A new method for the rapid identification of genes encoding restriction and modification enzymes.
We have constructed derivatives of Escherichia coli that can be used for the rapid identification of recombinant plasmids encoding DNA restriction enzymes and methyltransferases. The induction of the DNA-damage inducible SOS response by the Mcr and Mrr systems, in the presence of methylated DNA, is used to select plasmids encoding DNA methyltransferases. The strains of E. coli that we have constructed are temperature-sensitive for the Mcr and Mrr systems and have been further modified to include a lacZ gene fused to the damage-inducible dinD locus of E. coli. The detection of recombinant plasmids encoding DNA methyltransferases and restriction enzymes is a simple, one step procedure that is based on the induction at the restrictive temperature of the lacZ gene. Transformants encoding DNA methyltransferase genes are detected on LB agar plates supplemented with X-gal as blue colonies. Using this method, we have cloned a variety of DNA methyltransferase genes from diverse species such as Neisseria, Haemophilus, Treponema, Pseudomonas, Xanthomonas and Saccharopolyspora.
Piekarowicz
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Institute of Microbiology, Warsaw University, Poland.
Yuan
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Stein
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DC
D003001:Cloning, Molecular; D004249:DNA Damage; D015254:DNA Modification Methylases / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000235:genetics*; D004274:DNA, Recombinant / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005821:Genetic Techniques; D010957:Plasmids; D013014:SOS Response, Genetics; D013696:Temperature; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000235:genetics
D004274:DNA, Recombinant; D015254:DNA Modification Methylases; D004262:DNA Restriction Enzymes; D001616:beta-Galactosidase