The long term goals of this research are to understand how trans-acting regulatory factors interact with each other and with cis-acting gene sequences to fine tune the level of expression of muscle-specific actin genes in muscle cells. The experiments in this proposal are designed to investigate the contribution of trans-acting factors to cell type specific differences in the regulation of expression of vascular smooth and skeletal muscle actin genes. One of two potentially convergent approaches investigates the effects of increasing the number of expressed myogenesis determination genes both on the pattern of accumulation of vascular smooth and skeletal muscle actin mRNAs and on the relative amounts of the myogenesis determination gene mRNAs in differentiating BC3H1 cells. BC3H1 cells normally express two myogenesis determination genes, myf-5 and myogenin, at appreciable levels. Cell lines expressing in addition myo-D and/or herculin will be derived by gene transfer. Blot hybridization analysis and ribonuclease protection assays will be used to determine and compare the patterns of expression of the actin and myogenesis determination mRNAs during differentiation in these and the parent cell line. The second approach is to look among various myogenic cell lines known to differ both in the way they regulate vascular smooth muscle actin gene expression and in the complement of myogenesis determination genes they express for in vivo differences in the specific sequences to which proteins bind in the 5' flanking regulatory region of the vascular smooth muscle actin gene. The technique to be used is in vivo footprinting facilitated by ligation mediated polymerase chain reaction. The goal for student participation is to involve students in the excitement of bench research, particularly that which explores the general questions surrounding regulation of eukaryotic gene expression in the specific context of myogenesis, and in so doing to expose students to the power of recombinant DNA approaches and train them in recombinant DNA techniques. This work is basic research which, by virtue of its contribution to the understanding of muscle cell development, may one day contribute to the understanding and cure of diseases of the muscular system.
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