Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase.
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.
Czank
A
A
Institut für Molekularbiologie II, Universität Zürich, Switzerland.
Häuselmann
R
R
Page
A W
AW
Leonhardt
H
H
Bestor
T H
TH
Schaffner
W
W
Hergersberg
M
M
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
D011993:Recombinant Fusion Proteins; D015257:DNA-Cytosine Methylases