It is our goal to continue our molecular and genetic analysis of acetylcholine synthesis and flow in the soil nematode Caenorhabditis elegans. These studies focus on-the cha-1 - unc-17 gene complex; cha-1 is the structural gene for choline acetyltransferase (ChAT), the enzyme which synthesizes the neurotransmitter acetylcholine, and we have recently shown that unc-17 is the gene which encodes the synaptic vesicle acetylcholine transporter. C. elegans is being used for these studies because of its simple nervous system, its ease of genetic analysis, and the availability of DNA-mediated transformation techniques. The cloned acetylcholine transporter will be expressed in mammalian cells, and the biochemical and pharmacological properties of acetylcholine transport will be characterized. Mutants of acetylcholine transport (unc-17 mutants) will similarly be analyzed, in order to correlate specific protein alterations with observed functional defects. Antibodies which bind to ChAT and the transporter protein will be used to identify all of the cholinergic neurons in C. elegans. The regulation of cha-1 - unc-17 expression and alternative splicing will be studied by DNA-mediated transformation experiments using sets of deleted or substituted derivatives. We will also attempt to identify additional genes whose products interact with ChAT and/or the unc-17 protein by isolating extragenic suppressors of cha- 1 or unc-17 and by using the newly-developed two-hybrid system. The goal of these studies is to increase our knowledge of the mechanisms of neurotransmitter synthesis and packaging, as well as the regulation of these processes. Although this is basic research, it is clearly relevant to health, since alterations in acetylcholine metabolism and/or cholinergic function have been identified in many neurological and psychiatric disorders, including familial infantile myasthenias, Alzheimer's disease, and depression.
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