FIG. 3.

Assay of DNA replication by slot blot assays and DpnI digestion. (A) Slot blot analysis of transfected cells with the indicated bacmids. The DNA from 10⁴ transfected cells (at 0, 12, 24, 48, 72, 96, and 120 h p.t.) was applied onto nylon membranes and hybridized with a labeled gus for a probe. The names of bacmids used for transfections are shown at the top of the lanes. (B) Quantification of slot blot analysis for viral DNA replication. The images were analyzed with ImageQuant 5.2 software. Data are presented as relative intensities from triplicate experiments. (C) Slot blot analysis of viral DNA detected from the supernatant of transfected cells with the indicated bacmids. Virions were isolated from the supernatants (at 0, 24, 48, 72 and 96 h p.t.), and the viral DNA was extracted, applied to a nylon membrane, and hybridized with a labeled gus probe. The names of bacmids used for transfections are shown at the top of the lanes. Mock, mock infected. (D) DpnI assay of DNA from vAcAN-KO/GUS- and vAcIEGUS-transfected cells. Duplicate 20-μl aliquots from the transfected DNA (at 0, 12, and 24 h p.t.) and duplicate 5-μl aliquots from the DNA (at 48 and 72 h p.t.) were digested with XhoI (−) or XhoI-DpnI (+), separated on gels, and blotted onto membranes. The membranes were hybridized with a labeled gus probe. Part of the membrane (lanes 19 to 22) was exposed for a quarter of the time of exposure of the other part. The names of the bacmids used for transfections are shown at top of the lanes. Lane M, vAcIEGUS bacmid DNA purified from E. coli for controls.