High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M.EcoR124.
We have cloned the genes coding for the two subunits (HsdM and HsdS) of the type-I DNA methyltransferase (MTase), M.EcoR124, into the specially constructed expression vector, pJ119. These subunits have been synthesized together as an intact MTase. We have also cloned the individual subunit-encoding genes under the control of the T7 gene 10 promoter or the lacUV5 promoter. High levels of expression have been obtained in all cases. While HsdM was found to be soluble, HsdS was insoluble. However, in the presence of the co-produced HsdM subunit, HsdS was found in the soluble fraction as part of an active MTase. We have partially purified the cloned multi-subunit enzyme and shown that it is capable of DNA methylation both in vivo and in vitro.
Patel
J
J
Biophysics Laboratories, School of Biological Sciences, Portsmouth Polytechnic, U.K.
Taylor
I
I
Dutta
C F
CF
Kneale
G
G
Firman
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D015254:DNA Modification Methylases / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D008745:Methylation; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics
D007544:Isopropyl Thiogalactoside; D004247:DNA; D015254:DNA Modification Methylases