FIG. 8.

Short recombinant AAV2 genomes are deficient in the accumulation of extended relative to turnaround replicative-form DNA. Ad5dl312-infected 293 cells were transfected with psubλdl1495 (A) or p015-AAV (B), infected cells were harvested 60 h later, and 10⁴ cell equivalents of the cell pellet (lanes C), the hypotonic lysate (lanes L), or a mixture of the two (lanes B) were subjected to electrophoresis in neutral agarose and analyzed by Southern blotting (left autoradiograms). Two-dimensional electrophoresis was carried out as described in the legend to Fig. 7 (right autoradiograms). Southern blots were hybridized to a probe specific for λDNA. Markers for panel A were 6.4-kb SfiI-to-Eco47III fragment of psubλdl1495 (lane D) and a 3.3-kb SmaI fragment of psubλdl1495 that was either untreated (lane M) or denatured (lane S). In panel B, both Southern blots were hybridized to a probe corresponding to the AAV2 terminal repeats. Markers for panel B are undigested p015-AAV (lane U), 11.4-kb p015-AAV NheI-linearized fragment (lane L), a 9.6-kb PpuMI-SwaI fragment from p015-AAV that is similar in size to AAV2 dimer-length replicative-form DNA (lane D), and a 4.7-kb PvuII fragment from psub201(−) that was either left untreated (lane M) or denatured (lane S). Markers for the second dimension were loaded in a well that was originally at the bottom of the first gel. The markers are not shown, but their positions are identified by size designations above the autoradiogram. They were a mixture of a 3.2-kb AseI-FspI fragment of pGET015, a 4.7-kb PvuII fragment from psub201(−), a 6.8-kb NotI-linearized pGET015, and a 9.6-kb PpuMI-SwaI fragment from p015-AAV. ssDNA, single-stranded DNA.