The kinetics of adenovirus recombination in homotypic and heterotypic genetic crosses

The kinetics of recombination between temperature-sensitive mutants has been studied in both homotypic (Ad5 x Ad5) and heterotypic (Ad5 x Ad2+NDI) crosses. There is a significant increase in the recombination frequency during the rise period of viral replication in a single-step replication cycle. This observation suggests that adenovirus DNA molecules can undergo progressive rounds of recombination before assembly into virions. To examine this possibility further, advantage was taken of the serotype-specific restriction endonuclease sites and polypeptide sizes of Ad5 and Ad2+ND1 to enumerate and to localize the sites of crossing over in ts+ recombinants isolated early and late in a heterotypic infection. The late recombinants exhibited, on average, more crossovers per genome than did the early recombinants. This finding is predicted if multiple rounds of recombination take place in some genomes. Using blot hybridization with specific probes, the production of recombinant molecules in the intracellular DNA replicating pool has been followed. Recombinants were found before the rise in infectious virus and increased in frequency relative to the parental molecules throughout the exponential period. These data confirm and extend the genetic observations, which were made on a selected set of infectious virus.