FIG. 5.
Effect of the rlrA/rorA genes on copy number of pSLA2-derived plasmids. (A) Agarose gel electrophoresis analysis of pSLA2-derived linear plasmids. Approximately the same volumes of Streptomyces mycelium from YEME liquid culture were used for DNA isolation (22). DNAs were diluted 0, 2, 4, and 8× in TE (10 mM Tris Hcl, 1 mM EDTA) and loaded in equal volumes in a 0.5% agarose gel at 65 V for 7 h. Gels were then stained with ethidium bromide. A 1-kb DNA ladder was used as a size marker. (B) Agarose gel analysis of pSLA2-derived circular plasmids. Transformants from colonies were picked from R2YE medium and added to a lysis solution (16). After proteinase K-sodium dodecyl sulfate treatment and phenol-chloroform extraction (22), supernatants were loaded in a 0.5% agarose gel at 65 V for 7 h. Gels were then stained with ethidium bromide. A 1-kb supercoiled DNA ladder was used as a size marker.