Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Jul;7(7):2397–2405. doi: 10.1128/mcb.7.7.2397

Genetic manipulation of centromere function.

A Hill, K Bloom
PMCID: PMC365371  PMID: 3302676

Abstract

A conditional centromere was constructed in Saccharomyces cerevisiae by placing the centromere of chromosome III immediately downstream from the inducible GAL1 promoter from S. cerevisiae. By utilizing growth conditions that favor either transcriptional induction (galactose-carbon source) or repression (glucose-carbon source) from the GAL1 promoter, centromere function can be switched off or on, respectively. With the conditional centromere we were able to radically alter the mitotic transmission pattern of both monocentric and dicentric plasmids. Moreover, it was possible to selectively induce the loss of a single chromosome from a mitotically dividing population of cells. We observed that the induction of chromosome III aneuploidy resulted in a dramatic change in cell morphology. The construction of a conditional centromere represents a novel way to create conditional mutations of cis-acting DNA elements and will be useful for further analysis of this important stabilizing element.

Full text

PDF
2398

Images in this article

Selected References

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

  1. Adams B. G. Induction of galactokinase in Saccharomyces cerevisiae: kinetics of induction and glucose effects. J Bacteriol. 1972 Aug;111(2):308–315. doi: 10.1128/jb.111.2.308-315.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bassel J., Mortimer R. Genetic order of the galactose structural genes in Saccharomyces cerevisiae. J Bacteriol. 1971 Oct;108(1):179–183. doi: 10.1128/jb.108.1.179-183.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bloom K. S., Amaya E., Carbon J., Clarke L., Hill A., Yeh E. Chromatin conformation of yeast centromeres. J Cell Biol. 1984 Nov;99(5):1559–1568. doi: 10.1083/jcb.99.5.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Brinkley B. R., Cartwright J., Jr Cold-labile and cold-stable microtubules in the mitotic spindle of mammalian cells. Ann N Y Acad Sci. 1975 Jun 30;253:428–439. doi: 10.1111/j.1749-6632.1975.tb19218.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Clarke L., Carbon J. Genomic substitutions of centromeres in Saccharomyces cerevisiae. Nature. 1983 Sep 1;305(5929):23–28. doi: 10.1038/305023a0. [DOI] [PubMed] [Google Scholar]
  8. Clarke L., Carbon J. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature. 1980 Oct 9;287(5782):504–509. doi: 10.1038/287504a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. DOUGLAS H. C., HAWTHORNE D. C. ENZYMATIC EXPRESSION AND GENETIC LINKAGE OF GENES CONTROLLING GALACTOSE UTILIZATION IN SACCHAROMYCES. Genetics. 1964 May;49:837–844. doi: 10.1093/genetics/49.5.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dutcher S. K., Hartwell L. H. The role of S. cerevisiae cell division cycle genes in nuclear fusion. Genetics. 1982 Feb;100(2):175–184. doi: 10.1093/genetics/100.2.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Fitzgerald-Hayes M., Clarke L., Carbon J. Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell. 1982 May;29(1):235–244. doi: 10.1016/0092-8674(82)90108-8. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Giniger E., Varnum S. M., Ptashne M. Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell. 1985 Apr;40(4):767–774. doi: 10.1016/0092-8674(85)90336-8. [DOI] [PubMed] [Google Scholar]
  17. Haber J. E., Thorburn P. C. Healing of broken linear dicentric chromosomes in yeast. Genetics. 1984 Feb;106(2):207–226. doi: 10.1093/genetics/106.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kiehart D. P. Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system. J Cell Biol. 1981 Mar;88(3):604–617. doi: 10.1083/jcb.88.3.604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liras P., McCusker J., Mascioli S., Haber J. E. Characterization of a mutation in yeast causing nonrandom chromosome loss during mitosis. Genetics. 1978 Apr;88(4 Pt 1):651–671. [PMC free article] [PubMed] [Google Scholar]
  23. Maine G. T., Surosky R. T., Tye B. K. Isolation and characterization of the centromere from chromosome V (CEN5) of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jan;4(1):86–91. doi: 10.1128/mcb.4.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mann C., Davis R. W. Instability of dicentric plasmids in yeast. Proc Natl Acad Sci U S A. 1983 Jan;80(1):228–232. doi: 10.1073/pnas.80.1.228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McClintock B. The Behavior in Successive Nuclear Divisions of a Chromosome Broken at Meiosis. Proc Natl Acad Sci U S A. 1939 Aug;25(8):405–416. doi: 10.1073/pnas.25.8.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Murray A. W., Szostak J. W. Pedigree analysis of plasmid segregation in yeast. Cell. 1983 Oct;34(3):961–970. doi: 10.1016/0092-8674(83)90553-6. [DOI] [PubMed] [Google Scholar]
  28. Neitz M., Carbon J. Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Nov;5(11):2887–2893. doi: 10.1128/mcb.5.11.2887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Panzeri L., Landonio L., Stotz A., Philippsen P. Role of conserved sequence elements in yeast centromere DNA. EMBO J. 1985 Jul;4(7):1867–1874. doi: 10.1002/j.1460-2075.1985.tb03862.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Panzeri L., Philippsen P. Centromeric DNA from chromosome VI in Saccharomyces cerevisiae strains. EMBO J. 1982;1(12):1605–1611. doi: 10.1002/j.1460-2075.1982.tb01362.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  32. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  33. Stinchcomb D. T., Mann C., Davis R. W. Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol. 1982 Jun 25;158(2):157–190. doi: 10.1016/0022-2836(82)90427-2. [DOI] [PubMed] [Google Scholar]
  34. Weisbrod S. Active chromatin. Nature. 1982 May 27;297(5864):289–295. doi: 10.1038/297289a0. [DOI] [PubMed] [Google Scholar]
  35. Wu C. The 5' ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. Nature. 1980 Aug 28;286(5776):854–860. doi: 10.1038/286854a0. [DOI] [PubMed] [Google Scholar]
  36. Yeh E., Carbon J., Bloom K. Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jan;6(1):158–167. doi: 10.1128/mcb.6.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES