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. 2002 Sep 1;30(17):3818–3830. doi: 10.1093/nar/gkf501

Figure 1.

Figure 1

Restriction protection analysis of in vivo methylated plasmid DNA. (A) Plasmids encoding targeted HhaI and HpaII were isolated from E.coli and subjected to restriction by restriction enzymes HhaI and HpaII respectively, as described in Materials and Methods. Lane 1, uncut control pGEX5X-3 vector; lane 2, R.HhaI restricted pGEX5X-3 vector; lane 3, uncut pGHhaI vector; lane 4, R.HhaI restricted pGHhaI vector; lane 5, uncut pGZfHhaI vector; lane 6, R.HhaI restricted pGZfHhaI vector; lane 7, as lane 1; lane 8, R.HpaII restricted pGEX5X-3 vector; lane 9, uncut pGHpaII vector; lane 10, R.HpaII restricted pGHpaII vector; lane 11, uncut pGZfHpaII vector; lane 12, R.HpaII restricted pGZfHpaII vector; lane m, 100 bp marker (NEB); lane kb, 1 kb ladder (NEB). (B) Plasmids encoding mutant targeted HpaII were isolated from E.coli and subjected to restriction by HpaII. Mutants are: Mut1, wild-type vector cut with EcoRI and filled in to generate HpaII out of frame with GST–Zf; Mut2, wild-type vector with first codon (methionine) of HpaII removed; Mut3, Mut2 vector cut with NdeI and filled in to generate Zf-HpaII out of frame with GST (for details see text). Lanes 1 and 2, uncut and R.HpaII cut wild-type targeted HpaII vector, respectively; lanes 3 and 4, uncut and R.HpaII cut Mut1 vector; lanes 5 and 6, uncut and R.HpaII cut Mut2 vector; lanes 7 and 8, uncut and R.HpaII cut Mut3 vector; lane 9, R.HpaII cut pGEX empty vector. (C) SDS–PAGE analysis of protein induction for wild-type and mutant targeted HpaII enzymes. Molecular weights are indicated on the right of the gel. Induced protein products are arrowed.