A novel bacterial reversion and forward mutation assay based on green fluorescent protein.
We report the first use of green fluorescent protein (GFP) for mutation detection. We have constructed a plasmid-based bacterial system whereby mutated cells fluoresce and non-mutated cells do not fluoresce. Fluorescence is monitored using a simple hand-help UV lamp; no additional cofactors or manipulations are necessary. To develop a reversion system, we introduced a +1 DNA frameshift mutation in the coding region of GFP and the resulting protein is not fluorescent in Escherichia coli. Treatment of bacteria containing the +1 frameshift vector with ICR-191 yields fluorescent colonies, indicating that reversion to the wild-type sequence has occurred. Site-directed mutagenesis was used to insert an additional cytosine into a native CCC sequence in the coding region of GFP in plasmid pBAD-GFPuv, expanding the sequence to CCCC. A dose-related increase in fluorescent colonies was observed when the bacteria were treated with ICR-191, an agent that induces primarily frameshift mutations. The highest dose of ICR-191 tested, 16 microg/ml, produced a mutant fraction of 16 x 10(-5) and 8.8 x 10(-5) in duplicate experiments. The reversion system did not respond to MNNG, an agent that produces mainly single-base substitutions. To develop a forward system, we used GFP under the control of the arabinose PBAD promoter; in the absence of arabinose, GFP expression is repressed and no fluorescent colonies are observed. When cells were treated with MNNG or ENNG, a dose-dependent increase in fluorescent colonies was observed, indicating that mutations had occurred in the arabinose control region that de-repressed the promoter. Treating bacteria with 100 microg/ml MNNG induced mutant fractions as high as 82 x 10(-5) and 40 x 10-5 in duplicate experiments. Treating bacteria with 150 microg/ml ENNG induced a mutant fraction of 2.1 x 10(-5) in a single experiment.
Cariello
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Medicines Safety Evaluation, Glaxo Wellcome, 5 Moore Drive, Research Triangle Park, NC 27709, USA. nfc20355@glaxowellcome.com
Narayanan
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Kwanyuen
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Muth
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Casey
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D000585:Aminacrine / Q000031:analogs & derivatives; D001089:Arabinose / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016368:Frameshift Mutation; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics; D008769:Methylnitronitrosoguanidine; D016297:Mutagenesis, Site-Directed; D009588:Nitrogen Mustard Compounds; D009876:Operon; D010957:Plasmids; D014170:Transformation, Genetic
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009588:Nitrogen Mustard Compounds; D008769:Methylnitronitrosoguanidine; D049452:Green Fluorescent Proteins; D000585:Aminacrine; C004294:acridine half-mustard; D001089:Arabinose