【摘要】 人工合成两条雌激素效应元件(ERE)重复的核苷酸序列,并在5′和3′端设计HindⅢand SphⅠ黏性末端。将两条互补的ERE溶液加热至95℃,然后冷却至室温使其形成双股DNA,将双股ERE插入到pERE-CYC-yEGFP载体的细胞色素C(CYC1)启动子相应的酶切位点,生成pERE-CYC-αyEGFP载体。pERE-CYC-yEGFP和pERE-CYC-αyEGFP载体的不同之处是ERE位于CYC1启动子的位置,前者ERE下游的CYC1启动子含有α和β-TATA框,而后只含有-αTATA框。将两者分别转化于可表达人雌激素受体的酵母细胞,构建成两种受雌激素调控的重组酵母细胞。将该重组酵母细胞经6种不同的雌激素化合物作用4 h,发现重组pERE-CYC-αyEGFP的酵母细胞的绿色荧光蛋白(GFP)表达量与受试物间剂量效应关系较差,而含有pERE-CYC-yEGFP载体重组酵母细胞,与受试物具有明显的剂量效应关系。因此,在用于快速、高通量筛选雌激素类化合物生物评价中,其CYC1基本启动子中的α和-βTATA框是必须的。更多还原

【Abstract】 The complementary oligonucleotides,each with two consensus estrogen response element(ERE)-sequences and 5′-Hind Ⅲ and 3′-Sph Ⅰsticky ends were artificially synthesized.A solution with both the complementary DNA sequences was heated to 95℃ and cooled down to room temperature to form double strand DNA(dsDNA).The set was cloned into the corresponding sites of CYC1 promoter of the pERE-CYC-yEGFP to yield pERE-CYCα-yEGFP vector.The two different reporter vectors,pERE-CYC-yEGFP and pERE-CYCα-yEGFP,the 2ERE,were placed in the CYC1 promoter.The former promoter downstream ERE contains α and β-TATA boxes and the latter has only α-TATA box.The two different reporter vectors were transformed into the yeast cells that express human estrogen receptor α(ERα).Incubation of the recombinant yeasts with the six estrogenic compounds for 4 hours showed that the recombinant cell containing pERE-CYCα-yEGFP would give very poor dose-response curves,in contrast to the recombinant cell containing pERE-CYC-yEGFP which produced well-shaped dose-response curves.So it is necessary for this bioassay that α and β-TATA boxes in the minimal CYC1 promoter when the promoter is used as a rapid and high throughput system for screening estrogenic chemical products.更多还原