Project: Research project

Project Details


We propose to investigate positional effect of mating type genes in the
yeast Saccharomyces cerevisiae. Three loci in Saccharomyces cerevisiae
carry one of two possible genetic elements that can determine cell type.
At one of these loci, MAT, this information is expressed to give rise to
the maitng type of the cell. At the other two loci, HML and HMR, this
information is phenotypically and transcriptionally silent, even though the
sequence of HML and HMR can be identical to that of MAT well in excess of
the coding regions contained within the element. Transcriptional
repression of HML and HMR requires the trans active gene products of four
unlinked loci, SIR1 through SIR4. In addition, repression the HML and HMR
requires the integrity of two sites (designated 'E' and 'I') at each locus,
which lie considerable distances from the promoters of the genes they

We plan to continue our analysis of the mechanism of transcriptional
repression at HML along two lines of experimentation that are well
developed in our laboratory. The first line involves extensive mutational
analysis of the 'E' site of HMl, coupled with isolation of second site
suppressors of such mutations. The second line involves analysis of the
SIR4 gene product. We have completely sequenced the gene and have raised
polyclonal and monoclonal antibodies against the protein encoded by it. We
plan to use these reagents to determine the physical interactions and
biochemical properties of this product. In addition, we are synthesizing
the products of the SIR2, SIR3, and SIR4 genes in vitro and will use this
material to determine the nature of the protein-protein and protein-DNA
interactions between these regulatory components. The results of these
experiments should provide significant insights into the role of enhancer
elements in controlling expression of eucaryotic genes. In addition these
results can provide a model system for evaluating positional effects
observed in higher cells as well as the role of alterations in chromatin
structure as a function of normal and abnormal development.

We have used the mating type system to develop a novel genetic selection
for mutations in essential genes required for maturation of tRNAs. We plan
to siolate and characterize such mutations, which should prove invaluable
in defining and analyzing the multitude of steps involved in synthesis of
mature tRNA molecules.
Effective start/end date12/31/8912/31/89


  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences


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