WO1998007838A1 - Proteine de telomerase d'animaux superieurs et gene codant pour elle - Google Patents

Proteine de telomerase d'animaux superieurs et gene codant pour elle Download PDF

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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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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

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Abstract

L'invention porte sur une protéine de type télomérase provenant d'animaux supérieurs dont l'homme. Ladite protéine et le gène codant pour elle peuvent par exemple servir à clarifier certains mécanismes de régulation biologique tels que la croissance et le vieillissement cellulaire et devraient pouvoir intervenir en particulier dans la mise au point de médicaments contre le cancer. L'invention porte également sur un procédé de criblage des substances agissant sur l'expression de l'activité enzymatique de la télomérase d'animaux supérieurs consistant à mesurer le poids moléculaire de la télomérase présente dans des cellules ou des tissus en contact avec une substance test en utilisant par exemple le procédé d'électrophorèse SDS sur polyacrylamide.
PCT/JP1997/002904 1996-08-21 1997-08-21 Proteine de telomerase d'animaux superieurs et gene codant pour elle WO1998007838A1 (fr)

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US7732402B2 (en) 1994-07-07 2010-06-08 Geron Corporation Mammalian telomerase
US6808880B2 (en) 1996-10-01 2004-10-26 Geron Corporation Method for detecting polynucleotides encoding telomerase
US6617110B1 (en) 1996-10-01 2003-09-09 Geron Corporation Cells immortalized with telomerase reverse transcriptase for use in drug screening
US6261836B1 (en) 1996-10-01 2001-07-17 Geron Corporation Telomerase
US7285639B2 (en) 1996-10-01 2007-10-23 Geron Corporation Antibody to telomerase reverse transcriptase
US6475789B1 (en) 1996-10-01 2002-11-05 University Technology Corporation Human telomerase catalytic subunit: diagnostic and therapeutic methods
US7056513B2 (en) 1996-10-01 2006-06-06 Geron Corporation Telomerase
US7005262B2 (en) 1996-10-01 2006-02-28 Geron Corporation Methods for detecting nucleic acids encoding human telomerase reverse transcriptase
US6927285B2 (en) 1996-10-01 2005-08-09 Geron Corporation Genes for human telomerase reverse transcriptase and telomerase variants
US7560437B2 (en) 1996-10-01 2009-07-14 Geron Corporation Nucleic acid compositions for eliciting an immune response against telomerase reverse transcriptase
US7585622B1 (en) 1996-10-01 2009-09-08 Geron Corporation Increasing the proliferative capacity of cells using telomerase reverse transcriptase
US5981707A (en) * 1996-11-15 1999-11-09 Amgen Inc. Genes encoding telomerase protein 1
US5919656A (en) * 1996-11-15 1999-07-06 Amgen Canada Inc. Genes encoding telomerase protein 1
US6174703B1 (en) 1996-11-15 2001-01-16 Amgen Inc. Genes encoding telomerase protein 1
US7390891B1 (en) 1996-11-15 2008-06-24 Amgen Inc. Polynucleotides encoding a telomerase component TP2
US8236774B2 (en) 1997-04-18 2012-08-07 Geron Corporation Human telomerase catalytic subunit
US7622549B2 (en) 1997-04-18 2009-11-24 Geron Corporation Human telomerase reverse transcriptase polypeptides
US7262288B1 (en) 1997-04-18 2007-08-28 Geron Corporation Nucleic acids encoding human telomerase reverse transcriptase and related homologs
US8709995B2 (en) 1997-04-18 2014-04-29 Geron Corporation Method for eliciting an immune response to human telomerase reverse transcriptase
US7413864B2 (en) 1997-04-18 2008-08-19 Geron Corporation Treating cancer using a telomerase vaccine
US7750121B2 (en) 1997-04-18 2010-07-06 Geron Corporation Antibody to telomerase reverse transcriptive
US6610839B1 (en) 1997-08-14 2003-08-26 Geron Corporation Promoter for telomerase reverse transcriptase
US7199234B2 (en) 1997-08-14 2007-04-03 Geron Corporation Regulatory segments of the human gene for telomerase reverse transcriptase
US7378244B2 (en) 1997-10-01 2008-05-27 Geron Corporation Telomerase promoters sequences for screening telomerase modulators
US6777203B1 (en) 1997-11-19 2004-08-17 Geron Corporation Telomerase promoter driving expression of therapeutic gene sequences
US7402307B2 (en) 1998-03-31 2008-07-22 Geron Corporation Method for identifying and killing cancer cells
US7091021B2 (en) 1998-03-31 2006-08-15 Geron Corporation Inactive variants of the human telomerase catalytic subunit
US7824849B2 (en) 1998-03-31 2010-11-02 Geron Corporation Cellular telomerase vaccine and its use for treating cancer
US6440735B1 (en) 1998-03-31 2002-08-27 Geron Corporation Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer
US8796438B2 (en) 1998-03-31 2014-08-05 Geron Corporation Nucleic acids encoding inactive variants of human telomerase
WO2003024997A1 (fr) * 2001-09-14 2003-03-27 Jun-Ping Liu Peptides inhibiteurs de la telomerase et leurs utilisations

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