Transfer of mlt mutations into polyomavirus intronless genomes by intramolecular recombination in bacteria

We describe a modification of the procedure of Weber and Weissmann [Nucl. Acids Res. 11 (1983) 5661-5669] for the formation of hybrid genes by in vivo recombination to introduce two separate mutations into the same gene. The mutants of interest are inserted as head-to-tail tandems in a bacterial plasmid in such a way that the 5'-proximal mutation is located upstream from the mutant with the more distal mutation. Propagation of the plasmid in a rec+ strain of Escherichia coli allows recombination between homologous sequences in the insert. DNA with the size expected for the recombinant plasmid is isolated by agarose gel electrophoresis, cloned in a recA strain, and characterized by restriction endonuclease mapping. Using this procedure, we have transferred the deletion from polyomavirus mutant dl-8 into other mutant genomes lacking the intervening sequences for either middle T or large T.