The T/t complex on mouse chromosome 17 contains a number of mutant genes that function during early embryonic development, and others that affect the differentiation of spermatozoa. Thus it has been intensively investigated as a model system for analyzing differentiation and its genetic control. Until recently, however, the T/t complex was not approachable in terms of molecular biology since the genetic and molecular markers necessary for investigation at the level of DNA structure were not available. New results from this laboratory, obtained with a combination of genetic, serological, and molecular techniques have now provided the necessary material for intensive molecular analysis. We have defined 7 t-lethal genes as non-allelic but functionally related, and have accurate information on their location relative to one another and to other chromosome markers, including the TL, K, I, anbd D regions of the MHC. Four of the lethal genes are interspersed with MHC genes. We have generated an extensive panel of recombinant chromosomes, derived by crossing over between two different t-haplotypes, and defined their breakpoints. Fourteen of these are intra H-2 recombinants, with breakpoints defined by restriction fragment polymorphisms for 7 MHC probes. Thus for the first time the opportunity exists for the molecular investigation of a gene complex that regulates development in a mammal. This project proposes three complementary strageties to define the molecular organization of the mutant T/t complex and compare it to its wild type counterpart, and to identify and clone mutant genes in that region. (1) Chromosomal walking in a cosmid genomic library of homozygous t-w5 DNA, initiated with probes to H-2 class I genes and with K region probes that map very close (0-0.1 cM) to the t-w5 lethal mutation, will produce a restriction map of the MHC that can be compared to wold type. (2) A similar chromosomal walk in a cosmid genomic library of t-12/t-w5 DNA will permit a comparison of the restriction maps of the MHC of t-12 with t-w5. This comparison will be especially interesting because it will give information on evolutionary changes in DNA structure that can occur in the virtual absence of recombination. It also may help to pinpoint structural aberrations associated with the t-12 and t-w5 lethal mutations. (3) Cloning of chromosome 17 sequences expressed during spermatogenesis in normal and mutant testes to identify and map genes responsible for T/t complex phenotypes.
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