Cloning the DdeI restriction-modification system using a two-step method.
DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli.
D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000235:genetics*; D004279:DNA, Viral / Q000378:metabolism; D015257:DNA-Cytosine Methylases; D015252:Deoxyribonucleases, Type II Site-Specific; D003901:Desulfovibrio / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation
D004279:DNA, Viral; C050391:DNA modification methylase DdeI; D015257:DNA-Cytosine Methylases; D004248:DNA (Cytosine-5-)-Methyltransferases; D004262:DNA Restriction Enzymes; C027908:endodeoxyribonuclease DdeI; D015252:Deoxyribonucleases, Type II Site-Specific