The control of gene expression underlies the development and maintenance of different cell types in eukaryotes, and often results from the complex interaction of different regulatory elements. An understanding of the molecular mechanisms of gene control will require analysis of systems in which the relevant cis- acting DNA elements and the trans-acting protein factors can be readily identified, isolated, and manipulated.
The aim of this proposal is to understand the mechanism of action of transcriptional 'silencers', novel regulatory elements which play a key role in controlling mating type in the yeast Saccharomyces cerevisiae. Yeast mating type (either a or alpha) is determined by genes at the MAT locus. Additional complete copies of mating-type genes are present at other loci (HML and HMR), but are not transcribed. These 'silent' loci act as donors of information in a mating-type switching process. The HML and HMR loci are repressed by flanking DNA sequences called 'silencers' which act over large distances (greater than 1 kb) to control transcription and transposition. The silencers contain multiple regulatory elements, and proteins which bind to two of these elements have been identified. One protein (RAP1) has been purified and its gene has been cloned. Paradoxically, RAP1 is also involved, in other contexts, in transcriptional activation. The specific (SBF-B) is involved both in silence function and DNA replication initiation. The other protein aims of this proposal are first to study the function of RAP1 by (1) mutation of the cloned gene, and (2) analysis of interacting molecules by both direct biochemical methods and by the isolation of suppressors of rap1 mutations. Other silencer binding factors (e.g. SBF-B) will be purified and cloned, and possible interactions between silencer binding factors will be examined biochemically. Finally, the DNA elements necessary and sufficient for silencer function will be determined by deletion analysis and reconstruction experiments.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040094-02
Application #
3297442
Study Section
Genetics Study Section (GEN)
Project Start
1988-04-01
Project End
1991-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Type
Schools of Medicine
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
Moretti, P; Shore, D (2001) Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Mol Cell Biol 21:8082-94
Vannier, D; Damay, P; Shore, D (2001) A role for Sds3p, a component of the Rpd3p/Sin3p deacetylase complex, in maintaining cellular integrity in Saccharomyces cerevisiae. Mol Genet Genomics 265:560-8
Marcand, S; Wotton, D; Gilson, E et al. (1997) Rap1p and telomere length regulation in yeast. Ciba Found Symp 211:76-93; discussion 93-103
Marcand, S; Gilson, E; Shore, D (1997) A protein-counting mechanism for telomere length regulation in yeast. Science 275:986-90
Shore, D (1997) Telomere length regulation: getting the measure of chromosome ends. Biol Chem 378:591-7
Wotton, D; Shore, D (1997) A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in Saccharomyces cerevisiae. Genes Dev 11:748-60
Shore, D (1997) Telomerase and telomere-binding proteins: controlling the endgame. Trends Biochem Sci 22:233-5
Marcand, S; Buck, S W; Moretti, P et al. (1996) Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap 1 protein. Genes Dev 10:1297-309
Chi, M H; Shore, D (1996) SUM1-1, a dominant suppressor of SIR mutations in Saccharomyces cerevisiae, increases transcriptional silencing at telomeres and HM mating-type loci and decreases chromosome stability. Mol Cell Biol 16:4281-94
Wotton, D; Freeman, K; Shore, D (1996) Multimerization of Hsp42p, a novel heat shock protein of Saccharomyces cerevisiae, is dependent on a conserved carboxyl-terminal sequence. J Biol Chem 271:2717-23

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