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McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning
E A Raleigh1, N E Murray, H Revel
1New England Biolabs, Beverly, MA 01915.
Abstract:
The McrA and McrB (modified cytosine restriction) systems of E. coli interfere with incoming DNA containing methylcytosine. DNA from many organisms, including all mammalian and plant DNA, is expected to be sensitive, and this could interfere with cloning experiments. The McrA and B phenotypes of a few strains have been reported previously (1-4). The Mcr phenotypes of 94 strains, primarily derived from E. coli K12, are tabulated here. We briefly review some evidence suggesting that McrB restriction of mouse-modified DNA does occur in vivo and does in fact interfere with cloning of specific mouse sequences.
Insights
The modified cytosine restriction (Mcr) systems in E. coli can hinder DNA cloning. This study tabulates Mcr phenotypes for 94 strains, finding they interfere with cloning mammalian DNA.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The modified cytosine restriction (Mcr) systems in *E. coli* target and degrade incoming DNA containing methylcytosine.
- This restriction can impede various molecular biology techniques, particularly DNA cloning, as DNA from many organisms, including mammals and plants, is methylated.
- Previous reports documented Mcr phenotypes for a limited number of *E. coli* strains.
Purpose of the Study:
- To systematically investigate and tabulate the modified cytosine restriction (Mcr) phenotypes of a large set of *E. coli* strains.
- To assess the potential interference of these Mcr systems with DNA cloning experiments, especially concerning methylated DNA from other organisms.
- To review existing evidence on McrB system activity in vivo and its impact on cloning specific sequences.
Main Methods:
- Phenotypic characterization of 94 *E. coli* strains, primarily derived from the K12 lineage, to determine their McrA and McrB restriction capabilities.
- Literature review to synthesize evidence regarding the in vivo activity of the McrB system and its effect on cloning mouse DNA.
- Analysis of Mcr phenotypes in relation to potential interference with DNA cloning experiments.
Main Results:
- Detailed tabulation of the Mcr phenotypes for 94 *E. coli* strains, providing a valuable resource for researchers.
- Evidence suggests that the McrB system can restrict mouse-modified DNA in vivo.
- The study confirms that Mcr systems can interfere with the cloning of specific methylated DNA sequences, such as those from mice.
Conclusions:
- The McrA and McrB systems represent a significant barrier in *E. coli* for cloning methylated DNA from various sources.
- Understanding the Mcr phenotypes of different *E. coli* strains is crucial for successful DNA cloning and manipulation.
- Further research and strain selection are necessary to overcome Mcr-mediated restriction in molecular cloning applications.