FIG. 4.

The pACYC184 DraI fragment is bent, and this fragment affects plasmid supercoiling. (A) Cesium-purified plasmid pACYC184 DNA was digested with DraI to release a 339-bp fragment. The digest was mixed with the 1-kb ladder and analyzed by two-dimensional gel electrophoresis. The DraI fragment, which falls off the linear diagonal generated by the ladder, shows retarded migration. (B) Chloroquine gel electrophoresis was conducted on pACYC184 and pACYCΔDra1 isolated from Y. enterocolitica cells grown at different temperatures. Lanes 1 and 2, pACYC184 isolated from Y. enterocolitica grown at 25 and 37°C, respectively; lanes 3 and 4, plasmid pACYCΔDra1 (339-bp deletion) isolated at the same temperatures. The band across the top in all four lanes is open-circle DNA, showing little difference in migration, whereas the deletion derivative topoisomers migrate much further in the gel.