Adenovirus infection of semipermissive and nonpermissive cells induces cellular DNA synthesis under culture conditions where DNA synthesis does not occur in uninfected cells. DNA synthesis induced by adenovirus occurs in the absence of several G1-phase events which normally accompany serum-stimulated DNA synthesis. This includes the expression of a set of cell cycle- dependent genes which are a subset of those expressed following serum stimulation. Although there are exceptions, the trend shows that Ad2 generally activates late G1- or S-phase genes, while early and mid G1-phase genes are not affected. These results suggest that: 1) Adenoviruses stimulate cellular DNA synthesis by a mechanism which is not the same (although it could be part of the) mechanism used by serum growth factors. 2) Ad2 infection of nonpermissive cells could be used to screen for cellular genes perferentially expressed in late G1/S-phase of the cell division cycle. Our immediate aim will be to identify a subset of late G1/S-phase cell cycle-dependent gene transcripts which are expressed in both adenovirus-infected and in serum- stimulated cells. The experimental approaches used will be as follows: 1) A complementary DNA (cDNA) bacterial plasmid library of cellular poly(A+) mRNA sequences expressed in a nonpermissive temperature-sensitive, G1-phase cell cycle mutant of Syrian hamster (tsAF8) infected with adenovirus 2 will be constructed. 2) Differential molecular hybridization will be used to identify from among the members of the cDNA library, those cDNA clones which correspond to cellular gene transcripts preferentially expressed in adenovirus-infected cells. 3) The cDNA inserts from these clones will be used as molecular probes to identify and characterize messenger RNA transcripts by Northern blot analysis and genomic DNA sequences by Southern blot analyses. 4) The cell cycle-dependent level of RNA expression of these genes will be ascertained in cells synchronized in different phases of the cell division cycle, and in other ts cell cycle mutants that arrest growth in different phases when serum- stimulated at restrictive temperature. Once this has been accomplished, the cellular genes encoding these sequences will be identified and isolated from a Syrian hamster genomic bacteriophage library. The long range goals of the project will be to analyze the structure of these genes, and to ascertain the critical importance of their expression as it relates to cell DNA replication, morphological transformation and immortalization of primary Syrian hamster dermal fibroblasts.
