Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1992 Sep;132(1):39–51. doi: 10.1093/genetics/132.1.39

A Colony Color Assay for Saccharomyces Cerevisiae Mutants Defective in Kinetochore Structure and Function

F Perier 1, J Carbon 1
PMCID: PMC1205128  PMID: 1398062

Abstract

We have designed a colony color assay for monitoring centromere DNA-protein interactions in yeast (Saccharomyces cerevisiae). The assay is based on the ability of centromere DNA sequences to block (in cis) transcription initiated from a hybrid CEN-GAL1 promoter. Using a IacZ reporter gene under control of the CEN-GAL1 promoter, we screened colonies derived from mutagenized cells for a blue color phenotype indicative of derepression of the hybrid construct. A limited screen in which a 61-bp CEN11 DNA fragment containing an intact CDEIII subregion plus flanking sequences was used as the ``pseudo-operator'' led to the identification of mutations (blu) in three complementation groups. The blu1 mutants exhibited a decrease in activity of the major CEN DNA-binding proteins in vitro. The BLU1 gene was shown to be identical to the previously isolated SPT3 gene, known to be involved in the transcriptional regulation of a subset of yeast genes. Our results indicate that the BLU1/SPT3 gene product may also be required to maintain optimal levels of functional centromere DNA-binding proteins.

Full Text

The Full Text of this article is available as a PDF (3.9 MB).

Selected References

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

  1. Baker R. E., Masison D. C. Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1. Mol Cell Biol. 1990 Jun;10(6):2458–2467. doi: 10.1128/mcb.10.6.2458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bram R. J., Kornberg R. D. Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor. Mol Cell Biol. 1987 Jan;7(1):403–409. doi: 10.1128/mcb.7.1.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brent R., Ptashne M. A bacterial repressor protein or a yeast transcriptional terminator can block upstream activation of a yeast gene. Nature. 1984 Dec 13;312(5995):612–615. doi: 10.1038/312612a0. [DOI] [PubMed] [Google Scholar]
  4. Burke D., Gasdaska P., Hartwell L. Dominant effects of tubulin overexpression in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Mar;9(3):1049–1059. doi: 10.1128/mcb.9.3.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carbon J., Clarke L. Centromere structure and function in budding and fission yeasts. New Biol. 1990 Jan;2(1):10–19. [PubMed] [Google Scholar]
  6. Clarke L. Centromeres of budding and fission yeasts. Trends Genet. 1990 May;6(5):150–154. doi: 10.1016/0168-9525(90)90149-z. [DOI] [PubMed] [Google Scholar]
  7. Cottarel G., Shero J. H., Hieter P., Hegemann J. H. A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Aug;9(8):3342–3349. doi: 10.1128/mcb.9.8.3342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cumberledge S., Carbon J. Mutational analysis of meiotic and mitotic centromere function in Saccharomyces cerevisiae. Genetics. 1987 Oct;117(2):203–212. doi: 10.1093/genetics/117.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dailey D., Schieven G. L., Lim M. Y., Marquardt H., Gilmore T., Thorner J., Martin G. S. Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity. Mol Cell Biol. 1990 Dec;10(12):6244–6256. doi: 10.1128/mcb.10.12.6244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fitzgerald-Hayes M., Buhler J. M., Cooper T. G., Carbon J. Isolation and subcloning analysis of functional centromere DNA (CEN11) from Saccharomyces cerevisiae chromosome XI. Mol Cell Biol. 1982 Jan;2(1):82–87. doi: 10.1128/mcb.2.1.82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fitzgerald-Hayes M. Yeast centromeres. Yeast. 1987 Sep;3(3):187–200. doi: 10.1002/yea.320030306. [DOI] [PubMed] [Google Scholar]
  13. Futcher B., Carbon J. Toxic effects of excess cloned centromeres. Mol Cell Biol. 1986 Jun;6(6):2213–2222. doi: 10.1128/mcb.6.6.2213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gaudet A., Fitzgerald-Hayes M. Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jan;7(1):68–75. doi: 10.1128/mcb.7.1.68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hegemann J. H., Shero J. H., Cottarel G., Philippsen P., Hieter P. Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jun;8(6):2523–2535. doi: 10.1128/mcb.8.6.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hieter P., Pridmore D., Hegemann J. H., Thomas M., Davis R. W., Philippsen P. Functional selection and analysis of yeast centromeric DNA. Cell. 1985 Oct;42(3):913–921. doi: 10.1016/0092-8674(85)90287-9. [DOI] [PubMed] [Google Scholar]
  17. Hirschhorn J. N., Winston F. SPT3 is required for normal levels of a-factor and alpha-factor expression in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Feb;8(2):822–827. doi: 10.1128/mcb.8.2.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hopper A. K., Hall B. D. Mating type and sporulation in yeast. I. Mutations which alter mating-type control over sporulation. Genetics. 1975 May;80(1):41–59. doi: 10.1093/genetics/80.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lechner J., Carbon J. A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell. 1991 Feb 22;64(4):717–725. doi: 10.1016/0092-8674(91)90501-o. [DOI] [PubMed] [Google Scholar]
  20. McGrew J., Diehl B., Fitzgerald-Hayes M. Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Feb;6(2):530–538. doi: 10.1128/mcb.6.2.530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Neigeborn L., Mitchell A. P. The yeast MCK1 gene encodes a protein kinase homolog that activates early meiotic gene expression. Genes Dev. 1991 Apr;5(4):533–548. doi: 10.1101/gad.5.4.533. [DOI] [PubMed] [Google Scholar]
  22. Ng R., Carbon J. Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Dec;7(12):4522–4534. doi: 10.1128/mcb.7.12.4522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ng R., Ness J., Carbon J. Structural studies on centromeres in the yeast Saccharomyces cerevisiae. Basic Life Sci. 1986;40:479–492. doi: 10.1007/978-1-4684-5251-8_36. [DOI] [PubMed] [Google Scholar]
  24. Pluta A. F., Cooke C. A., Earnshaw W. C. Structure of the human centromere at metaphase. Trends Biochem Sci. 1990 May;15(5):181–185. doi: 10.1016/0968-0004(90)90158-8. [DOI] [PubMed] [Google Scholar]
  25. Runge K. W., Wellinger R. J., Zakian V. A. Effects of excess centromeres and excess telomeres on chromosome loss rates. Mol Cell Biol. 1991 Jun;11(6):2919–2928. doi: 10.1128/mcb.11.6.2919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shero J. H., Hieter P. A suppressor of a centromere DNA mutation encodes a putative protein kinase (MCK1). Genes Dev. 1991 Apr;5(4):549–560. doi: 10.1101/gad.5.4.549. [DOI] [PubMed] [Google Scholar]
  27. Siliciano P. G., Tatchell K. Identification of the DNA sequences controlling the expression of the MAT alpha locus of yeast. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2320–2324. doi: 10.1073/pnas.83.8.2320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. St John T. P., Davis R. W. The organization and transcription of the galactose gene cluster of Saccharomyces. J Mol Biol. 1981 Oct 25;152(2):285–315. doi: 10.1016/0022-2836(81)90244-8. [DOI] [PubMed] [Google Scholar]
  29. Thomas J. H., Botstein D. A gene required for the separation of chromosomes on the spindle apparatus in yeast. Cell. 1986 Jan 17;44(1):65–76. doi: 10.1016/0092-8674(86)90485-x. [DOI] [PubMed] [Google Scholar]
  30. Tschumper G., Carbon J. Copy number control by a yeast centromere. Gene. 1983 Aug;23(2):221–232. doi: 10.1016/0378-1119(83)90054-9. [DOI] [PubMed] [Google Scholar]
  31. West R. W., Jr, Yocum R. R., Ptashne M. Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG. Mol Cell Biol. 1984 Nov;4(11):2467–2478. doi: 10.1128/mcb.4.11.2467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Winston F., Chaleff D. T., Valent B., Fink G. R. Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae. Genetics. 1984 Jun;107(2):179–197. doi: 10.1093/genetics/107.2.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Winston F., Durbin K. J., Fink G. R. The SPT3 gene is required for normal transcription of Ty elements in S. cerevisiae. Cell. 1984 Dec;39(3 Pt 2):675–682. doi: 10.1016/0092-8674(84)90474-4. [DOI] [PubMed] [Google Scholar]
  34. Winston F., Minehart P. L. Analysis of the yeast SPT3 gene and identification of its product, a positive regulator of Ty transcription. Nucleic Acids Res. 1986 Sep 11;14(17):6885–6900. doi: 10.1093/nar/14.17.6885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wood J. S. Genetic effects of methyl benzimidazole-2-yl-carbamate on Saccharomyces cerevisiae. Mol Cell Biol. 1982 Sep;2(9):1064–1079. doi: 10.1128/mcb.2.9.1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zirkle R. E. Ultraviolet-microbeam irradiation of newt-cell cytoplasm: spindle destruction, false anaphase, and delay of true anaphase. Radiat Res. 1970 Mar;41(3):516–537. [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES