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WO1998023759A2 - Identification d'inhibiteurs empechant l'acces de la telomerase aux terminaisons chromosomiques - Google Patents

Identification d'inhibiteurs empechant l'acces de la telomerase aux terminaisons chromosomiques Download PDF

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WO1998023759A2
WO1998023759A2 PCT/US1997/021272 US9721272W WO9823759A2 WO 1998023759 A2 WO1998023759 A2 WO 1998023759A2 US 9721272 W US9721272 W US 9721272W WO 9823759 A2 WO9823759 A2 WO 9823759A2
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peptide
protein
telomerase
antibody
gene
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Victoria Lundblad
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Baylor College Of Medicine
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Priority to JP52474398A priority patent/JP2001507570A/ja
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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Definitions

  • the present invention relates to the fields of cellular and molecular biology.
  • the present invention relates to genes encoding proteins that play a role in telomerase function and regulation.
  • Telomeres are specialized sequences present at the ends of linear eukaryotic chromosomes, and are required for genomic stability and complete replication of chromosomal termini (reviewed in Blackburn and Greider 1995; Zakian 1995; Greider 1996). Telomeres are characterized by a tandem array of short repeated DNA sequences; in humans, the telomeric repeat sequence is a d(TTAGGG) n .
  • 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 onto the 3' end of the G-rich strand of the telomere, and uses an internal template present in an RNA subunit to dictate the sequence of the added telomeric DNA.
  • the gene encoding the RNA component of telomerase has been identified in a number of species (Greider and Blackburn 1989; Shippen-Lentz and Blackburn 1990; Lingner, et al.
  • telomere enzyme complex Biochemical purification of telomerase 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.
  • telomere deficiency Two genes have been previously identified in Saccharomyces cerevisiae which, when deleted, result in a cell with phenotypes expected from the elimination of telomerase activity.
  • the first of these is the EST1 gene, which encodes a highly basic 82 kD protein 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 estl null mutant strains display two characteristics predicted for a telomerase deficiency: progressive loss of telomere sequences from chromosomal termini and a senescence phenotype (manifested as a steady decline in cell viability). Strong support that such phenotypes could be diagnostic for a defect in telomerase came from the demonstration that a yeast strain deleted for the TLCl gene, encoding the yeast telomerase RNA, exhibits the same set of phenotypes
  • telomerase activity is still present in chromatographic fractions prepared from an estl- A 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
  • Estl protein binds single-stranded yeast telomere sequences Estl may function as a telomerase component responsible for telomere recognition. Consistent with this, Estl protein is associated with the telomerase RNA and/or enzyme activity (Lin and Zakian 1995; Steiner, et al. 1996), although it was not possible to determine whether the association was quantitative in these experiments.
  • the properties of Estl binding to telomeric DNA are also consistent with a role as a telomere-end-binding protein acting as a positive regulator of telomerase function by directing telomerase onto the chromosomal terminus. In either model, the requirement for Estl function might be obviated in the in vitro experiments in which naked DNA is used as a substrate for telomerase elongation.
  • telomeres In mammalian cells, telomerase is highly regulated: it is present in the germ line but is absent or greatly reduced in most normal tissues. Consistent with the absence of this enzyme, telomeres shorten in these normal cells.
  • telomerase activity is up-regulated, relative to the levels present in normal cells. This up-regulation is presumed to be necessary in order to maintain telomere length and allow unregulated proliferation during tumor development.
  • This proposal is supported by a substantial survey of the status of enzyme activity in different tissues: telomerase activity is expressed in 851 out of 998 human tumor tissues, but is not present in most normal tissues, with the exception of the germ line and hematopoietic stem cells (Shay and Wright 1996, and references therein).
  • telomerase is expressed at higher levels in late-stage cancers as compared to early-stage cancers, with some data suggesting that the survival rate of patients correlated with the levels of telomerase activity (Hiyama, et al. 1995a. 1995b).
  • telomerase inhibition may have limited side effects when used as an anti-cancer therapeutic.
  • telomere activity is present in certain human cell lines such as hematopoietic cell lines
  • EST1, EST2 and EST3 are novel genes that function solely in the same pathway for telomere replication as defined by TLCl (Lendvay, et al., 1996).
  • the fourth gene (initially called EST4 but subsequently shown 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 for maintaining telomere integrity, as first suggested by a study from Lee Hartwell's laboratory showing that a rapid loss of one strand of the telomere occurs in the absence of CDC13 function (Garvik, et al. 1995).
  • CDC13 has a separate essential function that must be maintained in addition to the telomerase-based pathway.
  • purified CDC13 protein binds specifically to single-stranded yeast telomeric substrates. Based on these data, we proposed that CDC13 has two distinct functions at the telomere (Nugent, et al. 1996). The first of these proposed roles is to protect the end of the chromosome, which is essential for cell viability. Second, we proposed that
  • Cdcl3 protein regulates telomerase by mediating, either directly or indirectly, access of this enzyme to the chromosomal terminus, and that this access is now eliminated by the cdcl3-2— mutation.
  • the present invention provides the primary structure of two new telomerase-associated proteins Est2 and Est3.
  • An object of the present invention is to provide a method for identifying and isolating other telomerase-associated proteins using the Est2 or Est3 proteins or fragments thereof.
  • An object of the present invention is to provide a method for identifying and isolating other telomerase-associated proteins using the Est2 or Est3 proteins or fragments thereof to generate antibodies and to use the antibodies thus generated to purify telomerase-associated proteins.
  • An object of the present invention is to provide a method to identify telomerase-associated proteins using standard biochemical techniques.
  • An object of the present invention is to provide a novel method for screening for EST genes.
  • An object of the present invention is 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 provide a method for finding the human homologues of yeast telomerase-associated proteins by identifying conserved motifs in the yeast telomerase-associated proteins and searching human protein databases for such conserved motifs.
  • Figure 1 shows a schematic representation of the experimental protocol used to isolate the genes coding est mutations.
  • Figure 2A shows a Southern blot analysis of telomeric DNA of yeast strains having mutations in the EST genes.
  • Figure 2B shows the results of successive streak-outs of mutant yeast strains.
  • Figure 3A shows a Southern blot analysis of telomeric DNA of yeast strains having mutations in the EST genes.
  • Figure 3B shows the results of a viability assay of yeast strains with various mutations.
  • Figure 4A shows a Southern blot analysis of yeast strains with various mutations.
  • Figure 4B shows a Southern blot analysis of yeast strains with various mutations.
  • Figure 4C shows the results of a viability assay of yeast strains with various mutations.
  • Figure 5A shows a schematic representation of the strategy used to locate the EST2 open reading frame.
  • Figure 5B shows the primary structure of the EST2 gene product.
  • Figure 5C shows the results of a viability assay of yeast strains with various mutations.
  • Figure 6 shows the primary sequence of the EST3 gene product.
  • Figure 7 shows the nucleotide sequence of the EST3 gene including flanking regions and location of the + 1 ribosomal frame shift.
  • Figure 8 shows the transposon mapping of the EST3 gene.
  • Figure 9 shows a model for ribosome frame shifting in EST3.
  • Figure 10A-10H show various EST3 constructs.
  • 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 viability of a mutant strain containing
  • Figure 15A shows a schematic representation of the structure of the
  • Est3-fusion proteins constructs prepared and Figure 15B shows a Western blot analysis of the expression of the protein constructs.
  • Yeast strains and media ⁇ S. cerevisiae strains used in this study are shown in Table 1; YPH275 was the diploid parent of all strains used in these studies.
  • the mutant derivatives MVLl to MVL26 were isolated, as described below, from the haploid strain TVL227-1A, obtained by transformation of YPH275 with plasmid PVL106 (Lundblad and Szostak 1989) and subsequent sporulation and dissection.
  • TLV120 and TLV140 were described previously (Lundblad and Szostak 1989; Lundblad and Blackburn 1993); DLV131 was constructed from YPH275 by introducing a tlcl- ⁇ ::LEU2 disruption, constructed by PCR-mediated deletion (Baudin, et al. 1993) of sequences between 192 and 909. Strains were grown at 30 °C and in standard media (Guthrie and Fink 1991), except that chromosome loss assays were performed on media with limiting adenine (6 ⁇ g/ml) to enhance the detection of red Ade- sectors in colonies. Canavanine plates and 5-FOA SC-LEU plates were prepared as described in Ausubel, et al. 1987.
  • Yeast transformations were performed using the lithium acetate method (Schiestl and Gietz 1989); sporulation and tetrad dissections were carried out using standard techniques (Guthrie and Fink 1991).
  • EMS Mutagenesis TVL227-1A was grown overnight to saturation in SC-LEU-TRP, diluted ten-fold into YEPD media and grown to O.D. 0.8 to 1.0.
  • Mutant screen Colony sectoring screen: 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, cultures were plated on SC-LEU-TRP plates at a density of 500 - 1000 per plate and colonies allowed to grow at 30 ° C to full size, for approximately three to four days. Pools of ⁇ 7500 colonies were generated by re-suspending colonies from SC-LEU-TRP plates in sterile dH a O, and re-plating at a density of ⁇ 350 per plate on SC-LEU plates with limiting adenine.
  • the total number of colonies re-plated from each pool was approximately four-fold the size of the pool, to increase the odds that each colony in the original pool was represented. Plates were incubated for seven days at 30 ° C to allow full development of red Ade- sectors; in addition, overnight incubation at 4 ° C prior to scoring for chromosome loss helped enhance detection of red sectors (F. Spencer, personal communication). If two est mutants were isolated from a single pool and were later shown to map to the same complementation group, they were considered to be siblings of each other and only one was subsequently analyzed.
  • Plasmid linearization assay Colonies with increased numbers of Ade ⁇ sectors were picked into microtiter dish wells containing 0.2 ml SC-
  • LEU media to select for retention of pVL106 (Lundblad and Szostak 1989).
  • 5 ⁇ l aliquots from each well were transferred to SC-LEU-5-FOA plates, using a multiple-channel pipettor, and plates were subsequently incubated 3 more days at 30 °C to allow the growth of 5-FOA resistant micro-colonies.
  • a wild type un-mutagenized strain gives rise to ⁇ 10-15 micro-colonies on the 5-FOA plates in this assay; all mutants that showed more than a three-fold reduction were saved for further analysis.
  • telomere length was assayed with increasing numbers of generations of growth ( Figures 2 and 4), cultures were grown to saturation in YEPD and subsequently diluted by a factor of 10 "4 and allowed to re-grow to saturation; this process was repeated three to four times.
  • Haploid est spores of opposite mating type were used to generate diploids (also by un-selected matings) which were heterozygous for two different EST genes. Diploids were sporulated and dissected and spores analyzed for cellular senescence and telomere length. Initially, both tetrads with four viable spores as well as tetrads with less than four viable spores were analyzed; when no evidence for synthetic lethality was obtained (see Figure 4), subsequent analysis concentrated on tetrads with four viable spores.
  • ⁇ 8 tetrads were analyzed for telomere length and senescence, with at least four tetratype and non-parental ditype tetrads recovered and analyzed for each.
  • the resulting diploid strains were also used to construct the double and single mutant haploid strains analyzed in Figure 4.
  • the est2-l, est3-l and est4-l haploids used to generate these diploids were the result of at least three backcrosses to wild type, and the estl- A::HIS3 and tlcl- ⁇ ::LEU2 haploids were isolated from TLV120 and DVL131, respectively.
  • est survivor strains Each freshly generated est haploid strain was grown in liquid YEPD culture, with successive dilutions, for 120 to 150 generations and subsequently plated for single colonies on YEPD plates. After two days growth at 30 °C, large colonies were examined for both growth phenotype and telomere structure on Southern blots, as described above.
  • the healthy growth phenotype and wild type telomere length were shown in each case to be plasmid-dependent. Plasmids were subsequently recovered from each yeast strain by rescue through E. coli. A combination of restriction mapping and Southern analysis of the genomic inserts demonstrated that the four plasmids recovered from the 2 ⁇ LEU2 library were identical to each other, and had genomic inserts that overlapped with the inserts present in the two plasmids recovered from the URA3 CEN genomic libraries.
  • the est2- ⁇ 1::URA3 disruption mutation was constructed by deleting an internal 2.2 kb Hindlll fragment, removing aa 13 to aa 686 of the EST2 ORF, and inserting a 1.17 kb URA3 Hindlll fragment. This disruption was introduced into TVL140 to generate an EST2/est2- ⁇ l::URA3 heterozygous diploid (strain TVL228), which was confirmed by Southern analysis. Twelve tetrads were analyzed in detail for senescence and telomere phenotypes, as described above.
  • the original identification of the estl-1 mutant employed a screen that assayed the behavior of a circular plasmid containing inverted repeats of Tetrahymena telomeric sequences bracketing 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 yeast telomeric sequences onto the Tetrahymena termini in order to form functional telomeres.
  • mutants defective in the linearization process can be identified by screening for colonies that exhibit decreased frequencies of resistance to the drug 5-FOA (Ura + cells convert 5-FOA to a toxic intermediate, whereas the loss of URA3 prevents this; Boeke, et al. 1984). Potentially, one subset of mutants that could be identified by this assay would also be defective for the ability to form functional telomeres, once the plasmid was linearized; such mutants should also exhibit shorter chromosomal telomeres.
  • the estl-1 mutant was the one candidate that fell into this class, with a progressive reduction in telomere length with increasing growth of the culture (Lundblad and Szostak 1989).
  • one drawback to the plasmid linearization assay was that the process of testing individual colonies for their frequency of 5-FOA resistance was labor-intensive, which limited the original screening to only ⁇ 7000 mutagenized colonies.
  • estl-1 mutant and the subsequently constructed estl- ⁇ strain were shown to have two additional associated phenotypes: a senescence phenotype, manifested as a gradual decrease in cell viability, and an accompanying progressive increase in frequency of chromosome loss (Lundblad and Szostak 1989).
  • a senescence phenotype manifested as a gradual decrease in cell viability
  • an accompanying progressive increase in frequency of chromosome loss Loundblad and Szostak 1989.
  • all four of the phenotypes displayed by estl mutants were incorporated into an expanded screening protocol, presented in Figure 1. Due to the difficulty in screening large numbers of colonies in the plasmid linearization assay, this step was first preceded by an enrichment step for colonies that exhibited increased frequencies of chromosome loss.
  • Detection of alterations in chromosome stability relied on a previously developed color-based visual assay that monitored the presence/absence of a non-essential 150 kb artificial test chromosome (Spencer, et al. 1990).
  • a number of screens have been performed which use this assay, or similar variants, to detect mutants which affect chromosome fidelity (Meeks-Wagner, et al. 1986; Spencer, et al. 1990; Kouprina, et al. 1988; Runge and Zakian 1993). None of these previous screens, which have collectively identified a large number of genes involved in chromosome maintenance, detected mutations in either EST1 or the new EST genes identified in this work.
  • a total of 350,000 single colonies from 12 independently mutagenized cultures of yeast were screened by the four- tiered system shown in Figure 1. Individual colonies from cultures mutagenized with EMS to an average survival of ⁇ 50 % 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-fold and 50-fold, respectively. Southern blot analysis of telomere length of 375 candidates that passed these two tests led to the identification of 49 mutants with either decreases (ranging from ⁇ 50 bp to >300 bp) or increases (ranging from 150 bp to > 2 kb) in telomere length.
  • the new esf mutants map to four genes: Each of the est mutants, when crossed to wild type, were recessive for telomere length, chromosome loss and senescence (data not shown). The 19 mutants were also tested for whether they contained mutations in either EST1 or TLCl by crossing each mutant to an estl-A strain and an tlcl-A strain; the resulting diploids were examined for both telomere length and viability. All 19 mutant strains fully complemented both phenotypes of the tlcl- mutation, indicating that no new alleles of TLCl had been identified (data not shown).
  • est2, est3 and est4 mutants are phenotypically indistinguishable fromestl and tlcl strains: If the three new EST genes play a similar role in telomere replication as EST1 and TLCl, mutations in these new genes should display phenotypes similar to those exhibited by estl-A and tlcl-A strains.
  • tlcl, estl, est2 and est3 strains four successive sub-cultures, representing a total of ⁇ 65 - 70 generations of growth, are shown; due to the slightly weaker phenotype of est4-l, a fifth subculture (corresponding to ⁇ 13 - 15 additional generations of growth) is included.
  • Lanes 1 and 2 and lanes 24 and 25 show the first and fourth sub- culture, respectively, of two ESTVTLCV strains handled in parallel.
  • the bracket indicates a telomeric band that represents approximately 2/3 of the telomeres in this strain (those that are Y'-containing); arrows indicate restriction fragments corresponding to individual non-Y'-containing telomeres.
  • Panel B shows the viability ofestl- ⁇ 3::HIS3, tlcl- ⁇ ::LEU2, est2-l, est3-l and est4-l, shown as three successive streak-outs on YEPD plates, differing from each other by about 25 generations of growth; for est4-l, one additional successive streak-out is shown.
  • Figure 2A shows the telomere length decline that a representative mutant from each of the new complementation groups displayed over time, as compared to that displayed by estl- ⁇ and tlcl-O strains. Strains were grown for ⁇ 80 generations, with DNA prepared for Southern analysis every ⁇ 15 generations. Telomere length in each mutant declined with time, and did so to the same degree as observed in estl-A and tlcl- ⁇ strains. In parallel with the analysis of telomere length, we also analyzed the growth and chromosome stability over time in these new mutants.
  • each new est mutant In parallel with the senescence phenotype, each new est mutant also showed a striking 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 estl- ⁇ strains (Lundblad and Szostak 1989).
  • New est mutants utilize the same alternative pathway for telomere maintenance as estl-A or tlcl- A strains: Previous analysis has shown that late in the outgrowth of estl mutant cultures, a small proportion of cells are able to escape the lethal consequences of the absence of EST1 function. These estl- ⁇ survivors arise as a result of an bypass pathway for telomere maintenance that is activated in late estl cultures (Lundblad and Blackburn 1993). Activation of this alternative pathway occurs as the result of a global amplification and rearrangement of both telomeric G-rich repeats and sub- telomeric regions.
  • telomere function is restored and the survivors have re-gained a normal or near-normal growth phenotype.
  • This pathway requires the RAD52 gene, which mediates the majority of homologous recombination events in yeast; in the absence of
  • estl rad52 strains cannot generate late-culture survivors and instead die completely after ⁇ 40 to ⁇ 60 generations (Lundblad and Blackburn 1993). Consistent with the other similarities between EST1 and TLCl, a tlcl- ⁇ strain also exhibits alterations of telomeric and sub- telomeric DNA (Singer and Gottschling 1994) which is RAD52-dependent
  • est2, est3 and est4 strains were also capable of participating in this process.
  • two to three isolates for each mutant were grown in culture for 120 to 150 generations and plated for single colonies.
  • Several single colonies from each culture were analyzed for growth characteristics and telomere structure.
  • Panel A shows a Southern blot of genomic yeast DNA. Lanes 1 and 11, a mixture of ⁇ Hindlll and a single d(G 1 . 3 T)-containing 4.0 kb fragment detected by the probe; lanes 2 and 10, EST1 + TLC1 + ; lanes 3 and 9, early tlcl- ⁇ ::LEU2; lanes 4 - 8, survivors isolated from tlcl- ⁇ ::LEU2, estl- ⁇ 3::HIS3, est2-l, est3-l and est4-l, respectively.
  • brackets A shorter exposure of the region indicated in brackets is shown below, to demonstrate the degree of amplification of Y'-containing sub- telomeric bands (indicated by brackets) that occurs in survivor strains (Lundblad and Blackburn 1993). Note that the probe detects the yeast d(G x . 3 T) telomeric repeats, indicating that both Y' and d(G 1 . 3 T) sequences are highly amplified in survivors.
  • Panel B shows the viability of ESTV rad52::LEU2; tlcl- ⁇ ::LEU2 rad52::LEU2; estl- ⁇ 3::HIS3 rad52::LEU2; est2-l rad52::LEU2; est3-l rad52::LEU2; and est4-l rad52::LEU2, shown as two successive streak-outs.
  • Figure 3A shows the Southern blot of one such survivor from each of the five tlcl or est strains, probed with poly d(GT); all displayed the same type of telomere rearrangement originally observed in estl mutants, characterized by extensive amplification of both d(G 1 . 3 T) telomeric DNA and Y' sub-telomeric repeats (Lundblad and Blackburn 1993).
  • each of these est2, est3 and est4 survivors had now acquired a growth phenotype that closely approximated that of wild type (data not shown), similar to that previously observed for estl survivors.
  • TLCl and the four EST genes function in a single pathway for telomere replication:
  • the above comparison demonstrated that strains carrying mutations in the EST2, EST3 and EST4 genes are phenotypically similar to estl and tlcl strains; in fact, many of the new est isolates are indistinguishable from estl- ⁇ and tlcl- ⁇ strains, suggesting that these alleles may also be null mutations.
  • This similarity in phenotype argues that the new EST genes function in the same genetic pathway for telomere replication as originally defined by ESTl.
  • the alternative possibility is that these five genes operate in more than one pathway, each required to form a functional telomere.
  • each new est mutant was crossed against estl- ⁇ , tlcl- ⁇ or the other est mutant strains to generate diploids heterozygous at several EST/TLC1 loci. The subsequent diploids were sporulated and dissected to generate wild type, single mutant and double mutant spore products.
  • Each double mutant strain was analyzed for telomere length, senescence and chromosome loss ( Figure 4 and data not shown), in parallel with single mutants and wild type. In every case, no enhancement of phenotype was seen for double mutant combinations.
  • Figure 4. Epistasis analysis of tlcl and est mutants.
  • Figures 4A and 4B show telomere length for a representative set of double mutant strains compared with the appropriate single mutant strains; in each strain, telomere length declined over time to the same degree.
  • Figure 4C there was no alteration in the senescence phenotype in any of the double mutant strains, relative to single mutants. This is in striking contrast to the severe enhancement of senescence that occurred as a result of the introduction of a rad52 mutation into any of the est mutant strains ( Figure 3B and 5C).
  • the timecourse of the senescence phenotype of a haploid strain carrying only the tlcl- ⁇ mutation was accelerated when the tlcl- ⁇ strain was derived from DVL132 (TLCl * /tlcl- ⁇ ESTV/estl-A), compared to the same isogeneic haploid tlcl- ⁇ strain derived from DVL131 (TLCl + /tlcl- ⁇ EST /EST ); (compare the tlcl- ⁇ strains 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 the five genes in this pathway.
  • telomere length was slightly shorter in multiply heterozygous diploids as compared to single heterozygote (D.K.M., unpublished data). Note that the conclusions drawn from the data presented in Figure 4, that additive combinations of different mutations do not show enhancement of phenotype, were made from comparisons between sets of double and single mutant strains derived from the same diploid parent; comparisons to the single mutant haploids in Figure 2 are not valid. Although we do not understand the molecular basis for this phenomenon, it suggests that alterations in gene dosage may be disrupting a complex or a set of interacting complexes.
  • the EST2 gene encodes a novel, highly basic protein:
  • the wild type EST2 gene was cloned by complementation of the senescence phenotype of est2-l, from both high and low copy genomic libraries (see Materials and
  • telomere shortening and senescence phenotypes of an est2-l mutant strain were assayed for complementation of the telomere shortening and senescence phenotypes of an est2-l mutant strain; insertions which fully complement the Est- phenotype are indicated as an open triangle above the rectanglar box representing the DNA, and failure to complement is indicated by a solid triangle below the representation of the DNA.
  • One insertion, 74 bp upstream of the initiating AUG exhibited an intermediate phenotype, in that the senescence phenotype was complemented but a telomere shortening phenotype was observed (data not shown).
  • the positions of five small ORFs and a poly-A tract in the promoter region of EST2 are also indicated.
  • the promoter region of the EST2 gene exhibited several notable features found in common with that of ESTl. First, both genes diverge significantly from the consensus sequence found around the initiating AUG of many yeast genes (Hinnebusch and Liebman 1991; Galibert, et al. 1996). In addition, for both ESTl and EST2, the region just upstream of the AUG initiation codon has the potential to encode multiple small ORFs, a feature that is not usually observed in the promoter regions of yeast genes (Cigan and Donahue 1987). In the rare genes where such upstream ORFs have been analyzed, they have been shown to be involved in translational control of gene expression (Hinnebusch 1992).
  • EST2 promoter One feature of the EST2 promoter that was not found in ESTl was a 32 bp long poly (dA) tract just upstream of the start of the EST2 coding region.
  • Poly(dA-dT) tracts have previously been implicated intranscriptional activation (Struhl 1986; Lue and Kornberg 1987); the EST2 poly (dA) tract may also be a promoter element, although it is unusually close to the translational start.
  • an internal deletion within this ORF was removed and replaced with the URA3 gene.
  • This construct was introduced into a diploid strain by one-step gene replacement, and the resulting heterozygous strain was sporulated; >80% of the tetrads had four viable spores for each, indicating that this gene was not essential for immediate viability.
  • the haploid strains from 12 tetrads were further analyzed for senescence and telomere length; for all 12 tetrads, an est mutant phenotype co-segregated 100% of the time with the URA3 marker.
  • est2- ⁇ l The senescence phenotype of est2- ⁇ l was identical to that displayed by an estl- ⁇ strain, both in the presence or absence O ⁇ RAD52 gene function (Figure 5C); furthermore, consistent with the double mutant analysis conducted above with the est2-l point mutation, a strain deleted for both genes showed no enhanced phenotype with regard to senescence ( Figure 5C) or telomere length (data not shown).
  • an est2- ⁇ l strain was capable of giving rise to i?AD52-dependent survivors with a frequency comparable to that observed for estl- ⁇ and tlcl- ⁇ strains.
  • est2- ⁇ l mutation failed to complement the est2-l mutation for both telomere length and senescence, and the est2-l point mutation was shown by plasmid gap-repair to map within the EST2 coding region (data not shown), indicating that the correct gene had 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 motifs that provide clues as to its function. In particular, there was no sequence similarity between Est2p and either of the two protein sub-units of the Tetrahymena telomerase enzyme (Collins, et al. 1995). Like Estl, Est2 is an unusually basic protein: both proteins have predicted pis of 10.
  • TLCl and the four EST genes function in a single pathway for telomere replication:
  • the above comparison demonstrated that strains carrying mutations in the EST2, EST3 and EST4 genes are phenotypically similar to estl and tlcl strains; in fact, many of the new est isolates are indistinguishable from estl-A and tlcl- ⁇ strains, suggesting that these alleles may also be null mutations.
  • This similarity in phenotype argues that the new EST genes function in the same genetic pathway for telomere replication as originally defined by the TLCl gene.
  • RNALEU UAG
  • This tRNA can slip from CTT to TTA (thereby slipping to the +1 reading frame) as a consequence of a translational pause due to low availability of the tRNA decoding the next codon.
  • EST3 contains the DNA sequence CTT AGT TGA, where AGT is a rarely used codon for serine (and TGA is a nonsense codon). Therefore, by analogy with Ty, our hypothesis was that pausing at the CTT codon, due to the rarely used subsequent codon, allowed slippage to read the codon TTA, thereby changing the reading frame by +1.
  • Figure HA shows the structure of the EST3 gene
  • 11B shows a mutational analysis of the frameshift site
  • 11C shows the results of a
  • telomerase-associated proteins are defined as those proteins that are required for the normal in vivo 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 seen to include any peptide that contains 6 contiguous amino acids or more that are identical to 6 contiguous amino acids of either of the sequences shown in Figures 5,6 and 10. Fragments may be used to generate antibodies. Particularly useful fragments will be those that make up domains of the Est2 or Est3 protein.
  • Domains are defined as portions of the proteins having a discrete tertiary structure and that is maintained in the absence of the remainder of the protein. Such 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 then be transferred to a PVDF membrane and subjected to micro sequencing to determine the amino acid sequence of the N-terminal of the fragments.
  • telomerase-associated proteins Expression of telomerase-associated proteins.
  • EST2 and EST3 gene will permit the over expression of the gene in conventional expression systems.
  • Conventional expression systems are seen to include but are not limited to baculovirus, E. coli, vaccinia virus or other equivalent expression systems.
  • an equivalent expression system results in a protein that is either functionally active or antigenically similar to the native protein.
  • genes of the present invention may be expressed from the native promoters or may be expressed from heterologous promoters. Promoters may be modified to contain regulatory elements.
  • the proteins may be expressed in the form of fusion proteins contain heterologous protein sequences.
  • the coding sequence of the EST2 or EST3 or fragments thereof may be fused to glutathione-S- transferase (GST).
  • GST glutathione-S- transferase
  • the EST2 or EST3 gene or fragments thereof my be fused to other known protein or peptides that are useful to facilitate purification or to permit identification of the fusion protein.
  • Proteins and peptides are seen to include, but are not limited to, maltose binding protein, six histidine peptide, epitopes from hemagglutinin epitopes from 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-myc tag at the N-terminal as well as at an internal restriction site.
  • the same construct may contain more than one heterologous protein sequence.
  • the heterologous sequences may be the same or different and may be juxtaposed or may be separated by sequences derived from the telomerase-associated protein.
  • the Est2 or Est3 protein or fragments thereof may be fused to the heterologous proteins at the N-terminal or the C-terminal.
  • the peptide may be fused at the N-terminal or the C-terminal or the peptide may be fused to the interior of the Est2 or Est3 protein or fragment thereof.
  • the heterologous peptide is the hemagglutinin epitope or the c-myc epitope and the epitope is fused at the N-terminal, C-terminal, and at locations internal 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 fragment thereof.
  • the fusions proteins may be prepared such that the heterologous portion may be cleaved from the fusion protein by the action of a proteolytic enzyme. That is to say, a recognition site for a proteolytic enzyme may 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 may be subjected to proteolysis in order to remove the heterologous portion of the fusion protein.
  • fusion proteins preparation of fusion proteins, their expression, and purification of the resultant fusion proteins may be accomplished by techniques well known to those skilled in the art. Examples of such techniques can be found in
  • DNA constructs corresponding to the protein sequences shown in Figure 10A-10F were prepared.
  • the DNA sequence corresponding to the wild type Est3 protein was inserted into a plasmid containing the Gal4 activation domain coding sequence fused in frame to the coding sequence for the HA epitope. Expression was driven by the ADH promoter.
  • the plasmids were transfected into yeast strain AVL78 and 1.5 ml cultures were grown to mid- log phase.
  • FIG. 15B lane 1 shows the background plasmid expressing the Gal4-activation domain fused to the HA epitope.
  • Lanes 2 and 7 show the expression of the sequence shown in Figure 10A in which the wild type EST3 gene fused in frame to the Gal4 activation domain- HA construct. Two proteins are expressed corresponding to the non- frameshifted product of 93 amino acids (lower band) and a full length, frameshifted product of 181 amino acids (upper band).
  • Lane 3 shows the expression of a deletion construct having the sequence depicted in Figure 10C in which amino acids 34-164 of the wild type full length protein have been deleted.
  • Lane 4 shows the expression of a truncation construct having the sequence shown in Figure 10E in which tyrosine 35 has been mutated to a stop codon.
  • Lane 5 shows the expression of a truncation construct in which penylalanine 103 has been mutated to a stop codon.
  • Lane 6 shows the expression of a frame shifted construct that can only express the full length 181 amino acid Est3 protein.
  • EXAMPLE 5 A new screen in yeast to identify additional telomerase-associate proteins.
  • This Example proposes a new approach to recover additional genes required for telomere function in yeast.
  • We describe a new mutant screen which relies on a strain background that is sensitized to the absence of telomerase function.
  • the method described herein is technically much less complex than our previous screen for EST genes and allows as many as 10 ⁇ colonies to be screened for est-like defects.
  • This method relies upon the observation that a cdcl3-l- tlcl- ⁇ strain has two synthetic phenotypes: the maximum permissive temperature of a senescence phenotype as seen in Figure 14.
  • the procedure employs a plasmid-shuffle strategy, starting with a cdcl3-l- strain transfected with a pCDC13-URA3 plasmid.
  • 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.
  • CM-Ura complete media minus uracil
  • This primary set of mutagenized cultures will be tested for growth at 26° C on CM-Ura plates, in the presence of the pCDC13-URA3 plasmid (to rule out the possibility that the second mutation confers a general temperature sensitive phenotype) and re-tested more carefully for the 5-FOA phenotype at 26° C versus 23° C (to confirm that it is not due to an inability to lose the plasmid at any temperature or failure to grow on 5-FOA).
  • Those candidates that can lose the plasmid at 23° C will be tested for a potential senescence phenotype in the absence of the CDC13 plasmid; as shown in Figure 14B, this senescence phenotype should be evident after only one streak-out.
  • telomere length in the presence of the CDC 13 plasmid, to allow sufficient growth for Southern blots. Since this procedure involves only plating for single colonies and replica-plating, it is greatly simplified relative to our previous screen.
  • the two-hybrid system uses the restoration of transcriptional activation to indicate an interaction between two proteins.
  • a eukaryotic transcriptional activator generally the yeast GAL4 transcription factor
  • a transcriptional activation domain is divided into two domains, a transcriptional activation domain and a DNA-binding domain.
  • the two domains are part of the same protein.
  • a functional transcriptional activator can be assembled.
  • the EST2 or EST3 gene or fragments thereof may be cloned into a plasmid vector containing the coding sequence for the DNA-binding domain.
  • the gene or fragment may be inserted into the vector in such a fashion as to generate an in-frame fusion protein with the DNA-binding domain.
  • a DNA library will be constructed in a plasmid containing the activation domain. The library will be inserted into the plasmid adjacent to the binding domain coding sequence and will result in a fusion protein between the binding protein and the genes represented in the library.
  • the library may be constructed from random DNA fragments or may be constructed from cDNA fragments. The two plasmids will be cotransformed into an appropriate yeast strain and cotransformants screened for the expression of a functional GAL4 transcriptional activator.
  • This screen may be accomplished by placing a reporter gene, for example ⁇ -galactosidase, under the control of a promoter containing a GAL4 responsive element.
  • a reporter gene for example ⁇ -galactosidase
  • the two GAL4 functional domains will be brought into close proximity. This will result in the restoration of a functional transcriptional activator and expression of the reporter gene.
  • the colonies will be screened for the expression of the reporter gene and colonies that express the reporter gene will be isolated.
  • the plasmid containing the activation domain library fusion protein is recovered and the sequence of the library protein is determined.
  • Figure 10 shows the primary structure of a number of GAL4 activation domain fusions prepared with the EST3 gene.
  • Figure 10A shows a GAL4 activation domain - HA fusion expressing both the first open reading frame and the entire frame shifted gene product.
  • Figure 10B shows the GAL4-HA fusion with the frame shift corrected EST3.
  • Figure IOC shows a deletion derivative truncated after the first 50 amino acids of the first open reading frame.
  • Figure 10D shows truncation mutant where phenylalanine 103 was converted to a stop codon.
  • Figure 10E shows a GAL4-HA fusion protein where tyrosine 35 was converted to stop mutation.
  • Figure 10F shows an internal proteolytic fragment of approximately 70 amino acids.
  • Figure 10G shows an EST3 gene tagged at the C-terminal with three HA epitopes.
  • Figure 10H shows a construct prepared for baculovirus expression wherein the Est3 protein has been tagged on the C-terminal with six histidine amino acids.
  • fusion proteins that have been prepared include a GST-Est2 fusion protein.
  • the proteins or fragments may be used to generate antibodies specific to the proteins or fragments thereof.
  • the antibodies may be polyclonal or monoclonal.
  • the production of antibodies will be accomplished using conventional techniques well known to those of skill in the art. The techniques used can be found in a variety of references, for example, Harlow and Lane, Antibodies, A Laboratory Manual, the disclosure of which is specifically incorporated herein by reference.
  • the protein or fragment thereof may be purified from native sources or, alternatively, may be purified from a heterologous over expressing host.
  • the fragments may be prepared from purified protein by treatment with protease enzymes or alternatively, the fragments may be prepared synthetically using solid phase synthesis technology well known to those of skill in the art.
  • the purified protein or fragment may be mixed with an adjuvant, for example, Freund's complete adjuvant or Freund's incomplete adjuvant or other synthetic adjuvants known to those of skill in the art.
  • the adjuvant/protein mixture may be injected into a laboratory animal in order to produce an immune response.
  • the immune response may be boosted by the injection of additional protein material with or without adjuvant.
  • Injection schedules and regimens are well known to those skilled in the art.
  • the peptide may be conjugated to a carrier molecule such as keyhole limpet hemocyanin (KLH), or any other carrier protein known in the art.
  • KLH keyhole limpet hemocyanin
  • telomere binding protein that is required for telomerase access to the chromosome terminus.
  • telomere function Characterization of the yeast CDC13 gene (identified in our est mutant screen) has shown that it has a dual role in telomere function.
  • the CDC13 gene is an essential yeast gene that is required for maintaining telomere integrity, as first suggested by a study from Lee Hartwell's laboratory showing that a rapid loss of one strand of the telomere occurs in the absence of CDC13 function. Recent work from our laboratory has uncovered an additional role for CDC 13 in telomere maintenance.
  • cdcl3-2eSt a novel mutation that displayed a phenotype virtually identical to that of a telomerase-minus strain.
  • epistasis analysis between the cdcl3-2eSt mutation and tlcl-A demonstrated that cdcl3-2eSt perturbs a function of CDC13 that is required for the telomerase pathway.
  • genetic analysis of the previously isolated conditional lethal allele of CDC13 (cdcl3-lts) showed that CDC13 has a separate essential function that must be maintained in addition to the telomerase-based pathway.
  • Cdcl3 binds specifically to single-stranded yeast telomeric substrates. Based on these data, we propose that Cdcl3 has two distinct functions at the telomere. The first of these roles is to protect the end of the chromosome, which is essential for cell viability. Second, Cdcl3 protein regulates telomerase by mediating, either directly or indirectly, access of this enzyme to the chromosomal terminus, and that this access is now eliminated by the cdcl3-2eSt mutation.
  • This second role provides the basis for blocking access of the enzyme telomerase to the chromosomal terminus, either by mutation or potentially via the binding of a small molecule or peptide inhibitor to Cdcl3p, at a site defined by the cdcl3-2eSt mutation.
  • This provides an alternative means of inhibition of telomerase activity, via the identification of inhibitors that block access of the enzyme to the telomere.
  • Random peptide and small molecue libraries may be screened in a variety of ways known to those of skill in the art.
  • One preferred embodiment will be to screen a phage display library.
  • the technique employs fusion of random peptide sequences to either of two proteins that make up the phage coat of filamentous phage. Multiple rounds of enrichment for phage that exhibit tight binding to the selector protein is achieved via either affinity chromatography or panning.
  • telomerase assays An alternative method for screening both random peptide libraries and small molecule libraries is to use in vitro telomerase assays and test for inhibition of the enzymatic activity of the telomerase.
  • This type of assay can readily be automated using robotic instrumentation to rapidly assay large quantities of samples.
  • the ability to establish suitable enzymatic assays and the analysis of inhibition data are well within the purview of those of ordinary skill in the art.
  • High complexity libraries will be used.
  • the libraries may incorporate a number of recent modifications such as: monovalent display, that allow selection for higher affinity ligands; structurally constrained libraries; and libraries displaying D-peptide ligands.
  • monovalent display that allow selection for higher affinity ligands
  • structurally constrained libraries that allow selection for higher affinity ligands
  • libraries displaying D-peptide ligands In addition, more than one library will be screened in order to increase the odds of recovery and to expand the range of peptides recovered.
  • Peptides and small molecules identified as binding to the Cdcl3 protein will be screened for the ability to inhibit telomerase activity and such peptides and small molecules will be useful in vivo to prevent telomerase function, thereby preventing unlimited replication potential.
  • the selection protein is derived from a human telomerase complex
  • the peptides identified will find use as anti-tumor agents to suppress the unlimited replication of tumor cells.
  • the selection proteins are derived from pathogenic organisms, the peptides identified by this screen may be used to inhibit the growth of the pathogenic organism.
  • yeast expression vectors are the inducible yeast GAL promoter, providing regulated transcription, present on a high copy yeast shuttle vector (Ninomiya-Tsuji, et al., 1991); the second library was constructed using the constitutive yeast ADH1 promoter and is also present on a high copy yeast vector (Becker, et al., 1991).
  • yeast GAL promoter the inducible yeast GAL promoter
  • the second library was constructed using the constitutive yeast ADH1 promoter and is also present on a high copy yeast vector (Becker, et al., 1991).
  • two different cDNA libraries (with and without nuclear localization signals) from 24 K. lactis behind the S. cerevisiae ADH promoter in the pYES2 vector (Pearlman,
  • Becherer and Lundblad, unpublished) have been constructed for use in identifying homologs from more closely related species.
  • Freshly dissected haploid est2- rad52- ⁇ and est3- ⁇ rad52- strains will be transformed with either of these libraries and healthy-growing transformants identified; the presence of the rad52 mutation is necessary to prevent the appearance of a bypass pathway that gives rise to healthy growing derivatives (Lundblad and Blackburn, 1993).
  • the libraries will be screened for complementation of (i) the senescence phenotype of est4 rad52 , (ii) the ts phenotype of a cdcl3-ts mutation and (iii) the lethality of a est4/cdcl3- (this latter experiment will be performed using a plasmid shuffle type of strategy); complementation of these three different types of mutations may help the ability to recover a complementing cloneA second method to isolate homologs will be by cross-hybridization and degenerate PCR.
  • amino acid signature sequences can also be used to design degenerate PCR primers against highly conserved regions to be used for a substantial cross-species jump. If more than two highly conserved regions in a given EST gene are identified, nested degenerate PCR reactions will be performed. PCR products will be size-selected, cloned and sequenced, and sequence information will be evaluated to determine whether we have identified an EST homolog. Promising PCR clones will be used to identify and sequence full length cDNAs from the appropriate library. The source of the cDNA libraries used will be the two HeLa cDNA libraries described above.
  • each cDNA will be subsequently tested for whether it can confer a dominant effect on telomere replication in wild type yeast; in other words, over-expression of such a cDNA may inhibit yeast telomere replication by interfering with the formation of a necessary yeast complex (such as telomerase).
  • RNAs of different ciliates have a common secondary structure and a permuted template. Genes and Dev. 8: 1984-1998.
  • - pVL106 is an ARS1 LEU2 CEN3 plasmid used in the plasmid linearization assay; see Lundblad and Szostak 1989 for more details.

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Abstract

L'invention concerne de nouvelles protéines associées à la télomérase, ainsi que des procédés servant à isoler les gènes codant ces protéines. De nouveaux procédés de criblage permettent d'isoler des gènes supplémentaires associés à la télomérase, d'exprimer et de caractériser leurs produits de protéines. L'identification d'interactions entre protéines nécessaires au fonctionnement in vivo de la télomérase constitue la base d'un nouveau procédé de criblage afin de rechercher des agents anti-tumeur.
PCT/US1997/021272 1996-11-26 1997-11-26 Identification d'inhibiteurs empechant l'acces de la telomerase aux terminaisons chromosomiques WO1998023759A2 (fr)

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AU56868/98A AU5686898A (en) 1996-11-26 1997-11-26 Identification of inhibitors that prevent access of telomerase to chrom osomal terminus
JP52474398A JP2001507570A (ja) 1996-11-26 1997-11-26 染色体末端へのテロメラーゼのアクセスを阻害する阻害剤の同定

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US6440735B1 (en) 1998-03-31 2002-08-27 Geron Corporation Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer
US6808880B2 (en) 1996-10-01 2004-10-26 Geron Corporation Method for detecting polynucleotides encoding telomerase
US6927285B2 (en) 1996-10-01 2005-08-09 Geron Corporation Genes for human telomerase reverse transcriptase and telomerase variants
US7195911B2 (en) 1996-10-01 2007-03-27 Geron Corporation Mammalian cells that have increased proliferative capacity
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US7413864B2 (en) 1997-04-18 2008-08-19 Geron Corporation Treating cancer using a telomerase vaccine
US7517971B1 (en) 1996-10-01 2009-04-14 Geron Corporation Muteins of human telomerase reverse transcriptase lacking telomerase catalytic activity
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US6808880B2 (en) 1996-10-01 2004-10-26 Geron Corporation Method for detecting polynucleotides encoding telomerase
US6927285B2 (en) 1996-10-01 2005-08-09 Geron Corporation Genes for human telomerase reverse transcriptase and telomerase variants
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