが細胞核の辺縁に位置することが酵母を用いた形態学的研究で明か にされており、 テロメァが染色体を核の特定の位置に固定する 「錨」 として作用 し、 細胞核内で各染色体間の物理的相互作用を制御している可能性が示唆されて いる。 また、 以下のように、 真核細胞の線状二本鎖 DNA の複製ごとの短縮化による 染色体機能の不活化を防ぐ機能を有することが示唆されている。  Telomere structure is thought to have an important function in chromosome stabilization. For example, morphology studies using yeast have revealed that telomeres are located at the periphery of the cell nucleus, and telomeres act as "anchors" that anchor chromosomes to specific locations in the nucleus, and are located in the cell nucleus. Suggests that it may control physical interactions between chromosomes. It has also been suggested that it has a function to prevent inactivation of chromosomal functions by shortening each replication of eukaryotic linear double-stranded DNA as follows.
線状二本鎖 DNA の両鎖の同時複製の過程では、 一方の DNA鎮 （リーディング鎖） が 3 '末端をプライマ一として 5 '→3 'DNA ポリメラ一ゼにより連続的に複製されるの に対し、 他方の DNA鎖 （ラギング鎖） では小さい R プライマーを用いた断続的な ものになる。 従って、 新生鎖 （ラギング鎖） の 5 '末端の RNA プライマーは DNA に置 き換えられないので、 細胞分裂を繰り返す毎に一方の娘細胞の 5 '末端が次第に短縮 することになり、 最後には染色体が不安定になって細胞が死に至る。 しかしな力 ら、 生殖細胞系列では DNA の繰り返し複製によって染色体機能が損なわれるような 染色体 DNA の短縮ィ匕が生じないことが明らかにされており （Allsopp, R. C. et al. ,In the process of simultaneous replication of both strands of linear double-stranded DNA, one of the DNA strands (leading strand) is continuously replicated by the 5 '→ 3' DNA polymerase with the 3 'end as the primer. On the other hand, the other DNA strand (lagging strand) is intermittent using a small R primer. Therefore, since the RNA primer at the 5 'end of the nascent strand (lagging strand) cannot be replaced with DNA, the 5' end of one of the daughter cells will gradually decrease with each repetition of cell division, The chromosomes become unstable and cells die. But the power It has been shown that germline DNA does not cause chromosomal DNA truncation that would impair chromosomal function due to repeated DNA replication (Allsopp, RC et al.,
Proc. Natl. Acad. Sci. U. S. A. , 89, 10114, 1992) 、 テロメァやそれに隣接す る領域がヘアピン構造を採ったり、 短縮化に対する緩衝帯として機能している可 能性が示唆されている。 Proc. Natl. Acad. Sci. U. S.A., 89, 10114, 1992), suggesting that telomeres and the region adjacent to them may adopt a hairpin structure or function as a buffer band for shortening.
テロメァが染色体の短縮化を防ぐ機能を有することは、 細胞の老化 ·不死化と テロメァ繰り返し配列の平均長の変化との関係からも強く示唆されている。 多細 胞生物の線維芽細胞などをイン ' ビトロで継代培養すると、 継代を経るにつれて 増殖能が低下し、 最終的には増殖能を失った 「老化」 細胞となるカ^ 予め細胞内 にある種の癌遺伝子を導入しておく と永久増殖能を獲得した不死化細胞が得られ る場合がある。 これらは細胞レベル （イン ' ビトロ） での老化現象及び発癌のモ デルと理解されているが、 分子レベルでの研究により、 正常細胞では分裂回数の 増加につれてテロメァ繰り返し配列の平均長が短縮化し、 その平均長は継代可能 回数と相関すること、 並びに、 不死化細胞ではテロメァ繰り返し配列の平均長が 短いが、 継代中にその平均長が変化しないことが明らかにされた。  The fact that telomeres have the function of preventing chromosome shortening is strongly suggested from the relationship between cell aging and immortalization and changes in the average length of telomere repeats. When subcultured in vitro, such as fibroblasts of multicellular organisms, the proliferative capacity decreases as the cells pass, and eventually becomes “senescent” cells that have lost proliferative capacity. Immortalized cells that have acquired permanent growth ability may be obtained by introducing a certain oncogene into the cell. Although these are understood to be models of aging and carcinogenesis at the cellular level (in vitro), studies at the molecular level show that in normal cells the average length of telomere repeats decreases as the number of divisions increases. It was shown that the average length correlated with the number of passages, and that the average length of telomere repeats was shorter in immortalized cells, but the average length did not change during the passage.
テロメァ繰り返し配列平均長の制御機構の一つとして、 テロメァ繰り返し配列 を伸長させる RNA依存性 DNAポリメラ一ゼ （テロメラーゼ） が注目されている。 こ の酵素は、 原生動物テトラヒメナの大核抽出液中から、 テ卜ラヒメナのテロメァ 繰り返し配列由来の合成ォリゴヌクレオチド（TTGGGG)の 3 '端に同じ 6塩基の繰り返 し配列を付加する酵素として見い出されたものであり、 活性に必要なサブュニッ トとしてテロメァ DNA 配列の 5 ' - TTAGGG- 3 'に相補的な铸型 RNA を含み、 铸型 RNA を基にしてテロメァ DNA の一本鎖を延長する一種の逆転写酵素である。 テトラヒメ ナ .テロメラ一ゼ由来のテロメラーゼが精製され、 その cDNAがクローニングされた RNA-dependent DNA polymerase (telomerase), which extends telomere repeats, has attracted attention as one of the mechanisms for controlling the average length of telomere repeats. This enzyme is an enzyme that adds the same 6-base repeat sequence to the 3 'end of a synthetic oligonucleotide (TTGGGG) derived from the tetramer repeat sequence of Tetrahymena from the prokaryotic tetrahymena macronuclear extract. It has been discovered and contains a type I RNA complementary to the 5'-TTAGGG-3 'of the telomeric DNA sequence as a subunit required for activity, and elongates the single-stranded telomeric DNA based on the type I RNA Is a kind of reverse transcriptase. Telomerase from Tetrahymena telomerase was purified and its cDNA was cloned.
(Collins, K, et al. , Cell, 81， 677, 1995)。 このテロメラーゼは铸型 RNA と 結合する 80 kD のサブュニッ ト及びプライマーとなる DNA 末端に結合する 95 kD のサブュニッ トからなり、 RNA ウィルスの RNAポリメラ一ゼに比較的類似の一次構 造を有することが明かにされた。 (Collins, K, et al., Cell, 81, 677, 1995). This telomerase consists of an 80 kD subunit that binds to type I RNA and a 95 kD subunit that binds to the DNA end that serves as a primer, and may have a primary structure relatively similar to RNA virus RNA polymerase. It was revealed.
テロメラ一ゼの生物学的意義は、 テトラヒメナゃ酵母などの下等真核生物で明 らか ( された。 すなわち、 テトラヒメナ 'テロメラーゼ RNA遺伝子のテロメァ繰り 返し配列の铸型部分に点突然変異を導入した遺伝子で形質転換された個体では、 導入されたある種の点突然変異に対応する変異テロメァ繰り返し配列が生合成さ れると同時に増殖不可能になる。 また、 パン酵母 'テロメラ一ゼ RNA遺伝子である TLC1 が破壊されると、 継代を重ねるにつれてその酵母のテロメァ繰り返し配列 平均長が短くなり、 最終的には増殖不可能となる。 従って、 単細胞真核生物では テロメラーゼが細胞増殖に必須の酵素であると理解されている。 The biological significance of telomerase is apparent in lower eukaryotes such as Tetrahymena That is, in individuals transformed with a gene in which a point mutation has been introduced into the type II portion of the telomere repeat sequence of the Tetrahymena 'telomerase RNA gene, When the mutant telomere repeats are biosynthesized and become non-proliferable, the baker's yeast telomerase RNA gene, TLC1, is destroyed, and as the passage continues, the average length of the yeast telomere repeats increases. Therefore, telomerase is understood to be an essential enzyme for cell growth in unicellular eukaryotes.
イン . ビトロでのヒ ト細胞の不死化過程において、 テロメラーゼ活性が癌遺伝 子導入後の継代初期には認められず、 無限増殖能を獲得した細胞集団において検 出されることが明らかにされた。 また、 実際のヒ ト癌細胞のほとんどにテロメラー ゼ活性が検出される一方で、 多くの正常細胞ではテロメラーゼ活性は検出されな いと言われている。 これらの知見から、 癌細胞は、 その成立過程においてテロメ ラーゼ活性の発現によりテロメァ DNA の短縮化を免れ、 永久増殖能を獲得するので はないかとの推測が可能である。 従って、 テロメラ—ゼ阻害剤が選択性の高い抗 癌剤として有用であり、 テロメラーゼ活性の測定により癌の早期診断が可能にな ると予測される。  During in vitro immortalization of human cells, it was revealed that telomerase activity was not detected early in the passage after the introduction of the cancer gene and was detected in a cell population that had acquired infinite proliferative capacity. . It is also said that telomerase activity is detected in most of actual human cancer cells, but telomerase activity is not detected in many normal cells. From these findings, it is possible to speculate that cancer cells may escape from shortening of telomere DNA by expressing telomerase activity during the establishment process, and acquire permanent growth ability. Therefore, telomerase inhibitors are useful as highly selective anticancer agents, and it is expected that early diagnosis of cancer will be possible by measuring telomerase activity.
テロメラーゼ R N Aサブュニッ 卜の発現の程度は必ずしもテロメラーゼ活性に 相関しないという報告がある （A v i I o nら、 C a n c e r R e s . ， 5 6、 6 4 5、 1 9 9 6 ) 。 しかしながら、 現在のところ、 ヒ トを含めた高等動物におい てはテロメラーゼ自体が未だ分離 ·精製されておらず、 その物質的実態は不明の ままであり、 しかも、 実際にテロメラーゼ活性を検出するためには P C Rを用い た煩雑な検出法を用いる必要があるので、 テロメラ一ゼについての酵素学的研究 はほとんどなされていないのが現状である。 さらに、 病理切片などを用いてテロ メラーゼ活性の発現を個々の細胞レベルで判定することもできないため、 テロメ ラーゼと癌の悪性度との正確な関係を解析することは困難である。  It has been reported that the degree of expression of telomerase RNA subunits does not always correlate with telomerase activity (AviIon et al., Cancer Res., 56, 645, 1996). However, at present, in higher animals including humans, telomerase itself has not yet been separated and purified, and its actual substance state remains unknown. Since it is necessary to use a complicated detection method using PCR, almost no enzymological research has been conducted on telomerase. Furthermore, since the expression of telomerase activity cannot be determined at the individual cell level using pathological sections or the like, it is difficult to analyze the exact relationship between telomerase and cancer malignancy.
従って、 テロメラ一ゼ蛋白質を単離 ·同定することによって、 高等動物テロメ ラ一ゼの物質的特徴を解明するとともに、 酵素学的見地からテロメラーゼの阻害 剤の研究を行い、 テロメラーゼと癌の悪性度との関係を解明することが強く望ま /02 れている。 発明の開示 Therefore, by isolating and identifying the telomerase protein, we will elucidate the physical characteristics of telomerase in higher animals, and conduct research on telomerase inhibitors from an enzymatic point of view. It is highly desirable to clarify the relationship with / 02 Disclosure of the invention
そこで本発明者らは、 高等動物テロメラーゼ蛋白質を単離 ·同定するべく鋭意 検討を重ね、 高等動物テロメラ一ゼ蛋白質をコ一ドする遣伝子のクローニングに 初めて成功し、 さらにその遺伝子から遗伝子産物である高等動物テロメラ—ゼ蛋 白質を発現させることに成功した。 また、 この遺伝子産物を特異的に認識する抗 体を作製し、 これを用いてテロメラ一ゼ活性とこの遺伝子産物との密接な関係を 証明することにも成功した。 本発明はこれらの知見を基にして完成されたもので ある。 なお、 最近、 ヒ ト 'テロメラ一ゼ蛋白質の全長のアミノ酸配列が報告され たが （Science, 275, pp. 973-977, February 14, 1997)、 c-DNA の塩基配列及びァ ミノ酸配列は本発明者らが解明したものと多くの部分で相違している。  Accordingly, the present inventors have conducted intensive studies to isolate and identify a higher animal telomerase protein, and succeeded for the first time in cloning a gene encoding a higher animal telomerase protein, and further obtained a gene from the gene. We succeeded in expressing a higher product telomerase protein, a progeny product. They also produced an antibody that specifically recognizes this gene product and used it to prove the close relationship between telomerase activity and this gene product. The present invention has been completed based on these findings. Recently, the full-length amino acid sequence of human 'telomerase protein was reported (Science, 275, pp. 973-977, February 14, 1997), but the nucleotide sequence and amino acid sequence of c-DNA were It differs in many respects from what we have elucidated.
本発明は、 配列表の配列番号 1に記載のァミノ酸配列で特定されるポリべプチ ドを提供するものであり、 該ポリベプチドはラッ 卜由来テロメラ一ゼ蛋白質であ ることを特徴としている。 また、 本発明により、 配列表の配列番号 1に記載のァ ミノ酸配列に 1又は 2以上のアミ ノ酸残基による置換、 揷入、 及び/又は欠失が 存在しており、 実質的にヒ トを含む高等動物テロメラーゼ蛋白質として機能する ことを特徴とするポリペプチドが提供され、 その好ましい態様により、 ヒ 卜の生 体内でテロメラーゼ蛋白質として機能することができる上記ポリぺプチドが提供 される。  The present invention provides a polypeptide specified by the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, wherein the polypeptide is a rat-derived telomerase protein. Further, according to the present invention, substitution, insertion, and / or deletion by one or more amino acid residues are present in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence is substantially present. A polypeptide characterized by functioning as a higher animal telomerase protein including human is provided. According to a preferred embodiment, the above polypeptide capable of functioning as a telomerase protein in a human organism is provided.
また、 本発明の別の態様により、 配列表の配列番号 2に記載のアミ ノ酸配列で 特定されるポリべプチドが提供されるが、 このポリべプチドはヒト由来テロメラ一 ゼ蛋白質の部分ポリべプチドであることを特徴としている。 さらに本発明により、 配列表の配列番号 2に記載のァミノ酸配列に 1又は 2以上のアミ ノ酸残基による 置換、 挿入、 及び 又は欠失が存在しており、 実質的にヒ トを含む高等動物テロ メラーゼ蛋白質の部分ポリベプチドとして機能することを特徴とするポリぺプチ ドが提供される。  According to another embodiment of the present invention, there is provided a polypeptide identified by the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing, wherein the polypeptide is a partial polypeptide of a human-derived telomerase protein. It is characterized by being a peptide. Further, according to the present invention, substitution, insertion, and / or deletion by one or more amino acid residues is present in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and substantially contains human. There is provided a polypeptide characterized by functioning as a partial polypeptide of a higher animal telomerase protein.
さらに本発明の別の態様により、 配列表の配列番号 1 3に記載のァミノ酸配列 で特定されるポリペプチドが提供されるが、 該ポリペプチドはヒ 卜由来テロメラ一 ゼ蛋白質であることを特徴としている。 また、 本発明により、 配列表の配列番号According to still another embodiment of the present invention, an amino acid sequence represented by SEQ ID NO: 13 in Sequence Listing Is provided, wherein the polypeptide is a human-derived telomerase protein. In addition, according to the present invention,
1 3に記載のアミノ酸配列に 1又は 2以上のアミノ酸残基による置換、 挿入、 及 びノ又は欠失が存在しており、 実質的にヒ 卜を含む高等動物テロメラ一ゼ蛋白質 として機能することを特徴とするポリベプチドが提供され、 その好ましい態様に より、 ヒ 卜の生体内でテロメラ一ゼ蛋白質として機能することができる上記ポリ ぺプチドが提供される。 13. The amino acid sequence described in 13 includes substitution, insertion, and / or deletion by one or more amino acid residues, and substantially functions as a higher telomerase protein containing humans. The present invention provides a polypeptide characterized by the above-mentioned, and in a preferred embodiment thereof, the above-mentioned polypeptide which can function as a telomerase protein in a living body of a human.
さらに本発明の別の態様によれば、 上記の各ポリベプチドをコ一ドするヌクレ ォチド配列が提供される。 このヌクレオチド配列としては、 D N A配列又は R N A 配列を挙げることができ、 例えば、 その好ましい態様として、 配列表の配列番号 1に記載の D N A配列の核酸番号 1 9 9から核酸番号 8 0 8 5 (終始コドンを含ま ず） で特定される D N A、 又は配列表の配列番号 2に記載の D N A配列の核酸番 号 1から核酸番号 4 8 7で特定される D N A、 又は配列表の配列番号 1 3に記載 の D N A配列の核酸番号 1 5 6から核酸番号 8 0 3 0 (終始コ ドンを含まず） で 特定される D N Aが提供される。 以上に加えて、 上記 D N A配列を含む組み換え ベクター、 該組み換えベクターが導入された形質転換体、 及び、 該形質転換体を 培養した培養物から上記 D N A配列の遣伝子産物であるポリぺプチドを分離 ·採 取する工程を含む、 上記ポリぺプチドの製造方法も提供される。  According to still another aspect of the present invention, there is provided a nucleotide sequence encoding each of the above polypeptides. Examples of the nucleotide sequence include a DNA sequence and an RNA sequence.For example, in a preferred embodiment, nucleic acid numbers from 199 to nucleic acid numbers of the DNA sequence described in SEQ ID NO: 1 in the sequence listing are set to be from nucleic acid number (Excluding codons), or the DNA specified by nucleic acid No. 1 to nucleic acid No. 487 of the DNA sequence described in SEQ ID No. 2 in the sequence listing, or the DNA specified in SEQ ID No. 13 in the sequence listing Provided is a DNA specified by nucleic acid numbers 156 to 830 (not including the termination codon) of the DNA sequence of SEQ ID NO: 1. In addition to the above, a recombinant vector containing the DNA sequence, a transformant into which the recombinant vector has been introduced, and a polypeptide which is a gene product of the DNA sequence from a culture obtained by culturing the transformant. There is also provided a method for producing the above-mentioned polypeptide, comprising a step of separating and collecting.
本発明のさらに別の態様として、 上記の各ポリべプチドを特異的に認識するこ とができる抗体、 上記の各ヌクレオチド配列の一部又は全部に相補的に結合可能 なヌクレオチド配列を含む核酸プローブが提供されるが、 これらの抗体又は核酸 プローブは癌細胞検出用試薬として有用であり、 上記抗体又は核酸プローブを含 む癌診断用の医薬組成物が本発明の一態様として提供される。  As still another embodiment of the present invention, there are provided an antibody capable of specifically recognizing each of the above-mentioned polypeptides, a nucleic acid probe comprising a nucleotide sequence capable of binding complementarily to part or all of the above-mentioned nucleotide sequences These antibodies or nucleic acid probes are useful as reagents for detecting cancer cells, and a pharmaceutical composition for cancer diagnosis containing the above antibodies or nucleic acid probes is provided as one embodiment of the present invention.
これらの発明に加えて、 本発明の別の態様により、 S D S (ドデシル硫酸ナト リウム） 一ポリアクリルアミ ド電気泳動法 （P A G E ) による分子量が、 不活性 型では約 2 4 0 k D aであり、 活性型では約 2 3 0 k D aであることを特徴とす る上記ポリぺプチドと、 S D S —ポリアクリルアミ ド電気泳動法による分子量が 約 2 3 0 k D aであることを特徴とする活性型のポリぺプチドが提供される。 ま た、 高等動物テロメラーゼ蛋白質の酵素活性の発現に作用する物質のスク リ一二 ング方法であって、 被験物質と接触させた細胞又は組織に含まれる高等動物テロ メラ一ゼ蛋白質のサブュニッ トであるポリべプチドの分子量を測定する工程を含 む方法も提供される。 In addition to these inventions, according to another aspect of the invention, the molecular weight by SDS (sodium sodium dodecyl sulfate) -polyacrylamide electrophoresis (PAGE) is about 240 kDa for the inactive form. The above-described polypeptide, which is characterized by being about 230 kDa in the active form, and the molecular weight of about 230 kDa by SDS-polyacrylamide electrophoresis. Active polypeptides are provided. Ma A method of screening a substance that acts on the expression of the enzymatic activity of a higher animal telomerase protein, which is a subunit of a higher animal telomerase protein contained in a cell or tissue that has been brought into contact with a test substance. Also provided is a method comprising the step of measuring the molecular weight of the polypeptide.
上記方法の発明の好ましい態様によれば、 被験物質との接触工程を被験物質の 存在下における培養工程又は動物への被験物質の投与工程により行う上記方法； 分子量の測定を S D S —ポリアク リ ルア ミ ド電気泳動法で行う上記方法；約 2 4 0 k D aの不活性型ポリぺプチド及び約 2 3 0 k D aの活性型のポリぺプチ ドの存在比を測定する工程を含む上記方法 ； 被験物質の非存在下における 2 4 0 k D aのポリベプチドの存在比と比較して、 該ポリベプチドの存在比が被 験物質の存在下において実質的に増加している場合には、 該被験物質が高等動物 テロメラーゼ蛋白質の酵素活性の発現を阻害する物質であると判定する工程を含 む上記方法；並びに、 被験物質の非存在下における 2 3 0 k D aのポリペプチド の存在比と比較して、 該ポリべプチドの存在比が被験物質の存在下において実質 的に増加している場合には、 該被験物質が高等動物テロメラーゼ蛋白質の酵素活 性の発現を活性化する物質であると判定する工程を含む上記方法が提供される。 図面の簡単な説明  According to a preferred embodiment of the above method, the step of contacting with the test substance is carried out by a culturing step in the presence of the test substance or a step of administering the test substance to an animal; The molecular weight is measured by SDS-polyacrylamide. The above method comprising the steps of measuring the abundance ratio of an inactive polypeptide of about 240 kDa and an active polypeptide of about 230 kDa. If the abundance of the polypeptide is substantially increased in the presence of the test substance as compared to the abundance of the 240 kDa polypeptide in the absence of the test substance, The above method comprising the step of determining that the substance is a substance that inhibits the expression of the enzymatic activity of a higher animal telomerase protein; and comparing the abundance ratio of a 230 kDa polypeptide in the absence of the test substance And the polypeptide When the abundance ratio is substantially increased in the presence of the test substance, the above method including the step of determining that the test substance is a substance that activates the expression of the enzyme activity of a higher animal telomerase protein, Provided. BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 ラッ ト 'テロメラーゼ蛋白質遺伝子の c D N Aクローンの制限酵素 切断地図を示した図である。  FIG. 1 is a diagram showing a restriction enzyme cleavage map of a cDNA clone of a rat telomerase protein gene.
第 2図は、 P C Rによって増幅されたヒト ·テロメラ一ゼ蛋白質遺伝子の c D N A 断片の D N A配列と、 予想されるアミノ酸配列について、 それぞれラッ 卜のもの 又はテトラヒメナ p 8 0との相同性を比較した結果を示した図である。 図中、 R はラッ ト遺伝子、 Hはヒ ト遣伝子、 p 8 0はテ トラヒメナ p 8 0遺伝子を示す。 第 3図は、 組み換えラッ ト ·テロメラーゼ蛋白質断片に対する特異抗体をコー トしたビーズを用いて、 ヒト癌細胞 （P A— 1 ) またはラッ 卜癌細胞 （A H 6 6 F ) 抽出液由来のテロメラ一ゼ活性が免疫沈降させた結果を示した図である。 P C R と E L I S Aを組み合わせた方法を用いて検討した結果を示してあり、 縦軸はテ ロメラーゼ活性を表し、 「ビ一ズのみ」 は抗体をコートしていない陰性対照、 「P I 一 1」 は免疫前血清由来 I g Gをコートした陰性対照を示す。 「 1— 4 1 d」 と 「R 1 - 1 1 6 d」 は過免疫血清由来特異 I g Gをコートしたサンプルの結果を 示す。 Figure 2 compares the homology of the DNA sequence of the cDNA fragment of the human telomerase protein gene amplified by PCR with the predicted amino acid sequence of the rat and Tetrahymena p80, respectively. It is a figure showing a result. In the figure, R indicates the rat gene, H indicates the human gene, and p80 indicates the Tetrahymena p80 gene. Figure 3 shows telomerase derived from human cancer cell (PA-1) or rat cancer cell (AH66F) extract using beads coated with a specific antibody against the recombinant rat telomerase protein fragment. FIG. 4 shows the results of immunoprecipitation of activity. The results of studies using a method combining PCR and ELISA are shown. The expression of Romerase activity, "Beads only" indicates a negative control not coated with antibody, and "PI-11" indicates a negative control coated with IgG derived from preimmune serum. “1−41 d” and “R 1−116 d” show the results of samples coated with specific IgG derived from hyperimmune serum.
第 4図は、 ヒ ト ·テロメラ一ゼ蛋白質遺伝子の c D N Aクローンの制限酵素切 断地図を示した図である。 発明を実施するための最良の形態  FIG. 4 is a diagram showing a restriction enzyme cut map of a cDNA clone of a human telomerase protein gene. BEST MODE FOR CARRYING OUT THE INVENTION
本発明のポリベプチドの第一の態様は、 配列表の配列番号 1に記載のァミノ酸 配列で特定され、 マウス由来のテロメラーゼ蛋白質を構成するポリぺプチドに相 当するものである。 本発明により提供される上記ポリペプチドは、 配列番号 1に 記載された特定のポリぺプチドに限定されることはなく、 配列表の配列番号 1に 示されたアミノ酸配列に 1又は 2以上のアミノ酸残基による置換、 挿入、 及び/ 又は欠失が存在しており、 ヒ トを含む高等動物のテロメラ一ゼ蛋白質として実質 的に機能することができるポリペプチドも本発明の範囲に包含される。 また、 こ のようなポリぺプチドをサブュニッ トとして含む高等動物テロメラ一ゼ蛋白質も 本発明の範囲に包含される。  A first aspect of the polypeptide of the present invention is specified by the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and corresponds to a polypeptide constituting a mouse-derived telomerase protein. The polypeptide provided by the present invention is not limited to the specific polypeptide described in SEQ ID NO: 1, but may have one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. Polypeptides in which substitutions, insertions, and / or deletions by residues are present and which can substantially function as a telomerase protein of a higher animal including human are also included in the scope of the present invention. Higher animal telomerase protein containing such a polypeptide as a sub-unit is also included in the scope of the present invention.
本発明のポリべプチドの第二の態様は、 配列表の配列番号 2に記載のアミノ酸 配列で特定され、 ヒ 卜由来のテロメラーゼ蛋白質を構成するポリべプチドの部分 ポリぺプチドに相当するものである。 本発明により提供される上記ポリぺプチド は、 配列番号 2に記載された特定のポリペプチドに限定されることはなく、 配列 表の配列番号 2に示されたァミノ酸配列に 1又は 2以上のァミノ酸残基による置 換、 挿入、 及び/又は欠失が存在しており、 実質的に高等動物、 好ましくはヒ ト のテロメラ一ゼ蛋白質の部分ポリぺプチドとして機能することができるポリぺプ チドも本発明の範囲に包含される。  A second embodiment of the polypeptide of the present invention is specified by the amino acid sequence of SEQ ID NO: 2 in the sequence listing, and corresponds to a partial polypeptide of the polypeptide constituting the human-derived telomerase protein. is there. The polypeptide provided by the present invention is not limited to the specific polypeptide set forth in SEQ ID NO: 2, but may be one or more than one of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. A polypeptide which has a substitution, insertion and / or deletion by an amino acid residue and which can function as a partial polypeptide of a telomerase protein of a higher animal, preferably a human. Tides are also included in the scope of the present invention.
本発明のポリべプチドの第三の態様は、 配列表の配列番号 1 3に記載のァミノ酸 配列で特定され、 ヒ ト由来のテロメラーゼ蛋白質を構成するポリべプチドに相当 するものである。 本発明により提供される上記ポリペプチドは、 配列番号 1 3に 記載された特定のポリペプチドに限定されることはなく、 配列表の配列番号 1 3 に示されたァミ ノ酸配列に 1又は 2以上のァミノ酸残基による置換、 挿入、 及び 又は欠失が存在しており、 ヒ トを含む高等動物のテロメラ一ゼ蛋白質として実 質的に機能することができるポリべプチドも本発明の範囲に包含される。 また、 このようなポリぺプチドをサブュニッ トとして含む高等動物テロメラーゼ蛋白質 も本発明の範囲に包含される。 A third embodiment of the polypeptide of the present invention is specified by the amino acid sequence shown in SEQ ID NO: 13 in the sequence listing, and corresponds to a polypeptide constituting a human-derived telomerase protein. The polypeptide provided by the present invention is represented by SEQ ID NO: 13. It is not limited to the specific polypeptides described, but may be substituted, inserted, and / or deleted by one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 13 in the sequence listing. Is included in the scope of the present invention, wherein the polypeptide can substantially function as a telomerase protein of a higher animal including human. Higher animal telomerase proteins containing such polypeptides as a sub-unit are also included in the scope of the present invention.
本発明のポリぺプチドには、 上記の各ポリぺプチドを部分配列として含むポリ ペプチドも包含される。 例えば、 上記の各ポリペプチドに対してその発現効率を 向上させる性質を有する適宜のァミノ酸配列を結合させたポリべプチド、 上記の 各ポリべプチドに対してシグナル配列を結合させたポリべプチド、 上記ポリぺプ チドの発現を確認するために読み枠が変わらないように他の蛋白質と上記ポリべ プチドとを結合させた、 いわゆるタグ配列との融合蛋白質なども本発明の範囲に 包含される。  The polypeptide of the present invention also includes a polypeptide containing each of the above polypeptides as a partial sequence. For example, a polypeptide obtained by binding an appropriate amino acid sequence having a property of improving the expression efficiency to each of the above polypeptides, a polypeptide obtained by binding a signal sequence to each of the above polypeptides However, a fusion protein of a so-called tag sequence in which another protein is bound to the above polypeptide so that the reading frame does not change so as to confirm the expression of the above polypeptide is also included in the scope of the present invention. You.
上記のポリぺプチドのうちのいずれかをコ一 ドするヌクレオチド配列は、 いず れも本発明のヌクレオチド配列に包含される。 本発明のテロメラ一ゼ蛋白質をコ一 ドする遺伝子 （本明細書において 「テロメラーゼ蛋白質遣伝子」 という場合があ り、 テロメラ一ゼ蛋白質を構成するポリベプチドの全長又はその一部をコードす るヌクレオチド配列を意味するものとして用いる） としては、 上記の第一の態様、 第二の態様、 及び第三の態様に包含されるポリべプチドをコー ドするヌクレオチ ド配列、 好ましくは D N A配列を挙げることができる。  Any nucleotide sequence encoding any of the above polypeptides is encompassed by the nucleotide sequence of the present invention. Gene encoding the telomerase protein of the present invention (may be referred to as “telomerase protein gene” in the present specification, a nucleotide encoding the full length of the polypeptide constituting the telomerase protein or a part thereof) The term “sequence” is used to mean a nucleotide sequence encoding a polypeptide included in the first, second, and third embodiments, and preferably a DNA sequence. Can be.
本明細書において 「高等動物」 という用語は、 ヒ 卜を含む哺乳類動物を包含す る概念として用いる。 このような高等動物、 好ましくは哺乳類動物に由来するテ ロメラ一ゼ蛋白質を構成するポリぺプチドは、 それぞれ高い相同性を有している ことが期待される。 従って、 本明細書に詳細に開示されたマウス由来のテロメラー ゼ蛋白質遺伝子についてのクロ一ニング方法及びその遺伝子の情報を基にすれば、 当業者は高等動物由来のテロメラ一ゼ蛋白質を構成するポリべプチドをコ一ドす る遺伝子を容易に入手できるとともに、 その遺伝子産物を取得することが可能で あることはいうまでもない。 本発明のテロメラーゼ蛋白質遺伝子は、 例えば次のような方法によって得られ る。 本発明のテロメラーゼ蛋白質遺伝子を含有する DNAライブラリ一としては、 不死化した高等動物細胞株、 好ましくはヒ ト、 サル、 ゥマ、 ゥシ、 ヒッジ、 ィヌ、 ネコ、 ゥサギ、 ラッ ト、 マウスなどの細胞株から調製した RN Aを用いて公知の 常法により作成したプラスミ ド c DNAライブラリ一若しくはファージ c DNA ライブラリ一、 又はファージゲノ ミ ックライブラリ一などが利用できる。 As used herein, the term “higher animal” is used as a concept including mammals including humans. It is expected that polypeptides constituting the telomerase protein derived from such higher animals, preferably mammals, have high homology. Therefore, based on the method for cloning the mouse-derived telomerase protein gene disclosed in detail in the present specification and the information on the gene, those skilled in the art can understand the polymorphism of the telomerase protein derived from higher animals. It goes without saying that a gene encoding a peptide can be easily obtained and its gene product can be obtained. The telomerase protein gene of the present invention can be obtained, for example, by the following method. Examples of the DNA library containing the telomerase protein gene of the present invention include immortalized higher animal cell lines, preferably humans, monkeys, pomas, magpies, higgies, dogs, cats, puppies, rats, mice, and the like. A plasmid cDNA library, a phage cDNA library, a phage genomic library, or the like can be used which is prepared by a known conventional method using RNA prepared from the above cell line.
例えば、 ファージ c DNAライブラリ一を用いる場合には、 まず、 癌などの組 織、 あるいは不死化した高等動物細胞株を液体窒素中で粉砕し、 グァニジンイソ チオシァネー 卜水溶液等中でホモジナイズした後、 C h i r g w i nらの方法 For example, when a phage cDNA library is used, first, a tissue such as a cancer or an immortalized higher animal cell line is ground in liquid nitrogen, homogenized in an aqueous solution of guanidine isothiocyanate, and then used in Chirgwin. Their method
[B i o c h em i s t r y 1 8、 5294 - 5299 ( 1 979 ) ] に従つ て塩化セシゥム平衡密度勾配遠心法によって全 R N Aを沈澱として分離する。 R N A の分離には市販の RN A z o 1 (T e 1 T e s t社） などの抽出試薬を使用する こともできる。 RNAの分離後、 フヱノール抽出、 エタノール沈澱により全 RNA を精製し、 オリゴ （dT) セルロースカラムクロマ トグラフィーに付して精製す ることにより、 目的のテロメラ一ゼ蛋白質の mRNAを含むポリ （A) 含有 mRNATotal RNA is separated as a precipitate by cesium chloride equilibrium density gradient centrifugation according to [Biochemistry 18, 5294-5299 (11979)]. For separation of RNA, a commercially available extraction reagent such as RNAzo1 (Te1Test) can be used. After isolating the RNA, the total RNA is purified by phenol extraction and ethanol precipitation, and purified by oligo (dT) cellulose column chromatography to obtain poly (A) containing the mRNA of the target telomerase protein. Contained mRNA
(p o 1 y A+ mR A) 群を調製することができる。 (po 1 y A + mRA) groups can be prepared.
次に、 上記で調製した mRNA群に対して、 例えば、 デォキシチミジンが 1 2 個から 1 8個連結したいわゆる 0 1 i g o (d T) 配列自体、 又はネ一チヤ一 Next, for the mRNA group prepared above, for example, the so-called 01 igo (d T) sequence in which 12 to 18 deoxythymidines are linked, or
[Na t u r e 329、 836— 838 ( 1 987) ] に記載されているよう な 01 i g o (dT) 配列を含有するような合成 DNAにより構成されるプライ マ— DNAをハイプリダイズさせ、 逆転写酵素により 1本鎖 c DNAを合成する。 市販の c DNAの合成キッ トにもこれに類する配列が利用されているので、 その ような配列を用いてもよい。 その後、 市販プライマ—に対する P C R反応用の合 成 DNA (通常はキッ トに添付されているもの自体） を用いて PCR反応を行え ば良い。 また、 前記文献 [Na t u r e 329、 836— 838 ( 1 987) ] に記載されているようなプライマー DNAを用いる場合には、 その配列に相補的 な配列を設計し、 PCR反応用のプライマ一としてあらかじめ用意しておくこと が好ましい。 その後、 大腸菌の DNAポリメラ一ゼ I、 大腸菌の DNAリガーゼ、 R N a s e Hを用いて、 常法に従って 2本鎖 c DN Aを合成する。 次いで、 T 4 DN Aポリメラーゼにより c DN Aの末端を平滑ィ匕した後、 いわゆる E c o R I アダプタ一等の、 制限酵素により切断された形をなす DN Aの小断片を T 4 DNA リガーゼにより c DN A鎖の両末端に付加する。 Primer DNA composed of synthetic DNA containing the 01 igo (dT) sequence as described in [Nature 329, 836–838 (1987)] is hybridized, and reverse transcriptase is used. Synthesize single-stranded cDNA. Similar sequences are also used in commercially available cDNA synthesis kits, and such sequences may be used. Thereafter, the PCR reaction may be performed using a synthetic DNA for the PCR reaction with a commercially available primer (usually the one itself attached to the kit). When a primer DNA such as that described in the above-mentioned literature [Nature 329, 836-838 (1987)] is used, a sequence complementary to that sequence is designed and used as a primer for PCR reaction. It is preferable to prepare them in advance. Then, E. coli DNA polymerase I, E. coli DNA ligase, Using RNase H, double-stranded cDNA is synthesized according to a conventional method. Then, after terminating the end of the cDNA with T4 DNA polymerase, a small fragment of DNA, which is cut with a restriction enzyme, such as a so-called EcoRI adapter, is digested with T4 DNA ligase. Add to both ends of DNA chain.
この際、 例えば E c o R Iメチレース等の DN Aメチレースで c DN A中の制 限酵素切断点をメチル化し （例えば、 E c o R Iメチレースの場合は E c o R I 切断点のメチル化を行い） 、 制限酵素 E c o R Iの切断から c DN Aを保護して おき、 次に、 c DN Aの末端に、 いわゆる E c o R I リ ンカ一等を T 4 DNAリ ガーゼにより付加した後、 制限酵素 E c o R Iでリンカ— DN A部分のみを切断 しても同様な結果が得られる。 ベクターのクローニングサイ トとして、 例えば B a mH Iなどの他の制限酵素の切断点を選択する場合には、 前述の一連の末端 処理の操作を、 例えば B a mH Iアダプタ一の結合もしくは B a mH Iメチレー ス、 B amH I リ ンカ一、 B amH I等の組み合わせで処理にすることによって も同様な結果を得ることができる。  At this time, for example, a restriction enzyme cleavage point in cDNA is methylated with a DNA methylase such as EcoRI methylase (for example, in the case of EcoRI methylase, the EcoRI cleavage point is methylated). Protect the cDNA from cleavage of the EcoRI enzyme, add a so-called EcoRI linker to the end of the cDNA using T4 DNA ligase, and then use the restriction enzyme EcoRI. The same result can be obtained by cutting only the linker-DNA portion with the above. When selecting a cleavage site for another restriction enzyme such as BamHI as a vector cloning site, the above-described series of terminal treatment operations can be performed, for example, by binding one BamHI adapter or BamHI. Similar results can be obtained by processing with a combination of mHI methylase, BamHI linker, BamHI, and the like.
上記の様に末端処理された c DN A鎖を市販の； Iファージベクタ一、 例えば； I ZAP (P r o m e g a B i o t e c h社） 等のス ファージベクターまたは p G E M 2 (P r om e g a B i o t e c h社） 等のプラス ミ ドベクタ一の E c o R I切断部位に常法に従って揷入することにより、 組換えスファージ DNA 群または組換えプラスミ ド DN A群を製造することができる。 あるいは、 PCR 反応を用いて断片を取得する場合には、 P CR反応により増幅された DN Aの断 片の末端に特異的に 〔A〕 が付加されるために、 それに相補的な 〔T〕 を付加し たべク タ一、 例えば p C R I I ( I n V i t r o g e n社） や p T 7 (Ν ο ν a g e η社） などのベクターを用いて製造することができる。  The cDNA chain end-treated as described above is commercially available; I phage vector, for example; a phage vector such as IZAP (Promega Biotech) or pGEM2 (Promega Biotech) The recombinant sphage DNA group or the recombinant plasmid DNA group can be produced by inserting the plasmid into the EcoRI cleavage site of the plasmid vector according to a conventional method. Alternatively, when a fragment is obtained using a PCR reaction, since (A) is specifically added to the end of the DNA fragment amplified by the PCR reaction, the complementary (T) It can be produced using a vector to which, for example, pCRII (In Vitrogen) or pT7 (ogenονage η) is added.
このようにして得られた組換え λファージ DNA群を材料として、 市販のィン · ビトロ 'パッケージング 'キッ ト、 例えばギガパック · ゴールド （プロメガ · ' ィォテツク社） などを用いていわゆるィン · ビトロ 'パッケージングを行い、 組 換えスファージ DN Aを有するスファージ粒子を製造することができる。 パッケ一 ジングは、 一般には、 市販のキッ 卜の添付説明書の条件に従って行えばよい。 得 られた ス フ ァ 一 ジ粒子を常法、 例えば T . M a n i a t i s らの方法 ( 「 M o l e c u l a r C l o n i n g」 、 C o l d S p r i n g Ha r b o r L a b o r a t o r i e s 1 982年） に従い、 例えば大腸菌 などの宿主に形質導入し、 得られた形質転換体を増殖させることによってファー ジ c DN Aライブラリーを作ることができる。 また、 組換えプラスミ ド DNA群 では、 常法に従い、 例えば大腸菌などの宿主に形質転換し、 得られた形質転換体 を増殖させことによって、 プラスミ ド c DNAライブラリ一を得ることができる。 次に、 これらファージあるいは大腸菌などの形質転換体を増殖させ、 例えばジ一 ンスク リーンプラス （Du p 0 n t社） などのナイ口ン膜あるいは二トロセルロー ス膜上に移し取り、 アル力リ存在下で蛋白を除くことにより調製した； Iファージ DNAあるいはプラスミ ド DNAに対して、 後述の方法で増幅された高等動物テ 口メラ一ゼ蛋白質遺伝子の部分断片から作製した [32P] 標識プローブをハイプリ ダイズさせ、 プラークハイブリダィゼ一シヨン法によって選択し、 目的とする高 等動物テロメラ一ゼ蛋白質遺伝子をコードする c DN Aクローンの全部または一 部を得ることができる。 Using the recombinant λ phage DNA group obtained in this manner as a material, a commercially available in vitro 'packaging' kit, for example, Gigapack Gold (Promega, Inc.), is used for so-called in vitro 'Packaging can be performed to produce sphage particles having the recombinant sphage DNA. In general, packaging may be performed in accordance with the conditions in the instruction manual of a commercially available kit. Profit The obtained spheroid particles are transduced into a host such as E. coli according to a conventional method, for example, the method of T. Maniatis et al. (“Molecular Cloning”, Card Spring Laboratories, 1998). Then, a phage cDNA library can be prepared by growing the obtained transformant. In the group of recombinant plasmid DNAs, a plasmid cDNA library can be obtained by transforming a host such as Escherichia coli, for example, and growing the resulting transformant according to a conventional method. Next, these phages or transformants such as Escherichia coli are propagated, and transferred onto a Nymouth membrane or a ditrocellulose membrane such as GenScreen Plus (Dupnt), for example. A [ 32 P] -labeled probe prepared from a partial fragment of the higher animal lipoprotein melase protein gene amplified by the method described below was used for the phage DNA or plasmid DNA. The soybean is selected by the plaque hybridization method to obtain all or part of the cDNA clone encoding the higher animal telomerase protein gene of interest.
ファージ c DN Aライブラ リ一またはプラスミ ド c DNAライブラ リ一から目 的とする高等動物テロメラーゼ蛋白質遺伝子をコードする c DN Aクローンを選 択する為に用いるプローブは、 常法に従い、 例えば市販のキッ ト等を用いて調製 することができる。 例えば、 既知のテロメラ一ゼ蛋白質 （C o l 1 i n s ら、 C e 1 1、 81、 677— 686、 1995) をコ一ドする遣伝子に由来する D N A 配列や、 そのァミノ酸配列と相同性を有するアミノ酸配列をコ一ドし得る別の生 物の遺伝子の D N A配列を N a t i o n a l C e n t e r f o r B i o t e c h n o l o g y I n f o r m a t i o n (N C B I ) などの遺伝 子バンク中で TB LAS TNなどのプログラムを用いて検索し、 ある程度相同性 を有するァミノ酸配列について、 それをコ一 ドし得る DN A配列を参考にしてォ リゴヌクレオチドを合成してプローブとして用いることができる。 また、 同様な 遺伝子の D N A配列を基に P C Rプラ イ マーを設計 し、 いわゆ る d e g e n e r a t i v e PC R法によって、 より長い D N Aを取得してプロ一 ブとして用いてもよい。 この場合、 P C R法に用いる铸型には、 目的のプローブ DN Aを含む細胞由来のファージ c DNAライブラリ一、 プラスミ ド c DNAラ ィブラリ一、 または抽出した RNAから常法に従って合成した c DNAなどを用 いることができる。 Probes used to select a cDNA clone encoding the higher animal telomerase protein gene from the phage cDNA library or the plasmid cDNA library can be selected by a conventional method, for example, a commercially available kit. It can be prepared by using a method such as For example, a DNA sequence derived from a gene encoding a known telomerase protein (Col 1 ins et al., Ce 11, 81, 677-686, 1995), and homology to its amino acid sequence Using a program such as TB LAS TN in a gene bank such as the National Center for Biotechnology Information (NCBI) to search for the DNA sequence of a gene of another organism capable of encoding an amino acid sequence having An amino acid sequence having a certain degree of homology can be used as a probe by synthesizing an oligonucleotide with reference to a DNA sequence capable of coding the amino acid sequence. In addition, PCR primers are designed based on the DNA sequence of similar genes, and longer DNA is obtained by the so-called degenerative PCR method to obtain a professional DNA. It may be used as a bus. In this case, the type II used in the PCR method includes a phage cDNA library derived from cells containing the target probe DNA, a plasmid cDNA library, or cDNA synthesized from extracted RNA according to a conventional method. Can be used.
また、 上記のように遺伝子ライブラリ一をハイプリダイゼーション法でスクリ一 ニングせずに、 プローブ DNAを設計したように P C Rプライマーを設計し、 い わゆる P C R法で高等動物のテロメラーゼ蛋白質遺伝子の一部を取得することも できる。 その場合、 P C R法に用いる铸型としては、 前述のファージ c DNAラ イブラリー、 プラスミ ド c DN Aライブラリーの他、 不死化細胞より抽出した RN A から常法に従って合成した c DN Aを直接用いることができる。 P C R反応後、 反応液をァガ口—スゃポリアクリルアミ ドゲル電気泳動で解析し、 二種類のブラ イマ一により増幅される DN A断片の中から、 予想される大きさの断片を回収、 精製し、 例えば P CR— I Iの様な PC R断片を直接組み込むことができる市販 のベクターに結合し、 得られた組み換えベクターで大腸菌などの宿主を形質転換 して塩基配列の解析に用いることができる。 さらに、 得られた高等動物テロメラー ゼ蛋白質遺伝子の部分配列を基にして新たに P C Rプライマーを設計、 合成し、 高等動物テロメラ一ゼ蛋白質の配列を基に設計した P C Rプライマ一、 あるいは c DN Aを合成する際に用いるプライマ一に対して相補的な配列のプライマー、 または c DN Aの両端に付加したアンカ一配列に対応する P C R用プライマ一、 c DN Aが組み込まれたベクターに対するプライマーと、 新たに合成した上記プ ライマ一との間で DN Aの増幅を繰り返し行うことによって高等動物テロメラ一 ゼ蛋白質の全長をコー ドする遺伝子を取得することもできる。  Also, without screening the gene library by the hybridization method as described above, PCR primers were designed as if the probe DNA had been designed, and a part of the telomerase protein gene of higher animals was so-called PCR. Can also be obtained. In this case, as the type に used in the PCR method, in addition to the phage cDNA library and the plasmid cDNA library described above, and cDNA directly synthesized from RNA extracted from immortalized cells according to a conventional method, are used directly. be able to. After the PCR reaction, the reaction solution was analyzed by agarose-polyacrylamide gel electrophoresis, and fragments of the expected size were recovered from the DNA fragments amplified by the two primers. It can be purified, ligated to a commercially available vector that can directly incorporate a PCR fragment such as PCR-II, and transformed into a host such as Escherichia coli using the resulting recombinant vector for nucleotide sequence analysis. it can. Furthermore, a new PCR primer was designed and synthesized based on the obtained partial sequence of the higher animal telomerase protein gene, and a PCR primer or cDNA was designed based on the sequence of the higher animal telomerase protein. A primer having a sequence complementary to the primer used in the synthesis, or a primer for PCR corresponding to the anchor sequence added to both ends of the cDNA, and a primer for a vector in which the cDNA is incorporated; A gene encoding the full length of a higher animal telomerase protein can also be obtained by repeatedly amplifying DNA with the above-mentioned primer synthesized above.
PCR反応の終了後、 DN Aの断片をァガロース又はポリアクリルアミ ドゲル 電気泳動に付して常法に従って解析、 回収、 及び精製を行うことができる。 得ら れた精製 DN A断片を、 例えば p CR— I Iの様な P C R断片を直接組み込むこ とができるベクターに揷入し、 得られた組み換えベクターで大腸菌を形質転換し て常法に従って DN Aを調製し、 S a n g e r らのジデォキシ法 [P r o c. Na t l . A c a d. S c U SA, 74、 5463 , 1 977年] によって 目的 DNA断片の塩基配列を決定することができる。 配列の決定は AB I 373 A (アプライ ド 'バイオ ' システムズ社） の様な自動シークェンサ一によって行う こともできる。 After the completion of the PCR reaction, the DNA fragment can be subjected to agarose or polyacrylamide gel electrophoresis and analyzed, recovered, and purified according to a conventional method. The obtained purified DNA fragment is inserted into a vector into which a PCR fragment such as pCR-II can be directly incorporated, and E. coli is transformed with the obtained recombinant vector, and the DNA fragment is prepared according to a conventional method. Was prepared according to the dideoxy method of Sanger et al. [Proc. Natl. Acad. Sc USA, 74, 5463, 1977]. The base sequence of the target DNA fragment can be determined. Sequence determination can also be performed by an automated sequencer, such as ABI 373A (Applied 'Bio' Systems).
またファージライブラリーやプラスミ ドライブラリ一から得られたクローンの 場合、 一般的には、 自動シークェンサ一を用いて塩基配列を決定できる配列長に は限界があるため、 ベクターに挿入された c DN Aの全領域を一度に解析するこ とが困難な場合がある。 このような場合には、 断片を適当な制限酵素で切断した 後、 断片をゲル電気泳動で分離、 回収し、 さらに回収した断片を適宜のベクター に挿入し直すことにより解析を容易にすることができる。 このような操作 （サブ クローニング） の他、 自動シークェンサ一が決定した塩基配列の中から適当な配 列を選び、 新たなプライマ一を設計して、 そこから先を継続して解析することも できる。 このようにして決定される DN A断片の配列を互いに重なるようにつな ぎ合わせることにより、 例えば、 配列表の配列番号 1または 1 3に記載したよう な高等動物テロメラーゼ蛋白質を構成する全長ポリベプチドをコ一 ドするヌクレ ォチド配列、 又は配列表の配列番号 2に記載したような高等動物テロメラーゼ蛋 白質を構成する部分ポリべプチド配列をコードするヌクレオチド配列を決定する ことができる。  In the case of clones obtained from a phage library or a plasmid library, the length of a sequence that can be determined using an automatic sequencer is generally limited. It may be difficult to analyze all regions at once. In such a case, analysis can be facilitated by digesting the fragment with an appropriate restriction enzyme, separating and recovering the fragment by gel electrophoresis, and reinserting the recovered fragment into an appropriate vector. it can. In addition to such operations (sub-cloning), it is also possible to select an appropriate sequence from the base sequence determined by the automatic sequencer, design a new primer, and continue analysis from there. . By joining the sequences of the DNA fragments thus determined so as to overlap each other, for example, a full-length polypeptide constituting the higher animal telomerase protein as described in SEQ ID NO: 1 or 13 in the sequence listing can be obtained. The nucleotide sequence encoding the nucleotide sequence to be encoded or the partial polypeptide sequence constituting the higher animal telomerase protein as described in SEQ ID NO: 2 in the Sequence Listing can be determined.
本発明のヌクレオチドには DN A及び RN Aが包含されるが、 配列表の配列番 号 1、 1 3、 及び 2には、 それぞれ、 ラッ ト及びヒ 卜由来テロメラーゼ蛋白質を 構成する全長ポリぺプチドをコ一ドする DN A配列、 並びにヒ ト由来テロメラ一 ゼ蛋白質を構成する部分ポリべプチド配列をコードする DN A配列を好ましい態 様として記載した。 本発明のヌクレオチドには、 上記の配列番号 1、 1 3、 及び 2により特定される DN A配列のほか、 それらがコードするポリぺプチドのァミ ノ酸配列に対して 1又は 2以上のアミノ酸残基による置換、 挿入、 及び/又は欠 失が導入されており、 実質的に高等動物テロメラーゼ蛋白質の全長又は部分ポリ ぺプチドとして機能するポリベプチドをコ一ドするヌクレオチドが包含される。 このようなアミノ酸残基の置換、 挿入、 及びノ又は欠失等によるアミ ノ酸配列の 改変は、 例えば、 Nu c l e i c Ac i d Re s. , Vo l . 10, 6487 — 6500 (1 982) 、 Me t h o d s i n E n z ymo l . , Vo l . 2 1 7, 2 1 8 - 227 ( 1 9 9 3 ) , 同 V o し 2 1 7, 27 0 - 27 8 ( 1 993 ) 等に記載の部位特異的変異技術により行うことができるが、 これら の方法に限定されることはなく、 当業者に利用可能なものであればいかなる方法 を用いてもよい。 The nucleotides of the present invention include DNA and RNA, and SEQ ID NOs: 1, 13, and 2 in the sequence listing show full-length polypeptides constituting rat and human derived telomerase proteins, respectively. And a DNA sequence encoding a partial polypeptide sequence constituting a human-derived telomerase protein have been described as preferred embodiments. The nucleotides of the present invention include, in addition to the DNA sequences specified by SEQ ID NOs: 1, 13, and 2 above, one or more amino acids relative to the amino acid sequence of the polypeptide encoded by them. Residue substitutions, insertions, and / or deletions have been introduced and include nucleotides that encode a polypeptide that functions substantially as a full-length or partial polypeptide of a higher animal telomerase protein. Modification of the amino acid sequence by such substitution, insertion, and deletion or deletion of amino acid residues can be performed, for example, by the methods described in Nucleic Acid Res., Vol. 10, 6487. — 6500 (1 982), Methodsin Enzymo l., Vo l. 2 1 7, 2 18-227 (1993), and Vo 2 1 7, 27 0-278 (1993) ) And the like, but the method is not limited to these methods, and any method available to those skilled in the art may be used.
以上のようにして得られた高等動物テロメラーゼ蛋白質遺伝子 DN Aの少なく とも一部分をハイブリダィゼ一シヨン 'プローブまたは PCRプライマーとして 用いることにより、 他の種の高等動物テロメラーゼ蛋白質遺伝子を同様な方法で 単離することができる。 例えば、 テ トラヒメナ · テロメ ラーゼ蛋白質 （p 80) とラッ ト ·テロメラ一ゼ蛋白質のァミノ酸配列の相同性の最も高い部分に由来す る PCRプライマーを用いて、 対応する部分のヒ 卜 'テロメラーゼ蛋白質のァミ ノ酸配列を明らかすることも可能であり、 さらにはその全長 c DN Aを得ること もできる。  Using at least a portion of the higher animal telomerase protein gene DNA obtained as described above as a hybridization probe or PCR primer, a higher animal telomerase protein gene of another species is isolated in a similar manner. be able to. For example, using a PCR primer derived from the highest homology of the amino acid sequence of the Tetrahymena telomerase protein (p80) and the rat telomerase protein, the corresponding portion of the human telomerase protein is used. The amino acid sequence can be determined, and its full-length cDNA can also be obtained.
上記のようにして得られる高等動物テロメラーゼ蛋白質遺伝子 DN A又はその DNA断片は、 その両端あるいはどちらか一端を改変し、 またはそれ自体で、 公 知の発現べクタ一にそれ自体公知の方法でプロモーターの下流に挿入することが でき、 このようにして製造される遣伝子発現用の組み換えベクターを、 大腸菌、 酵母、 動物細胞宿主等、 公知の細胞中にそれ自体公知の方法により導入して形質 転換体を製造することができる。  The higher animal telomerase protein gene DNA or its DNA fragment obtained as described above may be modified at both ends or at one end thereof, or by itself, in a known expression vector in a manner known per se. The recombinant vector for gene expression thus produced is introduced into a known cell such as Escherichia coli, yeast, or an animal cell host by a method known per se to transform the gene. A transformant can be manufactured.
本発明の高等動物テロメラ一ゼ蛋白質の産生方法につき詳細に説明すると、 発 現用ベクターとしては、 上記のようにして得られた高等動物テロメラ一ゼ蛋白質 をコードする DN Aを転写できる位置にプロモータ一を含有しているものが使用 される。  The method for producing a higher animal telomerase protein of the present invention will be described in detail. As an expression vector, a promoter is located at a position at which DNA encoding the higher animal telomerase protein obtained as described above can be transcribed. Is used.
高等動物テロメラーゼ蛋白質の工業的生産のためには、 安定した宿主一ベクター 系を構築すること、 さらに生物学的に活性を有する高等動物テロメラーゼ蛋白質 を発現しうる系を用いる必要がある。 高等動物テロメラーゼ蛋白質は比較的大き な蛋白質であり、 そのリフォールディ ングが生理活性の獲得に重要である。 一般 的には、 リフオールディ ングを考慮した場合、 宿主として動物細胞を用いること が有利である。 高等動物テロメラ—ゼは、 数種の蛋白質及び R N Aサブュニッ ト からなる複合体として存在する可能性があり、 生理活性のある高等動物テロメラー ゼとして組み換え体から精製する場合には、 導入する高等動物テロメラ一ゼ蛋白 質の由来する生物種と宿主細胞の由来する生物主の一致することが好ましい。 も つとも、 高等動物テロメラーゼ蛋白質を大腸菌で生産させた後、 活性を有する複 合体として i n V i t r oで他の構成成分と再構成することが可能であること はいうまでもない。 For industrial production of higher animal telomerase proteins, it is necessary to construct a stable host-vector system and to use a system capable of expressing a biologically active higher animal telomerase protein. Higher animal telomerase proteins are relatively large proteins, and their refolding is important for obtaining biological activity. Generally, animal cells should be used as hosts when refolding is considered. Is advantageous. The higher animal telomerase may exist as a complex composed of several kinds of proteins and RNA subunits. When purified from a recombinant as a biologically active higher animal telomerase, the higher animal telomerase to be introduced is used. It is preferred that the species from which the protein is derived and the species from which the host cell is derived coincide. Needless to say, after producing a higher animal telomerase protein in Escherichia coli, it can be reconstituted with other components in vitro as an active complex.
動物細胞としては、 例えば C H O細胞 （生物種：ハムスター） 、 C O S細胞 （生 物種：サル） 、 N I H 3 T 3細胞 （生物種：マウス） 、 R a t— 1 (生物種： ラ ッ ト） 細胞、 V A— 1 3 (生物種： ヒ ト） 細胞等が挙げられる。 これらの細胞を 宿主とした発現用プラスミ ドは、 プロモーターとしては S V 4 0プロモータ一由 来またはウィルス遺伝子由来のプロモーターが好ましい。 この下流に高等動物テ 口メラ一ゼ蛋白質遺伝子を 5 ' 側から挿入する。 また高等動物テロメラ一ゼ蛋白 質の生産量を上げるために、 高等動物テロメラーゼ蛋白質遺伝子を 5 ' 側から 2 〜 3個つなげたものを揷入してもよいし、 各高等動物テロメラーゼ蛋白質遺伝子 の 5 ' 側に S V 4 0などのプロモータ一を挿入したものを 2〜 3個つなげてもよ い。 この高等動物テロメラーゼ蛋白質遣伝子の下流にポリアデニル化部位を含む ことが好ましく、 例えば S V 4 0 D N A、 /3—グロビン遺伝子またはメ夕ロチォ ネィン遺伝子由来のものを用いることができる。  Examples of animal cells include CHO cells (species: hamster), COS cells (species: monkey), NIH3T3 cells (species: mouse), Rat-1 (species: rat), VA-13 (organism: human) Cells and the like. The expression plasmid using these cells as a host is preferably a promoter derived from the SV40 promoter or a viral gene. Into this downstream, a higher animal lipomelase protein gene is inserted from the 5 'side. In order to increase the production of higher animal telomerase protein, two or three higher animal telomerase protein genes may be inserted from the 5 'side, or each higher animal telomerase protein gene You may connect two or three units with a promoter such as SV40 inserted on the 'side. It is preferable to include a polyadenylation site downstream of the higher animal telomerase protein gene. For example, those derived from the SV40 DNA, / 3-globin gene or the metrotionein gene can be used.
このような発現べクタ一は、 例えば C H 0細胞などの動物細胞に形質転換した 際の選択マーカ一を有していてもよい。 選択マーカーを用いる場合には、 例えば、 メ ト トレキセ一ト耐性を与える D H F R遺伝子、 ネオマイシン誘導体 G— 4 1 8 耐性遺伝子などを用いることができる。 各耐性遺伝子の 5 ' 側に例えば S V 4 0 由来のプロモーターが挿入されており、 各耐性遺伝子の 3 ' 側にポリアデニル化 部位が含まれていることが好ましい。 高等動物テロメラ一ゼ蛋白質の発現べクタ一 に対してこれらの耐性遺伝子を挿入する場合、 高等動物テロメ ラ一ゼ蛋白質遺伝 子のポリアデニル化部位下流に挿入すればよい。 また、 発現ベクターは形質転換 体の選択マーカ一を有していなくてもよい。 この場合には、 高等動物テロメラー CT/JP97/0204 ゼ蛋白質の発現ベクターと共に形質転換体選択のマーカーを有するベクタ—、 例 えば p SV 2 n e o、 p SV 2 g p t、 pMT V d h f rなどを用いて二重形質 転換することが好ましい。 Such an expression vector may have a selection marker when transformed into an animal cell such as a CH0 cell. When a selectable marker is used, for example, a DHFR gene that provides methotrexate resistance, a neomycin derivative G—418 resistance gene, or the like can be used. It is preferable that, for example, a promoter derived from SV40 is inserted on the 5 'side of each resistance gene, and a polyadenylation site is included on the 3' side of each resistance gene. When these resistance genes are inserted into the expression vector of the higher animal telomerase protein, they may be inserted downstream of the polyadenylation site of the higher animal telomerase protein gene. Further, the expression vector may not have the transformant selection marker. In this case, the higher animal telomere Double transformation is preferably performed using a vector having a marker for transformant selection together with an expression vector for the CT / JP97 / 0204 protein, for example, pSV2neo, pSV2gpt, pMTV dhfr and the like.
上記の高等動物テロメラーゼ蛋白質発現べクタ—、 またはそれに加えて形質転 換体選択マーカーを有するベクターにより形質転換した動物細胞を選択するため には、 該選択マーカ一の発現による表現形質を利用することができる。 さらに、 高等動物テロメラーゼ蛋白質の発現量の上昇を目的として、 高等動物テロメラ一 ゼ蛋白質の発現が確認された細胞に対し、 選択マ—力—を変更して形質転換を繰 り返してもよい。 発現べクタ一に使用されるプラスミ ドベクターの具体例として は、 S V 40初期プロモータ一、 ゥサギの /3—グロビン遺伝子に由来するスプラ ィス配列 D N A、 ゥサギの /9一グロビン遺伝子からのポリアデニル化部位、 S V 40 初期領域からのポリアデニル化部位、 並びに P BR 322由来の複製開始点およ びアンピシリ ン耐性遗伝子を含有する pKCR (P r o c. N a t 1. Ac a d. S c i . U SA, 78、 1 528、 （ 1 98 1 ) ) などが挙げられる。  In order to select an animal cell transformed with the above-mentioned vector for expressing a higher animal telomerase protein or a vector having a transformant selectable marker in addition thereto, it is necessary to utilize a phenotype by expression of the selectable marker. it can. Further, for the purpose of increasing the expression level of the higher animal telomerase protein, the cells in which the expression of the higher animal telomerase protein has been confirmed may be repeatedly transformed by changing the selection ability. Specific examples of the plasmid vector used in the expression vector include the SV40 early promoter, the splice sequence DNA derived from the ゥ 3 / 3-globin gene of the egret, and the polyadenylation from the 99 / globin gene of the egret. Site, a polyadenylation site from the early region of SV40, and a pKCR containing the origin of replication from PBR322 and the ampicillin resistance gene (Proc. U.S.A., 78, 1528, (1981)) and the like.
発現べクタ一の動物細胞への移入はリ ン酸カルシウムや c a t i o n i c Expression vectors can be transferred to animal cells using calcium phosphate or cationic.
1 i p i dを DNAのキャリアとして用いる トランスフヱクション法が一般的で ある。 形質転換された動物細胞の培養は、 常法により浮遊培養または付着培養で 行うことができる。 培地としては、 MEM、 R PM I 1 640などを用い、 5〜A transfection method using 1 ip id as a DNA carrier is common. Culture of the transformed animal cells can be performed by suspension culture or adherent culture according to a conventional method. Use MEM, RPMI 1640, etc.
1 0 %血清存在下もしくは適当量のィンシユリ ン、 デキサメサゾン、 卜ラ ンスフ ユリ ンの存在下で培養を行うか、 又は無血清下で培養を行うことができる。 高等 動物テロメラーゼ蛋白質を発現している動物細胞中には高等動物テロメラーゼ蛋 白質が大量に存在していると考えられるので、 この形質転換体の培養物から得た 蛋白抽出液を用いて高等動物テロメラーゼ蛋白質の分離精製を行うことが可能で ある。 生産された高等動物テロメラーゼ蛋白質を含む培養上清は各種クロマトグ ラフィ 一、 例えば、 へパリ ンセファロースもしくはブルーセファロース等を用い たクロマトグラフィーにより精製可能である。 The culture can be performed in the presence of 10% serum or in the presence of an appropriate amount of insulin, dexamethasone, or transfusion, or in the absence of serum. Since it is considered that a large amount of the higher animal telomerase protein is present in the animal cells expressing the higher animal telomerase protein, the higher animal telomerase is used by using a protein extract obtained from a culture of the transformant. It is possible to separate and purify proteins. The culture supernatant containing the produced higher animal telomerase protein can be purified by chromatography using various chromatographies, for example, heparin sepharose or blue sepharose.
また大腸菌、 枯草菌等の微生物を宿主として用いるときには、 発現ベクターは プロモータ一、 リボゾーム結合 （SD) 配列、 高等動物テロメラ—ゼ蛋白質遺伝 子、 転写終結配列、 およびプロモーターを制御する遺伝子を含むことが好ましい。 プロモーターとしては、 大腸菌、 ファージ等由来のもの、 例えばトリブトファン 合成酵素 （ t r p) 、 ラク トースオペ口ン （ 1 a c) 、 スファージ P L、 PR、 T 5ファ一ジの初期遺伝子のプロモータ一である Ρ 25、 Ρ 26プロモーター等 が挙げられる。 また、 これらは例えば p a cプロモータ一 [A g r i c. B i o l. C h e m. 52、 983— 988、 1 988年] のように独自に改変、 設計され た配列でも良い。 When a microorganism such as Escherichia coli or Bacillus subtilis is used as a host, the expression vector is a promoter, a ribosome binding (SD) sequence, a higher animal telomerase protein gene. It preferably contains a gene that controls a promoter, a transcription termination sequence, and a promoter. Examples of the promoter include those derived from Escherichia coli and phage, such as tributophan synthase (trp), lactose operin (1 ac), sphage PL, PR, and the promoter of the early gene of T5 phage. Ρ 26 promoters and the like. These may also be sequences that have been independently modified and designed, for example, the pac promoter [Agric. Biol. Chem. 52, 983-988, 1988].
リボゾ一ム結合配列としては、 大腸菌、 ファージ等由来のものでもよいが、 D N A 合成により作成した 1 6 Sリボソーム RNAの 3' 末端領域に相補的な配列を 4 塩基以上連続してもつコンセンサス配列を持ったものでもよい。 転写終結配列は 必ずしも必要ではないが、 /0非依存性のもの、 例えばリボプロテイ ンターミネ一 ター、 t r pオペロンターミネーター等を有している方が好ましい。  The ribosome binding sequence may be derived from Escherichia coli, phage, etc., but a consensus sequence having 4 or more consecutive bases complementary to the 3′-terminal region of 16S ribosomal RNA prepared by DNA synthesis may be used. You may have it. The transcription termination sequence is not necessarily required, but preferably has a / 0-independent sequence, such as a riboprotein terminator or a trp operon terminator.
発現に必要なこれらの因子の発現プラスミ ド上での配列順序は、 例えば、 5' 上流から、 プロモータ一、 SD配列、 高等動物テロメラーゼ蛋白質遺伝子、 転写 終結因子の順であることが望ましい。 また発現べクタ一上の SD配列と高等動物 テロメラーゼ蛋白質遺伝子とのュニッ トを複数個同方向に揷入することにより、 ベクター上の転写単位のコピー数を増加させる方法 （特開平 1— 95798号公 報などに記載の方法） を用いることもできる。  The sequence of these factors required for expression on the expression plasmid is preferably, for example, in the order of 5 'upstream, promoter, SD sequence, higher animal telomerase protein gene, and transcription termination factor. Also, a method of increasing the copy number of a transcription unit on a vector by inserting a plurality of units of the SD sequence on the expression vector and the higher animal telomerase protein gene in the same direction (Japanese Patent Laid-Open No. 1-95798) The method described in public announcements) can also be used.
発現した高等動物テロメラーゼ蛋白質又はその部分ポリペプチドを大腸菌など の形質転換体からの簡便に回収、 精製するために種々のァフィ二ティ一カラムを 利用することができる。 例えば、 ヒスチジンが 6個以上並んだアミノ酸配列、 い わゆるヒスチジンタグを有する蛋白質がキレ一トカラムに結合する性質を利用し て、 プロモーターの下流に例えばヒスチジンが 6個以上並んだアミノ酸配列をコー ドする DN Aを配置し、 さらにその下流に高等動物テロメラ一ゼ蛋白質遺伝子を 結合することにより、 ヒスチジンタグを含む高等動物テ口メラ一ゼ蛋白質又はそ の部分ポリぺプチドを発現させることができ、 発現した高等動物テロメ ラ一ゼ蛋 白質又はその部分ポリべプチドをキレートカラムにより容易に精製することがで きる。 さらに、 ヒスチジンタグと高等動物テロメラーゼ蛋白質を構成するポリベプチ ド又はその部分ポリペプチドとの間に、 例えばトロンビン、 TEVプロテア一ゼ、 又は第 X因子などのプロテア一ゼにより特異的に切断されるポリぺプチド配列を 組み込み、 キレートカラム精製後のポリぺプチドを対応のプロテア一ゼで処理す ることにより、 天然型の高等動物テロメラ一ゼ蛋白質又はその部分ポリぺプチド を回収することができる。 プロテアーゼによる切断後は H P L C等により分離、 精製することができる。 Various affinity columns can be used to easily recover and purify the expressed higher animal telomerase protein or its partial polypeptide from a transformant such as Escherichia coli. For example, an amino acid sequence in which six or more histidines are arranged downstream of a promoter is coded using the property of binding an amino acid sequence in which six or more histidines are arranged, that is, a protein having a histidine tag to a chelate column. A telomerase protein gene containing a histidine tag or a partial polypeptide thereof can be expressed by arranging a DNA to be expressed and further binding a higher animal telomerase protein gene downstream thereof. The expressed higher animal telomerase protein or its partial polypeptide can be easily purified using a chelate column. Furthermore, a polypeptide specifically cleaved by a protease such as thrombin, TEV protease, or factor X between the histidine tag and the polypeptide constituting the higher animal telomerase protein or a partial polypeptide thereof. By incorporating the peptide sequence and treating the polypeptide after purification by a chelate column with a corresponding protease, a natural higher animal telomerase protein or its partial polypeptide can be recovered. After cleavage with protease, it can be separated and purified by HPLC or the like.
上記の他、 発現べクタ一として使用できるものとして、 p UA I 2 (特開平 1 - 95798号公報） や市販の pKK 233— 2 (P h a r m a c i a社） 等を 挙げることができる。 また、 日本住血吸虫由来グルタチオン— S— トランスフエ ラーゼとの融合蛋白として発現させる発現べク夕一として p G E Xシ リ ーズ (P h a r m a c i a社） を利用することができ、 ヒスチジン配列を利用した精 製が可能なベクターとして P P r o E X— I (G i b c o BRL) を用いるこ とができる。 宿主の形質転換法は、 常法に従い行うことができる。 また、 昆虫細 胞としては、 例えば I n V i t r o g e n社のバキュロウィルス発現キッ トであ る マ ッ ク スノ ッ ク （ M A X B A CTm、 B A C U L O V I R U S EXPRE S S I ON SYSTEM MANUAL VERS I ON 1. 4) のマニュアルに従い、 このキッ 卜を使用することができる。 この時、 発現量を上 げるためにポリヘドリ ンのプロモーターから開始コ ドンまでの距離を変えること が好ましい。 In addition to the above, pUAI2 (Japanese Patent Application Laid-Open No. 1-95798), commercially available pKK233-2 (Pharmacia), and the like can be used as expression vectors. In addition, pGEX series (Pharmacia) can be used as an expression vector for expression as a fusion protein with glutathione-S-transferase derived from Schistosoma japonicum, and purification using a histidine sequence can be performed. PP ro EX-I (Gibco BRL) can be used as a vector that can be produced. Transformation of the host can be performed according to a conventional method. As insect cells, for example, according to the manual of Max Knock (MAXBAC Tm , BACULOVIRUS EXPRE SSI ON SYSTEM MANUAL VERS I ON 1.4), which is a baculovirus expression kit from Invitrogen, Inc. This kit can be used. At this time, it is preferable to change the distance from the polyhedrin promoter to the initiation codon in order to increase the expression level.
形質転換体の培養は、 当業者に利用可能な常法に従って行うことができる。 培 養温度としては、 28 °C~42 °Cが適当である。 ラク トースォペロン （ l a c) のプロモーターを利用する場合は、 菌体培養液の 600 nmの波長における吸光 度がおよそ 0. 5になったところで、 終濃度が 1 mM程度になるように I PTG を加えて発現誘導を行うことが必要である。  Culturing of the transformant can be performed according to a conventional method available to those skilled in the art. An appropriate cultivation temperature is 28 ° C to 42 ° C. When using the lactose operon (lac) promoter, add IPTG so that the final concentration will be about 1 mM when the absorbance of the bacterial cell culture at 600 nm becomes about 0.5. It is necessary to induce expression.
上記方法で単離 ·精製された高等動物テロメラ一ゼ蛋白質又はその部分ポリベ プチドを用いて、 サル、 ヒッジ、 ゥサギ、 ラッ ト、 マウスなどの哺乳類動物を免 疫することができ、 高等動物テロメラ一ゼ蛋白質を特異的に認識するポリクロー ナルまたはモノクローナル抗体を作製することができる。 その特異性の検討には、 高等動物テ口メラーゼ蛋白質遺伝子を含む発現べクタ一を導入した形質転換体の 培養液又は遺伝子産物の抽出液を用いることができる。 Using the higher animal telomerase protein or its partial polypeptide isolated and purified by the above method, mammals such as monkeys, sheep, rabbits, rats and mice can be immunized. Polyclones that specifically recognize zeoproteins Null or monoclonal antibodies can be made. In order to examine the specificity, a culture solution of a transformant or an extract of a gene product into which an expression vector containing a higher animal Tesla merase protein gene has been introduced can be used.
このような高等動物テロメラーゼ蛋白質又はその部分ポリべプチドに特異的な ポリクローナルまたはモノクロ一ナル抗体を固定化したァフィ二ティカラム用い て、 テロメラーゼ活性を有する不死化細胞株または形質転換体の抽出液から、 高 等動物テロメラーゼ複合体を濃縮 '精製することができる。 また、 テロメラーゼ 活性を有する真核動物不死化細胞株に対して、 高等動物テ口メラ一ゼ蛋白質とグ ルタチオン一 S— トランスフェラーゼ、 ポリ · ヒスチジンなどのいわゆる 「タグ 配列」 との融合蛋白質を発現するベクターを導入し、 得られた形質転換体の抽出 液をグルタチォン 'セファロ一ス （Ph a rma c i a社） 、 ニッゲル · N T A · ァガロース （Q I AGEN社） 等の 「タグ配列」 に特異的に結合するリガンドを 固定化したカラムに付して精製することにより、 高等動物テロメラーゼ複合体を 濃縮 ·精製することができる。 以上のような方法で得られた高等動物テロメラー ゼ複合体は、 高活性の高等動物テロメラ一ゼとして阻害剤の評価などに利用する ことができるほか、 新規な構成成分の解析、 及びそれらの単離 ·精製の材料とし て用いることが可能である。  Using an affinity column immobilized with a polyclonal or monoclonal antibody specific for such a higher animal telomerase protein or a partial polypeptide thereof, from an extract of an immortalized cell line or a transformant having telomerase activity, The higher animal telomerase complex can be concentrated and purified. In addition, it expresses a fusion protein of a higher animal teliptic melanase protein and a so-called “tag sequence” such as glutathione-1S-transferase and polyhistidine in an eukaryotic immortalized cell line having telomerase activity. The vector is introduced, and the resulting extract of the transformant is specifically bound to a “tag sequence” such as glutathione 'Sepharose (Pharmacia), Nigel NTA agarose (QIAGEN), etc. By purifying the ligand by immobilizing it on a column on which the ligand is immobilized, the higher animal telomerase complex can be concentrated and purified. The higher animal telomerase complex obtained by the above method can be used as a highly active higher animal telomerase for the evaluation of inhibitors, etc., as well as the analysis of novel components and the simple analysis of those components. It can be used as a material for separation and purification.
また、 いわゆる 「ツー ·ハイブリ ツ ド （Two— h y b r i d) 法」 に従い、 酵母を含む様々な形質転換体を用いて、 高等動物テロメラーゼ蛋白質に物理的に 強固に結合する蛋白質をコードする遣伝子を単離 ·同定することができる。 この ような目的のためには、 例えば C 1 o n t e c h社の 「Ma t c h Ma k e r キッ ト」 などを用いることができる。  In addition, according to the so-called “Two-hybrid method”, a gene encoding a protein that physically binds tightly to a higher animal telomerase protein is obtained using various transformants including yeast. Isolation · Can be identified. For such a purpose, for example, a “Mtch Maker Kit” of C1ontech can be used.
上記の高等動物テ口メラーゼ蛋白質の特異抗体を用いることにより上記遺伝子 の発現の程度を蛋白質レベルで観測することができ、 核酸プローブや PC Rブラ イマ一を用いて遺伝子レベルでの発現状況を観測することができる。 このような 方法によれば、 癌細胞の検出、 並びにテロメラ—ゼ活性の変化に起因する疾患及 びテロメラーゼ活性の変化を伴う疾患の診断が可能である。 例えば、 患者から分 離 ·採取された試料を適宜の方法で抽出した後、 特異抗体を用いた E L I SA法 もしくはウェスタン ' プロッ ト法、 核酸プローブを用いたサザンまたはノザン · プロッ ト法、 またはォリゴヌクレオチド · プライマ一を用いた P C R法により判 定を行うことができる。 従って、 本発明のポリべプチドを特異的に認識すること ができる抗体又は本発明のヌクレオチド配列の一部又は全部に相補的に結合可能 なヌクレオチド配列を含む核酸プローブは、 癌細胞の検出試薬、 又は癌診断用の 医薬組成物の有効成分として有用である。 The expression level of the above gene can be monitored at the protein level by using the above specific antibody for the higher animal tepa-merase protein, and the expression level at the gene level can be monitored using a nucleic acid probe or a PCR primer. can do. According to such a method, it is possible to detect cancer cells and diagnose a disease caused by a change in telomerase activity and a disease accompanied by a change in telomerase activity. For example, after extracting a sample separated and collected from a patient by an appropriate method, the ELI SA method using a specific antibody Alternatively, the determination can be performed by the Western 'plot method, the Southern or Northern plot method using a nucleic acid probe, or the PCR method using an oligonucleotide primer. Accordingly, an antibody capable of specifically recognizing the polypeptide of the present invention or a nucleic acid probe comprising a nucleotide sequence capable of binding complementarily to part or all of the nucleotide sequence of the present invention can be used as a reagent for detecting cancer cells, Or it is useful as an active ingredient of a pharmaceutical composition for cancer diagnosis.
なお、 後述の実施例で示したように、 ラッ 卜由来のテロメラ一ゼ蛋白質には、 S D S —ポリアクリルアミ ド電気泳動法による分子量が約 2 4 0 k D aの不活性 型ポリペプチ ドと、 S D S —ポリアク リルアミ ド電気泳動法による分子量が約 2 3 0 k D aの活性型ポリべプチドの存在が確認されている。 また、 約 2 4 0 k D a の不活性型ポリぺプチドが最初に発現し、 約 2 3 0 k D aの活性型ポリぺプチド に変換される機構の存在が証明されている。 従って、 他の高等動物においても、 同様な不活性型及び活性型のポリぺプチドが存在しており、 不活性型ポリぺプチ ドから活性型ポリベプチドに変換される同様な機構が存在していることが当業者 に自明である。 これらの分子種 （サブュニッ ト） はいずれも本発明の範囲に包含 される。  As shown in the examples below, rat-derived telomerase protein includes an inactive polypeptide having a molecular weight of about 240 kDa by SDS-polyacrylamide electrophoresis, The presence of an activated polypeptide with a molecular weight of about 230 kDa has been confirmed by SDS-polyacrylamide electrophoresis. In addition, the existence of a mechanism whereby an inactive polypeptide of about 240 kDa is first expressed and converted to an active polypeptide of about 230 kDa has been proved. Therefore, similar inactive and active polypeptides are present in other higher animals, and a similar mechanism exists for converting inactive to active polypeptides. It is obvious to those skilled in the art. All of these molecular species (subunits) are included in the scope of the present invention.
上記の活性型ポリぺプチド及び不活性型ポリぺプチドの存在比を測定すること により、 テロメラーゼの活性化機構に作用する物質をスクリーニングすることが できる。 このスクリーニング方法は、 典型的には、 被験物質を投与した後の高等 動物の組織や細胞、 又は培養系において被験物質の存在下で培養を行った高等動 物の組織や細胞に含まれる上記の活性型ポリべプチド及び不活性型ポリぺプチド の存在比を測定し、 被験物質の非存在下での存在比と比較する工程を含んでいる。 分子量の測定は、 一般的には、 S D S—ポリアクリルアミ ド電気泳動法で行えば よい。  By measuring the abundance ratio of the active polypeptide and the inactive polypeptide, it is possible to screen for a substance that acts on the telomerase activation mechanism. This screening method is typically carried out in tissues or cells of a higher animal after administration of a test substance, or tissues or cells of a higher animal cultured in the presence of the test substance in a culture system. Measuring the abundance ratio of active and inactive polypeptides and comparing the abundance ratio in the absence of the test substance. Generally, the molecular weight may be measured by SDS-polyacrylamide electrophoresis.
例えば、 被験物質と接触していない細胞や組織に含まれるテロメラーゼ蛋白質 のサブュニッ トの分子量を S D S —ポリアク リルアミ ド電気泳動で測定し、 約 2 4 0 k D aのポリぺプチドと約 2 3 0 k D aのポリぺプチドとの存在比をあら かじめ調べておく。 つぎに、 被験物質を投与し、 又は被験物質の存在下で培養を 行うことにより被験物質と接触させた細胞や組織に含まれるテロメラーゼ蛋白質 のサブュニッ 卜の分子量を同様に測定し、 約 24 0 k D aのポリぺプチドと約 23 0 kD aのポリべプチドの存在比を測定する。 被験物質と接触した細胞や組 織において約 24 0 kD aのポリべプチドの存在比が非接触時の場合に比べて実 質的に増加していれば、 被験物質はテロメラーゼの活性化機構を阻害すると判定 できる。 一方、 約 230 kD aの蛋白質の存在比が増加していれば、 被験物質は テロメラ一ゼの活性化を促進すると判定できる。 このようにしてテロメラーゼの 活性化機構に作用することが確認された物質も本発明の範囲に包含されることを 理解すべきである。 For example, the molecular weight of a telomerase protein subunit contained in cells or tissues that have not been contacted with the test substance is measured by SDS-polyacrylamide electrophoresis, and a polypeptide of about 240 kDa and about 230 Check the abundance ratio of kDa to the polypeptide in advance. Next, administer the test substance or culture in the presence of the test substance. By doing so, the molecular weight of the telomerase protein subunit contained in the cells and tissues contacted with the test substance was measured in the same manner, and the presence of about 240 kDa polypeptide and about 230 kDa polypeptide was observed. Measure the ratio. If the abundance of the approximately 240 kDa polypeptide is substantially increased in cells or tissues that have come into contact with the test substance as compared to the case without contact, the test substance will activate the mechanism of telomerase activation. It can be determined that it inhibits. On the other hand, if the abundance of the approximately 230 kDa protein is increased, it can be determined that the test substance promotes telomerase activation. It should be understood that substances confirmed to act on the telomerase activation mechanism in this way are also included in the scope of the present invention.
実施例 Example
以下、 本発明を実施例によりさらに具体的に説明するが、 本発明の範囲は以下 の実施例に限定されることはない。  Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to the following Examples.
実施例 1 ： ラッ ト ·テロメラーゼ蛋白質遺伝子の取得 Example 1: Obtaining rat telomerase protein gene
(1) テ トラヒメナ ' テロメラーゼ 'サブュニッ ト p 80遺伝子に相同な遺伝子の検 索  (1) Search for genes homologous to the Tetrahymena 'telomerase' subunit p80 gene
I n t e r n e t にて、 N a t i o n a l C e n t e r f o r B i o t e c h n o l o g y I n f o rma t i o nの h om e p a g eに アクセスし、 T B L A S TNプログラムにて、 テ 卜ラヒメナ · テロメラ一ゼ . サ ブュニッ ト p 80のアミノ酸配列に相同なァミノ酸配列をコ一ドし得る DNA配 列を検索した。 その結果、 E x p r e s s i o n S e q u e n c e T a g Access the National C enterfor Biotechnology Informa tion homepage on the Internet, and use the TBLAS TN program to obtain amino acids homologous to the amino acid sequence of Tetrahymena telomerase. Sabunit p80. A DNA sequence capable of coding the sequence was searched. As a result, E x pr e s s i o n S e q u e n c e T ag
(E ST) DNA配列のデータバンクに登録された、 ラッ ト P C 1 2細胞由来の 機能不明な mRNA配列に相補的な DNA配列 （配列表の配列番号 3) 、 p 80 の一部のア ミ ノ酸配列に弱い相同性 （H i g h S c o r e ： 9 4、 P r o b a b i l i t y : !!. 7 x 1 0 "3) を示すァミノ酸配列 （下記表 1 ： ラッ ト c DNA) をコードし得ることがわかった （表中、 アミノ酸は 1文字表記で示 し、 Xは終始コ ドンを表す） 。 表 1 p 80 (N末端側） AVII NE_L (EST) A DNA sequence (SEQ ID NO: 3 in the sequence listing) registered in the DNA sequence data bank that is complementary to the mRNA sequence of unknown function derived from rat PC12 cells, It is possible to encode an amino acid sequence (Table 1 below: rat cDNA) showing weak homology to the amino acid sequence (High S core: 94, Probability: !!. 7 x 10 " 3 ). Yes (in the table, amino acids are represented by single letter notation, and X represents a stop codon). Table 1 p80 (N-terminal side) AVII NE_L
ラッ ト c DNA (N末端側） XA S L YARQQL p 80 Y I R.TTTN.Y I VAF C VVH Rat cDNA (N-terminal) XA S L YARQQL p 80 Y I R.TTTN.Y I VAF C VVH
ラッ ト c DNA NLRD I AN I VLAVAALL p 80 KNTQP.F I EKYFNKAVL Rat cDNA NLRD I AN I VLAVAALL p 80 KNTQP.F I EKYFNKAVL
ラッ ト c DNA PACRPHVRRYYSA I VH p 80 LPNDL LEVCEFAQVLY Rat cDNA PACRPHVRRYYSA I VH p 80 LPNDL LEVCEFAQVLY
ラッ ト c DNA L P SDWNQVAE FYQVWY p 80 I (C末端側） Rat cDNA L P SDWNQVAE FYQVWY p 80 I (C-terminal)
ラッ ト c DN A L (C末端側） Rat c DN A L (C-terminal side)
(2) ラッ 卜 ·テロメラ―ゼ蛋白質遺伝子の部分断片の取得 (2) Obtaining rat and telomerase protein gene partial fragments
(1) で得られた p 80のアミノ酸配列に極めて弱い相同性を示すラッ ト由来の アミノ酸配列については、 その上流に終止コ ドンが存在し、 しかもその下流には 開始コ ドンとしてのメチォニンが存在しないことから、 このアミノ酸配列をコ一 ドする mRNAが実際に存在するものかどうか不明である。 また、 p 80に相同 性を有する蛋白質を生合成できるかどうか自体も不明である。 しかし、 データバ ンクに登録された DN A配列に相補的な DN A配列がこのァミノ酸配列をコ一ド する可能性があり、 実際転写された対応の mRNAはスプライシングを受けて、 配列が変化している可能性がある。 そこで、 ラッ 卜由来細胞に実際にこの mRNA が存在するか否かを検討した。  In the amino acid sequence derived from rat that shows very weak homology to the amino acid sequence of p80 obtained in (1), a stop codon is present upstream of it, and methionine as an initiation codon is located downstream of it. Since it does not exist, it is unknown whether the mRNA encoding this amino acid sequence actually exists. It is also unknown whether a protein having homology to p80 can be biosynthesized. However, a DNA sequence complementary to the DNA sequence registered in the data bank may encode this amino acid sequence, and in fact, the corresponding transcribed mRNA is spliced and the sequence changes. Could be. Therefore, it was examined whether this mRNA actually exists in rat-derived cells.
まず、 アデノウィルスで形質転換されたラッ ト 3 Y 1細胞由来 Z 1 9細胞から、 Ch omc z y n s k iの方法 (An a 1. B i o c h em. 、 1 62、 1 56 - 1 59. 1 987 ) によって RN Aを調製した。 すなわち、 Z 1 9細胞 1 08 個を、 グァニジンイソチオシァネ一 卜溶液 [4 Mグァニジンイソチオシァネート (和光純薬） 、 25 mMクェン酸ナ ト リウム （和光純薬） 、 0. 1 M 2—メル カプトエタノール、 0. 5%ザルコシン酸ナトリウム （和光純薬） ] 中でホモジ ナイズし、 0. 1容量の 2 M酢酸ナトリウム （p H4. 0) を加えて混和した。 こ のホモジュネートに等容量の水飽和フエノール （和光純薬） 及び 0. 2容量のク ロロホルム （和光純薬） イソアミルアルコール （和光純薬） 混合液 （49対1、 体積比） を加えて 1 0秒間激しく混和し、 1 0、 000 x g、 20分間の遠心分 離により上清の水層を回収した。 回収した水層に等容量のイソプロパノール （和 光純薬） を混和し、 一 20度で 1時間冷却した後、 1 5、 000 X g、 20分間 の遠心分離を行った。 得られた沈澱物を再びグァニジンイソチオシァネート溶液 に溶解し、 等容量のイソプロパノールを加え、 — 20度で 1時間冷却した後、 15、 000 X g、 20分間の遠心分離により総 RN Aを回収した。 First, from rat 3Y1 cell-derived Z19 cells transformed with adenovirus, RNA was prepared by the method of Ch omc zynski (An a 1. Biochem., 162, 156-159. 987). That is, 1 0 8 Z 1 9 cells, guaiacolsulfonate two gin isothiocyanate Xia Ne one Bok solution [4 M guaiacolsulfonate two gin isothiocyanate Xia sulfonate (Wako Pure Chemical), 25 mM Kuen Sanna preparative potassium (Wako Pure Chemical), 0 1 M 2-mercaptoethanol, 0.5% sodium sarcosinate (Wako Pure Chemical Industries, Ltd.)], and mixed with 0.1 volume of 2 M sodium acetate (pH 4.0). An equal volume of a mixture of water-saturated phenol (Wako Pure Chemical) and 0.2 volumes of chloroform (Wako Pure Chemical) isoamyl alcohol (Wako Pure Chemical) (49: 1, volume ratio) is added to this homogenate. The mixture was vigorously mixed for 10 seconds, and the aqueous layer of the supernatant was collected by centrifugation at 10,000 xg for 20 minutes. An equal volume of isopropanol (Wako Pure Chemical Industries) was mixed with the collected aqueous layer, cooled at 120 ° C for 1 hour, and centrifuged at 15,000 Xg for 20 minutes. The precipitate obtained was dissolved again in guanidine isothiocynate solution, an equal volume of isopropanol was added, and the mixture was cooled at 20 ° C. for 1 hour, and then centrifuged at 15,000 × g for 20 minutes to obtain total RNA. Was recovered.
RN Aの精製は以下のように行った。 すなわち、 0. 2mgの総 RNAを ImM E DT A、 20 mMトリス塩酸 （p H 7. 5) に溶解し、 70°C、 5分間の熱 処理後、 氷上で急冷した。 この溶液に 5 M N a C 1溶液を終濃度が 0. 5Mに なるように加えて、 01 i g o— dTセルロースカラム （ t y p e 7， 1 c m x l c m、 P h a r ma c i a社） に展開し、 1 mM EDTAおよび 0. 5M N a C 1を含む 2 OmMトリス塩酸緩衝液 （pH 7. 5) でカラムを洗浄後、 滅 菌脱塩水にて結合分画を溶出して 4 gの p o 1 y (A) + RNAを得た。  RNA was purified as follows. That is, 0.2 mg of total RNA was dissolved in ImMEDTA and 20 mM Tris-HCl (pH 7.5), heat-treated at 70 ° C. for 5 minutes, and quenched on ice. To this solution was added a 5 M NaC1 solution to a final concentration of 0.5 M. The solution was applied to a 01 igo-dT cellulose column (type 7, 1 cmxlcm, Pharmacia), and 1 mM EDTA and After washing the column with 2 OmM Tris-HCl buffer (pH 7.5) containing 0.5 M NaC1, the bound fraction was eluted with sterile deionized water, and 4 g of po 1 y (A) + RNA was obtained.
上記のようにして得られた p o 1 y (A) +RNA 1マイクロ gを铸型にして c DNAを合成し、 この cDNAに 1 O pmo 1 eのランダム · へキサマ一 . プ ライマ一と 200ュニッ 卜の MM LV逆転写酵素 （ 『SUPER SCR I P T』 、 G I B C O B R L) を加えて 1 s t s t r a n dを合成し、 次に、 1. 4ュ ニッ トの RNa s e H、 40ユニッ トの大腸菌 D N Aポリメラ一ゼ I及び 1 5ュ ニッ 卜の大腸菌 DN Aライゲ―スを加えて 2 n d s t r a n dを合成した。 反 応終了後、 フヱノ—ル /クロ口ホルム抽出を行い、 上清の水層を回収した。 回収 した水層と等容量の 5 M酢酸アンモニゥム溶液を添加後、 2倍容量のェタノール を混和した。 次に、 1 5、 000 x g、 1 0分間の遠心分離を行い、 エタノール 沈澱による c DN Aの回収を行った。 One microgram of po 1 y (A) + RNA obtained as described above was used as a template to synthesize cDNA, and this cDNA was randomized to 1 O pmo 1 e. MMLV reverse transcriptase ("SUPER SCR IPT", GIBCOBRL) was added to synthesize 1 ststrand, then 1.4 units of RNAse H and 40 units of E. coli DNA polymerase I were synthesized. And 15 units of E. coli DNA ligase were added to synthesize 2nd strand. After the completion of the reaction, phenol / chloroform extraction was performed, and the aqueous layer of the supernatant was recovered. Collection After adding an equal volume of a 5 M ammonium acetate solution to the aqueous layer, the mixture was mixed with twice the volume of ethanol. Next, centrifugation was performed at 15,000 xg for 10 minutes, and cDNA was recovered by ethanol precipitation.
上記のよ う に して得られた c D N Aについて、 R i 1 e y らの方法 (Ve c t o r e t t e法、 Nu c l e i c Ac i d Re s. 、 18、 2887 - 2890) を用いて、 （1) の工程で得られた cDN A配列 （配列番号 3 ) に対応 する部分のさらに 5' 側上流に位置する未知の c DN A配列を解析した。 まず、 60 n gの c DNAを T 4ポリメラ一ゼで処理して末端を平滑化し、 さらに 1 0 ュニッ トの制限酵素 P v u I I (東洋紡製、 緩衝液は添付のものを使用） と 37 てで 2時間ィ ンニュペー トした。 切断した DN Aをフヱノール/ク口口ホルム処 理及びエタノ—ル沈澱にて精製した後、 下記表 2に示す V e c t o r e t t e u n i t (v c t Aと v c t Bをァニールさせたもの） 3 pmo l eを DNAリ ガーゼを用いて連結した。 表 2  The cDNA obtained as described above was subjected to the step (1) using the method of Riey et al. (Vectorette method, Nucleic Acid Res., 18, 2887-2890). The unknown cDNA sequence located further 5 'upstream of the portion corresponding to the obtained cDNA sequence (SEQ ID NO: 3) was analyzed. First, 60 ng of cDNA was treated with T4 polymerase to blunt the ends, and then 10 units of the restriction enzyme Pvu II (manufactured by Toyobo, using the supplied buffer) and 37 I wrote for two hours. The digested DNA was purified by phenol / cohol-form treatment and ethanol precipitation, and then the vector unit shown in Table 2 below (annealed vct A and vct B) 3 pmoles was ligated to DNA ligase. Was connected using Table 2
V c t A ： 5 AAGGAGAGGACGCTG VctA: 5 AAGGAGAGGACGCTG
TCTGTCGAAGGTAAG GAACGGACGAGAGAA GGGAGAG- 3'  TCTGTCGAAGGTAAG GAACGGACGAGAGAA GGGAGAG-3 '
V c t B ： 5 CTCTCC CTTCTCGAA VctB: 5 CTCTCC CTTCTCGAA
TCGTAAC CGTTCGTA CGAGAATCGCTGTCC TCTCCTT一 3'  TCGTAAC CGTTCGTA CGAGAATCGCTGTCC TCTCCTT-1 3 '
V e c t o r e t t e u n i tを平滑末端に連結させた c DNAを铸型とし て、 下記表 3に示す V e c t o r e t t e u n i tの片方鎖にハイブリダイズ する v c t Gオリゴヌクレオチド · プライマーと、 配列番号 3に示す c DNA配 列にハイプリダイズする R a PC 5' オリゴヌクレオチド · プライマーとを用い た PC Rを行い、 R a PC 5' オリゴヌクレオチド ' プライマ一の結合する部分 から 5' 側上流の未知の部分を含む c DN Aを増幅した。 増幅反応は常法に従い、 P CR用サ一マルサイクラ一を用いて 93。Cで 1分間、 65 で 1分間、 及び 72 °Cで 2分間の保温サイクルを 35回繰り返した。 表 3 The cDNA in which the Vectoretteunit is connected to the blunt end is type- 铸 and hybridized to one strand of the Vectoretteunit shown in Table 3 below. PCR using a vct G oligonucleotide primer and a Ra PC 5 'oligonucleotide primer that hybridizes to the cDNA sequence shown in SEQ ID NO: 3 to perform Ra PC 5' oligonucleotide primer CDNA containing an unknown portion 5 ′ upstream from the binding site of was amplified. The amplification reaction was carried out according to a conventional method using a thermal cycler for PCR. A heat retention cycle of 1 minute at C, 1 minute at 65, and 2 minutes at 72 ° C was repeated 35 times. Table 3
V c t G ： 5 ' CGGTACCGAATCGTA VctG: 5 'CGGTACCGAATCGTA
ACCGTTCGTACGAGA ATCGCT— 3'  ACCGTTCGTACGAGA ATCGCT— 3 '
R a P C 5 ' ： 5' - CATAC CTGGT R a PC 5 ': 5'-CATAC CTGGT
AGAACTC GGCTA - 3'  AGAACTC GGCTA-3 '
P CR産物をフヱノールノクロ口ホルム処理及びエタノ一ル沈澱にて精製した 後、 一部を DNAリガ一ゼを用いて pT7B 1 u eTベクター （Ph a rma c i a 社） に連結し、 形質転換された組み換え大腸菌をアンピシリンで選択して、 ブラ スミ ド DN Aを調製した。 挿入された P C R産物の DN A配列を A B I 373 A シークェンサ一 （App l i e d B i o s y s t ems社） を用いた S a n g e r 法により決定した。 その結果、 配列表の配列番号 4に記載された塩基配列がブラ スミ ド R a P C 53に挿入された c DN Aに見出された。 After the PCR product was purified by treatment with phenolic noroform and ethanol precipitation, a portion was ligated to pT7B1ueT vector (Pharmacia) using DNA ligase, and the transformed recombinant was used. Escherichia coli was selected with ampicillin to prepare a plasmid DNA. The DNA sequence of the inserted PCR product was determined by the Sanger method using ABI373A Sequencer (Applied Biosystems). As a result, the nucleotide sequence described in SEQ ID NO: 4 in the sequence listing was found in cDNA inserted into the plasmid RaPC53.
R a P C 53の塩基配列を解析した結果、 相補鎖 DN Aから予想された配列表 の配列番号 3に記載の核酸番号 1〜 1 70までの塩基配列が、 実際のラッ 卜細胞 では配列表の配列番号 4の核酸番号 1〜 244までの塩基配列に対応しているこ とが認められた。 配列表の配列番号 3の核酸番号 1 63〜 1 72までの塩基配列 (5' — TCTC TCCTAG - 3' ) が s p l i c i n g a c c e p t o r s i t eのコンセンサス配列、 5' - P y P y P y P y P y P y N C AG - 3' に相当することから、 この結果はアーティファク トによるものではなく、 スプラ イシングによる RN Aの編集が行われた結果と考えられた。 従って、 配列表の配 列番号 3に記載の塩基番号 1 7 0の 〔T〕 は実際には配列番号 4の配列において は 〔Α〕 となっており、 終止コ ドン TAGがリジン A AGになっていた。 しかも 5 ' 側上流に向けてオープン · リーディ ング · フレイムがさらに伸びていること が判明した。 As a result of analyzing the nucleotide sequence of RaPC53, the nucleotide sequence of nucleic acid numbers 1 to 170 described in SEQ ID NO: 3 in the sequence listing predicted from the complementary strand DNA was found to be the same as that of the sequence listing in actual rat cells. It was confirmed that it corresponded to the nucleotide sequences of nucleic acid numbers 1 to 244 of SEQ ID NO: 4. The base sequence (5 '— TCTC TCCTAG-3') of nucleic acid number 163 to 172 of SEQ ID NO: 3 in the sequence listing is splicingacceptor Since the consensus sequence at site corresponds to 5'-PyPyPyPyPyPyNCAG-3 ', this result is not due to artifacts, but to RNA editing by splicing. It was thought that the result was done. Therefore, the [T] of base number 170 in SEQ ID NO: 3 in the sequence listing is actually [Α] in the sequence of SEQ ID NO: 4, and the stop codon TAG is lysine AAG. I was In addition, it was found that the open, reading, and flames were further increasing toward the 5 'upstream.
また、 そのオープン · リ一ディング ' フレイムのァミノ酸配列は、 工程（1) で予 想されたテトラヒメナ p 8 0のアミノ酸配列に相同性 （H i g h S c o r e ： 9 4、 P r o b a b i l i t y : l . 7 x 1 0一3) を示すラッ 卜由来のァミノ酸配 列に比べて、 さらに高い相同性を示すものであり （H i g h S c o r e ： 1 2 5 , P r o b a b i l i t y : l . 6 x 1 0— 18 ) 、 配列表の配列番号 3の 3 1 2番目 の 〔A〕 が 〔T〕 となっており、 対応するアミノ酸がァスパラギン （AA C) か らィソロイシン （A T C) に変異していることが判明した。 The amino acid sequence of the open reading frame is homologous to the amino acid sequence of Tetrahymena p80 predicted in step (1) (High S core: 94, Probability: 1.7). x 1 0 one 3) in comparison with the Amino acid sequence of rat Bok from showing a, which shows a higher homology (H igh S core: 1 2 5, P robability:. l 6 x 1 0- 18 ), The [A] at position 312 in SEQ ID NO: 3 is [T], indicating that the corresponding amino acid is mutated from asparagine (AA C) to isoloisin (ATC). .
(3) ラッ ト 'テロメラーゼ蛋白質全長 c DN Αの取得 (3) Obtain rat telomerase protein full-length c DN Α
まず、 S V4 0ウィルスで形質転換されたラッ ト 3 Y 1由来 S V— 3 Y 1 - C 6 6 細胞から、 工程（1) の方法と同様な方法で p o 1 y ( A) + R N Aを得、 S T R A T A G E N E社の c D N A合成キッ トを用いて c D N Aを調製した。 c D N Aの調製はマニュアルに従って行ったが、 1 s t s t r a n d合成反応 はプライマーとしてランダムへキサマー 'オリゴヌクレオチドとオリゴ d Tブラ イマ—の両方を最終濃度各 2 / M加えて行った。  First, po1y (A) + RNA was obtained from the rat 3Y1-derived SV-3Y1-C66 cells transformed with the SV40 virus in the same manner as in step (1). CDNA was prepared using a cDNA synthesis kit from STRATAGENE. Preparation of cDNA was performed according to the manual, but 1ststrand synthesis reaction was performed by adding both random hexamer 'oligonucleotide and oligo dT primer as primers at a final concentration of 2 / M each.
次に、 c D NAの末端にDN Aリガーゼによって E c o R Iアダプターを付加 した後、 反応産物を S e p h a c r y l S — 5 0 0力ラムに展開し、 未反応の E c o R Iアダプターとサイズの小さい c DNAを除いた。 素通り画分の c DNA をエタノ一ル沈澱で回収し、 予め制限酵素 E c o R Iで消化され、 さらに末端を 脱リン酸化されたス Z A Pファージ DN Aと上記の c DN AをDNAリガーゼで 結合した。 さらに、 c D N Aと結合した λ Z A Pファージ DNAをファージ粒子 へパッケージングした。 以上の作業は S TRATA G E N E社の G I GA P A C K GOLD I I Iキッ トを用い、 添付のマニュアルに従って行った。 得られたフ ァージ粒子を常法に従い大腸菌 C 600 h f 1 A株に感染させて増幅を行い、 フ ァージ粒子を回収した。 一連の操作により、 約 500万のファージクローンを得 た。 Next, after adding an Eco RI adapter to the end of the cDNA using DNA ligase, the reaction product was developed on Sephacryl S—500 column, and unreacted Eco RI adapter and small c DNA was removed. The cDNA of the flow-through fraction was recovered by ethanol precipitation, and the cDNA was digested with the restriction enzyme EcoRI in advance and the terminal dephosphorylated. . Further, λ ZAP phage DNA bound to cDNA was packaged into phage particles. The above work is performed by GI GA PACK of S TRATA GENE The test was performed using the GOLD III kit according to the attached manual. The obtained phage particles were infected with Escherichia coli C600hf1A strain according to a conventional method and amplified, and the phage particles were collected. Through a series of operations, about 5 million phage clones were obtained.
約 1 00万のファージクローンを常法に従い大腸菌 C 600 h f 1 A株に感染 させ、 プレート上の N Z Y寒天培地上で培養した。 ナイロン膜にファージ粒子を 写し取ったもののレプリカを 2枚作製し、 洗浄及びアルカリ処理した後、 工程 (2) で得られた R a P C 53を32 P標識してプローブとして用い、 このプローブにハ イブリダィズするファージ ' クローンをスクリーニングした。 その結果、 3つの 陽性シグナルを見出したので、 それらについてファージ粒子を回収し、 同様な方 法てク口一ンィ匕した後、 S t r a t a g e n e社のマニュアルに従って揷入され た c DN A部分を含むプラスミ ド （RET 1、 RET 2, RET3) を i n v i v o e x c i s i o n法にて回収した。 Approximately 1,000,000 phage clones were infected with E. coli C600hf1A strain according to a conventional method, and cultured on a NZY agar medium on a plate. Two replicas of the phage particles copied on a nylon membrane were prepared, washed and treated with alkali, and then the RaPC53 obtained in step (2) was labeled with 32 P and used as a probe, and the probe was hybridized. Phage clones were screened. As a result, three positive signals were found, and phage particles were collected from them. After cloning the phage particles in the same manner, the plasmid containing the cDNA fragment inserted according to the Stratagene manual was used. (RET1, RET2, RET3) were recovered by the invivoexcision method.
プラスミ ド RET 1、 RET 2、 RE T 3について制限酵素切断地図を作製し たところ、 各々 1. 3 k b p、 2. 4 k b p、 6. 5 k b pの c D N Aが挿入さ れており、 図 1に示すように各々の c DN Aが重複する位置にあることがわかつ た。 常法に従って欠失変異 c DN Aを作製し、 RE T 1の全長と RE T 2及び RE T 3の一部分の DNA配列を解読した。 それら DN A配列を制限酵素切断地 図に従って組み合わせたところ、 約 4. 6 k b pにわたる大きなオープン ' リー ディ ング · フレイムが見出された。 この中には、 工程 （2) で得られたテトラヒ メナ p 80のアミノ酸配列と相同性を示す R a P C 53のアミノ酸配列 （H i gh When restriction maps of plasmids RET1, RET2, and RET3 were prepared, cDNAs of 1.3 kbp, 2.4 kbp, and 6.5 kbp were inserted, respectively. As shown, each cDNA was found to be in an overlapping position. A deletion mutant cDNA was prepared according to a conventional method, and the full length of RET1 and the DNA sequence of a part of RET2 and RET3 were decoded. When the DNA sequences were combined according to the restriction enzyme cleavage map, a large open reading frame spanning about 4.6 kbp was found. Among them, the amino acid sequence of RaPC53 (High) showing homology to the amino acid sequence of Tetrahymena p80 obtained in step (2)
S c o r e : 125、 P r o b a b i l i t y : l. 6 x 1 0— 18 ) も含まれて おり、 ホモロジ一サーチによりテトラヒメナ p 80のァミノ酸配列とのさらに高 い相同性が明 ら かに な っ た （ H i g h S c o r e ： 2 3 4 、 P r o b a b i l i t y : :!. I x l 0_49 ) 。 S core: 125, P robability: . L 6 x 1 0- 18) also included are, even higher have homology with the Amino acid sequence of Tetrahymena p 80 by homology one search is Tsu name Akira et crab ( High S core: 234, Probability::!. Ixl0_49 ).
しかし、 上記のォ一プン ' リーディング ' フレイムの C末端には終止コ ドンが 見出されないこと、 またいくつかのテロメラ一ゼ活性陽性のラッ ト細胞から抽出 した mRN Aのノザン解析の結果から、 得られた。 DNAの由来する実際の mRNA は 1 0 k b近い大きなものと考えられたことから、 さらに 3' 側部分の c DN A の取得を試みた。 すなわち、 RET 3の 3' 端に近い、 配列表の配列番号 1に示 す DN A配列のうち核酸番号 4083〜521 6にあたる部分の DN A断片を"2 P 標識してプローブとして用い、 さらに約 1 00万のファージクローンをスクリー ニングした。 その結果、 新たに 1 3の陽性シグナルが見出された。 そのうち 6個 のクローンについてファージ粒子から挿入された c DNA部分を含むプラスミ ド ( R E Τ λ 0 1 , 0 7、 0 8、 0 9、 1 0、 1 3 ) を i n v i v o e x c i s i o n法にて回収した。 However, no termination codon was found at the C-terminus of the open 'reading' frame, and the results of Northern analysis of mRNAs extracted from some telomerase-positive rat cells showed that Obtained. Actual mRNA from DNA Was considered to be as large as 10 kb, so we tried to obtain 3'-side cDNA. In other words, close to the 3 'end of the RET 3, used as a probe to "2 P labeled DN A fragment of a portion corresponding to the nucleic acid number 4083-521 6 of shows to DN A sequence in SEQ ID NO: 1, even about One million phage clones were screened, and 13 new positive signals were found, of which six contained plasmids containing the cDNA portion inserted from phage particles (REλλ). 0 1, 0 7, 08, 09, 10, 13) were recovered by the invivoexcision method.
プラスミ ド RETス 01、 R Ε Τ λ 09 , R Ε Τ λ 1 3について制限酵素切断 地図を作製したところ、 各々 5. 0 kbp、 4. 9 kb p、 4. 9k bpの cDNA が挿入されており、 図 1に示すように各々の c DNAが重複する位置にあること が判明した。 これらのうち、 ΚΕΤ λ 1 3を新たに 「RET 7」 と命名し、 常法 に従って欠失変異 c DN Aを作製して、 RET 7の全長の DN A配列を解読した。 その結果と、 プラスミ ド RET 1、 RET 2及び RE T 3から得られた DNA配 列の情報とを組み合わせたところ、 終止コ ドンを含めて 7890 b pにわたる大 きなオープン ' リーディ ング ' フレイムが見出された （配列表の配列番号 1 ) 。 (4) ラッ ト ·テロメラーゼ蛋白質 c DNAの取得—上流配列の取得 （5' -RACE 法）  When restriction maps were prepared for plasmid RETs 01, RΕλλ09, and RΕλ13, cDNAs of 5.0 kbp, 4.9 kbp, and 4.9 kbp were inserted, respectively. As shown in FIG. 1, it was found that each cDNA was located at an overlapping position. Of these, Δλ13 was newly named “RET 7”, and a deletion mutant cDNA was prepared according to a conventional method to decode the full-length DNA sequence of RET7. Combining the results with the information on the DNA sequences obtained from the plasmids RET1, RET2 and RET3 revealed a large open 'reading' frame of 7890 bp including the stop codon. (SEQ ID NO: 1 in the sequence listing). (4) Obtaining rat telomerase protein cDNA—Obtaining upstream sequence (5'-RACE method)
工程（3) で得られた c DNAには、 最も 5' 端の ATGよりもさらに 5' 側に 同フレイムの終止コ ドンが見出せないため、 さらに 5' 側の mRNAの配列につ いて 5' — Ra p i d Amp l i f i c a t i on o f c D N A E n d s (RACE) 法を用いて検討した。  In the cDNA obtained in step (3), the termination codon of the same frame was not found on the 5 'side further than the ATG on the 5' end, so that the 5 ' — Investigations were performed using the Rapid Amplification of cDNA Ends (RACE) method.
5' -R A C E法は、 C 1 o n t e c h社の 5' — RACEキッ トを用い、 マ ニュアルに従い行った。 工程（3) において SV— 3 Y 1— C 66細胞から得られた p o 1 y (A) + RNA 2 gと、 配列表の配列番号 1の核酸番号 1 49 3〜 151 5の部分に相補的な DN A配列のオリゴヌクレオチドプライマ一 N c EX 3' 1 0 pmo 1 eとを混合し、 加熱した後に急冷した。 反応混合物に逆転写酵素 The 5'-RACE method was performed according to the manual using a 5'-RACE kit of C1onetech Inc. In step (3), 2 g of po1y (A) + RNA obtained from SV-3Y1-C66 cells is complementary to the nucleic acid number 1493-3151 of SEQ ID NO: 1 in the sequence listing Oligonucleotide primer NcEX 3′10 pmo1e having a unique DNA sequence was mixed, heated and quenched. Reverse transcriptase to reaction mixture
(G I B CO BRL社の S u p e r s c r i p t) 、 基質ヌクレオチドと緩衝 液を加えて 4 2°Cで 1時間反応させた。 E DT Aを加えて反応を停止させた後、 アル力リ処理で铸型 RNAを分解し、 ィソプロパノ—ル沈澱を行って単鎖 c DNA を単離した。 さらに、 この c DNAの半量に、 5' — R AC E用アンカープライ マ一 〔5' - P ( + ) ANC] 4 pmo l eを RNAリガーゼを用いて連結させ た。 反応は、 25 %P EG存在下に 37 °Cで 3時間行った。 (GIB CO BRL Superscript), substrate nucleotides and buffers The solution was added and reacted at 42 ° C for 1 hour. After terminating the reaction by adding EDTA, the type II RNA was decomposed by an aliquot treatment, and single-stranded cDNA was isolated by isopropanol precipitation. In addition, an anchor primer for 5'-RACE [5'-P (+) ANC] 4 pmoles was ligated to half of the cDNA using RNA ligase. The reaction was performed at 37 ° C. for 3 hours in the presence of 25% PEG.
次に、 N c EX 3' で逆転写プライムされ、 さらに 3' 端にアンカ一 DN A配 列を付加された単鎖 c DNAを铸型として、 アンカ一 DN Aに相補的なォリゴヌ クレオチドプライマ一 RACE— PRMと、 配列表の配列番号 1の核酸番号 1039 〜 1 0 5 6の部分に相捕的な D N A配列のオリゴヌク レオチ ドプライマ一 R a P C 5' とを用いて、 P C Rによる DN A増幅を行った。 反応には 20分の 1量の単鎖 c DNAと各々 1 O pmo 1 eのプライマーとを用い、 G I B C O BRL社の T a Qポリメラ一ゼを用いて添付のマニュアルに従って P C Rを行つ た。 ただし、 非特異的な DN A増幅を避けるために、 反応はマニュアル .ホッ ト - スタート法で開始した後、 94°Cで 30秒、 5 5°Cで 1分、 7 2°Cで 2分のサイ クルを 3 5回繰返した。  Next, an oligonucleotide primer complementary to the anchor DNA is used as a type I single-stranded cDNA that is reverse-transcribed with NcEX 3 'and further has an anchor DNA sequence added to the 3' end. RACE—PCR amplification of DNA using PRM and the oligonucleotide primer Ra PC 5 'of the DNA sequence complementary to nucleic acid numbers 1039 to 106 of SEQ ID NO: 1 in the sequence listing. went. In the reaction, a PCR was performed according to the attached manual using a 1/20 volume of single-stranded cDNA and a primer of 1 O pmo 1 e each, using TaQ polymerase of GIBCORBRL. However, to avoid non-specific DNA amplification, the reaction was started manually using a hot-start method, followed by 30 seconds at 94 ° C, 1 minute at 55 ° C, and 2 minutes at 72 ° C. This cycle was repeated 35 times.
P C R産物を p T 7 B 1 u e Tベクタ—に組み込み、 増幅 DN Aの挿入された ものについて DN A配列を解読した結果、 これらのうちの 1 0クローンが殆ど同 じ DN A配列を有していた。 これらのクローンのうち、 代表的なクローンである RAC E 3及び RACE 5は図 1に示すような位置に存在しており、 配列表の配 列番号 1の核酸番号 1 99〜20 1の八丁6の5' 側上流約 200 bpまで c DNA が逆転写及び伸長され得ることがわかった。 配列表の配列番号 1の塩基番号 1 99 〜20 1の ATGより 5' 側上流には、 配列番号 1のフレイムと合う終止コ ドン は見出されなかったが、 増幅された DN Aの長さがほぼ均一であることから、 実 際の mRNAの 5' 端に対応する c D N Aを得た可能性が高いと考えられた。 実施例 2 ： ヒ ト ·テロメラ一ゼ蛋白質遺伝子の取得  The PCR product was incorporated into the pT7B1ueT vector, and the DNA sequence of the inserted DNA was analyzed. As a result, 10 clones among these clones had almost the same DNA sequence. Was. Among these clones, RACE 3 and RACE 5, which are representative clones, are located at the positions shown in FIG. 1, and nucleic acid numbers 199 to 201 of SEQ ID NO: 1 in the sequence listing. It was found that cDNA could be reverse transcribed and extended to about 200 bp 5 ′ upstream of No termination codon matching the frame of SEQ ID NO: 1 was found 5 'upstream of the ATG at nucleotide numbers 199 to 201 in SEQ ID NO: 1, but the length of the amplified DNA It was highly likely that cDNA corresponding to the 5 'end of the actual mRNA was obtained because the DNA was almost uniform. Example 2: Acquisition of human telomerase protein gene
(1) ヒ ト 'テロメラーゼ蛋白質遺伝子の部分断片の取得 (1) Obtaining a partial fragment of the human telomerase protein gene
テトラヒメナ p 80のァミノ酸配列と実施例 1の工程（3) で得られたラッ 卜 'テロ メラーゼ蛋白質のァミノ酸配列との相同性を検討したところ、 同一のァミノ酸配 列がいくつか見出されたことから、 そのような領域が種を越えて広く保存されて いる可能性が考えられた。 そこで、 そのような領域のアミノ酸配列から、 いわゆ る d e g e n e r a t i v e PC Rプライマーを作製することにより、 このブラ イマ一を用いた P C R法によってテトラヒメナゃラッ ト以外の各動物種固有のテ ロメラーゼ蛋白質 c DN A断片を取得できると期待された。 The amino acid sequence of Tetrahymena p80 and the rat telo obtained in step (3) of Example 1 Examination of the homology with the amino acid sequence of the merase protein revealed several identical amino acid sequences, suggesting that such regions may be widely conserved across species. Was. Therefore, a so-called degenerative PCR primer is prepared from the amino acid sequence of such a region, and the PCR method using this primer is used to carry out the PCR method using this primer, which is specific to each animal species other than tetrahymenadrat c DN. It was expected that A fragment could be obtained.
まず、 センスプライマーとして、 配列表の配列番号 1のァミ ノ酸番号 3 7 9〜 384に対応する HP E T 5 (配列表の配列番号 5) 、 アンチセンスプライマー として、 配列表の配列番号 1のァミノ酸番号 532〜537に対応する HP E T 3 (配列表の配列番号 6) を用い、 実施例 1の工程 (3) で得られたラッ ト S V 3 Y 1 一 C 6 6細胞由来 c DN A及び同様な方法で取得されたヒ ト卵巣奇形腫由来 P A 一 1細胞由来 c DNAを铸型として P C Rを常法にて行ったが、 P A— 1細胞由 来 c DN A及び陽性対照としての SV— 3 Y 1 - C 6 6細胞由来 c DNAからも 目的の DN Aは増幅されなかった。  First, as a sense primer, HPET5 (SEQ ID NO: 5 in the sequence listing) corresponding to amino acid number 379-1384 of SEQ ID NO: 1 in the sequence listing, and as an antisense primer, Using the HP ET3 (SEQ ID NO: 6 in the sequence listing) corresponding to amino acid numbers 532 to 537, rat DN derived from step SV3Y1-C66 cells obtained in step (3) of Example 1 c DNA PCR was performed in the usual manner using cDNA derived from PA-11 cells derived from human ovarian teratoma obtained by the same method as described above, but PCR was performed using PA-1 cell-derived cDNA and SV as a positive control. — The target DNA was not amplified from cDNA derived from 3Y1-C66 cells.
次に、 センスプライマーとして、 配列表の配列番号 1のァミ ノ酸番号 3 76〜 385に対応する HPET 5 - 2 (配列表の配列番号 7 ) または配列表の配列番 号 1のアミノ酸番号 380 - 388に対応する HP ET 5 - 3 (配列表の配列番 号 8) 、 アンチセンスプライマ一として配列表の配列番号 1のァミノ酸番号 5 32 〜 540に対応する HP ET 3— 2 (配列表の配列番号 9 ) または配列表の配列 番号 1のアミノ酸番号 5 34〜542に対応する HP ET 3 - 3 (配列表の配列 番号 1 0) を用い、 SV— 3Y 1— C 66細胞由来 c DNA及びPA— 1細胞由 来 c DN Aの各々の铸型について 4通りのプライマ—の組合せの P CRを常法に て行った。  Next, as a sense primer, HPET 5-2 (SEQ ID NO: 7 in Sequence Listing) corresponding to amino acid number 376 to 385 in SEQ ID NO: 1 in Sequence Listing or amino acid number 380 in SEQ ID NO: 1 in Sequence Listing is used. HP ET 5-3 (SEQ ID NO: 8) corresponding to -388, HP ET 3-2 (SEQ ID NO: 5) corresponding to the amino acid number 532 to 540 of SEQ ID NO: 1 as an antisense primer Using SEQ ID NO: 9) or HP ET3-3 (SEQ ID NO: 10 in the sequence listing) corresponding to amino acid numbers 534 to 542 of SEQ ID NO: 1 in the sequence listing, cDNA derived from SV-3Y1-C66 cells For each type III of cDNA and PA-1 cell-derived cDNA, PCR was performed in the usual manner using four combinations of primers.
P C R産物をァガロース ·ゲル電気泳動した後、 臭化工チジゥムで DN Aを染 色したゲルを U Vイルミネ一タ一で観察したところ、 H P E T 5— 2または HP E T 5— 3と HPET 3— 2との組み合わせで S V - 3 Y 1 — C 6 6細胞由 来 c DNAを铸型とした P C Rを行った場合に、 予想された約 500 b pの DNA 断片が増幅された。 また、 P A— 1細胞由来 c DN Aを铸型とした場合には、 HPET 5— 2と HPET 3— 2との組み合わせのプライマーを用いることによ つて同様に約 5 0 0 b pの D N A断片が増幅された。 この D N A断片を pT 7 B l u eプラスミ ドにサブクローニングして DN A配列を解読したところ、 対応するラッ ト c DN A配列に塩基レベルで約 77%の相同性を持ち、 アミノ酸 レベルでも 76 %の相同性を示す DNA配列 （図 2、 配列表の配列番号 2) が得 られた。 After agarose gel electrophoresis of the PCR product, the gel stained with DNA by bromide chromatography was observed on a UV illuminator. The results showed that HPET 5-2 or HPET 5-2 and HPET 5-2 When PCR was performed using the cDNA derived from SV-3Y1-C66 cells in combination as the type III, the expected DNA fragment of about 500 bp was amplified. In addition, when PA-1 cell-derived cDNA is type II, Similarly, a DNA fragment of about 500 bp was amplified by using the primer of the combination of HPET5-2 and HPET3-2. When this DNA fragment was subcloned into pT7Blue plasmid and the DNA sequence was decoded, it had about 77% homology at the base level and 76% homology at the amino acid level to the corresponding rat cDNA sequence. A DNA sequence (FIG. 2, SEQ ID NO: 2 in the sequence listing) showing the sex properties was obtained.
そこで、 得られた D N A配列の情報を基にして、 ヒ ト ' テロメラ一ゼ蛋白質 c DNA断片を PCR増幅できるオリゴヌクレオチド · プライマーを設計した。 センスプライマーとして、 配列表の配列番号 2の核酸番号 92〜 1 14に対応す る h T P C 5 (配列表の配列番号 1 1) 、 アンチセンスプライマーとして、 配列 表の配列番号 2の核酸番号 433〜455に対応する h TP C 3 (配列表の配列 番号 1 2) を用い、 数種のヒ 卜細胞 mRNA由来 c DN Aを铸型として常法にて P C Rを行った。  Therefore, based on the obtained information on the DNA sequence, oligonucleotide primers capable of PCR-amplifying the human 'telomerase protein cDNA fragment were designed. H TPC5 (SEQ ID NO: 11 in the sequence listing) corresponding to nucleic acid numbers 92 to 114 of SEQ ID NO: 2 in the sequence listing as a sense primer, and nucleic acid number 433 to SEQ ID NO. 2 in the sequence listing as an antisense primer Using hTPC3 corresponding to 455 (SEQ ID NO: 12 in the sequence listing), PCR was carried out in the usual manner using several types of human cell mRNA-derived cDNA as type III.
まず、 ヒ ト胎盤由来総 RNA、 ヒ ト B細胞白血病由来 R a j i細胞由来総 RNA、 ヒ ト扁平上皮癌由来 A 4 3 1細胞由来 p o 1 y (A) + RNA、 ヒ ト乳癌由来 B T 474細胞、 SKB R 3細胞、 B SMZ細胞、 及び MC F 7細胞由来 p o 1 y (A) + RNAを Ch omc z yn s k iの方法 (An a 1. B i o c h em. 、 162、 1 56— 1 59、 1 987 ) 及び P h a r m a c i a社のキッ トを用い て取得し、 Ph a rma c i a社の F i r s t s t r a n d s y n t h e s i s k i tを用いて c DNAを合成した。 First, human placenta-derived total RNA, human B-cell leukemia-derived Raji cell-derived total RNA, human squamous cell carcinoma-derived A431 cell-derived po1y (A) + RNA, and human breast cancer-derived BT474 cells , SKB R3 cells, B SMZ cells, and MCF7 cells derived po 1 y (A) + RNA by the method of Ch omc z yn ski (An a 1. Biochem., 162, 156—159, 1987) and a kit from Pharmacia, and cDNA was synthesized using First Strandsynthesis kit from Pharmacia.
これら c DNAのおよそ 20分の 1量を铸型として、 hTPC 5と h TPC 3 をプライマ一として用いた P C Rを行った。 DNAポリメラ一ゼとしては、 Amp 1 i t a q Go l d (P e r k i n— E l m e r社） を用い、 95てで 1 0分間の熱処理の後、 95 で 30秒、 65 °Cで 30秒、 及び 72 °Cで 30秒 の保温サイクルを 35回繰り返した。 その結果、 予想された約 390 b pの DNA 断片がヒ 卜癌細胞由来 c NAを铸型としたときに増幅されてきたが、 铸型 （一） の陰性対照とヒ ト胎盤総 RN A由来 c DN Aを铸型とした場合には検出されなか つた。 O T PCR was performed using hTPC5 and hTPC3 as primers with about 1/20 of these cDNAs as type III. As a DNA polymerase, Amp 1 itaq Gold (Perkin-Elmer) was used, and after heat treatment at 95 ° C for 10 minutes, 95 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C The 30 second heat retention cycle was repeated 35 times. As a result, the expected DNA fragment of approximately 390 bp was amplified when the human cancer cell-derived cDNA was type III, but the type III (1) negative control and human placenta total RNA-derived cNA were amplified. When DNA was type II, it was not detected. OT
この結果、 hTPC 5と h TPC 3をプライマーとして用いればヒ ト 'テロメ ラーゼ蛋白質 c DN A断片を増幅できることが判明したので、 C 1 o n t e c h 社製ヒ ト胸腺由来 c DNAライブラリ一のうち、 1 0万個のファ一ジを铸型とし て用いて上記同様の方法で P C Rを行ったところ DN Aの増幅は認められなかつ たが、 100万個のファージを铸型として用いたときに予想された大きさの DNA が増幅された。  As a result, it was found that the h'TPC5 and hTPC3 could be used as primers to amplify the human telomerase protein cDNA fragment. When PCR was performed in the same manner as above using 10,000 phages as type I, no amplification of DNA was observed, but expected when one million phages were used as type II. Large size DNA was amplified.
そこで、 上記 c DNAライブラリーのベクタ一として用いられているス g t 10 の c DNA挿入部位の 5' 側及び 3， 側に対応する 2つのオリゴヌクレオチド ' プライマ一 （各々、 5' A g t 1 0及び 3' λ g t 1 0 ) (C l o n t e c h社 製） と h TPC 5と hTPC 3をプライマ一として用い、 h TPC 5の 5' 側上 流または h T P C 3の 3' 側下流の未知の部分の c DN A断片の取得を試みた。 c DNAライブラリーのうち 1 00万個のファージを铸型とし、 4通りのプライ マ一の組合せ （h T P C 5対 5' ス g t 10または 3' ス g t l O及び hTPC 3 対 5' A g t l Oまたは 3' ス g t 10) の P C Rを上記の方法に従って行った。 ただしァニール温度は 65 °Cの代わりに 55°Cにして行った。 その結果、 hTPC 5 の 5' 側上流約 1. 5 k b pに対応する部分の DN A断片が増幅された。  Therefore, two oligonucleotides 'primers' corresponding to the 5 'and 3' sides of the cDNA insertion site of gt10 used as the vector of the cDNA library (5'A gt10 And 3′λ gt10) (Clontech) and hTPC5 and hTPC3 as primers to determine the unknown portion upstream of 5 ′ side of hTPC5 or downstream of 3′side of hTPC3. An attempt was made to obtain a cDNA fragment. One million phages of the cDNA library were type I, and four combinations of primers (hTPC 5 vs. 5's gt10 or 3's gtl O and hTPC 3 vs. 5 'A gtl O Alternatively, 3's gt 10) PCR was performed according to the method described above. However, the annealing temperature was 55 ° C instead of 65 ° C. As a result, a DNA fragment corresponding to approximately 1.5 kbp upstream of the 5 ′ side of hTPC5 was amplified.
(2) ヒ ト ' テロメラーゼ蛋白質遣伝子全長 c DN Aの取得 (2) Acquisition of human full-length telomerase protein gene cDNA
まず、 R a j i細胞及び PA— 1細胞それぞれ約 1億個から、 RNA z o l溶 液 （T e 1 — T e s t社） を用いて C h omc z y n s k iの方法 （An a 1. B i o c h em. 、 1 62、 1 56 - 1 59、 1 987 ) により総 RN Aを取得 し、 得られた総 RNAを 0 1 i g o— d Tセルロースカラム （ t y p e 7、 l cmx l cm、 Ph a rma c i a社） に付してそれぞれ約 100/ gの p o l y (A) + RNAを得た。  First, the Chomc zynski method (An a 1. Bioch em., 1) was performed using RNA zol solution (T e1 — Test) from about 100 million Raji cells and PA-1 cells, respectively. 62, 156-159, 1987) to obtain total RNA, and apply the obtained total RNA to a 0.1 igo-d T cellulose column (type 7, lcmxlcm, Pharmacia). As a result, about 100 / g of poly (A) + RNA was obtained.
c DNAの合成には、 p o l y (A) + RNA 5 μ gを铸型に用いた。 反応には c DNA s y n t h e s i s mo d u l e (Ame r s h a m社) に添付さ れた逆転写酵素、 リボヌクリア一ゼ1^、 大腸菌 DN Aポリメラーゼを用い、 添付 の説明書に従って二本鎖 c DN Aを合成した。 次に、 cDNA s yn t h e s i s mo d u l e (Ame r s h a m社） に添付された T 4 DNAポリ メラ一ゼを 用いて c DN A末端の平滑化を行った。 反応終了後、 フヱノ ール/ク ロ口ホルム 抽出を行い、 上清の水層を回収した。 回収した水層と等容量の 5 M酢酸アンモニ ゥム溶液を添加後、 2倍容量のエタノールを混和した。 その後、 1 5、 0 0 0 X g、 1 0分間の遠心分離を行い、 エタノール沈澱により c DNAの回収を行った。 回収した c D N Aを乾燥して 2 0 1 の滅菌脱塩水に溶解した後、 1 0 1の c DNA (約 2 g) を分取して、 その末端に E c o R Iアダプタ一 （宝酒造） を付加した。 すなわち、 2 0〃 1 の T 4 DNAリガーゼ反応液 「 6 6 mM卜リス 塩酸緩衝液 （p H 7. 6) 、 6. 6 mM M g C 10 (和光純薬） 、 1 O mMジチ オスレィ トール （D T T、 和光純薬） 、 6 6 mMアデノ シン 5 ' —三リ ン酸For cDNA synthesis, 5 μg of poly (A) + RNA was used for type I. For the reaction, double-stranded cDNA was synthesized using reverse transcriptase, ribonuclease 1 ^ and Escherichia coli DNA polymerase attached to cDNA synthesis module (Amersham) according to the attached instructions. Next, the T4 DNA polymerase attached to the cDNA synthesis module (Amersham) was used. Was used to blunt the cDNA end. After the completion of the reaction, phenol / chloroform extraction was performed, and the aqueous layer of the supernatant was recovered. After adding an equal volume of a 5 M ammonium acetate solution to the recovered aqueous layer, 2 volumes of ethanol were mixed. Thereafter, centrifugation was performed at 15, 000 X g for 10 minutes, and cDNA was recovered by ethanol precipitation. The recovered cDNA is dried and dissolved in 201 sterile demineralized water. Then, 101 cDNA (approximately 2 g) is collected and Eco RI adapter 1 (Takara Shuzo) is added to the end. did. That is, 20〃1 of T4 DNA ligase reaction solution “66 mM Tris-HCl buffer (pH 7.6), 6.6 mM MgC 10 (Wako Pure Chemical Industries, Ltd.), 1 OmM dithiolysate Toll (DTT, Wako Pure Chemical), 66 mM adenosine 5'-triphosphate
(AT P、 S I GMA社） 」 中で 3 5 0単位の T 4 D NAリガーゼ （宝酒造） と ともに 1 6 °Cで 2時間ィンキュベ一 卜 し、 2 0 0 p m o 1 eの E c o R I ァダプ ターを c DN Aの末端に結合した。 (ATP, SI GMA) incubate with 350 units of T4 DNA ligase (Takara Shuzo) at 16 ° C for 2 hours to obtain a 200 pmo 1 e Eco RI adapter. Was attached to the end of cDNA.
反応物を常法に従い S e p h a c r y l S— 2 0 0カラム （l c m x 4 c m) に展開し、 I mM E D TAと 0. 5 mM N a C 1を含む 1 0 mMトリス塩酸 緩衝液 （p H 7. 5 ) を用いて、 末端に E c o R Iアダプタ一を付加した c DNA を溶出した。 溶出した c DN Aをエタノール沈澱で回収し、 沈澱を乾燥後、 2 β The reaction product was developed on a Sephacryl S-200 column (lcmx 4 cm) according to a conventional method, and a 10 mM Tris-HCl buffer solution (pH 7.0) containing ImM EDTA and 0.5 mM NaC1 was used. Using 5), the cDNA to which an EcoRI adapter was added at the end was eluted. The eluted cDNA was recovered by ethanol precipitation, and the precipitate was dried.
1の滅菌脱塩水に溶解した。 あらかじめ制限酵素 E c 0 R I (宝酒造） で消化後 に末端を脱リ ン酸ィ匕した λ Z A Pファージ DNA (S t r a t a g e n e社） 1Dissolved in 1 sterile demineralized water. ΛZAP phage DNA (Stratagene) with digestion with Ec0RI (Takara Shuzo) and dephosphorylation of the ends beforehand
/z gと、 E c o R Iアダプターを付加した上記の c D NA (4 0 0 n g) とを、/ z g and the above c DNA (400 ng) to which the E co R I adapter has been added,
1 6 の T 4 DNAリガーゼ反応液 （5 1 ) 中で 1 8時間ィンキュペートして 結合させた。 さ らに、 c D N A と結合した λ Ζ Α Ρフ ァ ージ D N AをIncubation was carried out for 18 hours in the T4 DNA ligase reaction solution (51) of 16 for binding. In addition, λ Ζ Α Α phage D N A combined with c D N A
G i g a p a c k l I G o l d (S t r a t a g e n e社） を用いてファージ粒 子へパッケージングした。 得られたファージ粒子を常法に従って大腸菌 C 6 0 0 h f 1 A株に感染及び增 幅させてファ一ジ粒子を回収した。 一連の操作により、 l O O n gの c DN Aあ たり約 2 0 0万のファージクローンを得た。 約 1 0 0万のファージクローンを常 法に従って大腸菌 C 6 0 0 h f 1 A株に感染させ、 プレート上の N Z Y寒天培地 上で培養した。 ナイ口ン膜にファージ粒子を写し取ってレプリカを 2枚作製し、 洗浄及びアルカリ処理した後、 実施例 2の工程（1) で得られたヒ ト ·テロメラーゼ 蛋白質 c DNA断片を32 P標識してプローブとして用い、 このプローブにハイプリ ダイズするファージ ■ クローンをスクリーニングした。 得られた陽性シグナルに ついて フ ァ ー ジ粒子を回収 し、 同様な方法で ク ロ ー ン化 した後、 S t r a t a g e n e社のマニュアルに従い、 揷入された c DNA部分を含むプ ラスミ ドを i n v i v o e x c i s i o n法にて回収した。 It was packaged into phage particles using Gigapackl IG old (Stratagene). The obtained phage particles were infected and spread in E. coli C600hf1A strain according to a conventional method, and phage particles were collected. Through a series of operations, about 200,000 phage clones were obtained per 100 ng of cDNA. About 100,000 phage clones were infected with E. coli C600hf1A strain according to a conventional method, and cultured on a NZY agar medium on a plate. Copy the phage particles onto the membrane to make two replicas, After washing and alkali treatment, the human telomerase protein cDNA fragment obtained in the step (1) of Example 2 was labeled with 32 P and used as a probe, and phage clones hybridizing to the probe were screened. The phage particles were collected from the obtained positive signal, cloned by the same method, and the plasmid containing the inserted cDNA was purified by the in vivooexcision method according to the manual of Stratagene. Collected at
(3) 完全長ヒ ト ' テロメラーゼ蛋白質 c DNA 3' 側下流配列の取得 （ 3' — R A C E法） (3) Acquisition of downstream sequence of full-length human 'telomerase protein cDNA 3' (3'-RACE method)
上記工程（2) で得られた mRN Aを铸型にして、 Ma r a t h o nTm c DNA Amp l i f i c a t i on k i t (C l on t e c h社） を用いて、 RACE 法による c DNAの増幅を行った。 以下の反応において、 合成 DNAプライマ— The mRNA obtained in the above step (2) was converted into type 铸, and cDNA was amplified by the RACE method using a Marathon Tm cDNA Amplification kit (Clontech). In the following reactions, synthetic DNA primers
TU  TU
は、 M a r a t h 0 n 1M cDNA Am l i f i c a t i o n k i tに添付 されたプライマ一以外は、 AB I 394 DN A合成機を用いて合成した。 反応は、 Ma r a t h o n c D N A Amp l i f i c a t i o n k i tに添付され た緩衝液および d NT Pを用いて行った。 Is, M arath 0 n 1M cDNA Am primer one except attached to lificationkit were synthesized using AB I 394 DN A synthesizer. The reaction was performed using the buffer and dNTP attached to the Rathonc DNA Amplification kit.
まず、 c DN Aの合成を行った。 精製した p o 1 y (A) + RNA l i gと c D N A 逆 転 写 プ ラ イ マ — 、 5 ' -  First, cDNA was synthesized. Purified p o 1 y (A) + RNA lig and c D NA reverse transcription primer —, 5'-
(G/A/C) (G/A/C/T) - 3' (52ヌクレオチド） を逆転写酵素に て 37°Cで処理し、 第 1鎖 c DNAを合成した。 第 2鎖伸長反応及び末端の平滑 化を行い、 c D N Aの両端に ア ダ プタ ー プラ イ マ 一 、 [ 5 ' — 一 3' (44ヌクレオチド） と 5' - P04 —ACCTGCC C— NH2 — 3' (8 ヌクレオチド） ] を結合させた。 最終反応の反応液 1 0 1を希釈して 50 1 とし、 以後の増幅反応に 1 H 1を用いた。 (G / A / C) (G / A / C / T) -3 ′ (52 nucleotides) was treated with reverse transcriptase at 37 ° C. to synthesize first-strand cDNA. Performing a second chain extension reaction and ends of smoothing, A da descriptor over plug Lee Ma foremost ends of the c DNA, [5 '- one 3' (44 nucleotides) and 5 '- P0 4 -ACCTGCC C- NH 2 — 3 '(8 nucleotides)]. The reaction solution 101 of the final reaction was diluted to 50 1, and 1 H1 was used for the subsequent amplification reaction.
増幅反応は、 ヒ ト 'テロメラーゼ蛋白質 c DNAの配列の一部と相補的なブラ イマ一および 3' 末端に付加したアダプタープライマーと相補的なプライマー 〔5' -C CATC CTAATACGACTCACTATAGGGC - 3' (27ヌク レオチド） 〕 、 並びに T a Q DN Aポリメラーゼを用いて行った。 反応液の全量 を 50 1とし、 94 °Cで 1分間のインキュベーションの後、 94 °Cで 30秒間、 60てで 30秒間、 及び 68°Cで 5分間のィンキュベーシヨンを 30サイクル行 い、 最後に 72 で 7分間のィンキュベ一ションを行って反応を終了した。 反応 液の 1 0分の 1量を 5 %P AGEにて解析した。 また、 上記反応液のうち 5 1 を 50倍希釈し、 その 5 / 1を用いて 2回目の増幅反応を行った。 The amplification reaction was carried out using a primer complementary to a part of the sequence of the human 'telomerase protein cDNA and a primer complementary to the adapter primer added to the 3' end (5'-C CATC CTAATACGACTCACTATAGGGC-3 '(27 nuclei Reotide)], and TaQDNA polymerase. Set the total volume of the reaction solution at 501, and incubate at 94 ° C for 1 minute, and then perform 30 cycles of incubation at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 5 minutes. Finally, incubation was performed at 72 for 7 minutes to complete the reaction. One-tenth volume of the reaction solution was analyzed by 5% PAGE. In addition, 51 of the above reaction solution was diluted 50-fold, and a second amplification reaction was performed using 5/1.
2回目の増幅反応は 1回目の増幅反応に準じて行った。 希釈反応液 5 1を铸 型とし、 ヒ ト 'テロメラーゼ蛋白質 c DN Aの配列の一部と相補的で 1回目の増 幅反応に用いたプライマーよ り内側に位置するプライマーおよび 5 ' — ACTCACTATAGGGCTCGAGCGGC- 3' (23ヌクレオチド） を用いて、 T a q DNAポリメラーゼでの増幅反応を行った。 反応液の全量を 50 〃 1 とし、 94 °Cで 1分間のインキュベーショ ンの後、 94°Cで 30秒間、 60 。Cで 30秒間、 及び 68°Cで 5分間のィンキュベ一シヨンを 30サイクル行い、 最後に 72°Cで 7分間のィンキュベーションを行って反応を終了した。 反応終了 後、 反応液の 1 0分の 1量を 5 %P AGEにて解析した。  The second amplification reaction was performed according to the first amplification reaction. The diluted reaction mixture 51 was made into type III, a primer complementary to a part of the sequence of human 'telomerase protein cDNA and located inside the primer used for the first amplification reaction, and 5' — ACTCACTATAGGGCTCGAGCGGC- An amplification reaction with Taq DNA polymerase was performed using 3 ′ (23 nucleotides). Bring the total volume of the reaction mixture to 50〃1 and incubate at 94 ° C for 1 minute followed by 60 at 94 ° C for 30 seconds. The incubation was performed at 30 ° C. for 30 seconds and at 68 ° C. for 5 minutes for 30 cycles, and finally, the reaction was completed at 72 ° C. for 7 minutes. After completion of the reaction, one-tenth volume of the reaction solution was analyzed by 5% PAGE.
次に、 ゲル断片から増幅した c DN A断片を回収して精製し、 T4DNAリガ一 ゼを用いてプラス ミ ドベクタ一 p CR I I ( I n V i t r o g e n社） のクロー ニング部位に挿入して、 得られた組み換えベクターで大腸菌 J M 1 09株を形質 転換した。 X— G a 1— I PTG— LB— Amp寒天培地上に出現した耐性菌で、 かつ X— G a 1により発色していない 3つの形質転換体について、 常法に従いプ ラスミ ド DN Aを調製し、 解析を行った。 さらに、 調製したプラスミ ド DNAを 用いて c DN Aの塩基配列を決定した。 その結果、 3' 非翻訳領域の塩基配列を 有する c DN A断片を得た。  Next, the cDNA fragment amplified from the gel fragment was recovered, purified, and inserted into the cloning site of plasmid vector pCRII (Invitrogen) using T4 DNA ligase to obtain the DNA fragment. Escherichia coli JM109 strain was transformed with the obtained recombinant vector. X—Ga1—I PTG—LB—Amplified DNA from three transformants that appeared on the agar medium and were not colored by X—Ga1 according to the standard method Then, the analysis was performed. Furthermore, the nucleotide sequence of cDNA was determined using the prepared plasmid DNA. As a result, a cDNA fragment having the nucleotide sequence of the 3 ′ untranslated region was obtained.
(4) 完全長ヒ ト ' テロメラ一ゼ蛋白質 c DN A 5' 側上流配列の取得 （5' — RAC E法）  (4) Acquisition of full-length human 'telomerase protein c DNA 5' upstream sequence (5 '— RACE method)
5 ' —RAC E法の反応は、 3' —RAC E法に準じて行った。 合成 DNAプ ライマーは、 Ma r a t h o n cDNA Amp l i f i c a t i o n k i t に添付されたプライマー以外は A B I 394 DNA合成機を用いて合成した。 反 応は、 Ma r a t h o nTM c DNA Amp l i f i c a t i o n k i tに添 付された緩衝液および d NT Pを用いた。 铸型としては、 3' —RAC E法の反 応と同様に、 両端にアダプタ一プライマーを付加した c DNAを用いた。 1回目 の増幅反応は、 ヒ ト 'テロメラーゼ蛋白質 c DN Aの配列の一部と相補的プライ マ一および 3' 末端に付加したアダプタープライマーと相補的な、 3' -RACE 法 の 反 応 の 際 に も 用 い た プ ラ イ マ — 、 5 ' — C CATC CTAATACGACTCACTATAGGGC— 3' (27ヌクレ ォチド） を用いた。 反応液は全量を 50 1 として、 T a q DN Aポリメラーゼ を用いて増幅反応を行った。 反応は、 94°Cで 1分間のイ ンキュベーショ ンの後、 94°Cで 3 0秒間、 60°Cで 30秒間、 及び 68 °Cで 5分間のィンキュベーショ ンを 30サイクル行い、 最後に Ί 2 °Cで 7分間のィンキュベ一シヨンを行って反 応を終了した。 反応後、 反応液の 1 0分の 1量を 5 %P AGEにて解析した。 ま た、 上記反応液のうち 5 ;u 1を 50倍希釈し、 その 5 1を铸型として用いて、 2回目の増幅反応を行った。 The reaction of the 5'-RAC E method was performed according to the 3'-RAC E method. Synthetic DNA primers were synthesized using an ABI 394 DNA synthesizer, except for the primers attached to the Rathon cDNA Amplification kit. Anti For the reaction, a buffer and dNTP attached to Marathon ™ cDNA Amplification kit were used. As type III, a cDNA to which an adapter-primer was added at both ends was used as in the reaction of the 3'-RACE method. The first amplification reaction was performed during the reaction of the 3'-RACE method, which was complementary to a part of the sequence of the human 'telomerase protein cDNA and a primer complementary to an adapter primer added to the 3' end. The primer used for the following step was 5'-C CATC CTAATACGACTCACTATAGGGC- 3 '(27 nucleotides). The total amount of the reaction solution was adjusted to 50 1 and an amplification reaction was carried out using Taq DNA polymerase. The reaction was performed at 94 ° C for 1 minute, followed by 30 cycles of incubation at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 5 minutes, and finally Ί2 The reaction was completed at 7 ° C for 7 minutes. After the reaction, one-tenth volume of the reaction solution was analyzed by 5% PAGE. In addition, 5; u1 of the above reaction solution was diluted 50-fold, and the second amplification reaction was performed using 51 as type III.
2回目の増幅反応は 1回目の増幅反応に準じて行った。 プライマーとしては、 、 ヒ ト .テロメラーゼ蛋白質 c DN Aの配列の一部と相補的で 1回目の増幅反応に 用いた プラ イ マ ー よ り 内側に位置す る プラ イ マ ーお よ び 5 ' - AC TCACTATAGGGCTC GAGC GGC— 3' (23ヌ ク レオチ ド） を用いて行った。 反応は、 94°Cで 1分間のインキュベーションの後、 94°Cで 30秒間、 60。Cで 30秒間、 及び 68 で 5分間のイ ンキュベーシ ョ ンを 3 0 サイクル行い、 最後に 7 2 で 7分間のィンキュベ一ションを行って反応を終了 した。 反応後、 反応液の 1 0分の 1量を 5 %P AGEで解析した。 ゲル断片から 増幅した c DN Aを回収して精製し、 プラスミ ドベクタ一 p CR I Iのクロー二 ング部位に挿入した後、 得られた組み換えべク一を用いて大腸菌 J M 1 09株を 形質転換した。 X - G a 1— I PTG— L B— Amp寒天培地上に出現した耐性 菌で、 かつ X— G a 1により発色していない 3つの形質転換体について、 常法に 従い、 プラスミ ド DNAを調製した。 調製したプラスミ ド DNAを用いて解析を 行い、 さらに、 塩基配列の決定を行った。 その結果、 ヒ ト 'テロメラ一ゼ蛋白質 の 5' 非翻訳領域の配列を有する c DNA断片を得た。 実施例 3 ： ヒ ト ' テロメラーゼ蛋白質遺伝子の取得 The second amplification reaction was performed according to the first amplification reaction. As primers, primers complementary to a part of the sequence of human telomerase protein cDNA and located inside the primer used in the first amplification reaction and 5 ' -Performed using AC TCACTATAGGGCTC GAGC GGC-3 '(23 nucleotides). The reaction was incubated at 94 ° C for 1 minute followed by 60 minutes at 94 ° C for 30 seconds. The incubation was performed for 30 cycles at 30 seconds at C and for 5 minutes at 68, followed by incubation at 72 for 7 minutes to complete the reaction. After the reaction, one-tenth volume of the reaction solution was analyzed with 5% PAGE. The cDNA amplified from the gel fragment was recovered, purified, inserted into the cloning site of plasmid vector pCRII, and the resulting recombinant vector was used to transform Escherichia coli JM109. . X-Ga1—I PTG—LB—Amplified plasmid DNA from 3 transformants that appeared on the agar medium and did not develop color due to X—Ga1 according to the standard method did. Analysis was performed using the prepared plasmid DNA, and the nucleotide sequence was determined. As a result, the human telomerase protein A cDNA fragment having the sequence of the 5 'untranslated region was obtained. Example 3: Obtaining human 'telomerase protein gene
(1) ヒ ト ' テロメラーゼ蛋白質遺伝子全長 c DN Aの取得  (1) Obtaining the full-length human telomerase protein gene cDNA
ラッ ト .テロメラーゼ蛋白質遺伝子を取得したのと同様、 まず、 PA— 1細胞 を用いて c DN Aライブラリーを作成した。 このライブラリ一を、 前述の hTPC 5 (配列表の配列番号 1 1 ) と前述の h TP C 3 (配列表の配列番号 1 2) をブラ イマ一として用いた PC R産物をプローブにしてスクリ一二ングを行い、 ヒ ト ■ テロメラーゼ蛋白質遺伝子全長 c DN Aを取得した。  As in the case of obtaining the rat telomerase protein gene, a cDNA library was first prepared using PA-1 cells. This library was screened using the PCR product obtained by using the aforementioned hTPC5 (SEQ ID NO: 11 in the sequence listing) and the aforementioned hTPC3 (SEQ ID NO: 12 in the sequence listing) as a primer. The human ■ telomerase protein gene full-length cDNA was obtained.
まず、 PA— 1細胞から p o 1 y (A) T RNAを得た。 即ち、 細胞 1 08個を、 グァニジンイソチオシァネート溶液中でホモジナイズし、 0. 1容量の 2 M酢酸 ナトリウム （pH4. 0) を加えて混和した。 このホモジュネートに等容量の水 飽和フヱノール及び 0. 2容量のク口口ホルム/ィソァミルアルコール混合液を 加えて激しく混和し、 遠心分離により上清の水層を回収した。 回収した水層に等 容量のイソプロパノールを混和し、 一 20度で 1時間冷却した後、 遠心分離を行 つた。 得られた沈殿物を再びグァニジンイソチオシァネート溶液に溶解し、 等容 量のイソプロパノールを加え、 一 20度で 1時間冷却した後、 遠心分離により総 RN Aを回収した。 First, po 1 y (A) T RNA was obtained from PA-1 cells. That is, 1 0 8 cells were homogenized in guaiacolsulfonate two gin isothiocyanate Xia sulphonate solution was added and mixed of 2 M sodium acetate 0.1 volume (pH 4. 0). An equal volume of water-saturated phenol and 0.2 volume of a mixed solution of formaldehyde / isoamyl alcohol were added to the homogenate, mixed vigorously, and the aqueous layer of the supernatant was recovered by centrifugation. An equal volume of isopropanol was mixed with the collected aqueous layer, cooled at 120 ° C for 1 hour, and centrifuged. The obtained precipitate was again dissolved in a guanidine isothiocyanate solution, an equal volume of isopropanol was added, the mixture was cooled at 120 ° C. for 1 hour, and then total RNA was recovered by centrifugation.
総 RNAを I mM E D T A、 20 mM卜リス塩酸 （ p H 7. 5) に溶解し、 70て、 5分間の熱処理後、 氷上で急冷した。 この溶液に N a C 1溶液を終濃度 が 0. 5 Mになるように加えて、 01 i g o— d Tセルロースカラム （ t y p e 7、 l cmx l cm、 Ph a rma c i a社） に展開し、 ImM EDTAぉょび0. 5M N a C 1を含む 2 OmMトリス塩酸緩衝液 （p H 7. 5) でカラムを洗浄 後、 滅菌脱塩水で結合分画を溶出して P o 1 y (A) + RNAを得た。 Total RNA was dissolved in ImM EDTA and 20 mM Tris-HCl (pH 7.5), heat treated for 70 minutes, and quenched on ice. NaC1 solution was added to this solution to a final concentration of 0.5 M, and developed on a 01 igo-d T cellulose column (type 7, lcmxlcm, Pharmacia). After washing the column with 2 OmM Tris-HCl buffer (pH 7.5) containing EDTA and 0.5 M NaC1, the bound fraction is eluted with sterile demineralized water and Po1y (A) + RNA was obtained.
この p o l y (A) + RNAから S t r a t a g e n e社の c DNA合成キッ ト を用いて c DN Aを調製した。 1 s t s t r a n d合成はプライマ一としてラ ンダムへキサマー ·ォリゴヌクレオチドとォリゴ d tプライマーの両方を最終濃 度各 2 /M加えて行った。 T 4 DN Aポリメラーゼを用いて c DN A末端の平滑 化を行った後、 末端に E c o R I アダプタ—を付加した。 反応産物を S e p h a c r y l S— 500力ラムに展開し、 未反応の E c o R Iアダプタ一 とサイズの小さい c DN Aを除いた。 c DNAをエタノール沈殿で回収し、 λ ΖΑΡ ファージ DN Αに挿入した。 From this poly (A) + RNA, cDNA was prepared using a cDNA synthesis kit from Stratagene. 1ststrand synthesis was performed by adding both random hexamer oligonucleotide and oligonucleotide dt primer as primers to a final concentration of 2 / M each. Blunt cDNA ends using T4 DNA polymerase After the conversion, an Eco RI adapter was added to the end. The reaction product was developed on Sephacryl S-500 force column to remove unreacted EcoRI adapter and small size cDNA. cDNA was recovered by ethanol precipitation and inserted into λλ phage DNΑ.
c D N Aと結合したス ZAPファージ DNAを、 S t r a t a g e n e社の G I GAPACK GOLD I I Iキッ トを用いて、 ファージ粒子へパッケージ ングした。 一連の操作により、 約 1 000万のファージクローンを得た。  The sZAP phage DNA bound to cDNA was packaged into phage particles using a GI GAPACK GOLD II kit from Stratagene. By a series of operations, about 10 million phage clones were obtained.
約 1 00万のファージクローンを常法に従い大腸菌 C 600 h f 1 A株に感染 させ、 プレー ト上の N Z Y寒天培地上で培養した。 ナイロン膜にファージ粒子を 写し取ったレプリカを 2枚作製し、 洗浄及びアル力リ処理した。 h T P C 5と hTPC 3をプライマーとして用いた PC R産物を32 P標識してプロ一ブとして用 い、 このプローブにハイプリダイズするファージ · クローンをスクリ一ニングし た。 その結果、 2つの陽性シグナルを見出したので、 それらについてファージ粒 子を回収し、 同様な方法でクローン化した後、 挿入された c DN A部分を含むプ ラスミ ド （pHB 0 1、 pHB 04) を i n v i v o e x c i s i o n法に て回収した。 Approximately 1 million phage clones were infected with E. coli C600hf1A strain according to a conventional method, and cultured on NZY agar medium on a plate. Two replicas in which the phage particles were copied onto a nylon membrane were prepared, washed, and washed. A PCR product using hTPC5 and hTPC3 as primers was labeled with 32 P and used as a probe, and phage clones hybridizing to this probe were screened. As a result, two positive signals were found, and phage particles were recovered from them, cloned in the same manner, and the plasmid containing the inserted cDNA portion (pHB01, pHB04) Was recovered by the invivoexcision method.
プラスミ ド pHB 0 1、 p HB 04について制限酵素切断地図を作製したとこ ろ、 各々 1. l k b p、 7. 4 k b pの c DNAが揷入されており、 図 4に示す ような、 重複する位置関係にあることがわかった。 常法に従って欠失変異 c DN A を作製し、 pHB 0 1、 p HB 04の DNA配列を解読した。 この DN A配列を 制限酵素切断地図に従って組み合わせたところ、 約 8. 1 k b pにわたる領域を カバーし、 この中に C末端側のストップ ' コ ドンを含む長大なオープン · リ一デ イ ング . フレイムが見出された。 このオープン · リ一ディ ング · フレイムから予 測されるァミノ酸配列がラッ ト ·テロメラ一ゼ蛋白質の C末端側のァミノ酸配列 と 70%以上の同一性という高い相同性を示したことから、 この配列がヒ 卜 ·テ ロメラ一ゼ蛋白質のものであると判断した。  When restriction maps were prepared for plasmids pHB01 and pHB04, cDNAs of 1.lkbp and 7.4 kbp were inserted, respectively, and overlapping positional relationships as shown in Fig. 4 It turned out to be. A deletion mutant cDNA was prepared according to a conventional method, and the DNA sequences of pHB01 and pHB04 were decoded. When the DNA sequences were combined according to a restriction map, a large open reading frame covering a region of about 8.1 kbp, including a C-terminal stop 'codon, was included. Was found. The amino acid sequence predicted from this open reading frame showed high homology of 70% or more identity with the amino acid sequence at the C-terminal side of rat telomerase protein. This sequence was determined to be that of the human telomerase protein.
(2) ヒ ト 'テロメラーゼ蛋白質 c DN Aの取得—上流配列の取得 （ 5' -RACE 法） 工程（1) で得られた DN A配列は配列表の配列番号 13に示す DN A配列のうち 核酸番号 756番目以降の配列であつたが、 ラッ ト 'テロメラ一ゼ蛋白質との一 次構造の比較から、 オープン ' リーディ ング ' フレイムが N末端側に向かって、 さらに伸びていると考えられた。 そこで、 さらに 5' 側の mRN Aの配列につい て 5' — R a p i d Amp l i r i c a t i o n o f c DNA E n d s(2) Obtaining human 'telomerase protein cDNA—Obtaining upstream sequence (5'-RACE method) The DNA sequence obtained in the step (1) was the sequence of the nucleic acid sequence from position 756 onwards of the DNA sequence shown in SEQ ID NO: 13 in the sequence listing, but it had the primary structure of rat 'telomerase protein. From the comparison, it was considered that the open 'leading' frame extended further toward the N-terminal. Therefore, 5'-Rapid Amp liricationofc DNA Ends
(RACE) 法を用いて検討した。 (RACE) method.
5 ' — R A C E法は、 C 1 o n t e c h社の 5' — RACEキッ 卜を用い、 マ ニュアルに従い行った。 工程（1) において PA— 1細胞から得られた p o 1 y (A) + RN A 2 gと、 配列表の配列番号 13の核酸番号 1 165〜 1 1 87番目の部 分に相補的な DN A配列のォリゴヌクレオチドプライマ一 T L P CM3  The 5'-RACE method was carried out according to the manual using a 5'-RACE kit of C1onetech. In step (1), 2 g of po1y (A) + RNA obtained from PA-1 cells and a DN complementary to the nucleic acid number 1165 to 1187 of SEQ ID NO: 13 in the sequence listing Oligonucleotide primers of A sequence TLP CM3
1 0 pm o 1 とを混合し、 加熱した後に急冷した。 反応混合物に逆転写酵素 After mixing with 10 pm o 1 and heating, the mixture was quenched. Reverse transcriptase to reaction mixture
(G I B CO BRL社の S u p e r S c r i p t I I ) 、 基質ヌクレオチド、 及び緩衝液を加えて 42 °Cで 1時間反応させた。 E DT Aを加えて反応を停止さ せた後、 アルカリ処理で铸型 RNAを分解し、 イソプロパノール沈殿を行って単 鎖 c DNAを単離した。 さらに、 この c DNAの半量に、 5' —RACE用アン 力一プライマ一 [5' - P ( + ) ANC] 4 pmo 1を RN Aリガーゼを用いて 連結させた。 (SUPER SCRIPTII from GIBCO BRL), a substrate nucleotide, and a buffer were added and reacted at 42 ° C. for 1 hour. After terminating the reaction by adding EDTA, type I RNA was decomposed by alkali treatment, and single-stranded cDNA was isolated by isopropanol precipitation. Further, half of this cDNA was ligated with 5′-RACE force primer [5′-P (+) ANC] 4 pmo 1 using RNA ligase.
次に、 TL PCM3で逆転写プライムされ、 さらに 3' 端にアンカ一 DNA配 列を付加された単鎖 c DNAを铸型として、 アンカー DN Aに相補的なォリゴヌ クレオチドプライマ一 RACE— PRM 2と、 配列表の配列番号 1 3の核酸番号 1024 - 1046の部分に相補的な DNA配列のオリゴヌクレオチドプライマ一 TLPNEとを用いて、 P C Rによる DNA増幅を行った。 反応には 20分の 1 量の単鎖 c DNAと各々 l O pmo lのプライマーとを用い、 G I BCO B RL 社の T a qポリメラーゼを用いて添付のマニュアルに従って P CRを行った。 た だし、 非特異的な DN A増幅を避けるために、 反応はマニュアル .ホッ ト .スター ト法で開始した後、 94 °Cで 30秒、 60 で1分、 72 °Cで 2分のサイクルを 35回繰り返した。  Next, a single-stranded cDNA that was reverse-transcribed with TL PCM3 and further added with an anchor DNA sequence at the 3 ′ end was type III, and the oligonucleotide primer RACE—PRM 2 complementary to the anchor DNA was used. The DNA was amplified by PCR using an oligonucleotide primer TLPNE having a DNA sequence complementary to the nucleic acid number 1024-1046 of SEQ ID NO: 13 in the sequence listing. In the reaction, PCR was performed using a 1/20 amount of single-stranded cDNA and each lOpmol primer, and using Taq polymerase from GICO BRL in accordance with the attached manual. However, to avoid non-specific DNA amplification, the reaction was started with a manual hot-start method and then cycled for 30 seconds at 94 ° C, 1 minute at 60, and 2 minutes at 72 ° C. Was repeated 35 times.
P C R産物を p T 7 B 1 u e Tベクターに組み込み、 増幅 DN Aの揷入された ものについて DN A配列を解読した結果、 これらのうちの 3クローンが殆ど同じ DNA配列を有していた。 これらのクローンのうち、 代表的なクローンである RACE— L 4は図 4に示す位置に存在するものであった。 配列表の配列番号 13 の核酸番号 1 56〜 1 58に開始コ ドンが存在し、 さらに上流の同じく核酸番号 144〜 146に同一フレームの終始コ ドンが存在した。 開始コ ドンの 5' 側上 流 1 57 b pまで、 増幅された DN Aの長さがほぼ均一であることから、 実際の mRN Aの 5 ' 端に対応する c D N Aを得た可能性が高いと考えられた。 実施例 4 ：組換えラッ ト ·テロメラーゼ蛋白質の取得及び特異抗体の作製 日本住血吸虫グルタチオン一 S— トランスフエラ一ゼとラッ ト ' テロメラーゼ 蛋白質 （配列表の配列番号 1のァミノ酸番号 2 1 7〜345番目に相当する部分 ポリペプチド） との融合蛋白質 （GST— p 80 h om) を大腸菌を用いて発現 させ、 精製した遣伝子産物を抗原としてゥサギを免疫した。 次に、 ラッ ト ·テロ メラーゼ蛋白質の同じ部分を別の発現べク夕一を用いてヒスチジン ·へキサマー との融合蛋白質 （6H i s - p 80 h om) として発現させ、 精製した遣伝子産 物を用いてァフィ二ティ ·カラムを作製し、 ゥサギ抗血清からラッ 卜 ' テロメラ一 ゼ蛋白質を認識するポリクローナル抗体 （配列表の配列番号 1のアミノ酸番号 217 〜345番目に相当する部分に特異的なポリクロ一ナル抗体） を取得した。 まず、 発現プラスミ ドベクター pGEX 2 T (P h a rma c i a社） を制限 酵素 Sm a Iで切断した後、 H i n d I I I切断部位を有するオリゴヌクレオチ ド · リ ンカーを挿入し、 発現べクタ一 p GEXH 12を作製した。 このベクター を制限酵素 E c 0 R Iで切断した後、 T 4ポリメラーゼ （東洋紡） を用いて末端 を平滑化し、 さらに制限酵素 H i n d I I Iで切断した。 次に、 ラッ ト ' テロメ ラーゼ蛋白質 c DN A断片含むプラスミ ド R a P C 53を制限酵素 B a mH Iで 切断し、 T 4ポリメラーゼ （東洋紡） を用いて末端を平滑化した。 その後、 制限 酵素 H i n d I I Iにてさらに切断して、 ポリアク リルァミ ド · ゲル電気泳動に 付してラッ ト ' テロメラーゼ蛋白質 c DNAの部分 DN A断片 （配列表の配列番 号 1の核酸番号 648〜； 1 034に相当する約 390 b pの H i n d i I I - B a mH I由来平滑末端の D N A断片） を単離した。 以上により得られた H i n d I I I—平滑末端の p GEXH 1 2ベクタ一とラッ ト ' テロメラ一ゼ蛋 白質 c DNA由来 DNA断片とを DNAライゲ一シヨ ン 'キッ ト （宝酒造） を用 いて連結させ、 得られた組み換えべクタ一を用いて大腸菌株 J M 109 (東洋紡） を形質転換した。 アンピシリ ン耐性のクローンについて各プラスミ ドの制限酵素 切断地図を作成し、 正しい組み換えプラスミ ドを保有している pGEXp 80hom / J M 1 09を選択した。 The PCR product was incorporated into the pT7B1ueT vector, and the amplified DNA was inserted. As a result of decoding the DNA sequence, three of these clones had almost the same DNA sequence. Among these clones, a representative clone, RACE-L4, was located at the position shown in FIG. An initiation codon was present at nucleic acid numbers 156 to 158 of SEQ ID NO: 13 in the sequence listing, and a termination codon of the same frame was present at nucleic acid numbers 144 to 146 upstream. Since the length of the amplified DNA is almost uniform up to 157 bp upstream of the start codon, it is highly likely that the cDNA corresponding to the 5 'end of the actual mRNA was obtained. It was considered. Example 4: Acquisition of recombinant rat telomerase protein and preparation of specific antibody Schistosoma japonicum glutathione-1 S-transferase and rat 'telomerase protein (amino acid number 2 17 to SEQ ID NO: 1 in the sequence listing) A fusion protein (GST-p80hom) with the 345th partial polypeptide was expressed using Escherichia coli, and the purified gene product was used as an antigen to immunize egrets. Next, the same portion of the rat telomerase protein was expressed as a fusion protein with histidine-hexamer (6His-p80hom) using another expression vector, and purified gene product was used. A polyclonal antibody that recognizes rat 'telomerase protein from egret antiserum (specific for the portion corresponding to amino acids 217 to 345 of SEQ ID NO: 1 in the sequence listing) Polyclonal antibody). First, the expression plasmid vector pGEX2T (Pharmacia) is cleaved with the restriction enzyme SmaI, and an oligonucleotide linker having a HindIII cleavage site is inserted into the expression vector pGEXH. 12 were produced. After digestion of this vector with the restriction enzyme Ec0RI, the ends were blunted using T4 polymerase (Toyobo), and further digested with the restriction enzyme HindIII. Next, the plasmid RaPC53 containing the rat telomerase protein cDNA fragment was cleaved with the restriction enzyme BamHI, and the ends were blunt-ended using T4 polymerase (Toyobo). Then, it is further digested with the restriction enzyme HindIII, subjected to polyacrylamide gel electrophoresis, and a partial DNA fragment of rat 'telomerase protein cDNA (nucleic acid No. 648- About 390 bp of Hindi II-corresponding to 1034; BamHI-derived blunt-ended DNA fragment) was isolated. The HindIII-blended pGEXH12 vector thus obtained is ligated to a rat DNA fragment derived from the telomerase protein cDNA using a DNA ligase kit (Takara Shuzo). E. coli strain JM109 (Toyobo) was transformed using the obtained recombinant vector. For the ampicillin-resistant clone, a restriction map of each plasmid was prepared, and pGEXp80hom / JM109 having the correct recombinant plasmid was selected.
p GEXp 80 h om/JMl 09を、 アンピシリ ンを含む 50 m 1の LB培 地に接種し、 37 で一晩振とう培養した。 翌日これを同じ培地で 10倍希釈し、 さらに 37 °Cで 1時間培養した後、 1 丁6を最終濃度0. 3 mMになるように 加え、 S DS— PAGEで分子量約 44 kD aの GST— p 80 h omを発現さ せた。 GST— p 80 h omを発現させた組み換え大腸菌は F r a n g o n iの 方法 （An a l . B i o c h em. 、 21 0、 1 79、 1993) に従って、 最 終濃度 1. 5 %ザルコシル酸ナ ト リウムを含む緩衝液中で溶解し、 最終濃度 2% トライ ト ン X— 1 0 0を加えた後、 グルタチオン ' セファロ一ス . ビーズ (P h a r m a c i a社） を加えて懸濁した。 4°Cで 40分懸濁しながら保温し た後、 ビーズを 1 % トライ トン X— 1 00を含むリン酸緩衝液 （P B S) で洗浄 してカラムに充填した。 ビーズに結合した G S T— p 80 h omを 25 mM還元 型グルタチオン及び 0. 1 %トライ トン X— 1 00を含む H e p e s緩衝液で溶 出した。  pGEXp80hom / JMl09 was inoculated into a 50 ml LB medium containing ampicillin, and cultured with shaking at 37 overnight. The next day, dilute this 10-fold with the same medium, further culture at 37 ° C for 1 hour, add 1/6 to a final concentration of 0.3 mM, and use GDS with a molecular weight of about 44 kDa by SDS-PAGE. — P80hom was expressed. Recombinant E. coli expressing GST-p80hom contains a final concentration of 1.5% sodium sarcosylate according to the method of Frangoni (An al. Biochem., 210, 179, 1993). After dissolving in a buffer solution and adding a final concentration of 2% Triton X-100, glutathione 'Sepharose beads (Pharmacia) were added and suspended. After incubating at 4 ° C for 40 minutes, the beads were washed with a phosphate buffer (PBS) containing 1% Triton X-100 and packed in a column. The GST-p80hom bound to the beads was eluted with a Hepes buffer containing 25 mM reduced glutathione and 0.1% Triton X-100.
典型的には、 1 0 0 m 1培養分の組み換え体から 0. 7 m gの G S T— p 80 h omが得られた。 GST— p 80 h omをトロンビン処理することによ り、 融合蛋白質は、 SDS— PAGEにおける見かけの分子量が約 29 kDaの GSTと約 1 6 kD aのラッ ト 'テロメラーゼ蛋白質断片 （配列表の配列番号 1 に示すラッ ト 'テロメラ一ゼ蛋白質においてァミノ酸番号 2 1 7〜 345に相当 する部分） の 2つに切断された。 後者を PVDF膜に固定化処理して N末端のァ ミノ酸配列をェドマン法にて解析し、 予想されたァミノ酸配列と同一であること を確認した。 体重約 2. 6 k gの日本在来種雄ゥサギ 2羽 （尺 1及び!^ 2) を常 法に従って 1回につき 1 O O ^ gの GST— p 80 h omとフロイン ト ' アジュ バン 卜の混合物で免疫して抗血清を得た。 Typically, 0.7 mg of GST-p80hom was obtained from a 100 ml culture of recombinant. By treating GST-p80hom with thrombin, the fusion protein was transformed into a GST with an apparent molecular weight of about 29 kDa and a rat 'telomerase protein fragment of about 16 kDa in SDS-PAGE (sequence in Sequence Listing). The portion of the rat's telomerase protein shown in No. 1 (corresponding to amino acids No. 217-345) was cut into two. The latter was immobilized on a PVDF membrane, and the amino acid sequence at the N-terminus was analyzed by the Edman method, and it was confirmed that the amino acid sequence was the same as the expected amino acid sequence. Two Japanese native male herons (shake 1 and! ^ 2) weighing approximately 2.6 kg According to the method, antiserum was obtained by immunization with a mixture of 100 g of GST-p80hom and Freund's adjuvant at one time.
上記抗血清からラッ ト 'テロメラ一ゼ蛋白質特異的な抗体を精製するためのァ ワイ二ティ · カラムを作製するため、 GS Tの代わりにヒスチジン 'へキサマ一 をタグ配列として用いて同じ部分の抗原を発現させ、 同様に精製した。 まず、 プ ラスミ ド R a PC 53を制限酵素 H i n d I I I及び B amH Iで切断し、 ラッ ト .テロメラ一ゼ蛋白質 c DNAの約 390 b pの H i n d i I I—B amH I の DNA断片 （配列表の配列番号 1で核酸番号 648 ~ 1 034に相当する） を 単離し、 この断片を p B l u e S c r i p t (東洋紡） の H i n d I I I 一 B a mH I部位にサブクローニングした。 制限酵素 X h o I及び N o t Iを用い て、 このプラスミ ドからラッ ト ' テロメラ一ゼ蛋白質 c DN Aの核酸番号 648 - 1 0 34 (配列表の配列番号 1 ) に相当する DN A断片を含む X h o I 一 N o t I DNA断片を単離し、 制限酵素 S a 1 I及び N o t Iで切断した発現プ ラスミ ドベクタ一 p P r oEX— l (G i b c o BRL社） と DNAライゲー シヨン 'キッ ト （宝酒造） を用いて連結させた。 得られた組み換えベクターを用 いて大腸菌株 JM 1 09 (東洋紡） を形質転換した。 アンピシリ ン耐性のクロ一 ンについて各プラスミ ドの制限酵素切断地図を作成し、 正しい組み換えプラスミ ドを保有している P P r o EXp 80 h om/J M 109を選択した。  To prepare an affinity column for purifying rat 'telomerase protein-specific antibody from the above antiserum, use histidine' hexamer instead of GST as the tag sequence to The antigen was expressed and purified similarly. First, Plasmid RaPC53 was digested with restriction enzymes HindIII and BamHI, and the approximately 390 bp HindiII-BamHI DNA fragment of rat telomerase protein cDNA (sequence listing). (Corresponding to nucleic acid numbers 648 to 1034 in SEQ ID NO: 1), and this fragment was subcloned into HindIII-BamHI site of pBlueScript (Toyobo). Using the restriction enzymes XhoI and NotI, a DNA fragment corresponding to the nucleic acid number 648-1034 of rat telomerase protein cDNA (SEQ ID NO: 1 in the sequence listing) was digested from this plasmid using the restriction enzymes XhoI and NotI. The XhoI-NotI DNA fragment was isolated, and the expression plasmid vector pProEX-I (Gibco BRL) cut with the restriction enzymes Sa1I and NotI and DNA ligation kit (Takara Shuzo). The resulting recombinant vector was used to transform E. coli strain JM109 (Toyobo). For the ampicillin-resistant clone, a restriction map of each plasmid was prepared, and PProEXp80hom / JM109 having the correct recombinant plasmid was selected.
p P r o EXp 80 h o m/ J M 109をァンピシリ ンを含む 50 m 1の L B 培地に接種し、 37 °Cで一晩振とう培養した。 翌日この培養物を同じ培地で 1 0 倍希釈し、 さらに 37°Cで 1時間培養した後、 I PTGを最終濃度 I mMになる ように加えて、 SD S— PAGEで分子量約 18 kDaの 6H i s— p 80 h om を発現させた。 6 H i s - p 8 0 h o mを発現させた組み換え大腸菌を、 Q i a g e n社のプロ トコ—ルに従って 6 Mグァニジン塩酸を含む結合緩衝液に 溶解し、 N i—NTA—ァガロース （Q i a g e n社） で展開した。 ビーズを洗 浄した後、 結合した 6 H i s - p 80 h o mを 6 M尿素を含む p H 4 · 3の T r i s Zリン酸緩衝液で溶出した。 精製された 6 H i s - p 80 h omを含む画分を中 和した後、 PB Sに対して透析して尿素を希釈し、 不溶性物質を遠心分離で除い た。 上清にァフィゲル 10 (B i o r a d社） を懸濁させ、 6H i s -p 80 h om をクロスリンクしたァフィ二ティ · ビーズを作製した。 典型的には、 1 00m l 分の p P r o EXp 80 h om/J Ml 09の培養菌体から 0. 7 m gの可溶性 の 6H i s - 80 h o mが得られ、 その 95 %以上がァフィゲル 1 0にクロス リ ンクされた。 pProEXp80hom / JM109 was inoculated into a 50 ml LB medium containing ampicillin, and cultured with shaking at 37 ° C overnight. The next day, the culture was diluted 10-fold with the same medium, and further cultured at 37 ° C for 1 hour. IPTG was added to a final concentration of ImM, and 6H with a molecular weight of about 18 kDa was determined by SDS-PAGE. is—p80hom was expressed. Recombinant Escherichia coli expressing 6His-p80hom was dissolved in a binding buffer containing 6M guanidine hydrochloride according to the protocol of Qiagen, and the solution was dissolved in Ni-NTA-agarose (Qiagen). Expanded. After washing the beads, the bound 6His-p80hom was eluted with a pH 4.3 Tris Z phosphate buffer containing 6M urea. Neutralize the fraction containing the purified 6His-p80hom, dilute urea by dialyzing against PBS, and remove insoluble substances by centrifugation. Was. Affigel 10 (Biorad) was suspended in the supernatant to produce affinity beads crosslinked with 6His-p80hom. Typically, 100 mg of pProEXp80hom / JMl09 cultured cells yield 0.7 mg of soluble 6His-80 hom, of which 95% or more is Affigel 10 Was cross-linked.
「 An t i b o d y」 （E d Ha r l owら編、 C o l d S p r i n g Ha r bo r L a b o r a t o r y P r e s s) に記載の方法に従って、 GST - p 80で免疫された R 1の 7週間目の過免疫血清 2 m 1から 1 75 g (R 1 — 4 I d) 、 R 2の 7週間目の過免疫血清 2m lから 86 g (R 2 - 4 1 d) の抗体を得た。 これらの精製抗体が G S Tに対しては反応せず、 ラッ 卜 'テロメ ラーゼ蛋白質 （配列表の配列番号 1に示すラッ ト 'テロメラ一ゼ蛋白質のうち、 ァミノ酸番号 2 1 7〜345に相当する部分） にのみ反応することを、 ゥヱスタ ン ·ブロッ 卜法で確認した。 実施例 5 ：免疫沈降法及びテロメラ一ゼ活性測定による、 抗ラッ ト 'テロメラ一 ゼ蛋白質特異抗体の評価  Hyperimmune serum of R1 immunized with GST-p80 at 7 weeks according to the method described in "Antibody" (Edited by Ed Harlow, et al., Cold Spring Harboratory Press). Antibodies from 2 ml to 175 g (R 1 — 4 Id) and 86 ml (R 2 -41 d) from 2 ml of hyperimmune serum at 7 weeks of R2 were obtained. These purified antibodies did not react with GST, and the rat telomerase protein (corresponding to amino acid numbers 217 to 345 of the rat telomerase protein shown in SEQ ID NO: 1 in the sequence listing) It was confirmed by the Western blotting method that the reaction only occurred in part). Example 5: Evaluation of anti-rat 'telomerase protein-specific antibody by immunoprecipitation and telomerase activity measurement
実施例 1から実施例 3で得られたラッ トまたはヒ ト由来のテロメラーゼ蛋白質 c DNAが、 実際にラッ トまたはヒ ト 'テロメラーゼ蛋白質をコ一ドしているこ とを以下のように証明した。 すなわち、 実施例 4で得られた組み換えラッ 卜 'テ ロメラーゼ蛋白質断片に対する特異抗体を用いて、 ラッ トまたはヒ ト紬胞抽出液 中のテロメラーゼ活性が免疫沈降されるかどうかを検討した。  It was proved as follows that the rat or human-derived telomerase protein cDNA obtained in Examples 1 to 3 actually encoded a rat or human 'telomerase protein. . That is, using a specific antibody against the recombinant rat'telomerase protein fragment obtained in Example 4, it was examined whether or not telomerase activity in a rat or human cell extract was immunoprecipitated.
まず、 R 1の免疫前血清からプロテイン Aセファロース （P h a rma c i a 社） を用いて総 I g Gを精製し （P I— 1) 、 この I gGと R 1の過免疫血清か ら得られた精製 I g G、 R 1 - 4 1 d (免疫開始後 7週後血清由来） 及び R 1一 1 1 6 d (免疫開始後 1 6週後血清由来） の 3種類の I gGを予めプロテイン A セファロースにコ一 トした。 ヒ 卜卵巣奇形腫由来 PA— 1細胞及びラッ ト肝癌由 来 AH 6 6 F細胞から、 C o u n t e rらの方法 （EMB 0 J . 、 1 1、 1 92 1、 1 992 ) に従って S 1 00抽出液を調製した。 この抽出液に等容量 の 1 %CHAP SZ1 x H y p o緩衝液 （C o u n t e rら、 上掲論文） を加え た混合物 1 50 1に、 の I gGをコートした上記プロティン Aセファロー ス . ビーズを加え、 4 で1. 5時間保温した。 その後、 0. 5%CHAP SZ 1 xHy p o緩衝液で洗浄した各々のビーズをテロメラーゼ反応液に懸濁して、 テロメラ一ゼ活性を測定した。 First, total IgG was purified from R1 pre-immune serum using protein A sepharose (Pharmacia) (PI-1), and obtained from the IgG and R1 hyperimmune serum. Purified IgG, R 1-41 d (derived from serum 7 weeks after the start of immunization) and R 1-11 d (derived from serum 16 weeks after the start of immunization) Coated on Sepharose. From a human ovarian teratoma-derived PA-1 cell and a rat liver cancer-derived AH66F cell, an S100 extract was prepared according to the method of Counter et al. (EMB 0 J., 11, 1921, 1992). Was prepared. Equal volume of this extract To a mixture 1501 containing 1% CHAP SZ1xHypo buffer (Counter et al., Supra), the IgG-coated Protein A Sepharose beads were added, and the mixture was added for 1.5 hours. Insulated. Thereafter, each bead washed with 0.5% CHAP SZ 1 xHypo buffer was suspended in a telomerase reaction solution, and telomerase activity was measured.
テロメラーゼの活性は、 T a t ema t s uらの方法 （On c o g e n e、 13， 2265— 2274, 1 996 ) に従い、 ジゴキシゲニン標識 d U T Pと抗ジゴ キシゲニン抗体を用いた E L I S A法で測定した。 ただし、 テロメラーゼによる 伸長反応の際のプライマーとして 5' 端をピオチン標識したォリゴヌクレオチド b p T G 3 ( B i o t i n y l a t e d 5 ' 一  Telomerase activity was measured by the ELISA method using digoxigenin-labeled dUPP and an anti-digoxigenin antibody according to the method of Tatema tsu ssu et al. (Oncgene, 13, 2265—2274, 1996). However, as a primer for the extension reaction by telomerase, an oligonucleotide bpTG 3 (Bioti n y l a t e d 5 '
—3' ) を用い、 各々 0. 8mMのモノデォキシヌクレオチド （TTP、 d ATP, d GTP) を基質として、 30°Cで 1時間反応させた。 酵素反応は過剰の EDTA を加えることにより停止させた。 Using 3 ′), the reaction was carried out at 30 ° C. for 1 hour using 0.8 mM each of monodeoxynucleotides (TTP, dATP, dGTP) as substrates. The enzymatic reaction was stopped by adding excess EDTA.
一方、 EDC (S i gma社製） を用いてス 卜レプトアビジン （G I B CO B R L社製） をポリカーボネート製 96穴マイクロタイタ一プレート （タカラ） にクロスリ ンクさせ、 ブロッキング剤 （ベ一リンガーマンハイム山之内社製） を 用いて 37 °Cで 2時間ブロッキングした。 上記の各ゥヱルに、 TB Sで希釈した テロメラーゼ伸長反応産物 25 1を加えて、 37°Cで 30分保温してプレート 上のストレプトアビジンに結合させた。 サンプル溶液を捨てた後、 過剰量のピオ チン溶液を加えて 37°Cで 30分保温し、 余剰のストレブトアビジンをブロッキ ングした。  On the other hand, streptavidin (GIB CO BRL) was cross-linked to a 96-well polycarbonate microtiter plate (Takara) using EDC (Sigma), and a blocking agent (Belinger Mannheim Yamanouchi) Was blocked at 37 ° C for 2 hours. The telomerase extension reaction product 251 diluted with TBS was added to each of the above-mentioned tubes, and the mixture was incubated at 37 ° C. for 30 minutes to bind to streptavidin on the plate. After discarding the sample solution, an excess amount of a biotin solution was added, and the mixture was incubated at 37 ° C for 30 minutes, and excess streptavidin was blocked.
各ゥエルを洗浄した後、 20 mMT r i s— HC 1 (p H 8. 3) 、 75mM KC 1、 0. 005 %W- 1 , 1. 5mM Mg C 12 , 4 ;uMの b pTG 3、 1 Mのオ リ ゴヌ ク レオチ ドプライマ一、 p T A Gガンマ （ 5 ' — CAGGAAACAGCTATGACCCCTAACCCTAACCCTAACCCT After washing each Ueru, 20 mMT ris- HC 1 (p H 8. 3), 75mM KC 1, 0. 005% W- 1, 1. 5mM Mg C 1 2, 4; uM of b pTG 3, 1 M oligonucleotide primer, p TAG gamma (5 '— CAGGAAACAGCTATGACCCCTAACCCTAACCCTAACCCT
一 3' ) 、 各々 50 Mの dATP、 dCTP、 d GTP、 25 /Mの TTP、 1 /Μのジゴキシゲニン— dUTP (ベ一リンガーマンハイム山之内社製） 、 及 びタック .スタート .アンチボディ （東洋紡社製） 処理した 1ュニッ 卜のタック ポリメラーゼ （G I B C 0 B R L社製） を含む P C R反応液を加え、 タカラ' P CRサ—マルサイクラ一を用いて P CR増幅を行った（ 93 °Cで 30秒、 69 °C で 3 0秒、 72 °Cで 1分の条件で 34サイクル） 。 1 3 '), 50 M each of dATP, dCTP, d GTP, 25 / M TTP, 1 / ジ digoxigenin—dUTP (manufactured by Behringer Mannheim Yamanouchi), and Add a PCR reaction solution containing 1 unit of Tac polymerase (GIBC 0 BRL) treated with Antibody (Toyobo Co., Ltd.) and perform PCR amplification using Takara's PCR thermal cycler. (30 cycles at 93 ° C, 30 seconds at 69 ° C, and 1 cycle at 72 ° C for 34 cycles).
5 OmM炭酸ナ卜リゥム緩衝液 （pH 9. 6 ) を用いて 5 m g Zm 1 に調製し たストレプトアビジンを、 白色ポリスチレン製 96穴マイクロタイタ一プレー ト に 1 00 1 Zゥヱルの割合で分注後、 37 で 1時間保温してストレブトアビ ジンをコートした。 ストレプトアビジン溶液を捨てた後、 ブロッキング剤を 1 50 〃 1 /ゥヱルの割合で分注し、 37°Cで 2時間ブロッキングした。 このゥヱルに、 TB Sで 20倍に希釈した P C R産物を 1 00 ^ 1 Zゥエルずつ加え、 3 Ί。Cで 30分保温してプレートに結合させた。 さらに、 各ゥエルを 1 5 0 1 Zゥエル の 0. 05 %Twe e n 20ZTB Sで 5回洗浄した後、 T B Sで 5000倍希 釈したアル力リホスファタ一ゼ標識抗ジゴキシゲニン抗体 （ベ一リンガーマンハ ィム山之内） を加えて 37°Cで 3 0分保温した。 プレー トを 1 50〃 1ノゥエル の 0. 05 %Tw e e n 2 OZTB Sで 5回洗浄した後、 0. 1 Mジエタノール ァミン緩衝液 （p H 9. 5 ) で 1 00倍希釈した C S PD (D i s o d i um 3 - (4 -me t h o x y s p i r o 〔1， 2— d i o x e t a n e— 3， 2— (5 一 c h l o r o) t r i c y c l o 〈3. 3. 1. 1 3, 7> d e c a n) - 4 - y 1 ) p h e n y l p h o s p h a t e) (T r o p i x社製） を加え、 室 温で 30分間化学発光させてルミノメーター （ベル卜一ルド' ジャパン） で発光量 を定量した。  5 Streptavidin prepared to 5 mg Zm 1 using OmM sodium carbonate buffer (pH 9.6) was dispensed into white polystyrene 96-well microtiter plates at a rate of 1001 Z Thereafter, the cells were kept warm at 37 for 1 hour and coated with streptavidin. After discarding the streptavidin solution, a blocking agent was dispensed at a ratio of 150 11 / ゥ ヱ and blocking was performed at 37 ° C for 2 hours. To this vial, add the PCR product diluted 20-fold with TBS in 100 ^ 1 Zells, and add 3 μl. C was incubated for 30 minutes to bind to the plate. Further, each well was washed 5 times with 1.51% Zell's 0.05% Tween 20ZTBS, and then diluted with TBS 5000-fold with an anti-digoxigenin-labeled anti-lipoxyphosphatase antibody (Beilinger Mannheim). (Yamanouchi) and kept at 37 ° C for 30 minutes. The plate was washed five times with 150〃 1 noel of 0.05% Tween 2 OZTB S, and then diluted CS-PD (D) 100-fold with 0.1 M diethanolamine buffer (pH 9.5). isodi um 3-(4 -me thoxyspiro [1, 2-dioxetane-3, 2-(5-chloro) tricyclo <3.3.1.1, 3, 7> decan)-4-y 1) phenylphosphate) (T (produced by Ropix), and the mixture was allowed to emit chemiluminescence at room temperature for 30 minutes, and the luminescence was quantified using a luminometer (Berthold's Japan).
この結果、 ラッ 卜癌細胞抽出液またはヒ 卜癌細胞抽出液のいずれを用いた実験 においても、 図 3に示すように I g Gをコートしていないビーズ、 免疫前血清由 来 I gG (P I — 1) をコートしたビーズにはテロメラーゼ活性が殆ど認められ なかったが、 実施例 4で得られた組み換えラッ 卜 'テロメラーゼ蛋白質断片に対 する特異抗体の 2ロッ 卜のいずれかをコ一トしたビーズには明らかに高いテロメ ラ一ゼ活性が ¾1められた。 実施例 6 :35S—メチォニン標識ラッ ト癌細胞抽出液の免疫沈降による、 抗ラッ ト ' テロメラ—ゼ蛋白質特異抗体の評価 As a result, in the experiments using either the rat cancer cell extract or the human cancer cell extract, as shown in Fig. 3, beads not coated with IgG, IgG derived from pre-immune serum (PI Although almost no telomerase activity was observed in the beads coated with —1), any one of two lots of the specific antibody against the recombinant rat telomerase protein fragment obtained in Example 4 was coated. The beads had clearly higher telomerase activity. Example 6: Evaluation of anti-rat 'telomerase protein-specific antibody by immunoprecipitation of 35 S-methionine-labeled rat cancer cell extract
5 0 0万個のラ ッ ト肝癌由来 A H 6 6 F細胞を、 透析済みゥシ胎児血清 (d F C S) 1 0 %を含むメチォニン欠乏ダルベッコ変法 MEM (DMEM) で 洗浄した後、 35S—メチォニンを加えた同培地中で培養して35 S標識し、 実施例 5 で用いた 0. 5 %C H AP S/ 1 X H y p o緩衝液中で抽出した。 ゥサギ R 1の 免疫前血清由来 I gG、 または組み換えラッ ト ·テロメラーゼ蛋白質断片による 過免疫血清由来 I g Gを予めコー卜したプロテイン Aセファロ一ス ' ビーズを同 細胞数に相当する抽出液に加えて 4°Cで 2時間保温した。 洗浄後、 L a emm l i の S D S変性緩衝液を加えて加熱 '変性し、 6%SD S - PAGEで展開した。 ゲルを酢酸で固定した後、 ENHANCE (NEN社製） 処理し、 乾燥後にフル ォログラフィ一に付した。 その結果、 過免疫血清由来 I gG処理したサンプルに のみ、 約 300 kD aの明確なバン ドが観察された。 実施例 7 ： ヒ 卜 ' テロメラーゼ蛋白質 mRNAのヒ ト癌細胞及び正常組織におけ る発現 5 0 0 million pieces of rat liver cancer-derived AH 6 6 F cells, washed with dialyzed © Shi fetal serum (d FCS) Mechionin deficiency Dulbecco's modified MEM containing 1 0% (DMEM), 35 S- The cells were cultured in the same medium supplemented with methionine, labeled with 35 S, and extracted in the 0.5% CHAPS / 1 X Hypo buffer used in Example 5.加 え Protein A Sepharose 'beads, pre-coated with IgG from pre-immune serum of heron R1 or IgG from hyperimmune serum with recombinant rat telomerase protein fragment, are added to the extract corresponding to the same number of cells. And kept at 4 ° C for 2 hours. After washing, Laemmli's SDS denaturation buffer was added and the mixture was denatured by heating and developed with 6% SDS-PAGE. The gel was fixed with acetic acid, treated with ENHANCE (manufactured by NEN), dried, and subjected to fluorography. As a result, a clear band of about 300 kDa was observed only in the sample treated with IgG from hyperimmune serum. Example 7: Expression of human 'telomerase protein mRNA in human cancer cells and normal tissues
C l o n t e c h社の Mu l t i p l e T i s s u e No r t h e r n B l o t及び、 Huma n C a n c e r C e l l L i n e Mu l t i l e M l t i p l e T i s s s u e No R t h e r n B l o t and Human C a n c e r C e l l L i n e Mul t i l e
T i s s u e o r t h e r n B l o tを用いて、 ヒ ト .テロメラ一ゼ蛋 白質 mRNAのヒ ト癌細胞及び正常組織における発現を検討した。 プローブとし ては実施例 2の工程（1) で得られたヒ ト 'テロメラ一ゼ蛋白質遺伝子 cDN A断片 (配列表の配列番号 2) を"2 P標識して用い、 ハイブリダィゼーシヨンは 50%フ オルムアミ ド存在下に 42 °Cで一昼夜行った。 各プロッ ト膜を 0. 1 %SDSを 含む 1 X及び 0· 1 X S S P E緩衝液で洗浄した後に、 ォ一トラジォグラフィー に付した。 Using T issueorthern B lot, expression of human .telomerase protein mRNA in human cancer cells and normal tissues was examined. Is a probe used in the "2 P-labeled (SEQ ID NO: 2) human 'Teromera Ichize protein gene cDNA A fragment obtained in the step of Example 2 (1), hybrida I See Chillon is This was performed overnight at 42 ° C in the presence of 50% formamide.Plotted membranes were washed with 1X and 0.1 XSSPE buffer containing 0.1% SDS and then subjected to autoradiography. .
その結果、 脾、 胸腺、 脬、 精巣、 卵巣、 小腸、 大腸、 心臓、 胎盤、 肺、 肝、 骨 格筋、 及び肾などのヒ ト正常組織由来の p o 1 y (A) + RNAには明確な 10. 7 k bのバン ドが検出された。 また、 ヒ ト癌由来細胞株由来の p o 1 y (A) + RNAのブロッ トでは、 1 0. 7 k bのバン ドに加えて、 8. 6 k bの短い分子 種が観察された。 実施例 8 ： ラッ 卜 'テロメラーゼ蛋白質の精製と分子種の同定 As a result, po 1 y (A) + RNA from human normal tissues such as spleen, thymus, 脬, testis, ovary, small intestine, large intestine, heart, placenta, lung, liver, skeletal muscle, and 肾 is clear A new 10.7 kb band was detected. In addition, po 1 y (A) + In the RNA block, a short 8.6 kb species was observed in addition to the 10.7 kb band. Example 8: Purification of rat telomerase protein and identification of molecular species
3 x 1 0。 個のラッ ト肝癌由来細胞株 AH 66 F細胞から Co u n t e rらの 方法 （EMBO. J、 1 1、 1 92 1、 1 995) に従って S 1 00抽出液を調 製した。 これを、 TMG緩衝液 （ 1 OmM T r i s—酢酸 p H 8. 0、 1 mM 塩化マグネシウム、 ImM ジチオスレイ ト一ル、 1 0%グリセロール） で飽 和したへパリ ンセファロース C L— 6 Bカラム （フアルマシア社） に供し、 塩化 力リゥムを用いた段階溶出を行った。 各溶出画分中のテロメラ一ゼ活性を実施例 5で用いた方法で測定し、 活性を含む画分を集めた。 これを 5 OmM 塩化カリ ゥムを含む TMG緩衝液で飽和したハイ ドロキシァパタイ トカラム （バイオラッ ド社） に供し、 5mM KP緩衝液 （0. 25mM リ ン酸二水素一カリウム、 4. 75 mM リ ン酸一水素二カリウム、 5 OmM 塩化カリウム、 I mM 塩化マグネシウム、 I mM ジチオスレィ トール、 1 0 %グリセロール） で洗浄 後、 0. 5M ! ?緩衝液 （25111]^ リ ン酸二水素一カリウム、 475 mM リン酸一水素二カリウム、 5 OmM 塩化カリウム、 ImM 塩化マグネシウム、 ImM ジチオスレイ ト一ル、 10%グリセロール） を用いた段階溶出を行った。 テロメラーゼ活性を有する画分を集め、 5 OmM 塩化カリウムを含む TMG 緩衝液 （ジチオスレィ トール不含） で飽和した陰イオン交換カラム （商品名リ ソー ス Q、 フアルマシア社） に供し、 塩化カリウムを用いた段階溶出を行った。 次い で、 テロメラーゼ活性を有する画分を集め、 0. 5M 塩化カ リウムと I mM イ ミダゾ—ルを含む TMG緩衝液 （ジチオスレィ トール不含） で飽和した金属 (Z n2+) キレー トァフィ二ティカラム （商品名ハイ トラップ キレ一ティ ング、 フアルマシア社） に供し、 イ ミダゾールを用いた段階溶出を行った。 テロメラー ゼ活性を有する溶出画分を 1 5— 40%グリセロール濃度勾配遠心分離 （ベック マン社 SW28口一ター、 25000回転、 2°C、 24時間） に供した。 その結 果、 テロメラーゼ活性と相関のある蛋白質として 44 Sの沈降係数を示すものが P97/0204 得られ、 その分子量は約 1 500 k D aと計算された。 3 x 10 An S100 extract was prepared from the rat hepatoma-derived cell line AH66F cells according to the method of Counter et al. (EMBO. J, 11, 1921, 1995). Heparin Sepharose CL-6B column (Pharmacia) saturated with TMG buffer (1 OmM Tris—pH 8.0, 1 mM magnesium chloride, ImM dithiothratel, 10% glycerol) And elution was performed in a stepwise manner using a chlorinated rim. Telomerase activity in each eluted fraction was measured by the method used in Example 5, and fractions containing the activity were collected. This was applied to a hydroxyapatite column (BioRad) saturated with TMG buffer containing 5 OmM potassium chloride, and 5 mM KP buffer (0.25 mM monopotassium dihydrogen phosphate, 4.75 mM phosphoric acid) After washing with dipotassium monohydrogen, 5 OmM potassium chloride, ImM magnesium chloride, ImM dithiothreitol, 10% glycerol), 0.5 M!? Buffer (25111] ^ monopotassium dihydrogen phosphate, 475 mM Step elution was performed using dipotassium monohydrogen phosphate, 5 OmM potassium chloride, ImM magnesium chloride, ImM dithiothreitol, and 10% glycerol). The fractions having telomerase activity were collected, applied to an anion exchange column (trade name: Resource Q, Pharmacia) saturated with a TMG buffer (containing no dithiothreitol) containing 5 OmM potassium chloride, and potassium chloride was used. Step elution was performed. Next, the fractions having telomerase activity were collected, and the metal (Zn 2+ ) chelate was saturated with a TMG buffer (containing no dithiothreitol) containing 0.5 M potassium chloride and ImM imidazole. The solution was applied to a T-column (trade name: High Trap Cleaning, Pharmacia), and stepwise elution was performed using imidazole. The eluted fraction having telomerase activity was subjected to 15-40% glycerol concentration gradient centrifugation (Beckman SW28, 25,000 rpm, 2 ° C, 24 hours). As a result, a protein with a sedimentation coefficient of 44 S was found to be a protein correlated with telomerase activity. P97 / 0204 was obtained and its molecular weight was calculated to be approximately 1500 kDa.
さらに、 グリセロール濃度勾配遠心分離で生じた各画分を 6%SDS— PAGE で分離し、 実施例 4で取得した組換えラッ 卜 'テロメラーゼ蛋白質に対する特異 抗体でゥヱスタンブロッ トを行ったところ、 テロメラ一ゼ活性を示す蛋白質画分 には 3つの抗体反応性のバンド （SDS— PAGE上の分子量は約 240 kDa、 230 kD a、 55 k D a) が観察された。 このうち 55 kD aのバン ドは熱処 理実験により 240 kD aまたは 230 kD aの蛋白質の分解産物であることを 確認した。 この結果より、 ラッ ト 'テロメラ一ゼ蛋白質には 240 k D aの蛋白 質 （以下 「p 240」 と称することもある） を成分として構成されたものと、 23 O kD aの蛋白質 （以下 「p 230」 と称することもある） を成分として構 成されたもの二種類が存在すると推測された。 実施例 9 ： ラッ トテロメラーゼ分子種の生成と活性化  Furthermore, the fractions generated by glycerol concentration gradient centrifugation were separated by 6% SDS-PAGE, and subjected to pestic blot with a specific antibody against the recombinant rat telomerase protein obtained in Example 4. Three antibody-reactive bands (molecular weights on SDS-PAGE of about 240 kDa, 230 kDa and 55 kDa) were observed in the protein fraction showing activity. Of these, the band of 55 kDa was confirmed to be a 240 kDa or 230 kDa protein degradation product by heat treatment experiments. These results indicate that the rat telomerase protein was composed of a 240 kDa protein (hereinafter sometimes referred to as “p240”) as a component, and a 23 OkDa protein (hereinafter “p240”). p230 ”) as a component. Example 9: Generation and activation of rat telomerase species
p 240と p 230の生成過程を調べるため、 細胞のパルス ' チヱイス実験を 行った。 10 cmプラスチックディッシュに蒔いたラッ 卜肝癌由来細胞株 AH66 F 細胞を 250〃 C i Zm 1の [^S] メチォニン （商品名 T r a n 35 S - 1 a b e 1、 I C N社） と 1 0 %牛胎児血清 （ J RH バイオサイエンス社） を 含む 1 m 1の DMEM培地 （メチォニン、 システィ ン不含、 ライフテックオリエ ンタル社） 中で 30分間パルスラベルし、 次いで大過剰の非放射性メチォニンを 培地中に添加した。 非放射性メチォニン添加後 0、 1、 3、 6時間後に細胞を回 収し、 組換えラッ ト ·テロメラーゼ蛋白質に対する特異抗体を用い、 実施例 4と 同様に免疫沈降を行った。  In order to examine the production process of p240 and p230, we performed a pulsed-thirds experiment on cells. Rat hepatoma-derived cell line AH66F cells sown on a 10 cm plastic dish were incubated with 250 ^ CiZm1 [^ S] methionine (trade name Tran 35 S-1 abe 1, ICN) and 10% fetal calf Pulse label for 30 minutes in 1 ml DMEM medium (without methionine and cystine, Lifetech Oriental) containing serum (JRH Biosciences), then add a large excess of non-radioactive methionine to the medium did. At 0, 1, 3, and 6 hours after the addition of non-radioactive methionine, the cells were collected and immunoprecipitated in the same manner as in Example 4 using a specific antibody against the recombinant rat telomerase protein.
得られた免疫沈降物を 6%SDS— PAGE、 次いでォ一 トラジオグラフィ一 に供した。 その結果、 パルスラベル直後 （0時間） では免疫沈降されたのは主に p 240であつた。 しかし、 経時的 （ 1、 3、 6時間） に p 24 0は減少し、 p 230が増加した。 このことから、 ラッ ト ' テロメ ラ一ゼ蛋白質は、 はじめ p 240を含む構成の蛋白質として発現され、 その後修飾を受けて p 230を含 む構成の蛋白質になると考えられる。 ラッ ト正常組織及びラッ ト肝癌由来細胞株 A H 6 6 F細胞中の p 2 4 0 / p 2 3 0 の存在比と、 テロメラーゼ活性との相関を調べた。 まず、 ラッ ト肝臓、 腎臓、 精 巣及び A H 6 6 F細胞から C o u n t e r らの方法 （E M B 0 . J、 1 1、 1 9 2 1、 1 9 9 5 ) に従って S 1 0 0抽出液を調製し、 これを実施例 8と同様 にへパリ ンセファロース C L— 6 Bカラムにて部分精製した。 各テロメラーゼ部 分精製画分について組換えラッ ト ·テロメラ一ゼ蛋白質特異抗体を用いたウェス タンブロッ ト法による p 2 3 0 Z p 2 4 0存在比、 および、 テロメラ一ゼ活性の 測定を行った。 その結果、 テロメラーゼ活性の強さの順は高いものより A H 6 6 F 細胞、 精巣、 肝臓であり、 腎臓では検出されなかった。 一方、 p 2 3 0の存在比 は多い順に A H 6 6 F細胞、 精巣、 肝臓であった。 腎臓ではほとんど p 2 3 0は 観察されなかった。 The obtained immunoprecipitate was subjected to 6% SDS-PAGE and then to autoradiography. As a result, p240 was mainly immunoprecipitated immediately after the pulse labeling (0 hour). However, over time (1, 3, 6 hours) p240 decreased and p230 increased. From this, it is considered that the rat telomerase protein is first expressed as a protein having p240, and then modified to become a protein having p230. The correlation between the abundance ratio of p240 / p230 in rat normal tissues and rat liver cancer cell line AH66F cells and telomerase activity was examined. First, an S100 extract was prepared from rat liver, kidney, testis, and AH66F cells according to the method of Counter et al. (EMB 0.J, 11, 1921, 1995). This was partially purified using a Heparin Sepharose CL-6B column in the same manner as in Example 8. The ratio of p230Zp240 and the telomerase activity of each telomerase-purified fraction were measured by Western blotting using a recombinant rat telomerase protein-specific antibody. . As a result, telomerase activity was higher in AH66F cells, testis and liver than in higher order, and was not detected in kidney. On the other hand, the abdominal ratio of p230 was AH66F cells, testis, and liver in descending order. Almost no p230 was observed in the kidney.
この結果は P 2 3 0の存在比とテロメラーゼ活性との強い相関を示しており、 P 2 3 0が活性型、 p 2 4 0は不活性型のラッ ト ·テロメラーゼ蛋白質を構成す る分子種であると考えられる。 以上より、 ラッ トテロメラ一ゼは、 はじめ不活性 型の p 2 4 0で構成された分子種として生成され、 その後修飾を受けて p 2 4 0 が p 2 3 0に変化し活性型分子種が生成することが確認された。 産業上の利用可能性  This result shows a strong correlation between the abundance ratio of P230 and telomerase activity, where P230 is the active type and p240 is the molecular species constituting the inactive rat telomerase protein. It is considered to be. As described above, rat telomerase is initially generated as a molecular species composed of inactive p240, and after modification, p240 is changed to p230 and the active molecular species is converted. It was confirmed to generate. Industrial applicability
本発明により、 細胞増殖に必須であり、 かつ癌細胞の増殖への関与が示唆され ている高等動物由来のテロメラ一ゼ蛋白質及びそれをコ一ドする遺伝子が提供さ れた。 本発明のテロメラーゼ蛋白質及びそれをコードする遺伝子は、 例えば、 細 胞増殖及び細胞の老化などの生体制御機構の解明に有用であり、 癌の治療薬の開 発に特に有用性が期待される。 また、 本発明のテロメラ一ゼ蛋白質を特異的に認 識する抗体は癌細胞の検出のための試薬として有用であり、 癌の早期発見を目的 とした臨床検査薬としての有用性が期待される。 さらに、 本発明のテロメラーゼ 蛋白質のサブュニッ 卜が活性型および不活性型では S D S -ポリアク リルアミ ド 電気泳動法における分子量が異なるという性質を利用して、 テロメラ一ゼ蛋白質 に作用する薬物のスクリ一ニングを行うことが可能になる。 4 配列表 配列番号： 1 According to the present invention, a telomerase protein derived from a higher animal, which is essential for cell growth and has been suggested to be involved in cancer cell growth, and a gene encoding the same are provided. The telomerase protein and the gene encoding the telomerase protein of the present invention are useful for elucidating, for example, biological control mechanisms such as cell growth and cell aging, and are expected to be particularly useful for the development of therapeutic drugs for cancer. Further, the antibody specifically recognizing the telomerase protein of the present invention is useful as a reagent for detecting cancer cells, and is expected to be useful as a clinical test agent for early detection of cancer. . Furthermore, by utilizing the property that the telomerase protein of the present invention differs in the molecular weight in the SDS-polyacrylamide electrophoresis between the active form and the inactive form, screening of a drug acting on the telomerase protein can be carried out. It is possible to do. 4 Sequence Listing SEQ ID NO: 1
配列の長さ ：核酸 = 8 2 1 5、 アミノ酸 = 2 6 2 9 Sequence length: nucleic acid = 8 2 15; amino acid = 26 29
配列の型：核酸及びァミノ酸 Sequence type: nucleic acid and amino acid
トポロジー ：直鎖状二本鎖  Topology: linear double-stranded
配列の種類： c D N A Sequence type: c D N A
起源：生物名 ラッ 卜 Origin: organism name rat
配列 Array
AGCTCCGCCC CTCCCCTTGC CCAGCCTCGC CCCTTCGCCT CTCTAGGGTG TTGGTTTCCT 60 TTCAGTTCTC TTTCTTCAAC CTATCCACTG GCTGACCTAG GCCGGTTTCT GCTCCTTGTT 120 GCGGAGAACC AACGCGCCCC TCACTGTGCA CAGCTTTTCC AGTCCCGAGC GCAGGCACAT 180 AGAGATTGTG CTGCCGCT ATG GAG AAA CTC TGT GGT TAT GTG CCT GTC 228  AGCTCCGCCC CTCCCCTTGC CCAGCCTCGC CCCTTCGCCT CTCTAGGGTG TTGGTTTCCT 60 TTCAGTTCTC TTTCTTCAAC CTATCCACTG GCTGACCTAG GCCGGTTTCT GCTCCTTGTT 120 GCGGAGAACC AACGCGCCCC TCACTGTGCA CAGCTTTTCC AGTCCCGATC GAGAGTCAG AGAG GCAATTCAGT GAG CAGTCAG GAGATTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCGCTCTC
Met Glu Lys Leu Cys Gly Tyr Val Pro Val  Met Glu Lys Leu Cys Gly Tyr Val Pro Val
1 5 10  1 5 10
CAC CCA GAC ATC CTC TCC TTG AAG AAT CGG TGC CTG ACC ATG CTC TCT 276 His Pro Asp l ie Leu Ser Leu Lys Asn Arg Cys Leu Thr Met Leu Ser  CAC CCA GAC ATC CTC TCC TTG AAG AAT CGG TGC CTG ACC ATG CTC TCT 276 His Pro Asp lie Leu Ser Leu Lys Asn Arg Cys Leu Thr Met Leu Ser
15 20 25  15 20 25
GAC ATC CAA CCC CTG GAG AAA ATA CAT GGA CAG AGA TCT GTC AAC CCA 324 Asp l ie Gin Pro Leu Glu Lys l ie His Gly Gin Arg Ser Val Asn Pro  GAC ATC CAA CCC CTG GAG AAA ATA CAT GGA CAG AGA TCT GTC AAC CCA 324 Asp lie Gin Pro Leu Glu Lys lie His Gly Gin Arg Ser Val Asn Pro
30 35 40  30 35 40
GAC ATC CTG TCC TTG GAG AAC CGG TGC CTG ACC TTG CTC CCT GAT CTC 372 Asp l ie Leu Ser Leu Glu Asn Arg Cys Leu Thr Leu Leu Pro Asp Leu  GAC ATC CTG TCC TTG GAG AAC CGG TGC CTG ACC TTG CTC CCT GAT CTC 372 Asp lie Leu Ser Leu Glu Asn Arg Cys Leu Thr Leu Leu Pro Asp Leu
45 50 55  45 50 55
CAG CCC ATG GAG AAA ATA CAT GGA CAG AGA TCT GTC CAC CCA GAC ATC 420 Gin Pro Met Glu Lys l ie His Gly Gin Arg Ser Val His Pro Asp l ie  CAG CCC ATG GAG AAA ATA CAT GGA CAG AGA TCT GTC CAC CCA GAC ATC 420 Gin Pro Met Glu Lys l ie His Gly Gin Arg Ser Val His Pro Asp l ie
60 65 70  60 65 70
CTC TCC TCA GAG AAC CGG TGT CTG ACC TTG CTC CCT GAC CTC CAG TCC 468 19 jq丄 ngq s ngq Ι ΒΛ OJJ BTV ^ TO Ϊ^Λ ΐ^Λ " T O " TO "ΐΰ ^TO ^ TO 006 V3V 3X0 VVV VXD 313 033 V33 000 010 010 VVO VVO VVO VV9 V90 000 CTC TCC TCA GAG AAC CGG TGT CTG ACC TTG CTC CCT GAC CTC CAG TCC 468 19 jq 丄 ngq s ngq Ι Λ Λ OJJ BTV ^ TO Ϊ ^ Λ ΐ ^ ΐ Λ "TO" TO "ΐΰ ^ TO ^ TO 006 V3V 3X0 VVV VXD 313 033 V33 000 010 010 VVO VVO VVO VV9 V90 000
912 Q\Z m  912 Q \ Z m
"91 s nsq J3S ^ χ nsq OJJ OJJ ηχο BTV "TO sA ηχο "91 s nsq J3S ^ χ nsq OJJ OJJ ηχο BTV" TO sA ηχο
2S8 OIX 30V VXD XOV 0V1 3X1 130 OXV 333 DIO VVO VDO 30V VVO VVV VVO 2S8 OIX 30V VXD XOV 0V1 3X1 130 OXV 333 DIO VVO VDO 30V VVO VVV VVO
002 96T 061  002 96T 061
"TO "TO na ηχο sA Αχο ηχο jas Ι^Λ SAQ S y UT "^l ^TV Λ 08 VVO 3V3 Oil VVO OVV V30 VVO VOX DID 301 030 OVO 100 3X1 130 VIO  "TO" TO na ηχο sA Αχο ηχο jas Ι ^ Λ SAQ S y UT "^ l ^ TV Λ 08 VVO 3V3 Oil VVO OVV V30 VVO VOX DID 301 030 OVO 100 3X1 130 VIO
S8T 081 5iT  S8T 081 5iT
niO ui9 ΤΒΛ 3·ΐγ ^TV nM s J3S sAq nsq BI V Say QJd Αχο 95A VVO 0V3 113 V03 IDS VDV 130 131 OXV 331 VVV DID 039 OOV 333 V00 Oil 591 091 Q5I niO ui9 ΤΒΛ 3ΐ ^ γ ^ TV nM s J3S sAq nsq BI V Say QJd Αχο 95A VVO 0V3 113 V03 IDS VDV 130 131 OXV 331 VVV DID 039 OOV 333 V00 Oil 591 091 Q5I
JMI OJJ naq dsy naq ηχ^ sAq nsq 9i SAQ OJ^ BIV ηχο 9χι jqi OJJ JMI OJJ naq dsy naq ηχ ^ sAq nsq 9i SAQ OJ ^ BIV ηχο 9χι jqi OJJ
80i 13V V33 113 OVO VIO VVO 3VV Oil 311 191 V33 130 3V0 VIV 33V 133 80i 13V V33 113 OVO VIO VVO 3VV Oil 311 191 V33 130 3V0 VIV 33V 133
091 m OH 091 m OH
OJJ n^ ) OJJ 9i naq SAQ usy S-iy 9lld usy OJJ dsy S y nsq dsy BTVOJJ n ^) OJJ 9i naq SAQ usy S-iy 9l ld usy OJJ dsy S y nsq dsy BTV
099 133 0V3 133 3X1 013 331 3VV 303 ill OVV 933 OVO 103 9X3 IVO 130 099 133 0V3 133 3X1 013 331 3VV 303 ill OVV 933 OVO 103 9X3 IVO 130
98ΐ οετ ΐ 98ΐ οετ ΐ
UTO ^TV sin J3S S^V S H nsq s nsq nsq OJJ Λχθ JSSUTO ^ TV sin J3S S ^ VSH nsq s nsq nsq OJJ Λχθ J S S
219 VV3 VDO 03V XVO 131 VOV 3V3 IXD I3X 0V3 313 Oil 333 309 I9V 03X 219 VV3 VDO 03V XVO 131 VOV 3V3 IXD I3X 0V3 313 Oil 333 309 I9V 03X
021 5Π Oi l  021 5Π Oi l
ΐ^Λ 3JV sAq τ¾ -JMX 0Jd na J¾ BTV n SAQ Sjy usy ηχο nsq IIO 13V VOV OVV VIO IOV 333 313 33V 130 113 101 V03 3VV OVO Oil ΐ ^ Λ 3JV sAq τ¾ -JMX 0Jd na J¾ BTV n SAQ Sjy usy ηχο nsq IIO 13V VOV OVV VIO IOV 333 313 33V 130 113 101 V03 3VV OVO Oil
50ΐ 00Ϊ 56  50ΐ 00Ϊ 56
J9S naq ¾ dsy OJJ S T H J3S J9§ a¾ S T¾ Α ^ SAQ nsq sAq η ^ naq  J9S naq ¾ dsy OJJ S T H J3S J9§ a¾ S T¾ Α ^ SAQ nsq sAq η ^ naq
915 131 3X3 3X0 OVO VOO DVO IOV IDI OXV XV3 V30 101 VIO OVV OVO 013 06 58 08 52,915 131 3X3 3X0 OVO VOO DVO IOV IDI OXV XV3 V30 101 VIO OVV OVO 013 06 58 08 52,
J9S UX9 ri9i dsy O-ij na naq JIJ naq SAQ Sjy usy JSS ュ naq f06iO/ .6df/13d 8£8ム 0/86 OAV 220 225 230 J9S UX9 ri9i dsy O-ij na naq JIJ naq SAQ Sjy usy JSS naq f06iO / .6df / 13d 8 £ 8m 0/86 OAV 220 225 230
TCT GGA GAC TCT GAC TCT CAC CCT GAA ACC ACT GAC CAG ATC CTG CAG 948 TCT GGA GAC TCT GAC TCT CAC CCT GAA ACC ACT GAC CAG ATC CTG CAG 948
Ser Gly Asp Ser Asp Ser His Pro Glu Thr Thr Asp Gin l ie Leu GinSer Gly Asp Ser Asp Ser His Pro Glu Thr Thr Asp Gin lie Leu Gin
235 240 245 250235 240 245 250
GAG AAG AAG ATG GCT CTC TTG ACC TTG CTG TGC TCA GCT ATG GCC TCA 996GAG AAG AAG ATG GCT CTC TTG ACC TTG CTG TGC TCA GCT ATG GCC TCA 996
Glu Lys Lys Met Ala Leu Leu Thr Leu Leu Cys Ser Ala Met Ala Ser Glu Lys Lys Met Ala Leu Leu Thr Leu Leu Cys Ser Ala Met Ala Ser
255 260 265  255 260 265
AGT GTG AAT GTG AAA GAT GCC TCC GAT CCT ACC CGG GCA TCT ATC CAT 1044 AGT GTG AAT GTG AAA GAT GCC TCC GAT CCT ACC CGG GCA TCT ATC CAT 1044
Ser Val Asn Val Lys Asp Ala Ser Asp Pro Thr Arg Ala Ser l ie His Ser Val Asn Val Lys Asp Ala Ser Asp Pro Thr Arg Ala Ser lie His
270 275 280 270 275 280
GAA GTC TGC AGT GCG CTG GCC CCC TTG GAA CCT GAG TTC ATC CTT AAG 1092GAA GTC TGC AGT GCG CTG GCC CCC TTG GAA CCT GAG TTC ATC CTT AAG 1092
Glu Val Cys Ser Ala Leu Ala Pro Leu Glu Pro Glu Phe l ie Leu Lys Glu Val Cys Ser Ala Leu Ala Pro Leu Glu Pro Glu Phe lie Leu Lys
285 290 295  285 290 295
GCA TCT TTG TAT GCT AGG CAG CAG CTT AAC CTC CGG CAC ATA GCC AAT 1140 GCA TCT TTG TAT GCT AGG CAG CAG CTT AAC CTC CGG CAC ATA GCC AAT 1140
Ala Ser Leu Tyr Ala Arg Gin Gin Leu Asn Leu Arg Asp l ie Ala Asn Ala Ser Leu Tyr Ala Arg Gin Gin Leu Asn Leu Arg Asp lie Ala Asn
300 305 310  300 305 310
ATA GTG TTG GCC GTG GCT GCC CTC TTG CCA GCC TGC CGC CCC CAT GTA 1188 l ie Val Leu Ala Val Ala Ala Leu Leu Pro Ala Cys Arg Pro His Val ATA GTG TTG GCC GTG GCT GCC CTC TTG CCA GCC TGC CGC CCC CAT GTA 1188 l ie Val Leu Ala Val Ala Ala Leu Leu Pro Ala Cys Arg Pro His Val
315 320 325 330 315 320 325 330
CGA CGG TAT TAC TCT GCC ATT GTT CAC CTG CCT TCA GAC TGG ATC CAG 1236 CGA CGG TAT TAC TCT GCC ATT GTT CAC CTG CCT TCA GAC TGG ATC CAG 1236
Arg Arg Tyr Tyr Ser Ala l ie Val His Leu Pro Ser Asp Trp l ie Gin Arg Arg Tyr Tyr Ser Ala lie Val His Leu Pro Ser Asp Trp lie Gin
335 340 345  335 340 345
GTA GCC GAG TTC TAC CAG AGC CTG GCA GAA GGG GAT GAG AAG AAG TTG 1284 GTA GCC GAG TTC TAC CAG AGC CTG GCA GAA GGG GAT GAG AAG AAG TTG 1284
Val Ala Glu Phe Tyr Gin Ser Leu Ala Glu Gly Asp Glu Lys Lys Leu Val Ala Glu Phe Tyr Gin Ser Leu Ala Glu Gly Asp Glu Lys Lys Leu
350 355 360 350 355 360
GTG CCC CTG CCT GCC TGC CTC CGT GCT GCC ATG ACT GAC AAA TTT GCC 1332GTG CCC CTG CCT GCC TGC CTC CGT GCT GCC ATG ACT GAC AAA TTT GCC 1332
Val Pro Leu Pro Ala Cys Leu Arg Ala Ala Met Thr Asp Lys Phe Ala Val Pro Leu Pro Ala Cys Leu Arg Ala Ala Met Thr Asp Lys Phe Ala
365 370 375 89 365 370 375 89
ZUl 930 0X3 OXV 330 OXV Oil 033 3X3 VVV 000 IVV OVO VIV 313 VV3 OVO ZUl 930 0X3 OXV 330 OXV Oil 033 3X3 VVV 000 IVV OVO VIV 313 VV3 OVO
029 9Ϊ9 0 Ϊ9  029 9Ϊ9 0 Ϊ9
dJI Τ^Λ s BIV SJV usy gjy n^l -iss n3l nT0 3ュ V nW il OOX 0X0 LOl lO V3V OVV VOO 100 VIX 30V 313 OVO 090 0V3 001 33V dJI Τ ^ Λ s BIV SJV usy gjy n ^ l -iss n3 l n T0 3 u V n W il OOX 0X0 LOl lO V3V OVV VOO 100 VIX 30V 313 OVO 090 0V3 001 33V
509 005 56^  509 005 56 ^
"TO OJJ 3jy "TO nsq sA 9j¾ gjy u\ Λχο BJV 3ュ V JSS J9S dsy dュ丄"TO OJJ 3jy" TO nsq sA 9j¾ gjy u \ Λχο BJV 3 ュ V JSS J 9 S dsy d 丄
91丄 ΐ OVO V33 09V VV3 313 OVV OXV 903 WO 009 130 OOV 30V ID1 3V3 001 91 丄 ΐ OVO V33 09V VV3 313 OVV OXV 903 WO 009 130 OOV 30V ID1 3V3 001
981^ 08 S ^ 981 ^ 08 S ^
OJd χο 。 naq STH JSS S^V ^Ί. "TO Η9Ί J J3S OJd ιΑχOJd χο. naq STH JSS S ^ V ^ Ί. "TO Η9 Ί J J3 S OJd ιΑχ
8991 V3D 900 130 013 1V3 13V 003 X3I 111 313 3V0 V13 33V 331 V33 3VX 8991 V3D 900 130 013 1V3 13V 003 X3I 111 313 3V0 V13 33V 331 V33 3VX
0 99^ 09， 0 99 ^ 09,
SJV
Figure imgf000055_0001
na naq Βχγ STH UTO Βχγ OJJ ηχ STH ΘΠ SIH SJ V
Figure imgf000055_0001
na naq Βχγ STH UTO Βχγ OJJ ηχ STH ΘΠ SIH
0291 09V 3VI 339 310 010 330 0V3 3X9 IVD OVD 030 130 0V9 1V3 DIV XV3 0291 09V 3VI 339 310 010 330 0V3 3X9 IVD OVD 030 130 0V9 1V3 DIV XV3
S OQ^ ^ n3q u 9
Figure imgf000055_0002
naq sAq s i ns jq aqj §jy o-td n91 Say sAq sAqS OQ ^ ^ n3q u 9
Figure imgf000055_0002
naq sAq si ns jq aqj §jy o -td n9 1 Say sAq sAq
OID WD OVO VIO 911 3VV OVV 0X0 IDV 311 OOV V33 VIO DOV VVV 3VV οε OID WD OVO VIO 911 3VV OVV 0X0 IDV 311 OOV V33 VIO DOV VVV 3VV οε
"TO -las ΤΒΛ BTV usy JA BTV ^τν ηχο aqj JSS an uig ηχο usy sAq ^51 9V0 VOX 910 V30 XVV IVl 130 VD3 WO Oil 031 UV OVO WO DVV VVV  "TO-las ΤΒΛ BTV usy JA BTV ^ τν ηχο aqj JSS an uig ηχο usy sAq ^ 51 9V0 VOX 910 V30 XVV IVl 130 VD3 WO Oil 031 UV OVO WO DVV VVV
^ s  ^ s
nsq oュ d dJi τ Jas sAq OJJ s j sAo sA Αχ J3S "TO 34d oュ dnsq o d dJi τ Jas sAq OJJ sj sAo sA Αχ J3S "TO 3 4d o d
9 H XIO 033 391 110 30V OVV VOO III 131 VVV 330 XOV 0V3 VOX III V33 01 00^ 5689 H XIO 033 391 110 30V OVV VOO III 131 VVV 330 XOV 0V3 VOX III V33 01 00 ^ 568
OJd s Jii Sjy OJJ 3ュ y oュ d OJJ u g 3jy 3 J¾ sAq JSSOJd s Jii Sjy OJJ 3 yo yo d d OJJ ug 3jy 3 J¾ sAq JSS
8 I X33 VVV V3V 30V WO 133 300 300 V33 OVO 303 331 133 VDV 9VV 031 8 I X33 VVV V3V 30V WO 133 300 300 V33 OVO 303 331 133 VDV 9VV 031
068 988 088 068 988 088
SJV SIH s q 3jy OJJ usy Αχ sAq BIV naq u^o "^丄 ηχ dsy sqj uxgSJV SIH s q 3jy OJJ usy Αχ sAq BIV naq u ^ o "^ 丄 ηχ dsy sqj uxg
0881 VOO OVO VVV 33D V33 OVV OVl OVV 033 VI3 OVD 3V1 0V3 IVO Hi 3V30881 VOO OVO VVV 33D V33 OVV OVl OVV 033 VI3 OVD 3V1 0V3 IVO Hi 3V3
»D6lO/ .6df/XDd 9£SL0I86 OAV Glu Glu Leu l ie Asp Asn Gly Lys Leu Pro Phe Met Ala Met Leu Arg »D6lO / .6df / XDd 9 £ SL0I86 OAV Glu Glu Leu lie Asp Asn Gly Lys Leu Pro Phe Met Ala Met Leu Arg
525 530 535  525 530 535
AAC CTG TGT AAC CTG CTG CGG ACT GGG ATC AGT GCC CAC CAC CAT GAA 1860 AAC CTG TGT AAC CTG CTG CGG ACT GGG ATC AGT GCC CAC CAC CAT GAA 1860
Asn Leu Cys Asn Leu Leu Arg Thr Gly l ie Ser Ala His His His Glu Asn Leu Cys Asn Leu Leu Arg Thr Gly lie Ser Ala His His His Glu
540 545 550  540 545 550
CTC GTT CTC CAG AGA CTC CAG CAT GAG AAA TCT GTG ATT CAC AGT CGG 1908 CTC GTT CTC CAG AGA CTC CAG CAT GAG AAA TCT GTG ATT CAC AGT CGG 1908
Leu Val Leu Gin Arg Leu Gin His Glu Lys Ser Val He His Ser Arg Leu Val Leu Gin Arg Leu Gin His Glu Lys Ser Val He His Ser Arg
555 560 565 570 555 560 565 570
CAG TTT CCA TTC AGA TTC CTT AAT GCT CAC GAC TCT CTC GAT AGA CTC 1956 CAG TTT CCA TTC AGA TTC CTT AAT GCT CAC GAC TCT CTC GAT AGA CTC 1956
Gin Phe Pro Phe Arg Phe Leu Asn Ala His Asp Ser Leu Asp Arg Leu Gin Phe Pro Phe Arg Phe Leu Asn Ala His Asp Ser Leu Asp Arg Leu
575 580 585  575 580 585
GAG GCT CAG CTC AGA AGT AAA GCA TCG CCC TTC CCT TCC AAT ACA ACA 2004 GAG GCT CAG CTC AGA AGT AAA GCA TCG CCC TTC CCT TCC AAT ACA ACA 2004
Glu Ala Gin Leu Arg Ser Lys Ala Ser Pro Phe Pro Ser Asn Thr Thr Glu Ala Gin Leu Arg Ser Lys Ala Ser Pro Phe Pro Ser Asn Thr Thr
590 595 600  590 595 600
TTG ATG AAG CGG ATA ATG ATT AGA AAC TCA AAA AAA ATC AAG AGA CCT 2052 TTG ATG AAG CGG ATA ATG ATT AGA AAC TCA AAA AAA ATC AAG AGA CCT 2052
Leu Met Lys Arg l ie Met l ie Arg Asn Ser Lys Lys l ie Lys Arg Pro Leu Met Lys Arg lie Met lie Arg Asn Ser Lys Lys lie Lys Arg Pro
605 610 615  605 610 615
GCC AAC CCG AGG TAC CTG TGC ACC CTG ACG CAG CGG CAG CTT CGG GCG 2100 GCC AAC CCG AGG TAC CTG TGC ACC CTG ACG CAG CGG CAG CTT CGG GCG 2100
Ala Asn Pro Arg Tyr Leu Cys Thr Leu Thr Gin Arg Gin Leu Arg Ala Ala Asn Pro Arg Tyr Leu Cys Thr Leu Thr Gin Arg Gin Leu Arg Ala
620 625 630  620 625 630
GCA ATG GCT ATC CCG GTG ATG TAT GAG CAT CTC AAG CGG GAG AAA CTG 2148 GCA ATG GCT ATC CCG GTG ATG TAT GAG CAT CTC AAG CGG GAG AAA CTG 2148
Ala Met Ala l ie Pro Val Met Tyr Glu His Leu Lys Arg Glu Lys Leu Ala Met Ala lie Pro Val Met Tyr Glu His Leu Lys Arg Glu Lys Leu
635 640 645 650 AGG CTG CAC AAG GCC AGA CAG TGG ACC TGT GAC CTT GAG TTG CTG GAG 2196635 640 645 650 AGG CTG CAC AAG GCC AGA CAG TGG ACC TGT GAC CTT GAG TTG CTG GAG 2196
Arg Leu His Lys Ala Arg Gin Trp Thr Cys Asp Leu Glu Leu Leu Glu Arg Leu His Lys Ala Arg Gin Trp Thr Cys Asp Leu Glu Leu Leu Glu
655 660 665  655 660 665
CGG TAT CGC CAG GCC CTG GAA ACG GCC GTG AAC ATC TCT GTA AAG CAC 2244 CGG TAT CGC CAG GCC CTG GAA ACG GCC GTG AAC ATC TCT GTA AAG CAC 2244
Arg Tyr Arg Gin Ala Leu Glu Thr Ala Val Asn l ie Ser Val Lys His 670 675 680Arg Tyr Arg Gin Ala Leu Glu Thr Ala Val Asn lie Ser Val Lys His 670 675 680
AAC CTA CCC CCG CTG CCA GGC CGA ACC CTC TTG GTC TAT CTC ACA GAT 2292AAC CTA CCC CCG CTG CCA GGC CGA ACC CTC TTG GTC TAT CTC ACA GAT 2292
Asn Leu Pro Pro Leu Pro Gly Arg Thr Leu Leu Val Tyr Leu Thr Asp Asn Leu Pro Pro Leu Pro Gly Arg Thr Leu Leu Val Tyr Leu Thr Asp
685 690 695  685 690 695
GCA AAT GCC AAC AGA CTT TGT CCC AAG AGT CAC TTG CAA GGG CCT CCC 2340 GCA AAT GCC AAC AGA CTT TGT CCC AAG AGT CAC TTG CAA GGG CCT CCC 2340
Ala Asn Ala Asn Arg Leu Cys Pro Lys Ser His Leu Gin Gly Pro Pro Ala Asn Ala Asn Arg Leu Cys Pro Lys Ser His Leu Gin Gly Pro Pro
700 705 710  700 705 710
CTG AAC TAT GTG CTG CTG TTG ATC GGG ATG ATG ATG GCT CGG GCG GAG 2388 CTG AAC TAT GTG CTG CTG TTG ATC GGG ATG ATG ATG GCT CGG GCG GAG 2388
Leu Asn Tyr Val Leu Leu Leu He Gly Met Met Met Ala Arg Ala Glu 715 720 725 730Leu Asn Tyr Val Leu Leu Leu He Gly Met Met Met Ala Arg Ala Glu 715 720 725 730
CAG ACG ACA GTT TGG CTG TGT GGG ACA GGA ACT GTG AAG ACA CCA GTA 2436CAG ACG ACA GTT TGG CTG TGT GGG ACA GGA ACT GTG AAG ACA CCA GTA 2436
Gin Thr Thr Val Trp Leu Cys Gly Thr Gly Thr Val Lys Thr Pro Val Gin Thr Thr Val Trp Leu Cys Gly Thr Gly Thr Val Lys Thr Pro Val
735 740 745  735 740 745
CTT ACA GCC GAC GAA GGT ATC CTG AAG ACT GCC ATC AAA CTT CAG CCT 2484 CTT ACA GCC GAC GAA GGT ATC CTG AAG ACT GCC ATC AAA CTT CAG CCT 2484
Leu Thr Ala Asp Glu Gly l ie Leu Lys Thr Ala He Lys Leu Gin Ala Leu Thr Ala Asp Glu Gly lie Leu Lys Thr Ala He Lys Leu Gin Ala
750 755 760 750 755 760
CAA GTC CAG GAG TTA GAA GAA AAT GAT GAG TGG CCC CTG GAA ACT TTT 2532CAA GTC CAG GAG TTA GAA GAA AAT GAT GAG TGG CCC CTG GAA ACT TTT 2532
Gin Val Gin Glu Leu Glu Glu Asn Asp Glu Trp Pro Leu Glu Thr Phe Gin Val Gin Glu Leu Glu Glu Asn Asp Glu Trp Pro Leu Glu Thr Phe
765 770 775  765 770 775
GAG AAG TAC CTG CTA TCT CTG GCT GTG CGA AGG ACC CCT ATT GAC AGG 2580 GAG AAG TAC CTG CTA TCT CTG GCT GTG CGA AGG ACC CCT ATT GAC AGG 2580
Glu Lys Tyr Leu Leu Ser Leu Ala Val Arg Arg Thr Pro l ie Asp Arg Glu Lys Tyr Leu Leu Ser Leu Ala Val Arg Arg Thr Pro lie Asp Arg
780 785 790  780 785 790
GTC ATC CTG TTC GGC CAA AGG ATG GAT ACG GAG CTG CTG AAT GTA GCC 2628 GTC ATC CTG TTC GGC CAA AGG ATG GAT ACG GAG CTG CTG AAT GTA GCC 2628
Val l ie Leu Phe Gly Gin Arg Met Asp Thr Glu Leu Leu Asn Val Ala 795 800 805 810Val lie Leu Phe Gly Gin Arg Met Asp Thr Glu Leu Leu Asn Val Ala 795 800 805 810
AAA CAG ATT ATC TGG CAG CAT GTG AAT TCC AAG TGC CTC TTC GTC AGT 2676AAA CAG ATT ATC TGG CAG CAT GTG AAT TCC AAG TGC CTC TTC GTC AGT 2676
Lys Gin l ie l ie Trp Gin His Val Asn Ser Lys Cys Leu Phe Val Ser Lys Gin lie lie Trp Gin His Val Asn Ser Lys Cys Leu Phe Val Ser
815 820 825 99 815 820 825 99
9518 D13 31V 030 010 311 013 0V3 I3X OVV OVO 310 0V3 000 1X3 331 010 0L6 596 096 556 niO nsq u^ usy Siy m nT9 ηχο ηχ^ ι¾ 9U Λχ dj丄 v9518 D13 31V 030 010 311 013 0V3 I3X OVV OVO 310 0V3 000 1X3 331 010 0L6 596 096 556 niO nsq u ^ usy Siy m n T9 ηχο ηχ ^ ι¾ 9U Λχ dj 丄 v
801 S VVO 0X3 VVO V3V OVV 03V 333 33V OVO VVO 0V9 03V 3iV VOO 031 300 801 S VVO 0X3 VVO V3V OVV 03V 333 33V OVO VVO 0V9 03V 3iV VOO 031 300
096 m na dsy an BTV STH Π3Ί ュ θΐ Ι Sjy S TH 0 d 3Md BW 3jV ^IV uTO096 m na dsy an B TV S TH Π3 θ θ ΐ Ι Sjy S TH 0 d 3 Md B W 3j V ^ IV u TO
090S OID DV9 ilV 330 0V3 113 30V OIV 303 OVD 303 311 030 V03 330 OVO 090S OID DV9 ilV 330 0V3 113 30V OIV 303 OVD 303 311 030 V03 330 OVO
986 086 6 naq B-[V OJJ nsq -[BA JSS 3ュ V naq na dsy 3 n^g Α ^ sfH I9)ij  986 086 6 naq B- [V OJJ nsq-[BA JSS 3 ュ V naq na dsy 3 n ^ g Α ^ sfH I9) ij
21 OS 010 030 V33 3X3 110 101 V93 OXV OXD Oil 3V0 VOO VVO 300 XV3 OXV 21 OS 010 030 V33 3X3 110 101 V93 OXV OXD Oil 3V0 VOO VVO 300 XV3 OXV
026 5ΐ 6 0Ϊ6  026 5ΐ 6 0Ϊ6
dsy V 3Md J3S ュ as 3i【 aqj ngq 3iy 3Π usy 8jy dJ丄 Αχο S TH 962 DVO V33 311 13V 331 331 UV 0X1 XXO 003 OiV 3VV 333 001 VOO IVO  dsy V 3Md J3S asas 3i 【aqj ngq 3iy 3Π usy 8jy dJ 丄 Sο S TH 962 DVO V33 311 13V 331 331 UV 0X1 XXO 003 OiV 3VV 333 001 VOO IVO
506 006 968  506 006 968
UT9 J3s 3T I OJd ΙΒΛ 9Md OJd Λχο OJJ usy usy ηχ^ ηχο naq OJJ 3ay UT9 J3s 3T I OJd ΙΒΛ 9Md OJd Λχο OJJ usy usy ηχ ^ ηχο naq OJJ 3ay
9162 OVD 331 IlV 133 1X3 IL 330 IDO 133 OVV OVV OVO OVO 013 333 ODD 068 588 088 9189162 OVD 331 IlV 133 1X3 IL 330 IDO 133 OVV OVV OVO OVO 013 333 ODD 068 588 088 918
"97 OJd ass ΙΒΛ sA J¾ s q A[o OJJ OJJ OJJ OI^ d\[ s 9¾j an "97 OJd ass ΙΒΛ sA J¾ s q A [o OJJ OJJ OJJ OI ^ d \ [s 9¾j an
8982 313 133 VOX 310 9VV V3V 3VV VOO VOO ODD VDO I3D OIV OVV 311 VXV 8982 313 133 VOX 310 9VV V3V 3VV VOO VOO ODD VDO I3D OIV OVV 311 VXV
0L2 998 098 sAq dsy nsi UTO ^TO ΐ¾ STH ηχο na na Sjy J8S ^TV AI3 S TH ηχο0L2 998 098 sAq dsy nsi UTO ^ TO ΐ¾ STH ηχο na na Sjy J8S ^ TV A I3 S TH ηχο
OVV IV9 VX3 VVO 399 0X0 XV3 VVO 9X3 113 103 Ldl 033 VOO XVO 9V0 OVV IV9 VX3 VVO 399 0X0 XV3 VVO 9X3 113 103 Ldl 033 VOO XVO 9V0
958 058 9^8  958 058 9 ^ 8
^TV θΐ ΐ 9¾j sA nsq an A TO dsy J¾ SAQ Α η3Ί J 八 dsy^ TV θΐ ΐ 9¾j sA nsq an A TO dsy J¾ SAQ η η3 Ί J eight dsy
Zlll 909 IlV ail OVV 913 OXV 030 3V3 13V 301 309 031 DID 03V 0X0 IVO Zlll 909 IlV ail OVV 913 OXV 030 3V3 13V 301 309 031 DID 03V 0X0 IVO
0^8 968 0£8  0 ^ 8 968 0 £ 8
usy OJJ usy naq usy OJJ JSS αΑ .9¾ sXq 8jy nsq[ na 八 ί^Ζ 2 XVV 330 XVV Ll IVV VOO VOL 9XV 3VX 3VD OXV VVV 303 VXD OXD Old usy OJJ usy naq usy OJJ JSS αΑ .9¾ sXq 8jy nsq [na 8ί ^ Ζ 2 XVV 330 XVV Ll IVV VOO VOL 9XV 3VX 3VD OXV VVV 303 VXD OXD Old
P06Z0/L6drilDd 8£ 0/86 OAV L P06Z0 / L6drilDd 8 £ 0/86 OAV L
UT3 BIV ^ΐθ ojj uio naq αΑχ §jy sAq ujg an ュ 9S 丄 τ^Λ dsyUT3 BIV ^ ΐθ ojj uio naq αΑχ §jy sAq ujg an Interview 9 S丄τ ^ Λ dsy
0V3 330 009 133 OVO 010 3V1 100 OVV 9VD OXV 3X0 30V 001 013 IVO 0V3 330 009 133 OVO 010 3V1 100 OVV 9VD OXV 3X0 30V 001 013 IVO
ΟΠ Ϊ 50Π OOU uiO naq A n9q Αχ^ sqj ηχο ηχ9 nsq Αχο λτθ ュ ェ OJJ 3jy ΟΠ Ϊ 50Π OOU uiO naq A n9q Αχ ^ sqj ηχο ηχ9 nsq Αχο λτθ O OJJ 3jy
0^9ε VV3 313 1X9 OIX 0V3 VOO III OVO OVO 013 D90 9D3 I3V IVl 333 930 0 ^ 9ε VV3 313 1X9 OIX 0V3 VOO III OVO OVO 013 D90 9D3 I3V IVl 333 930
5601 0601 980Ϊ 5601 0601 980Ϊ
^TO BTV BTV Τ^Λ ^TO ^TO dJI ηχο SAQ JSS JA丄 3jy 3^V s J¾ 1¾^ TO BTV BTV Τ ^ Λ ^ TO ^ TO dJI ηχο SAQ JSS JA 丄 3jy 3 ^ V s J¾ 1¾
Ζ6^ε 300 030 VOO VIO 330 V39 OOX WO 101 DDI DVl 30V 303 301 33V 丄丄3 Ζ6 ^ ε 300 030 VOO VIO 330 V39 OOX WO 101 DDI DVl 30V 303 301 33V 丄 丄 3
080Ϊ 9iOI OiOI  080Ϊ 9iOI OiOI
ηΐθ sAq uio ηχο UT naq sqj 3jy sAq ngq ηχ^ id SJV S TH BTV nn 3vo vvv ova wo m via an vov ovv oio wo VDI axo ooa voo ηΐθ sAq uio ηχο UT naq sqj 3jy sAq ngq ηχ ^ id SJV S TH BTV nn 3vo vvv ova wo m via an vov ovv oio wo VDI axo ooa voo
■ 0901 9501 ■ 0901 9501
BIV ηΐΰ ηχ9 JSS ητο 19$ d i sqj dsy OJJ sA djx T^A dsy OJJ τ^Λ BIV ηΐΰ ηχ9 JSS ητο 19 $ d i sqj dsy OJJ sA djx T ^ A dsy OJJ τ ^ Λ
9626 130 OVO WO VOX OVO OOI IIV III 3V0 133 VVV 931 3X3 iVO VDO 9139626 130 OVO WO VOX OVO OOI IIV III 3V0 133 VVV 931 3X3 iVO VDO 913
0501 9^οΐ o \ geoi0501 9 ^ οΐ o \ geoi
J3S "31 slid Λχο OJJ dsy Siy 9Md ユ ^TV "TO dsV ュ 3SJ3S "31 slid Λχο OJJ dsy Siy 9Md Yu ^ TV" TO ds V Interview 3 S
8^8 IDX 39V 113 Oil 193 133 IVO VOD OIL 3VI DIV Old LOO WD OVO 131 8 ^ 8 IDX 39V 113 Oil 193 133 IVO VOD OIL 3VI DIV Old LOO WD OVO 131
080Ϊ S20I 0201 080Ϊ S20I 0201
OJd ηχο JSS Sjy UTO A13 gjy usy na 9¾j ui 3« I^A MS 9« ηΐ0 οοεε 330 wo οαι οοο ννα οοο χοο ονν οια an ΰνο οιν οιο ονο οιν ονο OJd ηχο JSS Sjy UTO A13 gjy usy na 9¾j ui 3 «I ^ A MS 9« η ΐ0 οοεε 330 wo οαι οοοοννα οοο χοο ονν οια an ΰνο οιν οιο ονονον
9ΐ01 Οΐθΐ S00T jiU ΤΒΛ S 3·ΐν ^ΤΟ -I3S QJd i Sjy uio jqx dJi S TH sqd S TH OJJ 9ΐ01 Οΐθΐ S00T jiU ΤΒΛ S 3ΐ ΤΟν ^ ΤΟ -I3S QJd i Sjy uio jqx dJi S TH sqd S TH OJJ
VOV VXD X3X 003 000 001 I3D DVJ, V9D OVO 33V 001 OVO ill 3V3 ODD VOV VXD X3X 003 000 001 I3D DVJ, V9D OVO 33V 001 OVO ill 3V3 ODD
000Ϊ 566 066 000Ϊ 566 066
STH dsy ojj n3i dsy J ^9S o-^d OJJ jqx Αχ Ατ ^丄 Sjy ュ 3S ^ΐθ οζε ova Dvo xao oio ivo ivx aov DDO DOO ιαν IVI 330 ivi ooo oax ooo STH dsy ojj n3i dsy J ^ 9 S o- ^ d OJJ jqx Αχ Ατ ^丄Sjy Interview 3 S ^ ΐθ οζε ova Dvo xao oio ivo ivx aov DDO DOO ιαν IVI 330 ivi ooo oax ooo
S86 086 S 6  S86 086 S 6
nsi 9Π io χΒΛ aqj ng UTO S usy ηχ "TO ^TO η91 sA0 T¾ ム 6dTA M 8£8 0/86 OAV 83 nsi 9Π io χΒΛ aqj ng UTO Susy ηχ "TO ^ TO η9 1 sA 0 T¾ m 6dTA M 8 £ 8 0/86 OAV 83
Ολ2Ι 9921 092ΐ η3Ί JMI "13 Αιο OJd uio naq dj uio Βχγ J9S sAq na nsq nsq sA m on xov ovo ooo vao vvo oxa ooi ovo oao ioi vvv DID OIO OIO ovv Ολ2Ι 9921 092ΐ η 3 Ί JMI "13 Αιο OJd uio naq dj uio Βχγ J9S sAq na nsq nsq sA m on xov ovo ooo vao vvo oxa ooi ovo oao ioi vvv DID OIO OIO ovv
99ΖΪ 0 ΐ 5^1  99ΖΪ 0 ΐ 5 ^ 1
"TO UTO na ηχο c 丄 \B naq Say 丄 J3S 0Jd nal BTV ュ3 S z OVD ovo oia vvo ooi oio oia aoo vov DVI ιαν aov OOD OIO XDD OOV "TO UTO na ηχο c丄\ B naq Say丄J3 S 0J d na l B TV Interview 3 S z OVD ovo oia vvo ooi oio oia aoo vov DVI ιαν aov OOD OIO XDD OOV
0^1 9821  0 ^ 1 9821
OJJ η ο ng sA siy ns STJI jq SAQ 3ュ v n3l ndl fZ^ ODD 0V9 VOO OID VVV VVD IV3 DID IVD D3V 101313303 V3V 313310 m\ m\ 9121 OJJ η ο ng sA siy ns STJI jq SAQ 3 ュ v n3 l nd l fZ ^ ODD 0V9 VOO OID VVV VVD IV3 DID IVD D3V 101313303 V3V 313310 m \ m \ 9121
usv 9qd EIV η9Ί sAo UTO dsy OJJ Sjy ^TV BTV ュ 9S 9Md stH sqd sMd usv 9qd EIV η9 Ί sAo UTO dsy OJJ Sjy ^ TV BTV Interview 9 S 9 Md stH sqd s Md
9 8S OVV 3X113D XX3 ΙΰΙ OVO OVO 133303300 V39 VOI IIX 3V3311 Oil9 8S OVV 3X113D XX3 ΙΰΙ OVO OVO 133 303 300 V39 VOI IIX 3V3311 Oil
0Ϊ2Ϊ 5021 OOZI S6I10Ϊ2Ϊ 5021 OOZI S6I1
ISA 3Md OJd BIV ΤΒΛ usv oad uio dsy OJJ I¾A sAq ngq ejv s 八ISA 3Md OJd BIV ΤΒΛ usv oad uio dsy OJJ I¾A sAq ngq ejv s
8288 110 Oil 333330010 IVV 333 OVO DV9 ODD 110 OVV 01333903X 013 8288 110 Oil 333330010 IVV 333 OVO DV9 ODD 110 OVV 01333903X 013
06Π 98Π 08Π t 9i J9S BIV "31 3ild BTV J sA Αχ9 uio BTV UTO AJO ΘΠ ΐ 06Π 98Π 08Π t 9i J9S BIV "31 3ild BTV J sA Αχ9 uio BTV UTO AJO ΐ ΐ
0818 110 DDI VDO OID Oil 339 IOV OVV V33 OVO V33 VOO OVO 09011V 019 0818 110 DDI VDO OID Oil 339 IOV OVV V33 OVO V33 VOO OVO 09011V 019
51Π OiTI 99Π  51Π OiTI 99Π
nsi J9g nsi Sjy AIO STH OJd na sw naq uif) uio I^A ュ DSV "TOnsi J9g nsi Sjy AIO STH OJd na sw naq uif) uio I ^ A DS V "TO
VI330V 013 OOV 000 OVO 333013 OIV 313 OVD VVO OIO 33V IVO OVO VI330V 013 OOV 000 OVO 333013 OIV 313 OVD VVO OIO 33V IVO OVO
09Π ¾9ίΐ 05Π  09Π ¾9ίΐ 05Π
naq na^ 8jy oュ d 3jy BTV OJJ J3S OJJ OJJ as sAq na ufj) u g 9iid 89S ilD 113333 VOO 303 V30 ODD 10V 033 V3303V 3VV Old 9V30V3 illnaq na ^ 8jy o d 3jy B TV OJJ J3S OJJ OJJ as sAq na ufj) ug 9iid 89S ilD 113333 VOO 303 V30 ODD 10V 033 V3303V 3VV Old 9V30V3 ill
\ m\ Gen  \ m \ Gen
S v UTO ΘΤ Ι naq dsy JQS 3T I Αχο OJJ ητο naq 9898 30V 333 OVO OIV 3111V30V9 VVO VOX 3XV 331 V93 V3D 0V33V0 Oil Οεΐΐ ¾2Π ΟΖΠ 9Πΐ S v UTO Ι Ι naq dsy JQS 3T I Αχο OJJ ητο naq 9898 30V 333 OVO OIV 3111V30V9 VVO VOX 3XV 331 V93 V3D 0V33V0 Oil Οεΐΐ ¾2Π ΟΖΠ 9Πΐ
P06Z0/L6dr/L3d 9£8L0/96 OAS. GTC CTT ATT ATC GAC GGG GCA GAT AAG TTG GTG GAC CAT AAT GGA CAG 4068 Val Leu l ie l ie Asp Gly Ala Asp Lys Leu Val Asp His Asn Gly Gin 1275 1280 1285 1290P06Z0 / L6dr / L3d 9 £ 8L0 / 96 OAS. GTC CTT ATT ATC GAC GGG GCA GAT AAG TTG GTG GAC CAT AAT GGA CAG 4068 Val Leu lie lie Asp Gly Ala Asp Lys Leu Val Asp His Asn Gly Gin 1275 1280 1285 1290
CTG ATT TCA GAC TGG ATC CCC AAG TCT CTT CCG CGG CGA GTA CAC CTG 4116 Leu He Ser Asp Trp l ie Pro Lys Ser Leu Pro Arg Arg Val His Leu CTG ATT TCA GAC TGG ATC CCC AAG TCT CTT CCG CGG CGA GTA CAC CTG 4116 Leu He Ser Asp Trp lie Pro Lys Ser Leu Pro Arg Arg Val His Leu
1295 1300 1305  1295 1300 1305
GTG CTG AGT GTG TCT AGT GAC TCA GGC CTG GGA GAG ACC CTT CAG CAA 4164 Val Leu Ser Val Ser Ser Asp Ser Gly Leu Gly Glu Thr Leu Gin Gin  GTG CTG AGT GTG TCT AGT GAC TCA GGC CTG GGA GAG ACC CTT CAG CAA 4164 Val Leu Ser Val Ser Ser Asp Ser Gly Leu Gly Glu Thr Leu Gin Gin
1310 1315 1320  1310 1315 1320
AGT CAG AGT GCT TAT GTG GTG GCC TTG GGG TCT TTG GTC CCG TCT TCA 4212 Ser Gin Ser Ala Tyr Val Val Ala Leu Gly Ser Leu Val Pro Ser Ser  AGT CAG AGT GCT TAT GTG GTG GCC TTG GGG TCT TTG GTC CCG TCT TCA 4212 Ser Gin Ser Ala Tyr Val Val Ala Leu Gly Ser Leu Val Pro Ser Ser
1325 1330 1335  1325 1330 1335
AGG GCT CAG CTT GTG AGA GAA GAG CTA GCA CTG TAT GGG AAA CGG CTG 4260 Arg Ala Gin Leu Val Arg Glu Glu Leu Ala Leu Tyr Gly Lys Arg Leu  AGG GCT CAG CTT GTG AGA GAA GAG CTA GCA CTG TAT GGG AAA CGG CTG 4260 Arg Ala Gin Leu Val Arg Glu Glu Leu Ala Leu Tyr Gly Lys Arg Leu
1340 1345 1350  1340 1345 1350
GAG GAG TCA CCT TTT AAC AAC CAG ATG CGG CTG CTG CTG GCA AAG CAG 4308 Glu Glu Ser Pro Phe Asn Asn Gin Met Arg Leu Leu Leu Ala Lys Gin 1355 1360 1365 1370 GAG GAG TCA CCT TTT AAC AAC CAG ATG CGG CTG CTG CTG GCA AAG CAG 4308 Glu Glu Ser Pro Phe Asn Asn Gin Met Arg Leu Leu Leu Ala Lys Gin 1355 1360 1365 1370
GGG TCA AGC CTG CCA CTG TAC CTG CAC CTC GTC ACT GAC TAC CTG AGG 4356 Gly Ser Ser Leu Pro Leu Tyr Leu His Leu Val Thr Asp Tyr Leu Arg GGG TCA AGC CTG CCA CTG TAC CTG CAC CTC GTC ACT GAC TAC CTG AGG 4356 Gly Ser Ser Leu Pro Leu Tyr Leu His Leu Val Thr Asp Tyr Leu Arg
1375 1380 1385  1375 1380 1385
CTT TTC ACA CTG TAC GAA CAG GTG TCT GAG AGA CTT CGA ACC CTG CCC 4404 Leu Phe Thr Leu Tyr Glu Gin Val Ser Glu Arg Leu Arg Thr Leu Pro  CTT TTC ACA CTG TAC GAA CAG GTG TCT GAG AGA CTT CGA ACC CTG CCC 4404 Leu Phe Thr Leu Tyr Glu Gin Val Ser Glu Arg Leu Arg Thr Leu Pro
1390 1395 1400  1390 1395 1400
GCC ACT CTC CCA CTG CTG CTG CAG CAC ATC CTG AGC ACC TTG GAG CAA 4452 Ala Thr Leu Pro Leu Leu Leu Gin His l ie Leu Ser Thr Leu Glu Gin  GCC ACT CTC CCA CTG CTG CTG CAG CAC ATC CTG AGC ACC TTG GAG CAA 4452 Ala Thr Leu Pro Leu Leu Leu Gin His lie Leu Ser Thr Leu Glu Gin
1405 1410 1415  1405 1410 1415
GAG CAT GGC CAT AAC GTC CTT CCT CAA GCT TTG ACT GCC CTT GAG GTC 4500 09 naq usy JiU ri3i sqj sAq BTV ndT. nsq 13 STH usy J8S naqGAG CAT GGC CAT AAC GTC CTT CCT CAA GCT TTG ACT GCC CTT GAG GTC 4500 09 naq usy JiU ri3i sqj sAq BTV nd T.nsq 13 STH usy J8S naq
Z96i^ 313 DVV 33V 113 Oil OVV V30 113 313 100 IVO DVV 300 OOV 0V3 313 Z96i ^ 313 DVV 33V 113 Oil OVV V30 113 313 100 IVO DVV 300 OOV 0V3 313
09GI 5551 0551  09GI 5551 0551
na^ sty JA OJJ nsq dsy sAq naq eiy OJJ OJJ SAQ JSS 3jy 3Mdna ^ sty JA OJJ nsq dsy sAq naq eiy OJJ OJJ SAQ JSS 3jy 3 Md
^88^ 010 OVO 3V1 133 Vil IVO VVV 013 100 OVO ODD 133 331 I3V V03 Oil ^ 88 ^ 010 OVO 3V1 133 Vil IVO VVV 013 100 OVO ODD 133 331 I3V V03 Oil
0^91 9S5I  0 ^ 91 9S5I
jqェ χο JSS BTV dsy OJJ dsy s ； sAq dJi ns SIH BTV ¾TV 9"Πjq ェ χο JSS BTV dsy OJJ dsy s; sAq dJin s SIH BTV ¾TV 9 "Π
33V 309 VOX 030 IV9 133 OVO 101 OiV 9VV 001 310 OVO 100 V33 丄丄 V 0851 5Z9I 0Ζ9ΐ 5ΐ5ΐ nai ISA S IR e^y -tqi sA na Λχ na-j Sjy sAq Αχο 3jy 333V 309 VOX 030 IV9 133 OVO 101 OiV 9VV 001 310 OVO 100 V33 丄 丄 V 0851 5Z9I 0Ζ9ΐ 5ΐ5ΐ nai ISA S IR e ^ y -tqi sA na Λχ na-j Sjy sAq Αχο 3jy 3
313 310 IVO ODD 13V OVV OVO VXD 039 913 90V VVV 000 IVI 303 130 313 310 IVO ODD 13V OVV OVO VXD 039 913 90V VVV 000 IVI 303 130
OiQl 9091 0051 sAq TEA B V ュ1 U 3JV ndl OJJ Α ^ dsy J9S naq SAQ naq 8jy ^TV 人 Ϊ3OiQl 9091 0051 sAq TEA BV 11 U 3JV nd l OJJ Α ^ dsy J9S naq SAQ naq 8jy ^ TV people Ϊ3
Οί'^ VVV 1X0 V33 V3V OOV 0X3 XDO 300 IVO X3X 3X3 30X 3X3 103 330 30i) ΐ 06 S8 Οί '^ VVV 1X0 V33 V3V OOV 0X3 XDO 300 IVO X3X 3X3 30X 3X3 103 330 30i) ΐ 06 S8
0Jd SJV T^A ojj nio Aio naq ns J9S Sjy η3Ί J9S "TO ΐ^Λ0Jd SJV T ^ A ojj nio Aio naq ns J9S Sjy η3 Ί J9 S "TO ΐ ^ Λ
269^ 100 Odd OVO 9X0 330 330 3V0 300 VI3 VU I0V 393 VXD XOV 9V3 310 269 ^ 100 Odd OVO 9X0 330 330 3V0 300 VI3 VU I0V 393 VXD XOV 9V3 310
08^1 SAH ΰί  08 ^ 1 SAH ΰί
naq JAx BTV 0Jd BI V OJJ 丄 naq usy ；） SJH S BTV 9 110 OVI 330 111 V33 130 Oil 333 OVI 313 DVV VOO I0V DVO 10V 330  naq JAx BTV 0Jd BI V OJJ 丄 naq usy;) SJH S BTV 9 110 OVI 330 111 V33 130 Oil 333 OVI 313 DVV VOO I0V DVO 10V 330
59^1 09 ΐ 99^1  59 ^ 1 09 ΐ 99 ^ 1
BTV ΪΒΛ BTV ηΐΟ ηχο (Ιιχ J9g sX Jtqi ηχο sAq OJJ naq J¾ naq (ΙαχBTV ΪΒΛ BTV η ΐΟ ηχο (Ιιχ J9g sX Jtqi ηχο sAq OJJ naq J¾ naq (Ιαχ
965^ 130 0X0 VOO OVO WO OOX 30V OVV I3V 0V3 OVV 330 Oil 13V 911 001965 ^ 130 0X0 VOO OVO WO OOX 30V OVV I3V 0V3 OVV 330 Oil 13V 911 001
05^1 i om ^zn ュ jas na BT V ST H naq uio dsy I^A J η3Ί Λΐ0 J9S STH aqi 8^ ^ 03V 30V 013 3ID V39 1V3 313 OVO OVO 910 I3V OID 100 I0V 3VD 03V 05 ^ 1 i om ^ zn Interview jas na BT V ST H naq uio dsy I ^ A J η3 Ί Λ ΐ0 J9 S STH aqi 8 ^ ^ 03V 30V 013 3ID V39 1V3 313 OVO OVO 910 I3V OID 100 I0V 3VD 03V
οε ι ^zn i ΐ^Λ " TO Η9Ί BTV -iMI nsq BIV "19 OJJ nsq χΒΛ usy S T H ATO SIR ni;) οε ι ^ zn i ΐ ^ Λ "TO Η9 Ί B TV -iMI nsq BIV" 19 OJJ nsq χΒΛ usy STH ATO SIR ni;)
IO6Z0/iL6dUr/X3d 8£8 .0/86 OAV ΐ9IO6Z0 / iL6dUr / X3d 8 £ 8.0 / 86 OAV ΐ9
ZL l 5U1 OUT  ZL l 5U1 OUT
ηΐΟ ηχο uio djx J¾ Say ndl usy naq ngq 丄 an ュ ^ld ^TV OVO OVO 3VD OOI 03V VOV 311 3VV Oil 013 DVI LLV VOV 000 103 130ηΐΟ ηχο uio djx J¾ Say nd l usy naq ngq 丄 an ^ ^ ld ^ TV OVO OVO 3VD OOI 03V VOV 311 3VV Oil 013 DVI LLV VOV 000 103 130
50 ΟΟΑΐ 569Ϊ 50 ΟΟΑΐ 569Ϊ
Jm ^ΐθ ΤΒΛ BTV BIV "TO usy οα^ s sqd ^TV Ι^Λ ¾TV J¾ 918 13V 900 DIO X30 VOO VOV WO 300 IVV IDD IDI OIL DDO OLD 133 IDV 0691 5891 089 ΐ 9191 ojd jag jas J3S 3 Π OJJ nsi s n S "TO UID 13 sA naq J¾ V03 031 V3I 331 IIV VD3 013 131 Oil 30V VV3 3V3 133 OVV Oil 33V J m ^ ΐθ ΤΒΛ BTV BIV "TO usy οα ^ s sqd ^ TV Ι ^ Λ ¾TV J¾ 918 13V 900 DIO X30 VOO VOV WO 300 IVV IDD IDI OIL DDO OLD 133 IDV 0691 5891 089 ΐ 9191 ojd jag jas J3S 3 Π OJJ nsi s n S "TO U ID 13 sA naq J¾ V03 031 V3I 331 IIV VD3 013 131 Oil 30V VV3 3V3 133 OVV Oil 33V
0Z9I 9991 0991 0Z9I 9991 0991
"TO oュ d sAi usy m dJi sAq naq 3χ [ SAQ UTO usy S T H dュ丄 8jy "TO"TO o d sAi usy m dJi sAq naq 3χ [SAQ UTO usy S T H d u 8jy" TO
0Z29 OVD 333 VVV IVV IIV 301 VVV 9ID VXV DOI OVO DVV 3V0 001 000 OVD 0Z29 OVD 333 VVV IVV IIV 301 VVV 9ID VXV DOI OVO DVV 3V0 001 000 OVD
959Ϊ 0991 m\ 959Ϊ 0991 m \
■ill丄 ns Π3ΐ OJJ BTV "TO SAQ SA] IBA OJJ J9S nio OJJ S■ ill 丄 ns Π3ΐ OJJ BTV "TO SAQ SA] IBA OJJ J9S nio OJJ S
ZL \ ^ 33V 313 013 ODD 330 3V3 DOI 301 110 13D VDl OVO WO IDD 0V3 33V ZL \ ^ 33V 313 013 ODD 330 3V3 DOI 301 110 13D VDl OVO WO IDD 0V3 33V
0^9ΐ 089Ϊ  0 ^ 9ΐ 089Ϊ
BTV BTV "TO uio nsq ngq naq naq OJJ αΛχ UTO am ns nsq aas BTV i^IS 130 VOO 0V3 OVD 3X3 9X3 3X3 Oil X3D JLV丄 3V3 3DV 113 313 VOL 133  BTV BTV "TO uio nsq ngq naq naq OJJ αΛχ UTO am ns nsq aas BTV i ^ IS 130 VOO 0V3 OVD 3X3 9X3 3X3 Oil X3D JLV 丄 3V3 3DV 113 313 VOL 133
529ΐ 029ΐ 9Ϊ9Ϊ  529ΐ 029ΐ 9Ϊ9Ϊ
"TO "TO sXq ns aqj usy S TH 3i T^A ¾TV T¾ DSV ^TV "TO c d Η9Ί 9Z09 3V3 WD VVV 0X3 Oil DVV OVD 311 VIO I3D 113 XVO V30 OVO 333 313 0ΐ9ΐ 5091 0091 5651 s UTO usy T^A NTO OJJ sAq J9S JGS BTV ュ入丄 Η9Ί nT0 ュ 丄 BIV nT0 8205 OVV OVO DVV 310 WO IDD OVV VDl 131 130 XVI 313 9V3 3VX X30 OVO "TO" TO sXq ns aqj usy S TH 3i T ^ A ¾TV T¾ DS V ^ TV "TO cd Η9 Ί 9Z09 3V3 WD VVV 0X3 Oil DVV OVD 311 VIO I3D 113 XVO V30 OVO 333 313 0ΐ9ΐ 5091 0091 5651 s UTO usy T ^ A N TO OJJ sAq J9S JGS BTV 丄Η9 Ί n T0 丄B IV n T0 8205 OVV OVO DVV 310 WO IDD OVV VDl 131 130 XVI 313 9V3 3VX X30 OVO
0691 9891 089Ϊ ri9i dsy OJ^ T^A "31 ^ TO T^A " TO ュ BT V ^TV I^A Ι^Λ STH O 0X1 3X3 OVO 033 DID VXD 130 0X0 VV3 013 IVl V30 X30 010 9X3 丄 V 0691 9891 089Ϊ ri9i dsy OJ ^ T ^ A "31 ^ TO T ^ A" TO BT V ^ TV I ^ A Ι ^ Λ S TH O 0X1 3X3 OVO 033 DID VXD 130 0X0 VV3 013 IVl V30 X30 010 9X3 丄 V
¾L9i 0Ζ5Ϊ 59GT  ¾L9i 0Ζ5Ϊ 59GT
P06Z0/L6dr/J d 8£8ん 0/86 O P97/02 04P06Z0 / L6dr / J d 8 £ 8 0/86 O P97 / 02 04
AAG GCT CTG GTG AGT GGC TGT GAT GGG ATT TCC TCT TTC GCG TTC CTG 5412AAG GCT CTG GTG AGT GGC TGT GAT GGG ATT TCC TCT TTC GCG TTC CTG 5412
Lys Ala Leu Val Ser Gly Cys Asp Gly l ie Ser Ser Phe Ala Phe Leu Lys Ala Leu Val Ser Gly Cys Asp Gly lie Ser Ser Phe Ala Phe Leu
1725 1730 1735  1725 1730 1735
TCA GAC ACT GCT CTT TTC CTT ACC ACC TTC GAT GGG CTC CTG GAG CTT 5460 TCA GAC ACT GCT CTT TTC CTT ACC ACC TTC GAT GGG CTC CTG GAG CTT 5460
Ser Asp Thr Ala Leu Phe Leu Thr Thr Phe Asp Gly Leu Leu Glu Leu Ser Asp Thr Ala Leu Phe Leu Thr Thr Phe Asp Gly Leu Leu Glu Leu
1740 1745 1750  1740 1745 1750
TGG GAC CTG CAA CAT GGT TGT TGG GTG TTC CAG ACC AAG GCC CAC CAG 5508 TGG GAC CTG CAA CAT GGT TGT TGG GTG TTC CAG ACC AAG GCC CAC CAG 5508
Trp Asp Leu Gin His Gly Cys Trp Val Phe Gin Thr Lys Ala His Gin Trp Asp Leu Gin His Gly Cys Trp Val Phe Gin Thr Lys Ala His Gin
1755 1760 1765 1770 1755 1760 1765 1770
TAC CAA ATC ACT GGC TGC TGC CTG AGC CCA GAC CGC CGC CTG CTG GCC 5556 TAC CAA ATC ACT GGC TGC TGC CTG AGC CCA GAC CGC CGC CTG CTG GCC 5556
Tyr Gin l ie Thr Gly Cys Cys Leu Ser Pro Asp Arg Arg Leu Leu Ala Tyr Gin l ie Thr Gly Cys Cys Leu Ser Pro Asp Arg Arg Leu Leu Ala
1775 1780 1785  1775 1780 1785
ACC GTG TGT TTG GGA GGA TAC GTA AAG CTG TGG GAC ACA GTC CAG GGC 5604 ACC GTG TGT TTG GGA GGA TAC GTA AAG CTG TGG GAC ACA GTC CAG GGC 5604
Thr Val Cys Leu Gly Gly Tyr Val Lys Leu Trp Asp Thr Val Gin Gly Thr Val Cys Leu Gly Gly Tyr Val Lys Leu Trp Asp Thr Val Gin Gly
1790 1795 1800  1790 1795 1800
CAG CTG GCT TTC CAG TAC ACC CAT CCC AAG TCT CTA AAC TGC ATC ACC 5652 CAG CTG GCT TTC CAG TAC ACC CAT CCC AAG TCT CTA AAC TGC ATC ACC 5652
Gin Leu Ala Phe Gin Tyr Thr His Pro Lys Ser Leu Asn Cys l ie Thr Gin Leu Ala Phe Gin Tyr Thr His Pro Lys Ser Leu Asn Cys lie Thr
1805 1810 1815  1805 1810 1815
TTC CAC CCA GAG GGG CAG GTG GTA GCC ACA GGC AAC TGG TCT GGC ATC 5700 TTC CAC CCA GAG GGG CAG GTG GTA GCC ACA GGC AAC TGG TCT GGC ATC 5700
Phe His Pro Glu Gly Gin Val Val Ala Thr Gly Asn Trp Ser Gly l ie Phe His Pro Glu Gly Gin Val Val Ala Thr Gly Asn Trp Ser Gly lie
1820 1825 1830  1820 1825 1830
GTG ACC TTC TTC CAG GCA GAT GGA CTC AAA GTC ACC AAG GAA CTA GGG 5748 GTG ACC TTC TTC CAG GCA GAT GGA CTC AAA GTC ACC AAG GAA CTA GGG 5748
Val Thr Phe Phe Gin Ala Asp Gly Leu Lys Val Thr Lys Glu Leu Gly Val Thr Phe Phe Gin Ala Asp Gly Leu Lys Val Thr Lys Glu Leu Gly
1835 1840 1845 1850 1835 1840 1845 1850
GGC CCA GGA CCC TCT GTT CGT ACG CTG GCA TTC AGT GCA CCC GGG AAG 5796 GGC CCA GGA CCC TCT GTT CGT ACG CTG GCA TTC AGT GCA CCC GGG AAG 5796
Gly Pro Gly Pro Ser Val Arg Thr Leu Ala Phe Ser Ala Pro Gly Lys Gly Pro Gly Pro Ser Val Arg Thr Leu Ala Phe Ser Ala Pro Gly Lys
1855 1860 1865  1855 1860 1865
GTT GTG GCT CTA GGC CGG ATA GAT GGG ACA GTG GAG CTG TGG GCC TGG 5844 S9 ΐ^Λ QJd sAq uio sAo TB J3S S "91 dj丄 naq J9S naq jas usy 010 133 OVV OVD 301 010 331 VOX 313 031 0X0 331 0V3 113 331 OVV 0102 9002 0002 566ΪGTT GTG GCT CTA GGC CGG ATA GAT GGG ACA GTG GAG CTG TGG GCC TGG 5844 S9 ΐ ^ Λ QJd sAq uio sAo TB J3S S "91 dj 丄 naq J9S naq jas usy 010 133 OVV OVD 301 010 331 VOX 313 031 0X0 331 0V3 113 331 OVV 0102 9002 0002 566Ϊ
SJV SJV nsi 9j[ ί Ατθ STH ngq J9§ Ατθ DSV nT3 ^TV ^ΐθ -iaS ΐ^Λ 8229 VOV OOV 010 9IV 001 300 1V3 013 331 300 XVO VVO V30 109 IOV 0X3 SJ V SJV nsi 9j [ί Ατθ STH ngq J9§ Ατθ DS V n T3 ^ TV ^ ΐθ -iaS ΐ ^ Λ 8229 VOV OOV 010 9IV 001 300 1V3 013 331 300 XVO VVO V30 109 IOV 0X3
0661 9861 086Ϊ η9Ί Ι^Λ J3S 0Jd J3g na IBA naq eiv τ^Λ BTV T¾ usy nsq 08 ΐ 9 311 0X0 30V 303 IOV 913 001 013 OID VOO IDX 013 300 310 IVV VI3 0661 9861 086Ϊ η 9 Ί Ι ^ Λ J3 S 0Jd J3g na IBA naq eiv τ ^ Λ B TV T¾ usy nsq 08 ΐ 9 311 0X0 30V 303 IOV 913 001 013 OID VOO IDX 013 300 310 IVV VI3
5i6T 0Α6Ϊ G96I  5i6T 0Α6Ϊ G96I
"TO "TO sAo ufo BTV ητο αχο OJJ Αχ^ s J9S 9T I §JV 丄 8TI s q 2819 OVO WD 391 VV 130 OVO OVD 333 100 VOX 131 IIV VOV 3VX 3XV VVV  "TO" TO sAo ufo BTV ητο αχο OJJ Αχ ^ s J9S 9T I §JV 丄 8TI s q 2819 OVO WD 391 VV 130 OVO OVD 333 100 VOX 131 IIV VOV 3VX 3XV VVV
0961 9961 0961  0961 9961 0961
3T I ^TO dsy Αχο 3ュ V ュ Λτ I^A BTV T^A ui3 dsy χο dsy OJJ usy 809 XIV 390 IVO VOO VOD 3VX 309 1X0 139 0X0 OVO 3V3 100 OVO VOD OVV  3T I ^ TO dsy Αχο 3 V Λτ I ^ A BTV T ^ A ui3 dsy χο dsy OJJ usy 809 XIV 390 IVO VOO VOD 3VX 309 1X0 139 0X0 OVO 3V3 100 OVO VOD OVV
5^61 01^61 596Ϊ  5 ^ 61 01 ^ 61 596Ϊ
nsi eiv ΤΒΛ J9S "91 ^TV OJJ J3S na JA丄 na J9S AID naq SAQ Αχο nsi eiv ΤΒΛ J9S "91 ^ TV OJJ J3S na JA 丄 na J9S AID naq SAQ Αχο
9S09 313 130 010 X3I OID 030 I3D 131 XID 1VX 113 131 303 0X3 331 100 0861 5261 0261 SI619S09 313 130 010 X3I OID 030 I3D 131 XID 1VX 113 131 303 0X3 331 100 0861 5261 0261 SI61
3JV oュ d 3jy ^Τΰ naq sq^ A\ J9S dj丄 nsq uia BTV sAq A13 dsy ηχο 8865 OOV 333 033 303 1X3 XIX VOO VDI 031 VIX 0V3 130 OVV 003 XVO VVO 3JV o d 3jy ^ Τΰ naq sq ^ A \ J9S dj 丄 nsq uia BTV sAq A13 dsy ηχο 8865 OOV 333 033 303 1X3 XIX VOO VDI 031 VIX 0V3 130 OVV 003 XVO VVO
0I6I 5061 0061 0I6I 5061 0061
^TO BTV J¾ "31 sqd S-iV ^ΤΟ χο εχν sin naq aqd nai χ^Λ ュ s 0^65 030 100 9DV 0X3 Oil 903 300 VOO 130 IVD 911 Oil 113 110 33V 331 ^ TO BTV J¾ "31 sqd S-iV ^ ΤΟ χο εχν sin naq aqd nai χ ^ Λ s 0 ^ 65 030 100 9DV 0X3 Oil 903 300 VOO 130 IVD 911 Oil 113 110 33V 331
568Ϊ 068Ϊ 5881 ΐ^Λ ^10 s Q ui3 0Jd 9qd BTV ^TV η3Ί Say J¾ 3 "TO "TO 2685 310 130 393 X3X DVO VOO 130 Oil 330 V30 0X3 003 VOV 300 0V3 VVO 568Ϊ 068Ϊ 5881 ΐ ^ Λ ^ 10 s Q ui3 0Jd 9qd BTV ^ TV η3 Ί Say J¾ 3 "TO" TO 2685 310 130 393 X3X DVO VOO 130 Oil 330 V30 0X3 003 VOV 300 0V3 VVO
0881 9Ζ8ΐ 0A81  0881 9Ζ8ΐ 0A81
dュ丄 eiv dJX naq TBA ^χο dsy o\ [ 3jy 3 ns^ BTV TBA ΐ^Λ 2015 2020 2025d 丄 eiv dJX naq TBA ^ χο dsy o \ [3jy 3 ns ^ BTV TBA ΐ ^ Λ 2015 2020 2025
CTG GGG CTG GCT GCC TCC CAG GAG TTC TTG GCT TCT GCC TCA GAG GAC 6324 Leu Gly Leu Ala Ala Ser Gin Glu Phe Leu Ala Ser Ala Ser Glu Asp CTG GGG CTG GCT GCC TCC CAG GAG TTC TTG GCT TCT GCC TCA GAG GAC 6324 Leu Gly Leu Ala Ala Ser Gin Glu Phe Leu Ala Ser Ala Ser Glu Asp
2030 2035 2040  2030 2035 2040
TTC ACG GTG CGA CTG TGG CCA AGA CAG CTG CTG ACA CAG CCA CAT GCA 6372 Phe Thr Val Arg Leu Trp Pro Arg Gin Leu Leu Thr Gin Pro His Ala  TTC ACG GTG CGA CTG TGG CCA AGA CAG CTG CTG ACA CAG CCA CAT GCA 6372 Phe Thr Val Arg Leu Trp Pro Arg Gin Leu Leu Thr Gin Pro His Ala
2045 2050 2055  2045 2050 2055
GTA GAA GAG TTG CCC TGT GCG GCT GAA CTC CGG GGA CAC GAG GGG CCG 6420 Val Glu Glu Leu Pro Cys Ala Ala Glu Leu Arg Gly His Glu Gly Pro  GTA GAA GAG TTG CCC TGT GCG GCT GAA CTC CGG GGA CAC GAG GGG CCG 6420 Val Glu Glu Leu Pro Cys Ala Ala Gla Leu Arg Gly His Glu Gly Pro
2060 2065 2070  2060 2065 2070
GTG TGC TGC TGT AGC TTC AGC CCG GAT GGA CGC ATC TTG GCC ACA GCG 6468 Val Cys Cys Cys Ser Phe Ser Pro Asp Gly Arg l ie Leu Ala Thr Ala 2075 2080 2085 2090 GTG TGC TGC TGT AGC TTC AGC CCG GAT GGA CGC ATC TTG GCC ACA GCG 6468 Val Cys Cys Cys Ser Phe Ser Pro Asp Gly Arg lie Leu Ala Thr Ala 2075 2080 2085 2090
GGC AGG GAT CGG AAT CTC CTC TGC TGG GAC GTC AAG GTA GCC CAA GCC 6516 Gly Arg Asp Arg Asn Leu Leu Cys Trp Asp Val Lys Val Ala Gin Ala GGC AGG GAT CGG AAT CTC CTC TGC TGG GAC GTC AAG GTA GCC CAA GCC 6516 Gly Arg Asp Arg Asn Leu Leu Cys Trp Asp Val Lys Val Ala Gin Ala
2095 2100 2105  2095 2100 2105
CCT CTC CTG ATT CAC ACG TTC TCG TCC TGT CAT CGA GAC TGG ATC ACT 6564 Pro Leu Leu l ie His Thr Phe Ser Ser Cys His Arg Asp Trp l ie Thr  CCT CTC CTG ATT CAC ACG TTC TCG TCC TGT CAT CGA GAC TGG ATC ACT 6564 Pro Leu Leu lie His Thr Phe Ser Ser Cys His Arg Asp Trp lie Thr
2110 2115 2120  2110 2115 2120
GGC TGT ACG TGG ACC AAA GAC AAC ATC CTG ATC TCC TGC TCT AGT GAT 6612 Gly Cys Thr Trp Thr Lys Asp Asn l ie Leu l ie Ser Cys Ser Ser Asp  GGC TGT ACG TGG ACC AAA GAC AAC ATC CTG ATC TCC TGC TCT AGT GAT 6612 Gly Cys Thr Trp Thr Lys Asp Asn l ie Leu lie Ser Cys Ser Ser Asp
2125 2130 2135  2125 2130 2135
GGC TCT GTG GGA CTC TGG AAC CCA GAG GCA GGA CAG CAA CTT GGC CAG 6660 Gly Ser Val Gly Leu Trp Asn Pro Glu Ala Gly Gin Gin Leu Gly Gin  GGC TCT GTG GGA CTC TGG AAC CCA GAG GCA GGA CAG CAA CTT GGC CAG 6660 Gly Ser Val Gly Leu Trp Asn Pro Glu Ala Gly Gin Gin Leu Gly Gin
2140 2145 2150  2140 2145 2150
TTC CCA GGT CAC CAG AGT GCC GTG AGC GCT GTG GTT GCT GTG GAG GAA 6708 Phe Pro Gly His Gin Ser Ala Val Ser Ala Val Val Ala Val Glu Glu 2155 2160 2165 2170 CAC ATT GTA TCT GTG AGT CGG GAT GGG ACC TTG AAA GTG TGG GAC CGT 6756 His l ie Val Ser Val Ser Arg Asp Gly Thr Leu Lys Val Trp Asp Arg TTC CCA GGT CAC CAG AGT GCC GTG AGC GCT GTG GTT GCT GTG GAG GAA 6708 Phe Pro Gly His Gin Ser Ala Val Ser Ala Val Val Ala Val Glu Glu 2155 2160 2165 2170 CAC ATT GTA TCT GTG AGT CGG GAT GGG ACC TTG AAA GTG TGG GAC CGT 6756 His lie Val Ser Val Ser Arg Asp Gly Thr Leu Lys Val Trp Asp Arg
2175 2180 2185  2175 2180 2185
CAG GGT GTG GAG CTG ACC AGC ATC CCT GCC CAT TCC GGA CCC ATT AGC 6804 Gin Gly Val Glu Leu Thr Ser l ie Pro Ala His Ser Gly Pro l ie Ser  CAG GGT GTG GAG CTG ACC AGC ATC CCT GCC CAT TCC GGA CCC ATT AGC 6804 Gin Gly Val Glu Leu Thr Ser lie Pro Ala His Ser Gly Pro lie Ser
2190 2195 2200 2190 2195 2200
CAG TGT GCG GCT GCT CTG GAA CCC CGT CCA GCT GGA CAG CCT GGA TCA 6852 Gin Cys Ala Ala Ala Leu Glu Pro Arg Pro Ala Gly Gin Pro Gly Ser CAG TGT GCG GCT GCT CTG GAA CCC CGT CCA GCT GGA CAG CCT GGA TCA 6852 Gin Cys Ala Ala Ala Leu Glu Pro Arg Pro Ala Gly Gin Pro Gly Ser
2205 2210 2215  2205 2210 2215
GAG CTT ATG GTG GTG ACT GTT GGA CTG GAT GGG GCC ACA AAG CTG TGG 6900 Glu Leu Met Val Val Thr Val Gly Leu Asp Gly Ala Thr Lys Leu Trp  GAG CTT ATG GTG GTG ACT GTT GGA CTG GAT GGG GCC ACA AAG CTG TGG 6900 Glu Leu Met Val Val Thr Val Gly Leu Asp Gly Ala Thr Lys Leu Trp
2220 2225 2230  2220 2225 2230
CAT CCC CTG TTG GTG TGC CAA ATA CAT ACC CTG CAG GGA CAC AGT GGT 6948 His Pro Leu Leu Val Cys Gin l ie His Thr Leu Gin Gly His Ser Gly 2235 2240 2245 2250 CAT CCC CTG TTG GTG TGC CAA ATA CAT ACC CTG CAG GGA CAC AGT GGT 6948 His Pro Leu Leu Val Cys Gin lie His Thr Leu Gin Gly His Ser Gly 2235 2240 2245 2250
CCA GTC ACA GCT GCT GCT GCT TCA GAG GCC TCA GGC CTC CTG CTG ACC 6996 Pro Val Thr Ala Ala Ala Ala Ser Glu Ala Ser Gly Leu Leu Leu Thr CCA GTC ACA GCT GCT GCT GCT TCA GAG GCC TCA GGC CTC CTG CTG ACC 6996 Pro Val Thr Ala Ala Ala Ala Ser Glu Ala Ser Gly Leu Leu Leu Thr
2255 2260 2265  2255 2260 2265
TCA GAC AAT AGC TCT GTA CGA CTC TGG CAG ATC CCT AAG GAA GCA GAT 7044 Ser Asp Asn Ser Ser Val Arg Leu Trp Gin l ie Pro Lys Glu Ala Asp  TCA GAC AAT AGC TCT GTA CGA CTC TGG CAG ATC CCT AAG GAA GCA GAT 7044 Ser Asp Asn Ser Ser Val Arg Leu Trp Gin lie Pro Lys Glu Ala Asp
2270 2275 2280  2270 2275 2280
GAT ACC TGC AAA CCT AGG AGT TCT GCG GTC ATC ACC GCT GTG GCG TGG 7092 Asp Thr Cys Lys Pro Arg Ser Ser Ala Val l ie Thr Ala Val Ala Trp  GAT ACC TGC AAA CCT AGG AGT TCT GCG GTC ATC ACC GCT GTG GCG TGG 7092 Asp Thr Cys Lys Pro Arg Ser Ser Ala Val lie Thr Ala Val Ala Trp
2285 2290 2295  2285 2290 2295
GCA CCA GAT GGT TCT CTG GTG GTG TCT GGA AAT GAA GCT GGG GAA CTA 7140 Ala Pro Asp Gly Ser Leu Val Val Ser Gly Asn Glu Ala Gly Glu Leu  GCA CCA GAT GGT TCT CTG GTG GTG TCT GGA AAT GAA GCT GGG GAA CTA 7140 Ala Pro Asp Gly Ser Leu Val Val Ser Gly Asn Glu Ala Gly Glu Leu
2300 2305 2310  2300 2305 2310
ACG CTG TGG CAG AAA GCG CAG GCT GTG GCT ACG GCA CGG GCT CCA GGC 7188 Thr Leu Trp Gin Lys Ala Gin Ala Val Ala Thr Ala Arg Ala Pro Gly 2315 2320 2325 2330ACG CTG TGG CAG AAA GCG CAG GCT GTG GCT ACG GCA CGG GCT CCA GGC 7188 Thr Leu Trp Gin Lys Ala Gin Ala Val Ala Thr Ala Arg Ala Pro Gly 2315 2320 2325 2330
CGC GTC AGT GAC CTC ATC TGG TGC TCC GCA AAT GCA TTC TTT GTT CTC 7236CGC GTC AGT GAC CTC ATC TGG TGC TCC GCA AAT GCA TTC TTT GTT CTC 7236
Arg Val Ser Asp Leu l ie Trp Cys Ser Ala Asn Ala Phe Phe Val Leu Arg Val Ser Asp Leu lie Trp Cys Ser Ala Asn Ala Phe Phe Val Leu
2335 2340 2345  2335 2340 2345
AGT GCT AAT GAA AAT GTC AGT GAG TGG CAA GTG GAA CTG AGG AAA GGT 7284 AGT GCT AAT GAA AAT GTC AGT GAG TGG CAA GTG GAA CTG AGG AAA GGT 7284
Ser Ala Asn Glu Asn Val Ser Glu Trp Gin Val Glu Leu Arg Lys Gly Ser Ala Asn Glu Asn Val Ser Glu Trp Gin Val Glu Leu Arg Lys Gly
2350 2355 2360  2350 2355 2360
TCA ACA TGC ACC AAT TTC AGA CTT TAT CTG AAG AGA GTT CTG CAG GAG 7332 TCA ACA TGC ACC AAT TTC AGA CTT TAT CTG AAG AGA GTT CTG CAG GAG 7332
Ser Thr Cys Thr Asn Phe Arg Leu Tyr Leu Lys Arg Val Leu Gin Glu Ser Thr Cys Thr Asn Phe Arg Leu Tyr Leu Lys Arg Val Leu Gin Glu
2365 2370 2375  2365 2370 2375
GAC TTG GGA GTC TTG ACA GGT ATG GCC CTG GCC CCT GAC GCC CAG TCT 7380 GAC TTG GGA GTC TTG ACA GGT ATG GCC CTG GCC CCT GAC GCC CAG TCT 7380
Asp Leu Gly Val Leu Thr Gly Met Ala Leu Ala Pro Asp Gly Gin Ser Asp Leu Gly Val Leu Thr Gly Met Ala Leu Ala Pro Asp Gly Gin Ser
2380 2385 2390  2380 2385 2390
CTC ATT TTG ATG AAA GAG GAT GTA GAA TTG CTA CAG ATG AAG CCC GGG 7428 CTC ATT TTG ATG AAA GAG GAT GTA GAA TTG CTA CAG ATG AAG CCC GGG 7428
Leu l ie Leu Met Lys Glu Asp Val Glu Leu Leu Gin Met Lys Pro Gly 2395 2400 2405 2410Leu lie Leu Met Lys Glu Asp Val Glu Leu Leu Gin Met Lys Pro Gly 2395 2400 2405 2410
TCT ACT CCA TCT TCG ATC TGC AGG AGG TAT GCA GTG CAT TCT TCT ATA 7476TCT ACT CCA TCT TCG ATC TGC AGG AGG TAT GCA GTG CAT TCT TCT ATA 7476
Ser Thr Pro Ser Ser He Cys Arg Arg Tyr Ala Val His Ser Ser l ie Ser Thr Pro Ser Ser He Cys Arg Arg Tyr Ala Val His Ser Ser lie
2415 2420 2425  2415 2420 2425
CTG TGC ACC AGC AAA GAC TAT GGC CTG TTT TAC CTG CAG CAG GGA AAC 7524 CTG TGC ACC AGC AAA GAC TAT GGC CTG TTT TAC CTG CAG CAG GGA AAC 7524
Leu Cys Thr Ser Lys Asp Tyr Gly Leu Phe Tyr Leu Gin Gin Gly Asn Leu Cys Thr Ser Lys Asp Tyr Gly Leu Phe Tyr Leu Gin Gin Gly Asn
2430 2435 2440  2430 2435 2440
TCT GGA TCT CTT TCT ATC TTG GAG CAG GAG GAG TCA GGG AAG TTT GAA 7572 TCT GGA TCT CTT TCT ATC TTG GAG CAG GAG GAG TCA GGG AAG TTT GAA 7572
Ser Gly Ser Leu Ser He Leu Glu Gin Glu Glu Ser Gly Lys Phe Glu Ser Gly Ser Leu Ser He Leu Glu Gin Glu Glu Ser Gly Lys Phe Glu
2445 2450 2455  2445 2450 2455
AAG ACC CTG GAC TTC AAT CTG AAC TTA AAT AAT CCT AAT GGG TCC CCA 7620 AAG ACC CTG GAC TTC AAT CTG AAC TTA AAT AAT CCT AAT GGG TCC CCA 7620
Lys Thr Leu Asp Phe Asn Leu Asn Leu Asn Asn Pro Asn Gly Ser Pro L9 z 0I9Z Lys Thr Leu Asp Phe Asn Leu Asn Leu Asn Asn Pro Asn Gly Ser Pro L9 z 0I9Z
dsy ^ΐθ ΐ¾Λ BTV ngq ui9 naq OJJ ュ 35 s OJJ 3f[ dュ丄 OJJ ηχο dsy ^ ΐθ ΐ¾Λ BTV ngq ui9 naq OJJ 35 35 s OJJ 3f [d 丄 OJJ ηχο
Z IVO VOO 013 I00 IIO OVO 0X0 003 131 39V 333 3V0 9IV 001 133 VVO Z IVO VOO 013 I00 IIO OVO 0X0 003 131 39V 333 3V0 9IV 001 133 VVO
0092 9692 0692  0092 9692 0692
n91 sAo jag TSA 0Jd Αχο ηχο SAQ Siy aqd na Αχο na nsq υχ 9» 008 013 XOI DOV 0X0 D3D 933 VV3 101 V30 311 311 303 313 313 OVO OIV n 9 1 sAo jag TSA 0Jd Αχο ηχο SAQ Siy aqd na Αχο na nsq υχ 9 »008 013 XOI DOV 0X0 D3D 933 VV3 101 V30 311 311 303 313 313 OVO OIV
9852 0892  9852 0892
J9s ojd SJV nT3 CIJI naq sA 八 dsy 3jy dsy n^ J9S ^TV Τ^ΛJ9s ojd SJV n T3 CIJI naq sA eight dsy 3jy dsy n ^ J9S ^ TV Τ ^ Λ
9G6i I9V ODD VOV OVO 031 3ID OVV IIO IVO V3V DVO 3V3 VOX 133 XOV 0X09G6i I9V ODD VOV OVO 031 3ID OVV IIO IVO V3V DVO 3V3 VOX 133 XOV 0X0
Z 09 595Z nsi rial Aio ojj ng 八 S TH naq εχν α¾ T^A J^S ^10 nsl S TH ST IZ 09 595Z nsi rial Aio ojj ng eight S TH naq εχν α¾ T ^ AJ ^ S ^ 10 ns l S TH ST I
806 OID 911 VOO 333 0X3 010 IV3 013 339 03V 313 131 303 Oil 3V3 11V 806 OID 911 VOO 333 0X3 010 IV3 013 339 03V 313 131 303 Oil 3V3 11V
0592 5^2 mZ sAq sAi SAQ ux3 3jy sAq naq S TH J¾ OJJ ηχο ΤΒΛ 丄 J3S dsy 0592 5 ^ 2 mZ sAq sAi SAQ ux3 3jy sAq naq S TH J¾ OJJ ηχο ΤΒΛ 丄 J3S dsy
098 9VV VVV XOI 0V3 OOD VOO OVV Vll 1V3 VOV OOD VVO VIO 001 33V 1V3 098 9VV VVV XOI 0V3 OOD VOO OVV Vll 1V3 VOV OOD VVO VIO 001 33V 1V3
9852 0852  9852 0852
sAo 9¾j n9i Αΐ9 OJJ J9S J3S dsy qi Αχο OJJ J¾ 3ュ V -isS sAq sAo 9¾j n9i Αΐ9 OJJ J9S J3S dsy qi Αχο OJJ J¾ 3 V -isS sAq
OXV 301 3X1 VII 300 VOO 331 93X IVO V3V 909 333 X3V VOO 10V VVV OXV 301 3X1 VII 300 VOO 331 93X IVO V3V 909 333 X3V VOO 10V VVV
0292 5192 0192  0292 5192 0192
ojd usy Siy J3S sAq sAq ux dj丄 ΘΠ usy dsy Τ^Λ ΐ^Λ 丄 ηχο Αχΰ ^ 丄 ID3 OVV VOV VOX VVV VVV OVD 001 OIV OVV IVO VIO 0X3 031 OVO V03 ojd usy Siy J3 S sAq sAq ux dj 丄 ΘΠ usy dsy Τ ^ Λ ΐ ^ Λ 丄 ηχο Αχΰ ^ 丄 ID3 OVV VOV VOX VVV VVV OVD 001 OIV OVV IVO VIO 0X3 031 OVO V03
90S2 0052 %U  90S2 0052% U
"TO OJJ jqx SAQ Π13 jas naq usy ΰαχ naq ΘΚ TO dsy J3S -iqi BTV "TO OJJ jqx SAQ Π13 jas naq usy ΰαχ naq ΘΚ TO dsy J3S -iqi BTV
9 i VVO V33 03V 101 OVO I3X VII OVV ODi 010 OXV 000 IVO 131 33V 133 06^ 58^2 08^2 S 2 s o nsq naq s ュ 95 Αχ J9S 。13 OJJ ηχ BTV "TO 9ΐΙ ΐ¾9 i VVO V33 03V 101 OVO I3X VII OVV ODi 010 OXV 000 IVO 131 33V 133 06 ^ 58 ^ 2 08 ^ 2 S 2 so nsq naq s 95 95 J9S. 13 OJJ ηχ BTV "TO 9 ΐΙ ΐ¾
899 XOI Oil IIO 03X 331 000 I3J, 0V3 133 VVO 130 OVO 丄 3V 3XV V3I VX3 899 XOI Oil IIO 03X 331 000 I3J, 0V3 133 VVO 130 OVO 丄 3V 3XV V3I VX3
0 Z 59^2 09^2 tO6Z0 L6dr/ D<I 8C8 .0/86 ΟΛλ GCA CAA GGA AAC TTG TAT TTT CTA TCT TGG GAA TGAAGATGAA 8095 Ala Gin Gly Asn Leu Tyr Phe Leu Ser Trp Glu *** 0 Z 59 ^ 2 09 ^ 2 tO6Z0 L6dr / D <I 8C8 .0 / 86 ΟΛλ GCA CAA GGA AAC TTG TAT TTT CTA TCT TGG GAA TGAAGATGAA 8095 Ala Gin Gly Asn Leu Tyr Phe Leu Ser Trp Glu ***
2620 2625 2629  2620 2625 2629
GAATCAGGAC AAAGATGGTG TCACCGGATG ATGGTCACCT GAAGACACCA GTGTCTATAT 8155 TCTTAATAAG GTTATAAAAT AAAGTGTTGG AAGATCTAAA AAAAAAAAAA AAAAAAAAAA 8215 配列番号： 2  GAATCAGGAC AAAGATGGTG TCACCGGATG ATGGTCACCT GAAGACACCA GTGTCTATAT 8155 TCTTAATAAG GTTATAAAAT AAAGTGTTGG AAGATCTAAA AAAAAAAAAA AAAAAAAAAAAA 8215 SEQ ID NO: 2
配列の長さ ：核酸 = 4 8 7、 アミノ酸 = 1 6 2 Sequence length: nucleic acid = 487, amino acid = 162
配列の型：核酸及びァミノ酸 Sequence type: nucleic acid and amino acid
トポロジー ：直鎖状二本鎖  Topology: linear double-stranded
配列の種類： c D N A Sequence type: c D N A
起源：生物名 ヒ ト Origin: organism name human
配列 Array
AAG TTC GCG CAG TTT GAC GAG TAC CAG CTG GCT AAG TAC AAC CCT CGG 48 Lys Phe Ala Gin Phe Asp Glu Tyr Gin Leu Ala Lys Tyr Asn Pro Arg  AAG TTC GCG CAG TTT GAC GAG TAC CAG CTG GCT AAG TAC AAC CCT CGG 48 Lys Phe Ala Gin Phe Asp Glu Tyr Gin Leu Ala Lys Tyr Asn Pro Arg
1 5 10 15  1 5 10 15
AAG CAC CGG GCC AAG AGA CAC CCC CGC CGG CCA CCC CGC TCT CCA GGG 96 Lys His Arg Ala Lys Arg His Pro Arg Arg Pro Pro Arg Ser Pro Gly  AAG CAC CGG GCC AAG AGA CAC CCC CGC CGG CCA CCC CGC TCT CCA GGG 96 Lys His Arg Ala Lys Arg His Pro Arg Arg Pro Pro Arg Ser Pro Gly
20 25 30 20 25 30
ATG GAG CCT CCA TTT TCT CAC AGA TGT TTT CCA AGG TAC ATA GGG TTT 144 Met Glu Pro Pro Phe Ser His Arg Cys Phe Pro Arg Tyr l ie Gly Phe ATG GAG CCT CCA TTT TCT CAC AGA TGT TTT CCA AGG TAC ATA GGG TTT 144 Met Glu Pro Pro Phe Ser His Arg Cys Phe Pro Arg Tyr lie Gly Phe
35 40 45  35 40 45
CTC AGA GAA GAG CAG AGA AAG TTT GAG AAG GCC GGT GAT ACA GTG TCA 192 Leu Arg Glu Glu Gin Arg Lys Phe Glu Lys Ala Gly Asp Thr Val Ser  CTC AGA GAA GAG CAG AGA AAG TTT GAG AAG GCC GGT GAT ACA GTG TCA 192 Leu Arg Glu Glu Gin Arg Lys Phe Glu Lys Ala Gly Asp Thr Val Ser
50 55 60  50 55 60
GAG AAA AAG AAT CCT CCA AGG TTC ACC CTG AAG AAG CTG GTT CAG CGA 240 Glu Lys Lys Asn Pro Pro Arg Phe Thr Leu Lys Lys Leu Val Gin Arg 65 70 75 80 CTG CAC ATC CAC AAG CCT GCC CAG CAC GTT CAA GCC CTG CTG GGT TAC 288 Leu His l ie His Lys Pro Ala Gin His Val Gin Ala Leu Leu Gly Tyr GAG AAA AAG AAT CCT CCA AGG TTC ACC CTG AAG AAG CTG GTT CAG CGA 240 Glu Lys Lys Asn Pro Pro Arg Phe Thr Leu Lys Lys Leu Val Gin Arg 65 70 75 80 CTG CAC ATC CAC AAG CCT GCC CAG CAC GTT CAA GCC CTG CTG GGT TAC 288 Leu His lie His Lys Pro Ala Gin His Val Gin Ala Leu Leu Gly Tyr
85 90 95  85 90 95
AGA TAC CCC TCC AAC CTA CAG CTC TTT TCT CGA AGT CGC CTT CCT GGG 336 Arg Tyr Pro Ser Asn Leu Gin Leu Phe Ser Arg Ser Arg Leu Pro Gly  AGA TAC CCC TCC AAC CTA CAG CTC TTT TCT CGA AGT CGC CTT CCT GGG 336 Arg Tyr Pro Ser Asn Leu Gin Leu Phe Ser Arg Ser Arg Leu Pro Gly
100 105 110  100 105 110
CCT TGG GAT TCT AGC AGA GCT GGG AAG AGG ATG AAG CTG TCT AGG CCA 384 Pro Trp Asp Ser Ser Arg Ala Gly Lys Arg Met Lys Leu Ser Arg Pro  CCT TGG GAT TCT AGC AGA GCT GGG AAG AGG ATG AAG CTG TCT AGG CCA 384 Pro Trp Asp Ser Ser Arg Ala Gly Lys Arg Met Lys Leu Ser Arg Pro
115 120 125  115 120 125
GAG ACC TGG GAG CGG GAG CTG AGC CTA CGG GGG AAC AAA GCG TCG GTC 432 Glu Thr Trp Glu Arg Glu Leu Ser Leu Arg Gly Asn Lys Ala Ser Val  GAG ACC TGG GAG CGG GAG CTG AGC CTA CGG GGG AAC AAA GCG TCG GTC 432 Glu Thr Trp Glu Arg Glu Leu Ser Leu Arg Gly Asn Lys Ala Ser Val
130 135 140  130 135 140
TGG GAG GAA CTC ATT GAA AAT GGG AAG CTT CCC TTC ATG GCC ATG CTC 480 Trp Glu Glu Leu l ie Glu Asn Gly Lys Leu Pro Phe Met Ala Met Leu 145 150 155 160 TGG GAG GAA CTC ATT GAA AAT GGG AAG CTT CCC TTC ATG GCC ATG CTC 480 Trp Glu Glu Leu lie Glu Asn Gly Lys Leu Pro Phe Met Ala Met Leu 145 150 155 160
AGC ATC T 487 Ser l ie AGC ATC T 487 Ser lie
162 配列番号： 3  162 SEQ ID NO: 3
配列の長さ ： 3 4 7 Array length: 3 4 7
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー ：直鎖状  Topology: linear
起源：生物名 ラッ ト Origin: organism name rat
配列 Array
CGGATGTAGA TGGTCAGGAG ATTAAGTGAT GCTGCCTGCA CCTTATCTTT GCAGGTATTG 60 GGTCATTCTC AGAAATTATT TTATCAGGCA GATTCAATAA GCAAGATAGT TTAGGGCGTG 120 AAGCTATGGG ATCAGTGACC AAGATGCCAA TCTCTTCTAC CCTCTCTCC TAG GCA TCT 178 CGGATGTAGA TGGTCAGGAG ATTAAGTGAT GCTGCCTGCA CCTTATCTTT GCAGGTATTG 60 GGTCATTCTC AGAAATTATT TTATCAGGCA GATTCAATAA GCAAGATAGT TTAGGGCGTG 120 AAGCTATGGG ATCAGTGACC AAGATGCCAA TCTCTTCTCTAC CCTCTCTCC TAG GCA TCT 178
Ala Ser  Ala Ser
1  1
TTG TAT GCT AGG CAG CAG CTT AAC CTC CGG GAC ATA GCC AAT ATA GTG 226 Leu Tyr Ala Arg Gin Gin Leu Asn Leu Arg Asp l ie Ala Asn l ie Val  TTG TAT GCT AGG CAG CAG CTT AAC CTC CGG GAC ATA GCC AAT ATA GTG 226 Leu Tyr Ala Arg Gin Gin Leu Asn Leu Arg Asp lie Ala Asn l ie Val
5 10 15  5 10 15
TTG GCC GTG GCT GCC CTC TTG CCA GCC TGC CGC CCC CAT GTA CGA CGG 274 Leu Ala Val Ala Ala Leu Leu Pro Ala Cys Arg Pro His Val Arg Arg  TTG GCC GTG GCT GCC CTC TTG CCA GCC TGC CGC CCC CAT GTA CGA CGG 274 Leu Ala Val Ala Ala Leu Leu Pro Ala Cys Arg Pro His Val Arg Arg
20 25 30  20 25 30
TAT TAC TCT GCC ATT GTT CAC CTG CCT TCA GAC TGG AAC CAG GTA GCC 322 Tyr Tyr Ser Ala l ie Val His Leu Pro Ser Asp Trp Asn Gin Val Ala  TAT TAC TCT GCC ATT GTT CAC CTG CCT TCA GAC TGG AAC CAG GTA GCC 322 Tyr Tyr Ser Ala lie Val His Leu Pro Ser Asp Trp Asn Gin Val Ala
35 40 45 50 35 40 45 50
GAG TTC TAC CAG GTA TGG TAC TTA G 347 Glu Phe Tyr Gin Val Trp Tyr Leu GAG TTC TAC CAG GTA TGG TAC TTA G 347 Glu Phe Tyr Gin Val Trp Tyr Leu
55 配列番号： 4  55 SEQ ID NO: 4
配列の長さ ： 4 0 8 Array length: 4 0 8
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー ：直鎖状  Topology: linear
起源：生物名 ラ ッ 卜 Origin: organism name rat
直接の起源： プラス ミ ド R a P C 5 3 Direct Origin: Plus Mid-RaPC 53
配列 Array
AGC TTG GGG GGA GAA GAA GAA GAA GTG GTG GGG GCA CCG GTC CTA AAA 48 Ser Leu Gly Gly Glu Glu Glu Glu VAl Val Gly Ala Pro Val Leu Lys  AGC TTG GGG GGA GAA GAA GAA GAA GTG GTG GGG GCA CCG GTC CTA AAA 48 Ser Leu Gly Gly Glu Glu Glu Glu VAl Val Gly Ala Pro Val Leu Lys
1 5 10 15  1 5 10 15
CTC ACA TCT GGA GAC TCT GAC TCT CAC CCT GAA ACC ACT GAC CAG ATC 96 Leu Thr Ser Gly Asp Ser Asp Ser His Pro Glu Thr Thr Asp Gin l ieCTC ACA TCT GGA GAC TCT GAC TCT CAC CCT GAA ACC ACT GAC CAG ATC 96 Leu Thr Ser Gly Asp Ser Asp Ser His Pro Glu Thr Thr Asp Gin lie
20 25 3020 25 30
CTG CAG GAG AAG AAG ATG GCT CTC TTG ACC TTG CTG TGC TCA GCT ATG 144 Leu Gin Glu Lys Lys Met Ala Leu Leu Thr Leu Leu Cys Ser Ala Met CTG CAG GAG AAG AAG ATG GCT CTC TTG ACC TTG CTG TGC TCA GCT ATG 144 Leu Gin Glu Lys Lys Met Ala Leu Leu Thr Leu Leu Cys Ser Ala Met
35 40 45  35 40 45
GCC TCA AGT GTG AAT GTG AAA GAT GCC TCC GAT CCT ACC CGG GCA TCT 192 Ala Ser Ser Val Asn Val l ie Tyr Ala Ser Asp Pro Thr Arg Ala Ser  GCC TCA AGT GTG AAT GTG AAA GAT GCC TCC GAT CCT ACC CGG GCA TCT 192 Ala Ser Ser Val Asn Val lie Tyr Ala Ser Asp Pro Thr Arg Ala Ser
50 55 60  50 55 60
ATC CAT GAA GTC TGC AGT GCG CTG GCC CCC TTG GAA CCT GAG TTC ATC 240 He His Glu Val Cys Ser Ala Leu Ala Pro Leu Glu Pro Glu Phe l ie 65 70 75 80 ATC CAT GAA GTC TGC AGT GCG CTG GCC CCC TTG GAA CCT GAG TTC ATC 240 He His Glu Val Cys Ser Ala Leu Ala Pro Leu Glu Pro Glu Phelie 65 70 75 80
CTT AAG GCA TCT TTG TAT GCT AGG CAG CAG CTT AAC CTC CGG GAC ATA 288 Leu Lys Ala Ser Leu Tyr Ala Arg Gin Gin Leu Asn Leu Arg Asp l ie CTT AAG GCA TCT TTG TAT GCT AGG CAG CAG CTT AAC CTC CGG GAC ATA 288 Leu Lys Ala Ser Leu Tyr Ala Arg Gin Gin Leu Asn Leu Arg Asp l ie
85 90 95  85 90 95
GCC AAT ATA GTG TTG GAA GTG GCT GCC CTC TTG CCA GCC TGC CGC CCC 336 Ala Asn l ie Val Lys Ala Val Ala Ala Leu Leu Pro Ala Cys Arg Pro  GCC AAT ATA GTG TTG GAA GTG GCT GCC CTC TTG CCA GCC TGC CGC CCC 336 Ala Asn lie Val Lys Ala Val Ala Ala Leu Leu Pro Ala Cys Arg Pro
100 105 110 100 105 110
CAT GTA CGA CGG TAT TAC TCT GCC ATT GTT CAC CTG CCT TCA GAC TGG 384 His Val Arg Arg Tyr Tyr Ser Ala l ie Val His Leu Pro Ser Asp Trp CAT GTA CGA CGG TAT TAC TCT GCC ATT GTT CAC CTG CCT TCA GAC TGG 384 His Val Arg Arg Tyr Tyr Ser Ala lie Val His Leu Pro Ser Asp Trp
115 120 125  115 120 125
ATC CAG GTA GCC GAG TTC TAC CAG 408 l ie Glu Val Ala Glu Phe Tyr Gin  ATC CAG GTA GCC GAG TTC TAC CAG 408 l ie Glu Val Ala Glu Phe Tyr Gin
130 135 配列番号： 5  130 135 SEQ ID NO: 5
配列の長さ ： 1 7 Array length: 1 7
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 トポロジー ：直鎖状 Number of chains: single strand Topology: linear
配列の種類：他の核酸 合成 D N A Sequence type: Other nucleic acids Synthetic DNA
他の情報： R は A または G、 Y はじ または T を示す。 Other information: R indicates A or G, Y or T.
配列 Array
CARTTYGAYG ARTAYCA 17 配列番号： 6  CARTTYGAYG ARTAYCA 17 SEQ ID NO: 6
配列の長さ ： 1 7 Array length: 1 7
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー ：直鎖状  Topology: linear
配列の種類：他の核酸 合成 D N A Sequence type: Other nucleic acids Synthetic DNA
他の情報： R は A または G、 N は A、 G、 C または T、 W は A または T を示す。 配列 Other information: R indicates A or G, N indicates A, G, C or T, W indicates A or T. Array
ARCATNGCCA TRffANGG 17 配列番号： 7  ARCATNGCCA TRffANGG 17 SEQ ID NO: 7
配列の長さ ： 2 3 Array length: 2 3
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー ：直鎖状  Topology: linear
配列の種類：他の核酸 合成 D N A Sequence type: Other nucleic acids Synthetic DNA
他の情報： R は S A または G、 Y はじ または T、 I は inosine を示す。 Other information: R for S A or G, Y for J or T, I for inosine.
配列 Array
AARTTYGCIC ARTTYGAYGA RTA 23 配列番号： 8  AARTTYGCIC ARTTYGAYGA RTA 23 SEQ ID NO: 8
配列の長さ ： 2 6 配列の型：核酸 Array length: 2 6 Sequence type: nucleic acid
鎖の数：一本鎖  Number of chains: single strand
トポロジー ：直鎖状  Topology: linear
配列の種類：他の核酸 合成 D N A Sequence type: Other nucleic acids Synthetic DNA
他の情報： R は A または G、 Y は または T、 I は inosine を示す。 Other information: R for A or G, Y for or T, I for inosine.
配列 Array
TTYGAYGART AYCARYTIGC 1AARTA 26 配列番号： 9  TTYGAYGART AYCARYTIGC 1AARTA 26 SEQ ID NO: 9
配列の長さ ： 2 6 Array length: 2 6
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー ：直鎖状  Topology: linear
配列の種類：他の核酸 合成 D N A Sequence type: Other nucleic acids Synthetic DNA
他の情報： R は A または G、 I は inosine、 K は G または T を示す。 Other information: R for A or G, I for inosine, K for G or T.
配列 Array
ARRTTICKIA RCATIGCCAT RAAIGG 26 配列番号： 1 0  ARRTTICKIA RCATIGCCAT RAAIGG 26 SEQ ID NO: 10
配列の長さ ： 2 6 Array length: 2 6
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー ：直鎖状  Topology: linear
配列の種類：他の核酸 合成 D N A Sequence type: Other nucleic acids Synthetic DNA
他の情報： R は A または G、 1 は inosine、 K は G または T を示す。 Other information: R indicates A or G, 1 indicates inosine, K indicates G or T.
配列 Array
TTRCAIARRT TICKIARCAT IGCCAT 26 配列番号： 1 1 TTRCAIARRT TICKIARCAT IGCCAT 26 SEQ ID NO: 1 1
配列の長さ ： 2 3 Array length: 2 3
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎮 Number of chains: single
トポロジー ：直鎖状  Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
CAGGGATGGA GCCTCCATTT TCT 23 配列番号： 1 2  CAGGGATGGA GCCTCCATTT TCT 23 SEQ ID NO: 1 2
配列の長さ ： 2 3 Array length: 2 3
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー ：直鎖状  Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
TCAATGAGTT CCTCCCAGAC CGA 23  TCAATGAGTT CCTCCCAGAC CGA 23
配列番号： 1 3 SEQ ID NO: 1 3
配列の長さ ：核酸 = 88 39、 アミノ酸 = 262 5 Sequence length: nucleic acid = 88 39, amino acid = 262 5
配列の型：核酸及びァミノ酸 Sequence type: nucleic acid and amino acid
トポロジー ：直鎖状二本鎖  Topology: linear double-stranded
配列の種類： c DN A Sequence type: c DN A
起源：生物名 ヒ ト Origin: organism name human
配列 Array
AGATCCGCAT CCGGCGCCTC CCCCGGCTGC CACCCTTCCC ACCGGCAGAA TCCAGAGCGA 60 AGTTTCTGCT TCCTGCTGCG GGAATCGGAC GCCCCAGGTC AGGCACCCAG GGTTTCCAGC 120 CCCAGTCTAA GGCATATACA AGCTGAGTTT CAGCC ATG GAA AAA CTC CAT 170 AGATCCGCAT CCGGCGCCTC CCCCGGCTGC CACCCTTCCC ACCGGCAGAA TCCAGAGCGA 60 AGTTTCTGCT TCCTGCTGCG GGAATCGGAC GCCCCAGGTC AGGCACCCAG GGTTTCCAGC 120 CCCAGTCTAA GGCATATACA AGCTGAGTTT CAGCC ATG GAA AAA CTC CAT 170
Met Glu Lys Leu His  Met Glu Lys Leu His
1 5  1 5
GGG CAT GTG TCT GCC CAT CCA GAC ATC CTC TCC TTG GAG AAC CGG TGC 218 Gly His Val Ser Ala His Pro Asp He Leu Ser Leu Glu Asn Arg Cys  GGG CAT GTG TCT GCC CAT CCA GAC ATC CTC TCC TTG GAG AAC CGG TGC 218 Gly His Val Ser Ala His Pro Asp He Leu Ser Leu Glu Asn Arg Cys
10 15 20  10 15 20
CTG GCT ATG CTC CCT GAC TTA CAG CCC TTG GAG AAA CTA CAT CAG CAT 266 Leu Ala Met Leu Pro Asp Leu Gin Pro Leu Glu Lys Leu His Gin His  CTG GCT ATG CTC CCT GAC TTA CAG CCC TTG GAG AAA CTA CAT CAG CAT 266 Leu Ala Met Leu Pro Asp Leu Gin Pro Leu Glu Lys Leu His Gin His
25 30 35 25 30 35
GTA TCT ACC CAC TCA GAT ATC CTC TCC TTG AAG AAC CAG TGC CTA GCC 314 Val Ser Thr His Ser Asp l ie Leu Ser Leu Lys Asn Gin Cys Leu Ala GTA TCT ACC CAC TCA GAT ATC CTC TCC TTG AAG AAC CAG TGC CTA GCC 314 Val Ser Thr His Ser Asp lie Leu Ser Leu Lys Asn Gin Cys Leu Ala
40 45 50  40 45 50
ACG CTT CCT GAC CTG AAG ACC ATG GAA AAA CCA CAT GGA TAT GTG TCT 362 Thr Leu Pro Asp Leu Lys Thr Met Glu Lys Pro His Gly Tyr Val Ser  ACG CTT CCT GAC CTG AAG ACC ATG GAA AAA CCA CAT GGA TAT GTG TCT 362 Thr Leu Pro Asp Leu Lys Thr Met Glu Lys Pro His Gly Tyr Val Ser
55 60 65  55 60 65
GCC CAC CCA GAC ATC CTC TCC TTG GAG AAC CAG TGC CTG GCC ACA CTT 410 Ala His Pro Asp l ie Leu Ser Leu Glu Asn Gin Cys Leu Ala Thr Leu  GCC CAC CCA GAC ATC CTC TCC TTG GAG AAC CAG TGC CTG GCC ACA CTT 410 Ala His Pro Asp lie Leu Ser Leu Glu Asn Gin Cys Leu Ala Thr Leu
70 75 80 85  70 75 80 85
TCT GAC CTG AAG ACC ATG GAG AAA CCA CAT GGA CAT GTT TCT GCC CAC 458 Ser Asp Leu Lys Thr Met Glu Lys Pro His Gly His Val Ser Ala His  TCT GAC CTG AAG ACC ATG GAG AAA CCA CAT GGA CAT GTT TCT GCC CAC 458 Ser Asp Leu Lys Thr Met Glu Lys Pro His Gly His Val Ser Ala His
90 95 100  90 95 100
CCA GAC ATC CTC TCC TTG GAG AAC CGA TGC CTG GCC ACC CTC TCT ACT 506 Pro Asp 】le Leu Ser Leu Glu Asn Arg Cys Leu Ala Thr Leu Ser Ser  CCA GAC ATC CTC TCC TTG GAG AAC CGA TGC CTG GCC ACC CTC TCT ACT 506 Pro Asp) le Leu Ser Leu Glu Asn Arg Cys Leu Ala Thr Leu Ser Ser
105 110 115  105 110 115
CTA AAG AGC ACT GTG TCT GCC AGC CCC TTG TTC CAG ACT CTA CAG ATA 554 Leu Lys Ser Thr Val Ser Ala Ser Pro Leu Phe Gin Ser Leu Gin He  CTA AAG AGC ACT GTG TCT GCC AGC CCC TTG TTC CAG ACT CTA CAG ATA 554 Leu Lys Ser Thr Val Ser Ala Ser Pro Leu Phe Gin Ser Leu Gin He
120 125 130  120 125 130
TCT CAC ATG ATG CAA GCT GAT TTG TAC CGT GTG AAC AAC AGC AAT TGC 602 9L TCT CAC ATG ATG CAA GCT GAT TTG TAC CGT GTG AAC AAC AGC AAT TGC 602 9L
J3S BIV sAq 3Md nio B"[V n3l "10 S-^V 隨 Oil 131 V30 OVV 3X3 31V III OVO XD3 3V0 DID 3X3 330 113 VVO 103 sAo 9Ϊ Ι "TO dsyュ 3Sュ q丄 usy usv ϋJ3S BIV sAq 3Md nio B "[V n3 l" 10 S- ^ V Exclusive Oil 131 V30 OVV 3X3 31V III OVO XD3 3V0 DID 3X3 330 113 VVO 103 sAo 9Ϊ Ι "TO dsy 3 S q 丄 usy usv ϋ
986 101 DIV VVO 丄丄丄 IIV D30 130 OXD DOV 333 OVO 131 VOV IVV OVV ΰΐν 986 101 DIV VVO 丄 丄 丄 IIV D30 130 OXD DOV 333 OVO 131 VOV IVV OVV ΰΐν
092  092
usy ΤΒΛ "TO s ュ s nai Π9ΐ nsq usy ΤΒΛ "TO s s nai Π9ΐ nsq
8C6 OVV VI3 VVO V3I 310 0X3 IOV X3I DOX 313 3X1 33V 3X3 VX3 133 9XV s ηχ uio S TH dsyュ oュ d STH "TO J9S8C6 OVV VI3 VVO V3I 310 0X3 IOV X3I DOX 313 3X1 33V 3X3 VX3 133 9XV s ηχ uio S TH dsy new d STH "TO J9 S
068 OVV ovv vvo m IV3 OVO IOV 133 9V0 V33 1V3 X3I VVO 丄)丄 dsy ュ as jqi naq dsy ΠΤ0 "TO ΠΤ0 "TO 8 OVO V90 X31 33V Old OVV OXO 300 OXD XVO OVO 910 OVO 0V9 068 OVV ovv vvo m IV3 OVO IOV 133 9V0 V33 1V3 X3I VVO 丄) 丄 dsy as as jqi naq dsy ΠΤ0 "TO ΠΤ0" TO 8 OVO V90 X31 33V Old OVV OXO 300 OXD XVO OVO 910 OVO 0V9
90Z 002 nsi ns "TO  90Z 002 nsi ns "TO
6 VOO Oli 30V 913 IOV IVI OIV VVO 33V OVO VOO 090 VVV 3VV  6 VOO Oli 30V 913 IOV IVI OIV VVO 33V OVO VOO 090 VVV 3VV
96Ϊ 061 S8i  96Ϊ 061 S8i
"TO ni3 -tas dsy 9Md d-τχ 3ュ V ^TO eiV "TO jqi "TO VV9 VOX 1V0 100 100 OIX X3V V3D VVO OVD 130 V3V OVO 081 5ZI Oil  "TO ni3 -tas dsy 9Md d-τχ 3 ュ V ^ TO eiV" TO jqi "TO VV9 VOX 1V0 100 100 OIX X3V V3D VVO OVD 130 V3V OVO 081 5ZI Oil
ュ¾ ^TV J9S 3Π J3S sA 3T I OJJ s Jqj, J3S nsq dsy na ¾ ^ TV J9S 3Π J3S sA 3T I OJJ s Jqj, J3S nsq dsy na
869 V3V 330 IDI OIV 331 VVV OXD 300 ViV 103 301 DOV VDI 113 OVO VXD S9I 091 05Ϊ869 V3V 330 IDI OIV 331 VVV OXD 300 ViV 103 301 DOV VDI 113 OVO VXD S9I 091 05Ϊ
^TD sA jas 9i S H "TO BTV 3JV dユエ J3S ojj ojd ri"[9ュ 9S naq naq^ TD sA jas 9i S H "TO BTV 3JV d Yue J3S ojj ojd ri" [9u 9S naq naq
059 V03 OVV 131 DII 1V3 OVO 130 00V 301 13V VDD 133 OVO 101 010 9XD ΐ 059 V03 OVV 131 DII 1V3 OVO 130 00V 301 13V VDD 133 OVO 101 010 9XD ΐ
s usy J3S usy usy ΪΒΛ 3·^ 丄 naq dsy Βχν ujj) 3J( 3)1( STH J9S s usy J3S usy usy ΪΒΛ 3 · ^ 丄 naq dsy Βχν ujj) 3J (3) 1 (STH J9S
P06Z0/L6ar/ 3d 8£8ム 0/86 OAV LL P06Z0 / L6ar / 3d 8 £ 8m 0/86 OAV LL
οε  οε
OJJ oュ d usy sAq •iqX dsy Ai9 ητο sqd s i OJJ o d usy sAqiqiq dsy Ai9 ητο sqd s i
99H V33 133 IVV OVV VVV OVO V3J, 010 VOV IVO 109 330 OVV 0V9 丄丄丄 OVV ς 0 99H V33 133 IVV OVV VVV OVO V3J, 010 VOV IVO 109 330 OVV 0V9 丄 丄 丄 OVV ς 0
V "TO ^IO ηχθ 9¾ί 9T I 丄 SJV SAQ Say S H V "TO ^ IO ηχθ 9¾ί 9T I 丄 SJV SAQ Say S H
VOV OVO OVO WO VOV 313 VIV 3VX 90V V33 III XOI VOV OVO VOV OVO OVO WO VOV 313 VIV 3VX 90V V33 III XOI VOV OVO
00， 56ε 068 00, 56ε 068
J3S ^Md OJd OJJ ^I OJd J9S 3jy 0Jd gjy OJJ S TH i3i 丄丄丄 νοα ιοο 9V9 OIV 903 vaa LOl D93 ODD V33 003 303 ODD 3V3 J3S ^ Md OJd OJJ ^ I OJd J9S 3jy 0J d gjy OJJ S TH i3i 丄 丄 丄 νοα ιοο 9V9 OIV 903 vaa LOl D93 ODD V33 003 303 ODD 3V3
988 088  988 088
3jy s S T H sAq gjy oュ d usy ュ 丄 sAq UT3 丄 "ID VOV OVV 330 000 OVD OVV 903 133 VV V丄 OVV 130 0X3 0V3 3VX 0V9
Figure imgf000079_0001
3jy s STH sAq gjy o d usy u sAq UT3 丄 "ID VOV OVV 330 000 OVD OVV 903 133 VV V 丄 OVV 130 0X3 0V3 3VX 0V9
Figure imgf000079_0001
dsy siid "TO BTV BTV Jqi sAo BTV OJd OVO 丄丄丄 OVD 330 ILL VVV OVO ODV 3IV 300 I3V 100 013 IDI 330 333  dsy siid "TO BTV BTV Jqi sAo BTV OJd OVO 丄 丄 丄 OVD 330 ILL VVV OVO ODV 3IV 300 I3V 100 013 IDI 330 333
058  058
Π97 OJJ Λ nsq sAq usy sAq dsy ^TO ηΐ3 BTV "T9 丄 Π97 OJJ Λ nsq sAq usy sAq dsy ^ TO ηΐ3 BTV "T9 丄
01D 303 0X0 0X3 9VV XVV OVV IVO V39 OVO 133 3X0 33V OVD OV丄 1X3 01D 303 0X0 0X3 9VV XVV OVV IVO V39 OVO 133 3X0 33V OVD OV 丄 1X3
οεε  οεε
"TO BTV ΤΒΛ "TO 9T I 丄 dsy J3S OJd ngq UTO BTV sAo aqj 3V0 130 019 9V3 31V 001 3V3 丄 OL 133 010 3V3 310 IIV 330 101 311  "TO BTV ΤΒΛ" TO 9T I 丄 dsy J3S OJd ngq UTO BTV sAo aqj 3V0 130 019 9V3 31V 001 3V3 丄 OL 133 010 3V3 310 IIV 330 101 311
02S οιε 02S οιε
•ΐΛ丄 S:iv S H OJd 3ay s B"[V oュ d naq BTV ail BTV οεπ ivi νοα V30 OXD 3V3 ODD 303 X3I 039 933 311 IDO 11V 330 • ΐΛ 丄 S: iv S H OJd 3ays B "[V o d naq BTV ail BTV οεπ ivi νοα V30 OXD 3V3 ODD 303 X3I 039 933 311 IDO 11V 330
508 οοε  508 οοε
nsq 9f j sAq usy usy Sjy χΒΛ usy na 3^V ^TV J nsq 9f j sAq usy usy Sjy χΒΛ usy na 3 ^ V ^ TV J
2801 Oil 31V VVV IVV 339 010 XVV 003 310 OVV 013 ova OVO 90V 330 IVl 2801 Oil 31V VVV IVV 339 010 XVV 003 310 OVV 013 ova OVO 90V 330 IVl
062 982 082  062 982 082
P06ZWL6dr/lDd 8£8 .0/86 OM. SIP06ZWL6dr / lDd 8 £ 8.0 / 86 OM. SI
\ VIV 003 00V OIV OIO VOV VIV IVV 331 103 III D33 Oil V30 VV3 IVV  \ VIV 003 00V OIV OIO VOV VIV IVV 331 103 III D33 Oil V30 VV3 IVV
3JV naq UT BTV B"[v dsy STH ^ V usv n9i aqd3JV naq UT BTV B "[v dsy STH ^ V usv n9i aqd
8681 VOV DID WD 130 OVO 313 339 丄 V3 丄丄 V 339 XVO IVO 330 3VV 8681 VOV DID WD 130 OVO 313 339 丄 V3 丄 丄 V 339 XVO IVO 330 3VV
59¾ 095 99S  59¾ 095 99S
3jy 3Md OJd sqj s SIH 9ΐ Ι Ι^Λ S s 3jy 3Md OJd sqj s SIH 9 ΐ Ι Ι ^ Λ S s
0Q8T VOV Oil V33 丄丄丄 OVO 000 13V OVO 3IV 010 031 OVV 009 1V3 3VD 313 gjy "TO η3Ί 9Π STH STH 3-iV ュ as •I9S 311 0Q8T VOV Oil V33 丄 丄 丄 OVO 000 13V OVO 3IV 010 031 OVV 009 1V3 3VD 313 gjy "TO η3 Ί 9 Π STH STH 3-iV as
Ζ08Ϊ VOV 9V3 313 丄丄 V 3X3 9V0 iv3 I0V 3IV VOO 110 903 013  Ζ08Ϊ VOV 9V3 313 丄 丄 V 3X3 9V0 iv3 I0V 3IV VOO 110 903 013
0B9 529 ozs usy s naq usy SJV ΑΐΟ usv m\ 0X3 OVV 301 313 OVV 000 113 01V 330 01V Oil DDD 113 OVV 000 IVV 0B9 529 ozs usy s naq usy S JV ΑΐΟ usv m \ 0X3 OVV 301 313 OVV 000 113 01V 330 01V Oil DDD 113 OVV 000 IVV
0Ϊ5  0Ϊ5
ai l "91 ηχ9 "TO dJi usv H jag naq  ai l "91 ηχ9" TO dJi usv H jag naq
90 m XiV DID WO OVO 001 310 031 930 VVV OVV 000 093 VXD 30V 013 90 m XiV DID WO OVO 001 310 031 930 VVV OVV 000 093 VXD 30V 013
009 06  009 06
"TO V "TO 丄 人 If) BTV "TO V" TO 丄 人 If) BTV
8591 OVO 000 3V3 301 33V 3V0 VDO OOV OVV 3IV 90V OVV 000 X30 8591 OVO 000 3V3 301 33V 3V0 VDO OOV OVV 3IV 90V OVV 000 X30
08^ ς 0 jaS jas dsy 丄 OJd ATO old ュ as aqj  08 ^ ς 0 jaS jas dsy 丄 OJd ATO old as as aqj
0Ϊ9Ϊ VOV 33V 131 XVO 031 133 003 133 110 333 IOV V03 丄 丄丄丄 0X3 9V3  0Ϊ9Ϊ VOV 33V 131 XVO 031 133 003 133 110 333 IOV V03 丄 丄 丄 丄 0X3 9V3
09  09
usy J9S OJJ j gjy 丄 ^ΐθ BTV "TO  usy J9S OJJ j gjy 丄 ^ ΐθ BTV "TO
2991 VXD OVV 331 333 3VI VOV OVX XOO 313 913 330 WD 113 3V3 0V3 330 s q STH an 3¾ v n^l 130 OVV 3V3 OIV 3V3 013 V03 9V3 9VV OVV 0X3 3DV 3IX OOV P06Z0/L6d£llDd 61 2991 VXD OVV 331 333 3VI VOV OVX XOO 313 913 330 WD 113 3V3 0V3 330 sq STH an 3¾ vn ^ l 130 OVV 3V3 OIV 3V3 013 V03 9V3 9VV OVV 0X3 3DV 3IX OOV P06Z0 / L6d £ lld 61
^TO "TO "TO BIV sAq nsi ΙΒΛ BTV dsy TO ^ TO "TO" TO BIV sAq nsi ΙΒΛ BTV dsy TO
013 3IV 399 WO WO VDO 3VV 113 010 VDO I3V OVV OID I3V 3V0 193 013 3IV 399 WO WO VDO 3VV 113 010 VDO I3V OVV OID I3V 3V0 193
O U  O U
Τ^Λ dsy ΙΒΛ UTO "TO ^TV 3JV jq丄 an 19N 18W VOO 101 910 313 310 OVO 019 OVO OVO 300 99V OOV OIV 3IV 9IV 990  Τ ^ Λ dsy ΙΒΛ UTO "TO ^ TV 3JV jq 丄 an 19N 18W VOO 101 910 313 310 OVO 019 OVO OVO 300 99V OOV OIV 3IV 9IV 990
50A 002, 569 50A 002, 569
3J I nsq ¾TV 丄 usy naq OJJ OJJ ^TO "TO OJJ usy J3S sAq IIV Oil DI3 013 VDO IVl OVV 0X3 ODD D30 030 VV3 VDO 3VV 30V 9VV 3J Insq ¾TV 丄 usy naq OJJ OJJ ^ TO "TO OJJ usy J3S sAq IIV Oil DI3 013 VDO IVl OVV 0X3 ODD D30 030 VV3 VDO 3VV 30V 9VV
069 589 089  069 589 089
V33 101 3X3 OOV 3
Figure imgf000081_0001
V3 VOO IVV 130 IVO VOV OID IVl 310 I3V V33 101 3X3 OOV 3
Figure imgf000081_0001
V3 VOO IVV 130 IVO VOV OID IVl 310 I3V
519 0A9 S99  519 0A9 S99
3JV la nsq ojj naq jag sfH sXq Λ JSS nsq usy 3JV la nsq ojj naq jag sfH sXq Λ JSS nsq usy
9812 303 309 VOO 313 0X3 333 OID OOV DV3 OVV JL 丄J L did OVV 010 139 9812 303 309 VOO 313 0X3 333 OID OOV DV3 OVV JL 丄 J L did OVV 010 139
099 GS9 059  099 GS9 059
"TO v -ΐ χ 3jy usy nsq JAJL sAq "TO v -ΐ χ 3jy usy nsq JAJL sAq
8812 VOV ovo VID 3D0 0V3 V90 3VI OOV OVV 013 3IV OVO 100 IVO IVX VVV 8812 VOV ovo VID 3D0 0V3 V90 3VI OOV OVV 013 3IV OVO 100 IVO IVX VVV
0 9 989 089 丄 UTO sAq STH τ^Λ gjy naq sAq "TO n^o0 9 989 089 丄 UTO sAq ST H τ ^ Λ gjy naq sAq "TO n ^ o
0602 301 OVO VOV 030 OVV 0V3 VIO V3V 013 9VV OVO 30V OVV 313 9V3 OVO 0602 301 OVO VOV 030 OVV 0V3 VIO V3V 013 9VV OVO 30V OVV 313 9V3 OVO
929 029 919 m 3JV SW BTV 9W Say Π91 uio uio Sjy J9S Π91 z ivx oix 010 133 VIV OOV 31V V30 3IV 903 1X3 OVO OVO 103 D3V via 019 509 009 929 029 919 m 3JV SW BTV 9 W Say Π91 uio uio Sjy J9S Π91 z ivx oix 010 133 VIV OOV 31V V30 3IV 903 1X3 OVO OVO 103 D3V via 019 509 009
3ュ 3jy 3jy OJJ 3jy usy sA-i org usy 3JV ュ4丄 η3Ί 丄 JLO OOV 303 90V 303 130 DVV OVV WO IVV VOV I3V VIO 565 069 G85 3 u 3jy 3jy OJJ 3jy usy sA-i org usy 3JV u4 丄 η3 Ί 丄 JLO OOV 303 90V 303 130 DVV OVV WO IVV VOV I3V VIO 565 069 G85
9T I S-iV i9K nsi jqi θΐ ΐ usy J3S oュ d 3Md 0Jd nsq Βχγ uio USV *O6ZO/I6df/I3d P 9T I S-iV i9K nsi jqi θΐ ΐ usy J3S o d 3Md 0Jd nsq Βχγ uio US V * O6ZO / I6df / I3d P
730 735 740  730 735 740
AAG ACT GCC ATC AAG CTC CAG GCT CAA GTC CAG GAG TTT GAT GAA AAT 2426 Lys Thr Ala He Lys Leu Gin Ala Gin Val Gin Glu Phe Asp Glu Asn  AAG ACT GCC ATC AAG CTC CAG GCT CAA GTC CAG GAG TTT GAT GAA AAT 2426 Lys Thr Ala He Lys Leu Gin Ala Gin Val Gin Glu Phe Asp Glu Asn
745 750 755  745 750 755
GAT GGA TGG TCC CTG AAT ACT TTT GGG AAA TAC CTG CTG TCT CTG GCT 2474 Asp Gly Trp Ser Leu Asn Thr Phe Gly Lys Tyr Leu Leu Ser Leu Ala  GAT GGA TGG TCC CTG AAT ACT TTT GGG AAA TAC CTG CTG TCT CTG GCT 2474 Asp Gly Trp Ser Leu Asn Thr Phe Gly Lys Tyr Leu Leu Ser Leu Ala
760 765 770  760 765 770
GGC CAA AGG GTT CCT GTG GAC AGG GTC ATC CTC CTT GGC CAA AGC ATG 2522 Gly Gin Arg Val Pro Val Asp Arg Val He Leu Leu Gly Gin Ser Met  GGC CAA AGG GTT CCT GTG GAC AGG GTC ATC CTC CTT GGC CAA AGC ATG 2522 Gly Gin Arg Val Pro Val Asp Arg Val He Leu Leu Gly Gin Ser Met
775 780 785  775 780 785
GAT GAT GGA ATG ATA AAT GTG GCC AAA CAG CTT TAC TGG CAG CGT GTG 2570 Asp Asp Gly Met l ie Asn Val Ala Lys Gin Leu Tyr Trp Gin Arg Val 790 795 800 805 GAT GAT GGA ATG ATA AAT GTG GCC AAA CAG CTT TAC TGG CAG CGT GTG 2570 Asp Asp Gly Met lie Asn Val Ala Lys Gin Leu Tyr Trp Gin Arg Val 790 795 800 805
AAT TCC AAG TGC CTC TTT GTT GGT ATC CTC CTA AGA AGG GTA CAA TAC 2618 Asn Ser Lys Cys Leu Phe Val Gly l ie Leu Leu Arg Arg Val Gin Tyr  AAT TCC AAG TGC CTC TTT GTT GGT ATC CTC CTA AGA AGG GTA CAA TAC 2618 Asn Ser Lys Cys Leu Phe Val Gly lye Leu Leu Arg Arg Val Gin Tyr
810 815 820  810 815 820
CTG TCA ACA GAT TTG AAT CCC AAT GAT GTG ACA CTC TCA GGC TGT ACT 2666 Leu Ser Thr Asp Leu Asn Pro Asn Asp Val Thr Leu Ser Gly Cys Thr  CTG TCA ACA GAT TTG AAT CCC AAT GAT GTG ACA CTC TCA GGC TGT ACT 2666 Leu Ser Thr Asp Leu Asn Pro Asn Asp Val Thr Leu Ser Gly Cys Thr
825 830 835  825 830 835
GAT GCG ATA CTG AAG TTC ATT GCA GAG CAT GGG GCC TCC CAT CTT CTG 2714 Asp Ala l ie Leu Lys Phe l ie Ala Glu His Gly Ala Ser His Leu Leu  GAT GCG ATA CTG AAG TTC ATT GCA GAG CAT GGG GCC TCC CAT CTT CTG 2714 Asp Ala lie Leu Lys Phe lie Ala Glu His Gly Ala Ser His Leu Leu
840 845 850  840 845 850
GAA CAT GTG GGC CAA ATG GAC AAA ATA TTC AAG ATT CCA CCA CCC CCA 2762 Glu His Val Gly Gin Met Asp Lys l ie Phe Lys He Pro Pro Pro Pro  GAA CAT GTG GGC CAA ATG GAC AAA ATA TTC AAG ATT CCA CCA CCC CCA 2762 Glu His Val Gly Gin Met Asp Lys lie Phe Lys He Pro Pro Pro Pro
855 860 865  855 860 865
GGA AAG ACA CGG GTC CAG TCT CTC CGG CCA CTG GAA GAG GAC ACT CCA 2810 Gly Lys Thr Gly Val Gin Ser Leu Arg Pro Leu Glu Glu Asp Thr Pro 870 875 880 885 AGC CCC TTG GCT CCT GTT TCC CAG CAA GGA TGG GGC AGC ATC CGG CTT 2858 Ser Pro leu Ala Pro Val Ser Gin Gin Gly Trp Gly Ser l ie Arg Leu GGA AAG ACA CGG GTC CAG TCT CTC CGG CCA CTG GAA GAG GAC ACT CCA 2810 Gly Lys Thr Gly Val Gin Ser Leu Arg Pro Leu Glu Glu Asp Thr Pro 870 875 880 885 885 AGC CCC TTG GCT CCT GTT TCC CAG CAA GGA TGG GGC AGC ATC CGG CTT 2858 Ser Pro leu Ala Pro Val Ser Gin Gin Gly Trp Gly Ser lie Arg Leu
890 895 900  890 895 900
TTC ATT TCA TCC ACT TTC CGA GAC ATG CAC CGG GGA GCG GAC CTG CTG 2906 Phe He Ser Ser Thr Phe Arg Asp Met His Arg Gly Ala Asp Leu Leu  TTC ATT TCA TCC ACT TTC CGA GAC ATG CAC CGG GGA GCG GAC CTG CTG 2906 Phe He Ser Ser Thr Phe Arg Asp Met His Arg Gly Ala Asp Leu Leu
905 910 915  905 910 915
CTG AGG TCT GTG CTG CCA GCA CTG CAG GCC CGA GCG GCC CCT CAC CGT 2954 Leu Arg Ser Val Leu Pro Ala Leu Gin Ala Arg Ala Ala Pro His Arg  CTG AGG TCT GTG CTG CCA GCA CTG CAG GCC CGA GCG GCC CCT CAC CGT 2954 Leu Arg Ser Val Leu Pro Ala Leu Gin Ala Arg Ala Ala Pro His Arg
920 925 930  920 925 930
ATC AGC CTT CAC CGA ATC GAC CTC CGC TGG GGC GTC ACT GAG GAG GAG 3002 l ie Ser Leu His Arg l ie Asp Leu Arg Trp Gly Val Thr Glu Glu Glu  ATC AGC CTT CAC CGA ATC GAC CTC CGC TGG GGC GTC ACT GAG GAG GAG 3002 l ie Ser Leu His Arg lie Asp Leu Arg Trp Gly Val Thr Glu Glu Glu
935 940 945  935 940 945
ACC CGT AGG AAC AGA CAA CTG GAA GTG TGC CTT GGG GAG GTG GAG AAC 3050 Thr Arg Arg Asn Arg Gin Leu Glu Val Cys Leu Gly Glu Val Glu Asn ACC CGT AGG AAC AGA CAA CTG GAA GTG TGC CTT GGG GAG GTG GAG AAC 3050 Thr Arg Arg Asn Arg Gin Leu Glu Val Cys Leu Gly Glu Val Glu Asn
950 955 960 965 950 955 960 965
GCA CAG CTG TTT GTG GGG ATT CTG GGC TCC CGT TAT GGA AAC ATT CCC 3098 Ala Gin Leu Phe Val Gly l ie Leu Gly Ser Arg Tyr Gly Asn l ie Pro  GCA CAG CTG TTT GTG GGG ATT CTG GGC TCC CGT TAT GGA AAC ATT CCC 3098 Ala Gin Leu Phe Val Gly lie Leu Gly Ser Arg Tyr Gly Asn lie Pro
970 975 980  970 975 980
CCC AGC TAC AAC CTT CCT GAC CAT CCA CAC TTC CAC TGG GCC CAG CAG 3146 Pro Ser Tyr Asn Leu Pro Asp His Pro His Phe His Trp Ala Gin Gin  CCC AGC TAC AAC CTT CCT GAC CAT CCA CAC TTC CAC TGG GCC CAG CAG 3146 Pro Ser Tyr Asn Leu Pro Asp His Pro His Phe His Trp Ala Gin Gin
985 990 995  985 990 995
TAC CCT TCA GGG CGC TCT GTG ACA GAG ATG GAG GTG ATG CAG TTC CTG 3194 Tyr Pro Ser Gly Arg Ser Val Thr Glu Met Glu Val Met Gin Phe Leu  TAC CCT TCA GGG CGC TCT GTG ACA GAG ATG GAG GTG ATG CAG TTC CTG 3194 Tyr Pro Ser Gly Arg Ser Val Thr Glu Met Glu Val Met Gin Phe Leu
1000 1005 1010  1000 1005 1010
AAC CGG AAC CAA CGT CTG CAG CCC TCT GCC CAA GCT CTC ATC TAC TTC 3242 Asn Arg Asn Gin Arg Leu Gin Pro Ser Ala Gin Ala Leu l ie Tyr Phe  AAC CGG AAC CAA CGT CTG CAG CCC TCT GCC CAA GCT CTC ATC TAC TTC 3242 Asn Arg Asn Gin Arg Leu Gin Pro Ser Ala Gin Ala Leu lie Tyr Phe
1015 1020 1025  1015 1020 1025
CGG GAT TCC AGC TTC CTC AGC TCT GTG CCA GAT GCC TGG AAA TCT GAC 3290 dsy ojj BIV UT3 "31 ¾TV Λ "91 BTV n3l 9Md ^TV ュ1 U sACGG GAT TCC AGC TTC CTC AGC TCT GTG CCA GAT GCC TGG AAA TCT GAC 3290 dsy ojj BIV UT3 "31 ¾TV Λ" 91 BTV n3 l 9 Md ^ TV 11 U sA
IVO 133 130 OVO 013 330 V31 310 IID 131 VOO 013 Oil 330 VDV OVV IVO 133 130 OVO 013 330 V31 310 IID 131 VOO 013 Oil 330 VDV OVV
QLU 99Π 09 Π  QLU 99Π 09 Π
^TO uio λιο J9S "TO J¾ Τ^Λ η9Ί 3jV H sin ojj^ TO uio λιο J9S "TO J¾ Τ ^ Λ η9 Ί 3j VH sin ojj
300 OVD VOO VOI OVD OOO ODV 010 Old 30V 013 09V VOO DVO 333 313 300 OVD VOO VOI OVD OOO ODV 010 Old 30V 013 09V VOO DVO 333 313
59Π OQU 5^ΐ ΐ  59Π OQU 5 ^ ΐ ΐ
naq gjy iqj) ュ1 U dsy ui;) na ngq Sjy c d 3jV ^TV o^d J3Snaq gjy iqj) Interview 1 U dsy ui;) na ngq Sjy cd 3j V ^ TV o ^ d J3S
OIV OID 003 WD 010 V3V 3V0 OVO UD 113 303 VD3 903 300 IDD 10VOIV OID 003 WD 010 V3V 3V0 OVO UD 113 303 VD3 903 300 IDD 10V
ΟΗ Ϊ m i οεπ ΟΗ Ϊ m i οεπ
OJd OJd sA uio n3i ϋχο UTO 9Md jqi "TO Τ^Λ dsy dsy dsy OJd OJd sA uio n3i ϋχο UTO 9Md jqi "TO Τ ^ Λ dsy dsy dsy
8i58 033 V33 OVV OVD 013 OVO 0V3 3X1 33V 330 9VD 310 Oil 3V0 IVO OVO 8i58 033 V33 OVV OVD 013 OVO 0V3 3X1 33V 330 9VD 310 Oil 3V0 IVO OVO
0211 9Π ΐ ΟΤ Π ojd an J as Ι^Λ OJ^ ui n^l na \ λιο OJJ uia nsq Αχ naq 0211 9Π ΟΤ ΟΤ Π ojd an J as Ι ^ Λ OJ ^ ui n ^ l na \ λιο OJJ uia nsq Αχ naq
0δ9ε VOO 31V 3DI 010 VOO OVO OVO OID 013 339 000 133 OVO 013 OVI DID 0δ9ε VOO 31V 3DI 010 VOO OVO OVO OID 013 339 000 133 OVO 013 OVI DID
9011 00Π 5601 sAi uxo an ism usv d-ri T^A dsV uio NA1 T^A η9Ί "TO ^TO sqd ητο 28^9 OVV 0V3 31V 3IV IVV 931 VIO IVO OVO 013 110 Oil OVD 303 III 9V0 9011 00Π 5601 sAi uxo an ism usv d-ri T ^ A ds V uio NA 1 T ^ A η9 Ί `` TO ^ TO sqd ητο 28 ^ 9 OVV 0V3 31V 3IV IVV 931 VIO IVO OVO 013 110 Oil OVD 303 III 9V0
0601 080Ϊ nT0 "91 ^ΐθ ^ΐθ ΐ^Λ J人丄 ojj Hay ΐί) BIV ^TV ΐ^Λ ^TO ^το cLi丄 ε½ OVO 0X3 000 300 110 IVl 333 003 309 130 V30 010 100 000 001 9V0  0601 080Ϊ nT0 "91 ^ ΐθ ^ ΐθ ΐ ^ Λ J people 丄 ojj Hay ΐί) BIV ^ TV ΐ ^ Λ ^ TO ^ το cLi 丄 ε½ OVO 0X3 000 300 110 IVl 333 003 309 130 V30 010 100 000 001 9V0
5A0I ΟΖΟΐ 續  5A0I 續 Continue
sA3 ojj JAX gjy Sjy SAQ jqi 9ΐ ΐ Λτο sA iq;) Sjy S nsq αλχ J3S sA3 ojj JAX gjy Sjy SAQ jqi 9ΐ ΐ Λτο sA iq;) Sjy S nsq αλχ J3S
988£ XOI 333 3V1 VOV 303 30X 33V VXV 000 VVV OVD V3V 39V VI3 3VX 30V 988 £ XOI 333 3V1 VOV 303 30X 33V VXV 000 VVV OVD V3V 39V VI3 3VX 30V
090ΐ 990ΐ 090Ϊ  090ΐ 990ΐ 090Ϊ
sAq naq nio jas 9χι Sjy s Βχν BTV nj9 ηχο s ηχο jss ΙΒΛ sAq naq nio jas 9χι Sjy s Βχν BTV nj9 ηχο s ηχο jss ΙΒΛ
8998 013 VV3 VOL DXV 003 131 VOO 333 OVO WO 131 0V3 131 110 1118998 013 VV3 VOL DXV 003 131 VOO 333 OVO WO 131 0V3 131 110 111
^01 o oi 9εοτ οεοι dsy J9S sAq 丄 dsy OJJ IBA jas ^as and J3S dsv M)6rO/ .6df lDd 8£8ム 0/86 OAV S8
Figure imgf000085_0001
^ 01 o oi 9εοτ οεοι dsy J9S sAq 丄 dsy OJJ IBA jas ^ as and J3S dsv M) 6rO / .6df lDd 8 £ 8m 0/86 OAV S8
Figure imgf000085_0001
"TO ηχο 3jy
Figure imgf000085_0002
S na OJJ Αχο nsq"TO ηχο 3jy
Figure imgf000085_0002
S na OJJ Αχο nsq
9V3 OVO V3V 010 013 900 039 930 XOO IDX 33D OVO DID 133 000 Oil 9V3 OVO V3V 010 013 900 039 930 XOO IDX 33D OVO DID 133 000 Oil
9 ΐ6ΐ 0Ι8ΐ 508Ϊ  9 ΐ6ΐ 0Ι8ΐ 508Ϊ
BTV ns ΤΒΛ STH BTV ^1 "TO s uio naq jqi ηχο Αχο naq χ^ B TV ns ΤΒΛ STH BTV ^ 1 "TO s uio naq jqi ηχο Αχο naq χ ^
90 330 013 DIO OVO 303 XOO OVO OOV 3VD OVO Π3 D3V OVO 030 VX3 330 90 330 013 DIO OVO 303 XOO OVO OOV 3VD OVO Π3 D3V OVO 030 VX3 330
0081 G62i 0621  0081 G62i 0621
Ηχν dsy J9S J9S Ϊ ΒΛ 3S nsl ΐΒΛ naq
Figure imgf000085_0003
s Say OJJ ns sAqΗχν dsy J9S J9S Ϊ ΒΛ 3 S ns l ΐ Β Λ naq
Figure imgf000085_0003
s Say OJJ ns sAq
850^ VaO IVO X3V X3I 010 XOV OID 3X3 013 3V3 VIO 101 003 333 11D OVV850 ^ VaO IVO X3V X3I 010 XOV OID 3X3 013 3V3 VIO 101 003 333 11D OVV
982Ϊ 0821 UZ\ OLZl sA oュ d d i 丄 dsy JSS sn naq u g Αχο usy u^g dsy ΙΒΛ ns §jy 982Ϊ 0821 UZ \ OLZl sA o d d i 丄 dsy JSS sn naq u g Αχο usy u ^ g dsy ΙΒΛ ns §jy
Ο ΐ OVV V33 3XV 091 OVO VDX IIV 013 9V3 039 IVV 0V3 OVO 919 VII 03V Ο ΐ OVV V33 3XV 091 OVO VDX IIV 013 9V3 039 IVV 0V3 OVO 919 VII 03V
5921 0921  5921 0921
dsy BTV ^10 dsv 3T I 3T I ns ΙΒΛ UTO -ΐ¾ "TO ^TO oュ d STH na J3S 296S IVO 130 300 IVO 3IV OIV 9X3 313 OVO 33V OVO 300 IOD IVO 913 DDI dsy BTV ^ 10 dsv 3T I 3T Ins ΙΒ Λ UTO -ΐ¾ "TO ^ TO o d STH na J3S 296S IVO 130 300 IVO 3IV OIV 9X3 313 OVO 33V OVO 300 IOD IVO 913 DDI
052Ϊ 9^1 0^1 niO en ュ 9S sAq OJJ naq naq Sjy uio uig na ηχο ά χ Λ ng J9S H 6S OVO 130 131 OVV 33D 0X3 313 OOV 0V3 OVO 013 OVO 001 010 013 30V 052Ϊ 9 ^ 1 0 ^ 1 niO en u 9 S sAq OJJ naq naq Sjy uio uig na ηχο ά Λ ng ng J9S H 6S OVO 130 131 OVV 33D 0X3 313 OOV 0V3 OVO 013 OVO 001 010 013 30V
S ΐ i 5221  S ΐ i 5221
SJV J^L JMI -I3S OJJ nsi BTV ^TO J^S sAq naq uio 3ay n 9988 VOD 3VI 33V OOV 333 313 339 100 VOL OVO VVV V13 VV3 303 133 OID SJV J ^ L JMI -I3S OJJ nsi BTV ^ TO J ^ S sAq naq uio 3ay n 9988 VOD 3VI 33V OOV 333 313 339 339 100 VOL OVO VVV V13 VV3 303 133 OID
OZZl 512Ϊ 0\Z\  OZZl 512Ϊ 0 \ Z \
ュ A丄 Ji|丄 SAQ nsq Sjy Say naq naq jqx na Βχν naq Αχο U\Q dsy OJJ A 丄 Ji | i SAQ nsq Sjy Say naq naq jqx na Βχν naq Αχο U \ Q dsy OJJ
8188 IVl OOV I3X 3X3 333 VOV 3X3 313 X3V 313 339 113 100 OVO 3V9丄 ) 8188 IVl OOV I3X 3X3 333 VOV 3X3 313 X3V 313 339 113 100 OVO 3V9 丄
902ΐ 002ΐ 96Ι ΐ 06Π V ^TV χ J3S 9¾ι STH 3¾d 3Md ΐ ns^ OJJ BTV ΐ^Λ sAq εχν χο 0LL2, IOD XOO 303 131 III 3V3 311 3X1 DI3 VXl V33 V33 0X0 OVV 333 300  902ΐ 002ΐ 96Ι ΐ 06Π V ^ TV χ J3S 9¾ι STH 3¾d 3Md ΐ ns ^ OJJ BTV ΐ ^ Λ sAq εχν χο 0LL2, IOD XOO 303 131 III 3V3 311 3X1 DI3 VXl V33 V33 0X0 OVV 333 300
98Π 08Π SAU06ZOIL6d£llDd 8ε 0/86 OAV 8 98Π 08Π SAU06ZOIL6d £ llDd 8ε 0/86 OAV 8
^9^ 000 IVO 133 313 301 010 003 300 100 130 ODD OVO 313 133 309 DVO ^ 9 ^ 000 IVO 133 313 301 010 003 300 100 130 ODD OVO 313 133 309 DVO
( H ¾9^ΐ  (H ¾9 ^ ΐ
^TO nsi nsi ュ Sjy naq s naq JAJ, BIV 3iy OJJ Aio 9W ^ TO nsi nsi Sjy naq s naq JAJ, BIV 3iy OJJ Aio 9W
985^ 300 VXD Oil 10V 303 313 I0V OVO 310 013 3VX 330 III 333 300 OIV 985 ^ 300 VXD Oil 10V 303 313 I0V OVO 310 013 3VX 330 III 333 300 OIV
09^1 99^1 OS  09 ^ 1 99 ^ 1 OS
OJd J人丄 ojj dsy ^TO J9S usv Αΐο Βχγ BTV ΐ^Λ ^TV "TO "10 dJi jqx OJd J people 丄 ojj dsy ^ TO J9S usv Αΐο Βχγ BTV ΐ ^ Λ ^ TV "TO" 10 dJi jqx
885^ 333 3V1 333 3V0 V30 I9V OVV 1 0 133 133 310 V30 WO WO 331 33V885 ^ 333 3V1 333 3V0 V30 I9V OVV 1 0 133 133 310 V30 WO WO 331 33V
^ i m\ Qg^i οε^ΐ s jqi Aio sAq OJJ ns aq 3jy djJL ΐ^Λ ュ 9S η3Ί T^A S H n9l^ Im \ Qg ^ i οε ^ ΐ s jqi Aio sAq OJJ ns aq 3jy dj JL ΐ ^ Λ Interview 9 S η3 Ί T ^ ASH n9 l
06^ OVV IDV 900 OVV 933 VIO VDV 003 301 013 IOV 013 919 VOO OVO 013 06 ^ OVV IDV 900 OVV 933 VIO VDV 003 301 013 IOV 013 919 VOO OVO 013
52H 0 I 5ΐ^ΐ 52H 0 I 5ΐ ^ ΐ
UTO dsy Ι^Λ ュ iU nsi s Sjy J¾ ΐ ηχο ns Βχγ J¾ nsq BIVUTO dsy Ι ^ Λ i iU nsi s Sjy J¾ ΐ ηχο ns Βχγ J¾ nsq BIV
OVO OVO 910 13V Oil 100 IOV 003 V3V 313 WO VIO 330 IDV Oil 333 OVO OVO 910 13V Oil 100 IOV 003 V3V 313 WO VIO 330 IDV Oil 333
ΟίίΊ 1 00^1  ΟίίΊ 1 00 ^ 1
UT9 OJd ngq 八 dsy OJJ Λχ SJH ητο sAq nio ns jqi J9S n9q 9χι 6^ 3VD 333 IIO DID IVO 133 009 3V3 OVO DVV 0V9 013 V3V 30V 013 OIV  UT9 OJd ngq eight dsy OJJ Λχ SJH ητο sAq nio ns jqi J9S n9q 9χι 6 ^ 3VD 333 IIO DID IVO 133 009 3V3 OVO DVV 0V9 013 V3V 30V 013 OIV
56CI 068Ϊ  56CI 068Ϊ
STH uio naq ns OJJ χΒΛ ^丄 ^TV c d naq jqi Sjy nsq Sjy Mj) J3S STH uio naq ns OJJ χΒΛ ^ 丄 ^ TV cd naq jqi Sjy nsq Sjy Mj) J3S
9 DVO OVO 010 013 303 319 IDV 030 133 013 3DV 003 313 V3V OVO Idl9 DVO OVO 010 013 303 319 IDV 030 133 013 3DV 003 313 V3V OVO Idl
Figure imgf000086_0001
Figure imgf000086_0001
Ϊ ΒΛ "TO ηχο JAI Π3 jq 3¾j nsq gjy naq s丁 H dsy J¾ T ¾ nsq Sjy 86^ 010 OVO 9V3 IVI 013 D3V OIX 3X3 00V 013 OVO IVO 30V 3X0 Oil 393  Ϊ ΒΛ "TO ηχο JAI Π3 jq 3¾j nsq gjy naq s cho H dsy J¾ T ¾ nsq Sjy 86 ^ 010 OVO 9V3 IVI 013 D3V OIX 3X3 00V 013 OVO IVO 30V 3X0 Oil 393
098T gggl 0981 naq αΑ na OJJ gjy χο J9S rifj) 3iy sAi ns nsq ng 3jy 098T gggl 0981 naq αΑ na OJJ gjy χο J9S rifj) 3iy sAi ns nsq ng 3jy
092^ 01D OVI 013 033 0D3 ODD VOX VV3 030 9VV 010 310 013 013 V03 3丄 V mi on\ ¾εει092 ^ 01D OVI 013 033 0D3 ODD VOX VV3 030 9VV 010 310 013 013 V03 3 丄 V mion \ ¾εει
"ΐθ usv usv 3Md OJJ J3S ηχο naq Say sA 人 13 αΑχ nsq δχγ ns 20^ OVO DVV 3VV XXX V3D VOX OVO 9V0 0X3 303 OVV 009 3VX 313 330 iX 0/ム 6df/JLOd 8£8^0/86 OAV Glu Gly Pro Leu Glu Arg Pro Gly Ala Arg Leu Cys Leu Pro Asp Gly "ΐθ usv usv 3Md OJJ J3S ηχο naq Say sA person 13 αΑχ nsq δχγ ns 20 ^ OVO DVV 3VV XXX V3D VOX OVO 9V0 0X3 303 OVV 009 3VX 313 330 iX 0 / m 6df / JLOd 8 £ 8 ^ 0/86 OAV Glu Gly Pro Leu Glu Arg Pro Gly Ala Arg Leu Cys Leu Pro Asp Gly
1480 1485 1490  1480 1485 1490
CCC CTG AGA ACA GCA GCT AAA CGT TGC TAT GGG AAG AGG CCA GGG CTA 4682 Pro Leu Arg Thr Ala Ala Lys Arg Cys Tyr Gly Lys Arg Pro Gly Leu  CCC CTG AGA ACA GCA GCT AAA CGT TGC TAT GGG AAG AGG CCA GGG CTA 4682 Pro Leu Arg Thr Ala Ala Lys Arg Cys Tyr Gly Lys Arg Pro Gly Leu
1495 1500 1505  1495 1500 1505
GAG GAC ACG GCA CAC ATC CTC ATT GCA GCT CAG CTC TGG AAG ACA TGT 4730 Glu Asp Thr Ala His l ie Leu He Ala Ala Gin Leu Trp Lys Thr Cys  GAG GAC ACG GCA CAC ATC CTC ATT GCA GCT CAG CTC TGG AAG ACA TGT 4730 Glu Asp Thr Ala His lie Leu He Ala Ala Gin Leu Trp Lys Thr Cys
1510 1515 1520 1525 1510 1515 1520 1525
GAC GCT GAT GCC TCA GGC ACC TTC CGA ACT TGC CCT CCT GAG GCT CTG 4778 Asp Ala Asp Ala Ser Gly Thr Phe Arg Ser Cys Pro Pro Glu Ala Leu  GAC GCT GAT GCC TCA GGC ACC TTC CGA ACT TGC CCT CCT GAG GCT CTG 4778 Asp Ala Asp Ala Ser Gly Thr Phe Arg Ser Cys Pro Pro Glu Ala Leu
1530 1535 1540  1530 1535 1540
GGA GAC CTG CCT TAC CAC CTG CTC CAG AGC GGG AAC CGT GGA CTT CTT 4826 Gly Asp Leu Pro Tyr His Leu Leu Gin Ser Gly Asn Arg Gly Leu Leu  GGA GAC CTG CCT TAC CAC CTG CTC CAG AGC GGG AAC CGT GGA CTT CTT 4826 Gly Asp Leu Pro Tyr His Leu Leu Gin Ser Gly Asn Arg Gly Leu Leu
1545 1550 1555  1545 1550 1555
TCG AAG TTC CTT ACC AAC CTC CAT GTG GTG GCT GCA CAC TTG GAA TTG 4874 Ser Lys Phe Leu Thr Asn Leu His Val Val Ala Ala His Leu Glu Leu  TCG AAG TTC CTT ACC AAC CTC CAT GTG GTG GCT GCA CAC TTG GAA TTG 4874 Ser Lys Phe Leu Thr Asn Leu His Val Val Ala Ala His Leu Glu Leu
1560 1565 1570  1560 1565 1570
GGT CTG GTC TCT CGG CTC TTG GAG GCC CAT GCC CTC TAT GCT TCT TCA 4922 Gly Leu Val Ser Arg Leu Leu Glu Ala His Ala Leu Tyr Ala Ser Ser  GGT CTG GTC TCT CGG CTC TTG GAG GCC CAT GCC CTC TAT GCT TCT TCA 4922 Gly Leu Val Ser Arg Leu Leu Glu Ala His Ala Leu Tyr Ala Ser Ser
1575 1580 1585  1575 1580 1585
GTC CCC AAA GAG GAA CAA AAG CTC CCC GAG GCT GAC GTT GCA GTG TTT 4970 Val Pro Lys Glu Glu Gin Lys Leu Pro Glu Ala Asp Val Ala Val Phe  GTC CCC AAA GAG GAA CAA AAG CTC CCC GAG GCT GAC GTT GCA GTG TTT 4970 Val Pro Lys Glu Glu Gin Lys Leu Pro Glu Ala Asp Val Ala Val Phe
1590 1595 1600 1605 1590 1595 1600 1605
CGC ACC TTC CTG AGG CAG CAG GCT TCA ATC CTC AGC CAG TAC CCC CGG 5018 Arg Thr Phe Leu Arg Gin Gin Ala Ser l ie Leu Ser Gin Tyr Pro Arg  CGC ACC TTC CTG AGG CAG CAG GCT TCA ATC CTC AGC CAG TAC CCC CGG 5018 Arg Thr Phe Leu Arg Gin Gin Ala Ser lie Leu Ser Gin Tyr Pro Arg
1610 1615 1620  1610 1615 1620
CTC CTG CCC CAG CAG GCA GCC AAC CAG CCC CTG GAC TCA CCT CTT TGC 5066 Leu Leu Pro Gin Gin Ala Ala Asn Gin Pro Leu Asp Ser Pro Leu Cys 98 CTC CTG CCC CAG CAG GCA GCC AAC CAG CCC CTG GAC TCA CCT CTT TGC 5066 Leu Leu Pro Gin Gin Ala Ala Asn Gin Pro Leu Asp Ser Pro Leu Cys 98
08Π \ Q I 08Π \ Q I
sAo Aio IO nsq S Q Jqi BTV naq 8jy s dsy OJJ jas naq sAo Aio IO nsq S Q Jqi BTV naq 8jy s dsy OJJ jas naq
86^9 391 V30 V90 Oil 391 010 33V 330 V13 010 9D3 301 3V0 V03 30V 013 59Π 09ΑΪ 59ii 05il86 ^ 9 391 V30 V90 Oil 391 010 33V 330 V13 010 9D3 301 3V0 V03 30V 013 59Π 09ΑΪ 59ii 05il
S G SAQ Ai3 jqx 9{[ α uto st{{ Biy sAq jq uio [ ΒΛ SjyS G SAQ Ai3 jqx 9 {[α uto st {{Biy sAq jq uio [ΒΛ Sjy
OS ζ 001 331 300 I3V 31V WO DVI OVO 3V3 130 OVV 13V OVO 013 010 003 OS ζ 001 331 300 I3V 31V WO DVI OVO 3V3 130 OVV 13V OVO 013 010 003
ς I oni sen sA3 Ai STH \ ΠΘΊ dsy d^i nsq ηχο ns naq Αχο dsy aqd ^TV JMX 20^9 101 100 IVO OVO 913 OVO 301 DID OVO 010 DIO 000 OVO Oil 330 I3V  ς I oni sen sA3 Ai STH \ ΠΘΊ dsy d ^ i nsq ηχο ns naq Αχο dsy aqd ^ TV JMX 20 ^ 9 101 100 IVO OVO 913 OVO 301 DID OVO 010 DIO 000 OVO Oil 330 I3V
08Ai \ \ naq sqj nsq j dsy dsy Jas ns 9qj naq SAQ efv J9S λχο dsy 08Ai \ \ naq sqj nsq j dsy dsy Jas ns 9qj naq SAQ efv J9S λχο dsy
½S5 113 111 313 VOV OVO IVO 331 313 Oil Oil 101 133 ID! 31V V09 IVO ½S5 113 111 313 VOV OVO IVO 331 313 Oil Oil 101 133 ID! 31V V09 IVO
5 1 OUT SOLI  5 1 OUT SOLI
s^3 ΤΟ -ISS ΐ^Λ 1¾ J3S sAi ητο ηχο u^o dj丄 jq gjy dsy 097s ^ 3 ΤΟ -ISS ΐ ^ Λ 1¾ J3 S sAi ητο ηχο u ^ o dj 丄 jq gjy dsy 097
9085 101 300 IOV 910 013 X3X OVV 3V0 3V0 OVO 001 IDV VOV 0X3 3V0 OH 9085 101 300 IOV 910 013 X3X OVV 3V0 3V0 OVO 001 IDV VOV 0X3 3V0 OH
ΟΟΑΐ 69ΐ 0691  ΟΟΑΐ 69ΐ 0691
nai JAI Ι ΒΛ Jqi Ala usy BTV ュ ^13 ΐ^Λ eiV ^TV V "TO ^ΐθ usy 013 3VI 110 VDV ODO IVV 339 X3V 090 010 100 VOO V3V VV3 003 IVV 589Ϊ 089ΐ 0i91 ュ J9S sqd BTV I^A ^TV ^ΜΙ OJJ Jd^ J9S "^S ΐ^Λ ¾TV n^l s nsq 0129 33V 301 Oil 33D 013 130 IDV 133 DDI V3I 301 H VOO 913 131 313  nai JAI Ι ΒΛ Jqi Ala usy BTV ^^ 13 ΐ ^ Λ eiV ^ TV V "TO ^ ΐθ usy 013 3VI 110 VDV ODO IVV 339 X3V 090 010 100 VOO V3V VV3 003 IVV 589Ϊ 089ΐ 0i91 JJ9S sqd BTV I ^ A ^ TV ^ ΜΙ OJJ Jd ^ J9S "^ S ΐ ^ Λ ¾TV n ^ ls nsq 0129 33V 301 Oil 33D 013 130 IDV 133 DDI V3I 301 H VOO 913 131 313
S99T 099T 959Ϊ S99T 099T 959Ϊ
J3S S s uio uxo usy sA 3« J¾ 8jy o^d sA usy nsq dJ 3jyJ3S S s uio uxo usy sA 3 «J¾ 8jy o ^ d sA usy nsq dJ 3jy
29 Ϊ9 39V GDI 30V VV3 OVO IVV VVV OIV D3V OOD DOD VVV IVV 1X3 901 VOO 29 Ϊ9 39V GDI 30V VV3 OVO IVV VVV OIV D3V OOD DOD VVV IVV 1X3 901 VOO
0591 9^91 0^9ΐ na jq STH
Figure imgf000088_0001
J3S naq JSS BTV "TO S TH vio v v m OLO OVO OOI VOV OOD OOI OLD IO 03i oao ova0591 9 ^ 91 0 ^ 9ΐ na jq STH
Figure imgf000088_0001
J3S naq JSS BTV "TO S TH vio vvm OLO OVO OOI VOV OOD OOI OLD IO 03i oao ova
5891 0891 9291 5891 0891 9291
XH6Z0IL6d£llDd 8C8 .0/86 OAV 12 XH6Z0IL6d £ llDd 8C8 .0 / 86 OAV 12
8 6S 0X0 033 XV3 100 Π0 VD3 30V 313 V33 010 131 313 330 133 101 313 8 6S 0X0 033 XV3 100 Π0 VD3 30V 313 V33 010 131 313 330 133 101 313
6ΐ 0261 3Ι6ΐ 0ΐ6ΐ 6ΐ 0261 3Ι6ΐ 0ΐ6ΐ
•I3S Π9ΐ J9S Αχθ nsq S IH Αχο 3jy oid Sjy χ na jas Αΐ9 J3S dJi• I3S Π9ΐ J9S Αχθ nsq S IH Αχο 3jy oid Sjy χ na jas Αΐ9 J3S dJi
0869 X31 XIO 331 103 013 3V0 000 133 303 003 103 3XD X31 309 V3I 031 0869 X31 XIO 331 103 013 3V0 000 133 303 003 103 3XD X31 309 V3I 031
9061 0061 5681 9061 0061 5681
Τ ΒΛ "TO ΐ^Λ sA ATO dsy πχο Αχο ¾TV J¾ na ns u\ sA^ A\ BIVTO ΒΛ "TO ΐ ^ Λ sA ATO dsy πχο Αχο ¾TV J¾ na ns u \ sA ^ A \ BIV
2885 010 OVD 110 OVV 000 IVO OVO V33 130 00V 010 VII OVD 331 133 030 2885 010 OVD 110 OVV 000 IVO OVO V33 130 00V 010 VII OVD 331 133 030
0681 5881 0881 stH nsq aqj nsq εχγ Βχν BTV T^A 3Md ^χο STH S IH BTV OJJ 9i ^χν iva oia on xxo ooo IDO iao no in ooo ivo ova oao xoa on aao 0681 5881 0881 stH nsq aqj nsq εχγ Βχν BTV T ^ A 3Md ^ χο STH S IH BTV OJJ 9i ^ χν iva oia on xxo ooo IDO iao no in ooo ivo ova oao xoa on aao
SA8I 0 8T 5981 SA8I 08T 5981
BTV naq 3JV BTV
Figure imgf000089_0001
ュ dsy 98 5 130 310 000 V33 330 VVO VOO 03J, 030 091 9X3 OVO 010 01V I0V 3V0 BTV naq 3JV BTV
Figure imgf000089_0001
Dsy 98 5 130 310 000 V33 330 VVO VOO 03J, 030 091 9X3 OVO 010 01V I0V 3V0
098Ϊ 998ΐ 098Ϊ ri9i Say Αχ ΐ^Λ Biv Ι ΒΛ ΐΒΛ ^ΤΟ ^ι οα^ ΙΒΛ usy sqd BTV ュ 013 003 ODD 0X3 X00 310 XXO 000 000 133 010 XVV 3X1 330 Oil 30V 9^8ΐ 0^8ΐ 9221 0881 V an J3S EIV Ατο ojj Αχο Αχ OJ^ sAq J¾ T^A sAq naq Αχο 0695 1D3 3IV 131 030 VOO 033 303 000 133 VOO OVV 33V 310 VVV 313 003  098Ϊ 998ΐ 098Ϊ ri9i Say ΐ ΐ ^ Λ Biv Ι ΐΒΛ ΤΟ ^ ΤΟ ^ ι οα ^ ΙΒΛ usy sqd BTV B 013 003 ODD 0X3 X00 310 XXO 000 000 133 010 XVV 3X1 330 Oil 30V 9 ^ 8ΐ 0 ^ 8ΐ 9221 0881 V an J3S EIV Ατο ojj Αχο Αχ OJ ^ sAq J¾ T ^ A sAq naq Αχο 0695 1D3 3IV 131 030 VOO 033 303 000 133 VOO OVV 33V 310 VVV 313 003
9281 0281 5Ϊ81 dsv ΐ^Λ uio 9 d Jas 9TI S ^ ^TV 丄 J8S AIQ Jqi BTV 9TI Zm IVO 310 3V3 311 Oil 39V DXV 30V 300 133 001 30V 303 VOV 33D VIV 9281 0281 5Ϊ81 dsv ΐ ^ Λ uio 9 d Jas 9TI S ^ ^ TV 丄 J8S AIQ Jqi BTV 9 TI Zm IVO 310 3V3 311 Oil 39V DXV 30V 300 133 001 30V 303 VOV 33D VIV
0Ι8Ϊ 9081 0081  0Ι8Ϊ 9081 0081
ΪΕΛ uio Αχο ηχο OJJ S TH aqd BTV ΐ^Λ SAQ usy nsq s sAq OJJ JA丄 ΪΕΛ uio Αχο ηχο OJJ S TH aqd BTV ΐ ^ Λ SAQ usy nsq s sAq OJJ JA 丄
VI9 0V3 903 3V0 VOO 3V3 3X1 000 119 131 OVV 3X3 001 OVV 000 3VX VI9 0V3 903 3V0 VOO 3V3 3X1 000 119 131 OVV 3X3 001 OVV 000 3VX
06ΑΪ  06ΑΪ
•iqi S TH uio si BIV nsq Aio 3jy T^A D SV 丄 Η 9Ί sAl n9l• iqi S TH uio si BIV nsq Aio 3jy T ^ A DS V丄Η 9 Ί sA l n9 l
9^S5 ODV 3VD OVO Oil 330 Old OVO 000 I0D 310 V V OVO 001 Old OVV VIO *O6rO/i6Jf/13d 8£SL0IS6 O Leu Ser Pro Ala Leu Ser Val Ala Leu Ser Pro Asp Gly Asp Arg Val 9 ^ S5 ODV 3VD OVO Oil 330 Old OVO 000 I0D 310 VV OVO 001 Old OVV VIO * O6rO / i6Jf / 13d 8 £ SL0IS6 O Leu Ser Pro Ala Leu Ser Val Ala Leu Ser Pro Asp Gly Asp Arg Val
1930 1935 1940  1930 1935 1940
GCT GTT GGA TAT CGA GCG GAT GGC ATT AGG ATC TAC AAA ATC TCT TCA 6026 GCT GTT GGA TAT CGA GCG GAT GGC ATT AGG ATC TAC AAA ATC TCT TCA 6026
Ala Val Gly Tyr Arg Ala Asp Gly l ie Arg l ie Tyr Lys l ie Ser Ser Ala Val Gly Tyr Arg Ala Asp Gly lie Arg lie Tyr Lys lie Ser Ser
1945 1950 1955  1945 1950 1955
GGT TCC CAG GGG GCT CAG GGT CAG GCA CTG GAT GTG GCA GTG TCG GCC 6074 GGT TCC CAG GGG GCT CAG GGT CAG GCA CTG GAT GTG GCA GTG TCG GCC 6074
Gly Ser Gin Gly Ala Gin Gly Gin Ala Leu Asp Val Ala Val Ser Ala Gly Ser Gin Gly Ala Gin Gly Gin Ala Leu Asp Val Ala Val Ser Ala
1960 1965 1970  1960 1965 1970
CTG GCC TGG ATA AGC CCC AAG GTA TTG GTG AGT GGT GCA GAA GAT GGG 6122 CTG GCC TGG ATA AGC CCC AAG GTA TTG GTG AGT GGT GCA GAA GAT GGG 6122
Leu Ala Trp l ie Ser Pro Lys Val Leu Val Ser Gly Ala Glu Asp Gly Leu Ala Trp lie Ser Pro Lys Val Leu Val Ser Gly Ala Glu Asp Gly
1975 1980 1985  1975 1980 1985
TCC TTG CAG GGC TGG GCA CTC AAG GAA TGC TCC CTT CAG TCC CTC TGG 6170 TCC TTG CAG GGC TGG GCA CTC AAG GAA TGC TCC CTT CAG TCC CTC TGG 6170
Ser Leu Gin Gly Trp Ala Leu Lys Glu Cys Ser Leu Gin Ser Leu Trp Ser Leu Gin Gly Trp Ala Leu Lys Glu Cys Ser Leu Gin Ser Leu Trp
1990 1995 2000 2005 1990 1995 2000 2005
CTC CTG TCC AGA TTC CAG AAG CCT GTG CTA GGA CTG GCC ACT TCC CAG 6218 CTC CTG TCC AGA TTC CAG AAG CCT GTG CTA GGA CTG GCC ACT TCC CAG 6218
Leu Leu Ser Arg Phe Gin Lys Pro Val Leu Gly Leu Ala Thr Ser Gin Leu Leu Ser Arg Phe Gin Lys Pro Val Leu Gly Leu Ala Thr Ser Gin
2010 2015 2020  2010 2015 2020
GAG CTC TTG GCT TCT GCC TCA GAG GAT TTC ACA GTG CAG CTG TGG CCA 6266 GAG CTC TTG GCT TCT GCC TCA GAG GAT TTC ACA GTG CAG CTG TGG CCA 6266
Glu Leu Leu Ala Ser Ala Ser Glu Asp Phe Thr Val Gin Leu Trp Pro Glu Leu Leu Ala Ser Ala Ser Glu Asp Phe Thr Val Gin Leu Trp Pro
2025 2030 2035  2025 2030 2035
AGG CAG CTG CTG ACG CGG CCA CAC AAG GCA GAA GAC TTT CCC TGT GGC 6314 AGG CAG CTG CTG ACG CGG CCA CAC AAG GCA GAA GAC TTT CCC TGT GGC 6314
Arg Gin Leu Leu Thr Arg Pro His Lys Ala Glu Asp Phe Pro Cys Gly Arg Gin Leu Leu Thr Arg Pro His Lys Ala Glu Asp Phe Pro Cys Gly
2040 2045 2050  2040 2045 2050
ACT GAG CTG CGG GGA CAT GAG GGC CCT GTG AGC TGC TGT AGT TTC AGC 6362 ACT GAG CTG CGG GGA CAT GAG GGC CCT GTG AGC TGC TGT AGT TTC AGC 6362
Thr Glu Leu Arg Gly His Glu Gly Pro Val Ser Cys Cys Ser Phe Ser Thr Glu Leu Arg Gly His Glu Gly Pro Val Ser Cys Cys Ser Phe Ser
2055 2060 2065  2055 2060 2065
ACT GAT GGA GGC AGC CTG GCC ACC GGG GGC CGG GAT CGG AGT CTC CTC 6410 ACT GAT GGA GGC AGC CTG GCC ACC GGG GGC CGG GAT CGG AGT CTC CTC 6410
Thr Asp Gly Gly Ser Leu Ala Thr Gly Gly Arg Asp Arg Ser Leu Leu 2070 2075 2080 2085Thr Asp Gly Gly Ser Leu Ala Thr Gly Gly Arg Asp Arg Ser Leu Leu 2070 2075 2080 2085
TGC TGG GAC GTG AGG ACA CCC AAA ACC CCT GTT TTG ATC CAC TCC TTC 6458TGC TGG GAC GTG AGG ACA CCC AAA ACC CCT GTT TTG ATC CAC TCC TTC 6458
Cys Trp Asp Val Arg Thr Pro Lys Thr Pro Val Leu l ie His Ser Phe Cys Trp Asp Val Arg Thr Pro Lys Thr Pro Val Leu lie His Ser Phe
2090 2095 2100  2090 2095 2100
CCT GCC TGT CAC CGT GAC TGG GTC ACT GGC TGT GCC TGG ACC AAA GAT 6506 Pro Ala Cys His Arg Asp Trp Val Thr Gly Cys Ala Trp Thr Lys Asp  CCT GCC TGT CAC CGT GAC TGG GTC ACT GGC TGT GCC TGG ACC AAA GAT 6506 Pro Ala Cys His Arg Asp Trp Val Thr Gly Cys Ala Trp Thr Lys Asp
2105 2110 2115  2105 2110 2115
AAC CTA CTG ATA TCC TGC TCC AGT GAT GGC TCT GTG GGG CTC TGG GAC 6554 Asn Leu Leu l ie Ser Cys Ser Ser Asp Gly Ser Val Gly Leu Trp Asp  AAC CTA CTG ATA TCC TGC TCC AGT GAT GGC TCT GTG GGG CTC TGG GAC 6554 Asn Leu Leu lie Ser Cys Ser Ser Asp Gly Ser Val Gly Leu Trp Asp
2120 2125 2130  2120 2125 2130
CCA GAG TCA GGA CAG CGG CTT GGT CAG TTC CTG GGT CAT CAG AGT GCT 6602 Pro Glu Ser Gly Gin Arg Leu Gly Gin Phe Leu Gly His Gin Ser Ala  CCA GAG TCA GGA CAG CGG CTT GGT CAG TTC CTG GGT CAT CAG AGT GCT 6602 Pro Glu Ser Gly Gin Arg Leu Gly Gin Phe Leu Gly His Gin Ser Ala
2135 2140 2145  2135 2140 2145
GTG AGC GCT GTG GCA GCT GTG GAG GAG CAC GTG GTG TCT GTG AGC CGG 6650 Val Ser Ala Val Ala Ala Val Glu Glu His Val Val Ser Val Ser Arg  GTG AGC GCT GTG GCA GCT GTG GAG GAG CAC GTG GTG TCT GTG AGC CGG 6650 Val Ser Ala Val Ala Ala Val Glu Glu His Val Val Ser Val Ser Arg
2150 2155 2160 2165 2150 2155 2160 2165
GAT GGG ACC TTG AAA GTG TGG GAC CAT CAA GGC GTG GAG CTG ACC AGC 6698 Asp Gly Thr Leu Lys Val Trp Asp His Gin Gly Val Glu Leu Thr Ser  GAT GGG ACC TTG AAA GTG TGG GAC CAT CAA GGC GTG GAG CTG ACC AGC 6698 Asp Gly Thr Leu Lys Val Trp Asp His Gin Gly Val Glu Leu Thr Ser
2170 2175 2180  2170 2175 2180
ATC CCT GCT CAC TCA GGA CCC ATT AGC CAC TGT GCA GCT GCC ATG GAG 6746 He Pro Ala His Ser Gly Pro l ie Ser His Cys Ala Ala Ala Met Glu  ATC CCT GCT CAC TCA GGA CCC ATT AGC CAC TGT GCA GCT GCC ATG GAG 6746 He Pro Ala His Ser Gly Pro lie Ser His Cys Ala Ala Ala Met Glu
2185 2190 2195  2185 2190 2195
CCC CGT GCA GCT GGA CAG CCT GGG TCA GAG CTT CTG GTG GTA ACC ATC 6794 Pro Arg Ala Ala Gly Gin Pro Gly Ser Glu Leu Leu Val Val Thr He  CCC CGT GCA GCT GGA CAG CCT GGG TCA GAG CTT CTG GTG GTA ACC ATC 6794 Pro Arg Ala Ala Gly Gin Pro Gly Ser Glu Leu Leu Val Val Thr He
2200 2205 2210  2200 2205 2210
GGG CTA GAT GGG GCC ACA CGG TTA TGG CAT CCA CTC TTG GTG TGC CAA 6842 Gly Leu Asp Gly Ala Thr Arg Leu Trp His Pro Leu Leu Val Cys Gin  GGG CTA GAT GGG GCC ACA CGG TTA TGG CAT CCA CTC TTG GTG TGC CAA 6842 Gly Leu Asp Gly Ala Thr Arg Leu Trp His Pro Leu Leu Val Cys Gin
2215 2220 2225 06 2215 2220 2225 06
ZZZL IVD V30 VVV 333 Oil DiV 313 ill OVO 130 XVO 133 130 001 IVO 013 ZZZL IVD V30 VVV 333 Oil DiV 313 ill OVO 130 XVO 133 130 001 IVO 013
ZZ ^ZZ 09CZ  ZZ ^ ZZ 09CZ
J3S Jiu naq dsy rifg naq Q\ \ SJV usy naq sin naq J3S Jiu naq dsy rifg naq Q \ \ SJV usy naq sin naq
I9V VOV 013 DIO 000 Vll 3V0 OVO OVO V IIV V93 OVV 010 OVO 丄丄3 I9V VOV 013 DIO 000 Vll 3V0 OVO OVO V IIV V93 OVV 010 OVO 丄 丄 3
99£2 05SZ 5^2  99 £ 2 05SZ 5 ^ 2
J3S "31 usy AID OJJ BT V ^TO sAq 3jy naq sAq 八 u^o (Ια ηχο J3S "31 usy AID OJJ BT V ^ TO sAq 3jy naq sAq eight u ^ o (Ια ηχο
I0V Oil IVV VOO 333 VOO 031 100 3V3 VOO 013 VVV 010 VV3 001 OVOI0V Oil IVV VOO 333 VOO 031 100 3V3 VOO 013 VVV 010 VV3 001 OVO
0^82 9682 Q 0 ^ 82 9682 Q
J9S 3Ϊ Ι sA ηχο dsy BTV s na Ι^Λ 9Md 9qd J I S H ^TV ュ 3S J3S 8 U 39V 3IV VVV OVO 1V3 IDO I3V 3X3 310 III III 33V 3V3 V39 931 331J9S 3Ϊ Ι sA ηχο dsy BTV s na Ι ^ Λ 9Md 9qd JISH ^ TV Interview 3 SJ 3 S 8 U 39V 3IV VVV OVO 1V3 IDO I3V 3X3 310 III III 33V 3V3 V39 931 331
Z2Z mZ 0182 dJi 9T i noq Βχν Αχο 9χ ΐ STH λχο OJJ B V "TO ^TV ュ1 ^TV Τ^Λ ^TV 08U 001 31V 3X3 130 100 VIV DVD 300 VOO 130 OVO V39 VOV 339 0X3 130 Z2Z mZ 0182 dJi 9T i noq Βχν Αχο 9χ ΐ STH λχο OJJ BV "TO ^ TV 11 ^ TV Τ ^ Λ ^ TV 08U 001 31V 3X3 130 100 VIV DVD 300 VOO 130 OVO V39 VOV 339 0X3 130
00£2 5632 sAq Biv nio uia djx nsq 3χι naq ηχ ^10 ^TV "TO USV J3S ΐ^Λ 2801 OVV IDO WO 9V3 001 OH 31V VI3 WO 309 IDO VV3 IVV VOO 131 VI9 00 £ 2 5632 sAq Biv nio uia djx nsq 3χι naq ηχ ^ 10 ^ TV "TO US VJ 3 S ΐ ^ Λ 2801 OVV IDO WO 9V3 001 OH 31V VI3 WO 309 IDO VV3 IVV VOO 131 VI9
0622 mz 0822  0622 mz 0822
BIV 19W S ^TO dsv OJJ BTV Li丄 BT V Ϊ¾Λ ^TV ュ ΐ^Λ ^χγ BTV ュ HQL VDO OXV 331 309 IVO V33 V33 001 X39 0X0 139 I3V 310 300 V3D 丄:)丄  BIV 19W S ^ TO dsv OJJ BTV Li 丄 BT V Ϊ¾Λ ^ TV ΐ ^ ΐ ^ χγ BTV H HQL VDO OXV 331 309 IVO V33 V33 001 X39 0X0 139 I3V 310 300 V3D 丄 :) 丄
UZZ QLZZ ^ZZ  UZZ QLZZ ^ ZZ
J9S Say ojd an SAQ J¾ dsy dsy BTV n g sA OJJ χΒΛ "TO dJi Η3ΊJ9S Say ojd an SAQ J¾ dsy dsy BTV ng sA OJJ χΒΛ "TO dJ i Η3 Ί
9869 10V OOV VDO VIV LOl VOV 3V3 XVO V39 WO OVV 133 110 9V0 001 313 9869 10V OOV VDO VIV LOl VOV 3V3 XVO V39 WO OVV 133 110 9V0 001 313
0922 0922  0922 0922
§JV ΪΒΛ J3S ni9 J3S BTV -iqi ns 3f[ na εχν J^S JMI "TO s 8S69 003 V19 13X OXV OVO 131 330 33V 010 9IV DID 330 VOl 33V VV3 VOL
Figure imgf000092_0001
§JV ΪΒΛ J3S ni9 J3S BTV -iqi ns 3f [na εχν J ^ S JMI "TO s 8S69 003 V19 13X OXV OVO 131 330 33V 010 9IV DID 330 VOl 33V VV3 VOL
Figure imgf000092_0001
ΐΒΛ εχν BTV ^iv SJV Τ^Λ O-^d ^io aas S TH Αχο na naq J¾ STH ュ ¾ 0689 1X3 130 130 130 103 310 V30 300 OOV 3V3 VOO 013 3X0 D3V 3V3 33V ΐΒΛ εχν BTV ^ iv SJV Τ ^ Λ O- ^ d ^ io aas S TH Αχο na naq J¾ STH ¾ 0689 1X3 130 130 130 103 310 V30 300 OOV 3V3 VOO 013 3X0 D3V 3V3 33V
P06Z0IL6dr/lDd 9£SL0(86 OAS. Leu Asp Trp Ala Pro Asp Gly His Phe Leu l ie Leu Ala Lys Ala AspP06Z0IL6dr / lDd 9 £ SL0 (86 OAS. Leu Asp Trp Ala Pro Asp Gly His Phe Leu lie Leu Ala Lys Ala Asp
2375 2380 2385 2375 2380 2385
TTG AAG TTA CTT TGC ATG AAG CCA GGG GAT GCT CCA TCT GAA ATC TGG 7370 Leu Lys Leu Leu Cys Met Lys Pro Gly Asp Ala Pro Ser Glu l ie Trp 2390 2395 2400 2405 TTG AAG TTA CTT TGC ATG AAG CCA GGG GAT GCT CCA TCT GAA ATC TGG 7370 Leu Lys Leu Leu Cys Met Lys Pro Gly Asp Ala Pro Ser Glu lie Trp 2390 2395 2400 2405
AGC AGC TAT ACA GAA AAT CCT ATG ATA TTG TCC ACC CAC AAG GAA TAT 7418 Ser Ser Tyr Thr Glu Asn Pro Met l ie Leu Ser Thr His Lys Glu Tyr AGC AGC TAT ACA GAA AAT CCT ATG ATA TTG TCC ACC CAC AAG GAA TAT 7418 Ser Ser Tyr Thr Glu Asn Pro Met lie Leu Ser Thr His Lys Glu Tyr
2410 2415 2420  2410 2415 2420
GGC ATA TTT GTC CTG CAG CCC AAG GAT CCT GGA GTT CTT TCT TTC TTG 7466 Gly He Phe Val Leu Gin Pro Lys Asp Pro Gly Val Leu Ser Phe Leu  GGC ATA TTT GTC CTG CAG CCC AAG GAT CCT GGA GTT CTT TCT TTC TTG 7466 Gly He Phe Val Leu Gin Pro Lys Asp Pro Gly Val Leu Ser Phe Leu
2425 2430 2435  2425 2430 2435
AGG CAA AAG GAA TCA GGA AAG TTT GAA GAG AGG CTG AAC TTT GAT ATA 7514 Arg Gin Lys Glu Ser Gly Lys Phe Glu Glu Arg Leu Asn Phe Asp l ie  AGG CAA AAG GAA TCA GGA AAG TTT GAA GAG AGG CTG AAC TTT GAT ATA 7514 Arg Gin Lys Glu Ser Gly Lys Phe Glu Glu Arg Leu Asn Phe Asp l ie
2440 2445 2450  2440 2445 2450
AAC TTA GAG AAT CCT AGT AGG ACC CTA ATA TCG ATA ACT CAA GCC AAA 7562 Asn Leu Glu Asn Pro Ser Arg Thr Leu l ie Ser l ie Thr Gin Ala Lys  AAC TTA GAG AAT CCT AGT AGG ACC CTA ATA TCG ATA ACT CAA GCC AAA 7562 Asn Leu Glu Asn Pro Ser Arg Thr Leu lie Ser lie Thr Gin Ala Lys
2455 2460 2465  2455 2460 2465
CCT GAA TCT GAG TCC TCA TTT TTG TGT GCC AGC TCT GAT GGG ATG CTA 7610 Pro Glu Ser Glu Ser Ser Phe Leu Cys Ala Ser Ser Asp Gly Met Leu  CCT GAA TCT GAG TCC TCA TTT TTG TGT GCC AGC TCT GAT GGG ATG CTA 7610 Pro Glu Ser Glu Ser Ser Phe Leu Cys Ala Ser Ser Asp Gly Met Leu
2470 2475 2480 2485 2470 2475 2480 2485
TGG AAC CTG GCC AAA TGC AGC CCA GAA GGA GAA TGG ACC ACA GGT AAC 7658 Trp Asn Leu Ala Lys Cys Ser Pro Glu Gly Glu Trp Thr Thr Gly Asn  TGG AAC CTG GCC AAA TGC AGC CCA GAA GGA GAA TGG ACC ACA GGT AAC 7658 Trp Asn Leu Ala Lys Cys Ser Pro Glu Gly Glu Trp Thr Thr Gly Asn
2490 2495 2500  2490 2495 2500
ATG TGG CAG AAA AAA GCA AAC ACT CCA GAA ACC CAA ACT CCA GGG ACA 7706 Met Trp Gin Lys Lys Ala Asn Thr Pro Glu Thr Gin Thr Pro Gly Thr  ATG TGG CAG AAA AAA GCA AAC ACT CCA GAA ACC CAA ACT CCA GGG ACA 7706 Met Trp Gin Lys Lys Ala Asn Thr Pro Glu Thr Gin Thr Pro Gly Thr
2505 2510 2515  2505 2510 2515
GAC CCA TCT ACC TGC AGG GAA TCT GAT GCC AGC ATG GAT AGT GAT GCC 7754 Asp Pro Ser Thr Cys Arg Glu Ser Asp Ala Ser Met Asp Ser Asp Ala Z6 GAC CCA TCT ACC TGC AGG GAA TCT GAT GCC AGC ATG GAT AGT GAT GCC 7754 Asp Pro Ser Thr Cys Arg Glu Ser Asp Ala Ser Met Asp Ser Asp Ala Z6
0898 νθΙΟΟΟΙΰΟΙ V30900300V 90V9V0D3VI 3VV0V313X1 IXOOOOVaVO V0V3V3I0I3 0258 1II01VI33V OVVVOViXlV 3000VDIV33 iVDIOVOIOV V1VV9V0G9V 000V9IVV0V 09^8 VV0003XV3I I3V31IX33V 0VV00XV30V 9V3VV01IXV VOIIVDWOO 001I0IV313 00^8 V0V0IVV03V 09V30V030I I3IIV3001V 30VI333V90 V10D399033 DlDVOIVOOl 0½8 I31IVV0V1I II00VI3VVV OIXIIDiaVI OOVIOVIIIO IVVVOIVVOI 0VVV0VVDV3 0828 OVVDViaVXI 33IVV1XV0V V3I1VII31I 3131010101 VV13I1V3VI OIVXVXOIVI 3I01V1D11V IVOOVOOVDV VOVIIIVOOI i V UiXL 3ID3010300 VI0I0VVDI3 0918 IVVVVVVOXO 10VVOIVVVV VIVIIVVIVO 丄丄 C LUVVIV VI1VVVVIVI 丄 vv丄 VVJIU 0018 33VI03I3V3 V0VV0ID3DV VV30XV3V0V I39I0II33D 3V1V9XVVIV V0393IDV30 z z0898 νθΙΟΟΟΙΰΟΙ V30900300V 90V9V0D3VI 3VV0V313X1 IXOOOOVaVO V0V3V3I0I3 0258 1II01VI33V OVVVOViXlV 3000VDIV33 iVDIOVOIOV V1VV9V0G9V 000V9IVV0V 09 ^ 8 VV0003XV3I I3V31IX33V 0VV00XV30V 9V3VV01IXV VOIIVDWOO 001I0IV313 00 ^ 8 V0V0IVV03V 09V30V030I I3IIV3001V 30VI333V90 V10D399033 DlDVOIVOOl 0½8 I31IVV0V1I II00VI3VVV OIXIIDiaVI OOVIOVIIIO IVVVOIVVOI 0VVV0VVDV3 0828 OVVDViaVXI 33IVV1XV0V V3I1VII31I 3131010101 VV13I1V3VI OIVXVXOIVI 3I01V1D11V IVOOVOOVDV VOVIIIVOOI i V UiXL 3ID3010300 VI0I0VVDI3 0918 IVVVVVVOXO 10VVOIVVVV VIVIIVVIVO 丄 丄 C LUVVIV VI1VVVVIVI 丄 vv 丄 VVJIU 0018 33VI03I3V3 V0VV0ID3DV VV30XV3V0V I39I0V33V
*** niO djx usy naq _i ΤΒΛ usy ^TO uio 人 dsy*** niO djx usy naq _i ΤΒΛ usy ^ TO uio people dsy
0 08 010IV VV3X VV3 001 IVV 310 111 3V1 310 IVV 300 OVD 010 OVO 0 08 010IV VV3X VV3 001 IVV 310 111 3V1 310 IVV 300 OVD 010 OVO
0192 509Z 0092  0192 509Z 0092
^IO T¾ BTV Η3Ί "TO nsq am ュ 35 usy BTV Ατο nsq dュ丄 OJJ ηχ^ naq 66 V30 0X0 330 LLd OVO 013 D3V 331 OVV XOO 009 013 ΟΰΙ ID3 VVO 9X0 ^ IO T¾ BTV Η3 Ί "TO nsq am 35 35 usy BTV Ατο nsq d 丄 OJJ ηχ ^ naq 66 V30 0X0 330 LLd OVO 013 D3V 331 OVV XOO 009 013 ΟΰΙ ID3 VVO 9X0
56S2 06S2 9852  56S2 06S2 9852
s ass Ϊ ΒΛ s χο nig SXQ 3jy 3¾ ΠΘΊ ΑΪΟ naq na^ uio a« J9S s ass Ϊ ΒΛ s χο nig SXQ 3jy 3¾ ΠΘΊ ΑΪΟ naq na ^ uio a «J9S
9 6 391 03V 913 VOX 330 VVO 001 V33 311 0X3 3D0 013 013 9V3 01V 10V 9 6 391 03V 913 VOX 330 VVO 001 V33 311 0X3 3D0 013 013 9V3 01V 10V
0892 9i9Z 0 2  0892 9i9Z 0 2
ojd gjy "TO dュ丄 Π9ΐ sAq dsy Say dsy sAq za^ BJV ·ΐιΐ丄 ΐΕΛ η9Ί 868 333 VOV 3V0 001 VI3 OVV IIO IVO VOV 3V0 OVV 031 100 VDV 010 310 59S2 0992 9992 0592 nsi "TO ojj naq i si [ naq Βχγ Jqi J9S Ϊ3 siH 3 I sAq 0S8Z Oil 9V0 133 VIO 910 IVD 310 330 VDV 310 IDX 300 03X 3V3 IIV 9VV ojd gjy "TO d Interview丄Π9ΐ sAq dsy Say dsy sAq za ^ BJV · ΐιΐ丄 ΐ Ε Λ η9 Ί 868 333 VOV 3V0 001 VI3 OVV IIO IVO VOV 3V0 OVV 031 100 VDV 010 310 59S2 0992 9992 0592 nsi" TO ojj naq i si [naq Βχγ Jqi J9S Ϊ3 s iH 3 I sAq 0S8Z Oil 9V0 133 VIO 910 IVD 310 330 VDV 310 IDX 300 03X 3V3 IIV 9VV
5^92 Z 5852 5 ^ 92 Z 5852
SJV Sjy UTO Sjy J¾ sA na STH OJJ jqx OJJ ηχο aas dsy 3](( J9SSJV Sjy UTO Sjy J¾ sA na STH OJJ jqx OJJ ηχο aas dsy 3] ((J9S
208Z VOV 103 OVO 093 V3V DVV VI3 1V3 VOD VOV V33 9V9 I0V IVO OIV 30V 208Z VOV 103 OVO 093 V3V DVV VI3 1V3 VOD VOV V33 9V9 I0V IVO OIV 30V
0e92 0292  0e92 0292
JO6ZO/L6df/I3J 8€8 ,0/86 OAV - o o JO6ZO / L6df / I3J 8 € 8, 0/86 OAV -oo
o o
—j —j —J —j
C ACTTCC ACTTC
GTGAACCC GAGGCGGA CACCC G COTGTGAACCC GAGGCGGA CACCC G COT
AGGTGTGC GCG GGCTGGAAGGGGC CCGAGGC GG GTACCAGTATTGGAAAGT G AACCCATCT GGCTAAAAGTTACCC GCTCTCA ATACA ¾GAAATTATA AAA AGGTGTGC GCG GGCTGGAAGGGGC CCGAGGC GG GTACCAGTATTGGAAAGT G AACCCATCT GGCTAAAAGTTACCC GCTCTCA ATACA ¾GAAATTATA AAA

Claims

請 求 の 範 囲 The scope of the claims
1 . 配列表の配列番号 1に記載のアミノ酸配列で特定されるポリべプチド。1. A polypeptide specified by the amino acid sequence of SEQ ID NO: 1 in the sequence listing.
2 . ラッ ト由来テロメラーゼ蛋白質である請求の範囲第 1項に記載のポリベプチ 2. The polypeptide of claim 1 which is a telomerase protein derived from rat.
3 . 配列表の配列番号 1に記載のァミノ酸配列に 1又は 2以上のァミノ酸残基に よる置換、 挿入、 及びノ又は欠失が存在しており、 実質的にヒ トを含む高等動物 テロメラーゼ蛋白質として機能することを特徴とするポリべプチド。 3. Substitution, insertion, and / or deletion due to one or more amino acid residues in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, and a higher animal substantially containing human A polypeptide characterized by functioning as a telomerase protein.
4 . ヒ トの生体内でテロメラーゼ蛋白質として機能することができる請求の範囲 第 3項に記載のポリべプチド。  4. The polypeptide according to claim 3, which can function as a telomerase protein in a human body.
5 . 配列表の配列番号 2に記載のァミノ酸配列で特定されるポリべプチド。 5. A polypeptide specified by the amino acid sequence described in SEQ ID NO: 2 in the sequence listing.
6 . ヒ 卜由来テロメラーゼ蛋白質の部分ポリべプチドである請求の範囲第 5項に 記載のポリぺプチド。 6. The polypeptide according to claim 5, which is a partial polypeptide of a telomerase protein derived from human.
7 . 配列表の配列番号 2に記載のアミノ酸配列に 1又は 2以上のアミ ノ酸残基に よる置換、 挿入、 及び 又は欠失が存在しており、 実質的にヒ トを含む高等動物 テロメラ一ゼ蛋白質の部分ポリぺプチドとして機能することを特徴とするポリべ プチド。  7. Higher animal telomeres substantially containing human, in which one or more amino acid residues have substitutions, insertions, and / or deletions in the amino acid sequence of SEQ ID NO: 2 in the sequence listing. A polypeptide characterized by functioning as a partial polypeptide of a protein.
8 . 配列表の配列番号 1 3に記載のアミノ酸配列で特定されるポリペプチ ド。 8. A polypeptide identified by the amino acid sequence of SEQ ID NO: 13 in the sequence listing.
9 . ヒ ト由来テロメラーゼ蛋白質である請求の範囲第 8項に記載のポリべプチ ド。9. The polypeptide according to claim 8, which is a human-derived telomerase protein.
10. 配列表の配列番号 1 3に記載のアミノ酸配列に 1又は 2以上のアミノ酸残基に よる置換、 挿入、 及びノ又は欠失が存在しており、 実質的にヒ トを含む高等動物 テロメラーゼ蛋白質として機能することを特徴とするポリべプチド。 10. Higher animal telomerase substantially containing human, wherein substitution, insertion, and deletion or deletion by one or more amino acid residues are present in the amino acid sequence of SEQ ID NO: 13 in the sequence listing. A polypeptide characterized by functioning as a protein.
11. ヒ 卜の生体内でテロメラーゼ蛋白質として機能することができる請求の範囲第 10項に記載のポリぺプチド。  11. The polypeptide according to claim 10, which can function as a telomerase protein in a living body of a human.
12. 請求の範囲第 1項ないし 11項のいずれか 1項に記載のポリぺプチドをコ一ドす るヌク レオチ ド配列。  12. A nucleotide sequence encoding the polypeptide according to any one of claims 1 to 11.
13. D N Α配列又は R N Α配列である請求の範囲第 12項に記載のヌクレオチド配列。 13. The nucleotide sequence according to claim 12, which is a DN N sequence or an RNΑ sequence.
14. 請求の範囲第 13項に記載の D N A配列を含む組み換えベクター。 14. A recombinant vector comprising the DNA sequence according to claim 13.
15. 請求の範囲第 14項に記載の組み換えベクターが導入された形質転換体。 15. A transformant into which the recombinant vector according to claim 14 has been introduced.
16. 請求の範囲第 15項に記載の形質転換体を培養した培養物から請求の範囲第 13 項に記載の D N A配列の遺伝子産物であるポリぺプチドを分離 ·採取する工程を 含む、 請求の範囲第 1項ないし 11項のいずれか 1項に記載のポリベプチドの製造方 法。  16. A step of separating and collecting a polypeptide which is a gene product of the DNA sequence according to claim 13 from a culture obtained by culturing the transformant according to claim 15. 12. The method for producing a polypeptide according to any one of Items 1 to 11.
17. 請求の範囲第 1項ないし 11項のいずれか 1項に記載のポリぺプチドを特異的に 認識することができる抗体。  17. An antibody capable of specifically recognizing the polypeptide according to any one of claims 1 to 11.
18. 請求の範囲第 12項に記載のヌクレオチド配列の一部又は全部に相補的に結合可 能なヌクレオチドを含む核酸プローブ。  18. A nucleic acid probe comprising a nucleotide capable of complementarily binding to part or all of the nucleotide sequence according to claim 12.
19. 請求の範囲第 17項に記載の抗体又は請求の範囲第 18項に記載の核酸プローブを 含む癌細胞検出用試薬。  19. A reagent for detecting a cancer cell, comprising the antibody according to claim 17 or the nucleic acid probe according to claim 18.
20. 請求の範囲第 17項に記載の抗体又は請求の範囲第 18項に記載の核酸プローブを 含む癌診断用の医薬組成物。  20. A pharmaceutical composition for cancer diagnosis, comprising the antibody according to claim 17 or the nucleic acid probe according to claim 18.
21. 請求の範囲第 3項又は 10項に記載のポリペプチドをサブュニッ トとして含む高 等動物テロメラーゼ蛋白質。  21. A higher animal telomerase protein comprising the polypeptide according to claim 3 or 10 as a submit.
22. S D S —ポリアク リルアミ ド電気泳動法による分子量が、 不活性型では約 2 4 0 k D aであり、 活性型では約 2 3 0 k D aであることを特徴とする請求の 範囲第 3項に記載のポリぺプチド。  22. SDS—Polyacrylamide electrophoresis has a molecular weight of about 240 kDa for the inactive form and about 230 kDa for the active form. The polypeptide according to Item.
23. S D S —ポリアクリルァミ ド電気泳動法による分子量が約 2 3 0 k D aである ことを特徴とする活性型の請求の範囲第 3項に記載のポリべプチド。  23. The activated polypeptide according to claim 3, wherein the molecular weight is about 230 kDa as determined by SDS-polyacrylamide gel electrophoresis.
24. 高等動物テロメラ一ゼ蛋白質の酵素活性発現に作用する物質のスクリーニング 方法であって、 被験物質と接触させた細胞又は組織に含まれるテロメラ—ゼ蛋白 質またはそのサブュニッ 卜の分子量を測定する工程を含むスクリーニング方法。 24. A method for screening a substance that acts on the expression of an enzyme activity of a higher animal telomerase protein, which comprises the step of measuring the molecular weight of a telomerase protein or a subunit thereof contained in a cell or tissue contacted with a test substance. A screening method comprising:
25. 被験物質との接触工程を被験物質の存在下における培養工程又は動物への被験 物質の投与工程により行う請求の範囲第 24項に記載のスクリーニング方法。 25. The screening method according to claim 24, wherein the step of contacting with the test substance is performed by a culture step in the presence of the test substance or a step of administering the test substance to an animal.
26. 分子量の測定を S D S -ポリアクリルアミ ド電気泳動法で行う請求の範囲第 24 項又は 25項に記載のスクリ一ニング方法。  26. The screening method according to claim 24 or 25, wherein the molecular weight is measured by SDS-polyacrylamide electrophoresis.
27. 約 2 4 0 k D aの不活性型及び約 2 3 0 k D aの活性型のポリベプチドの存在 比を測定する工程を含む請求の範囲第 26項に記載のスク リ—ニング方法。 27. Presence of an inactive form of about 240 kDa and an active form of about 230 kDa 27. The screening method according to claim 26, comprising a step of measuring a ratio.
28. 被験物質の非存在下における 2 4 0 k D aのポリべプチドの存在比と比較して、 該ポリベプチドの存在比が被験物質の存在下において実質的に増加している場合 には、 該被験物質が高等動物テロメラーゼ蛋白質の酵素活性の発現を阻害する物 質であると判定する工程を含む請求の範囲第 26項又は 27項に記載のスク リーニング 方法  28. If the abundance of the polypeptide is substantially increased in the presence of the test substance, as compared to the abundance of the 240 kDa polypeptide in the absence of the test substance, 28. The screening method according to claim 26, comprising a step of determining that the test substance is a substance that inhibits the expression of the enzyme activity of a higher animal telomerase protein.
29. 被験物質の非存在下における 2 3 0 k D aのポリべプチドの存在比と比較して、 該ポリぺプチドの存在比が被験物質の存在下において実質的に増加している場合 には、 該被験物質が高等動物テロメラーゼ蛋白質の酵素活性の発現を活性化する 物質であると判定する工程を含む請求の範囲第 26項又は 27項に記載のスク リ一ニン グ方法。  29. If the abundance of the polypeptide is substantially increased in the presence of the test substance compared to the abundance of the 230 kDa polypeptide in the absence of the test substance 28. The screening method according to claim 26, further comprising a step of determining that the test substance is a substance that activates the expression of the enzyme activity of a higher animal telomerase protein.
30. 請求の範囲第 1項又は第 3項に記載のポリべプチドの分子量を測定する工程を 含む請求の範囲第 24項ないし 29項のいずれか 1項に記載のスクリ一二ング方法。  30. The screening method according to any one of claims 24 to 29, comprising a step of measuring the molecular weight of the polypeptide according to claim 1 or 3.
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