Overproduction of the EcoRII endonuclease and methylase by Escherichia coli strains carrying recombinant plasmids constructed in vitro.
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 Bam HI-EcoRI-fragment of the plasmid pBR322 conferring resistance to ampicillin and the Bam HI-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 ApRSuR-resistant clones, which restricted and modified phage lambda 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 degrees C is increased by 10--50-fold.
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000235:genetics*; D004274:DNA, Recombinant / Q000378:metabolism*; D015257:DNA-Cytosine Methylases; D015252:Deoxyribonucleases, Type II Site-Specific; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008780:Methyltransferases / Q000235:genetics*; D009154:Mutation; D010957:Plasmids; D013424:Sulfanilamides / Q000494:pharmacology; D013696:Temperature
D004274:DNA, Recombinant; D013424:Sulfanilamides; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D008780:Methyltransferases; D004262:DNA Restriction Enzymes; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific