The genetic analysis of recombination using adenovirus overlapping terminal DNA fragments

Abstract

We have studied the consequences of genetic recombination between overlapping terminal fragments of adenovirus genomes with respect to markers in the overlapping sequence. The findings are consistent with general recombination occurring approximately isotonically within the interval. In particular, single markers within the overlap, scored nonselectively, showed frequencies of recovery dependent on the position of their locus in relation to the ends of the overlap. Pairs of ts markers recombined to form ts+ progeny in proportion to their distance apart, provided the markers were oriented so that a single crossover between them would produce a full-length genome bearing both ts+ alleles. In the opposite orientation, where such a single crossover would be expected to produce ts/ts recombinants, the ts+ frequency was much lower, indicating that multiple recombination events are rare in this system. These findings rule out site-specific recombination, recombination occurring exclusively at the cleaved ends of the overlap, and recombination by means of mismatch repair of a heteroduplex the length of the overlap. They also indicate either that any heteroduplex junction region formed in the course of this reaction is quite short or that it is not subject to heteroduplex repair. Finally, our results demonstrate the efficacy of overlap recombination as a genetic and physical mapping tool and as a method of strain construction, and they suggest other applications, such as using overlap recombination to demonstrate that closely spaced pairs of markers (e.g., putative second-site reversions and their accompanying ts lesions) can be segregated.