Czank, Andreas; Häuselmann, Remo; Page, Andrea W.; Leonhardt, Heinrich; Bestor, Timothy H.; Schaffner, Walter; Hergersberg, Martin
Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase.
In: Gene, Vol. 109, Nr. 2: S. 259-263
Mammalian DNA cytosine-5-methyltransferase (MTase, EC 18.104.22.168) is an essential component for establishing and maintaining cell-type specific methylation patterns in the genome. The cDNAfor the murine enzyme was previously cloned in segments. We have reconstructed the entire gene, encoding a protein of 1517 amino acids, from a set of overlapping CDNA clones. We report the assembly of two expression constructs in bacterial/mammalian shuttle vectors. Transcription in the first construct (pEMT) is driven by the cytomegalovirus enhancer/promoter and encodes a fusion protein with 15 additional aa at the N terminus, while the second construct (pJMT) is driven by the simian virus 40 early promoter/enhancer upstream from the natural ATG codon. Immunofluorescence microscopy and immunoblot analysis have shown that both constructs direct the synthesis of MTase in COS-1 cells. Enzyme activity in whole-cell lysates of transfected COS-1 cells transfected with pEMT and pJMT are on average tenfold and fivefold higher than in control, respectively. The specific activities of the recombinant and endogenous mouse-cell enzyme are similar. These expression constructs will be of use in studies of DNA methylation in mammals.