We wish to continue our chromatin studies on active, potentially active, and inactive mammalian genes by addressing the following four major aims: 1. Ribosomal Chromatin We wish: (a) To determine the distribution of non-nucleosomal and nucleosomal ribosomal chromatin in human-mouse somatic cell hybrids in which the expression of either human or mouse rRNA genes is allelically excluded; (b) To determine the distribution of non-nucleosomal and nucleosomal ribosomal chromatin during the course of rRNA gene activation in regenerating liver; and (c) To elucidate the molecular structure of non-nucleosomal ribosomal chromatin. These studies will employ nucleic acid hybridization, recombinant DNA technology, and specific murine biological systems. 2. Immunoglobulin Chromatin (The Kappa light chain gene) We wish: (a) To identify the proteins that are associated with active nucleosomes; (b) To elucidate the biochemistry of the putative association of active chromatin with the nuclear matrix; (c) To assess the role of an enhancer element in determining the active chromatin phenotype; (d) To define the biochemical basis of a chromatin boundary; and (e) To determine the role of cell differentiation and/or gene rearrangement on these parameters. These studies will employ nucleic acid hybridization, recombinant DNA technology, DNA mediated gene transfer, and specific murine cell lines. 3. Metallothionein-I Chromatin We wish to determine the chromatin phenotypes of the MT-I gene: (a) in its non-induced, potentially active state; (b) in its induced, active state; and (c) in its non-expressed, inactive state. These studies will employ nucleic acid hybridization and specific cell lines. 4. Methylation Sites Along Specific Genes We wish to develop techniques to map every 5-methylcytosine residue along any specific gene by employing antibody isolation and nuclease protection procedures, as well as DNA sequencing strategies.
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