Investigators' Abstract) The goal of this project is the construction of high resolution genetic and physical maps of the murine X chromosome from clotting factor 9 to Duchenne muscular dystrophy. This region contains one of the sites of chromosomal rearrangement between the human and murine X chromosomes as well as loci corresponding to several human disease genes. The region covers 10-12 cM or approximately 12-15 million base pairs (Mb). Recombinant backcross progeny from an interspecific mating of Mus domesticus and M. spretus will be typed for 8 anchor loci spanning the murine X chromosome and form a genetic mapping panel. 270 backcross progeny have been produced, and a total of 400 animals will be generated. DNA probes spaced 0.3 to 0.5 cM apart spanning the region from Cf-9 to Dmd will be identified. Existing human and murine probes which will be mapped include (a) random murine X """"""""linking clones"""""""" that map proximal to Pgk-1 and (b) human YAC (yeast artificial chromosome) clones from Xq27-Xq28. An adaptation of an efficient method to generate probes from human YACs is proposed to enable cross-species hybridization and mapping. Additional random murine X probes will be generated from mouse X:human hybrids using polymerase chain reaction (PCR) amplification of DNA between mouse repetitive sequences, a technique called interspersed repetitive sequence (IRS) PCR. Preliminary mapping of PCR-generated probes from a hybrid containing a fragment of the mouse X demonstrates the feasibility of the method. Additional deleted-X hybrids will be generated by X-irradiation of the mouse X:human hybrids. A physical map of the region will be compiled using the deleted X hybrids, pulsed field gel electrophoresis (PFGE), and alignment of overlapping YAC clones. Genetic and physical maps will be compared. Maps of the region will be compared with the corresponding region of the human X (Xq27-Xq28).
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