Towards validation of the Big Blue transgenic mouse mutagenesis assay: the mutational spectrum of ex vivo pinpoint mutant plaques.
To explore further the origin of spontaneous mutations recovered with the Big Blue transgenic mouse mutagenesis assay, the spectrum of ex vivo mutations from pinpoint mutant plaques was determined and compared with the spectrum of putatively mouse-derived mutations from circular, mutant plaques. The entire lacI gene and lacZ operator region from 62 pinpoint blue plaques was sequenced. The observed mutational spectrum of pinpoint mutants differed significantly from that seen in circular mutants (p < 0.0001). Only four percent of the mutations were transitions at CpG sites whereas this type of mutation was the most common (35%) in circular mutants. Microdeletions/microinsertions were seen more frequently in pinpoint mutants relative to circular mutants. Four base pair deletion/insertion events at the E. coli hotspot tandem repeats were seen in 10 of 62 (16%) pinpoint mutants and minor hotspots of mutation were observed at bp 141 and 1110. The mutational spectrum of pinpoint mutants provides further evidence that most circular mutants originate in mouse.
Nishino
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Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.
Buettner
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Sommer
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D000367:Age Factors; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D011994:Recombinant Proteins; D012097:Repressor Proteins; D001616:beta-Galactosidase