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. 1987 Dec;7(12):4522–4534. doi: 10.1128/mcb.7.12.4522

Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

R Ng 1, J Carbon 1
PMCID: PMC368137  PMID: 2830498

Abstract

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

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Selected References

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

  1. Avila J., Montejo de Garcini E., Wandosell F., Villasante A., Sogo J. M., Villanueva N. Microtubule-associated protein MAP2 preferentially binds to a dA/dT sequence present in mouse satellite DNA. EMBO J. 1983;2(8):1229–1234. doi: 10.1002/j.1460-2075.1983.tb01574.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blackburn E. H. The molecular structure of centromeres and telomeres. Annu Rev Biochem. 1984;53:163–194. doi: 10.1146/annurev.bi.53.070184.001115. [DOI] [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. Bloom K. S., Fitzgerald-Hayes M., Carbon J. Structural analysis and sequence organization of yeast centromeres. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):1175–1185. doi: 10.1101/sqb.1983.047.01.133. [DOI] [PubMed] [Google Scholar]
  6. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  7. Bram R. J., Kornberg R. D. Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor. Mol Cell Biol. 1987 Jan;7(1):403–409. doi: 10.1128/mcb.7.1.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brenner S., Pepper D., Berns M. W., Tan E., Brinkley B. R. Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients. J Cell Biol. 1981 Oct;91(1):95–102. doi: 10.1083/jcb.91.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Carbon J. Yeast centromeres: structure and function. Cell. 1984 Jun;37(2):351–353. doi: 10.1016/0092-8674(84)90363-5. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Cumberledge S., Carbon J. Mutational analysis of meiotic and mitotic centromere function in Saccharomyces cerevisiae. Genetics. 1987 Oct;117(2):203–212. doi: 10.1093/genetics/117.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fangman W. L., Dujon B. Yeast mitochondrial genomes consisting of only A.T base pairs replicate and exhibit suppressiveness. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7156–7160. doi: 10.1073/pnas.81.22.7156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Frei E., Baumgartner S., Edström J. E., Noll M. Cloning of the extra sex combs gene of Drosophila and its identification by P-element-mediated gene transfer. EMBO J. 1985 Apr;4(4):979–987. doi: 10.1002/j.1460-2075.1985.tb03727.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Gorbsky G. J., Sammak P. J., Borisy G. G. Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends. J Cell Biol. 1987 Jan;104(1):9–18. doi: 10.1083/jcb.104.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Levinger L., Varshavsky A. Protein D1 preferentially binds A + T-rich DNA in vitro and is a component of Drosophila melanogaster nucleosomes containing A + T-rich satellite DNA. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7152–7156. doi: 10.1073/pnas.79.23.7152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Margolis R. L., Wilson L. Microtubule treadmills--possible molecular machinery. Nature. 1981 Oct 29;293(5835):705–711. doi: 10.1038/293705a0. [DOI] [PubMed] [Google Scholar]
  28. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Mitchison T. J., Kirschner M. W. Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J Cell Biol. 1985 Sep;101(3):766–777. doi: 10.1083/jcb.101.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mitchison T., Evans L., Schulze E., Kirschner M. Sites of microtubule assembly and disassembly in the mitotic spindle. Cell. 1986 May 23;45(4):515–527. doi: 10.1016/0092-8674(86)90283-7. [DOI] [PubMed] [Google Scholar]
  32. Moroi Y., Peebles C., Fritzler M. J., Steigerwald J., Tan E. M. Autoantibody to centromere (kinetochore) in scleroderma sera. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1627–1631. doi: 10.1073/pnas.77.3.1627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Newman A. J., Lin R. J., Cheng S. C., Abelson J. Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro. Cell. 1985 Aug;42(1):335–344. doi: 10.1016/s0092-8674(85)80129-x. [DOI] [PubMed] [Google Scholar]
  35. Newman A. J., Ogden R. C., Abelson J. tRNA gene transcription in yeast: effects of specified base substitutions in the intragenic promoter. Cell. 1983 Nov;35(1):117–125. doi: 10.1016/0092-8674(83)90214-3. [DOI] [PubMed] [Google Scholar]
  36. Ng R., Ness J., Carbon J. Structural studies on centromeres in the yeast Saccharomyces cerevisiae. Basic Life Sci. 1986;40:479–492. doi: 10.1007/978-1-4684-5251-8_36. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tschumper G., Carbon J. Copy number control by a yeast centromere. Gene. 1983 Aug;23(2):221–232. doi: 10.1016/0378-1119(83)90054-9. [DOI] [PubMed] [Google Scholar]
  42. Wallace R. B., Johnson M. J., Hirose T., Miyake T., Kawashima E. H., Itakura K. The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA. Nucleic Acids Res. 1981 Feb 25;9(4):879–894. doi: 10.1093/nar/9.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wallace R. B., Johnson P. F., Tanaka S., Schöld M., Itakura K., Abelson J. Directed deletion of a yeast transfer RNA intervening sequence. Science. 1980 Sep 19;209(4463):1396–1400. doi: 10.1126/science.6997991. [DOI] [PubMed] [Google Scholar]
  44. Wu C. An exonuclease protection assay reveals heat-shock element and TATA box DNA-binding proteins in crude nuclear extracts. Nature. 1985 Sep 5;317(6032):84–87. doi: 10.1038/317084a0. [DOI] [PubMed] [Google Scholar]

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