Europe PMC

This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy.

The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae.

Author information

Affiliations

  • 1. Swiss Institute for Experimental Cancer Research, Epalinges/Lausanne, Switzerland.
      Authors
    • Gotta M 1
    (1 author)

ORCIDs linked to this article

The Journal of Cell Biology, 01 Sep 1996, 134(6):1349-1363
https://doi.org/10.1083/jcb.134.6.1349 PMID: 8830766 PMCID: PMC2121006

This article is in the Europe PMC Open access subset. Refer to the copyright information in the article for licensing details.
Free full text in Europe PMC

Abstract 

We have developed a novel technique for combined immunofluorescence/in situ hybridization on fixed budding yeast cells that maintains the three-dimensional structure of the nucleus as monitored by focal sections of cells labeled with fluorescent probes and by staining with a nuclear pore antibody. Within the resolution of these immunodetection techniques, we show that proteins encoded by the SIR3, SIR4, and RAP1 genes colocalize in a statistically significant manner with Y' telomere-associated DNA sequences. In wild-type cells the Y' in situ hybridization signals can be resolved by light microscopy into fewer than ten foci per diploid nucleus. This suggests that telomeres are clustered in vegetatively growing cells, and that proteins essential for telomeric silencing are concentrated at their sites of action, i.e., at telomeres and/or subtelomeric regions. As observed for Rap1, the Sir4p staining is diffuse in a sir3- strain, and similarly, Sir3p staining is no longer punctate in a sir4- strain, although the derivatized Y' probe continues to label discrete sites in these strains. Nonetheless, the Y' FISH is altered in a qualitative manner in sir3 and sir4 mutant strains, consistent with the previously reported phenotypes of shortened telomeric repeats and loss of telomeric silencing.

Free full text 

Logo of jcellbiolLink to Publisher's site
J Cell Biol. 1996 Sep 2; 134(6): 1349–1363.
PMCID: PMC2121006
PMID: 8830766

The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae

Copyright and License information Disclaimer

Abstract

We have developed a novel technique for combined immunofluorescence/in situ hybridization on fixed budding yeast cells that maintains the three-dimensional structure of the nucleus as monitored by focal sections of cells labeled with fluorescent probes and by staining with a nuclear pore antibody. Within the resolution of these immunodetection techniques, we show that proteins encoded by the SIR3, SIR4, and RAP1 genes colocalize in a statistically significant manner with Y' telomere- associated DNA sequences. In wild-type cells the Y' in situ hybridization signals can be resolved by light microscopy into fewer than ten foci per diploid nucleus. This suggests that telomeres are clustered in vegetatively growing cells, and that proteins essential for telomeric silencing are concentrated at their sites of action, i.e., at telomeres and/or subtelomeric regions. As observed for Rap1, the Sir4p staining is diffuse in a sir3- strain, and similarly, Sir3p staining is no longer punctate in a sir4- strain, although the derivatized Y' probe continues to label discrete sites in these strains. Nonetheless, the Y' FISH is altered in a qualitative manner in sir3 and sir4 mutant strains, consistent with the previously reported phenotypes of shortened telomeric repeats and loss of telomeric silencing.

Full Text

The Full Text of this article is available as a PDF (8.2M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Aparicio OM, Billington BL, Gottschling DE. Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae. Cell. 1991 Sep 20;66(6):1279–1287. [Abstract] [Google Scholar]
  • Bell SP, Mitchell J, Leber J, Kobayashi R, Stillman B. The multidomain structure of Orc1p reveals similarity to regulators of DNA replication and transcriptional silencing. Cell. 1995 Nov 17;83(4):563–568. [Abstract] [Google Scholar]
  • Chung HM, Shea C, Fields S, Taub RN, Van der Ploeg LH, Tse DB. Architectural organization in the interphase nucleus of the protozoan Trypanosoma brucei: location of telomeres and mini-chromosomes. EMBO J. 1990 Aug;9(8):2611–2619. [Europe PMC free article] [Abstract] [Google Scholar]
  • Cockell M, Palladino F, Laroche T, Kyrion G, Liu C, Lustig AJ, Gasser SM. The carboxy termini of Sir4 and Rap1 affect Sir3 localization: evidence for a multicomponent complex required for yeast telomeric silencing. J Cell Biol. 1995 May;129(4):909–924. [Europe PMC free article] [Abstract] [Google Scholar]
  • Conrad MN, Wright JH, Wolf AJ, Zakian VA. RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability. Cell. 1990 Nov 16;63(4):739–750. [Abstract] [Google Scholar]
  • Cremer T, Kurz A, Zirbel R, Dietzel S, Rinke B, Schröck E, Speicher MR, Mathieu U, Jauch A, Emmerich P, et al. Role of chromosome territories in the functional compartmentalization of the cell nucleus. Cold Spring Harb Symp Quant Biol. 1993;58:777–792. [Abstract] [Google Scholar]
  • de Lange T. Human telomeres are attached to the nuclear matrix. EMBO J. 1992 Feb;11(2):717–724. [Europe PMC free article] [Abstract] [Google Scholar]
  • Devlin C, Tice-Baldwin K, Shore D, Arndt KT. RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene. Mol Cell Biol. 1991 Jul;11(7):3642–3651. [Europe PMC free article] [Abstract] [Google Scholar]
  • Doye V, Wepf R, Hurt EC. A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution. EMBO J. 1994 Dec 15;13(24):6062–6075. [Europe PMC free article] [Abstract] [Google Scholar]
  • Ekwall K, Javerzat JP, Lorentz A, Schmidt H, Cranston G, Allshire R. The chromodomain protein Swi6: a key component at fission yeast centromeres. Science. 1995 Sep 8;269(5229):1429–1431. [Abstract] [Google Scholar]
  • Fleig UN, Pridmore RD, Philippsen P. Construction of LYS2 cartridges for use in genetic manipulations of Saccharomyces cerevisiae. Gene. 1986;46(2-3):237–245. [Abstract] [Google Scholar]
  • Funabiki H, Hagan I, Uzawa S, Yanagida M. Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast. J Cell Biol. 1993 Jun;121(5):961–976. [Europe PMC free article] [Abstract] [Google Scholar]
  • Gasser SM, Laroche T, Falquet J, Boy de la Tour E, Laemmli UK. Metaphase chromosome structure. Involvement of topoisomerase II. J Mol Biol. 1986 Apr 20;188(4):613–629. [Abstract] [Google Scholar]
  • Gilson E, Roberge M, Giraldo R, Rhodes D, Gasser SM. Distortion of the DNA double helix by RAP1 at silencers and multiple telomeric binding sites. J Mol Biol. 1993 May 20;231(2):293–310. [Abstract] [Google Scholar]
  • Gottlieb S, Esposito RE. A new role for a yeast transcriptional silencer gene, SIR2, in regulation of recombination in ribosomal DNA. Cell. 1989 Mar 10;56(5):771–776. [Abstract] [Google Scholar]
  • Gottschling DE, Aparicio OM, Billington BL, Zakian VA. Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell. 1990 Nov 16;63(4):751–762. [Abstract] [Google Scholar]
  • Guacci V, Hogan E, Koshland D. Chromosome condensation and sister chromatid pairing in budding yeast. J Cell Biol. 1994 May;125(3):517–530. [Europe PMC free article] [Abstract] [Google Scholar]
  • Hardy CF, Balderes D, Shore D. Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing. Mol Cell Biol. 1992 Mar;12(3):1209–1217. [Europe PMC free article] [Abstract] [Google Scholar]
  • Hecht A, Laroche T, Strahl-Bolsinger S, Gasser SM, Grunstein M. Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast. Cell. 1995 Feb 24;80(4):583–592. [Abstract] [Google Scholar]
  • Hochstrasser M, Mathog D, Gruenbaum Y, Saumweber H, Sedat JW. Spatial organization of chromosomes in the salivary gland nuclei of Drosophila melanogaster. J Cell Biol. 1986 Jan;102(1):112–123. [Europe PMC free article] [Abstract] [Google Scholar]
  • Kennedy BK, Austriaco NR, 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. [Abstract] [Google Scholar]
  • Kimmerly WJ, Rine J. Replication and segregation of plasmids containing cis-acting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes. Mol Cell Biol. 1987 Dec;7(12):4225–4237. [Europe PMC free article] [Abstract] [Google Scholar]
  • Klein F, Laroche T, Cardenas ME, Hofmann JF, Schweizer D, Gasser SM. Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast. J Cell Biol. 1992 Jun;117(5):935–948. [Europe PMC free article] [Abstract] [Google Scholar]
  • Konkel LM, Enomoto S, Chamberlain EM, McCune-Zierath P, Iyadurai SJ, Berman J. A class of single-stranded telomeric DNA-binding proteins required for Rap1p localization in yeast nuclei. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5558–5562. [Europe PMC free article] [Abstract] [Google Scholar]
  • Kyrion G, Boakye KA, Lustig AJ. C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Nov;12(11):5159–5173. [Europe PMC free article] [Abstract] [Google Scholar]
  • Kyrion G, Liu K, Liu C, Lustig AJ. RAP1 and telomere structure regulate telomere position effects in Saccharomyces cerevisiae. Genes Dev. 1993 Jul;7(7A):1146–1159. [Abstract] [Google Scholar]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [Abstract] [Google Scholar]
  • Liu C, Mao X, Lustig AJ. Mutational analysis defines a C-terminal tail domain of RAP1 essential for Telomeric silencing in Saccharomyces cerevisiae. Genetics. 1994 Dec;138(4):1025–1040. [Europe PMC free article] [Abstract] [Google Scholar]
  • Louis EJ, Haber JE. The structure and evolution of subtelomeric Y' repeats in Saccharomyces cerevisiae. Genetics. 1992 Jul;131(3):559–574. [Europe PMC free article] [Abstract] [Google Scholar]
  • Louis EJ, Naumova ES, Lee A, Naumov G, Haber JE. The chromosome end in yeast: its mosaic nature and influence on recombinational dynamics. Genetics. 1994 Mar;136(3):789–802. [Europe PMC free article] [Abstract] [Google Scholar]
  • Lundblad V, Szostak JW. A mutant with a defect in telomere elongation leads to senescence in yeast. Cell. 1989 May 19;57(4):633–643. [Abstract] [Google Scholar]
  • Lustig AJ, Kurtz S, Shore D. Involvement of the silencer and UAS binding protein RAP1 in regulation of telomere length. Science. 1990 Oct 26;250(4980):549–553. [Abstract] [Google Scholar]
  • Lustig AJ, Liu C, Zhang C, Hanish JP. Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae. Mol Cell Biol. 1996 May;16(5):2483–2495. [Europe PMC free article] [Abstract] [Google Scholar]
  • Marcand S, Buck SW, Moretti P, Gilson E, Shore D. Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap 1 protein. Genes Dev. 1996 Jun 1;10(11):1297–1309. [Abstract] [Google Scholar]
  • Marshall M, Mahoney D, Rose A, Hicks JB, Broach JR. Functional domains of SIR4, a gene required for position effect regulation in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Dec;7(12):4441–4452. [Europe PMC free article] [Abstract] [Google Scholar]
  • Mathog D, Hochstrasser M, Gruenbaum Y, Saumweber H, Sedat J. Characteristic folding pattern of polytene chromosomes in Drosophila salivary gland nuclei. Nature. 308(5958):414–421. [Abstract] [Google Scholar]
  • Messmer S, Franke A, Paro R. Analysis of the functional role of the Polycomb chromo domain in Drosophila melanogaster. Genes Dev. 1992 Jul;6(7):1241–1254. [Abstract] [Google Scholar]
  • Moretti P, Freeman K, Coodly L, Shore D. Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev. 1994 Oct 1;8(19):2257–2269. [Abstract] [Google Scholar]
  • Chien CT, Buck S, Sternglanz R, Shore D. Targeting of SIR1 protein establishes transcriptional silencing at HM loci and telomeres in yeast. Cell. 1993 Nov 5;75(3):531–541. [Abstract] [Google Scholar]
  • Palladino F, Laroche T, Gilson E, Pillus L, Gasser SM. The positioning of yeast telomeres depends on SIR3, SIR4, and the integrity of the nuclear membrane. Cold Spring Harb Symp Quant Biol. 1993;58:733–746. [Abstract] [Google Scholar]
  • Paro R. Mechanisms of heritable gene repression during development of Drosophila. Curr Opin Cell Biol. 1993 Dec;5(6):999–1005. [Abstract] [Google Scholar]
  • Rae MM, Franke WW. The interphase distribution of satellite DNA-containing heterochromatin in mouse nuclei. Chromosoma. 1972;39(4):443–456. [Abstract] [Google Scholar]
  • Sandell LL, Zakian VA. Telomeric position effect in yeast. Trends Cell Biol. 1992 Jan;2(1):10–14. [Abstract] [Google Scholar]
  • Scherthan H, Cremer T, Arnason U, Weier HU, Lima-de-Faria A, Frönicke L. Comparative chromosome painting discloses homologous segments in distantly related mammals. Nat Genet. 1994 Apr;6(4):342–347. [Abstract] [Google Scholar]
  • Shore D, Nasmyth K. Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements. Cell. 1987 Dec 4;51(5):721–732. [Abstract] [Google Scholar]
  • Stavenhagen JB, Zakian VA. Internal tracts of telomeric DNA act as silencers in Saccharomyces cerevisiae. Genes Dev. 1994 Jun 15;8(12):1411–1422. [Abstract] [Google Scholar]
  • Stone EM, Swanson MJ, Romeo AM, Hicks JB, Sternglanz R. The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants. Mol Cell Biol. 1991 Apr;11(4):2253–2262. [Europe PMC free article] [Abstract] [Google Scholar]
  • Verdier JM, Stalder R, Roberge M, Amati B, Sentenac A, Gasser SM. Preparation and characterization of yeast nuclear extracts for efficient RNA polymerase B (II)-dependent transcription in vitro. Nucleic Acids Res. 1990 Dec 11;18(23):7033–7039. [Europe PMC free article] [Abstract] [Google Scholar]
  • Vignais ML, Huet J, Buhler JM, Sentenac A. Contacts between the factor TUF and RPG sequences. J Biol Chem. 1990 Aug 25;265(24):14669–14674. [Abstract] [Google Scholar]
  • Vourc'h C, Taruscio D, Boyle AL, Ward DC. Cell cycle-dependent distribution of telomeres, centromeres, and chromosome-specific subsatellite domains in the interphase nucleus of mouse lymphocytes. Exp Cell Res. 1993 Mar;205(1):142–151. [Abstract] [Google Scholar]
  • Walker CL, Cargile CB, Floy KM, Delannoy M, Migeon BR. The Barr body is a looped X chromosome formed by telomere association. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6191–6195. [Europe PMC free article] [Abstract] [Google Scholar]
  • Weiler KS, Wakimoto BT. Heterochromatin and gene expression in Drosophila. Annu Rev Genet. 1995;29:577–605. [Abstract] [Google Scholar]
  • Wimmer C, Doye V, Grandi P, Nehrbass U, Hurt EC. A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1. EMBO J. 1992 Dec;11(13):5051–5061. [Europe PMC free article] [Abstract] [Google Scholar]
  • Wright JH, Gottschling DE, Zakian VA. Saccharomyces telomeres assume a non-nucleosomal chromatin structure. Genes Dev. 1992 Feb;6(2):197–210. [Abstract] [Google Scholar]
Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

Full text links 

Read article at publisher's site: https://doi.org/10.1083/jcb.134.6.1349

Read article for free, from open access legal sources, via Unpaywall: https://rupress.org/jcb/article-pdf/134/6/1349/1256435/1349.pdfUnpaywall

Citations & impact 

Impact metrics

311
Citations
Jump to Citations
4
Data citations
Jump to Data

Citations of article over time

Alternative metrics

Altmetric item for https://www.altmetric.com/details/20984960
Altmetric
Discover the attention surrounding your research
https://www.altmetric.com/details/20984960

Article citations

Go to all (311) article citations

Data 

Similar Articles 

To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.