Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances.
Yeast strains transformed with high copy number plasmids carrying the gene encoding a green fluorescent protein optimised for yeast (yEGFP3) under the control of the RAD54 or RNR2 promoter were used to investigate the activity of potentially DNA-damaging substances. The assays were performed on 96-well microtitre plates in the presence of different concentrations of the test substances. The synthesis of GFP protein was measured through the fluorescence signal and cell growth was monitored by absorption. Here, we demonstrate that this system can be used as a biosensor to assess the genotoxic potential of drugs and other chemical substances. The use of microtitre plates will enable full automation of the system and allows the inclusion of internal reference standards in each assay.
Afanassiev
V
V
Hans-Knöll-Institut für Naturstoff-Forschung, Abteilung Zell- und Molekularbiologie, Beutenbergstr. 11, D-07745, Jena, Germany.
Sefton
M
M
Anantachaiyong
T
T
Barker
G
G
Walmsley
R
R
Wölfl
S
S
D015374:Biosensing Techniques; D015153:Blotting, Western; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000633:toxicity; D005069:Evaluation Studies as Topic; D005453:Fluorescence; D005656:Fungal Proteins / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000187:drug effects; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D008741:Methyl Methanesulfonate / Q000633:toxicity; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity; D011237:Predictive Value of Tests; D011401:Promoter Regions, Genetic / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012680:Sensitivity and Specificity; D014162:Transfection
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008164:Luminescent Proteins; D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D008741:Methyl Methanesulfonate; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes