Isolation of mutants in a DNA methyltransferase through mcrB-mediated restriction.
A procedure has been developed that permits the positive selection of mutants in a DNA methyltransferase (MTase) gene. The stringency of this selection can be varied so as to yield null mutants only, or a mixture of null and partially defective mutants. The procedure was developed with the PvuII MTase gene (pvuIIM), which was subcloned into a bacteriophage lambda vector. Growth of this lambda pvuIIM construct on an mcrB+ host selected for non-methylating mutants, and the stringency of selection was proportional to the number of consecutive lytic cycles. Many cytosine MTases have been found to generate substrates for mcrB-mediated restriction, and this procedure should be applicable to a number of cytosine MTase genes.
Blumenthal
R M
RM
Department of Microbiology, Medical College of Ohio, Toledo 43699.
Cotterman
M M
MM
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant; D015257:DNA-Cytosine Methylases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005821:Genetic Techniques; D008745:Methylation; D009154:Mutation; D010957:Plasmids
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; C047084:DNA modification methylase PvuII; D015257:DNA-Cytosine Methylases; D004262:DNA Restriction Enzymes; C059678:mcrB protein, E coli