A NOVEL BACTERIAL REVERSION AND FORWARD MUTATION ASSAY BASED ON GREENFLUORESCENT PROTEIN

We report the first use of green fluorescent protein (GFP) for mutatio n detection. We have constructed a plasmid-based bacterial system wher eby mutated cells fluoresce and non-mutated cells do not fluoresce. Fl uorescence is monitored using a simple hand-help UV lamp; no additiona l cofactors or manipulations are necessary. To develop a reversion sys tem, we introduced a fl DNA frameshift mutation in the coding region o f GFP and the resulting protein is not fluorescent in Escherichia coli . Treatment of bacteria containing the +1 frameshift vector with ICR-1 91 yields fluorescent colonies, indicating that reversion to the wild- type sequence has occurred. Site-directed mutagenesis was used to inse rt an additional cytosine into a native CCC sequence in the coding reg ion of GFP in plasmid pBAD-GFPuv, expanding the sequence to CCCC. A do se-related increase in fluorescent colonies was observed when the bact eria were treated with ICR-191, an agent that induces primarily frames hift mutations. The highest dose of ICR-191 tested, 16 mu g/ml, produc ed a mutant fraction of 16 x 10(-5) and 8.8 x 10(-5) in duplicate expe riments. The reversion system did not respond to MNNG, an agent that p roduces mainly single-base substitutions. To develop a forward system, we used GFP under the control of the arabinose P-BAD 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 do se-dependent increase in fluorescent colonies was observed, indicating that mutations had occurred in the arabinose control region that de-r epressed the promoter. Treating bacteria with 100 mu g/ml MNNG induced mutant fractions as high as 82 x 10(-5) and 30 x 10(-5) in duplicate experiments. Treating bacteria with 150 mu g/ml ENNG induced a mutant fraction of 2.1 x 10(-5) in a single experiment. (C) 1998 Elsevier Sci ence B.V. All rights reserved.