Physical mapping of temperature-sensitive mutations of adenoviruses

Temperature-sensitive mutants of the adenovirus 2-simian virus 40 hybrid, Ad2+ ND1‡, were isolated and crossed with temperature-sensitive mutants of adenovirus type 5 (Ad5). Forty-five wild type recombinants were selected and their DNAs were separately digested with five restriction endonucleases, EcoRI, HpaI, BamI, HindIII and SmaI. There are 15 sites cleaved by these enzymes that are unique to one parental DNA or the other. The fragments obtained by digestion of each of the recombinant DNAs were separated by gel electrophoresis and compared with those obtained from the parental genomes. The recombinant DNAs were found to consist of sequences derived both from Ad5 and from Ad2+ND1. Knowing the positions at which the five restriction enzymes cleave the genomes of the parental serotypes, it was possible to decide which regions of each recombinant DNA are composed of Ad5 and which of Ad2+ND1 sequences. In this way the cross-over events that generated the recombinants were mapped and the physical locations of the ts mutations in the parental genomes were determined.