Fig. 2.

Distributions of plasmids supercoiled to different degrees. (A and B) Plasmid samples were prepared from E. coli DM800 ΔtopA cells by a rapid lysis procedure, and were analyzed by 2D electrophoresis in 0.7% agarose gel slabs. The electrophoresis buffer contained 0.089 M Tris·borate, 2.5 mM EDTA, and 20 and 80 μg/ml of chloroquine diphosphate for 1D and 2D electrophoresis, respectively. Lanes 1 and 2 in A and B contained pVS1 and pVS1Δ2, respectively. Lane 3 in A contained pVS1Δ3, and lane 3 in B contained a control plasmid pUC18. The radiolabeled probe for detection of the DNA rings, other than the sample analyzed in lane 3 of B, was prepared from a fragment within the tetA region; thus, DNA molecules devoid of the tetA region, including the plasmid pASLR2 that carries ampicilin resistance and was used to introduce an inducible site-specific recombinase gene into strain DM800, were not seen in the autoradiograms shown. For detection of pUC18 (B, lane 3), a radiolabeled probe for the bla region was used. In each lane, the “substrate” refers to the monomeric form of the plasmid, and the “excision product” refers to the product of site-specific recombination that contains the tetA region.