title: Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase.
abstract: Mammalian DNA cytosine-5-methyltransferase (MTase, EC 2.1.1.37) is an essential component for establishing and maintaining cell-type specific methylation patterns in the genome. The cDNA for 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 controls, 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.
authors.affiliation: Institut für Molekularbiologie II, Universität Zürich, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Czank
authors.forename: R
authors.initials: R
authors.lastname: Häuselmann
authors.forename: A W
authors.initials: AW
authors.lastname: Page
authors.forename: H
authors.initials: H
authors.lastname: Leonhardt
authors.forename: T H
authors.initials: TH
authors.lastname: Bestor
authors.forename: W
authors.initials: W
authors.lastname: Schaffner
authors.forename: M
authors.initials: M
authors.lastname: Hergersberg
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics; D015257:DNA-Cytosine Methylases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013539:Simian virus 40 / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D015257:DNA-Cytosine Methylases