Journal: Nature Communications

Article Title: Bacterial N4-methylcytosine as an epigenetic mark in eukaryotic DNA

doi: 10.1038/s41467-022-28471-w

Figure Lengend Snippet: a Immuno-dot blot of total DNA extracted from E. coli Rosetta 2(DE3) strain transformed with different variants of recombinant N4CMT. DNA from non-transformed Rosetta 2(DE3) was used as a control. DNA was extracted after 4 h of IPTG-induced N4CMT expression; 400 ng of each DNA was spotted in one dot. Methyl marks are indicated on the right. b Methylcytosine-sensitive digestion of E. coli gDNA. Total DNA from Rosetta 2(DE3) strain, either transformed with recombinant N4CMTs (as shown on the top) or untransformed, was extracted 4 h post-induction; DNA (6.3 μg) was treated with McrBC. DNA from HepG2 liver cell line ( H. sapiens ) was used as a positive control (500 ng). Two independent experiments were performed with similar results. c Immuno-dot blot of total E. coli DNA showing the role of the SPPY motif in N4CMT methylation. Same designations as in ( a ). d , Immuno-dot blot of total E. coli DNA extracted from different strains and treated with N4CMT. Rosetta 2(DE3) and dam−/dcm− , 950 ng per dot; BL21-AI and M28, 500 ng per dot. e Immuno-dot blot with anti-4mC antibody for sAvL1-451 substrate treated with N4CMT allozymes and their catalytic site mutants in vitro.

Article Snippet: After the final OD check, expression of recombinant proteins was induced by supplying the growth medium with IPTG (Gold Bio) to 500 μM, and the culture was grown for an additional 4 h at 32 °C, 350 rpm for N4CMT versions or for an additional 3 h at 34 °C, 300 rpm for AvMBD’s.

Techniques: Dot Blot, Transformation Assay, Recombinant, Expressing, Positive Control, Methylation, In Vitro