Figure 2

Functional and molecular analysis of extrachromosomal homologous recombination. The upper panel is a schematic of the left and right-hand deletion substrates (DL and DR) showing the actin5C promoter (blue), luciferase coding sequence (red) and SV40 termination sequence (yellow). The 5' and 3' luciferase deletions are shown as dotted lines and the pUC18 plasmid backbone as a continuous line. The intact luciferase coding sequence used as a probe for Southern analysis is shown in green. All relevant restriction sites are indicated (B, Bam HI; S, Sac I; H, Hin dIII and Sc, Sca I). The central panel shows the various combinations of plasmids transfected into mosquito cells together with a Southern analysis of the reaction products in An. gambiae (Ag55) cells. Transfections involved either circular (C) or linear (L) forms of the parent plasmid (p[ACT-LUC]) and deletion substrates (DL; DR), as shown, with superscripts indicating linearization by the relevant restriction enzyme (L^B, Bam HI; L^S, Sac I; L^H, Hin dIII; L^Sc, Sca I). For Southern analysis, total cellular DNA was isolated from An. gambiae (Ag55) cells 48 hours post-transfection, digested with both Bam HI and Bgl II, resolved on 1.5% agarose and blotted onto nitrocellulose. The membrane was probed with a ³²P labelled luciferase fragment, washed at high stringency (1 × SSC; 0.1 % SDS; 65°C) and exposed overnight at -70°C to X-ray film against an intensifying screen. The size of relevant signals was determined by comparison to standard markers (MBI Kilobase Ladder) and is shown in kilobase pairs (Kb). The lower panel shows the mean luciferase activities (log₁₀ counts per minute) recovered from the various transfections into An. gambiae (Ag55) cells. Values were plotted following subtraction of the assay background and the error bars represent standard deviations. A logarithmic scale was employed so that all values could be represented on the same figure.