One of the key events that takes place during development of the central nervous system is the formation of synapses. Numerous lines of evidence indicate that expression of neurotransmitter sensitivity is fundamental to synaptogenesis. It is clear that change in sensitivity to neurotransmitters are due to changes in the molecular understanding of the formation of cholinergic synapses in the nervous system with the isolation of a family of genes encoding subunits of the receptors of acetylcholine (ACh). Although complementary DNA clones encoding ACh receptor subunits have been isolated and used to demonstrate functional diversity generated by instinct combinations of subunits in vitro, and the temporally- and spatially-restricted patterns of expression of the subunits in vivo, the cellular and molecular mechanisms controlling the expression of these genes are unknown. Therefore, as part of a long term goal to understand theses mechanisms, the specific aims of this proposal focus on identifying the regulatory mechanisms controlling the expression of, initially, one of these genes, that encoding the beta-4 subunit. Genetic elements required for the cell-type-specific expression of this gene will be identified and characterized, taking advantage of conferring cell-type-specific expression to a reporter gene in transfected mammalian cells. Identification of cellular factors that interact with these sequence elements will be accomplished using a variety of in vitro techniques. This information will be used to clone the genes encoding the factors. The clones will subsequently be used for the characterization of the factors. Finally, the mechanisms underlying the activation of the beta-4 subunit gene during neuronal differentiation will be investigated in PC12 cells, which, upon treatment with nerve growth factor, exhibit an increase in ACh receptor expression and an increase in the transcriptional activity of the beta-4 gene. The results of these studies will provide valuable information regarding not only ACh receptor gene expression in the nervous system, but also general principles of genetic regulation of neurotransmitter receptor diversity.
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