Structural Studies on Centromeres in the Yeast Saccharomyces Cerevisiae
In the yeast Saccharomyces cerevisiae, circular or linear plasmids containing a functional centromere (CEN) and a chromosomal replicator (ARS) are mitotically stable and segregate as ordinary yeast chromosomes in the first and second meiotic divisions. A centromere in S. cerevisiae consists of a region of DNA, approximately 150 bp in length, containing three important sequence elements, which are folded with proteins into a specific conformation in the chromatin (the yeast kinetochore). Each of the functional CEN sequences contains a high (91% to 95%) AT region (element II), 78 to 86 bp in length, flanked on one side by the common sequence PuTCACPuTG (element I), and on the other by the sequence TGTTT.TG.TTTCCGAAA…. AAA (element III). Deletions in the element II region partially inactivate mitotic function and cause precocious separation of the sister chromatids in meiosis I. Element III appears to be a protein binding site, as evidenced by the following observations. Various point mutations in element III inactivate centromere function, especially in the central CCG (17). One or more protein binding sites in the element III region can be demonstrated by an exonuclease III blocking assay. Wild-type CEN sequences compete strongly in this binding assay, whereas certain functionally inactive mutant CEN sequences do not. In addition, various DNA segments containing either CEN3 or the element III region strongly repress expression of the yeast GAL1 gene when inserted immediately upstream from the transcriptional start site. Helical DNA segments containing CEN3 or CEN14 are shown to be bent or distorted in shape in the high-AT element II region.
KeywordsProtein Binding Site XhoI Site Centromere Function Upstream Activator Sequence Centromere Sequence
Unable to display preview. Download preview PDF.
- 5.Bram, R.J., and R.D. Romberg (1986) Isolation of a yeast centromere DNA-binding protein, its human homologue, and its possible role as a transcription factor. Mol. Cell. Biol, (in press). Google Scholar
- 7.Broach, J.R. (1981) The yeast plasmid 2μ circle. In The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, J. Strathern, E. Jones, and J.R. Broach, eds. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 445–478.Google Scholar
- 8.Carbon, J., and L. Clarke (1984) Structural and functional analysis of a yeast centromere (CEN3). J. Cell Sci. (Suppl.) 1:43–58.Google Scholar
- 13.Guarente, L. (1983) Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. In Methods in Enzymology, Vol. 101, L. Grossman and K. Moldave, eds. Academic Press, Inc., New York, pp. 181–191.Google Scholar
- 17.McGrew, J., B. Diehl, and M. Fitzgerald-Hayes (1986) Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae. Mol. Cell. Biol. 6:530–538.Google Scholar
- 19.Miller, J.H. (1972) Assay of β-galactosidase. In Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 352–355.Google Scholar
- 22.Ng, R., S. Cumberledge, and J. Carbon (1986) Structure and function of centromeres. In Yeast Cell Biology, J. Hicks, ed. Alan R. Liss, Inc., New York (in press).Google Scholar
- 24.Rose, M., and D. Botstein (1983) Construction and use of gene fusions to lacZ (3-galactosidase) that are expressed in yeast. In Methods in Enzymology, Vol. 101, L. Grossman and K. Moldave, eds. Academic Press, Inc., New York, pp. 167–180.Google Scholar