Abstract
We have investigated the chromatin structure of Kluyveromyces lactis centromeres in isolated nuclei of K. lactis and Saccharomyces cerevisiae by using micrococcal nuclease and DNAse I digestion. The protected region found in K. lactis is approximately 270 bp long and encompasses the centromeric DNA elements, KlCDEI, KlCDEII, and KlCDEIII, but not KlCDE0. Halving KlCDEII to 82 bp impaired centromere function and led to a smaller protected structure (210 bp). Likewise, deletion of 5 bp from KlCDEI plus adjacent flanking sequences resulted in a smaller protected region and a decrease in centromere function. The chromatin structures of KlCEN2 and KlCEN4 present on plasmids were found to be similar to the structures of the corresponding centromeres in their chromosomal context. A different protection pattern of KlCEN2 was detected in S. cerevisiae, suggesting that KlCEN2 is not properly recognized by at least one of the centromere binding proteins of S. cerevisiae. The difference is mainly found at the KlCDEIII side of the structure. This suggests that one of the components of the ScCBF3-complex is not able to bind to KlCDEIII, which could explain the species specificity of K. lactis and S. cerevisiae centromeres.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baker RE, Masison DC (1990) Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1. Mol Cell Biol 10: 2458–2467
Bloom KS, Carbon J (1982) Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes. Cell 29: 305–317
Bullock WO, Fernandez JM, Short M (1987) XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Bio Techniques 5: 376–380
Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138: 179–207
Dower WJ (1988) Transformation of Escherichia coli to extremely high efficiency by electroporation. Mol Biol Rep 6: 3–4
Fishel B, Amstutz H, Baum M, Carbon J, Clarke L (1988) Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe. Mol Cell Biol 8: 754–763
Fitzgerald-Hayes M (1987) Yeast centromeres. Yeast 3: 187–200
Funk M, Hegemann JH, Philippsen P (1989) Chromatin digestion with restriction endonucleases reveals 150–160 bp of protected DNA in the centromere of chromosome XIV in Saccharomyces cerevisiae. Mol Gen Genet 219: 153–160
Futcher B, Carbon J (1986) Toxic effects of excess cloned centromeres. Mol Cell Biol 6: 2213–2222
Hegemann JH, Shero JH, Cottarel G, Philippsen P, Hieter P (1988) Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae. Mol Cell Biol 8: 2523–2535
Heus JJ, Zonneveld BJM, Steensma HY, Van denBerg JA (1990) Centromeric DNA of Kluyveromyces lactis. Curr Genet 18: 517–522
Heus JJ, Zonneveld BJM, Steensma HY, Van denBerg JA (1993) The consensus sequence of Kluyveromyces lactis centromeres shows homology to functional centromeric DNA from Saccharomyces cerevisiae. Mol Gen Genet 236: 355–362
Hieter P, Pridmore D, Hegemann JH, Thomas M, Davis RW, Philippsen P (1985) Functional selection and analysis of yeast centromeric DNA. Cell 42: 913–921
Hyman AA, Middleton K, Centola M, Mitchison TJ, Carbon J (1992) Microtubule-motor activity of a yeast centromere-binding protein complex. Nature 359: 533–536
Johnston HM, Davis RW (1984) Sequences that regulate the divergent GAL1-GAL10 promoter. Mol Cell Biol 4: 1440–1448
Lechner J, Carbon J (1991) A 240-kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell 64: 717–725
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: 4017–4026
Murakami S, Matsumoto T, Niwa O, Yanagida M (1991) Structure of the fission yeast centromere cen3 — Direct analysis of the reiterated inverted region. Chromosoma 101: 214–221
Noll M (1974a) Subunit structure of chromatin. Nature 251: 249–251
Noll M (1974b) Internal structure of the chromatin subunit. Nucleic Acids Res 1: 1573–1578
Pluta AF, Cooke CA, Earnshaw WC (1990) Structure of the human centromere at metaphase. Trends Biochem Sci 15: 181–185
Polizzi C, Clarke L (1991) The chromatin structure of centromeres from fission yeast — Differentiation of the central core that correlates with function. J Cell Biol 112: 191–201
Runge KW, Wellinger RJ, Zakian VA (1991) Effects of excess centromeres and excess telomeres on chromosome loss rates. Mol Cell Biol 11: 2919–2928
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Saunders M, Fitzgerald-Hayes M, Bloom K (1988) Chromatin structure of altered yeast centromeres. Proc Natl Acad Sci USA 85: 175–179
Sherman F, Fink G, Lawrence C (1979) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19–27
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517
Sreekrishna K, Webster TD, Dickson RC (1984) Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gen of Tn903. Gene 28: 73–81
Takahashi K, Murakami S, Chikashige Y, Funabiki H, Niwa O, Yanagida M (1992) Mol Biol Cell 3: 819–835
Wu C (1980) The 5′ ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. Nature 286: 854–860
Zonneveld BJM (1986) Cheap and simple yeast media. J Microbiol Methods 4: 287–291
Author information
Authors and Affiliations
Additional information
Communicated by: J. Huberman
Rights and permissions
About this article
Cite this article
Heus, J.J., Bloom, K.S., Zonneveld, B.J.M. et al. Chromatin structures of Kluyveromyces lactis centromeres in K. lactis and Saccharomyces cerevisiae . Chromosoma 102, 660–667 (1993). https://doi.org/10.1007/BF00352314
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00352314