The utilization of a Saccharomyces cerevisiae HUG1P-GFP promoter-reporter construct for the selective detection of DNA damage.
In this study, we report the creation and characterization of a yeast-based promoter-reporter construct for the detection of genotoxic compounds within a cell's local environment. We have synthesized a fusion containing the HUG1 promoter and GFP and incorporated this cassette into the yeast genome creating a stable, sensitive genotoxicity indicator. To quantify biosensor performance, HUG1P-GFP cells were exposed to multiple doses of a wide variety of genotoxins, including alkylating agents, an oxidative agent, a ribonucleotide reductase inhibitor, a UV mimetic agent, an agent that causes double strand breaks, a topoisomerase I inhibitor, and ionizing radiation, all of which triggered a detectable and reproducible level of GFP production by the HUG1P-GFP strain. Furthermore, GFP was not induced by general cell stresses including starvation, heat shock, and acidic pH. These results suggest this system will be a valuable supplement to traditional genotoxicity assays.
Benton
Michael G
MG
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
Glasser
Nathaniel R
NR
Palecek
Sean P
SP
D015374:Biosensing Techniques; D004249:DNA Damage; D004271:DNA, Fungal / Q000187:drug effects*; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity; D011401:Promoter Regions, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; C403293:HUG1 protein, S cerevisiae; D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins