The Effect of DNA Homology on General Recombination in Cultured Mammalian Cells and Bacteria

Abstract

Plasmid DNA substrates (ptk²) that contain two defective HSV tk genes with overlapping homologous sequences have been used to study homologous recombination during the transfer of DNA into LMtk⁻ cells. Recombination between the homologous sequences of those two defective genes produces an intact tk gene whose function can be detected by conversion of Tk⁻ cells to Tk⁺ in selective media. Using this system, some aspects of a model of recombination proposed by Lin et al. (1984) have been tested. Plasmids with restriction sites located asymmetrically between the two defective genes were constructed to study the effects on recombination of altered symmetry between the cut ends and the two directly-repeated tk genes. Results indicate that increasing the asymmetry of cutting adversely affects recombination in mammalian cells. In addition, plasmids containing various amounts of overlapping homology were constructed and used to study the relationship between recombination frequency and DNA homology in both cultured mammalian cells and bacteria. The frequency of recombination in mammalian cells was shown to be dependent on the amount of tk homology in a manner that suggests that different recombination mechanisms work above and below 145 bp of homology. In bacteria, it could be shown that the only recombination pathway capable of cyclizing linearized ptk² DNA is the recE pathway.