We wish to continue our studies on the molecular architecture of eukaryotic chromosomes by addressing the following four major aims with regard to nucleosome structure: 1. Binding Sites on Nucleosomes. We wish: (a) to map the points of contact between HMG-17 and histone octamers when either the first or second HMG binding site is occupied on nucleosomes; (b) to determine the polarity of adjacent histone H1 molecules along polynucleosomes; and (c) to map the points of contact between H1 and the histone octamer at the second H1 binding site on nucleosomes. These studies will employ techniques of zero-length protein crosslinking and peptide mapping. 2. DNA Methylation and the Nucleosome. We wish: (a) to determine the positions of 5-methylcytosine along the path of nucleosomal DNA; (b) to map the locations of methyl groups at higher levels of chromatin organization; and (c) to determine if a fraction of methylated DNA is organized in Z-form in isolated nuclei. Immunologic, radiochemical, and electron microscopic techniques will be employed. 3. Unusual Histone H2A Variants. We wish: (a) to determine the complete amino acid sequence of H2A.Z; and (b) to study the in vitro role of this protein in histone octamer formation. Protein chemistry and nucleosome reconstitution techniques will be used. 4. DNase I Hypersensitive Sites. We wish to identify, by in vitro mutagenesis, the in vivo base sequence determinants of the hypersensitive sites of a yeast heat shock gene. Standard procedures of recombinant DNA technology will be employed in conjunction with mapping hypersensitive sites by the indirect end labeling procedure.
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