Cloning and characterization of genes for the Pvul restriction and modification system

Abstract

The genes encoding the endonuclease and the methylase of the Pvul 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 Pvul 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 promotor on a high copy plasmid. The methylase did not completely protect plasmid DNA from R·Pvul digestion until the methylase gene was placed under lac promotor control in a multicopy plasmid. In the absence of the M·Pvul methylase, expression of the R·Pvul 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·Pvul endonuclease under λpL promotor control resulted in complete digestion of the E.coli chromosome by R·Pvul. © 1992 Oxford University Press.