CAREER: Tagged Transposon-Mediated Mutagenesis in C.elegans

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Jorgensen 9733685 Abstract Exocytosis in neurons has become specialized in order to allow for the rapid secretion of neurotransmitter. A number of proteins involved in synaptic vesicle trafficking have been identified by biochemical purification from synaptosomes. Nevertheless, current models for neurosecretion are greatly limited because there is only an incomplete list of the proteins that are required for these processes. Genetic studies in the nematode, Caenorhabditis elegans, could potentially identify the complete complement of genes required for synaptic function. C. elegans is particularly advantageous for such studies because it is possible to select for mutants defective in secretion. Using such screens, over 38 genes have been identified which are required for normal neurotransmission. The efficiency of these screens has generated more mutants than can be easily characterized using standard genetic techniques. Techniques to rapidly map and clone these genes lags far behind the ability to obtain mutants. Mutagenesis strategies using transposons modified to contain foreign plasmid sequences could allow the immediate cloning of mutant genes without the need to map the mutations. The aims of this proposal are to: 1) Develop methods to mobilize recombinant Tc3 transposons in the germ line, 2) Develop techniques to mobilize a heterologous mariner element in C. elegans, 3) Train undergraduates to screen for neurotransmission mutants in C. elegans using a recombinant mariner element and to directly sequence the mutant genes in a single semester course. The tools developed by this work will benefit the larger scientific community and accelerate experiments designed to analyze neurotransmission. The techniques developed will greatly increase the efficiency of forward genetic methods. Additionally, the development of these tools and reagents will provide students with a more complete laboratory experience.