Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase.
The gene coding for the pneumococcal DNA adenine methylase that recognizes the sequence 5'-GATC-3' was cloned in a strain of Streptococcus pneumoniae that lacked both restriction endonucleases DpnI and DpnII. The gene was cloned as a 3.7-kilobase fragment of chromosomal DNA from a DpnII-containing strain inserted in both possible orientations in the multicopy plasmid vector pMP5 to give recombinant plasmids pMP8 and pMP10. Recombinant plasmids were selected by their resistance to DpnII cleavage. Cells carrying the recombinant plasmids modified phage in vivo so that it was restricted by DpnI- but not DpnII-containing hosts. They also showed levels of DNA methylase activity five times higher than that in cells of the original DpnII strain. No DpnII activity was observed in the clones; therefore, it was concluded that the insert did not contain an intact DpnII endonuclease gene and that methylation of host DNA did not turn on a latent form of the gene.
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008745:Methylation; D008780:Methyltransferases / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics*
D004269:DNA, Bacterial; D008780:Methyltransferases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases