The objectives of this proposal are to elucidate the molecular mechanisms which coordinate the activation of contractile protein synthesis during skeletal muscle differentiation. We will use recombinant DNA techniques to examine the chromosomal and sequence organization of skeletal and cardiac muscle troponin, tropomyosin and the coordinately expressed skeletal muscle contractile protein genes are linked in these genomes, share homologous nucleotide sequences in their protein coding, intervening or flanking regions, or undergo DNA rearrangement during myoblast differentiation. Transcription and processing of skeletal and cardiac contractile protein mRNAs also will be examined in cultures of quail myoblasts and in embryonic fast and slow embryonic muscles to distinguish whether the coordinate regulation of contractile protein mRNA accumulation is controlled at the level of transcription or RNA processing during myoblast and fiber-type differentiation. The involvement of chromatin structure in this coordinate regulation also will be explored by analysis of the DNase I sensitivity of contractile protein genes in chromatin in relation to the transcription of these genes during myogenesis. Finally, in vitro transcription systems will be developed using cloned DNA segments of genes and isolated muscle nuclei to examine the function of specific DNA sequences and cellular molecules in the transcription of the contractile protein genes.
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