This proposal deals with the developmental regulation of the myosin heavy chain (MHC) gene family during embryogenesis and terminal muscle differentiation in the chick limb. The MHC genes are expressed in a developmental-stage-specific and tissue-specific manner. The overall aim of this project is to understand the structural modifications of the myosin genes which accompany, and may be responsible for their differential transcriptional activation. A MHC gene expressed specifically in the embryonic fast white muscle will be compared with a gene which is expressed only in the adult fast white fibers. Using DNAse I treatments of chromatin the nuclease hypersensitivity of the 5'-regions of the expressed and non-expressed genes during development in the different tissues will be measured in order to catalogue the hypersensitive sites, and correlate them with the transcriptional activation or inactivation of the genes. These data will then be used to map subdomains of sequences within the hypersensitive regions that are resistant to exonuclease III, in order to discern the binding of protein factors during the activation and deactivation of these genes. The degree of methylation will be measured, first on a gross level by using restriction enzymes that do, or do not recognize methylated sequences. This analysis will be followed by """"""""genomic sequencing"""""""" (Church & Gilbert, PNAS 81: 1991) which is able to detect the degree of methylation at all of the possible sites in the chosen sequence. These studies are directed toward the goal of understanding the molecular basis for the specific and regulated expression of the myosin gene family. By cataloging these basic structural parameters it becomes possible to anticipate the functional consequences and undertake a rational search for the factors which regulate the MHC genes' expression.
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