We have been engaged in a multifaceted investigation of the expression of the histone H3 and H4 genes in the yeast Saccharomyces cerevisiae. During the last period our experiments on regulation of the genes identified an upstream activation site (UAS) in the promoter region of the H3-H4 genes responsible for cell cycle control at the level of transcription. Gene deletions demonstrated a lack of dosage compensation among the H3-H4 gene sets indicating post-transcriptional regulation pathways as well. During the next period we will search for additional regulatory sites in the promoter region by linker substitution mutagenesis. We will attempt to identify and characterize the putative cell cycle activation protein and its gene by biochemical assays and genetic mutational analysis of the UAS. Post-transcriptional regulatory mechanisms will be examined by placing expression of histone mRNA under constitutive transcription control. Also during the last period nuclease mapping experiments demonstrated a set of cell cycle stage specific changes in the chromatin structure of the H3-H4 genes. The replication dependence of these changes will be investigated in cell division cycle mutants blocked in DNA replication. Future experiments will examine changes in hypersensitive sites at the sequence level by analysis of partial chromatin digestions and by in vivo methylation protection assays. We will also examine the relationship between hypersensitive sites and regulatory sequences such as the UAS by mutational analysis. Our previous genetic analyses of histone H4 have generated mutants with distinct phenotypes including increased mitotic chromosome loss, phenotypic sterility, and loss of function. During the next project period we plan to construct a tagged histone H4 by inserting the coding sequence for an antigenic oligopeptide. This will permit us to follow mutant H4 proteins by immunological techniques and determine expression levels of the protein, cellular localization, and interaction with chromatin. We have developed a rapid genetic screen for the efficient detection of functional H4 derivatives. A mutational map of permissible amino acid substitutions in the histone H4 will be derived from the sequence of these clones. Our analysis of mitotic stability H4 mutants will be extended by defining the amino acid substitutions that produce the defect. We plan to investigate the molecular basis for the phenotypic sterility histone H4 mutants. The hypothesis that these H4 mutants disrupt chromatin structure and prevent repression of silent mating type information will be tested by constructing double mutants with other genes involved in mating phenotype. Both the transcription of these related genes and the chromatin structure of the mating type genes will be assayed.
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