Modified-cytosine restriction-system-induced recombinant cloning artefacts in Escherichia coli.
We have tested whether, and to what extent, recombinant clones from DNA segments with 5-methylation of cytosines recovered in methylation-restrictive (mcr+) hosts contain mutations. We constructed a model system in which the tetracycline-resistance-encoding gene (tet) from pBR322 was cloned into the plasmid pGEM3Zf+. The central region of tet was removed from the construct, methylated in vitro and then religated back into the unmethylated remainder of the construct. The central region of tet was either (1) methylated with a combination of four bacterial methyltransferases (M.AluI, M.HaeIII, M.HpaII plus M.HhaI) or (2) methylated with M.SssI which methylates at all CpG dinucleotides. These two protocols generated theoretical levels of DNA methylation in the central fragment of 10.5% and 33%, respectively. The construct was transformed into a series of isogenic (recA+) bacterial strains that were mcrA+ mcrB+C+, mcrA+ mcrB-C+, mcrA- mcrB+C+, mcrA- mcrB-C+ or mcrA- delta mcrBC, and also into a set of isogenic recA- derivatives of these strains. With the two methylation protocols, there was an average 48- and 141-fold reduction, respectively, in the number of transformants recovered from the recA+ mcr+ hosts compared with a methylation-tolerant host (mcr-). Of the clones recovered in recA+mcr+ hosts, > 20% of clones had an inactivating mutation in tet. The majority of such mutant clones contained deletions that frequently extended into the unmethylated portion of tet and even into the plasmid sequences beyond the end of the polylinker. With the recA- mcr+ hosts, effective restriction was much more stringent, rendering the plasmid containing the methylated segment effectively unclonable.(ABSTRACT TRUNCATED AT 250 WORDS)
Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
M R
MR
Williamson
J P
JP
Doherty
D M
DM
Woodcock
D044503:5-Methylcytosine; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003596:Cytosine / Q000031:analogs & derivatives*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005838:Genotype; D009154:Mutation; D010957:Plasmids; D011995:Recombination, Genetic; D013753:Tetracycline Resistance / Q000235:genetics
D044503:5-Methylcytosine; D003596:Cytosine; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial; D015226:Dinucleoside Phosphates; C015772:CpG dinucleotide