The long-term goal of this project is to test the efficacy of expanding and detailing the genetic and functional maps of the entire mouse genome with the techniques of experimental mutagenesis. High-efficiency mutagenesis of mouse spermatogonial stem cells with the supermutagen N-ethyl-N-nitrosourea (EMU) now puts the creation of such functional maps of the mammalian genome within experimental reach. A continually evolving, detailed functional map of the model mouse genome, based on a series of """"""""point"""""""" mutations with specific phenotypic effects, will be an important and necessary complement to the advances anticipated in the physical mapping of the human (and model) genomes, and will provide vehicle for direct correlations between cloned segments of DNA and specific developmental phenotypes. For this type of strategy to succeed, however, it is critical that additional data be obtained that diminish current basic uncertainties about the functional composition of the genome, the relative mutability of loci, the density of essential genes, the detectability of mutant phenotypes, and the ability to correlate a highly detailed functional (mutation) map with a physical/molecular map throughout the mammalian genome. The experiments outlined in this proposal are designed to address some of these questions and uncertainties by concentrating on the """"""""saturation"""""""" mutagenesis (with respect to specific phenotypes) of two genetically well-characterized regions of mouse chromosome 7 associated with long, radiation-induced deletion mutations. New recessive lethal and visible point mutations mapping to this 15% of chromosome 7 will be selected by a hemizygosity-screening protocol of highly mutagenized, ENU-treated gametes. A sufficient number of gametes will be screened to allow for multiple independent mutations to be recovered at each locus, with the goal of approaching saturation mutagenesis for detectable phenotypes within each region. The mutations will be fine-structure mapped with respect to panels of deletion mutations that exist for these regions; this fine-structure genetic mapping of point mutations will coincide with fine-structure molecular mapping ongoing within these same regions to test whether it will be possible to correlate detailed functional and physical maps. Moreover, as a prelude to distribution of mutations and their subsequent detailed phenotypic characterization, each mutation will be placed onto an inbred genetic background, and the time of death of new lethal mutations will be determined. This project should provide a basic framework on which to build strategies for expanding the functional map, by mutagenesis, of a larger portion of, or the entire, mouse genome, as well as for relating the physical DNA map of the mammalian genome to the functional map.
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