In previous work done in collaboration with J. Fleming and others, three genes (unc-29, unc-38, and lev-1) have been cloned and sequence encoding structural subunits of a nicotinic acetylcholine receptor from the nematode Caenorhabditis elegans. Polyclonal antibodies were raised in rabbits against N- and C-terminal regions of receptor peptides expressed as fusion proteins in E. coli. Three mouse monoclonal antibodies capable of precipitating receptor binding from crude homogenates were also generated in previous work.
The aim of this proposal is to generate additional monoclonal antibodies and to use the antibodies in Western blot analysis of peptide expression and in histochemical analysis of receptor placement in the nervous system to understand how mutants of 6 receptor genes and mutants of 5 other genes deficient in response to cholinergic agonists affect receptor expression. The work will provide a better understanding of a receptor that has been an important target for parasitic nematode chemotherapy and will facilitate the identification and in vitro analysis of equivalent receptor subunits from important parasitic nematodes, heretofore difficult to study. Antibodies against the receptor should also be useful for binding and studying mutants that affect synapse formation in the nematode. Monoclonal antibodies will be raised against purified receptor preparations or synthetic peptides homologous to regions containing conformationally insensitive epitopes in the vertebrate acetylcholine receptor. The normal receptor peptide produced by each gene will be identified by Western blot analysis will be performed on extracts of mutants in 6 receptor genes and of mutants in 5 other genes affecting cholinergic response. The same mutants will be compared to wild-type nematodes by histochemically screening whole mounts using alkaline- phosphatase- and immunofluorescent-conjugated secondary antibody.
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