Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay).
The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene. The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor. The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay. This test procedure enables screening for mutations within one working day. Incubation for 16 h reveals high sensitivity.
Schmid
Claudia
C
Department of Environmental and Molecular Genotoxicity (AMMUG), University of Mainz, Obere Zahlbacher Strasse 63, 55101 Mainz, Germany.
Arndt
Christian
C
Reifferscheid
Georg
G
D000595:Amino Acid Sequence; D000668:Ampicillin Resistance / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics*; D016368:Frameshift Mutation; D017930:Genes, Reporter / Q000187:drug effects*; D007763:Lac Operon / Q000187:drug effects; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D013753:Tetracycline Resistance / Q000235:genetics
D004269:DNA, Bacterial; D009153:Mutagens