The long-term objective of this proposal are to study the organization, evolution and function of the histone multigene family, paying special attention to evolutionarily conserved histone variants. Antibodies to the macronuclear-specific Tetrahymena thermophila H2A variant hv1 and to mouse H2A.X will be used to confirm the identity of a conserved epitope that is localized to nuclear chromatin in mammalian cells and is associated with developmentally active loci in Drosophila and to test the hypothesis that shuffling of conserved epitopes occurs during evolution of histone H2A. Immunoprecipitation of formaldehyde crosslinked macronuclear chromatin with antibodies to hv1, to the macronuclear- specific basal H3 variant hv2 and to RNA polymerase II will be used to test the hypotheses that hv1 is associated with transcriptionally active or competent genes and that hv2 replaces the major H3 molecules that turn over during transcription. The crosslinking-immunoprecipitation method will also be used to study the proteins associated with these histone variants. Chromatin reconstitution will be used to determine whether variant containing core particles have an altered structure. The function of a small conserved sequence found in the carboxy termini of yeast H2As an of some H2A variants will be studied by mutating the element and studying its function in yeast. A Drosophila H2A variant whose existence is predicted from our studies of Tetrahymena and Drosophila H2A variants will be cloned and analyzed. These studies should shed light on the function of histone variants in regulation of gene expression in eukaryotes. This function must be understood in normal cells before a complete understanding of aberrant gene activity during abnormal development and neoplastic transformation can be achieved.
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