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. 2007 Mar 7;81(10):5349–5361. doi: 10.1128/JVI.02624-06

FIG. 3.

FIG. 3.

Analyses of the junctions between hamster genomic DNA and Ad12 DNA in TR12 and T637 cells. (A) DNA probe R. Lanes 1 to 3 show results for three independent isolates of TR12 genomic DNA (30 μg) cleaved with PvuII. The sequence of the cloned A-A and A-C junctions (Fig. 2) (18) predicted that probe R would hybridize with a 2,137-bp PvuII fragment from the A-A junction and a 1,260-bp fragment from the A-C junction. These two fragments were indeed observed. However, there was no additional fragment originating from the C-A junction. The 1,260-bp band produced a hybridization signal twice as strong as the 2,137-bp band, since it likely comprised both the identical A-C and C-A fragments. (B) DNA probe K. Lanes 1 and 2 show results for TR12 genomic DNA (30 μg), with EcoRI and MunI (lane 1) and XbaI (lane 2) cleavage. The predicted lengths of the EcoRI/MunI and XbaI fragments that span both the C-A and A-A junctions and hybridize to the K probe, are 6,685 bp and 7,563 bp, respectively. This calculation was based on the assumption that the cellular preinsertion site sequence is duplicated and inverted on the left side of the Ad12 transgenome. The observed fragment sizes are in agreement with these calculations. (C) DNA probe R. Lanes 1 and 2 show results for TR12 genomic DNA (30 μg), with EcoRI and MunI (lane 1) and XbaI (lane 2) cleavage. In addition to the C-A and A-A fragments (Fig. 2), probe R hybridized to an 11,227-bp EcoRI/MunI fragment and a 5,321-bp Xba fragment from the A-C junction (marked with asterisks). The lengths of these fragments were derived from the previously published sequence (18). (D) DNA probe C. Lanes 1 and 2, BHK21 genomic DNA (30 μg); lanes 3 to 6, TR12 genomic DNA (30 μg); lane 7, T637 genomic DNA (30 μg). Lanes 1, 3, 6, and 7 show results for SacI cleavage; lanes 2 and 4 show results for EcoRI cleavage; and lane 5 shows results for KpnI cleavage. Lanes M, lambda DNA/Eco130I (StyI) and MluI, marker 17 (MBI Fermentas, Vilnius, Lithuania), 3′ end labeled with [α-32P]dCTP. The lengths of the marker fragments are indicated on the right.