JPH10513343A - Telomerase protein components - Google Patents

Abstract

  (57) [Summary] Disclosed are genes encoding the amino acid sequence of the protein component of Tetrahymena telomerase and methods for their preparation and use. The protein component consists of two subunit polypeptides, the 80 kD and 95 kD polypeptides. Methods for detecting the protein component of human telomerase and uses thereof are also disclosed. The protein component of telomerase can be used to develop diagnostic tools for the detection of telomerase activity in cancer, microbial diseases and other disorders and produce therapeutic compounds that inhibit or enhance telomerase activity.

Description

DETAILED DESCRIPTION OF THE INVENTION                          Telomerase protein components Background of the Invention   Chromosome stability is essential for cell viability. Eukaryotic cells stain linearly It has a telomere that covers the body and ends that protect the chromosome from degradation and recombination. Loss of telomeric DNA during cell growth plays a key role in aging and cancer. I'm sorry. Counter (C.M.) et al., EMBO, 11: 1921-1929 (1992).   Telomere sequences are highly conserved in eukaryotes. The DNA sequence is It contains a single tandem repeat of a specific GT-rich motif. The exact sequence depends on the individual organism D) (TTGG in Tetrahymena). GG), d (TTTTGGGG) in Oxytricha and In humans, it is d (TTAGGG). The number of repeats at any given chromosome end Also appear to be heterogeneous or apparently "ambiguous" in Southern blots. Grant to telomeres. In addition to sequence preservation, telomere function is also eukaryotic Has been saved. Tetrahymena and human telomeres are derived from the yeast Saccharomyces It functions in Saccharomyces cerevisiae and yeast telomeres are used in other fungi. Function. Szostak, J.W. and E.H. Blackburn ) (1982) Cell, 29: 245-255. Therefore, the mechanism for maintaining stable ends is diverse eukaryotic They must share essential features in living things.   The telomere sequence is a highly specialized DNA polymerase called telomerase. Synthesized at the chromosome end by lyse. Telomerase is an RNA and protein component Both are ribonucleoprotein enzymes that are essential for telomerase activity. RNA synthesis The minute provides a template for telomere repeat synthesis. Blackburn (Blackburn E.H.) , (1992) Annu. Rev. Biochem., 61: 113-129.   Telomere replication establishes an equilibrium between telomere shortening and telomere elongation Including. DNA-template dependent DNA polymerase is the true end of the DNA molecule Since DNA cannot be replicated, DNA replication leads to shortening of telomeres. Telomera It extends chromosomes through de novo sequence addition. Then the DNA-template Double-stranded synthesis by dependent DNA polymerase and primers leads to complementary C Fill the rich chain.   The RNA component of telomerase is human (Feng, J., et al. (1995) Science 269: 12). 36-1241), mice and some mammalian species (Greider, C., unpublished data). Saccharomyces cerevisiae, Tetrahymena, Euplotes and And oxytrisha. Singer and Gotshling and Gottschling), (1994) Science, 266: 404-409; Lingner et al., (199 4) Genes & Development, 8: 1984-1988; Romero and Blackburn (Romero, D.P. and E. H. Blackburn), (1994) Cell, 67: 343-353. Terrorism of any species The protein component of merase has not yet been sequenced or cloned.Summary of the Invention   A gene encoding a telomerase protein component of eukaryotic origin including mammals, Telomerase protein encoded by the gene, encoding the polypeptide RNAs and sequences that hybridize to these genes are described herein. I have. As described herein, a genomic gene encoding a telomerase protein component The sequence was determined by the applicant. RNA and protein synthesis of telomerase Minutes are essential for maintaining chromosome telomere length. The protein component of telomerase is In diagnostic or therapeutic methods and in the assay of telomerase, It can be used in combination with the NA component.   As described herein, the telomerase protein component of Tetrahymena is The isolated Tetrahymena gene was isolated and sequenced. By these genes, The polypeptides loaded are the 80 kD and 95 kD polypeptides (respectively , P80 and p95). Polypeptides combine with the RNA component Chromosome telomeres that bind and act to add telomere TTGGGGG repeats Consists of a protein that stabilizes length. The present invention also relates to the disclosed polypeptides. DNA sequences and parts thereof, sequences complementary to these DNA sequences and High with either a sense or complementary (antisense) sequence or fragment thereof A sequence is provided that hybridizes, eg, a probe.   In particular, the 80 kD and 95 kD telomerase protein components of Tetrahymena Were isolated and sequenced. 80kD and 95k of protein component The amino acid sequence of the polypeptide of D is disclosed herein, and is 80 kD and 95 kD. The same applies to the DNA (nucleic acid) sequence encoding the kD protein.   In addition, p80 and p95 telomeres translated by most eukaryotic cells Disclosed are nucleotide sequences encoding ze polypeptides. These DNA sequences The rows are incorporated into a plasmid, which is transformed into a vector. This Host cells containing these vectors produce the recombinant telomerase protein component. Provided to build.   Also, DNA and polypeptide sequences substantially equivalent to the disclosed sequences Both sequences are also provided by the present invention.   Using DNA sequences encoding the protein components of Tetrahymena, other non-vertebrate animals And protein components of vertebrate species, especially humans, mice, rats, dogs, cats, Encoding a telomerase protein component of a mammalian species such as A method for determining a DNA sequence is also included.   This study also showed that abnormal telomerase activity in mammals, especially human Or to determine if it is likely to have a causative disorder or disease Create a method. Methods for detecting telomerase expression can be used to immortalize humans or other animals. It is provided as a means for diagnosing the tendency of cells or cancer cells to grow. In one aspect DNA or RNA present in a cell or tissue sample DNA and RNA probes complementary to all or part of the white matter component gene Bridging. As used herein, "telomerase protein component gene" The term refers to a gene whose sequence is described herein, said gene or a portion thereof. And humans, mice, rats, dogs, cats, pigs, Includes equivalent genes from other species such as chimpanzees, monkeys or Tetrahymena. The detection of hybridization is the growth of cancer or other disorders that result in immortalized cells. , Or an indicator of existence.   An important feature of the present invention is that telomerase is produced in cells using the protein component of telomerase. Telomerase inhibitors that can be used to inhibit the expression and / or activity of lyase Can be screened. Within the individual's own cells and tissues, Abnormal in either the foreign cells of the invading parasite or in a diseased organism that is eukaryotic As a basis for identifying and treating individuals affected by telomerase activity, Protein components can be used.   Thus, the present invention can test and improve telomerase activity inhibitors. Diagnosis that can diagnose diseases such as cancer or infectious diseases such as yeast or protozoan disease Provide tools.   Other aspects of the invention include, for example, one of the 80 kD or 95 kD polypeptides. One or both, synthetic telomerase polypeptide sequences or their polypeptides Antibody against a telomerase protein component such as an antibody against a part of the drug. these Is, for example, any of the 80 kD and 95 kD polypeptides of the invention or Polyclonal and monoclonal antibodies that bind to both And monoclonal antibodies. Such anti-telomerase antibodies can be used in cells and And is useful for detecting telomerase activity in tissues and tissues.   A further embodiment is that either or both of the genes for the telomerase subunit One alone or another gene such as a gene encoding the RNA component of telomerase Comprising recombinant and / or transformed cells containing in combination with And treatment methods. Recombinant or transformed cells producing anti-telomerase antibodies Vesicles are included as well. Such a method may be useful for treating disorders resulting from abnormal telomerase activity. Applicable for therapy and also increases telomerase expression to prevent cell death Or can be used to trigger.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the nucleotide sequence of the Tetrahymena 80 kD protein gene (SEQ ID NO: 1). Nucleotide sequences are derived from genomic and cDNA clones .   FIG. 2 shows the amino acid sequence of the 80 kD protein deduced from the nucleotide sequence shown in FIG. This is an acid sequence (SEQ ID NO: 2).   FIG. 3 shows the nucleotide sequence of the Tetrahymena 95 kD protein gene (SEQ ID NO: 3).   FIG. 4 shows the amino acid sequence of the 95 kD protein deduced from the nucleotide sequence shown in FIG. This is an acid sequence (SEQ ID NO: 4).   FIG. 5 shows the results from 12 deoxyribonucleotide sequences (primers 1 to 12). Primer set 1   FIG. 6 shows the sequence of 12 deoxyribonucleotide sequences (primers 13 to 24). Primer Set 2 consisting of   FIG. 7 shows the sequence of 10 deoxyribonucleotide sequences (primers 25 to 34). Primer Set 3 consisting of   8A-8B show 18 deoxyribonucleotide sequences (primers F1- F9 and R1 to R9).   FIG. 9 shows ten deoxyribonucleotide sequences (primers F10 to F14). And R10 to R14).   FIG. 10 shows the DNA sequence of the genetically engineered p80 gene (SEQ ID NO: 8).   FIG. 11 shows the DNA sequence of the genetically engineered p95 gene (SEQ ID NO: 9).Detailed description of the invention   The present invention relates to genes encoding eukaryotic telomerase protein components, such genes A polypeptide encoded by the offspring as well as an RNA encoding the polypeptide, To the complementary nucleotide sequence and the sense and complementary portions of the nucleotide sequence For a hybridizing probe.   In addition, synthetic genes encoding 80 kD and 95 kD telomerase protein components Genes, recombinant polypeptides encoded by these genes, the polypeptides RNA encoding the primers, primers used to synthesize these genes and And complementary nucleotide sequences or fragments thereof are provided.   One skilled in the art will recognize that many different DNA sequences may encode a single protein. You will recognize what you can do. In addition to the gene and other nucleotide sequences DNA sequences encoding catalytically active telomerase protein components and their DNA sequences Nucleotide sequences that hybridize to NA sequences are also considered to be within the scope of the present invention. It is. Generally, they will hybridize under moderately stringent conditions. I do. According to the present invention, the term “stringent conditions” refers to 62 ° to 6 ° Hybridizer consisting of a salt concentration of 4 × SSC (NaCl-citrate buffer) at 6 ° C. The conditions of the immobilization are referred to as “highly stringent conditions” at 68 ° C. Hybridization conditions consisting of 1 × SSC salt concentration. Aus Ausubel et al., (1994) Current Protocols in Molecular Biology, John Wile. y & Sons, Inc.   Methods for inferring other telomerase components using these sequences have been described. You. The protein component of telomerase, the nucleotide sequence encoding the protein component, etc. Diagnostic and therapeutic methods using or a part thereof are also included.   The following examples, which describe aspects of the invention as well as experimental procedures, The principle of the present invention will be described. All references to materials and methods are by reference Incorporated herein.   The present invention also relates to a protein of eukaryotic origin that includes the polypeptides described herein. Also encompassed are polypeptides composed of the romerase protein component. Telomerase protein synthesis All polypeptides that are active as telomerase components And the term telomerase protein component as used herein.   Telomerase protein components are produced in "substantially pure" form by the following method. It is. "Substantially pure" refers to a telomerase that binds to a telomerase RNA component. It is defined as the minimum amino acid sequence that exhibits the activity of the enzyme.Tetrahymena protein component gene   Using readily available chromatography matrices, tetrahymena The telomerase enzyme was purified. The following enzyme purification was performed using the two criteria. First, standard telomerase measurement method (Greider, CW (1987) Cell, 51: 887-898) The activity was measured by using, and the incorporation of 32 P-dGTP was determined by Spots were quantified by determining the incorporated counts (Greider, C . W. (1987) Ph. D. Thesis, Univ. Calif. Berekley). Second, telomerase RNA was subjected to Northern blot analysis and a known amount of synthetic telomerase RNA standard Was determined by comparison with a titer. Hydroxyapatite, spermine aga Loose, Sepharose CL-6B sizing column, Phenyl-Sepharose, DEA E-agarose (or Q-Sepharose) and 15 to 20-35% glyce Purification of the roll gradient provided a highly purified telomerase fraction. Active fraction In which, two predominant proteins of 80 and 95 kD were identified and telomerase activity And was stoichiometrically equivalent to telomerase RNA.   Two samples of the material purified as described above were separated on a native gel. gel One of the lanes was subjected to Northern blotting to identify the position of telomerase RNA. And the other lanes were excised from the native gel and two-dimensionally run on an SDS PAGE gel. Electrophoresed. Most proteins stayed near the wells of the first dimension native gel, Telomerase RNA and both p80 and p95 proteins are native gels The electrophoresis was performed about one-third of the time, and was at the same position. This means that p80 and p9 5 suggests that it is a component of telomerase. 300L or 1.2 × 10¹¹ Starting from the cells, the active fraction in the final glycerol gradient was 1 microgram Telomerase RNA. This allows co-purified polypeptides It was shown that there was enough material to sequence the code.   The p80 and p95 fractions consist of telomerase activity or have similar properties Telomerase is used to determine whether it is a migrating contaminant. Treated. Previous experiments showed that limited cleavage of telomerase RNA was completely It has been shown not to inactivate the merase activity. Greider, C. W. and E. H.  Blackburn (1989) Nature, 337: 331-337. Purified for glycerol gradient analysis Two fractions of telomerase were prepared and the cleavage of RNA was performed by RNP on a glycerol gradient. Was determined whether to change the mobility. Micrococcus nucleus And the other was incubated with buffer only. Group these samples A lyserol gradient was settled and fractions were collected from each gradient. Measure protein activity Phil decided. In the untreated fraction, fraction 8 with p80 and p95 and At 9, the activity peaked. In the Micrococcus nuclease-treated fraction, the fraction 10 shows a weak activity peak, and peaks of p80 and p95 are also shifted to fraction 10. That these proteins are expected to behave as telomerase components. Suggests that Most other protein precipitates in the gradient are telomerase Remained unchanged compared to changes in sediment.   The partial peptide sequence was determined from both p80 and p95. Two polypeps The complete amino acid sequence of the tide can be determined in a similar manner. 344L Telomerase from Tetrahymena cells by native gel electrophoresis and SDSP Add a DEAE agarose concentration step prior to AGE electrophoresis and follow the above procedure Purified. Cut out to avoid problems with direct N-terminal sequencing of proteins Digested protein band with lysyl endopeptidase from Achromobacter did. Peptide fragments were extracted from the gel and separated on a C18 reverse phase HPLC column. Several well-defined peptide peaks were analyzed on an Applied Biosystems automated system. -Sequencer, subjected to continuous Edman degradation. Two separate preparations of telomerase From the product to 7 peptides from p80 and 25 peptides from p95. The amino acid sequence was determined. Using tetrahymenacodon bias as a guide , A degenerate oligonucleotide was designed. Martindale, D.W. 1989). Protozol. 36: 29-34.   Using oligonucleotides in two sets, reverse transcribed RNA or A PCR product derived from DNA was obtained. Two PCR products for each protein gene was gotten. Three of the four PCR products have the same sequence depending on the protein sequence. Encoded a peptide that was defined but not used as a PCR primer ( Figures 2 and 4). PCR products for either p80 or p95 proteins The gene was found to be tetrahymenageno by genomic Southern blot used for lobes. It was suggested that it could exist as a single copy in the system. Actively growing Approximately 3.0 kb for p95 by Northern blot analysis from And a 2.5 kb single band for p80 mRNA Indicated. RNA derived from quiescent cells uses the 5 'part of the 95 kD gene as a probe. When used, two bands were shown. This allows the selective selection of this gene. Processing is suggested.   To obtain the full-length protein sequence, the cloned PCR product was For both Tetrahymena cDNA and genomic libraries Used as lobe. Positive clones were obtained, subcloned and sequenced. I did it. Since this sequence is different from that of other eukaryotes, these protein sequences For the estimation, the Tetrahymena genetic code was used. Prescott, D . M.) (1994) Microbiol. Rev., 58: 233-267. Applicants have identified p80 and p9 The entire open reading frame (ORF) for both of the five proteins was sequenced (respectively, FIG. 1, SEQ ID NO: 1 and FIG. 3, SEQ ID NO: 3). The nucleotide sequence is Polyadenylation of mRNA from cDNA and cDNA clones. Occurs near the 3 'end of the sequence. First, the p80 and p95 mRNA Southern blot analysis suggests sizes of about 2.47 and 2.9 kb. Applicants have determined that more than 2.4 and 2.8 kb of sequence for these mRNAs. Obtained. Second, all reliable peptide sequences were found in the ORF (p80 7/7; 25/25 for p95). Third, the first in the longest ORF Suggested translation of mRNA from methionine codon results in SDS-PAGE Putative protein product equivalent to or slightly larger than the molecular weight estimated from protein analysis by can get. Fourth, the sequence outside the region translated as a coding region is Tetrahymena It contains a higher A / T content than the coding region, characteristic of the gene. Presco (Prescott, DM) (1994) Microbiol. Rev., 58: 233-267. Any heredity Children are also Genbank, EKMBL, PIR and Swissprot databases. The source has no counterpart.   To show that the 80 kD and 95 kD proteins are components of telomerase A polyclonal antibody against the two proteins was prepared. Two from each protein Synthetic peptides corresponding to different regions were synthesized. For 80 kD protein peptide Two polyclonal antibodies (designated A81 and A82) and a 95 kD protein Four antibodies against white matter peptides (A83, A84, A85 and A86 and Name) showed good titers for each protein.   Table 1 describes the antibodies obtained against the various peptide sequences used. Pe The peptide sequence listing was obtained directly from the protein sequence of the PCR product. First pep The tide is from a preliminary test of the sequence and the gene has been cloned. Was determined to be incorrect. Later, this peptide and its antibody Was used as a control. Rabbit injected to make A85 and A86 The peptide has one error (second position) compared to the cloned sequence. However, an antibody against this peptide crossed with the 95 kD protein. react. To attach the peptide to the carrier protein, the N-terminal C Residues were added to each peptide.   Among these antibodies, those with the highest affinity for the 80 kD protein ( A82) and those with the highest affinity for the 95 kD protein (A86) And that the 80 kD and 95 kD polypeptides both have telomerase activity Have been demonstrated to be co-purified and these proteins are telomerase components It has been suggested. The results of immunoprecipitation studies on the 80 kD protein were consistent. This suggests that the 80 kD protein is a functional component of telomerase.Synthetic protein component gene   Creating genes encoding telomerase protein components in other eukaryotes In order to change the Tetrahymena genetic code, most eukaryotes, Organisms with the genetic code used / translated in mammals, such as And a synthetic gene sequence that enabled transcription was constructed. Oligonucleotide overlap (Primer set), the gene is synthesized as a gene fragment, Cloned into a plasmid. By joining the fragments in a plasmid, A full-length gene was constructed. The p80 gene was transferred to plasmid Bluescri pt and the p95 gene was constructed in plasmid pSE280. Can also be used.   To express the p80 and p95 proteins, the synthetic gene was transformed into a pRSET vector. And pBlueBac vectors were cloned into different restriction sites. P By transcription and translation of the genes in RSET and pBlueBac, respectively Recombinant proteins were made in E. coli and baculovirus. Each recombinant protein The His-tag and cleavage site at the end of white matter facilitates protein purification.   Vectors such as E. coli and pRSET containing p80 and p95 gene constructs Or alternatively, a pBlu containing baculovirus and the same construct Using vectors such as eBac, recombinant p80 and p95 can be expressed. Accordingly, Applicants have been able to express p80 and p95 telomerase protein components. The first known bacterial strain or expression vector was produced. One aspect of the present invention Is the production of one or more recombinant telomerase protein components in a host cell. One method involves providing a host cell containing a gene encoding a protein or analog thereof. Culturing under conditions that allow the production of the protein. In one aspect The method further comprising a step of recovering a certain amount of the protein and a purification step It is. Those skilled in the art can prepare various recombinant proteins of the present invention. Will recognize how.   Recombinant proteins and their fragments stimulate or inhibit telomerase catalytic activity It is useful for detecting drugs that do. They are responsible for telomerase activity in tumor cells Or stimulation of telomerase in response to exposure to a compound or It is also useful for producing antibodies for screening methods such as detection of inhibition production .   Also according to the invention, other telomerase proteins and RNAs of the polypeptide Genomics without affecting the binding capacity of the components or the enzyme activity of telomerase Amino acid sequence derived from telomerase polypeptide encoded by Different telomerase polypeptides (ie, Tetrahymena telomerase polypeptides) (Unlike natural telomerase polypeptides such as peptides). these Mutations can result in additions, deletions, substitutions and other changes to the amino acid sequence (eg, (A modification of the acid residue).   A gene encoding a telomerase protein component of Tetrahymena, a synthetic gene or Is a human telomere using a primer and a known method described herein. Cloning zeoprotein components and other mammalian telomerase protein components Can be.   Using Tetrahymena synthetic or primer sequences using two approaches Thus, the human telomerase protein gene can be cloned. these The technique is described in detail in the following examples.   In the first approach, using DNA sequence hybridization, Can identify and clone human homologues of Trachymena protein . A series of stringency-enhancing conditions using the Tetrahymena gene as a probe Human genomic DNA and mRNA blots are performed. Specific band identified CDNA or genome cloned into a phage lambda vector Libraries are searched for with similar stringency, and Identify the offspring. Create a restriction map of the positive phage, subclone it, To sequence.   The second approach is to address various regions of both p80 and p95 proteins. To produce a series of antibodies. Using the antibodies (A82 and A86) Can be. Additional antibodies were also generated as synthetic peptides and fusion proteins, and It can be used to identify human telomerase proteins by responsiveness. Protein Antibodies can be obtained from human or other mammalian cDNA libraries that express a portion of the quality. Used as a probe, human or mammalian The gene can be cloned. Sambrook et al., (1989) Mole cular Cloning -A Laboratory Manual, Cold Spring Harbor Press, Cold Sprin g See Harbor Laboratory, NY.   Human telomerase is an excellent target for anti-cancer therapy. Tetrahime The availability of the protein component of the enzyme facilitates a thorough understanding of telomerase biochemistry. To help identify specific anti-telomerase drugs.   Telomerase activity has been found in over 70 human immortalized cell lines and cancer tissues. But are rarely found in primary human somatic cells or tissues. Kim (Kim) et al. (1994) Science 266: 2011-2015. Maintaining telomere length is limited in lifespan It does not occur in primary human somatic cells. Primary culture in vitro or in vivo When the feeder divides, the telomere length becomes shorter. Germ cells show this shortening Absent. Allsopp et al., (1992) PNAS 89: 10114-10118; Harley (1990) Nature, 337: 331-337; Vaziri et al., (1993) Amer. J. Hu m. Genet. 52: 661-667. Telomerase activity is present in immortalized human HeLa cells. Although present (Morin, GB (1989) Cell 59: 521-529), telomerase is Not detected in primary fibroblast cultures. Applicants have determined from primary human cultured cells An SV40 immortalized cell line was established to establish a telomerase and telomere shortening. Investigated the relationship. In both primary cultures and human fetal kidney cells transformed with SV40 When cells were passaged, telomeres were shortened but telomerase was not detected. Was not. When a culture is in crisis, most cell lines die, Telomerase activity is detected in surviving immortalized clones and telomeres are short Was maintained stable. Counter et al., (1992) EMBO J .; 11: 1921-1929. Similar results were obtained with primary mouse cells in culture. Plows and glider -(Prowse, KR and CW Greider) (1995) PNAS 92: 4818.   Based on these results, primary cultured cells exhibited little or no telomerase activity. Does not appear, but immortalized cancer cells reactivate telomerase and maintain telomere length It is suggested that In fact, telomerase activity has been demonstrated in human ovarian cancer cells Has not been demonstrated in normal cervical endothelial cells. Counter (1994) PNAS 91: 2900-2904. Is shortening of pre-crisis telomeres fatal? These cells, which may be able to reactivate telomerase, may maintain telomere length. And live despite the crisis. This model makes telomerase immortal If necessary for the growth of metaplastic cells, telomerase inhibitors will be excellent anticancer agents.   This study shows that telomeres in somatic cells that do not normally express telomerase Diagnosis of cancer before or during clinical manifestation by detecting enzyme activity Provide a way to do it. Expression of telomerase mRNA in somatic cell or tissue samples At present it is detectable using DNA or RNA probes, which It is an indicator of the expression of merase, somatic cells do not normally produce telomerase, Next is an index of immortalized cancer cells. Detection of hybridisation s It is an indication of a tendency to die or cancer, or the presence of cancer or immortalized cells.   By hybridization, DNA and / or RNA molecules or their The fragments were used for hybridisation analysis, Ausubel et al., (1994) Current Protocols in Molecular Biology, (Suppl. 26), John Wiley & Sons, Inc. Under moderate stringency conditions, according to the method described in It is shown to detect polynucleotides.   Presence or immortalization of immortalized cells in a eukaryotic tissue sample or eukaryotic cell sample In one embodiment for detecting the tendency of the nucleic acid, the nucleic acid as a probe or primer Used. This embodiment comprises the following steps: a) obtaining a tissue or cell sample from a eukaryote; b) determining the presence of telomerase in the sample, wherein the sample determines the presence of telomerase; When indicated, there is an immortalized cell or a tendency to immortalization. Using the same method, A tendency for cancer or the presence of cancer cells or tissue may be detected.   In addition, expression of mammalian telomerase is dependent on p80 or p95 polypeptide Polyclonal or mono-subunit, both subunits or fragments thereof It can be detected using a clonal antibody. One antibody combines both subunits Using two antibodies, each detecting a different subunit. You can also. For example, a sample of somatic cells or tumor cells from an individual (if present If necessary, process or treat the telomerase to make it available for binding to the antibody. After that, it can be contacted with an anti-telomerase antibody. Antibody binding, telomera Is indicative of the presence of the enzyme, thus immortalizing the cell or prone to cancer or cancer cells Or it is an indicator of the presence of immortalized cells.   Use antibodies to detect telomerase in eukaryotic tissue samples or eukaryotic cell samples The method may comprise the following steps: a) obtaining a tissue or cell sample from a eukaryote; b) processing the sample to allow telomerase to bind to the anti-telomerase antibody; Producing a processed sample by doing so; c) Treating the treated sample against telomerase (polyclonal or monoclonal) Contacting with an anti-telomerase antibody; and d) detecting the binding of the telomerase antibody, if the binding occurs, the telomerase There is. Antibody detection can be, for example, the growth of cancer cells and cancer or immortalized cells. Not only to detect immortalization of cells caused by the presence of vesicles, but also as follows: It is also useful for detecting the presence of foreign eukaryotic cells in cells and tissues of multicellular organisms. is there.   The present invention relates to agents that disrupt or inactivate or interfere with telomerase activity and Methods for developing pharmaceutical compounds are also provided. Inhibits telomerase activity in Tetrahymena Or inactivating compounds may be responsible for their effects on mammalian telomerase Can also be evaluated. Telomerase protein with or without RNA component Drugs and pharmaceutical compounds effective as the following anticancer agents and antibacterial agents using white matter components Can be screened.   Furthermore, another telomerase activity has an anti-aging effect, and stabilizes telomere length. Stimulates or induces telomerase activity as it may result in more cellular recovery Compounds can be screened for the potential to do so. Protein components Linked to the RNA component of telomerase and delivered to cells in the usual manner A functional telomerase molecule can be produced. Alternatively, DNA encoding telomerase molecules can be obtained by recombinant DNA techniques and transformation techniques. It can be introduced into target cells by surgery. Extra of a functional telomerase molecule Replication uptake of host cells by stabilizing telomere length by copy incorporation May be extended. Accordingly, the present invention provides a method for gene therapy in mammals. Including.   Another application of the invention is directed to organisms that have developed eukaryotic disease in mammalian somatic cells and tissues. Detection and treatment of the resulting disease. Invades mammalian cells and tissues, There are also many fungi, protozoa and even algae that contribute to the disease of the stomach. Such diseases Examples of the disease include aspergillosis, histoplasmosis, candidiasis, paracoccosis Sidioid mycosis, malaria, trichinosis, filariasis, trypanosomiasis (Sleep sickness), schistosomiasis, toxoplasmosis and leishmaniasis But not limited to these. These organisms require telomerase and are normal Expresses this enzyme when grown in host cells that do not produce telomerase . Early detection of these eukaryotic microbial parasites using the method for detecting telomerase Detection and diagnostic methods can be developed.   Is disease caused by eukaryotic microorganisms in tissue samples or eukaryotic cell samples from individuals? Examples of such detection methods may include the following steps: a) obtaining a tissue or cell sample from the individual; b) determining the presence of telomerase in the sample, wherein the sample is a telomere of a eukaryotic microorganism. If it does, then there is a disease caused by a eukaryotic microorganism. If a nucleic acid probe Primers or antibodies that bind to the telomerase protein component. Telomerase in a sample can be determined by the methods described herein. It is not limited to.   In addition, mammalian somatic cells do not require telomerase, so telomerase inhibition The use of antibiotics for drugs and telomerase non-toxic to the host? There is provided a method for treating such diseases which shows little toxicity. For example, trypanoso Most drugs used to treat diseases caused by Use and wake up to death. 80 kD or 95 telomerase of Trypanosoma sp. Antisense RNA against kD protein component or drug against telomerase Inhibits telomerase and thus affects host somatic cells and tissues Without preventing the growth of this parasite species in the individual. Telomere An antisense nucleic acid that inhibits the translation of mRNA encoding the protein component of Included in these medicines.   Compounds that inhibit or destroy telomerase activity can be made using standard materials and methods. Formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier and / or other excipients can do. They are diseases that result from abnormal levels of telomerase activity. Alternatively, it can be administered to a human or non-human animal to treat the condition. Administration can be carried out by any of the usual routes (non- Oral, oral, inhalation, etc.). Compound reacts non-toxicly with active derivatives Pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral administration And excipients.   Specializations utilizing substantially preferred amounts of active compounds in certain cases The compound, the specific composition prescribed, the method of application, the specific location of the application and the treatment It is evaluated to change according to the individual. The dose for a given recipient is Based on individual characteristics such as length, weight, age and type and severity of condition being treated Is determined.   The formulations described herein may be used for veterinary disease treatment as well as for human applications Note that the terms “individual” or “host” should not be construed as limiting. Should be aware. These terms refer to human and non-human vertebrates, particularly mammals. Including.   In a further aspect, the invention provides a recombinant product comprising the steps of: Provide a way: (A) Step of producing an expression vector containing a DNA encoding a telomerase molecule ; (B) transforming or infecting a host cell with the vector; and (C) telomerase molecules encoded by culturing transformed or infected cell lines (Recombinant telomerase). Using standard techniques for molecular biology, Encodes the RNA and protein components of Romerase and regulates expression of the enzyme in host cells. DNA sequence for construction of a vector having an appropriate promoter for Can be Suitable host cell / vector systems, transformation or infection methods and Culture methods are known in the art. Using these systems, telomerase Can also be produced.   Using the method described above to study the role of telomerase in eukaryotes Transgenic cells, tissues and organisms for medical and therapeutic purposes. Is also evaluated. Accordingly, the present invention relates to telomeres derived from eukaryotes, including mammals. The present invention provides a transgenic biological material comprising a protein component of a protease.   Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited thereto. Absent.                                   Example 1 Purification of telomerase   Tetrahymena thermophila la) with PPYS (2% proteose peptone, 0.2% yeast extract) G, 10 μM FeCl_Three)) With a density of 4.0 × 10^Five/ Ml The cells were collected by centrifugation using a GSA rotor (manufactured by Sorvall) and dried (1.7 m). M NaC₆H₈O₇, 1.2 mM NaH_TwoPO_Four, 1.3 mM Na_TwoHPO_Four, 2 mM CaCl_Two) Was starved for 18 hours. Harvest the starved cells again And T2MG buffer (20 mM Tris-HCl pH 8.0, 1 mM Mg Cl_Two, 10% glycerin, 2 mM DTT or β-mercaptoethanol , 0.1 mM PMSF, 2 μg / ml leupeptin, 1 μg / ml pep (Statin) and NP-40 at a final concentration of 0.2% was added and dissolved. The lysed cells were centrifuged at 130,000 × g for 50 minutes at 4 ° C. to give S-100 An extract was obtained. The following steps were all performed at 4 ° C.   1-2 × 10¹¹S-100 obtained from a single cell (approximately 300 L of PPYS culture solution) The extract was filtered coarsely and applied to a ceramic HAP (AIC) equilibrated with T2MG. I plied. Telomerase at 0.2 M K in T2MG_TwoHPO_FourEluted with a gradient . Fractions with the highest activity were pooled and diluted with 3 volumes of T2MG to give 0.15 M Potassium taminate (KC_FiveH₈NO_Four, KG)) containing T2MG Spermine agarose (Sigma). Telomerase 0.65M Eluted with T2MG containing KG. Fractions with the highest activity were pooled and 20 mM KG and 3 mM NaN_Three1L Sepharose equilibrated with T2MG containing The solution was loaded on a CL-6B (Pharmacia) column, and the column was flushed with the same solution. Activity peaks Pool the minutes and adjust to 0.4M KG, T2 containing 0.4M KG It was applied to phenyl sepharose (Pharmacia) equilibrated with MG. Kara Was washed with T2MG, and then telomerase was added to 1% Triton X-1. Eluted with T2MG containing 00. Fractions with the highest activity were pooled and flattened with T2MG. It was applied to equilibrated DEAE agarose (manufactured by BioRad). Telomerase to T2 Gradient elution to 0.4M KG in MG or step elution to 0.4M KG Was. The fraction with the highest activity is sometimes diluted with distilled water to 15-35% or 20%. Loaded on a ~ 35% glycerin gradient. Gradient with SW41 rotor (Beckman) Centrifuged for 20 hours. Glycerin gradient purified telomerase described in the present invention Used for some experiments.   Telomerase is exposed before proteolytic digestion for peptide sequencing. Was purified. Glycerin gradient fractions with peak activity are pooled and equilibrated with T2MG And applied to DEAE agarose. Telomerase containing 0.4M KG Eluted with T2MG. The peak fraction was dialyzed against T2MG and then 6% Lilamide was applied to a 50 mM Tris-acetate gel, and 50 mM Tris was added. -Electrophoresis in acetate buffer, pH 8.0. Run the native gel for about 12 hours at about 250V Electrophoresed. Cut out the lane of the native gel containing telomerase and perform 2 × SDS test. Buffer (0.125 M Tris-HCl pH 6.8, 4% SDS, 10 % Β-mercaptoethanol, 20% glycerin, bromophenol blue ) And denatured 7% with 0.1% agarose in mM Tris-acetic acid Inserted into the wells of an acrylamide gel. SDS-PAGE to Tris-grid Syn-SDS buffer (25 mM Tris-HCl pH 8.3, 192 mM Lysine, 0.1% SDS).                                   Example 2 Analytical scale two-dimensional gel analysis   Fractions were adjusted to at least 10% glycerin and 6% acrylamide, 50% Loaded on a native gel, a minigel of mM Tris-acetic acid. 5 for native gel Electrophoresis was performed in 0 mM Tris-acetate buffer, pH 8.0. Contains telomerase Cut the native gel lane, immerse briefly in 2 × SDS sample buffer, add 25 mM   Denatured 5-15% with 0.1% agarose in Tris-acetic acid, or 5-15% Inserted into wells of a 20% gradient acrylamide minigel. SDS-PAGE Tris-glycine-SDS buffer (25 mM Tris-HCl pH 8.3 , 192 mM glycine, 0.1% SDS). After electrophoresis, run the gel It was immersed twice in 50% methanol for 10 minutes and equilibrated with 5% methanol. 0.1m Incubate the gel in 1 mg / ml silver nitrate for 20 minutes in M DTT for 20 minutes. And silver staining, 0.28M Na_TwoCO_Three, 0.0185% formua Deployed in Rudehid. The staining reaction was stopped with citric acid.                                   Example 3 Proteolytic digestion of telomerase subunits and peptide sequencing   The 80 kD and 95 kD subunits of telomerase were purified as described above. Preparing the SDS gel containing telomerase with 0.05% Coomassie Brilliant Blue (Manufactured by Aldrich), stained in 20% methanol and 0.5% acetic acid, and Destained in acid. After soaking in distilled water for 10 minutes, the polypeptide is cut out of the gel. Was. Crush the gel slice, dip it twice in 50% methanol for 20 minutes, decant And dried briefly under reduced pressure. Approximately 300 ng of Acromobacter Pro 0.1M Tris-HCl pH 9.0, 0.01% Tween Digested for 24 hours at 37 ° C. in en-20. Spin filtration of peptides from gel fragments Separated by filtration, concentrated with Speed-Vac, and applied to a C-18 column (manufactured by Vydac). I applied. The peptide was dissolved in 0.09% trifluoroacetic acid in acetonitrile: Elution was carried out with a sopanol (3: 1) gradient. Collect the absorbance peak at 214 nm, It was freeze-dried and applied to a protein sequencer (ABI).                                   Example 4 Cloning of 80 kD and 95 kD telomerase subunit genes   Considering the codon usage of Tetrahymena, degenerate primers Designed from columns. Martindale, D.W. (1989) Protozol . 36: 29-34. These primers can be used in multiple combinations under various PCR conditions. Used in The template for PCR is the genomic DNA of the macronucleus of Tetrahymena Alternatively, total RNA from Tetrahymena grown and starved as described above, or Is poly-A⁺RNA (Ausubel et al., (1992) Current Protocols in Mol ecular Biology, John Wiley & Sons, Inc. and Sambrook et al. Prepared as described in Ref. Purify PCR amplification products , Cloned into E. coli and sequenced according to standard protocols. PC R product encodes an additional peptide sequence not specified by the PCR primers If the PCR product is a p80 or P95 gene or cDNA Specified as internal peptide of or by degenerate PCR primers used in the reaction It was confirmed that it was obtained as a sequence adjacent to the sequence to be performed.   Λgt10cDN of Tetrahymena to which oligo dT was added using a PCR product A library (Takemasa et al. (1989) J. Biol. Chem. 264: 19293- 19301) was screened. Only incomplete clones (less than 0.8kb) are obtained Did not. Genomic DNA of Tetrahymena digested with EcoRI or ClaI Using genomic library with Bluescript KS + (Stratagene) Was built. A 3.2 kb clone containing the majority of the p80 gene was obtained. p A 1.1 kb clone containing the internal region of the sequence encoding 95 genes was obtained. . The p95 gene was identified by Southern blot of genomic DNA digested with EcoRI. Fragments containing other parts were detected, but completely missing in the constructed library Was. To obtain the 5 'ends of the cDNAs of both genes, Poly-A derived from mena⁺A RACE protocol was performed using RNA (Gibco   # 18374-025). To determine the 3 'end of the p95 cDNA, The sequence of the lambda clone was compared to the sequence obtained by 3'RACE. The above Based on protocol, poly-A of mRNA for reverse transcription⁺Priming from the end Using the oligo dT to be cloned from within the known sequence of the genomic clone for PCR. 3 'RACE was performed in combination with the priming. p80 cDNA Compare the sequence of the lambda and genomic clones to determine the 3 'end of Was. The lambda clones obtained for p80 are four genomic clones. Terminates at the 3 'end with a poly-A sequence in which only nin residues are present. at p80 The 3 'RACE results support that this region is the site of polyadenylation. I have.   In a previous determination of the p95 nucleic acid sequence, nucleotide 405 was a "G" and 9 It was shown that nucleotides 77-979 were "CGT". this child Means "R" instead of "Q" and "A" in the encoded protein, respectively. Was the result.                                   Example 5Production of antibodies against 80 kD and 95 kD polypeptides   Synthetic peptides corresponding to two different regions were synthesized from each protein. these Peptides were obtained from Genosys Biotechnologies. These peptides are labeled Using a standard protocol, Keyhole Lymphet Hemocia A cystine residue added to the amino terminus is added to the nin (KLH) carrier protein. Coupled via Harlow et al. (1988) Antibodies-A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. For KLH protein Each bound peptide is converted into 2 using standard protocols to periodically give antigen. The animals were injected into separate rabbits. Harlow et al., Supra. Rabbit every few weeks Serum was collected from the sample. Injections into animals were made at Hazelton Corporation. antigen 3-4 times before obtaining serum from all rabbits and first injecting each rabbit An ELISA assay was performed on the synthetic peptide used to perform the assay. Crude serum Some specifically recognized the peptide. Then, the antibody is The test was performed on all tetrahymena and the purified telomerase fraction. One type Two rabbits, immunized with a peptide, have very high power on 80 kD protein Had value. Antibodies obtained from each of these rabbits were designated A81 and A82. I named it. Similarly, four rabbits immunized with the two peptides had a 95 kD protein. He had a high titer for white matter. These antibodies are called A83, A84, A85 Named A86. A82 has the highest affinity for the 80 kD protein, and A8 6 had the highest affinity for the 95 kD protein.   Then, by binding to a column to which a specific peptide is bound, the antibody is Affinity purified. First, an acetate buffer (0.1 M NaOAc, pH 4. 0) elute the antibody from the column, followed by glycine buffer (0.1 M glycine, At pH 2.7) the more tightly bound antibody was removed. Affinity purified antibody Was used for both Western blot and immunoprecipitation. A82 and A86 antibodies Western analysis of the containing sera revealed a polypeptide of 80 kD and a polypeptide of 95 kD. Both peptides have telomerase activity throughout the column purification scheme It was shown to be co-purified as is (see Example 1). 95 kD protein and 80 Both levels of kD protein parallel the doubling of enzyme activity at each stage of purification. , Consistent with the fact that these proteins are telomerase components.   Telomerase activity is the highest affinity antibody (A82) for the 80 kD protein. ) Specifically immunoprecipitated. Immunoprecipitation is a standard technique (Harlow, et al., Supra). ). Aga in which affinity-purified antibody is bound to G protein The plate was incubated with the loin beads (Pharmacia). After the first binding reaction, Highly purified fractions from the non-peak region of the serine gradient (see Example 1) at 3-4 For 4 hours at 4 ° C. to allow telomerase to bind. then, The beads were spun at very low speed in an Eppendorf tube and the supernatant was removed. The beads were washed three times with T2MG containing 0.1 M KG, 0.5% NP-40, Resuspended in the same buffer. Then, the telomerase activity was measured in the supernatant fraction, final washing. Fractions and pellet fractions were assayed for each antibody. By antibody A82, Telomerase activity in the pellet and supernatant showed reduced activity. Con As trolls, affinity purified 80 kD and 9 by Western blot. Antibodies that did not recognize either of the 5 kD were used for immunoprecipitation. Other 80 kD polypep Antibodies (A83, A84) with lower affinity for the polypeptide and the 95 kD polypeptide As with A85 and A86), with this antibody the activity remains in the supernatant. Was. These results indicate that the 80 kD polypeptide is a functional component of telomerase It is shown that.                                   Example 6 Synthesis of 80 kD and 95 kD telomerase subunit genes   In addition to ciliates, all organisms express Tetrahymena proteins. To use UAA and UAG to encode glutamine Codon (Martindale, D.W. (1989) J. Protozol. 36:29). -34) must be replaced. In most eukaryotes, these codons Stands for "halt", so that these translations express the full-length protein Is hindered. 44 glutamine codons in the p95 gene These genes make each substitution because 18 glutamine sites require substitution. Rather than using site-specific mutations, de novo synthesis was performed. Build synthetic genes First determine which codons will be used to encode each amino acid. Was. Escherichia coli and Baki, two effective systems for expressing recombinant proteins Codon usage in urovirus (Rohrmann, GF (1986) J. Gen. V irol. 67: 1499-1513; Zhang, G. et al. (1991) Gene 105: 61-72). Based on these, these codons were selected. A list of selected codons is shown in Table 2.   Using the sequences of both p80 and p90 proteins as the genetic code using the codons in Table 2 Geneworks (IntelliGenetics) computer for "reverse translation" -Program was used. This resulted in some degeneracy due to the degeneracy of the selected genetic code A DNA sequence was created. Then, every 300 bp in each gene sequence, Restriction enzyme map predicted from this degenerate sequence to find the optimal restriction enzyme sites Was examined. Here, if necessary, select different codons that code for a particular amino acid. The restriction enzyme sites were individually deleted by selection. Has restriction enzyme sites at appropriate positions This process was repeated until a unique DNA sequence was obtained. Used for cloning Figure 5 shows the final DNA sequence with restriction enzyme sites created at the 5 'and 3' ends. 10 and 11 show.   To synthesize the two genes shown in FIGS. 10 and 11, 28 A set of overlapping oligonucleotides was synthesized (primer set 3 and 4, respectively, FIGS. 7 and 8), 34 sets were synthesized for p95 ( Primer sets 1, 2, and 3, respectively, Figures 5, 6, and 7), and duplicate genes Constructed by extension PCR. Prodromou (C.) and Pearl (LH Pear) l) (1992) Protein Engineering 5: 827-829; with Bambot, SB A. Russell (1993) PCR Meths. and Apps. 2: 266-271. Oligonu Creotide was purchased from Bioserve Biotechnologies (Laurel, MD). Each oligo Nucleotides are about 100 nt long and overlap with their complement , 20 base pairs were designed to hybridize. Each oligonucleotide is Also, at the 3 'end of the oligonucleotide, usually at the position where the phosphodiester is, It has a suholothioate linkage. This phosphorothioate is an early polymer Exonuclease with 20 bp hybrid overlap during the elongation step Hinders removal. Skehr, A. (1992) Nucl. Acid Res. 20: 3551 -3554.   First set of oligonucleotide pairs (primer set 4, FIGS. 8A-8B) And then using PCR to amplify the entire region as described , The p80 gene was constructed in two fragments. The second half is primer set 5 (FIG. 9) and a similar method was used. Each half of the gene was replaced with plasmid Blue Cloning into script to confirm that no new mutations were introduced The full length was sequenced for: The 5 'half was cloned into a BamHI-EcoRI fragment. And the 3 'half was cloned into the EcoRI-KpnI fragment. Or that Then, the two fragments were ligated to pBluescript (manufactured by Stratagene) in an appropriate order. Then, a full-length gene was constructed.   Combine each set of oligonucleotide pairs and then amplify the entire region The p95 gene was assembled into three fragments using PCR. The first fragment is Constructed using Primer Set 1 and the second fragment uses Primer Set 2. And the third was constructed using primer set 3. gene Were cloned into plasmid pSE280 (manufactured by Invitrogen). And sequenced the full length. The 5 'fragment (fragment and primer set 1) is Nc oI-BstBI fragment and cloned into an internal fragment (fragment and primer set 2) was cloned into the BstBI-EcoRI fragment and the 3 'fragment (fragment and plasmid) Immerset 3) was cloned into the EcoRI-HindIII fragment. So Then, fragments 1 and 2 were first ligated into the pSE280 plasmid and then The 3 'fragment was added to complete the gene to construct the full length gene.                                   Example 7 Expression and purification of recombinant p80 and p95 proteins from cells   Methods known to those skilled in the art (eg, Ausubel, supra; (See Sambrook, supra), each of the full-length constructs was pRSE T and pBlueBac vector (Invitrogen) and cloned into p80 and p 95 protein is expressed in E. coli and baculovirus. With these vectors And expression and purification of the recombinant protein. p80 to pRSET and pBlueBac Was cloned into the BamHI and HindIII sites of And HindIII sites. By transcription and translation of these constructs A series of 6 histidines (His-tags) at the rear produce recombinant proteins. And is separated by an EK (enterokinase) protease cleavage site. This As a result, each protein becomes Ni⁺⁺Purified by a process through a chelating column You. The purified protein is His by digestion with protease and enterokinase.   Removed from tag.                                   Example 8 Cloning of human telomerase protein components   Two general approaches to cloning human genes, DNA sequences And antibody-based approaches. First approach Utilizes the DNA sequence of the Tetrahymena gene to directly identify human homologs. One of skill in the art will recognize that three cloning homologs based on DNA sequences can be used. Recognizing different methods: (1) the human genome or c A method of directly hybridizing to a DNA library; Identify conserved regions of the telomerase protein in species and base these regions on (3) Telomerase residue by PCR probe Fill the entire region of the gene and “walk” along the gene length using PCR to A systematic way to identify mologs. All of the methodology is standard molecular genetics laboratory Based on the method (Sambrook et al., Supra).   In the first method, a series of increased stringency using the Tetrahymena gene Examine the human genomic DNA blot and mRNA blot. Specific ba If a cDNA or genomic library is identified, a similar string Investigate and identify genes for human homologs. Then positive phage A restriction map is generated, subcloned, and sequenced.   In the second method, the telomerase protein is located between human and Tetrahymena At the DNA sequence level, they are only slightly conserved, First involves the cloning of a telomerase protein from another ciliate. And then Milk analogs are cloned using information obtained from these cDNAs. this Thus, using the cloned tetrahymena gene, In order to identify the gene to be immobilized, distant tetrahymena or oxy Libraries from Trisha and Euplotes at moderate stringency You can find out at Ciliate Oxytrisha and Euplotes are Tetrahymena Has a telomerase enzyme functionally similar to that of (Lingner) et al. (1994) Genes Dev. 8: 1984-1998; Shippen-Lentz -Lentz, D.) and EH Blackburn (1990) Science 247: 546- 552), and probably homologous proteins could be identified in this way. Both ciliate insects Complete sequencing of both the p95 and p80 homologous genes And identify the most highly similar regions between different species it can. Reverse transcriptase PCR-based method for cloning human genes Choose three conserved regions for each protein for use in p80 gene and p Using the same approach to clone genes for both of the 95 genes You. Using human translation codon bias, tetrahymena, oxytrisha and Degenerate oligonucleotides encoding regions conserved in the telomerase protein of Protes Synthesize leotide. Starting PCR with two of the three oligonucleotides Do. The 3'-most oligonucleotide (oligo 1) is complementary to mRNA . Thus, from the mRNA isolated using oligo 1 as a primer, cDN A is synthesized. The oligo at the 5 'side (oligo 2) is 5' in the opposite direction to oligo 1. And the sequence is identical to the mRNA strand. Then this Oligonucleotides were used together with Oligo 1 in the PCR step, and Amplify the region between Finally, the region targeted by oligos 1 and 2 The third primer (oligo 3) faces the conserved region of the protein. Oligo Will be complementary to the mRNA. The PCR products amplified by Oligos 1 and 2 are Amplify again using go2 and oligo3. This ensures that all specific products produced It is confirmed that the telomerase has three conserved regions of the telomerase protein. PCR production The products are sequenced and those containing protein homologs are identified.   The third approach is a systematic and detailed approach, in which the Tetrahymena gene and Regions of homology between the genes, and then these regions are determined by PCR. It amplifies the area. Using the protein sequence of the Tetrahymena gene as a guide Further exploiting the human codon bias in DNA sequences, Tetrahymena A series of primer oligonucleotides are created that encode regions of the gene. at first Primer sets with different regions that hybridize by 10 amino acids Make to the 3 'end. These are used in the RT PCR reaction to produce cDNA. Used. Next, a primer set from the 5 'end to the 3' end of the gene was prepared. Used to amplify cDNA. Promising support for the Tetrahymena protein region Two PCR primers from the 5 'and 3' ends to identify All possible combinations of the mer can be used together. Specific product is produced If so, they should be annealed within the first two primers. Amplify again using primer. No primer can be used alone The specific product is subcloned and sequenced.                                   Example 9 An antibody approach to cloning human telomerase homologs.   This method utilizes conserved epitopes on the protein surface that are recognized by the antibody. To use. A series of antibodies against various regions of both p80 and p95 proteins create. Antibodies as described in Example 5 can be used. Antibody against antibody production The raw material is produced as a synthetic peptide or as a fusion protein. First, the fusion protein Producing synthetic peptides is faster because white matter does not have to be made; however, However, these peptides may not be as potent as synthetic peptides . About 10 to 15 residues of the peptide, preferably non-structural and therefore antigenic Select the 5 'and 3' ends of the protein. In addition, it is non-structural and close to the protein surface Determine the hydrophilic structure of the protein and the expected secondary structure to select likely regions Computer analysis can be applied to Use synthetic peptides as carriers such as KLH Used to inoculate rabbits. Suitable for binding to carriers If no amino acid is present in the peptide, the linker cysteine residue is added to the N-terminus. Add.   The fusion protein was produced using the T7 polymerase system (Studier et al. (1990) Me. th. Enzymol. 185: 60-89), purified for inoculation into rabbits and mice. So Then, an extract or purification from E. coli expressing the cloned protein is performed. Using the obtained fractions, sera were screened by Western blot. Positive antibody Recognizes a protein of 95 kD or 80 kD on Western blot and / or Specifically immunoprecipitates telomerase RNA, or otherwise telomerase The ability to inhibit the activity of the enzyme is tested. As controls, peptide and plain The ability of anti-peptide antibodies to precipitate telomerase RNA during cuvettes Should be extinguished. Mouse and rabbit sera and monoclonal media are terror Inhibits merase activity (L. Harrington, unpublished results) Use purified IgG to test the ability of antibodies to inhibit telomerase activity I have.   Polyclonal Antibodies and Monoclonal Antibodies against Tetrahymena Telomerase Protein Telomerase protein from human and mouse cells that cross-react with both null antibodies Can be used to identify by Western blot. Find a positive signal If activated, determine if the reactive band has been co-purified with telomerase activity. For the determination, the purified fraction of human telomerase is used. For evidence of co-refining This indicates that the cross-reacting band is a component of human telomerase. Antibodies that give the best signal, and then a lambda GT11 expression library Used to check. If using monoclonal antibodies, prepare an expression library. Pool several different antibodies for viewing. For polyclonal antibodies, 2 One or three different antibodies are used on duplicate plates. Emit light with both probes Only those phages that do not are considered positive. Purify these plaques and insert Subclones and sequences.                                 Example 10 Cloning and use of mouse homologs   The same two methods described above, DNA homology or antibody cross-reactivity, were used to It can also be used simultaneously to identify the components of the Romerase protein. of mouse Telomerase clones can be used to test cancer treatments or to produce mammalian telomerase. It can help you understand the physics. Identification of mouse telomerase Transgenic mice were tested for telomere length and telomere in vivo. It can also be used to test the role of ose. Once, mouse or human homo If any of the logs are identified, one of the clones will be sequenced to another organism Can be applied. High sequence similarity between human and mouse genes Thus, obtaining one clone using a probe derived from another clone is simple. It is a process. Then, load both the genomic and cDNA libraries. Rated and examined at moderate stringency for cross-hybridization Plaques to be identified. Then select positive plaques and map restriction enzyme Were sequenced and the telomerase protein homolog was cloned. Determine whether or not. Then, further functional analysis such as reconstruction and gene disruption Applies to human and mouse clones.Equivalent   Those skilled in the art will recognize, by mere routine experimentation, the invention described herein. Many equivalents to a particular embodiment can be recognized or confirmed. Would. Such equivalents are within the scope of the invention as set forth in the following claims. It is rare.

[Procedure for Amendment] Article 184-8, Paragraph 1 of the Patent Act [Date of Submission] December 18, 1996 [Content of Amendment] Claims 1. (A) an isolated DNA that encodes a telomerase protein component and is identical or substantially homologous to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 2. A DNA that hybridizes with the DNA of claim 1 under moderately stringent conditions. 3. An isolated RNA transcribed from the DNA of claim 1 or complementary to the DNA of claim 1. 4. An isolated RNA transcribed from the DNA of claim 2 or complementary to the DNA of claim 2. 5. A polypeptide encoded by the DNA of claim 1. 6. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 7. A therapeutic or diagnostic compound comprising an amino acid sequence encoded by the isolated DNA of claim 1 in combination with a pharmaceutically acceptable carrier, diluent or excipient. 8. A pharmaceutical composition comprising all or substantially all of one or both of the polypeptides of claim 1 in combination with a pharmaceutically acceptable diluent, excipient or carrier. 9. A transformed eukaryotic cell or eukaryote comprising the DNA sequence of claim 1 or a sequence complementary thereto. 10. A transformed prokaryotic cell comprising the DNA sequence of claim 1 or a sequence complementary to said sequence. 11. A transformed eukaryotic cell or eukaryote comprising the nucleotide sequence of claim 1. 12. A DNA sequence comprising SEQ ID NO: 8 or SEQ ID NO: 9. 13. A synthetic telomerase protein component encoded by the DNA sequence of claim 12. 14． A DNA or RNA sequence that hybridizes with the DNA sequence of claim 12. 15. An expression vector comprising DNA selected from the group consisting of primers 1 to 34, F1 to F14, and R1 to R14. 16. An anti-telomerase antibody that binds to a peptide comprising an amino acid sequence selected from the group consisting of AEGYSDINVRG, QNEFQFNNVK, and EFGLEPNI LT. 17． Substantially pure telomerase protein component. 18. 18. The substantially pure protein component of claim 17, which is a Tetrahymena telomerase protein component. 19. An anti-telomerase antibody that binds to all or a part of the telomerase protein component according to claim 17. 20. A method for detecting the presence or propensity of a cell to immortalize in a eukaryotic tissue or eukaryotic cell sample, comprising: a) obtaining a tissue or cell sample from a eukaryote; b) determining the presence of telomerase in the sample, if the sample shows the presence of telomerase, there is an immortalized cell or a tendency to immortalization. 21. If the presence of a disease (such as cancer) or disease tendency in a eukaryotic tissue sample or eukaryotic cell sample is detected by determining the presence of telomerase in the sample, and the sample indicates the presence of telomerase, the disease or disorder is detected. 21. The method of claim 20, wherein the tendency exists. 22. 22. The method of claim 21, wherein the disease is caused by a eukaryotic microorganism, and wherein the sample exhibits eukaryotic microbial telomerase, the disease caused by the eukaryotic microorganism is present. 23. 23. The method according to any of claims 20, 21 and 22, comprising the steps of: a) treating the sample to allow telomerase to bind to the anti-telomerase antibody, thereby producing a treated sample; b. A) contacting the treated sample with an anti-telomerase antibody; and c) detecting binding of the antibody to telomerase, if binding occurs, telomerase is present. 24. Administering the compound to cells of Tetrahymena, measuring the telomerase activity of Tetrahymena in the cells, if the telomerase activity of Tetrahymena is reduced, the compound is a telomerase inhibitor, telomerase activity in eukaryotic cells Methods for identifying compounds that inhibit, destroy or interfere. 25. Administering the compound to a eukaryotic cell that expresses telomerase activity, and measuring the telomerase activity.If the telomerase activity is reduced, the compound or compounds are identified as telomerase inhibitors, in mammalian cells, including humans. A method for identifying a compound that inhibits, destroys, or interferes with telomerase activity of a plant. 26. A therapeutic or diagnostic compound comprising a telomerase activity inhibitor in combination with a pharmaceutically acceptable carrier, diluent or excipient. 27. A method for preparing a therapeutic or diagnostic composition comprising combining an anti-telomerase compound with a pharmaceutically acceptable excipient, diluent or carrier. 28. A method for the therapeutic treatment of a mammal (such as a human) suffering from an abnormal level of telomerase activity, wherein the telomerase production is inhibited if the level is too high, and telomerase is administered if the level is too low The method that consists of doing. 29. 29. The method of claim 28, wherein the disease is caused by a eukaryotic microorganism in the mammal, and the disease is treated by administering to the mammal an effective amount of a telomerase inhibitor to inhibit telomerase activity in the microorganism. 30. Claims for inhibiting the activity of eukaryotic microbial parasites, especially fungal and protozoan parasites, comprising administering to a mammal an effective amount of a telomerase inhibitor to inhibit the activity of eukaryotic microbial parasites. 28. The method of claim 28. 31. A method for producing a recombinant telomerase or anti-telomerase antibody comprising the following steps: a) a step of producing an expression vector containing a DNA encoding a telomerase molecule or an anti-telomerase antibody, respectively; b) transforming or infecting a host cell with the vector; Producing a transformed or infected host cell by doing so; and c) culturing the transformed or infected cell to produce the encoded telomerase or anti-telomerase antibody. 32. A host cell comprising an expression vector comprising a DNA encoding a telomerase protein component or a fragment thereof. 33. 33. A method for producing a telomerase protein component or a fragment thereof, comprising the step of culturing the host cell according to claim 32 under conditions capable of producing a telomerase protein component or a fragment thereof. 34. 34. The method of claim 33, wherein two or more telomerase protein components or fragments thereof are produced in the same cell. 35. 34. The method of claim 33, further comprising the step of purifying the telomerase protein component or a fragment thereof.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 39/395 AGZ A61K 45/00 ADX 45/00 ADX 48/00 ADU 48/00 ADU C07K 16/40 C07K 16/40 C12N 1 / 21 C12N 1/21 9/12 5/10 C12P 21/08 9/12 G01N 33/573 A C12P 21/08 A01K 67/027 G01N 33/573 C12N 5/00 B // A01K 67/027 A61K 37 / 64 ADY (C12N 1/21 C12R 1:19) (C12N 9/12 C12R 1:19) (72) Collins, Kathleen United States of America New York 11743 Huntington, Prime Avenue 111 (72) Inventor Kobayashi, Ryuji United States of America New York 11791 Shiosset, Cove Edge Road 1152 (72) Inventor Yang, The Aohon Helena United States New York 11740 Green Lawn, Cuba Hill Road 90 (72) Inventor Hemis, Jill Em. United States New York 11746 Huntington Station, Fenwood Road 4 (72) Inventor Oteguzier, Chantal United States of America New York 11731 East Northport, Vernon Ba Leh Road 188

Claims (1)

[Claims] 1. Substantially pure telomerase protein component. 2. The substantially pure protein according to claim 1, which is a tetrahymena telomerase protein component. Protein component. 3. SEQ ID NO: 1 or SEQ ID NO: 3, which encodes a telomerase protein component An isolated DNA that is identical or substantially homologous to a nucleotide sequence. 4. 4. Hybridization with the DNA of claim 3 under moderately stringent conditions. DNA. 5. A DNA transcribed from the DNA of claim 3 or complementary to the DNA of claim 3. Isolated RNA. 6. A DNA transcribed from the DNA of claim 4 or complementary to the DNA of claim 4. Isolated RNA. 7. A polypeptide encoded by the DNA of claim 3. 8. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 5. 9. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 7. 10. Identical or substantially identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 Isolated DNA encoding such polypeptides. 11. AEGYSDINVRG, QNEFQFNNVK and EFGLEPNI Anti-telomere that binds to a peptide consisting of an amino acid sequence selected from the group consisting of LT Rase antibody. 12. An anti-protein that binds to all or a portion of a substantially pure telomerase protein component Romerase antibody. 13. Immortalized in a eukaryotic tissue sample or eukaryotic cell sample, comprising: Methods for detecting the presence of immobilized cells or their propensity to immortalize:   a) obtaining a tissue or cell sample from a eukaryote;   b) determining the presence of telomerase in the sample, wherein the sample is the presence of telomerase Indicates an immortalized cell or a tendency to immortalization. 14． Eukaryotic in a eukaryotic tissue sample or eukaryotic cell sample, comprising: Methods for detecting diseases caused by microorganisms:   a) obtaining a tissue or cell sample from a eukaryote;   b) determining telomerase in the sample, wherein the sample is telomerase of a eukaryotic microorganism; Indicates a disease caused by a eukaryotic microorganism. 15. Telomere in a eukaryotic tissue sample or eukaryotic cell sample, comprising: Detecting method:   a) obtaining a tissue or cell sample from a eukaryote;   b) processing the sample to allow telomerase to bind to the anti-telomerase antibody Producing a processed sample by doing so;   c) contacting the treated sample with an anti-telomerase antibody;   d) detecting the binding of the antibody to telomerase; Merase is present. 16. 2. The method of claim 1, wherein the presence of telomerase is indicative of a propensity for cancer or the presence of cancer. 5. The method according to 5. 17． The presence of telomerase is an indicator of the presence or propensity for immortalized cells. 16. The method of claim 15, comprising: 18. Presence of eukaryotic microbial telomerase is the presence of diseases caused by eukaryotic microbes 16. The method according to claim 15, which is an indicator of: 19. Administering the compound to the cells of Tetrahymena, Reduces telomerase activity in Tetrahymena by measuring merase activity Is a compound that is a telomerase inhibitor, telomerase in eukaryotic cells A method for identifying a compound that inhibits, destroys or interferes with an activity. 20. Administer the compound to eukaryotic cells that express telomerase activity, The telomerase activity is reduced, the compound or A group of compounds identified as telomerase inhibitors are found in mammalian cells, including humans. A method for identifying a compound that inhibits, destroys, or interferes with Romerase activity. 21. Telomere in combination with a pharmaceutically acceptable carrier, diluent or excipient A therapeutic or diagnostic compound comprising an inhibitor of the activity of an enzyme. 22. 2. The method of claim 1, wherein the pharmaceutically acceptable carrier, diluent or excipient is combined. 0. A therapeutic comprising the amino acid sequence encoded by the isolated DNA of claim 1 or Diagnostic compound. 23. SEQ ID NO: in combination with a pharmaceutically acceptable diluent, excipient or carrier : 2 or all or one of the polypeptides of SEQ ID NO: 4 A pharmaceutical composition comprising qualitatively all. 24. Anti-telomerase with pharmaceutically acceptable excipient, diluent or carrier A method for preparing a therapeutic or diagnostic composition comprising combining compounds. 25. Telomerase inhibition in an amount effective to inhibit telomerase activity in microorganisms A disease in a mammal caused by a eukaryotic microorganism, comprising administering the agent to the mammal. How to treat the disease. 26. An effective amount of a telomerase inhibitor to inhibit the activity of eukaryotic microbial parasites Eukaryotic microbial parasites in mammals, especially fungi and And methods of inhibiting protozoan parasite activity. 27. Treatment of a damaged human or animal with abnormal levels of telomerase activity A method of treatment, wherein if the level is too high, telomerase production is inhibited and A method comprising administering telomerase if the level is too low. 28. A transformant comprising the DNA sequence of claim 3 or a sequence complementary to said sequence. Nuclear cell or eukaryote. 29. A transformant comprising the DNA sequence according to claim 3 or a sequence complementary thereto. Nuclear cells. 30. A transformed eukaryote comprising the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3 Cell or eukaryote. 31. A method for producing a recombinant telomerase comprising the following steps:   (A) a process for producing an expression vector containing a DNA encoding a telomerase molecule; About;   (B) transforming or infecting a host cell with the vector; and   (C) culturing the transformed or infected cell line and encoding the encoded telomerase Manufacturing process. 32. A method for producing a recombinant anti-telomerase antibody comprising the following steps:   (A) Producing an expression vector containing a DNA encoding an anti-telomerase antibody Process;   (B) transforming or infecting a host cell with the vector, Producing a transformed or infected host cell; and   (C) culturing the transformed or infected cells to produce an anti-telomerase antibody Process. 33. A DNA sequence comprising SEQ ID NO: 8 or SEQ ID NO: 9. 34. A synthetic telomerase protein encoded by the DNA sequence of claim 33. component. 35. A DNA or RNA sequence that hybridizes with the DNA sequence of claim 33. Column. 36. Selected from the group consisting of primers 1-34, F1-F14 and R1-R14 An expression vector comprising the isolated DNA. 37. Comprising DNA encoding a telomerase protein component or a fragment thereof A host cell comprising an expression vector. 38. Claims under conditions that allow the production of telomerase protein components or fragments thereof. Telomerase protein component or fragment thereof comprising the step of culturing 37 host cells Manufacturing method. 39. Two or more telomerase protein components or fragments thereof are produced in the same cell. 39. The method according to claim 38. 40. Further comprising a step of purifying the telomerase protein component or a fragment thereof. 39. The method according to claim 38.