FIG. 2.

BPV-1 DNA detected by Southern blot in a BPV-infected S. cerevisiae culture over 5 days. S. cerevisiae (15 ml; 5 × 10⁷cells/ml) was infected with 0.9 μg of BPV-1 virus. At 24 h, 10 ml of S. cerevisiae was collected, with 5 ml each being used for Hirt DNA and RNA preparation. Then 10 ml of fresh medium was added for continuing culture. Thereafter, 10 ml of S. cerevisiae was collected for Hirt DNA preparation and 10 ml of fresh medium was added every 24 h until 120 h. A total of 15 ml of medium was added to 0.9 μg of BPV-1 virus and similarly sampled and diluted. Hirt DNA prepared from 5 ml of S. cerevisiae cultures for each time point was resuspended in 200 μl of TE buffer. Then 20 μl of Hirt DNA, after digesting partially with HindIII for about 2 h, was electrophoresed on a 1% agarose gel and blotted onto a nylon membrane. DNA blots were hybridized with whole BPV-1 DNA probe. As controls, 5 ml of medium infected with BPV-1 virus was pelleted for viral DNA extraction at high speed (10,000 rpm) for 30 min. The pellets were resuspended in 400 μl of lysate buffer and incubated with 100 μg of proteinase K at 37°C for 1 h. The DNA was extracted with Tris-buffered phenol and chloroform. The extracted DNA was also resuspended in 200 μl of TE buffer. Then 20 μl of the DNA suspensions was run on an agarose gel and blotted onto nylon membrane for BPV-1 DNA hybridization. The time point and effective culture dilution are shown above each lane. *1 indicates that the Hirt DNA contained the viral DNA from 0.03 μg of BPV-1 virions. The viral DNA dilution at the different time point is shown above each lane.