Fig. 4

Validation of ^5mCGATCG methylation status by Southern blot hybridization. a Scheme of the probed region from the CRISPR1 system of Synechocystis 6803 plasmid pSYSA and percentage of methylated cytosine residues at the indicated positions (first number, position on forward strand; second number, position on the reverse strand) according to bisulfite analysis. Undermethylated sites are bold-faced. The location of primers to generate the 600 bp probe for hybridization to slr7010 DNA fragments is indicated by arrows. b Gel image of the DNA from wild type (WT) and the two slr0214 mutants (A1 and B1) with no treatment (n.t.) and after restriction by PvuI, DpnI or Sau3AI and separation by agarose gel electrophoresis. Three different markers were used as size standards, a 1 kb (M1) and a 100 nt –ladder (M3) and DNA of bacteriophage λ after restriction by PstI (M2). c Image of the blot resulting from the gel in panel (b) after Southern transfer and hybridization to the probe indicated in panel (a). The lengths of the two additional bands in PvuI-digested DNA from the wild type correspond to the predicted lengths indicated in panel (A) for the products of partial digestion between the sites at positions 4053/4058 or 7392/7397 and 7998/8003 due to the different methylation levels