The native EcI5 intron is mobile in E. coli. (A) PCR-based mobility assays. Donor plasmids expressing the full-length Ll.LtrB and EcI5 introns (pACD-LtrB and pACDF-EcI5, respectively) were transformed into E. coli HMS174(DE3) together with their respective recipient plasmids, pBRR3-ltrB and pBRR-EcI5. After growing the cells at 37°C to early log phase and incubating with 0, 100, or 500 μM IPTG for 2 h to induce intron expression, plasmid DNA was isolated, and integration of the intron into the target site was detected by PCR of the 5′-integration junction, using a primer (RECSEQ01R), which anneals to the recipient plasmid vector backbone, and an intron-specific primer (LtrBAS2.2 and EcI5MOB01 for Ll.LtrB and EcI5, respectively; see Supplemental Table 2 for primer sequences). For each PCR, the right-hand lane (“Mix”) shows a control using the same primers and 100 ng each of the donor and recipient plasmids. The arrows indicate the 603- and 570-bp PCR products corresponding to the 5′-integration junctions of the Ll.LtrB and EcI5 introns, respectively. The larger bands in the “Mix” lanes are due to nonspecific priming on the plasmid DNA templates, which are present at relatively high concentrations, and those at the bottom of the gel are primer dimers. (B) Dependence of EcI5 intron mobility on the catalytic activity of the intron RNA and IEP expression. A mobility assay was carried out as in panel A with pACDF-EcI5 expressing wild-type (WT) EcI5 or mutant introns with a deletion of intron domain V (ΔDV) or a point mutation in the initiation codon of the intron ORF (ATG → ATT; −IEP). The arrow indicates the 570-bp PCR product corresponding to the 5′-integration junction. The lighter bands in the lanes for EcI5 mutants were sequenced and found to be PCR artifacts, presumably due to nonspecific annealing of the primers.
