Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Jul 1;107(1):9–15. doi: 10.1083/jcb.107.1.9

Selective excision of the centromere chromatin complex from Saccharomyces cerevisiae

PMCID: PMC2115176  PMID: 2839524

Abstract

We have taken advantage of the known structural parameters associated with centromere DNA in vivo to construct a CEN fragment that can be selectively excised from the chromatin DNA with restriction endonucleases. CEN3 DNA is organized in chromatin such that a 220-250- bp region encompassing the elements of centromere homology is resistant to nuclease digestion. Restriction enzyme linkers encoding the Bam HI- recognition site were ligated to a 289 base pair DNA segment that spans the 220-250-bp protected core (Bloom et al., 1984). Replacement of this CEN3-Bam HI linker cassette into a chromosome or plasmid results in formation of a complete structural and functional centromeric unit. A centromere core complex that retains its protected chromatin conformation can be selectively excised from intact nuclei by restriction with the enzyme Bam HI. The centromeric protein-DNA complex is therefore not dependent upon the intact torsional constrains on linear chromosomes for its structural integrity. Isolation of this complex provides a novel approach to characterizing authentic centromeric proteins bound to DNA in their native state.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  1. 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]
  2. Bloom K. S., Anderson J. N. Fractionation of hen oviduct chromatin into transcriptionally active and inactive regions after selective micrococcal nuclease digestion. Cell. 1978 Sep;15(1):141–150. doi: 10.1016/0092-8674(78)90090-9. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. Forte M. A., Fangman W. L. Naturally occurring cross-links in yeast chromosomal DNA. Cell. 1976 Jul;8(3):425–431. doi: 10.1016/0092-8674(76)90155-0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Hegemann J. H., Pridmore R. D., Schneider R., Philippsen P. Mutations in the right boundary of Saccharomyces cerevisiae centromere 6 lead to nonfunctional or partially functional centromeres. Mol Gen Genet. 1986 Nov;205(2):305–311. doi: 10.1007/BF00430443. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Karin M., Najarian R., Haslinger A., Valenzuela P., Welch J., Fogel S. Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast. Proc Natl Acad Sci U S A. 1984 Jan;81(2):337–341. doi: 10.1073/pnas.81.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Larsen A., Weintraub H. An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin. Cell. 1982 Jun;29(2):609–622. doi: 10.1016/0092-8674(82)90177-5. [DOI] [PubMed] [Google Scholar]
  12. Lilley D. M. The inverted repeat as a recognizable structural feature in supercoiled DNA molecules. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6468–6472. doi: 10.1073/pnas.77.11.6468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. McGhee J. D., Wood W. I., Dolan M., Engel J. D., Felsenfeld G. A 200 base pair region at the 5' end of the chicken adult beta-globin gene is accessible to nuclease digestion. Cell. 1981 Nov;27(1 Pt 2):45–55. doi: 10.1016/0092-8674(81)90359-7. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Nelson R. G., Fangman W. L. Nucleosome organization of the yeast 2-micrometer DNA plasmid: a eukaryotic minichromosome. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6515–6519. doi: 10.1073/pnas.76.12.6515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Smart J. E., Bonner J. Selective dissociation of histones from chromatin by sodium deoxycholate. J Mol Biol. 1971 Jun 28;58(3):651–659. doi: 10.1016/0022-2836(71)90030-1. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Weintraub H. A dominant role for DNA secondary structure in forming hypersensitive structures in chromatin. Cell. 1983 Apr;32(4):1191–1203. doi: 10.1016/0092-8674(83)90302-1. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES