GENETIC AND MOLECULAR ANALYSES OF MUTATIONS

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DESCRIPTION: (Adapted from investigator's abstract) This is a competitive renewal for an RO1 in its third funding cycle that requests five years of support to identify and analyze genes uncovered by genetic rearrangements that affect the p gene. Previous work by the applicant has defined two complementation groups -- runty/jerky/sterile (rjs) and cleft palate -- which lie proximal and distal to p, respectively. In the previous funding cycle, the applicant identified a very strong candidate for the rjs gene, demonstrated that deletion of the Gabrb3 gene was responsible for cleft palate, and, in addition, described an inversion allele, p100H, that disrupts the Sox6 gene and causes an unusual muscle disease reminiscent of Emery-Dreyfuss muscular dystrophy. The current application proposes to extend work in all three areas. The molecular pathogenesis of rjs deficiency will be investigated by further characterization of gene and protein expression, by identification of interacting proteins, and, in collaboration with others, biochemical and/or cell biologic assays of rjs domains that may function in protein ubiquitination and guanine nucleotide exchange. In addition, a loxP-flanked rjs allele will be created to investigate whether the pleiotropic phenotype caused by rjs deficiency reflects the sum of several tissue-specific defects. Preliminary studies suggest that cleft palate caused by deficiency for Gabrb3 reflects a requirement outside the central nervous system, pointing to a potentially novel role for GABA signaling during palate morphogenesis. These data will be confirmed by further studies of a Gabrb3 transgene controlled by the neuron-specific enolase promoter. In addition, the sites of Gabrb3 gene and protein expression, and GABA binding sites, will be characterized in non-neuronal tissues with special attention to the developing palate. The pathogenesis of muscle disease caused by the p100H mutation will be investigated by further characterization of a newly recognized Sox6 isoform highly expressed in muscle, by development of myoblast/myocyte cell culture systems from mutant animals, and by Sox6 gene rescue experiments in vivo and/or in vitro. In addition, differential display, cDNA subtraction, and/or gene expression profiling will be used to compare mutant and non-mutant tissues in an attempt to identify Sox6 targets.