Construction, analysis, and application of a radiation hybrid mapping panel surrounding the mouse agouti locus

The region surrounding the agouti coat color locus on mouse Chromosome 2 contains several genes required for peri-implantation development, limb morphogenesis, and segmentation of the nervous system. We have applied radiation hybrid mapping, a somatic cell genetic technique for constructing long-range maps of mammalian chromosomes, to eight molecular markers in this region. Using a mathematical model to estimate the frequency of radiation-induced breakage, we have constructed a map that spans approximately 20 recombination units and 475 centirays8000. The predicted order of markers, Prn-p-Pygb-Emv-13-Psp-Xmv-10-Emv-15-Src-Ada, is consistent with a previously derived multipoint meiotic map for six of the eight markers and suggests that Xmv-10 may lie relatively close to one or more of the agouti recessive lethal mutations. The resolution of our map is approximately 40-fold higher than the meiotic map, but the median retention frequency of mouse DNA in hybrid cells, 0.12, is 4-fold lower than similar experiments with human chromosomes. From one of the radiation hybrid lines that contained a minimum amount of mouse DNA, 25 independent cosmids were isolated with a mouse-specific hybridization probe. Single-copy fragments from two of these cosmids were shown to originate from mouse Chromosome 2, and the meiotic map position of one was found to be within 10 recombination units of the region of interest. Our results indicate more precise map positions for Pygb and Xmv-10, demonstrate that radiation hybrid mapping can provide high-resolution map information for the mouse genome, and establish a new method for isolating large fragments of DNA from a specific subchromosomal region.