Figure 7.
pKMP2-derived transcripts and mutations of the Ampr gene in plasmids selected in ampicillin. (A) RT-PCR of RNA of E. coli transformed with unmutated (WT) or two mutated (Mut1 and Mut2) pKMP2 plasmids that confer ampicillin resistance (Fig. 5 F and Fig. 6, clones 1 and 7). Kanr and Ampr are transcripts of the respective genes in fourfold sequential dilutions. Equal amounts of cDNA were used for the respective Kanr and Ampr reactions. RT-PCR products without reverse transcription with RNA samples corresponded to the highest concentrations of WT (1), Mut1 (2), and Mut2 (3) RNAs, respectively. (B–D) Mutation patterns in pKMP2 after exposure to low amounts of AID for a short time. Treatments of the plasmids are indicated, with numbers of colonies obtained in E. coli selected in ampicillin shown in parenthesis. The y axes represent numbers of mutations. Relative to Fig. 5, the scales were expanded by factors of 7.25, 4.8, and 6.9 for B, C, and D, respectively, for easier comparison with the data in Fig. 5 F (for B) and Fig. 5 D (for C and D), where larger numbers of colonies were obtained. The sequences are shown in Figs. S9–S11. (E) AID acts processively/cooperatively. Selection of AID-treated plasmids in ampicillin. 2: no ampicillin resistance was seen with nucleosomal DNA and 5, 10, or 20 min of exposure to AID, T7 RNA polymerase, and BamHI. 5: no ampicillin resistance was seen with naked DNA and 5 min of exposure to AID and T7 RNA polymerase. The size markers are 100, 200, and 300 bp. The experiments were done four (E4), three (E1 and 2), two (A, C, D, and E5-7), and one (B and E3) times with similar results.