Recent data from a variety of organisms, including mammals, suggests that genetic recombination plays a role not only in classical genetic exchange but also in gene regulation and evolution of the genome. By exploiting the techniques of recombinant DNA and DNA-mediated gene transfer, homologous recombination in cultured mammalian cells will be studied. Plasmid vectors will be constructed so as to allow the study of homologous recombination between closely linked (repeated) genes residing in the genome. A distinction between reciprocal and non-reciprocal (or gene conversion) events can be made readily accomplished using this system. In brief, two different insertion mutants of the Herpes simplex virus thymidine kinase (HTK) gene will be placed into a vector called PSV2-spt which contains a bacterial gene (xanthine guanine phosphoribosyl transference, gpt) that is dominantly expressed in mammalian cells. Thymidine kinase deficient cells will be transformed to gpt+ with these plasmids. After integration of the plasmid sequences (including the two mutant HTK genes) selection for the TK+ phenotype will be applied. Putative HTK+ recombinants will be analyzed using DNA-DNA hybridization techniques to determine the nature of the events. The effect of the orientation of the HTK gene pair (direct vs. indirect) will also be examined. Finally these plasmids will be used to measure recombination frequencies in various radiation and/or mutagen-sensitive mammalian cell mutants in the hopes of uncovering lines with abnormal levels of recombination.
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