Isolation of deletion and substitution mutants of adenovirus type 5

The infectivity of adenovirus type 5 DNA can be increased to about 5 x 10³ plaque-forming units per μg DNA if the DNA is isolated as a DNA-protein complex. Utilizing this improved infectivity, a method was developed for the selection of mutants lacking restriction endonuclease cleavage sites. The procedure involves three steps. First, the DNA-protein complex is cleaved with a restriction endonuclease. The Eco RI restriction endonuclease was used here. It cleaves adenovirus type 5 DNA to produce three fragments: fragment A (1-76 map units), fragment C (76-83 map units) and fragment B (10-83 map units). Second, the mixture of fragments is rejoined by incubating with DNA ligase, and, third, the modified DNA is used to infect cells in a DNA plaque assay. Mutants were obtained which lacked the endonuclease cleavage site at 0.83 map units. Such mutant DNAs were selected by this procedure because they were cleaved by the Eco RI endonuclease to produce only two fragments: a normal A fragment and a fused B/C fragment. These two fragments could be rejoined to produce a viable DNA molecule as a result of a bimolecular reaction with one ligation event; this exerted a strong selection for such molecules since a trimolecular reaction (keeping the C fragment in its proper orientation) and two ligation events were required to regenerate a wild-type molecule. The alterations resulting in the loss of the Eco RI endonuclease cleavage site at 0.83 map units include both deletion and substitution mutations. The inserted sequences in the substitution mutations are cellular in origin.