The aim of this proposal is the identification and characterization of the mouse severe combined immunodeficiency (scid) gene. Mice carrying the scid mutation, SCID mice, are deficient in double strand DNA repair, representing one of the rare viable mammalian models of a DNA repair system. SCID mice are profoundly deficient in mature lymphocytes due to failure of DNA recombination of the antigen receptor genes. The mice have proven to be invaluable for studies of lymphoid ontogeny and establishment of models of infectious diseases for vaccine development. Detailed analysis of the function of the scid gene in disease and in health is clearly warranted, but has been hindered by the lack of success trying to isolate the gene. This proposal outlines an approach to isolate the scid gene by positional cloning. This approach has not been directly applied to the identification of the scid gene, but has been used successfully to isolate several important human disease genes. The applicant has established a high resolution linkage map of the genomic region containing the scid locus based on the analysis of nearly 300 backcross progeny. The applicant has identified three molecular markers that do not recombine with the scid phenotype and, therefore, must be physically close to the scid gene. The applicant has used these markers to screen several mouse and human Yeast Artificial Chromosome (YAC) libraries and has identified multiple DNA clones. The first goal of this proposal will be to identify a YAC clone that contains the scid gene. This will be achieved using alternative approaches. The applicant has a system by which YACs can be assayed directly for their ability to complement the scid defect in vitro. If none of the YACs contain scid by this assay, additional overlapping YACs will be isolated to increase the genomic coverage. These YACs will be used to generate a physical map of the region of the genome containing the scid locus. The physical map of the region will be extended until recombination points, which flank the scid locus, are crossed. Once this is achieved, the applicant will be confident that the scid locus is contained within the contiguous set of YAC clones. YACs that must contain the scid locus will be used to identify expressed genes by hybridization to cDNA libraries and Northern blots, and analysis for CpG islands. Homologous human YACs will be used to identify regions of conserved sequence between human and mouse, which are likely to be candidate genes. Once the scid gene is isolated, it will be characterized for sequence homology to other proteins and the expression in normal development and in response to DNA damage. In addition, the homologues of scid will be isolated from other species for comparative and evolutionary analysis.