Benton Michael G, Glasser Nathaniel R, Palecek Sean P
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
Mutat Res. 2007 Sep 1;633(1):21-34. doi: 10.1016/j.mrgentox.2007.05.002. Epub 2007 May 13.
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.
在本研究中，我们报告了一种基于酵母的启动子-报告基因构建体的创建及特性，用于检测细胞局部环境中的遗传毒性化合物。我们合成了一个包含HUG1启动子和绿色荧光蛋白（GFP）的融合体，并将该盒式结构整合到酵母基因组中，创建了一个稳定、灵敏的遗传毒性指示剂。为了量化生物传感器的性能，将HUG1P-GFP细胞暴露于多种剂量的多种遗传毒素中，包括烷基化剂、氧化剂、核糖核苷酸还原酶抑制剂、紫外线模拟剂、导致双链断裂的试剂、拓扑异构酶I抑制剂和电离辐射，所有这些都引发了HUG1P-GFP菌株产生可检测且可重复水平的GFP。此外，一般的细胞应激，包括饥饿、热休克和酸性pH值，均不会诱导GFP产生。这些结果表明，该系统将是传统遗传毒性检测的有价值补充。
