Welch J, Fogel S, Buchman C, Karin M
Department of Genetics, University of California, Berkeley 94720.
EMBO J. 1989 Jan;8(1):255-60. doi: 10.1002/j.1460-2075.1989.tb03371.x.
The yeast CUP1 gene codes for a copper-binding protein similar to metallothionein. Copper sensitive cup1s strains contain a single copy of the CUP1 locus. Resistant strains (CUP1r) carry 12 or more multiple tandem copies. We isolated 12 ethyl methane sulfonate-induced copper sensitive mutants in a wild-type CUP1r parental strain, X2180-1A. Most mutants reduce the copper resistance phenotype only slightly. However, the mutant cup2 lowers resistance by nearly two orders of magnitude. We cloned CUP2 by molecular complementation. The smallest subcloned fragment conferring function was approximately 2.1 kb. We show that CUP2, which is on chromosome VII, codes for or controls the synthesis or activity of a protein which binds the upstream control region of the CUP1 gene on chromosome VIII. Mutant cup2 cells produced extremely low levels of CUP1-specific mRNA, with or without added copper ions and lacked a factor which binds to the CUP1 promoter. Integrated at the cup2 site, the CUP2 plasmid restored the basal level and inducibility of CUP1 expression and led to reappearance of the CUP1-promoter binding factor. Taken collectively, our data establish CUP2 as a regulatory gene for expression of the CUP1 metallothionein gene product.
酵母CUP1基因编码一种类似于金属硫蛋白的铜结合蛋白。对铜敏感的cup1s菌株含有单个拷贝的CUP1基因座。抗性菌株（CUP1r）携带12个或更多的多个串联拷贝。我们在野生型CUP1r亲本菌株X2180-1A中分离出12个甲磺酸乙酯诱导的对铜敏感的突变体。大多数突变体仅略微降低了铜抗性表型。然而，突变体cup2使抗性降低了近两个数量级。我们通过分子互补克隆了CUP2。赋予功能的最小亚克隆片段约为2.1 kb。我们发现位于第七条染色体上的CUP2编码或控制一种与第八条染色体上CUP1基因上游控制区结合的蛋白质的合成或活性。无论有无添加铜离子，突变体cup2细胞产生的CUP1特异性mRNA水平极低，并且缺乏与CUP1启动子结合的因子。整合到cup2位点的CUP2质粒恢复了CUP1表达的基础水平和诱导性，并导致CUP1启动子结合因子重新出现。综合来看，我们的数据确定CUP2是CUP1金属硫蛋白基因产物表达的调控基因。
