The long-term goal of this proposal is to increase our understanding of the mechanisms that normally regulate homologous recombination in mammalian cells. Such an aim seems relevant in light of the often-made observation that cancer cells exhibit aberrant chromosomal rearrangements. To study intrachromosomal recombination using a molecular genetic approach, mouse Ltk- cell lines will be established that contain two defective Herpes thymidine kinase (tk) gene sequences residing as closely linked direct repeats within the L cell genome. Recombination events that reconstruct a functional tk gene will be monitored by the appearance of tk-positive segregants. This work will refine earlier measurements of the homology dependence of "normal" recombination. By studying recombination between imperfectly matched tk sequences, I will explore the question of whether genetic information is transferred as one contiguous block during homologous recombination. This proposal also includes studies of extrachromosomal recombination between defective tk sequences designed to elucidate what the rate-limiting step of recombination might be. Understanding the parameters governing homologous recombination processes has additional relevance to "gene targeting," or homologous recombination between a transfected DNA molecule and a specific chromosomal target sequence. Gene targeting offers a powerful genetic tool for understanding gene function as well as a potential methodology for eventual human gene therapy. Unfortunately, gene targeting in mammalian cells is usually quite inefficient. (Commonly, only one in 10/6 cells may be successfully targeted.) The experiments mentioned above are intended to yield insights that should have relevance to recombination mechanisms in general, including gene targeting. To gain additional insight into some of the obstacles that might hinder efficient gene targeting, this proposal addresses the question of whether transfection of DNA into mammalian cells induces genetic instability which may itself pose a problem in gene targeting strategies.