Simian virus 40 recombinants are produced at high frequency during infection with genetically mixed oligomeric DNA

Abstract

Classical approaches to analysis of mitotic recombination by use of simian virus 40 (SV40) are limited in usefulness because of low frequencies of recombination. To bypass the apparent rate-limiting step in normal SV40 recombination, oligomeric SV40 was constructed in vitro by ligation of mixtures of pairs of linear DNAs carrying genetically distinct temperature-sensitive mutations. Cultured monkey cells infected with the unfractionated ligation products yielded frequencies of nonparental recombinant progeny that were increased up to 500-fold relative to cells infected with a mixture of the untreated circular molecules. Pairwise crosses were performed with tsB4, tsB8, and tsBC11 DNAs, using unfractionated oligomers constructed from linear molecules cleaved by EcoRI or BamHI. In each cross the fraction of progeny with nonparental genotypes was roughly proportional to the physical distances between the mutant sites. These results suggest a random, rather than site-specific, conversion of oligomers to monomers. Somewhat surprisingly, nonligated mixtures of linear tsB4 and tsB8 DNAs, created by EcoRI digestion, produced a 40-t to 100-fold increase in the frequency of nonparental progeny. These results indicate that intermolecular associations must occur with fairly high efficiency between these linear molecules.