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. 2009 Jan 6;37(3):983–998. doi: 10.1093/nar/gkn1010

Figure 8.

Figure 8.

Methylation status of plasmid isolated from cells expressing varied levels of WT M.PvuII or its variants. The complementary strands for the symmetrized C-box region of the PvuII R-M system are shown at the top. The upper strand specifies M.PvuII. The two changes that symmetrize the C-boxes are shown in red, along with the resultant changes in the MTase sequence (N4S/S10R). Substitution of the OL spacer, from CAT to CAA, would result in an additional change (M8L). Escherichia coli TOP10 cells carried two plasmids: pUC × 7PvuII (with seven PvuII sites) and plasmids expressing either WT pvuIIM (pBadMTwt-kan) or one of its variants (MTase N4S/S10R–pBadMT2-kan; MTase N4S/S10R/M8L–pBadMT2-kan) under the arabinose inducible promoter PBAD. After induction with a range of arabinose concentrations, cells were pelleted and plasmid DNA was isolated. The extent of methylation was assessed by digestion with PvuII restriction enzyme. Results ranged from full cleavage (no protection as in control lane 1; asterisk shows the position of highest bands) to no cleavage (complete methylation as in control lane 2). Digests were resolved on 1% agarose gel. Lanes 4 to 8 for each MTase represent equivalent increasing arabinose concentration from 0.01 to 0.2%. The topmost band in lanes 4–8 for each MTase is the pBAD plasmid linearized with XhoI prior to digestion with PvuII indicated by arrow; pUC × 7PvuII lacks a XhoI site. M lane shows DNA markers (1 kB Plus ladder; Invitrogen).