Advertisement

FISH Targeting of Chromosomes and Subchromosomal Regions in Yeast

  • Harry Scherthan
Protocol
  • 1.6k Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Yeasts represent powerful genetic model organisms, due in part to their tiny genomes with very little repetitive DNA. However, one drawback of this feature is an ill-defined metaphase chromosome structure. Fluorescence in situ hybridization has overcome this cytological disadvantage by unequivocally delineating chromosomes and their subregions of interest throughout the cell cycle, irrespective of chromatin compaction. Consequently, chromosome painting and locus-specific yeast FISH are now indispensable tools in the analysis of chromosome organization and dynamics in wild-type and mutant yeast strains. In this chapter, protocols are described that allow nuclei and meiotic chromosomes suitable for FISH analysis to be prepared.

Keywords

Sporulation Medium Sediment Cell Antifade Solution Yeast Nucleus Nuclear Spreading 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

I wish to thank the previous members of my laboratory for a fruitful collaboration, and the Deutsche Forschungsgemeinschaft for partial support of the work in my lab.

References

  1. Admire A, Shanks L, Danzl N, Wang M, Weier U, Stevens W, Hunt E, Weinert T (2006) Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast. Genes Dev 20:159–173CrossRefPubMedGoogle Scholar
  2. Bystricky K, Heun P, Gehlen L, Langowski J, Gasser SM (2004) Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques. Proc Natl Acad Sci USA 101:16495CrossRefPubMedGoogle Scholar
  3. Dresser ME, Giroux CN (1988) Meiotic chromosome behavior in spread preparations of yeast. J Cell Biol 106:567–573CrossRefPubMedGoogle Scholar
  4. Esposito RS, Dresser ME, Breitenbach M (1990) Identifying sporulation genes, visualizing synap-tonemal complexes, and large scale spore and spore wall purification. Guide to yeast genetics and molecular biology. Methods Enzymol 194:110–131CrossRefGoogle Scholar
  5. Fuchs J, Loidl J (2004) Behaviour of nucleolus organizing regions (NORs) and nucleoli during mitotic and meiotic divisions in budding yeast. Chromosome Res 12(5):427–438CrossRefPubMedGoogle Scholar
  6. Goffeau A, et al. (1997) The yeast genome directory. Nature 387(Supp l):1–105Google Scholar
  7. Gotta M, Laroche T, Formenton A, Maillet L, Scherthan H, Gasser SM (1996) Colocalization of telomeric sequences with Sir3, Sir4 and Rap1 detected by combined immunofluorescence and FISH in S. cerevisiae. J Cell Biol 134:1349–1363CrossRefPubMedGoogle Scholar
  8. Gotta M, Laroche T, Gasser SM (1999) Analysis of nuclear organization in Saccharomyces cerevisiae. Methods Enzymol 304:663–672CrossRefPubMedGoogle Scholar
  9. Guacci V, Hogan E, Koshland D (1994) Chromosome condensation and sister chromatid pairing in budding yeast. J Cell Biol 125:517–530CrossRefPubMedGoogle Scholar
  10. Guacci V, Hogan E, Koshland D (1997) Centromere position in budding yeast: evidence for ana-phase A. Mol Biol Cell 8:957–972PubMedGoogle Scholar
  11. Jin Q-W, Trelles-Sticken E, Scherthan H, Loidl J (1998) Yeast nuclei display prominent centromere clustering that is reduced in nondividing cells and in meiotic prophase. J Cell Biol 141:21–29CrossRefPubMedGoogle Scholar
  12. Kane SM, Roth R (1974) Carbohydrate metabolism during ascospore development in yeast. J. Bacteriol 118:8–14PubMedGoogle Scholar
  13. Kater J, McA (1927) Cytology of Saccharomyces cerevisiae with special reference to nuclear division. Biol Bull 52:436–449CrossRefGoogle Scholar
  14. Kuroiwa T, Kojima H, Miyakawa I, Sando N (1984) Meiotic karyotype of the yeast Saccharomyces cerevisiae. Exp Cell Res 153:259–265CrossRefPubMedGoogle Scholar
  15. Lichter P, Cremer T (1992) Chromosome analysis by non isotopic in situ hybridization. In: Rooney DE, Czepulkowsky BH (eds) Human cytogenetics—a practical approach, vol I, 2nd edn. IRL Press, Oxford, pp. 157–192Google Scholar
  16. Loidl J (2003) Chromosomes of the budding yeast Saccharomyces cerevisiae. Int Rev Cytol 222:141–196CrossRefPubMedGoogle Scholar
  17. Loidl J, Nairz K, Klein F (1991) Meiotic chromosome synapsis in a haploid yeast. Chromosoma 100:221–228CrossRefPubMedGoogle Scholar
  18. Loidl J, Scherthan H, Kaback DB (1994) Physical association between nonhomologous chromosomes precedes distributive disjunction in yeast. Proc Natl Acad Sci USA 91:331–334CrossRefPubMedGoogle Scholar
  19. Loidl J, Klein F, Engebrecht J (1998) Genetic and morphological approaches for the analysis of meiotic chromosomes in yeast. In: Berrios M (ed) Nuclear structure and function. Academic, San Diego, CA, pp 257–285Google Scholar
  20. Lorenz A, Fuchs J, Trelles-Sticken E, Scherthan H, Loidl J (2003) Spatial organisation and behaviour of the parental chromosome sets in the nuclei of Saccharomyces cerevisiae × S. paradoxus hybrids. J Cell Sci 115:3829–3835CrossRefGoogle Scholar
  21. Nag DK, Scherthan H, Rockmill B, Bhargava J, Roeder GS (1995) Heteroduplex DNA formation and homolog pairing in yeast meiotic mutants. Genetics 141:75–86PubMedGoogle Scholar
  22. Padmore R, Cao L, Kleckner N (1991) Temporal comparison of recombination and synaptonemal complex formation during meiosis in S. cerevisiae. Cell 66:1239–1256CrossRefPubMedGoogle Scholar
  23. Pringle JR, Adams AE, Drubin DG, Haarer BK (1991) Immunofluorescence methods for yeast. Methods Enzymol 194:565–602CrossRefPubMedGoogle Scholar
  24. Scherthan H, Loidl J, Schuster T, Schweizer D (1992) Meiotic chromosome condensation and pairing in Saccharomyces cerevisiae studied by chromosome painting. Chromosoma 101: 590–595CrossRefPubMedGoogle Scholar
  25. Scherthan H, Schweizer D, Loidl J (1993) Delineation of individual chromosomes of Saccharomyces cerevisiae by two colour in situ hybridisation. Trends Genet 9:41CrossRefPubMedGoogle Scholar
  26. Scherthan H, Bähler J, Kohli J (1994) Dynamics of chromosome organization and pairing during meiotic prophase in fission yeast. J Cell Biol 127:273–285CrossRefPubMedGoogle Scholar
  27. Trelles-Sticken E, Loidl J, Scherthan H (1999) Bouquet formation in budding yeast: initiation of recombination is not required for meiotic telomere clustering. J Cell Sci 112:651–658PubMedGoogle Scholar
  28. Trelles-Sticken E, Loidl J, Scherthan H (2003) Increased ploidy and KAR3 and SIR3 disruption alter the dynamics of meiotic chromosomes and telomeres. J Cell Sci 116:2431–2442CrossRefPubMedGoogle Scholar
  29. Uzawa S, Yanagida M (1992) Visualization of centromeric and nucleolar DNA in fission yeast by fluorescence in situ hybridization. J Cell Sci 101:267–275PubMedGoogle Scholar
  30. Weiner BM, Kleckner N (1994) Chromosome pairing via multiple interstitial intractions before and during meiosis in yeast. Cell 77:977–991CrossRefPubMedGoogle Scholar
  31. Wintersberger U, Binder M, Fischer P (1975) Cytogenetic demonstration of mitotic chromosomes in the yeast Saccharomyces cerevisiae. Mol Gen Genet 142:13–17PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2009

Authors and Affiliations

  • Harry Scherthan
    • 1
  1. 1.Institut für Radiobiologie der Bundeswehr, Neuherbergstr. 11MunichGermany

Personalised recommendations