, Volume 101, Issue 3, pp 189–197 | Cite as

Analysis of centromere function in Saccharomyces cerevisiae using synthetic centromere mutants

  • Michael R. Murphy
  • Dana M. Fowlkes
  • Molly Fitzgerald-Hayes
Original Articles


We constructed Saccharomyces cerevisiae centromere DNA mutants by annealing and ligating synthetic oligonucleotides, a novel approach to centromere DNA mutagenesis that allowed us to change only one structural parameter at a time. Using this method, we confirmed that CDE I, II, and III alone are sufficient for centromere function and that A+T-rich sequences in CDE II play important roles in mitosis and meiosis. Analysis of mutants also showed that a bend in the centromere DNA could be important for proper mitotic and meiotic chromosome segregation. In addition we demonstrated that the wild-type orientation of the CDE III sequence, but not the CDE I sequence, is critical for wild-type mitotic segregation. Surprisingly, we found that one mutant centromere affected the segregation of plasmids and chromosomes differently. The implications of these results for centromere function and chromosome structure are discussed.


Developmental Biology Saccharomyces Cerevisiae Chromosome Segregation Chromosome Structure Meiotic Chromosome 
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.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Baker RE, Masison DC (1990) Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1. Mol Cell Biol 10:2458–2467Google Scholar
  2. Baker RE, Fitzgerald-Hayes M, O'Brien TC (1989) Purification of the yeast centromere binding protein CP1 and a mutational analysis of its binding site. J Biol Chem 264:10843–10850Google Scholar
  3. Bauer BF, Holmes WM (1989) Cloning of synthetic oligonucleotides may result in high frequency promoter mutations in Escherichia coli. Nucleic Acids Res 17:812Google Scholar
  4. Bloom K, Hill A, Kenna M, Saunders M (1989) The structure of a primitive kinetochore. Trends Biol Sci 14:223–227Google Scholar
  5. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346Google Scholar
  6. Bram RJ, Kornberg RD (1987) Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor. Mol Cell Biol 7:403–409Google Scholar
  7. Cai M, Davis RW (1990) Yeast centromere binding protein CBF1, of the helix-loop-helix protein family, is required for chromosme stability and methionine prototrophy. Cell 61:437–446Google Scholar
  8. Clarke L, Carbon J (1983) Genomic substitutions of centromeres in Saccharomyces cerevisiae. Nature 305:23–28Google Scholar
  9. Cottarel G, Shero JH, Hieter P, Hegemann JH (1989) A 125-basepair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae. Mol Cell Biol 9:3342–3349Google Scholar
  10. Cumberledge S, Carbon J (1987) Mutational analysis of meiotic and mitotic centromere function in Saccharomyces cerevisiae. Genetics 117:203–212Google Scholar
  11. Densmore L, Payne WE, Fitzgerald-Hayes M (1991) In vivo genomic footprint of a yeast centromere. Mol Cell Biol 11:154–165Google Scholar
  12. Gaudet A, Fitzgerald-Hayes M (1987) Alterations in the ademineplus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae. Mol Cell Biol 7:68–75Google Scholar
  13. Gaudet A, Fitzgerald-Hayes M (1989a) Mutations in CEN3 cause aberrant chromosome segregation during meiosis in Saccharomyces cerevisiae. Genetics 121:447–489Google Scholar
  14. Gaudet A, Fitzgerald-Hayes M (1989b) The function of centromeres in chromosome segregation. In: Strauss P, Wilson S (eds) The eukaryotic nucleus: molecular biochemistry and macromolecular assemblies. Telford Press, Caldwell, New Jersey, pp 845–881Google Scholar
  15. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580Google Scholar
  16. Hegemann JH, Pridmore RD, Schneider R, Philippsen P (1986) Mutations in the right boundary of Saccharomyces cerevisiae centromere VI lead to nonfunctional or partially functional centromeres. Mol Gen Genet 205:305–311Google Scholar
  17. Hegemann JH, Shero JH, Cottarel G, Philipssen P, Hieter P (1988) Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae. Mol Cell Biol 8:2523–2535Google Scholar
  18. Hieter P, Mann C, Snyder M, Davis RW (1985) Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss. Cell 40:381–392Google Scholar
  19. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168Google Scholar
  20. Lechner J, Carbon J (1991) A 240kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell 64:717–725Google Scholar
  21. Mandecki W, Bolling TJ (1988) FokI method of gene synthesis. Gene 68:101–107Google Scholar
  22. Maniatis T, Fritsch EF, Sambrook J (1983) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  23. McClain WH, Foss K, Mittelstadt KL, Schneider J (1986) Variants in clones in gene-machine synthesized oligodeoxynucleotides. Nucleic Acids Res 14:6770Google Scholar
  24. McGrew JT, Diehl B, Fitzgerald-Hayes M (1986) Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae. Mol Cell Biol 6:530–538Google Scholar
  25. McGrew JT, Xiao Z, Fitzgerald-Hayes M (1989) Saccharomyces cerevisiae mutants defective in chromosome segregation. Yeast 5:271–284Google Scholar
  26. Mellor J, Jiang W, Funk M, Rathjen J, Barnes CA, Hinz T, Hegemann JH, Philippsen P (1990) CPF1, a yeast protein which functions in centromeres and promoters. EMBO J 9:4017–4026Google Scholar
  27. Murphy M, Fitzgerald-Hayes M (1990) Cis- and trans-acting factors involved in centromere function in Saccharomyces cerevisiae. Mol Microbiol 4:329–336Google Scholar
  28. Ng R, Carbon J (1987) Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae. Mol Cell Biol 7:4522–4534Google Scholar
  29. Ng R, Ness J, Carbon J (1986) Structural studies on centromeres in the yeast Saccharomyces cerevisiae. In: Wickner RB, Hinnebusch A, Lambowitz AM, Gunsalus IC, Hollaender A (eds) Extrachromosomal elements in lower eukaryotes. Plenum Press, New York, pp 479–492Google Scholar
  30. Panzeri L, Landonio L, Stotz A, Philippsen P (1985) Role of conserved sequence elements in yeast centromere DNA. EMBO J 4:1867–1874Google Scholar
  31. Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251Google Scholar
  32. Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211Google Scholar
  33. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  34. Saunders MJ, Fitzgerald-Hayes M, Bloom K (1988) Chromatin structure of altered yeast centromeres. Proc Natl Acad Sci USA 85:175–179Google Scholar
  35. Sherman F, Fink G, Hicks JB (1983) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  36. Snouwaert J, Bunick D, Hutchison C, Fowlkes DM (1987) Large numbers of random point and cluster mutations within the adenovirus VA I gene allow characterization of sequences required for efficient transcription. Nucleic Acids Res 15:8293–8303Google Scholar
  37. Sokal RR, Rohlf FJ (1987) Biometry. W.H. Freeman, San Francisco, CaliforniaGoogle Scholar
  38. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  39. Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) Highfrequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039Google Scholar
  40. Travers AA (1989) DNA conformation and protein binding. Annu Rev Biochem 58:427–452Google Scholar
  41. Wosnick MA, Barnett RW, Vicentini AM, Erfle H, Elliot R, Summer-Smith M, Mantei N, Davies RW (1987) Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene. Gene 60:115–127Google Scholar
  42. Wu H-M, Crothers DM (1984) The locus of sequence-directed and protein-induced DNA bending. Nature 308:509–513Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Michael R. Murphy
    • 1
  • Dana M. Fowlkes
    • 2
  • Molly Fitzgerald-Hayes
    • 1
  1. 1.Department of Biochemistry, Program in Molecular and Cellular Biology, 901 Lederle Graduate Research CenterUniversity of Massachusetts at AmherstAmherstUSA
  2. 2.Department of Pathology, Curriculum in GeneticsUniversity of North Carolina at Chapel HillChapel HillUSA

Personalised recommendations