A novel yeast-based reporter assay system for the sensitive detection of genotoxic agents mediated by a DNA damage-inducible LexA-GAL4 protein.
Yeast-based genotoxicity testing systems can sensitively detect DNA damaging agents in the environment. We have developed a novel "indirect" reporter assay system based on a recombinant yeast containing both a sensor and a reporter plasmid. The sensor plasmid contains a gene encoding the artificial transcription factor of the Escherichia coli LexA DNA binding domain fused to the transcriptional activation domain of yeast Gal4p, which is regulated by the DNA damage-inducible RNR2 promoter. The reporter plasmid contains the E. coli lacZ gene with the LexA binding site in the 5'-upstream region, allowing transcriptional activation by the induced LexA-GAL4 protein. The activity of DNA damage-dependent beta-galactosidase (beta-gal) in the "indirect" reporter assay system was compared with that of a current yeast-based "direct" reporter system. The "indirect" system exhibited 1.5- to 5-fold greater beta-gal activity upon induction by alkylating agents or camptothecin. To increase the sensitivity of the new reporter system further, several deletion yeast strains were tested, and enhanced induction of reporter activity was observed in DNA repair-deficient mag1Delta cells. The "indirect" 96-well microtiter plate assay system is a potentially inexpensive and sensitive method for detecting genotoxic activities in a wide range of compounds, and in polluted environmental samples.
Ichikawa
Kohei
K
Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580.
Eki
Toshihiko
T
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D004268:DNA-Binding Proteins; D017930:Genes, Reporter; D009152:Mutagenicity Tests; D009153:Mutagens / Q000032:analysis / Q000633:toxicity*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C052130:GAL4 protein, S cerevisiae