This is the first competitive renewal of an RO1 application that requests five years of support to study notochord development by identifying the gene mutated in Sd as an entry point. In the three years since the grant was first awarded, the applicant used embryonic chimera studies to determine whether or not Sd and tc (another mutation that affects notochord development) act in a cell-autonomous manner, and initiated a series of experiments to isolate Sd on the basis of its map position. The current application proposes to complete these experiments and to begin characterizing the Sd gene product at the level of gene and protein expression. At present, Sd is localized to a 0.3 cM interval; one side of the interval is closed physically with a 650 kb BAC contig (of which 600 kb is non-recombinant), the other side is defined by a molecular marker, D2Ucl3, that lies 7/2919 recombinants away from Sd. In the first specific aim the applicant will complete the physical map by chromosome walking using BACs and YACs, and try to narrow the critical region by carrying out BAC transgenic rescue experiments and by generating additional recombinant animals in a new backcross. (The applicant provided an update prior to the grant review that an additional two steps of chromosome walking had closed the physical gap, narrowing the critical region to <750 kb). In the second specific aim directed at molecular identification of Sd, a search for candidate genes will proceed by sequence sampling of 1-2 BAC clones if the critical region can be narrowed to <150 kb; if the region is larger, cDNA selection will be carried out using tissue from E9.5-10.5 whole embryos. Initial verification of a candidate gene as mutated in Sd will be based on expression and sequence structure analyses that compare mutant and normal tissues, followed by transgenic approaches to rescue or reproduce the mutant phenotype. Finally, in a third specific aim, mechanistic studies of the Sd gene product will be initiated by isolating a full-length cDNA, generating antibodies, and carrying out a detailed analysis of RNA and protein expression.
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