The long term goals of research in my laboratory are to understand how trans-acting regulatory factors interact with cis-acting gene sequences to regulate the (co)expression of muscle- specific genes in both skeletal and smooth muscle cells, and ultimately to learn something about the structure and regulation of the genes which code in turn for the trans-acting factors affecting muscle-specific gene expression. A number of diseases in muscle systems-cardiac myopathy, atherosclerosis, muscular dystrophy--involve changes from normal gene expression. Clearly, knowledge of the mechanisms by which gene expression is normally controlled in these tissues has the potential of divulging clues to diagnosis, prevention, or treatment of derangements in gene expression. The scope of this proposal is to determine which sequences in the 5' and 3' flanking regions of the mouse skeletal and vascular smooth muscle actin genes bind specifically with trans-acting protein factors present in differentiated cells of the smooth muscle-like cell line, BC3H- 1. This will be accomplished by gel shift assays using cloned fragments from the flanking regions of these genes. We will further test those same sequences to determine whether they bind factors present in cells which do not express the gene in question by performing similar assays with nuclear extracts from nor- expressing cells. Finally, we will determine whether sequences in the two different genes bird to the same factors by performing competition gel shift assays. Sequences which specifically bind factors will be further defined by nuclease digestion assays. The goal for student participation is to involve students in the excitement of research exploring the general questions surrounding regulation of eukaryotic gene expression in the context of the specific research outlined above, and in the process expose students to the power of recombinant DNA approaches and train them in recombinant DNA techniques.
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