Figure 7

Evaluation of TsoI REase/MTase activities in cleared lysates. (A) Evaluation in the presence of SAM. Lanes M, DNA standard (Gene Ruler™ Ladder Mix (Thermo Fisher Scientific (Fermentas); selected bands marked); lane 1, λ DNA; lane 2, λ DNA, cleaved with wt TsoI; lanes 3, 6, 9, 12, λ DNA after incubation with indicated amounts of cleared lysates prepared from cultures: (i) before TsoI induction (3, 6) or (ii) after induction (9, 12) in the standard reaction buffer supplemented with SAM; lanes 4, 7, 10, 13, the same as in lanes 3, 6, 9, 12, but after chloroform extraction, isopropanol precipitation and subsequent incubation of dissolved samples with wt TsoI; lanes 5, 8, 11, 14, the same as in lanes 4, 7, 10, 13, except that λ DNA was added before incubation with wt TsoI in order to assess the putative inhibitory effect of the chloroform-extracted DNA on wt TsoI. (B) Evaluation in the absence of SAM. Description of lanes as in A, with the exception that SAM was not included into the reaction mixtures used to evaluate activities of crude lysates.