MXPA99004853A - Identification of inhibitors that prevent access of telomerase to chromosomal terminus - Google Patents

Abstract

The present invention discloses novel telomerase-associated proteins as well as methods for isolating the genes encoding telomerase-associated proteins. Using novel screening methods presented additional telomerase-associated genes may be isolated and their protein products expressed and characterized. Identification of protein-protein interactions required for the in vivo function of telomerase provides the basis for a novel screening method for anti-tumor agents.

Description

IDENTIFICATION OF INHIBITORS THAT PREVENT THE ACCESS OF THE TELOMERASA TO THE CROMOSOMAL TERM FIELD OF THE INVENTION The present invention relates to the fields of cellular and molecular biology. In particular, the present invention relates to genes that encode proteins that play a role in the function and regulation of telomerase. BACKGROUND OF THE INVENTION Telomeres are specialized sequences present at the ends of linear eukaryotic chromosomes, and are required for genomic stability and complete replication of chromosomal terms (reviewed in Blackburn and Greider 1995; Zakian 1995; Greider 1996). Telomeres are characterized by an online array of short repeated DNA sequences; in humans, the repeated telomeric sequence is a d (TTAGGG) n. In all eukaryotes examined to date, with the exception of Drosophila and several other dipterans, telomeres are replicated by a specialized DNA polymerase called telomerase (reviewed in Kipling 1995, Blackburn and Greider 1995, Zakian 1995). This enzyme is responsible for adding telomeric repeats on the 3 'end of the telomere G-rich strain, and uses an internal standard present in a subunit of an RNA to dictate the added telomeric DNA sequence. The gene that encodes the RNA component of the telomerase has been identified in several species (Greider and Blackburn 1989, Shippen-Lentz and Blackburn 1990, Lingner, and collaborators, 1994, McEachern and Blackburn 1995, Feng, et al 1995, Blasco, and collaborators 1995), including the TLC1 gene of S. cerevisiae (Singer and Gottschling 1994). However, in contrast the characterization of the protein components of this enzyme has lagged behind. Furthermore, little is known at the molecular level on how the regulation of telomerase activity is achieved. There are several approaches to identify additional components of the telomerase enzyme complex. The biochemical purification of telo erasa from the ciliate Tetrahymena has recently led to the identification of two proteins which, in addition to the RNA component, comprise subunits of the enzyme (Collins, et al. 1995); homologs of these genes have not yet been identified in other species, including the fully sequenced genome of S. cerevisiae. An alternative to the biochemical pathway is to identify genes which, when mutated, have a set of predicted phenotypes for a defect in telomerase in vivo. These two different protocols could presumably lead to the identification of at least some of the same factors. However, a genetic approach has the potential to identify components that may be critical for live function (for example, as positive regulators) but which is not required in vitro for enzymatic activity and therefore may not be co-purified with the enzyme. Two genes have been previously identified in Saccharomyces cerevisiae which, when suppressed, result in a cell with phenotypes expected from the elimination of telomerase activity. The first of these is the EST1 gene, which encodes a very basic 82 kD protein, which is hypothesized to be a subunit of telomerase (Lundblad and Szostak 1989, Lundblad and Blackburn 1991). This proposal has been based largely on the observation that mutant strains without this display two predicted characteristics for a telomerase deficiency: progressive loss of telomere sequences of the chromosomal terms and a phenotype of senescence (manifested as a stable decline in cell viability). A strong support that these phenotypes could be diagnostic of a telomerase defect comes from the demonstration that a yeast strain that is deleted from the TLC1 gene, which encodes yeast telomerase RNA, exhibits the same set of phenotypes (Singer and Gottschling 1994). Although telomerase activity is still present in chromatographic fractions prepared from an Estl-4 strain, suggesting that Estlp is not required in vitro for catalytic activity (Cohn and Blackburn 1995), this does not exclude the possibility that EST1 is a non-catalytic component of telomerase (Lin and Zakian 1995). Since the Estl protein binds to single chain yeast telomer sequences, Estl can function as a telomerase component responsible for telomere recognition. Consistent with this, the Estl protein is associated with telomerase RNA and / or enzyme activity (Lin and Zakian 1995; Steiner, et al 1996), although it was not possible to determine if the association was quantitative in these experiments. The properties of the ligand Est to telomeric DNA are also consistent with a role as an extreme telomere ligand protein acting as a positive regulator of the telomerase function by directing telomerase on the chromosomal term. In either of the two models, the requirement for Estl function could be obviated in in vitro experiments in which pure DNA is used as a substrate for telomerase lengthening. In mammalian cells, telomerase is highly regulated: it is present in the germ line but is absent or very reduced in most normal tissues. Consistent with the absence of this enzyme, telomeres shorten in these normal cells. During the process of tumor formation, the activity of telomerase is over-regulated, in relation to the levels present in normal cells. This over-regulation is presumed to be necessary with in order to maintain telomere length and allow unregulated proliferation during tumor development. This proposal is supported by a substantial investigation of the state of enzymatic activity in different tissues: telomerase activity is expressed in 851 of 998 human tumor tissues, but is not present in most normal tissues, with the exception of the germline and hematopoietic stem cells (Shay and Wright 1996, and references therein). In some tumors, telomerase is expressed at higher levels in advanced-stage cancers compared to early-stage cancers, with some data suggesting that the degree of survival of patients correlated with telomerase activity levels (Hiyama, et al 1995a, 1995b). Specific studies in human breast cancer have also shown a strong correlation between tumor development and telomerase activity, and it has been predicted based on an analysis of the rate-limiting step in most ethastatic breast cancers (Shay , and collaborators 1993). Initial data have shown that alterations in telomere length occur in breast cancer cell lines, and the extent of change in the length of the telomeric tract correlates with the histological aggressiveness of breast carcinoma (Rogalla, et al. 1994); Odagiri, and collaborators 1994).

Subsequently, a direct analysis of telomerase activity showed a remarkable correlation between enzymatic activity and cancer progression; activity was detected in more than 95 percent of advanced stage breast cancers, while it was present in 68 percent to 81 percent of less advanced tumors and absent in fibrocystic disease (Hiyama, et al. 1996). Collectively, these data strongly argue that the reactivation of telomerase is an event necessary for the sustained growth of the tumor. Therefore, anti-cancer agents targeting this enzyme can provide a novel approach to cancer treatment, including breast cancer (Harley, et al. 1995). In addition, because telomerase expression is restricted almost exclusively to tumor cells, this suggests that inhibition of telomerase has limited side effects when used as an anti-cancer therapy. The limited population of stem cells that are telomerase-plus and which could be affected by telomerase inhibitors compares favorably with the much wider range of proliferating normal tissues that are the unintended target of most chemotherapeutic agents. Thus, inhibition of telomerase has been proposed as a potential anti-cancer therapeutic target, with the presumed advantage that inhibition of telomerase may have few effects collateral, since it may not be necessary for normal somatic cell growth. Currently, most efforts are directed at trying to find compounds that inhibit the catalytic activity of the enzyme, defined as in vitro polymerase activity. However, several observations suggest that the activity of telomerase, defined as the maintenance of a functional telomere, can also be regulated at different levels of the catalytic activity of the enzyme. The catalytic activity of telomerase is present in certain human cell lines such as hematopoietic cell lines (Broccoli, et al., 1995; Counter, et al., 1995) which, however, exhibit telomere shortening (Cooke and Smith 1986; et al., 1988; de Lange, et al. 1990). This argues that other factors must also be involved in mediating the control of telomere length, presumably by regulating telomerase activity in some way. However, the identification of these components in mammalian systems has been problematic, due to the absence of a test system for their detection. SUMMARY OF THE INVENTION We have focused on the problem of identifying the telomerase components by looking for these factors in yeast. Specifically we are interested in looking for mutants of yeast that display the phenotypes predicted for a defect in telomerase (telomere shortening and phenotype of senescence). A strong support that these characteristics would be diagnostic for a telomerase defect comes from the demonstration that a suppressed strain for the yeast telomerase RNA gene, TLC1, also showed these same two phenotypes (Singer and Gottschling 1994). Our selection identified four genes; the previously identified EST1 gene (Lundblad and Szostak 1989) and three new genes (Lendvay, et al., 1996). Genetic analysis showed that mutations in these four genes interrupted the same route for telomere replication as a mutation in TLC1, a known component of telomerase (Lendvay, et al., 1996). Three of these genes (EST1, EST2 and EST3) are novel genes that work only in the same route for telomere replication as defined by the TLC1 (Lendvay, et al., 1996). However, we have shown that the fourth gene (initially called EST4 but later proved to be the same as the previously cloned CDC13 gene) has a dual role in telomere function (Nugent, et al. 1995). The CDC13 gene is an essential yeast gene that is required to maintain telomere integrity, as was first suggested by a study by Lee Hartwell's lab that shows a rapid loss of a telomere strain occurs in the absence of the function of CDC13 (Garvik et al. 1995). Recent work from our laboratory has revealed an additional role for Cdcl3 in telomere maintenance. This was the result of the discovery of a novel mutation, called cdcl3-2-, which exhibits a phenotype virtually identical to that of the telomerase-minus strain (although, as just discussed, enzyme levels were normal in prepared extracts of this strain) . In addition, the analysis of epistasis between the cdc! 3-2- mutation and the mutation of tlcl-? demonstrated that cdc! 3-2- disrupts a function of CDC13 that is required for the telomerase pathway. In comparison, genetic analysis of the conditional lethal allele previously isolated from CDC13 (cdc! 3-l-) showed that CDC13 has a separate essential function that must be maintained in addition to the telomerase-based pathway. We have also shown that the purified CDC13 protein binds specifically to telomeric substrates of simple chain yeast. Based on these data, we have proposed that CDC13 has two distinct functions in the telomere (Nugent, et al. 1996). The first of these proposed roles is to protect the end of the chromosome, which is essential for the viability of the cell. Second, we proposed that the Cdcl3 protein regulates telomerase mediating, either directly or indirectly, the access of this enzyme to the term chromosomal, and that this access is now eliminated by the cdc! 3-2- mutation. The present invention provides the primary structure of two new proteins associated with telomerase Est2 and Est3. An object of the present invention is to provide a method for identifying and isolating other proteins associated with telomerase using the Est 2 or Est 3 proteins or fragments thereof. An object of the present invention is to provide a method for identifying and isolating other proteins associated with telomerase using the Est 2 or Est 3 proteins or fragments thereof to generate antibodies and use the antibodies thus generated to purify the proteins associated with telomerase. It is an object of the present invention to provide a method for identifying proteins associated with telomerase using standard biochemical techniques. An object of the present invention is to provide a novel method for searching EST genes. It is an object of the present invention to provide an assay for the identification of a novel class of telomerase inhibitors that function through a mechanism not taught by the prior art. An object of the present invention is to provide a method to find the human homologs of the proteins associated with yeast telomerase identifying the conserved motifs in the proteins associated with yeast telomerase and looking in the databases of human proteins for these conserved motifs. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic representation of the experimental protocol used to isolate the genes that encode est mutations. Figure 2A shows a Southern blot analysis of the telomeric DNA from yeast strains that have mutations in the EST genes. Figure 2B shows the results of striations of mutant yeast strains. Figure 3A shows a Southern blot analysis of telomeric DNA from strains of yeast that have mutations in the EST genes. Figure 3B shows the results of a viability assay of yeast strains with different mutations. Figure 4A shows a Southern blot analysis of yeast strains with several mutations. Figure 4B shows a Southern blot analysis of yeast strains with several mutations. Figure 4C shows the results of a viability assay of yeast strains with different mutations.

Figure 5A shows a schematic representation of the strategy used to locate the open reading structure of EST2. Figure 5B shows the primary structure of the EST2 gene product. Figure 5C shows the results of a viability assay of yeast strains with different mutations. Figure 6 shows the primary sequence of the EST3 gene product. Figure 7 shows the nucleotide sequence of the EST3 gene that includes flanking regions and the location of the ribosomal structure change +1. Figure 8 shows the transposon mapping of the EST3 gene. Figure 9 shows a model for a ribosome structure that changes in EST3. Figures 10A-10H show various EST3 constructions. Figure 11 shows the structure of the EST3 gene. Figure 12 shows a plasmid map of a fusion construct of the EST2 protein. Figure 13 shows a plasmid map of a fusion construct of the EST2 protein. Figure 14 shows the effect on the viability of a mutant strain containing cdc! 3-l- and tlcl_.

Figure 15A shows a schematic representation of the structure of the Est3 fusion protein constructs prepared and Figure 15B shows a Western blot analysis of the expression of the protein constructs. DETAILED DESCRIPTION OF THE PREFERRED MODALITIES MATERIALS AND METHODS Yeast strains and media: The strains of S. cerevisiae used in this study are shown in Table 1; YPH275 was the diploid father of all the strains used in these studies. Mutant derivatives MVL1 to MVL26 were isolated, as described below, from the haploid strain TVL227-1A, obtained by the transformation of YPH275 with the plasmid PVL106 (Lundblad and Szostak 1989) and sporulation and subsequent dissection. TLV120 and TLV140 were previously described (Lundblad and Szostak 1989; Lundblad and Blackburn 1993); DLV131 was constructed from YPH275 by introducing an interruption tlcl-:: LEU2, constructed by polymerase chain reaction-mediated suppression (Baudin, et al. 1993) of the sequences between 192 and 909. Strains were cultured at 30 ° C and in standard medium (Guthrie and Fink 1991), except that the chromosome loss assays were performed in media with limiting adenine (6 μg / milliliter) to increase the detection of "red" colonies in A. Canavanina boxes and boxes were prepared of 5-FOA SC-LEU as described in Ausubel, and collaborators 1987. Transformations of the yeast using the lithium acetate method (Schiestl and Gietz 1989); sporulation and tetrad dissections were carried out using standard techniques (Guthrie and Fink 1991). Mutagenesis of ethyl methane sulfonate (SEM): TVL227-1A was grown overnight to saturation in SC-LEU-TRP, diluted ten times in YEPD medium and cultured until O.D. 0.8 to 1.0. Forty milliliters of cells were washed twice in sterile dH20 and resuspended in 12 milliliters of 0.1 molar sodium phosphate, pH 7.0; portions of 1.7 milliliters were incubated with 45 microliters of ethyl methane sulfonate for one hour at 30 ° C with slow aeration. Cells were diluted 10-fold in sterile 5 percent sodium thiosulfate, washed twice in 5 percent sodium thiosulfate, twice in sterile dAHOO, and resuspended in 2.0 milliliters of dH20. Aliquots were plated in canavanine boxes to monitor the increase in frequency of canavanine resistance and the dilutions were plated in YEPD boxes to determine the percentage of cells that survived the mutagenesis procedure; both values were determined in relation to a sample of cells handled in parallel but without added ethylmethane sulfonate. Due to the large number of colonies selected, the complete selection was made in three stages, managing between 80,000 and 150,000 colonies in each stage. A total of 12 independent mutagenesis were performed; the average survival varied from 30 to 60 percent and the increase in resistance of canavanine was 50 to 150 times. Note that since all the selection steps were performed at 30 ° C, this mutant analysis was not specifically designed to recover lethal conditional mutations. Selection of mutants: Selection of colonies sectors: To detect mutants which show a phenotypic delay in chromosome loss, mutagenized cells were processed through two rounds of growth as single colonies. After mutagenesis, the cultures were plated on SC-LEU-TRP plates at a density of 500-1000 per plate and the colonies were grown at 30 ° C at full size for approximately three to four days. Groups of approximately 7500 colonies were generated by resuspending colonies from SC-LEU-TRP plates in sterile dH20, and replating at a density of approximately 350 per plate in SC-LEU plates with limited adenine. The total number of replanted colonies of each group was approximately four times the size of the group, to increase the singularities that each colony represented in the original group. The plates were incubated for seven days at 30 ° C to allow full development of red ade sectors, in addition, incubation overnight at 4 ° C before qualifying chromosome loss helped to increase the detection of red sectors (F Spencer, personal communication.) If two mutants are they were isolated from a single group and then showed that they mapped the same complementation group; they considered each other brothers and only one was analyzed later. Plasmid linearization assay: Colonies with increased numbers of Ade sectors were collected in wells of microtitre plates containing 0.2 milliliters of SC-LEU medium, to select the retention of pVL106 (Lundblad and Szostak 1989). two days at 30 ° C, 5 μl aliquots were transferred from each well to SC-LEU-5-FOA plates, using a multichannel pipette, and the plates were subsequently incubated 3 more days at 30 ° C to allow the growth of micro-colonies resistant to 5-FOA. A non-mutagenized wild type strain gave rise to approximately 10-15 micro-colonies on the 5-FOA plates in this assay; all mutants that showed more than one three-fold reduction were saved for further analysis. Each candidate was retrieved from the microtiter tray well and striated to look for unique colonies on the SC-LEU plates, and three single colonies for each were tested again in the plasmid linearization assay. Candidates who passed this second test were tested for alterations in telomere length by Southern analysis. In addition, candidates who reached this stage were transferred to glycerol at 15 percent and stored at 70 ° C, to avoid the loss of rapidly senescent strains.

Telomere length analysis: One to two single colonies of the SC-LEU striates for each candidate that passed the plasmid linearization assay were cultured to saturation in 8 milliliters of YEPD and genomic DNA was prepared as previously described ( Guthrie and Fink 1991). Samples of approximately 1 μg of DNA digested with Xhol enzyme (New England Biolabs) were separated into 20 centimeters of 0.7 percent agarose gels, transferred to nylon membranes and tested with poly d (GT / CA) as described previously (Lundlad and Szostak 1989). When telomere length was tested with increasing numbers of growing generations (Figures 2 and 4), the cultures were grown to saturation in YEPD and subsequently diluted by a factor of 10"4 and allowed to grow back to saturation This process was repeated three to four times Cell Senescence Assays: All mutants with decreases in telomere length greater than 250 base pairs were tested to see if they exhibited a phenotype associated with senescence by crossing back each mutant with a natural haploid derivative of YPH275 and sporulating and dissecting the posterior diploid strain For each mutant, the haploid spore products of four to six tetrads were striated to determine single colonies on YEPD, incubated at 30 ° C for 48 hours and scored for determine their growth characteristics in parallel with products of haploid spores of estl-41:: HIS3 generated from the dissection of TVL120. Simple colonies of these striated "IX" were subsequently striated and similarly analyzed; this process was repeated up to four times. For the data presented in Figures 2, 3, and 4, each of the successive striatum in YEPD was stored at 23 ° C and was used to reset a time course on a single plate. Genetic manipulations: Complementation analis: The diploids were isolated from non-selected matings, to avoid selection by reversion or suppression of the senescence phenotype. Haploid strains of the opposite mating type overlapped each other on YEPD plates and were allowed to mate for four to six hours. Four zygotes were collected for each cross by micromanipulation with a Zeiss dissection microscope and allowed to grow at 30 ° C for two days. The resulting strains were purified once via the isolation of a single colony and tested to determine the type of mating and any nutritional marker that differed between two haploid parents. To test complementation of telomere length, a single colony of two to three diploids was cultured and tested by Southern analysis together with each of the two haploid parents. The growth characteristics and stability of multiple diploid chromosomes for a cross given were analyzed similarly in parallel with the haploid parents. Linkage analysis and construction of double mutant haploid strains: Haploid spores of the opposite mating type were used to generate diploids (also by non-selective matings) which were heterozygous for two different EST genes. The diploids were sporulated and dissected and the spores were analyzed to determine cell senescence and telomere length. Initially both tetrads with four viable spores as well as tetrads with less than four viable spores were analyzed; when lack of evidence of synthetic lethality was obtained (see Figure 4), the subsequent analysis concentrated in tetrads with four viable spores. The following crosses were made to demonstrate that the new complementation groups (EST2, EST3 and EST4) were different from each other and from EST1 and TLC1: (i) est2-l x estl-4:: HIS3; (ii) est2-l x tlcl-4:: LEU2; (iii) est2-l x est 3-1; (iv) est3-l x est-4:: HIS3; (v) est3-l x tlcl-4:: LEU2; (vi) est4-l x estl-? :: HIS3; (vii) est4-l x tlcl-4:: EU2. For each cross, they were analyzed > 8 tetrads to determine telomere length and senescence, with at least four tetratypes and non-parental ditty tetrads recovered and analyzed for each one. The resulting diploid strains were also used to construct the double and single mutant haploid strains analyzed in Figure 4. The haploids est2-l, est3-l and est4-l used to generate these diploids were the result of at least three backcrosses with wild type, and the haploids estl- / .:: HIS3 and tlcl-, 4:: LEU2 were isolated from TLV120 and DVL131, respectively. Identification of surviving strains est: Each newly generated haploid strain was cultured in liquid YEPD culture, with successive dilutions for 120 to 150 generations and subsequently plated for single colonies on YEPD plates. After two days of growth at 30 ° C, large colonies were examined both to determine the growth phenotype and the telomere structure on Southern blots, as described above. Cloning of the EST2 gene: Freshly dissected mutant st2-l strains were transformed with genomic libraries and selected for complementation of the senescence phenotype, using two approaches: (1) A strain est2-l rad52 :: LEU2 was transformed with three different libraries genomic URA3 CEN (Rose, et al. 1987), with approximately 2000 transformants recovered for each transformation (which was probably a substantial sub-estimate of the efficiency of the transformation, due to the severe growth defect of the strain est2-l rad52 :: LEU2). The complementary clones were apparently not among any of these early transformants; However, after scraping and replacing, They identified healthy colonies from two or three library transformations. A single colony from each library group was saved for future analysis; both showed that they had wild type telomeres when tested on Southern blotches. (2) A strain st2-l in an intermediate stage in a progression of senescence was transformed with a LEU2 library of 2 μ (Engebrecht, et al. 1990) and approximately 30,000 transformants were carefully selected after 40 to 45 hours to determine the transformants with a slight growth advantage. Fifty-seven candidates were selected and striated for single colonies twice in succession to the test for complementation of the senescence phenotype. Ten of the 57 candidates showed a healthy growth phenotype by this test; four also had wild-type telomere length and were saved for further analysis (the remaining ones demonstrated the telomere deviation route dependent on the recombination previously observed for the mutants estl; Lundblad and Blackburn 1993). For the six transformants recovered from the above three transformations of the library, the healthy growth phenotype and the wild-type telomere length were in each case shown to be plasmid-dependent. The plasmids were subsequently recovered from each strain of yeast by rescue through E. coli. A combination of restriction mapping and Southern analysis of the genomic inserts showed that the four plasmids recovered from the 2μ LEU2 library were identical to each other, and had genomic inserts that overlapped with the inserts present in the two plasmids recovered from the libraries genomic URA3 CEN. A subcloned fragment of 4.4 kb common to these three unique plasmids capable of complementing st2-l was subjected to insertion mutagenesis using the bacterial transposon of 8.7 < 5 (Strathmann, et al., 1991). Insertion mutations were mapped by polymerase chain reaction using primers against the transposon and polylinker sequences that bound the cloned insert. The inserts? < 5 subsequently provided the basis for sequence analysis, using unique primers for the right and left ends of the transposon 8.7 alt 235; the sequence was confirmed by comparison with that generated by the • Yeast Genome Sequencing Project. The interruption mutation is: URA3 was constructed by omitting a 2.2 kb HindIII fragment, removing amino acid 13 to amino acid 686 from the open reading structure of EST2, and inserting a URA3 HindIII fragment of 1.17 kb. This interruption was introduced into TVL140 to generate a heterozygous diploid EST2 / est2-β:: URA3 (strain TVL228), which was confirmed by Southern analysis. Twelve tetrads were analyzed in detail to determine senescence and telomere phenotypes, as described above. EXAMPLE 1 Identification of an expanded collection of S. cerevisiae mutants with a telomere replication defect and a senescence phenotype. The original identification of the estl-1 mutant used a selection that tested the behavior of a circular plasmid containing inverted repeats of Tetrahymena telomer sequences by attaching the yeast URA3 gene (Lundblad and Szostak 1989). At a low frequency in wild type cells, this circular plasmid can be converted into a stable linear form. This conversion requires the addition of telomeric yeast sequences on the terms of Tetrahymena in order to form functional telomeres. Since the conversion to linear results in the loss of the URA3 gene, mutants defective in the linearization process can be identified by selecting colonies that exhibit decreased frequencies of resistance to the drug 5-FOA (Ura + cells convert 5-FOA into a toxic intermediate, whereas the loss of URA3 avoids this, Boeke, et al. 1984). Potentially, a subset of mutants that could be identified by this assay would also be defective by the ability to form functional telomeres, as soon as the plasmid is linearized; these mutants must also exhibit shorter chromosomal telomeres. From this original selection, the estl-1 mutant was the only candidate that fell within this class, with a progressive reduction in telomere length with increasing crop growth (Lundblad and Szostak 1989). However, a disadvantage of the plasmid linearization assay was that the process of testing individual colonies to determine their frequency of resistance to 5-FOA was labor intensive, which limited the original selection to only about 70,000 mutagenized colonies. As soon as they were identified, both the estl-1 mutant and the stl-? subsequently constructed it was shown that they had two additional associated phenotypes: a senescence phenotype, manifested as a gradual decrease in cell viability, and a progressive accompanying increase in the frequency of chromosome loss (Lundblad and Szostak 1989). In order to isolate additional yeast-like mutants, the four phenotypes exhibited by the mutants were incorporated into an expanded selection protocol, presented in Figure 1. Due to the difficulty of selecting large numbers of colonies in the assay of linearization of plasmids, this step was preceded first by a step of enrichment of colonies that presented increased frequencies of chromosome loss. Candidates who showed both an increase in chromosome loss and a defect in the plasmid linearization assay were subsequently selected to look for alterations in telomere length by Southern spotting analysis. Finally, these isolates with short telomeres were tested for an associated senescence phenotype, examining newly generated haploid mutants to determine if they showed the decline in characteristic viability previously observed in both imit and tlcl mutants. Due to a single aspect of the effect of mutations on chromosome instability, the standard chromosome loss assay used by others required modification. The detection of alterations in chromosome stability depends on a previously discovered color-based visual test that monitored the presence / absence of a non-essential 150 kb artificial test chromosome (Spencer, et al. 1990). Several selections have been made using this test, or similar variants, to detect mutants that affect chromosome fidelity (Meeks-Wagner, et al 1986, Spencer, et al 1990, Koupri na, et al 1988, Runge and Zakian 1993). . None of these previous selections, which have collectively identified a large number of genes involved in the maintenance of the chromosome, detected mutations either in EST1 or in the new EST genes identified in this work. This is presumably due to the fact that the effect of Mutations in the chromosome loss shows a phenotypic delay; no substantial increase in chromosome instability was observed in a mutant strain until approximately 40-60 generations of growth (Lundblad and Szostak 1989). Therefore, selecting mutants that show similar delayed effects on chromosome stability required faster growth after mutagenesis. Since the desired class of mutants was also expected to have a senescence phenotype, growth in liquid would exert a substantial selective disadvantage for these mutants. To avoid this, the mutagenized cultures were processed through two rounds of growth as single colonies before selecting them for chromosome instability (see materials and Methods for more details). A total of 350,000 single colonies of 12 cultures mutagenized independently of yeast by the four-row system shown in Figure 1. Individual colonies of cultures mutagenized with ethyl methane sulfonate at an average survival of approximately 50 percent were processed in batches of 80,000 to 150,000 colonies. The first two steps (chromosome loss and the plasmid linearization assay) resulted in enrichment steps of approximately 20 times to 50 times, respectively. Southern staining analyzes of the telomere length of 375 candidates who These two tests led to the identification of 49 mutants both with decreases (variants of approximately 50 base pairs at> 300 base pairs) or increases (variants of 150 base pairs at 2 kb) in telomere length. Of the 36 mutants with shorter telomeres, 19 they also had an associated senescence phenotype. These 19 est mutants, plus three additional short telomere mutants that had no detectable senescence phenotype but were subsequently demonstrated to map EST genes, are the subject of this report. The new mutants stipulate four genes: Each of the est mutants, when crossed with the wild type, were recessive for telomere length, chromosome loss and senescence (data not shown). The 19 mutants were also tested to determine whether they contained mutations in either EST1 or TLC1 by crossing each mutant with a strain J-st and a strain tlcl-i.; the resulting diploids were examined to determine both the telomere length and its viability. The 19 mutant strains completely complemented both phenotypes of the tlcl- mutation, indicating that no new alleles of TLC1 were identified (data not shown). However, eleven mutants failed to complement the phenotypes of both senescence and short telomere of the strain estl-? . Two additional mutants, with short telomeres but no obvious senescence phenotype, were also shown subsequently by complementation analysis that contained weak mutations. These complementation data were confirmed for five of these new alleles by demonstrating that the mutation could be repaired by gaps on a plasmid containing EST1 with a gap encompassing the wild-type EST1 gene (C. Nugent and V.L., unpublished data). The identification of 13 alleles showed that this expanded selection was able to detect est mutant strains. The remaining eight strains were crossed against each other in pairs combinations and the resulting diploids were analyzed to determine telomere length, chromosome loss and senescence. This analysis indicated that these eight mutants defined three new complementation groups: EST2, EST3 and EST4. Four mutations mapped in EST2, three mapped in EST3 and one isolated mapped in EST4. An additional mutant, with short telomeres but an apparently wild-type growth phenotype, was subsequently shown to contain a non-senescent allele of EST3. To confirm the complementation analysis, linkage analysis was performed on one of two isolates of each new complementation group to establish that EST2, EST3 and EST4 defined three genes that were different from each other and from TLC1 and EST1 (data not shown; and methods for more details). In addition, products from six to eight were analyzed tetrads with four viable spores of crosses. In all cases, the reduced telomere length and senescence showed 2: 2 segregation, these two phenotypes exhibiting 100 percent co-segregation, demonstrating that a single recessive nuclear mutation was responsible (data not shown). The lack of recovery of the tlcl mutants of this selection was surprising given the fact that a defect in the TLC1 gives rise to the same set of phenotypes as those observed in est mutant strains (Singer and Gottschling 1994; Figures 2, 3). One possible explanation is that many mutations induced by ethyl methane sulfonate in a gene whose product is an RNA would be expected to be phenotypically silent. However, the absence of tlcl mutations from our collection combined with the fact that only one isolate was recovered, suggests that there may be additional EST genes still to be identified. The mutants est2, est3 and est4 are phenotypically indistinguishable from the estl and tlcl strains: If the three new EST genes play a similar role in telomere replication than EST1 and TLC1, mutations in these new genes should display phenotypes similar to those exhibited. for the strains stl-zl and tlcl-? . To test this, several mutants from each new complementation group crossed back to the wild type and sporulated. The newly generated spore products were examined in time to determine their growth phenotype and telomere length, in parallel with a haploid strain estl-? newly generated As shown in Figure 2, the new mutant isolates are exhibiting the same phenotypes as the stl-4 and tlcl-, 4 strains. In panel A, Southern blotting of genomic yeast DNA grows successively in culture, tested with a telomere-specific probe (poly d [GT]). The rows 1, 2, 24 and 25 ESTl ± TLCl-; rows 3-6, tlcl-4:: LEU2; lanes 7-10, this: - HIS3, rows 11-14, est2-l; rows 15-18, est3-l; rows 19-23, est4-l. For strains tlcl estl est2 est3 and est3, four successive subcultures are shown, representing a total of approximately 65-70 generations of growth, due to the slightly weaker phenotype of est4-l, a fifth subculture is included (corresponding to approximately 13-15 additional generations of growth). 1 and 2 and rows 24 and 25 show the first and fourth subcultures, respectively, of two ESTl ^ TLC strains operated in parallel.The bracket indicates a teloeric band representing approximately 2/3 of the telomeres in this strain (those which are Y -continents), the arrows indicate restriction fragments corresponding to telomeres that do not contain Y. Panel B shows the viability of estl-43:: HIS3, tlcl-4:: LEU2, est2-l, est3-ly est4-l, shown as three cultures in line on YEPD plates, differing from each other by approximately 25 generations of growth; for est4- 1, an additional successive online crop is shown. Figure 2A shows the decline in telomere length that a representative mutant of each of the new complementation groups displays over time, as compared to that displayed by the strains estl-? and tlcl-_j. Strains were cultured for approximately 80 generations, with DNA prepared for Southern analysis every approximately 15 generations. The telomere length in each mutant declines over time, and thus the same degree as observed in the stl- ^ and tlcl-4 strains. In parallel with the analysis of telomere length, we also analyzed the growth and stability of the chromosome over time in these new mutants. Freshly dissected mutant haploid strains were cultured in line to give single colonies three times successively in YEPD plates, in parallel with newly generated haploid stl-4 and tlcl-4 strains. Each of the new mutants showed a dramatic decline in viability after their rapid growth. The growth phenotype for each of the new alleles est2 and est3 was qualitatively indistinguishable from that exhibited by null mutations in EST1 and TLC1 (Figure 2B and data not shown). The mutant est4-l also showed a clear senescence phenotype but in this case, senescence was delayed compared to the other est mutants, possibly because the simple allele est4 may be somewhat permeable. In Parallel to the phenotype of senescence, each new mutant was also shown a marked increase in the frequency of chromosome loss (data not shown); the appearance of this increased chromosome instability was delayed, similar to the delay previously exhibited by the estl-? (Lundblad and Szostak 1989). New mutants are using the same alternative route for telomere maintenance that the strains are? or tlcl-; Previous analyzes have shown that the after rapid growth of mutant cultures is stable. a small proportion of cells are able to escape the lethal consequences of the absence of the EST1 function. These survivors are? arise as a result of a deviation trajectory for telomere maintenance that is activated in late cultures (Lundblad and Backburn 1993). The activation of this alternative route is presented as the result of a global amplification and rearrangement of both telomeric repeats rich in G and subtelomeric regions. This amplification can be quite substantial: in some survivors, the number of telomeric repeats to G ^ _ - sT) increases by as much as 40 times, so that 4 percent of the genome consists of telomeric DNA (VL, unpublished data) . As a consequence of this genomic reorganization, the telomere function is restored and the survivors have regained a phenotype of Normal or almost normal growth. This route requires the RAD52 gene, which mediates most homologous recombination events in the yeast; in the absence of the function of the RAD52 gene, the rad52 strains can not generate ultimate culture survivors and instead disappear completely after approximately 40 to approximately 60 generations (Lundblad and Blackburn 1993). Consistent with the other similarities between EST1 and TLC1, a strain tlcl-? it also exhibits alterations of telomeric and subtelomeric DNA (Singer and Gottschling 1994) which is dependent on RAD52 (Figure 3). This alternative route has not been shown to give rise to other, non-senescent, telomeric replication strains of S. cerevisiae. To test if strains est2, est3 and est4 were also able to participate in this process, two or three isolates of each mutant are cultured for 120 to 150 generations and plated for single colonies. Several simple colonies of each culture were analyzed to investigate the growth characteristics and structure of telomeres. As shown in Figure 3, new mutants are showing the alternative route dependent on RAD52 for telomere maintenance. Panel A shows a Southern staining of genomic yeast DNA. The rows 1 and 11, a mixture of? HindIII and a single 4.0 kb fragment containing d (G., _ 3T) detected by the probe; rows 2 and 10, EST1 ± TLC ±; rows 3 and 9, tlcl-4:: LEU2 early; the rows 4-8 isolated survivors of tlcl-d:: LEU2, estl-id3:: HIS3, est2-l, est3-l and est4-l, respectively. A shorter exposure of the region indicated in brackets is shown below, to demonstrate the degree of amplification of subtelomeric bands containing Y '(indicated by brackets) that occur in surviving strains (Lundblad and Blackburn 1993). Note that the probe detects the telomeric repeats d (G1.3T), indicating that both the Y * and d (G., _ 3T) sequences are greatly amplified in the survivors. Panel B shows the viability of ESTl rad52:: LEU2; tlcl-4:: LEU2 rad52:: LEU2; estl-43:: HIS3 rad52 :: LEU2; est2-l rad52 :: LEU2; est3-l rad52 :: LEU2; and est4-l rad52 :: LEU2, shown as two successive striations. Figure 3A shows Southern blotting of one of these survivors from each of the five tlcl strains or is tested with poly d (GT); all exhibit the same type of telomere rearrangement originally observed in this mutant, characterized by the extensive amplification of both telomeric DNA d (G1.3T) and subtelomeric Y1 repeats (Lundblad and Blackburn 1993). In addition, each of these survivors is now, and has now acquired a growth phenotype that closely approximates that of the wild type (data not shown), similar to that previously observed for the survivors. The appearance of the Survivors were not specific for the specific alleles used in Figure 3, as all 19 of the mutants are isolated in this selection of mutants that exhibited a senescence phenotype that resulted in survivors (data not shown). To test if the process of generating survivors of the mutants est2, est3 and est4 was also dependent on RAD52, each mutant was crossed with a strain rad52:: LEU2. The resulting diploids were sporulated and dissected, and the haploid stra and est rad52 strains were analyzed in parallel. Figure 3B shows that the presence of a rad52 mutation conferred lethality on each strain after approximately 40 to 50 generations. Furthermore, the degree of increase of the senescence phenotype in the presence of a rad47 mutation was the same in the estl, est2, est3 and tlcl mutants, providing another point of similarity between mutations in these different genes. Consistent with the phenotype of delayed senescence exhibited in some way by the mutant est4-l in a strain RAD52 ± (Figure 2B) the appearance of lethality in a strain est4-l rad52 :: LEU2 was slightly delayed (Figure 3B) in relation to the other mutant strains are rad52 and tlcl rad52. TLC1 and the four EST genes work in a single pathway for telomere replication: The previous comparison showed that strains carrying mutations in the EST2, EST3 and EST4 genes are phenotypically similar to the estl and tlcl; in fact, many of the new isolates are indistinguishable from the estl-4 and tlcl strains, suggesting that these alleles may also be null mutations. This similarity in phenotype argues that the new EST genes work in the same genetic route for telomere replication as the one originally defined by EST1. The alternative possibility is that these five genes operate in more than one route, each required to form a functional telomere. These two possibilities can be distinguished by examining the behavior of strains that have multiple mutant combinations. The prediction is that if all five genes work in a single route necessary to replicate telomeres, multiple mutant strains should not show increased phenotype. This has already been demonstrated for EST1 and TLC1 (Virta-Pearlman, et al., Proposal), showing that EST1 works in the same route in vivo as defined by a known component of telomerase. However, if one or more genes function in an additional, separate pathway required for telomere replication, certain multiple combinations of mutants could be expected to show a more severe phenotype. The substantial increase in the phenotype of mutant strains is that it occurs in the presence of a rad52 mutation (Figure 3B), which eliminates the 1 deviation route of support for telomere maintenance discussed above, is an example of the latter possibility.

To distinguish between these two formal possibilities, each new mutant was crossed against estl-zl, tlcl-4 or the other mutant strains est to generate diploid heterozygotes at various EST / TLC1 sites. Subsequent diploids were sporulated and dissected to generate products of wild type spores, single mutants and double mutants. Each double mutant strain was analyzed for telomere length, senescence and chromosome loss (Figure 4 and data not shown), in parallel with single and wild type mutants. In any case, no phenotype increase was seen for combinations of double mutants. Figure 4, Analysis of epistasis of the tlcl and est mutants. A. Southern blotting of genomic yeast DNA. The rows 1, 2, 15, 16 STL ± TLC ± (representing four successive subcultures, handled as in Figure 2A); rows 2-14, three rows each (three successive subcultures) of estl-43:: HIS3; estl-, 43:: HIS3 est2-l; estl-43:: HIS3 est3-l; and est-J3:: HIS3 est4-l. B. Southern blotting of genomic yeast DNA.

The rows 1, 2, 21, 22, ESTl ± TLC ^; rows 3-20 three rows each (three successive subcultures) of tlcl-4:: LEU2; tlcl-4 :: LEU2 est2-l; tlcl-4:: LEU2 est3-l; tlcl-4:: LEU2 est4-l, est3-1; est2-l est3-l. C. The viability of double mutants shown as two successive flutes. Top: (counterclockwise from the top left) wild type; estl-43:: HIS3; estl- ^ 3:: HIS3 est2-l; estl-43 :: HIS3 est3-l; estl- ^ 3:: HIS3 est4-l; est2-l est3-l; Bottom: wild type; tlcl-4:: LEU2; tlcl-4:: LEU2 estl¿3:: HIS3; tlcl-d:: LEU2 est2-l; tlcl-zl:: LEU2 est3-l; tlcl-? :: LEU2 est4-l. Figures 4A and 4B show the telomere length for a representative set of double mutant strains compared to the appropriate single mutant strains; in each strain, the length of the telomere declined in time to the same degree. Similarly, as shown in Figure 4C, there was no alteration in the senescence phenotype in any of the double mutant strains, relative to the single mutants. This is in marked contrast to the severe increase in senescence that occurred as a result of the introduction of a rad52 mutation in any of the est mutant strains (Figure 3B and 5C). These pairwise combinations argue that each of the new EST genes work in the same route for replication as defined by EST1 and TLC1. The analysis of several combinations of double mutants and their haploid counterparts revealed an unexpected observation, which was that the phenotypes of senescence and telomere shortening phenotypes of simple mutant strains was somewhat increased when these haploids were derived from a diploid parent that was multiple heterozygous for genes in the EST / TLC1 pathway. For example, the time course of the senescence phenotype of a haploid strain carrying only the tlcl-4 mutation was accelerated when the tlcl-, 4 strain was derived from the DVL132 (TLClVtlcl- /! ESTVestl-?), Compared to the same haploid isogenic strain tlcl-i derived from DVL131 (TLClVtlcl-, 4 ESTlVESTl1); (compare strains tlcl-J in Figure 2B and 4C). This additive haplo insufficiency was not specific for the EST1 / TLC1 combination and was observed for every possible combination of five genes in this pathway. Consistent with the accelerated phenotype observed in haploids of these diploids, the telomere length was slightly shorter in multiply heterozygous diploids compared to simple heterozygous (D.K.M., unpublished data). Note that the conclusions drawn from the data presented in Figure 4, that the additive combinations of different mutations do not show an increase in the phenotype, were made of comparisons between sets of double and simple mutant strains derived from the same diploid father; Comparisons of simple mutant haploids in Figure 2 are not valid. Although we do not understand the molecular basis of this phenomenon, it suggests that alterations in the gene dose can interrupt a complex or a set of interactor complexes. The EST2 gene encodes a very basic, novel protein: the EST2 gene from the wild type was cloned by complementation of the se2-1 senescence phenotype of both high and low copy genomic libraries (see materials and Methods for more details). Three independent genomic clones with overlapping inserts were identified which complemented both the growth phenotypes and the telomere length of the st2-l mutation; no high copy suppressors were identified. The subcloning identified a genomic segment of 4.4 kb common to the three inserts which was able to complement st2-l. This fragment was sequenced and also subjected to mutagenesis with the transposable element d (Strathmann, et al., 1991), in order to identify the genetic boundaries of the EST2 gene. Figure 5. Cloning of the EST2 gene. A. Mutagenesis of insertion of the genomic clone 4. 4 kb, with the position of the indicated open reading structure of EST2. Each of the inserts 13? 5 were tested to determine their complementation of the telomere shortening and senescence phenotypes of a mutant strain st2-l; the insertions that completely complemented the Est phenotype are indicated as an open triangle above the rectangular box that represents the DNA, and the failure to complement is indicated by a solid triangle below the representation of the DNA One insert, 74 base pairs upstream of the start AUG, exhibited a phenotype intermediate, because the phenotype of senescence was complemented but a phenotype of telomer shortening was observed (data not shown). The positions of the five open reading structures stick us and a poly-A tract in the promoter region of EST2 are also indicated. B. The sequence of the EST2 open reading frame of 884 amino acids. C. Comparison of the viability of the strains estl-? , est2-4 and estl-4 est2-, 4 (both the RAD52 and rad52 versions), shown as two successive striations. A total of 13 insertion mutations were isolated, mapped by both polymerase chain reaction and sequence analysis, and tested for their ability to complement st2-l (Figure 5A). These data, combined with the sequence of the 4.4 kb fragment, showed that the complementation activity was due to an open reading structure of 884 amino acids (Figure 5B); no open reading structure greater than 65 amino acids was found within this sequenced insert. The promoter region of the EST2 gene exhibited several remarkable features in common with that of EST1. First, both genes diverged significantly from the consensus sequence found around the initial AUG of most yeast genes (Hinnebusch and Liebman 1991; Galibert, et al. 1996). In addition, for both EST1 and EST2, the region just upstream of the start codon AUG has the potential to encode multiple small open reading structures, a feature that is not usually observed in the promoter regions of the yeast genes (Cigan and Donahue 1987). In the rare genes where these upstream open reading structures have been analyzed, they have been shown to be involved in the translation control of gene expression (Hinnebusch 1992). Within 140 base pairs of the beginning of the coding sequence of the Estl protein, there are five overlapping open reading structures, ranging in size from 3 to 39 codons (Lundblad and Szostak 1989). Similarly, within the first 170 base pairs of the promoter region of the EST2 gene, there are five small open reading structures, from 4 to 11 codons in length (Figure 5A). Although these small upstream open reading structures have not been functionally dissected in detail in any gene, an interruption inserted into the core of three open reading structures upstream was partially defective for EST2 activity (Figure 5A and no data). shown), suggesting that this region can play an important role in the expression of EST2. One characteristic of the EST2 promoter that was not found in EST1 was a poly (dA) tract of 32 base pairs long just upstream of the beginning of the EST2 coding region. The poly (dA-dT) tracts have previously been implicated in activation of transcription (Struhl 1986; Lúe and Kornberg 1987); the poly (dA) tract of EST2 can also be a promoter element, although it is unusually near the beginning of translation. To determine the null phenotype and confirm that the EST2 gene has been cloned, an internal deletion within this open reading structure was removed and replaced with the URA3 gene. This construct was introduced into a diploid strain by a one-step gene replacement, and the resulting heterozygous strain was sporulated; > 80 percent of the tetrads had four viable spores for each, indicating that this gene was not essential for immediate viability. The haploid strains of 12 tetrads were subsequently analyzed to determine senescence and telomere length; for all 12 tetrads, a mutant phenotype was co-secreted 100 percent of the time with the URA3 marker. The senescence phenotype of st2-4l was identical to that exhibited by a stl-zl strain, both in the presence and absence of the RAD52 gene function (Figure 5C); in addition consistent with the double mutant analysis conducted previously with the st2-l point mutation, a suppressed strain for both genes showed no increased phenotype with respect to senescence (Figure 5C) or telomere length (data not shown). In addition, a strain st2-4l was able to give rise to RAD52-dependent survivors with a frequency comparable to that observed for strains estl- and tlcl-4. The ST2-4I mutation failed to complement the ST2-L mutation for both telomere length and senescence, and the ST2-1 point mutation was demonstrated by repairing the plasmid gap for mapping within the EST2 coding region (data not shown), indicating that the correct gene has been cloned. The EST2 gene encodes a novel 103 kD protein with no similarity to other sequences in the database, nor does it possess any motif that provides an indication of its function. In particular, there is no sequence similarity between Est2p and neither of the two subunits of the telomerase enzyme of Tetrahymena (Collins, et al. 1995). Like Estl, Est2 is an unusually basic protein; both proteins have predicted pls of 10. TLC1 and the four EST genes work in a single pathway for telomere replication: The above comparison showed that strains carrying mutations in the EST2, EST3 and EST4 genes are phenotypically similar to strains and tlcl; in fact, many of the new isolates are indistinguishable from the estl-4 and tlcl-4 strains, suggesting that these alleles can also be null mutations. This similarity in phenotype argues that the new EST genes work in the same genetic route for telomere replication as originally defined by the TLC1 gene. EXAMPLE 2 Cloning and sequencing of wild-type EST3. The cloning and sequencing of the wild-type EST3 gene revealed a gene structure with a usual DNA sequence that did not provide immediate indications about the nature of the EST3 gene product. The only two open reading structures contained within the genetic boundaries of the gene were each less than 100 amino acids (Figures 7 and 8). This suggested that either: (i) one or both of the open reading structures were required for the function of the gene; or (ii) the product of the est3 gene was an RNA minus the open reading structure required for telomere replication. To distinguish between these two possibilities, it was shown that a series of nonsense mutations constructed in any of the two open reading structures eliminate function. It was further shown that several of these nonsense mutations were suppressed in an ancestor strain that contained an ocher suppressor transfer RNA. These data definitely show that the translation of both open reading structures was required for the function of the gene. However, the ectopic expression of the two trans-open reading structures of the separated promoters did not restore the function of the gene. In addition, one of the EMS mutations recovered from our selection was mapped in the region between the two open reading structures, indicating that genetic information in this region was important for the function. After a more careful re-examination of this region of the EST3 sequence, we identified a sequence of four nucleotides (CTTA) in common with the +1 structure change site found in the elements of yeast retrotransposons, Tyl, Ty2 and Ty4 , suggesting a similar mechanism to generate the protein Est3. To determine if the EST3 gene similarly used ribosome shift structure (abbreviated RF below) to generate a protein product, an EST3 analysis was carried out to demonstrate that several components that were regulated for the Ty ribosome change structure were also essential for the change structure of EST3. To summarize the structure of Tyl ribosome change, this process depends on an unusual leucine isoaceptor, tRNALEU (UAG) which is able to recognize the six leucine codons (Belcourt and Farabaugh 1990). This tRNA can slide from CTT to TTA (thereby sliding to the reading structure +1) as a consequence of a translation pause due to the low availability of the tRNA decoding to the next codon. EST3 contains the CTT AGT TGA DNA sequence, where AGT is a codon rarely used for serine (and TGA is a nonsense codon). Therefore, by analogy with Ty, our hypothesis was that the pause in the CTT codon, due to the rarely used subsequent codon, allowed the glide to read the TTA codon, thereby changing the structure of the TTA codon. reading by +1. To test this, three experimental observations have been made (shown in Figure 11B): i. changing the CTT codon to another codon that encodes leucine removes the EST3 function. ii. changing from a low serine codon (AGT) to a high (TCT) eliminates the function. iii. suppressing any nucleotide in the CTTA sequence (to eliminate the need for ribosome exchange structure) results in a functional EST3 gene. These genetic results were confirmed by Western analysis demonstrating the production of both the total length 181 amino acid protein product Est3, as well as the truncated 93 amino acid product which did not undergo structural change (Figure 11C). Figure HA shows the structure of the EST3 gene, 11B shows a mutation analysis of a site of the structure change and 11C shows the results of a Western analysis of the Gal4 activation domain (row 1) and the Est3 protein fused to the activation domain Gal4 (row 2), showing the full-length fusion protein and the truncated version ending after the first open reading structure. EXAMPLE 3 Protein Structure Having determined the primary structure of the Est2 and Est3 proteins, the use of whole proteins and fragments of proteins is allowed to isolate other proteins associated with telomerase. The proteins associated with telomerase are defined as the proteins that are required for the normal live functioning of telomerase. These proteins may be associated with the catalytic activity of telomerase or, alternatively, these proteins may be associated with the ability of the telomerase complex to gain access to the telomere. Fragments of the protein are shown to include any peptide that contains 6 or more contiguous amino acids that are identical to 6 contiguous amino acids of any of the sequences shown in Figures 5, 6 and 10. The fragments can be used to generate antibodies. Particularly useful fragments will be those that conform domains of the Est2 or Est3 protein. Domains are defined as portions of proteins that have a discrete tertiary structure and that are maintained in the absence of the rest of the protein. These structures can be found by techniques known to those skilled in the art. The protein is partially digested with a protease such as subtilisin, trypsin, chymotrypsin or the like and then subjected to polyacrylamide gel electrophoresis to separate the protein fragments. The fragments can be transferred then to a PVDF membrane and subjecting to microsequencing to determine the N-terminal amino acid sequence of the fragments. EXAMPLE 4 Expression of proteins associated with telomerase Knowledge of the primary structure of the EST2 and EST3 gene will allow over expression of the gene in conventional expression systems. Conventional expression systems are seen to include but are not limited to crossover virus, E. coli, vaccinia virus or other equivalent expression systems. For the purposes of this invention, an equivalent expression system results in a protein that is either functionally active or antigenically similar to the native protein. The genes of the present invention can be expressed from the native promoters or can be expressed from heterologous promoters. The promoters can be modified to contain regulatory elements. In order to facilitate the purification of the recombinantly expressed proteins, the proteins can be expressed in the form of fusion proteins containing heterologous protein sequences. For example, the coding sequence of EST2 or EST3 or fragments thereof can be fused to glutathione-S-transferase (GST). The EST2 or EST3 gene or fragments thereof can be fused into another known protein or peptides that are useful to facilitate purification or allow identification of the fusion protein. Examples of these proteins and peptides are seen to include, but are not limited to, a protein that binds to maltose, six histidine peptides, epitopes of hemagglutinin epitopes of c-myc and any other protein or peptide known in the art for these purposes. Figures 12 and 13 show plasmid maps of specific constructs illustrating the inclusion of a c-mvc label at the N terminus as well as an internal restriction site. The same construct may contain more than one heterologous protein sequence. When more than one heterologous sequence is included in a construct, the heterologous sequences may be the same or different and may be juxtaposed or separated by sequences derived from a protein associated with telomerase. The Est 2 or Est 3 protein or fragments thereof can be fused with the heterologous proteins at the N terminal or at the C terminal. When small peptide fusions are made the peptide can be fused at the N-terminal or C-terminal or the peptide can be fused to the interior of the Est2 or Est3 protein or fragment thereof. In preferred embodiments the heterologous peptide is the hemagglutinin epitope or the c-myc epitope and the epitope is fused at the N-terminus, the C-terminus, and at sites internal to the Est2 or Est3 protein or fragment thereof. When the epitope melts internally to the Est2 or Est3 protein or fragment thereof, the location of the epitope will be selected so as not to interfere with the normal functioning of the protein or the fragment thereof. The fusion proteins can be prepared so that the heterologous portion can be dissociated from the fusion protein by the action of a proteolytic enzyme. That is, a recognition site for a proteolytic enzyme can be incorporated between the heterologous portion of the fusion protein and the portion derived from the Est2 or Est3 protein or fragment thereof. After purification of the fusion protein, the fusion protein can be subjected to proteolysis in order to remove the heterologous portion of the fusion protein. The preparation of the fusion proteins, their expression, and the purification of the resulting fusion proteins can be carried out by techniques well known to those skilled in the art. Examples of these techniques can be found in Sambrook, and collaborators, Molecular Cloning, 2nd edition and Ausubel, and collaborators, current protocols in Molecular Biology. The description of each of these publications is specifically incorporated herein by reference. DNA constructs corresponding to the protein sequences shown in Figure 10A-10F were prepared.

The DNA sequence corresponding to the wild type protein Est3 was inserted into a plasmid containing the Gal4 activation domain coding sequence fused in structure with the coding sequence for the HA epitope. The expression was conducted by the ADH promoter. The plasmids were transfected into yeast strain AVL78 and 1.5 milliliters of cultures were grown to the semilog phase. Cells were pelleted and resuspended in 30 μl of SDS gel loading buffer (125 mM Tris pH 6.8, 4 percent SDS, 20 percent glycerol, 2 percent 2-mercaptoethanol, 0.001 percent bromophenol blue), incubated at 95 ° C for 10 minutes. The samples were clarified by centrifugation at 10,000 rpm in a microcentrifuge and l-10μl samples were separated by SDS-PAGE on a 12 percent acrylamide gel. The size of the samples was adjusted to account for several levels of expression of the constructions. The proteins were transferred to nitrocellulose and examined with an anti-HA antibody. The proteins were visualized using horseradish peroxidase conjugated with goat anti-mouse antibody and increased chemiluminescence (Amersham). Figure 15B row 1 shows the background plasmid expressing the Gal4 activation domain fused to the HA epitope. The rows 2 and 7 show the expression of the sequence shown in Figure 10A in which the wild-type EST3 gene fused in the structure with the HA construct of activation domain Gal4. Two proteins corresponding to the product of unchanged structure of 93 amino acids (lower band) and a changed structure product, of full length of 181 amino acids (upper band), were expressed. Row 3 shows the expression of a deletion construct having the sequence depicted in Figure 10C in which amino acids 34-164 of the full-length wild-type protein have been deleted. Row 4 shows the expression of a truncation construct having the sequence shown in Figure 10E in which tyrosine 35 has mutated into a stop codon. Row 5 shows the expression of a truncation construct in which phenylalanine 103 has been mutated into a stop codon. Row 6 shows the expression of a change structure construct that can only express the full length Est3 protein of 181 amino acids. EXAMPLE 5 A new selection in yeast to identify additional proteins associated with telomerase. This example proposes a new approach to recover additional genes required for telomere function in yeast. We describe a new selection of mutants that is based on an ancestral strain that is sensitized to the absence of the telomerase function. The method described here is technically much less complex than our previous selection for EST genes and allows as many as 106 colonies to be selected to determine est-like defects. This method is based on the observation that a strain cdc! 3-l- tlcl-? has two synthetic phenotypes: the maximum permissible temperature of a senescence phenotype as seen in Figure 14. This observation is not exclusive to the strain cdc! 3-l- tlcl-, 4, because our unpublished data have shown that strains cdc! 3-l- suppressed either EST1, EST2 or EST3 also exhibit this behavior. This indicates that this is a general consequence of the loss of the EST / TLC1 route on a cdc! 3-l- ancestor. Therefore, the increased temperature sensitivity can be used as the basis for selecting additional mutations which, when in the presence of a cdc! 3-l- mutation. It gives the same phenotype. The procedure employs a plasmid redistribution strategy, starting with a cdc! 3-l- strain transfected with a plasmid pCDC13-URA3. The presence of the plasmid containing a wild-type allele of the CDC13 gene compensates for the temperature-sensitive mutation in the chromosomal copy of the gene. After mutagenesis, single colonies will be plagued on CM-Ura plates (complete medium minus uracil) and incubated at 23 ° C. This selects colonies that retain the plasmid. As soon as they grow completely, the colonies will be plagued in replica on plates with 5-FOA and incubated at 26 ° C.

The presence of 5-FOA selects the absence of plasmid. As a result of this absence, the only allele of the cdc! 3 present is the mutant copy. As shown above in Figure 14, when the temperature-sensitive allele of cdc! 3 is combined with the presence of mutated gene in the EST / TLC1 pathway, the result is an exacerbation of the temperature-sensitive phenotype. Strains carrying only the cdc! 3 temperature sensitive allele grow at 26 ° C while strains carrying both the cdc! 3 temperature sensitive allele and a second mutation in a EST / TLC1 path gene are not viable at 26 ° C. Any isolate that fails to grow on plates with 5-FOA at 26 ° C will recover from the CM-Ura plates. This primary set of mutagenized cultures will be tested for growth at 26 ° C on CM-Ura plates, in the presence of plasmid PCDC13-URA3 (to decide the possibility that the second mutation confers a temperature-sensitive phenotype) and return to test more carefully for the 5-FOA phenotype at 26 ° C (to confirm that this is not due to an inability to lose the plasmid at any temperature or fail to grow in 5-FOA). Candidates that can lose the plasmid at 23 ° C will be tested to determine a phenotype of potential senescence in the absence of plasmid CDC13; As shown in Figure 14B, this phenotype of senescence should be evident after only one groove. Finally, candidates passing these steps will be examined to determine alterations in telomere length (in the presence of plasmid CDC13, to allow sufficient growth for Southern spotting). Since this procedure involves only plating simple colonies and replicas, it is much simplified in relation to our previous selection. EXAMPLE 6 Two-Hybrid System One method for using the EST2 or EST3 genes or fragments thereof in order to identify additional proteins associated with telomerase is through the use of the two-hybrid system. The two-hybrid system uses the restoration of transcriptional activation to indicate an interaction between two proteins. In this system, a eukaryotic transcription activator, generally the yeast GAL4 transcription factor, is divided into two domains, a transcription activation domain and a DNA binding domain. Under normal circumstances the two domains are part of the same protein. However, as long as the two domains can be brought into close contact, a functional transcription activator can be assembled. In use, the EST2 or EST3 gene or fragments thereof can be cloned into the plasmid vector which contains the coding sequence for the DNA binding domain. He The gene or fragment can be inserted into the vector in such a way that it generates a fusion protein in the structure with the domain that binds to the DNA. A DNA library will be constructed in a plasmid that contains the activation domain. The library will be inserted into the plasmid adjacent to the coding sequence of the binding domain and will result in a fusion protein between the binding protein and the genes represented in the library. The library can be constructed from random fragments of DNA or can be constructed from cDNA fragments. The two plasmids will be cotransformed into a suitable yeast strain and the cotransformants will be selected to determine the expression of a functional GAL4 transcription activator. This selection can be carried out by placing a reporter gene, for example 3-galactosidase, under the control of a promoter containing a GAL4 responsive element. When an interaction between the EST2 or EST3 protein or fragment thereof and a protein in the library occurs, the two functional GAL4 domains will be placed in close proximity. This will result in the restoration of a functional transcription activator and the expression of the reporter gene. The colonies will be selected for the expression of the reporter gene and the colonies expressing the reporter gene will be isolated. The plasmid containing the activation domain library fusion protein is recovered and the sequence determined of the library protein. The materials needed to make this two-hybrid selection system are commercially available, for example at Clontech. Figure 10 shows the primary structure of several GAL4 activation domain fusions prepared with the EST3 gene. Figure 10A shows an activation domain HA-HA expressing both the first open reading structure and the product of the complete structure change gene. Figure 10B shows the GAL4-HA fusion with the corrected change structure EST3. Figure 10C shows a truncated deletion derivative after the first 50 amino acids of the first open reading structure. Figure 10D shows the truncation mutant where phenylalanine 103 became a stop codon. Figure 10E shows a GAL4-HA fusion protein where tyrosine 35 became a stop mutation. Figure 10F shows an internal proteolytic fragment of about 70 amino acids. Figure 10G shows an EST3 gene marked at the C terminal with three HA epitopes. Figure 10H shows a construction prepared for the expression of baculovirus wherein the Est3 protein has been labeled at the C terminus with six amino acids histidine. Other fusion proteins that have been prepared include a GST-Est2 fusion protein.

EXAMPLE 7 Production of Antibodies After expression and purification of the Est2 and Est3 proteins or fragments thereof the proteins or fragments can be used to generate antibodies specific to the proteins or fragments thereof. The antibodies can be polyclonal or monoclonal. The production of antibodies will be carried out using conventional techniques well known to those skilled in the art. The techniques used can be found in a variety of references, for example Harlow and Lane, Antibodies, A Laboratory Manual, the description of which is specifically incorporated herein by reference. In summary, the protein or fragment thereof can be purified from native sources or, alternatively, it can be purified from heterologous envelope that expresses a host. When fragments are used, the fragments can be prepared from purified protein by treatment with protease enzymes or alternatively, the fragments can be prepared synthetically using solid phase synthesis technology well known to those skilled in the art. The purified protein or fragment can be mixed with an adjuvant, for example complete Freund's adjuvant or incomplete Freund's adjuvant or other known synthetic adjuvants for the experts in the art. The adjuvant / protein mixture can be injected into a laboratory animal in order to produce an immune response. The immune response can be raised by injecting additional protein material with or without adjuvant. Injection programs and regimens are well known to those skilled in the art. When antibodies specific to the peptides are being made, the peptide can be conjugated to a carrier molecule such as a key-lock type henicyanin, or any other carrier protein known in the art. EXAMPLE 8 Identification of peptides or small molecules that inhibit telomerase function. The current approach to the development of anti-telomerase drugs is to look for compounds that inhibit the activity of the enzyme, a tactic that is being followed in several academic and commercial laboratories. This example, however, focuses on an alternative and innovative means of inhibiting telomerase, inhibiting not the same enzyme but preventing the access of the enzyme to the telomere. This is a new concept for the inhibition of telomerase, based on recent research in our laboratory that has led to the identification and characterization of a protein that binds to the telomere that is required for telomerase access at the end of the chromosome. The parallel development of inhibitors of both enzyme activity and access to the enzyme may provide the opportunity for a more powerful anti-telomerase combination cancer therapy, based on the example of increased efficacy of the combined use of protease inhibitors and reverse transcriptase in the treatment of HIV infection. The characterization of the yeast CDC13 gene (identified in our selection of mutant est) has shown that it has a dual role in telomere function. The CDC13 gene is an essential yeast gene that is required to maintain the integrity of the telomere, as first suggested by a study by Lee Hartwell's laboratory showing that a rapid loss of a telomere chain occurs in the absence of the CDC13 function. Recent work from our laboratory has revealed an additional role for CDC13 in telomere maintenance. This resulted from the discovery of a novel mutation, called cdc! 3-2eSt. which displayed a phenotype virtually identical to that of a telomerase-minus strain. In addition, the analysis of epistasis between the cdc! 3-2eSt mutation and tlcl-A demonstrated that the cdc! 3-2eSt disturbs a CDC13 function that is required for the telomerase pathway. In contrast, genetic analysis of the previously isolated conditional lethal allele of CDC13 (cdc! 3-lts) showed that CDC13 has a separate essential function that must be maintained in addition to the telomerase-based pathway. We have also shown that the Purified Cdcl3 protein is specifically linked to single-chain yeast εerial substrates. Based on these data, we propose that Cdcl3 has two different functions in the telomere. The first of these roles is to protect the end of the chromosome, which is essential for the viability of the cell. Second, the Cdcl3 protein regulates telomerase mediating, either directly or indirectly, the access of this enzyme to the chromosomal term, and that this access is now eliminated by the cdc! 3-2eSt mutation. This second paper provides the basis for blocking the access of the telomerase enzyme to the chromosomal term, also by mutation or potentially via binding or a small molecule or peptide inhibitor to cdcl3p, at the site defined by the cdcl3-2eSt mutation. This then provides an alternative means of inhibiting telomerase activity, via the identification of inhibitors that block the access of the enzyme to the telomere. We propose to identify the human Cdcl3 protein and using it as a tool to identify peptides that bind to this proposed telomerase loading site, via the use of random peptide libraries deployed by phage to identify peptides that will have antagonistic effects on telomerase-related function of CDC13. The random peptide and small molecule libraries can be selected in a variety of ways known to those skilled in the art. One modality preferred will be to select a phage display library. The technique employs fusion of random peptide sequences to either one of two proteins that make up the filamentous phage coat. Multiple rounds of enrichment for the phage exhibiting strong binding to the selector protein is achieved via either affinity or sieving chromatography. An alternative method to select both random peptide libraries and libraries of sticky molecules is to use in vitro telomerase assays and test their inhibition of telomerase enzymatic activity. This type of test can easily be automated using robotic instrumentation to quickly test large numbers of samples. The ability to establish convenient enzymatic assays and the analysis of inhibition data are well known to those of ordinary skill in the art. Very complex libraries will be used. Libraries may incorporate several of the recent modifications such as, monovalent display, which allows the selection of ligands of higher affinity; structurally restricted libraries; and libraries that display peptide D ligands. In addition, more than one library will be selected in order to increase the recovery singularities and expand the range of peptides recovered.

When selected with the Cdcl3 protein, alternate rounds of selection and anti-selection will be used. First a round will be conducted using wild-type Cdcl3 protein. This will select peptides and / or small molecules that are capable of binding to the wild type of the Cdcl3 protein. After this step, a round of antiselection with the Cdcl3- protein, which contains a mutation that avoids binding to the telomerase complex, will eliminate the peptides and small molecules that bind at random sites on the Cdcl3 protein. Therefore, in the anti-selection step, small molecules, peptides and phages that do not bind to the Cdcl3- protein will be recovered. Each library will be selected two to four times to ensure full coverage. Peptides and small molecules identified as being bound to the Cdcl3 protein will be selected to determine the ability to inhibit teloerase activity and these peptides and small molecules will be useful in vivo to avoid telomerase function, thereby avoiding unlimited replication. When the selection protein was derived from a human telomerase complex the identified peptides will find use as anti-tumor agents to suppress the unlimited replication of the tumor cells. When the selection proteins are derived from pathogenic organisms, the peptides identified by this selection can be used to inhibit the growth of the pathogenic organism.

EXAMPLE 9 Identification of mammalian homologs of the four EST genes In this example, methods are described that employ various molecular and / or genetic approaches to use cloned yeast EST genes, as well as est- mutant strains, as reagents to obtain equivalent human genes . A method will be functional complementation. Examples of two human cDNA libraries in yeast expression vectors are the inducible yeast GAL promoter, which provides regulated transcription, present in a high copy yeast redistribution vector (Ninomiya-Tsuji, et al. 1991); the second library was constructed using the constitutive yeast ADH1 promoter and is also present in a high copy yeast vector (Becker, et al. 1991). In addition, two different cDNA libraries (with or without nuclear localization signals) of 24 K. lactis behind the ADH S. cerevisiae promoter in the vector pYES2 (Pearlman, Becherer and Lundblad, unpublished) have been constructed for use in the Identification of homologs for related species more closely. Newly dissected haploid strains est2: rad52: and est3: rad52: will be transformed with one of these libraries and transformants that grow healthy identified; the presence of the rad52 mutation is necessary to avoid the appearance of a deviation route that results in healthy growing derivatives (Lundblad and Blackburn, 1993). In the case of the EST4 / CDC13 gene, the libraries will be selected for complementation of (i) the senescence phenotype of est4 rad52, (ii) the ts phenotype of a cdc! 3-ts mutation and (iii) the lethality of est4. / cdc! 3- (this last experiment will be done using a redistribution type strategy); Complementation of these three different types of mutations may help the ability to recover a complement clone. A second method for isolating homologs will be through cross-hybridization and degenerate polymerase chain reaction. Based on the results we have obtained with EST1, as well as the preliminary results by selecting a spotted zoo of distantly related species with probes EST2, EST3 and EST4, it is likely that we need to collect and sequence a set of 3 to 4 homologs for each gene EST of somewhat related species. This information will be used to look for patterns of amino acid sequences that are signatures for the Est3 protein. The amino acid signature sequences will be used to search the protein sequence databases using a pattern recognition search program (Smith and Smith, 1990) for proteins containing these motifs. The protein sequences identified by this procedure will be analyzed to see if there are additional sequence characteristics that suggest that we have identified an Est3 candidate. Any promising candidate will be analyzed in greater Detail as described below. These amino acid signature sequences can also be used to designate degenerate polymerase chain reaction primers against highly conserved regions to be used for substantial species crossing. If more than two highly conserved regions are identified in a given EST gene, nested degenerate polymerase chain reactions will be performed. The products of the polymerase chain reaction will be selected by size, cloned and sequenced, and the sequence information will be evaluated to determine if we have identified an EST homolog. Polymerase chain reaction clones will be used to identify and sequence full-length cDNA from the appropriate library. The cDNA library source used will be the two libraries described above HeLa cDNA. Since both are in vectors that allow the expression of the cloned human gene in yeast, this has the advantage that candidate genes can be quickly selected to confer a phenotype in yeast as discussed below. Several types of experiments will be used to show if the correct gene has been isolated. Sequence comparisons will be used if the gene has been identified by a polymerase chain reaction jump of a homologous database. Second, each candidate cDNA will be tested to see if it exerts a genetic phenotype in yeast. Each cDNA will verify to determine the complementation of an est3 mutation. If there is no evidence of complementation, each cDNA will be subsequently tested to determine if it can confer a dominant effect on telomere replication in wild-type yeast, in other words, over-expression of this cDNA can inhibit replication of the yeast telomere interfering with the formation of a necessary yeast complex (such as telomerase).

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Guarente, 1995. Mutation in the silencing gene SIR4 can delay aging in S. cerevisiae. Cell 80: 485-496. Kim, N.W. , M.A. Piatyszek, K.R. Prowse, C.B. Harley, M.D. West, et al 1994. Specific association of human telomerase activity with iramortal cells and cancer. Science 266: 2011-2015. Kipling, D., 1995. The Telomere Oxford University Press IC. NY. Kouprina, N.Y., O.B. Pashina, N.T. Nikolaishwili, A.M. Tsouladze and V. L. Larionov, 1988 Genetic control of chromosome stability in the yeast Saccharomyces cerevisiae. Yeast 4: 257-269. Lin, J.J. and V.A. Zakian, 1995. An in vitro assay for Saccharomyces telomerase requires EST1. Cell 81: 1127-1135. Lingner, J., L.L. Hendrick and T.R. Cech, 1994.

Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. Genes and Dev. 8: 1984-1998. Lúe, N.F. and R.D. Kornberg, 1987 Accurate initiation at RNA polymerase II promoters in extracts from Saccharomvces cerevisiae. Proc. Nati Acad. Sci. 84: 8839-8843. Lúe, N.F. and J.C. Wang, 1995 ATP-dependent processivity of a telomerase activity from Saccharomyces cerevisiae. J. Biol. Chem. 270: 21453-21456. Lundblad, V. and J. W. Szostak, 1989. A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 57: 633-643. Lundblad, V. and E.H. Blackburn, 1990 RNA-dependent polymerase motifs in the EST1 protein: tentative identification of a component of an essential yeast telomerase. Cell 60: 529-560. Lundblad, V. and E.H. Blackburn, 1993. An alternative pathway for yeast telomere maintenance rescues estl senescence. Cell 73: 347-360. McEachern, M.J. , and E.H. Blackburn, 1995. Runaway telomerase elongation caused by telomerase RNA gene mutations. Science 376: 403-409. Meeks-Wagner, D., J.S. Wood, B. Garvik and L.H. Hartwell, 1986. Isolation of two genes that affect mitotic chromosome transmission in S. cerevisiae. Cell 44: 53-63. Olovnikov, A.M. , 1973. A theory of marginotomy. The incomplete copying of temperature margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J. Theoretical Biol. 41: 181-190. Rose, M.D., P. Novick, J.H. Thomas, D. Botstein and G.R. Fink, 1987. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60: 237-243. Runge, K.W. and V.A. Zakian, 1993 Saccharomyces cerevisiae linear chromosome stability (les) mutants increase the rate of artificial and natural linear chromosomes. Chromosome 102: 207-217. Schiestl, R.H., and R.D. Gietz, 1989. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Current Genetics 16: 339-346. Shippen-Lentz, D., and E.H. Blackburn, 1990. Functional evidence for an RNA témplate in telomerase. Science 247: 546-552. Singer, M.S. and of. Gottschling, 1994. TLC1: RNA component of Saccharomvces cerevisiae telomerase. Science 266: 404-409. Smeal, T., J. Claus, B. Kennedy, F. Colé and L. Guarente, 1996. Loss of transcriptional silencing causes sterility in oíd rather cells of S. cerevisiae. Cell 84: 633-642. Smith, J.R. and R.G. Whitney, 1980. Intraclonal variation in proliferative potential of human diploid fibroblasts: stochastic mechanisms for cellular aging. Science 207: 82-84. Spencer, F., S.L. Gerring, C. Connelly and P. Hieter, 1990. Mitotic chromosome transmission fidelity mutants in Saccharomyces cerevisiae. Genetics 124: 237-249. Steiner, B.R. , K. Hidaka and B. Futcher, 1996. Association of the Est protein with telomerase activity in yeast. Proc. natl. Acad. Sci. 93: 2817-2821. Strathmann, M., B.A. Hamilton, C.A. Mayeda, M.I. Simon, E.M. Meyerwitz, and collaborators 1991. Transposon-facilitated DNA sequencing. Proc. Natl. Acad. Sci. USA 88: 1247-1250. Struhl, K., 1986. Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms. Mol Cell. Biol. 6: 3847-3853. Wilkie, A.O.M., D.R. Higgs, K.A. Rack, V.J. Buckle, N.K. Spurr, et al., 1991. Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16. Cell 64: 595-606. Zakian, V.A., 1995. Telomeres: beginning to understand the end. Science 270: 1601-1607.

Table 1 Yeast strains All strains were isogenic derivatives of YPH275; the diploids also carry ura3-52 / ura3-52 lys2-801 / lys2-801 ade2-101ade2-101 trpl-4l / trpl-4l his3-j200 / his3-i200 Ieu2-4l / leu2-il and the haploids carry ura3- 52 lys2-801 ade2-101 trpl-4l his3-¿200 leu2-, l pVL106 is an ARS1 LEU2 CEN3 plasmid used in the plasmid linearization assay; see Lundblad and Szostak 1989 for more details.

Although MVL1-MVL26 are derived from TVL227-1A, during the course of selection, many mutant isolates lose either CF or pVL106 (or both); therefore, these two elements are not indicated in the genotypes of MVL1-MVL26.

Claims (45)

1. NOVELTY OF THE INVENTION Having described the foregoing invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property 1. An isolated protein having at least the sequence shown in Figure 5.
2. 2 A peptide having at least six contiguous amino acids identical to six contiguous amino acids of the protein sequence in Figure 5.
3. 3. The peptide according to claim 2, further comprising a GAL4 DNA binding domain.
4. 4. The protein according to claim 2, further comprising a GAL4 activation domain.
5. 5. The peptide according to claim 2, further comprising a peptide epitope.
6. 6. The peptide according to claim 5, characterized in that the epitope is an epitope of hemagglutinin.
7. 7. The peptide according to claim 5, characterized by the peptide epitope is a c-myc epitope.
8. 8. A peptide in accordance with the claim in the claim 5, characterized in that the epitope is fused with the N terminus of the peptide.
9. 9. The peptide according to claim 5, characterized in that the epitope is fused with the C-terminus of the peptide.
10. 10. The peptide according to claim 5, characterized in that the epitope is fused with the interior of the peptide.
11. 11. A DNA molecule encoding the protein sequence in Figure 5.
12. 12. A DNA molecule encoding a peptide having at least six contiguous amino acids identical to six contiguous amino acids of the protein sequence of Figure 5.
13. 13 An isolated protein having at least the sequence shown in Figure 6.
14. 14. A peptide having at least six identical amino acids to six contiguous amino acids of the protein sequence of Figure 6.
15. 15. The peptide in accordance with the claimed in claim 14, which further comprises a binding domain GAL4 DNA.
16. 16. The protein according to claim 14, further comprising a GAL4 activation domain.
17. 17. The peptide according to claim as claimed in claim 14, further comprising a peptide epitope.
18. 18. The peptide according to claim 17, characterized in that the epitope is an epitope of hemagglutinin.
19. 19. The peptide according to claim 17, characterized in that the peptide epitope is a c-myc epitope.
20. 20. A peptide according to claim 17, characterized in that the epitope is fused with the N-terminus of the peptide.
21. 21. The peptide according to claim 17, characterized in that the epitope is fused with the C-terminus of the peptide.
22. 22. The peptide according to claim 17, characterized in that the epitope is fused with the interior of the peptide.
23. 23. A DNA molecule encoding the protein sequence in Figure 6.
24. 24. A DNA molecule encoding a peptide having at least six contiguous amino acids identical to six contiguous amino acids of the protein sequence of Figure 6.
25. A method for identifying genes encoding proteins associated with telomerase comprising the steps of: transfecting a temperature-sensitive organism, the temperature-sensitive organism being in a first gene associated with telomerase, with a plasmid encoding a wild-type allele of the first gene associated with telomerase in which the organism is sensitive to temperature, the plasmid encoding a selection gene; put the body in contact with a mutagen; culturing the mutagenized organism at a permissive temperature in a medium containing a substance that selects for the presence of the plasmid; Cultivating the organism at a higher temperature than a permissible temperature in a medium containing a substance that selects against the presence of the plasmid; and identify organisms that do not grow at a temperature greater than a permissible temperature; identify a mutated gene, distinct from the first gene associated with telomerase, in the organism that does not grow at a temperature higher than the permissible temperature, wherein the mutated gene is a gene associated with telomerase.
26. 26. A method according to claim 25, characterized in that the orqanism is yeast and the gene in which the yeast is sensitive to temperature is CDC13.
27. 27. An antibody that binds the protein of claim 1.
28. 28. An antibody according to claim 27, characterized in that the antibody is a polyclonal antibody.
29. 29. An antibody according to claim 27, characterized in that the antibody is a monoclonal antibody.
30. 30. An antibody that is capable of binding the protein of claim 13.
31. 31. An antibody according to claim 30, characterized in that the antibody is a polyclonal antibody.
32. 32. An antibody according to claim 30, characterized in that the antibody is a monoclonal antibody.
33. 33. An antibody that binds to the peptide of claim 2.
34. 34. An antibody according to claim 33, characterized in that the antibody is a polyclonal antibody.
35. 35. An antibody according to claim 33, characterized in that the antibody is a monoclonal antibody.
36. 36. An antibody that binds to the peptide of claim 14.
37. 37. An antibody according to claim 36, characterized in that the antibody is a polyclonal antibody.
38. 38. An antibody according to claim 36, characterized in that the antibody is a monoclonal antibody.
39. 39. A method for isolating compounds that inhibit telomerase, comprising the steps of: contacting a library of compounds with a selection protein; and isolating a compound that binds to the selection protein, characterized in that the selection protein is a protein associated with telomerase.
40. 40. A method according to claim 39, wherein the selection protein is encoded by an EST1 gene.
41. 41. A method according to claim 39, wherein the selection protein is encoded by an EST2 gene.
42. 42. A method according to claim 39, wherein the selection protein is encoded by an EST3 gene.
43. 43. A method according to claim 39, wherein the selection protein is encoded by a CDC13 gene.
44. 44. A method according to claim 39, characterized in that the compounds are selected from the group consisting of peptides and small molecules.
45. 45. A method for identifying homologs to proteins associated with telomerase comprising the steps of: identifying proteins associated with telomerase in two or more organisms; identify at least one reason that is preserved in all organisms; and search in a database for proteins containing the conserved motifs where proteins having a conserved motif are telomerase-associated proteins.