Interactions of Polyoma and Mouse DNAs. IV. Time Course and Extent of Integration of Polyoma DNA into Mouse DNA During Lytic Infection

Abstract

The time course of covalent binding of polyoma viral DNA to mouse DNA was followed in mouse embryo cells that had been grown prior to infection in the presence of 5-bromodeoxyuridine. Density-labeled (HL) mouse DNA was separated from free polyoma DNA by CsCl isopycnic centrifugation. Polyoma DNA sequences present in HL mouse DNA were detected by hybridization with radioactive cRNA synthesized in vitro. In reconstruction experiments, the limit of detection was found to be, on the average, about 0.5 genome equivalent (g.e.) of polyoma DNA per cell. To find conditions for the isolation of HL mouse DNA and for its complete separation from free polyoma DNA, cultures infected at 4°C were used. HL mouse DNA extracted with sodium dodecyl sulfate and high salt concentrations (5 to 6 M CsCl) and then purified by three consecutive CsCl density gradient centrifugations was free from detectable amounts of polyoma DNA, whereas HL mouse DNA extracted with chloroform and phenol and purified in the same way always contained contaminating, noncovalently bound polyoma DNA. In lytically infected bromodeoxyuridine-prelabeled mouse embryo cultures, polyoma DNA bound to HL mouse DNA that had been extracted by the sodium dodecyl sulfate-CsCl procedure was first detected in small amounts (1 to 2 g.e. per cell) at 10 h after infection. In cultures incubated with medium containing thymidine (5 μg/ml), 4 to 6 g.e. of polyoma DNA per cell was detected at 14 and 18 h after infection. In these samples, practically all viral DNA was bound to high-molecular-weight HL mouse DNA. In cultures incubated with normal medium (no additions) and extracted between 17 and 20 h after infection, 20 to 350 g.e. of polyoma DNA per cell banded with HL mouse DNA. However, when DNA of one of these samples was subfractionated by sodium dodecyl sulfate-salt precipitation prior to isolation of HL mouse DNA, about 80% of the viral DNA banding at increased density was present in the low-molecular-weight DNA fraction. This observation suggests that in normal medium some progeny viral DNA of increased density was synthesized. Covalent binding of polyoma DNA to density-labeled mouse DNA was demonstrated by alkaline CsCl density gradient centrifugation: nearly equal amounts of polyoma DNA were found in the H and L strands, respectively, as is expected for linear integration of viral DNA. The results lead to the conclusions that (i) early polyoma mRNA is transcribed from free parental viral DNA; (ii) covalent linear integration is first detectable at the time when tumor (T)-antigen is synthesized; and (iii) only few copies (<10 g.e./cell) become integrated between 10 and 18 h after infection, i.e., during the period when cellular and viral DNA replication starts in individual cells.