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目的用RNR3调控的全发光酵母细胞高通量筛选化学诱变原。方法以含有酵母嗜好遗传密码子的绿色荧光蛋白(yEGFP)报告载体为基本框架,用PCR方法从酵母(W303-1A)基因组扩增RNR3启动子,将其插入到yEGFP的上游,从而构建成RNR3调控的yEGFP酵母报告载体。用醋酸锂方法将载体转化于酵母细胞,构建成RNR3调控的yEGFP发光酵母细胞,可在96孔板中对不同浓度的化学诱变原进行筛选,具有快速、方便和高通量特点。结果对数种不同的DNA损伤药物研究结果表明,DNA烷化剂(甲磺酸甲脂)、DNA断裂剂(顺铂)、DNA结合剂(放线菌素D)和DNA合成酶抑制剂(5-氟尿嘧啶和羟基脲)可诱导RNR3的表达,而苯丁酸氮芥和4-硝基-N-氧化喹啉由于细胞的毒性的作用,未使RNR3发生表达。结论某些与致癌性有关的DNA损伤化学物能诱导RNR3表达,故该发光酵母细胞可用于对某些致癌物的高通量筛选。
Objective To screen high-throughput chemical mutagens with full-blown yeast cells regulated by RNR3. Methods The green fluorescent protein (yEGFP) reporter vector containing yeast-preferred genetic code was used as the basic framework. The RNR3 promoter was amplified from the genome of yeast (W303-1A) by PCR and inserted into the upstream of yEGFP to construct RNR3 Regulated yEGFP yeast reporter vector. The vector was transformed into yeast cells by the method of lithium acetate, and was constructed into RNF3-regulated yEGFP luminescent yeast cells. The mutagenized mutagen can be screened in different concentrations in 96-well plates, which is fast, convenient and high-throughput. Results Several different DNA damaging drug studies have shown that DNA alkylating agents (methyl methanesulfonate), DNA cleaving agents (Cisplatin), DNA binding agents (actinomycin D), and DNA synthase inhibitors 5-fluorouracil and hydroxyurea) induced the expression of RNR3, whereas chlorambucil and 4-nitro-N-quinolone did not express RNR3 due to the cytotoxicity. Conclusion Some DNA damage chemicals related to carcinogenicity can induce RNR3 expression. Therefore, the luminescent yeast cells can be used for high-throughput screening of certain carcinogens.