Nf. Cariello et al., A NOVEL BACTERIAL REVERSION AND FORWARD MUTATION ASSAY BASED ON GREENFLUORESCENT PROTEIN, Mutation research. Genetic toxicology and environmental mutagenesis, 414(1-3), 1998, pp. 95-105
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.