title: Cloning and characterization of the TneDI restriction: modification system of Thermotoga neapolitana.
abstract: A putative Type II restriction-modification system of Thermotoga neapolitana, TneDI, was cloned into Escherichia coli XL1-Blue MRF' and characterized. Gene CTN_0339 specifies the endonuclease R.TneDI, while CTN_0340 encodes the cognate DNA methyltransferase M.TneDI. Both enzymes were purified simply by heating the cell lysates of E. coli followed by centrifugation. The enzymes were active over a broad range of temperatures, from 42°C to at least 77°C, with the highest activities observed at 77°C. R.TneDI cleaved at the center of the recognition sequence (CG↓CG) and generated blunt-end cuts. Overexpression of R.TneDI in BL21(DE3) was confirmed by both SDS-PAGE and Western blotting.
authors.affiliation: Department of Biological Sciences, Bowling Green State University, OH, USA. zxu@bgsu.edu
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Xu
authors.forename: Dongmei
authors.initials: D
authors.lastname: Han
authors.forename: Jingjing
authors.initials: J
authors.lastname: Cao
authors.forename: Uksha
authors.initials: U
authors.lastname: Saini
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D015254:DNA Modification Methylases / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D011995:Recombination, Genetic; D046949:Thermotoga neapolitana / Q000201:enzymology / Q000235:genetics*
chemical_list: D017931:DNA Primers; D015254:DNA Modification Methylases