The long-term goal of the research described in this application is the elucidation of how chemical and electrical excitation of the plasma membrane affect gene expression in the neural control of acetylcholine sensitivity. Analysis of acetylcholine receptor regulation is important for a better understanding of neurological and neuromuscular disorders such as Alzheimer's disease or myasthenia gravis. The denervation-induced increase in acetylcholine receptor synthesis rate will be investigated in chick skeletal muscle. It is now known that during differentiation of myogenic cells and upon denervation of mature muscle fibers the transcription of the genes coding for the receptor subunits is activated. Regulatory elements in these genes that mediate stage- and tissue-specific expression will be identified by transfection of constructs containing sequences of interest as well as suitable reporter genes. The response of transfected cells to drugs that stimulate or block membrane activity will be measured and will permit identification of regulatory elements have already been identified in the upstream flanking regions of the alpha- and delta-subunit genes; future work will focus on other noncoding regions of these genes and on the gamma-subunit. Identification, isolation, and analysis of beta- and alpha-subunit genes will also be undertaken. The initial deletion mutation analysis of cis elements will be followed by more precise delineation using point mutations and footprinting techniques. Eventually, the developmental as well as stimulus-induced coordinated regulation of all subunits will be investigated in vitro and in transgenic animals.
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