Overproduction of the EcoRII endonuclease and methylase by Escherichia coli strains carrying recombinant plasmids constructed in vitro

Abstract

Recombinant DNA molecules were constructed from the plasmid pIL203 and the EcoRI-fragment of N3 plasmid containing EcoRII endonuclease and methylase genes and also a gene for resistance to sulfanilamide. The pIL203 plasmid, used as a vector, consisted of the BamHI-EcoRI-fragment of the plasmid pBR322 conferring resistance to ampicillin and the BamHI-EcoRI-fragment of lambda phage containing promoters, a thermosensitive mutation in the cI gene and a suppressible amber mutation in the cro gene. Ampicillin-sulfanilamide-resistant clones were selected and tested for their restriction and modification phenotype. The recombinant plasmid DNA, isolated from Ap^RSu^R-resistant clones, which restricted and modified phage λ imm21 with EcoRII specificity, had the EcoRI-fragment with EcoRII genes in a single orientation. The recombinant plasmid pSK323 was transferred into E. coli strains with su⁻, su1, su2 or su3 phenotypes. The synthesis of products of EcoRII genes by these strains grown at 37°C is increased by 10–50-fold.