Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1996 Dec;144(4):1399–1412. doi: 10.1093/genetics/144.4.1399

Senescence Mutants of Saccharomyces Cerevisiae with a Defect in Telomere Replication Identify Three Additional Est Genes

T S Lendvay 1, D K Morris 1, J Sah 1, B Balasubramanian 1, V Lundblad 1
PMCID: PMC1207693  PMID: 8978029

Abstract

The primary determinant for telomere replication is the enzyme telomerase, responsible for elongating the G-rich strand of the telomere. The only component of this enzyme that has been identified in Saccharomyces cerevisiae is the TLC1 gene, encoding the telomerase RNA subunit. However, a yeast strain defective for the EST1 gene exhibits the same phenotypes (progressively shorter telomeres and a senescence phenotype) as a strain deleted for TLC1, suggesting that EST1 encodes either a component of telomerase or some other factor essential for telomerase function. We designed a multitiered screen that led to the isolation of 22 mutants that display the same phenotypes as est1 and tlc1 mutant strains. These mutations mapped to four complementation groups: the previously identified EST1 gene and three additional genes, called EST2, EST3 and EST4. Cloning of the EST2 gene demonstrated that it encodes a large, extremely basic novel protein with no motifs that provide clues as to function. Epistasis analysis indicated that the four EST genes function in the same pathway for telomere replication as defined by the TLC1 gene, suggesting that the EST genes encode either components of telomerase or factors that positively regulate telomerase activity.

Full Text

The Full Text of this article is available as a PDF (4.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allsopp R. C., Vaziri H., Patterson C., Goldstein S., Younglai E. V., Futcher A. B., Greider C. W., Harley C. B. Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10114–10118. doi: 10.1073/pnas.89.21.10114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baudin A., Ozier-Kalogeropoulos O., Denouel A., Lacroute F., Cullin C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Jul 11;21(14):3329–3330. doi: 10.1093/nar/21.14.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blasco M. A., Funk W., Villeponteau B., Greider C. W. Functional characterization and developmental regulation of mouse telomerase RNA. Science. 1995 Sep 1;269(5228):1267–1270. doi: 10.1126/science.7544492. [DOI] [PubMed] [Google Scholar]
  4. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  5. Bryan T. M., Englezou A., Gupta J., Bacchetti S., Reddel R. R. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J. 1995 Sep 1;14(17):4240–4248. doi: 10.1002/j.1460-2075.1995.tb00098.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Collins K., Kobayashi R., Greider C. W. Purification of Tetrahymena telomerase and cloning of genes encoding the two protein components of the enzyme. Cell. 1995 Jun 2;81(5):677–686. doi: 10.1016/0092-8674(95)90529-4. [DOI] [PubMed] [Google Scholar]
  7. Cooke H. J., Smith B. A. Variability at the telomeres of the human X/Y pseudoautosomal region. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):213–219. doi: 10.1101/sqb.1986.051.01.026. [DOI] [PubMed] [Google Scholar]
  8. D'Mello N. P., Jazwinski S. M. Telomere length constancy during aging of Saccharomyces cerevisiae. J Bacteriol. 1991 Nov;173(21):6709–6713. doi: 10.1128/jb.173.21.6709-6713.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Engebrecht J., Hirsch J., Roeder G. S. Meiotic gene conversion and crossing over: their relationship to each other and to chromosome synapsis and segregation. Cell. 1990 Sep 7;62(5):927–937. doi: 10.1016/0092-8674(90)90267-i. [DOI] [PubMed] [Google Scholar]
  10. Feng J., Funk W. D., Wang S. S., Weinrich S. L., Avilion A. A., Chiu C. P., Adams R. R., Chang E., Allsopp R. C., Yu J. The RNA component of human telomerase. Science. 1995 Sep 1;269(5228):1236–1241. doi: 10.1126/science.7544491. [DOI] [PubMed] [Google Scholar]
  11. Galibert F., Alexandraki D., Baur A., Boles E., Chalwatzis N., Chuat J. C., Coster F., Cziepluch C., De Haan M., Domdey H. Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X. EMBO J. 1996 May 1;15(9):2031–2049. [PMC free article] [PubMed] [Google Scholar]
  12. Greider C. W., Blackburn E. H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 1989 Jan 26;337(6205):331–337. doi: 10.1038/337331a0. [DOI] [PubMed] [Google Scholar]
  13. Harley C. B., Futcher A. B., Greider C. W. Telomeres shorten during ageing of human fibroblasts. Nature. 1990 May 31;345(6274):458–460. doi: 10.1038/345458a0. [DOI] [PubMed] [Google Scholar]
  14. Hastie N. D., Dempster M., Dunlop M. G., Thompson A. M., Green D. K., Allshire R. C. Telomere reduction in human colorectal carcinoma and with ageing. Nature. 1990 Aug 30;346(6287):866–868. doi: 10.1038/346866a0. [DOI] [PubMed] [Google Scholar]
  15. Hollingsworth N. M., Ponte L., Halsey C. MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair. Genes Dev. 1995 Jul 15;9(14):1728–1739. doi: 10.1101/gad.9.14.1728. [DOI] [PubMed] [Google Scholar]
  16. Kennedy B. K., Austriaco N. R., Jr, Zhang J., Guarente L. Mutation in the silencing gene SIR4 can delay aging in S. cerevisiae. Cell. 1995 Feb 10;80(3):485–496. doi: 10.1016/0092-8674(95)90499-9. [DOI] [PubMed] [Google Scholar]
  17. Kim N. W., Piatyszek M. A., Prowse K. R., Harley C. B., West M. D., Ho P. L., Coviello G. M., Wright W. E., Weinrich S. L., Shay J. W. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994 Dec 23;266(5193):2011–2015. doi: 10.1126/science.7605428. [DOI] [PubMed] [Google Scholar]
  18. Lin J. J., Zakian V. A. An in vitro assay for Saccharomyces telomerase requires EST1. Cell. 1995 Jun 30;81(7):1127–1135. doi: 10.1016/s0092-8674(05)80017-0. [DOI] [PubMed] [Google Scholar]
  19. Lingner J., Hendrick L. L., Cech T. R. Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. Genes Dev. 1994 Aug 15;8(16):1984–1998. doi: 10.1101/gad.8.16.1984. [DOI] [PubMed] [Google Scholar]
  20. McEachern M. J., Blackburn E. H. Runaway telomere elongation caused by telomerase RNA gene mutations. Nature. 1995 Aug 3;376(6539):403–409. doi: 10.1038/376403a0. [DOI] [PubMed] [Google Scholar]
  21. Meeks-Wagner D., Wood J. S., Garvik B., Hartwell L. H. Isolation of two genes that affect mitotic chromosome transmission in S. cerevisiae. Cell. 1986 Jan 17;44(1):53–63. doi: 10.1016/0092-8674(86)90484-8. [DOI] [PubMed] [Google Scholar]
  22. Nugent C. I., Hughes T. R., Lue N. F., Lundblad V. Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance. Science. 1996 Oct 11;274(5285):249–252. doi: 10.1126/science.274.5285.249. [DOI] [PubMed] [Google Scholar]
  23. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  24. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  25. Singer M. S., Gottschling D. E. TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science. 1994 Oct 21;266(5184):404–409. doi: 10.1126/science.7545955. [DOI] [PubMed] [Google Scholar]
  26. Smeal T., Claus J., Kennedy B., Cole F., Guarente L. Loss of transcriptional silencing causes sterility in old mother cells of S. cerevisiae. Cell. 1996 Feb 23;84(4):633–642. doi: 10.1016/s0092-8674(00)81038-7. [DOI] [PubMed] [Google Scholar]
  27. Smith J. R., Whitney R. G. Intraclonal variation in proliferative potential of human diploid fibroblasts: stochastic mechanism for cellular aging. Science. 1980 Jan 4;207(4426):82–84. doi: 10.1126/science.7350644. [DOI] [PubMed] [Google Scholar]
  28. Steiner B. R., Hidaka K., Futcher B. Association of the Est1 protein with telomerase activity in yeast. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2817–2821. doi: 10.1073/pnas.93.7.2817. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES