Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains.
Identifying and eliminating endogenous bacterial enzyme systems can significantly increase the efficiency of propagation of eukaryotic DNA in Escherichia coli. We have recently examined one such system which inhibits the propagation of lambda DNA rescued from transgenic mouse tissues. This rescue procedure utilizes lambda packaging extracts for excision of the lambda DNA from the transgenic mouse genome, as well as E. coli cells for subsequent infection and propagation. This assay, in combination with conjugal mating, P1 transduction, and gene cloning, was used to identify and characterize the E. coli locus responsible for this difference in efficiency. It was determined that the E. coli K-12 mcrB gene when expressed on a high-copy-number plasmid can cause a decrease in rescue efficiency despite the presence of the mcrB1 mutation, which inactivates the classic McrB restriction activity. (This mutation was verified by sequence analysis.) However, this McrB1 activity is not observed when the cloned mcrB1 gene is inserted into the E. coli genome at one copy per chromosome. A second locus was identified which causes a decrease in rescue efficiency both when expressed on a high-copy-number plasmid and when inserted into the genome. The data presented here suggest that this locus is mrr and that the mrr gene product can recognize and restrict cytosine-methylated sequences. Removal of this DNA region including the mrr gene from E. coli K-12 strains allows high rescue efficiencies equal to those of E. coli C strains. These modified E. coli K-12 plating strains and lambda packaging extract strains should also allow a significant improvement in the efficiency and representation of eukaryotic genomic and cDNA libraries.
Kretz
P L
PL
Stratagene Cloning Systems, La Jolla, California 92037.
Kohler
S W
SW
Short
J M
JM
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D002872:Chromosome Deletion; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D003360:Cosmids; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000378:metabolism*; D015257:DNA-Cytosine Methylases; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005784:Gene Amplification; D005798:Genes, Bacterial; D008745:Methylation; D051379:Mice; D008822:Mice, Transgenic; D015183:Restriction Mapping
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C054926:DNA modification methylase AluI; D015257:DNA-Cytosine Methylases