摘要
目的建立化学致突变物的体外快速筛选方法。方法用PCR法从酵母基因组中扩增RNR3启动子,将其插入到pMP206/ERE报告载体,将该载体转入W303-1A酵母细胞。当化学诱变原作用于酵母细胞时,会诱导β-半乳糖苷酶表达,可对具有DNA毒性的化学物进行筛选。结果许多可造成DNA损伤的化学诱变原可诱导RNR3的表达。使DNA发生烷基化的诱变原(甲磺酸甲脂和瘤可宁)、使DNA发生断裂的诱变原(顺铂、4-硝基-N-氧化喹啉、博来霉素和福来霉素)、抑制DNA合成酶或拓扑异构酶的诱变原(5-氟尿嘧啶、羟基脲和喜树碱)均可诱导RNR3的表达。结论 RNR3调控的重组Lac Z报告基因酵母可用于对许多化学诱变原的筛选,筛选过程可在96孔板中进行,因而具有高通量性。
Objective To develop a fast screening method for chemical mutagens.Methods RNR3 promoter was amplified using polymerase chain reaction （PCR） from yeast genome and inserted into the yeast report vector of pMP206 / ERE to construct yeast Lac Z gene report vector regulated by RNR3.This vector was transformed into the yeast cell of W303-1A.When the yeast cell was treated by the chemical mutagenesis,the expression of Lac Z gene was induced.So it can be used to screen the chemicals with DNA toxicity by measuring the beta-galactose enzyme activity.Results Various chemicals that cause DNA damaged can induce RNR3 expression.The mutagens alkylating DNA （methyl methanesulfonate and chlorambucil）,the cleavages of DNA （cisplatin,4-nitro-Noxidation of quinoline,bleomycin and phleomycin） and the inhibitor of DNA polymerase or topoisomerase （5-fluorouracil,hydroxyurea and camptothecin） can induce RNR3 expression.Conclusions Recombinant Lac Z report gene yeast cells regulating by RNR3 can be used for the screening of chemical mutagen and this screen can be carried out in 96 well micro plate as a high throughput technology.
出处
《基础医学与临床》
CSCD
北大核心
2014年第5期679-683,共5页
Basic and Clinical Medicine
