Chernaik M L, Huang P C
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3024-8. doi: 10.1073/pnas.88.8.3024.
A set of mutant coding sequences for Chinese hamster metallothionein (MT) 2 in which codons for individual cysteines were replaced by serine codons was cloned into a yeast expression system. MT gene expression was placed under control of a constitutive promoter on a multicopy Escherichia coli-yeast shuttle vector. MTs were expressed in a metal-sensitive host that lacks the endogenous MT gene. The expressed MTs conferred increased metal resistance to the yeast host. A sensitive assay for cadmium resistance was developed in which population doubling times were monitored in rich liquid medium supplemented with a sublethal dose of CdCl2. Measurements on mutants with single cysteine replacements at 12 positions revealed two mutant classes. One class (Cys----Ser at position 5, 13, 19, or 33) did not affect the detoxification capacity of MT. A second class (Cys----Ser at position 7, 15, 26, 29, 44, 48, 50, or 60) conferred to the host markedly less resistance to cadmium. Bridging cysteines were more critical to cadmium resistance. All five bridging cysteine mutants studied (at positions 7, 15, 44, 50, and 60) conferred lower cadmium resistance. In contrast, mutation of four out of seven terminal cysteines (at position 5, 13, 19, or 33) was shown to be inconsequential. Mutations tend to be more detrimental in the alpha domain than in the beta domain in conveying cadmium resistance, suggesting that the contribution of individual cysteine to the detoxification function of MT is site specific.
一组中国仓鼠金属硫蛋白（MT）2的突变编码序列被克隆到酵母表达系统中，其中单个半胱氨酸的密码子被丝氨酸密码子取代。MT基因表达受多拷贝大肠杆菌-酵母穿梭载体上组成型启动子的控制。MT在缺乏内源性MT基因的金属敏感宿主中表达。表达的MT赋予酵母宿主更高的金属抗性。开发了一种镉抗性敏感测定法，其中在补充亚致死剂量CdCl2的丰富液体培养基中监测群体倍增时间。对12个位置有单个半胱氨酸取代的突变体的测量揭示了两类突变体。一类（第5、13、19或33位的Cys----Ser）不影响MT的解毒能力。第二类（第7、15、26、29、44、48、50或60位的Cys----Ser）赋予宿主对镉的抗性明显降低。桥连半胱氨酸对镉抗性更为关键。研究的所有五个桥连半胱氨酸突变体（第7、15、44、50和60位）赋予较低的镉抗性。相比之下，七个末端半胱氨酸中的四个（第5、13、19或33位）的突变被证明是无关紧要的。在传递镉抗性方面，突变在α结构域往往比在β结构域更具危害性，这表明单个半胱氨酸对MT解毒功能的贡献是位点特异性的。
