Detection, rescue, and mapping of mutations in the adenovirus DNA binding protein gene

r(ts107)202 was isolated in HeLa cells as a temperature-independent revertant of H5ts107, an adenovirus mutant that maps in the structural gene of the viral DNA-binding protein. r(ts107)202 is a host-range temperature-conditional mutant: it is temperature independent for growth in HeLa cells but temperature sensitive for growth in 293 cells, a type 5 adenovirus-transformed human cell line. Marker rescue experiments using H5ts107 DNA and restriction enzyme fragments from r(ts107)202 DNA demonstrated that the mutations causing the r(ts107)202 phenotype were localized in HindIII fragment A containing the entire DNA-binding protein gene. To obtain a fine structure map of the r(ts107)202 mutations, overlap recombination between EcoRI fragment A (0-75.9 map units) from either Ad5wt or r(ts107)202 and BamHI fragment B (59.5-100 map units) from either Ad5wt or r(ts107)202 was performed. Segregation of the H5ts107 primary-site mutation away from the accompanying reversion mutation could be demonstrated in 5 of 200 plaques when r(ts107)202 EcoRI fragment A was crossed with the Ad5wt BamHI fragment. In the reciprocal cross, none of 200 plaques contained the H5ts107 mutant. These results permitted a determination of the order of the primary-site mutation (H5ts107) and secondary-site mutation in r(ts107)202, and the frequency of recombination predicted a distance of 50-340 base pairs between these two mutations. This experimental result agrees with the nucleotide sequence of the r(ts107)202 mutant which shows that 182 base pairs separate the primary-site and secondary-site mutations in r(ts107)202.