The long-term goal of this research is to identify the mechanisms by which chromosomal structure contributes to the down regulation of gene expression and to characterize this regulation during the cell cycle by using S. cerevisiae as a model for other eukaryotes. S. cerevisiae has three loci, HMR, HML, and MAT, that encode alternative alleles, a or alpha, which determine the mating type of yeast. The allele at MAT is expressed. However, the alleles at HMR and HML are not transcribed, or silenced by the combined action of specific sequences on the chromosome called silencers, and several proteins, including SIR1, SIR2, SIR3, and SIR4, which do not bind DNA directly. The point in the cell cycle when the establishment of silencing occurs will be defined by monitoring mRNA expression of alpha1 from a modified HMR regulated by a synthetic silencer in the presence of hydroxyurea, a temperature-sensitive mutant of CDC9, or nocodazole to block cells in S, G2 or M phase, respectively. The role of DNA replication in establishing silencing will be assessed by using FLP1 recombinase to excise this modified HMR from its chromosomal context. To bypass the role of SIR1 in establishing silencing, SIR3 and SIR4 will be localized to a synthetic silencer containing Ga14 DNA binding sites via their association with the amino terminal tails of histones H3 and H4 fused to the Ga14 DNA binding domain.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM019392-02
Application #
2910036
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Tompkins, Laurie
Project Start
1998-05-01
Project End
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Rusche, Laura N; Kirchmaier, Ann L; Rine, Jasper (2002) Ordered nucleation and spreading of silenced chromatin in Saccharomyces cerevisiae. Mol Biol Cell 13:2207-22
Kirchmaier, A L; Rine, J (2001) DNA replication-independent silencing in S. cerevisiae. Science 291:646-50