title: Cloning and characterization of genes for the PvuI restriction and modification system.
abstract: The genes encoding the endonuclease and the methylase of the PvuI restriction and modification system were cloned in E.coli and characterized. The genes were adjacent in tandem orientation spanning a distance of 2200 bases. The PvuI endonuclease was a single polypeptide with a calculated molecular weight of 27,950 daltons. The endonuclease was easily detectable when the gene was expressed from its endogenous promotor and present on a low copy plasmid, but expression was considerably enhanced when the endonuclease gene was placed under the control of a strong promoter on a high copy plasmid. The methylase did not completely protect plasmid DNA from R.PvuI digestion until the methylase gene was placed under lac promotor control in a multicopy plasmid. In the absence of the M.PvuI methylase, expression of the R.PvuI endonuclease from the lac promotor on a multicopy plasmid was not lethal to wild type E.coli, but was lethal in a temperature-sensitive ligase mutant at the non-permissive temperature. Moreover, induction of the R.PvuI endonuclease under lambda pL promotor control resulted in complete digestion of the E.coli chromosome by R.PvuI.
authors.affiliation: Life Technologies, Inc., Gaithersburg, MD 20878.
authors.forename: M D
authors.initials: MD
authors.lastname: Smith
authors.forename: M
authors.initials: M
authors.lastname: Longo
authors.forename: G F
authors.initials: GF
authors.lastname: Gerard
authors.forename: D K
authors.initials: DK
authors.lastname: Chatterjee
mesh_terms: D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000378:metabolism; D003001:Cloning, Molecular; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011514:Proteus vulgaris / Q000201:enzymology / Q000235:genetics; D015183:Restriction Mapping
chemical_list: D004269:DNA, Bacterial; D015254:DNA Modification Methylases; C061224:endodeoxyribonuclease PvuI; D015252:Deoxyribonucleases, Type II Site-Specific