Russel M, Holmgren A
Laboratory of Genetics, Rockefeller University, New York, NY 10021.
Proc Natl Acad Sci U S A. 1988 Feb;85(4):990-4. doi: 10.1073/pnas.85.4.990.
Deoxyribonucleotides, the precursors of DNA, are formed de novo by ribonucleotide reductase, and in vitro thioredoxin or glutathione plus glutaredoxin have been isolated as hydrogen donors. The in vivo hydrogen donor for ribonucleotide reductase is not known. To study this, the Escherichia coli glutaredoxin gene (255 base pairs) was inactivated by inserting a 2-kilobase kanamycin-resistance fragment into the coding sequence of the cloned gene. The inactivated gene was inserted into the E. coli chromosome and mapped to about 18.5 min. A gene replacement technique was used to obtain a strain, A407, that lacked glutaredoxin by radioimmunoassay and by enzymatic assay with ribonucleotide reductase. Glutaredoxin was found not to be essential for viability of E. coli. Thioredoxin is also not essential for viability, as had been shown earlier, but a double mutant lacking glutaredoxin and thioredoxin could not be obtained by P1 transduction on a defined medium, indicating that either thioredoxin or glutaredoxin is essential. In rich medium, very slowly growing, unstable transductants were obtained that at high frequency gave rise to better growing cells. One such isolate, A410, was shown to still lack glutaredoxin and thioredoxin.
脱氧核糖核苷酸是DNA的前体，由核糖核苷酸还原酶从头合成，在体外，硫氧还蛋白或谷胱甘肽加谷氧还蛋白已被分离出来作为氢供体。核糖核苷酸还原酶在体内的氢供体尚不清楚。为了研究这一点，通过将一个2千碱基的卡那霉素抗性片段插入克隆基因的编码序列中，使大肠杆菌谷氧还蛋白基因（255个碱基对）失活。将失活的基因插入大肠杆菌染色体并定位到约18.5分钟处。采用基因置换技术获得了一株A407菌株，通过放射免疫测定和核糖核苷酸还原酶的酶活性测定发现该菌株缺乏谷氧还蛋白。结果发现谷氧还蛋白对大肠杆菌的生存力并非必需。如先前所示，硫氧还蛋白对生存力也不是必需的，但在限定培养基上通过P1转导无法获得同时缺乏谷氧还蛋白和硫氧还蛋白的双突变体，这表明硫氧还蛋白或谷氧还蛋白是必需的。在丰富培养基中，获得了生长非常缓慢、不稳定的转导子，这些转导子在高频下会产生生长更好的细胞。其中一个这样的分离株A410被证明仍然缺乏谷氧还蛋白和硫氧还蛋白。
