Molecular and General Genetics MGG

, Volume 219, Issue 1–2, pp 153–160

Chromatin digestion with restriction endonucleases reveals 150–160 bp of protected DNA in the centromere of chromosome XIV in Saccharomyces cerevisiae

  • Martin Funk
  • Johannes H. Hegemann
  • Peter Philippsen
Article

Summary

Isolated nuclei of Saccharomyces cerevisiae were incubated with five restriction nucleases. Out of the twenty-one recognition sequences for these nucleases in the centromere region of chromosome XIV, only five are accessible to cleavage. These sites map 11 by and 74 by to the left and 27 bp, 41 by and 290 by to the right, respectively, of the boundaries of the 118 by functional CEN14 DNA sequence. The distance between the sites accessible to cleavage and closest to CEN14 is 156 bp, suggesting this is the maximal size of DNA protected in CEN14 chromatin. The DNA in CEN14 chromatin protected against cleavage with DNase I and micrococcal nuclease overlaps almost completely with this region. Hypersensitive regions flanking both sides are approximately 60 by long. Analyses of other S. cerevisiae centromeres with footprinting techniques in intact cells or nucleolytic cleavages in isolated nuclei are discussed in relation to our results. We conclude that structural data of chromatin obtained with restriction nucleases are reliable and that the structure of CEN14 chromatin is representative for S. cerevisiae centromeres.

Key words

Centromere Chromatin Hypersensitive sites Yeast 

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References

  1. Achstetter T, Emter O, Ehmann C, Wolf DH (1984) Proteolysis in eukaryotic cells. J Biol Chem 259:1334–1343Google Scholar
  2. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1983) Molecular biology of the cell. Garland Publishing Inc., New YorkGoogle Scholar
  3. Almer A, Rudolph H, Hinnen A, Hörz W (1986) Removal of positioned nucleosomes from the yeast PHO5 promoter upon PH05 induction releases additional upstream activating DNA elements. EMBO J 5:2689–2696Google Scholar
  4. Barnes G, Rine J (1985) Regulated expression of endonuclease EcoRI in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 82:1354–1358Google Scholar
  5. Bloom KS, Carbon J (1982) Yeast centromere DNA is in a highly ordered structure in chromosomes and small circular minichromosomes. Cell 29:305–317Google Scholar
  6. Bloom KS, Amaya E, Carbon J, Clarke L, Hill E, Yeh E (1984) Chromatin conformation of yeast centromeres. J Cell Biol 99:1559–1568Google Scholar
  7. Brain RJ, Kornberg RD (1987) Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human analog, and its possible role as a transcription factor. Mol Cell Biol 7:403–409Google Scholar
  8. Carle GF, Olson MW (1984) Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res 12:5647–5665Google Scholar
  9. Carle GF, Olson MW (1985) An electrophoretic karyotype of yeast. Proc Natl Acad Sci USA 83:3756–3760Google Scholar
  10. Chikashige Y, Kinoshita N, Nakaseko Y, Matsumoto T, Murakami S, Niwa O, Yanagida M (1989) Composite motifs and repeat symmetry in S. pombe centromeres: direct analysis by integration of NotI restriction sites. Cell 57:739–751Google Scholar
  11. Cottarel G, Shero JH, Hieter P, Hegemann JH (1989) A 125 by CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere function in Saccharomyces cerevisiae. Mol Cell Biol 9:3342–3349Google Scholar
  12. Dingwall C, Lomonossof P, Laskey RA (1981) High sequence specificity of micrococcal nuclease. Nucleic Acids Res 9:2659–2673Google Scholar
  13. Drew HR (1984) Structural specificities of five commonly used DNA nucleases. J Mol Biol 176:535–557Google Scholar
  14. Hahnenberger KM, Baum MP, Polizzi CM, Carbon J, Clarke L (1989) Construction of functional artificial minichromosomes in the fission yeast Schizosaccharomyces pombe. Proc Natl Acad Sci USA 86:577–581Google Scholar
  15. Hieter P, Pridmore D, Hegemann JH, Thomas M, Davis RW, Philippsen P (1985) Functional selection and analysis of yeast centromeric DNA. Cell 42:913–921Google Scholar
  16. Hörz W, Altenburger W (1981) Sequence specific cleavage of DNA by micrococcal nuclease. Nucleic Acids Res 9:2643–2657Google Scholar
  17. Kenna M, Amaya E, Bloom K (1988) Selective excision of the centromere complex from Saccharomyces cerevisiae. J Cell Biol 107:9–15Google Scholar
  18. Koshland DE, Mitchison TJ, Kirschner MW (1988) Polewards chromosome movement driven by microtubule depolymerization in vitro. Nature 331:499–504Google Scholar
  19. Kunkel GR, Martinson HG (1981) Nucleosomes will not form on double-stranded RNA or over poly(dA)*poly(dT) tracts in recombinant DNA. Nucleic Acids Res 9:6860–6880Google Scholar
  20. Linxweiler W, Hörz W (1984) Reconstitution of mono-nucleosomes: characterization of distinct particles that differ in the position of the histone core. Nucleic Acids Res 12:9395–9413Google Scholar
  21. Lohr D, Tatchell K, Kovacic RT, van Holde KE (1977) Comparative subunit structure of HeLa, yeast, and chicken erythrocyte chromatin. Proc Natl Acad Sci USA 74:78–83Google Scholar
  22. Mitchison TJ, Evans L, Schulz E, Kirschner M (1986) Sites of microtubule assembly and disassembly in the mitotic spindle. Cell 45:515–527Google Scholar
  23. Nedospasov SA, Georgiev GP (1980) Non-random cleavage of SV 40 DNA in the compact minichromosome and free in solution by micrococcal nuclease. Biochem Biophys Res Commun 92:532–539Google Scholar
  24. Newton CS (1988) Yeast chromosome replication and segregation. Microbiol Rev 52:568–601Google Scholar
  25. Pavlovic B, Hörz W (1988) The chromatin structure of a glyceraldehyde phosphate dehydrogenase gene from Saccharomyces cerevisiae reflects its functional state. Mot Cell Biol 8:5513–5520Google Scholar
  26. Peterson JB, Ris H (1976) Electron microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae. J Cell Sci 22:219–242Google Scholar
  27. Prunell A (1982) Nucleosomc reconstitution on plasmid-inserted poly(dA)*poly(dT). EMBO J 1:173–179Google Scholar
  28. Saunders M, Fitzgerald-Hayes M, Bloom K (1988) Chromatin structure of altered yeast centromeres. Proc Natl Acad Sci USA 85:175–179Google Scholar
  29. Wu C (1980) The 5′ ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. Nature 286:854–859Google Scholar
  30. Wu KC (1983) Nucleosome arrangement in Green Monkey α-satellite chromatin. J Mot Biol 170:93–117Google Scholar
  31. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Martin Funk
    • 1
  • Johannes H. Hegemann
    • 1
  • Peter Philippsen
    • 1
  1. 1.Institute of Microbiology and Molecular BiologyJustus Liebig UniversityGiessenGermany

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