Interactions of polyoma and mouse DNAs. I. Lytic infection of bromodeoxyuridine-prelabeled mouse embryo cells.

Abstract

On CsCl isopycnic centrifugation of the DNA extracted from secondary mouse embryo (ME) cultures grown in the presence of 5-bromodeoxyuridine (BUdR) and 5-fluorodeoxyuridine (FUdR) for 40 h, 10 to 25% of the DNA was found to be unsubstituted, 70 to 80% was bromouracil-hybrid DNA, and 5 to 10% was heavy DNA. These results together with cell number determinations, autoradiography, and Feulgen microspectrophotometry revealed three types of cells in these cultures: (i) 60 to 80% of the cells replicated their DNA once, divided, and then stopped mitotic activity, (ii) 5 to 10% were going through a second round of DNA replication; whereas (iii) 10 to 30% did not replicate DNA during the BUdR-FUdR exposure. After the transfer of these cultures to normal medium (without BUdR-FUdR), up to 20% of the cells resumed DNA synthesis asynchronously within 60 h, but no increase in cell number was observed. BUdR-FUdR-treated cultures, which were infected with polyoma virus in the absence of the thymidine analogues, supported a lytic infection to the same extent as did untreated ME cultures. This was concluded from the similar number of cells, which were induced to synthesize DNA, from the similar replication rate of the viral DNA, from the similar number of cells containing polyoma capsid proteins, and from the similar yields of progeny virus determined by hemagglutination and plaque formation. Thus, BUdR-prelabeled ME cultures are suitable for the investigation of interactions of the polyoma and mouse genomes during the lytic infection.