FIG. 1.

(A) Schematic diagram and restriction map of the BAC containing the Dnmt1 gene. Restriction sites are abbreviated as follows: N, NotI; P, PstI. PCR screening of a mouse 129/Sv BAC library identified a BAC clone containing the Dnmt1 gene. The 150-kb BAC clone was shown to contain the complete Dnmt1 gene by a combination of PCR and Southern hybridization techniques (data not shown). (B) Western blot analysis of ES cell extracts using an antibody which was generated against the catalytic domain of Dnmt1. Similar results were obtained with a polyclonal antibody against the N-terminal region of Dnmt1 and are therefore not shown. All protein extracts were controlled by Coomassie-stained gel to ensure equal loading. (C) In vitro MTase activity assay measuring incorporation of the methyl group from [³H]S-adenosyl-l-methionine into a synthetic poly(dIdC) substrate. The in vitro assay was performed with three independently derived sets of protein extracts and their protein concentration was normalized. All results within a set were normalized to wild-type activity, which was defined as 100%. Each value represents the mean ± SEM. (D) 5-Methyl-cytosine content of genomic DNA from different ES cells and from differentiated tissues (liver, kidney), as measured by HPLC. Each value represents the mean ± SEM.