Figure 7.

The effect of mutation in O_R on pvuIICR transcription and on PvuII R-M system establishment. The nonrepressing C-box mutation (WWWR: 5′-GACT-CAT-AGTC-TGTA-GACT-CAA-GATC-3′) was tested in two ways. (A) In vivo titration with C.PvuII on arabinose inducible plasmid pIM1, where mutated C-box was fused to lacZ gene (pIM9). Cells were grown in minimal media with 0.2% glycerol, 0.2% glucose and the indicated concentration of arabinose as described in Figure 2B. The transcriptional activity was measured as β-galactosidase specific activity as described in the Figure 2 legend. (B) Equal amounts of three plasmid DNAs were used to determine the efficiency of transformation (EOT) in each of two host strains. The three plasmids were pPvuRM3.4 (WT PvuII R-M system), pIM6 (symmetrized nonrepressing variant), and pBR322 (vector control). These were introduced into competent E. coli TOP10 cells that already carried either the gene for the PvuII MTase (pvuIIM; plasmid pPvuM1.9-ACYC) or a vector control (pACYC177). Relative EOT was determined as the fraction of M.PvuII⁻ transformants obtained relative to the number of transformants for the M.PvuIIM⁺ strain, and then normalized to the pBR322 EOT ratio. Error bars indicate the SD.