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. 1993 Jun;13(6):3156–3166. doi: 10.1128/mcb.13.6.3156

Genetic dissection of centromere function.

I G Schulman 1, K Bloom 1
PMCID: PMC359754  PMID: 8497246

Abstract

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.

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Selected References

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  1. Apostol B., Greer C. L. Copy number and stability of yeast 2 mu-based plasmids carrying a transcription-conditional centromere. Gene. 1988 Jul 15;67(1):59–68. doi: 10.1016/0378-1119(88)90008-x. [DOI] [PubMed] [Google Scholar]
  2. Baker R. E., Fitzgerald-Hayes M., O'Brien T. C. Purification of the yeast centromere binding protein CP1 and a mutational analysis of its binding site. J Biol Chem. 1989 Jun 25;264(18):10843–10850. [PubMed] [Google Scholar]
  3. Bernat R. L., Borisy G. G., Rothfield N. F., Earnshaw W. C. Injection of anticentromere antibodies in interphase disrupts events required for chromosome movement at mitosis. J Cell Biol. 1990 Oct;111(4):1519–1533. doi: 10.1083/jcb.111.4.1519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bernat R. L., Delannoy M. R., Rothfield N. F., Earnshaw W. C. Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis. Cell. 1991 Sep 20;66(6):1229–1238. doi: 10.1016/0092-8674(91)90045-z. [DOI] [PubMed] [Google Scholar]
  5. Bitoun R., Zamir A. Spontaneous amplification of yeast CEN ARS plasmids. Mol Gen Genet. 1986 Jul;204(1):98–102. doi: 10.1007/BF00330194. [DOI] [PubMed] [Google Scholar]
  6. Bloom K. S., Carbon J. Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes. Cell. 1982 Jun;29(2):305–317. doi: 10.1016/0092-8674(82)90147-7. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Cai M. J., Davis R. W. Purification of a yeast centromere-binding protein that is able to distinguish single base-pair mutations in its recognition site. Mol Cell Biol. 1989 Jun;9(6):2544–2550. doi: 10.1128/mcb.9.6.2544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cai M., Davis R. W. Yeast centromere binding protein CBF1, of the helix-loop-helix protein family, is required for chromosome stability and methionine prototrophy. Cell. 1990 May 4;61(3):437–446. doi: 10.1016/0092-8674(90)90525-j. [DOI] [PubMed] [Google Scholar]
  10. Carbon J., Clarke L. Structural and functional analysis of a yeast centromere (CEN3). J Cell Sci Suppl. 1984;1:43–58. doi: 10.1242/jcs.1984.supplement_1.4. [DOI] [PubMed] [Google Scholar]
  11. Chlebowicz-Sledziewska E., Sledziewski A. Z. Construction of multicopy yeast plasmids with regulated centromere function. Gene. 1985;39(1):25–31. doi: 10.1016/0378-1119(85)90103-9. [DOI] [PubMed] [Google Scholar]
  12. Clarke L., Baum M. P. Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences. Mol Cell Biol. 1990 May;10(5):1863–1872. doi: 10.1128/mcb.10.5.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Clarke L., Carbon J. The structure and function of yeast centromeres. Annu Rev Genet. 1985;19:29–55. doi: 10.1146/annurev.ge.19.120185.000333. [DOI] [PubMed] [Google Scholar]
  14. Erhart E., Hollenberg C. P. The presence of a defective LEU2 gene on 2 mu DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number. J Bacteriol. 1983 Nov;156(2):625–635. doi: 10.1128/jb.156.2.625-635.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hartwell L. H., Weinert T. A. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. doi: 10.1126/science.2683079. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hill A., Bloom K. Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Mar;9(3):1368–1370. doi: 10.1128/mcb.9.3.1368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hill A., Bloom K. Genetic manipulation of centromere function. Mol Cell Biol. 1987 Jul;7(7):2397–2405. doi: 10.1128/mcb.7.7.2397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jehn B., Niedenthal R., Hegemann J. H. In vivo analysis of the Saccharomyces cerevisiae centromere CDEIII sequence: requirements for mitotic chromosome segregation. Mol Cell Biol. 1991 Oct;11(10):5212–5221. doi: 10.1128/mcb.11.10.5212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kingsbury J., Koshland D. Centromere-dependent binding of yeast minichromosomes to microtubules in vitro. Cell. 1991 Aug 9;66(3):483–495. doi: 10.1016/0092-8674(81)90012-x. [DOI] [PubMed] [Google Scholar]
  22. Koshland D., Hieter P. Visual assay for chromosome ploidy. Methods Enzymol. 1987;155:351–372. doi: 10.1016/0076-6879(87)55024-8. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Mellor J., Jiang W., Funk M., Rathjen J., Barnes C. A., Hinz T., Hegemann J. H., Philippsen P. CPF1, a yeast protein which functions in centromeres and promoters. EMBO J. 1990 Dec;9(12):4017–4026. doi: 10.1002/j.1460-2075.1990.tb07623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moens P. B. Kinetochore microtubule numbers of different sized chromosomes. J Cell Biol. 1979 Dec;83(3):556–561. doi: 10.1083/jcb.83.3.556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Murphy M. R., Fowlkes D. M., Fitzgerald-Hayes M. Analysis of centromere function in Saccharomyces cerevisiae using synthetic centromere mutants. Chromosoma. 1991 Dec;101(3):189–197. doi: 10.1007/BF00355368. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Runge K. W., Zakian V. A. Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation. Mol Cell Biol. 1989 Apr;9(4):1488–1497. doi: 10.1128/mcb.9.4.1488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Saunders M., Fitzgerald-Hayes M., Bloom K. Chromatin structure of altered yeast centromeres. Proc Natl Acad Sci U S A. 1988 Jan;85(1):175–179. doi: 10.1073/pnas.85.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schulman I., Bloom K. S. Centromeres: an integrated protein/DNA complex required for chromosome movement. Annu Rev Cell Biol. 1991;7:311–336. doi: 10.1146/annurev.cb.07.110191.001523. [DOI] [PubMed] [Google Scholar]
  32. Shero J. H., Koval M., Spencer F., Palmer R. E., Hieter P., Koshland D. Analysis of chromosome segregation in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:749–773. doi: 10.1016/0076-6879(91)94057-j. [DOI] [PubMed] [Google Scholar]
  33. Simerly C., Balczon R., Brinkley B. R., Schatten G. Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes. J Cell Biol. 1990 Oct;111(4):1491–1504. doi: 10.1083/jcb.111.4.1491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Smith D. R., Smyth A. P., Moir D. T. Amplification of large artificial chromosomes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8242–8246. doi: 10.1073/pnas.87.21.8242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Spencer F., Hieter P. Centromere DNA mutations induce a mitotic delay in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8908–8912. doi: 10.1073/pnas.89.19.8908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]

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