Replication intermediates of adenovirus DNA apparently contain extensive stretches of single-stranded DNA. Such single-stranded viral DNA sequences homologous to different regions of the viral genome present in adenovirus-infected cells during viral DNA replication have therefore been characterized by hybridization to the separated strands of restriction endonuclease fragments of 32P-labeled adenovirus types 2 and 5 DNA. Saturation hybridization experiments with infected cell DNA extracted at late times suggest that all regions of the adenovirus genome are represented in the single-stranded fraction, but at unequal frequencies. This nonuniform representation has been characterized in more detail with self-annealed, total cell DNA extracted 18 hr after adenovirus type 2 infection: the concentration of single-stranded sequences homologous to different regions of the viral genome was determined by comparing the rates of hybridization of 32P-labeled, single-stranded DNA probes with such self-annealed 18 hr DNA to the rates of hybridization of the same probes with equal concentrations of their complements. This approach allows the concentration of single-stranded viral DNA sequences in excess of their complements to be determined. Such sequences can be represented by two concentration gradients across the viral genome: those homologous to the r strand increase in concentration from 27.8-40.9 units toward the right end, whereas sequences homologous to the I strand increase from an area 27.8-40.9 units toward the left end. The time course of synthesis of single-stranded viral DNA sequences relative to accumulation of total viral DNA during the productive cycle and their behavior following a shift of H5ts125-infected cells in which viral DNA replication has begun from a permissive to a nonpermissive temperature support the contention that these sequences are indeed generated as adenovirus DNA is replicated. These results are therefore discussed in terms of current models of adenovirus DNA replication.
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)