The mating type loci in Saccharomyces cerevisiae provide a striking example of position effect regulation. Mating type regulatory genes located at the ends of chromosome III of yeast in loci, designated HML and HMR, are repressed by a novel regulatory system comprised of """"""""silencers"""""""" that mediate the action of products of four unlinked genes, SIR1 through SIR4, to render DNA in the vicinity of the silencers generally refractory to transcription. We have shown that DNA spanning the silent cassettes is packaged in nucleosomes that are hypoacetylated relative to those in active portions of the genome and that this hypoacetylation is catalyzed by the products of the SIR genes themselves. In addition, we have defined the salient features of the silencers located at HML and have shown that they act in tandem probably to direct chromatin condensation across the silent cassettes. We propose (1) to use hypoacetylation as a tag to define the extent and organization of silenced chromatin and its relationship to the structure and orientation of the silencers; (2) to examine the role of histones H1 and H5 homologs in silencing and assess the facility with which chromatin from silenced regions can undergo transition into a condensed state; (3) to identify, clone and characterize novel genes involved in silencing. We also propose to examine the molecular mechanism underlying directionality of mating type interconversion in yeast. Activation of information within the two silent mating cassettes occurs by directed transposition to an expressor locus MAT. Selection of the donor cassette during any one transposition is not random but is dictated by the mating allele present at MAT. This yields a precisely patterned order for mating type interconversion. We plan to examine the mechanism underlying this directionality of mating type interconversion by identifying cis and trans components required for selection of the donor locus, through (1) characterization of trans-acting mutants we previously isolated that have lost the normal bias in selecting mating type cassettes; and (2) interchanging HML and HMR cassettes along with varying extents of their flanking sequences in order to identify sites used by the cell to distinguish the two loci.
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