Figure 2.
Transcription through the Sγ3 region in its physiological orientation induces DNA rearrangement in the chromosome of E. coli. (A) Schematic of chromosomes of E. coli strains lacZ::PT7-Sγ3-F (left panel) and lacZ::PT7-Sγ3-R (right panel). Relative positions of the probe-binding sites, NotI cleavage sites, T7 promoter (T7), and Sγ3 region (Sγ3-F or Sγ3-R) are indicated. (B) Southern blot analysis of the chromosomal regions surrounding the Sγ3 transcription unit. BW25113, lacZ::PT7-Sγ3-F, and lacZ::PT7-Sγ3-R strains were grown in the presence of 1 mM IPTG for the indicated times. Genomic DNAs from these strains were purified in agarose plugs and digested with NotI. Digested DNAs were subjected to PFGE and Southern blot analysis with probe 1 (top panel) and probe 2 (bottom panel), respectively. Sizes of targeted DNA fragments are indicated. The starlet band, a slowly migrating band detected by probe 1, is indicated by an asterisk. (C) RNA- and ssDNA-specific nuclease treatments have no effect on the migration of the starlet band. The lacZ::PT7-Sγ3-F strain was grown in the presence of 1 mM IPTG for 30 min. NotI-digested DNAs were subsequently treated with RNase A (RN-A), RNase H (RN-H), mung bean nuclease (MBn), T7 exonuclease (T7 exo), or exonuclease T (Exo T), respectively, for the indicated times prior to PFGE. Southern blot analysis was performed as described above. (D) The RecBCD recombination pathway is required for the appearance of the starlet band. NotI-digested DNAs from lacZ::PT7-Sγ3-F, lacZ::PT7-Sγ3-FΔrecBC, and lacZ::PT7-Sγ3-FΔrecBCD strains were subjected to PFGE and Southern blot analysis as described above. (E) A DNA rearrangement event appears in the Sγ3 region upon induction of Sγ3 transcription in its physiological orientation. The lacZ::PT7-Sγ3-F strain was grown in the presence of 1 mM IPTG for the indicated times. DNAs were digested with NdeI, NdeI plus HindIII, or NdeI plus EcoRV, respectively, and hybridized with probe 3 specific to the lacY gene located immediately upstream of the Sγ3 transcription unit in the chromosome of the lacZ::PT7-Sγ3-F strain.