Transfer of recombinant plasmids containing the gene for DpnII DNA methylase into strains of Streptococcus pneumoniae that produce DpnI or DpnII restriction endonucleases.
Plasmid transfer via the transformation pathway of Streptococcus pneumoniae was weakly restricted by the DpnI or DpnII restriction endonuclease, either of which gave a reduction only to 0.4, compared with phage infection, which was restricted to 10(-5). The greater sensitivity of plasmid transfer compared with chromosomal transformation, which was not at all restricted, can be attributed to partially double-stranded intermediates formed from two complementary donor fragments. However, clustering of potential restriction sites in the plasmids increased the probability of escape from restriction. The recombinant plasmid pMP10 , in which the gene for the DpnII DNA methylase was cloned, can be transferred to strains that contain neither restriction enzyme or that contain DpnII as readily as can the vector pMP5 . Introduction of pMP10 raised the level of methylase by five times the level normally present in DpnII strains. Transfer of pMP10 to DpnI -containing strains was infrequent, presumably owing to the suicidal methylation of DNA which rendered it susceptible to the host endonuclease. The few clones in which pMP10 was established had lost DpnI . Loss of the plasmid after curing of the cell eliminated the methylase but did not restore DpnI . Although this loss of DpnI could result from spontaneous mutation, its relatively high frequency, 0.1% suggested that the loss was due to a regulatory shift.
D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000502:physiology; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000235:genetics*; D004274:DNA, Recombinant / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D005796:Genes; D005798:Genes, Bacterial; D008780:Methyltransferases / Q000235:genetics*; D010957:Plasmids; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics*
D004274:DNA, Recombinant; 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