Cloning and characterization of genes for the PvuI restriction and modification system.
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.
Smith
M D
MD
Life Technologies, Inc., Gaithersburg, MD 20878.
Longo
M
M
Gerard
G F
GF
Chatterjee
D K
DK
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
D004269:DNA, Bacterial; D015254:DNA Modification Methylases; C061224:endodeoxyribonuclease PvuI; D015252:Deoxyribonucleases, Type II Site-Specific