The long-term objectives of this proposal are to identify and to study a locus that is required for normal murine development in utero and for normal hepatic function after birth. This locus is identified by an insertional mutation due to a transgene integration; homozygous transgenic mice are runted at birth, feed poorly, and usually die during the first two days of life with a severely necrotic liver. The morphology of the hepatic injury appears similar to that seen in toxic liver injury or fulminant hepatitis. The affected locus is called ple, for perinatal lethality. The transgene has been mapped to a region of mouse chromosome 15 whose genetic organization has been conserved during evolution, suggesting the likelihood that there is a human homolog of the ple locus. The insertion site of the transgene has been cloned, which will facilitate the characterization of the gene(s) whose expression is affected by the transgene integration.
The specific aims of the proposal are to use well-proven strategies for the identification of transcribed genes in cloned genomic DNA sequences to obtain a full-length cDNA to the locus (or loci) that are disrupted by the transgene. Since there is evidence (by cross-hybridization) that there are related murine loci and the suggestion (by conservation of synteny) that there is a human homolog, these genes will also be pursued. These will then be characterized by biochemical and expression analysis in order to gain understanding of their role in normal murine development and hepatic metabolism. Because the region of chromosome 15 to which the ple locus maps contains a number of mouse mutations, and because this region has been conserved in humans, it is furthermore a specific aim to generate a detailed physical and genetic map of this region using pulsed-field gel electrophoresis and interspecific recombination mapping analysis, respectively. Additionally, it should be feasible to use crosses of ple with recombinant inbred mouse strains to map loci which influence expression of the ple phenotype. Based on genetic and physical analysis, it is possible that the disrupted locus is a member of the type II cytokeratin gene family. While this class of intermediate filaments has been studied extensively biochemically, no specific mutations in a cytokeratin have been identified, and their role in cellular physiology is not well understood. If the ple insertional mutation proves to be a cytokeratin, it will serve as a valuable resource for the study of inter-mediate filament gene function. Finally, a number of abnormal phenotypes, some clearly due to spontaneous mutations that are unlinked to the transgene, have been identified in this transgenic line. This transgenic line may be genetically unstable and have an elevated mutation frequency. Since there is presently insufficient evidence to prove this hypothesis, it will be necessary to continue to characterize the morphology and heritable nature of the abnormal phenotypes.
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