An G, Justesen J, Watson R J, Friesen J D
J Bacteriol. 1979 Mar;137(3):1100-10. doi: 10.1128/jb.137.3.1100-1110.1979.
We have isolated five specialized transducing lambda bacteriophages (lambda dpyrE spoT) carrying the pyrE and spoT genes of Escherichia coli. A fragment from one of these phages was used as the source of DNA to clone the spoT and pyrE genes on a multicopy plasmid, pBR322. Insertions and deletions in this plasmid were obtained. These plasmids were used to transform a minicell-producing strain, and the gene products synthesized were determined. Our experiments demonstrate that the spoT and pyrE genes are separated by about 4 magadaltons and suggest that the spoT gene product is a protein whose molecular weight is 80,000. The strain in which the spoT+ allele is carried on a plasmid produced nine times more spoT gene activity than a normal spoT+ strain when assayed in crude extracts. This strain was used to prepare partially purified gene product, guanosine 5'-diphosphate, 3'-diphosphate pyrophosphatase. The enzyme has the following characteristics. (i) It hydrolyzes pyrophosphate from the 5'-pyrophosphate of guanosine 5'-diphosphate, 3'-diphosphate, yielding GDP and pyrophosphate. (ii) Its activity is strongly stimulated by Mn2+ and slightly stimulated by salt. (iii) Its activity is inhibited by uncharged tRNA. There are also two additional activities in the cell extract which degrade guanosine in 5'-diphosphate, 3'-diphosphate in vitro but which are not specified by the spoT gene.
我们分离出了5种携带大肠杆菌pyrE和spoT基因的特异性转导λ噬菌体（λdpyrE spoT）。其中一个噬菌体的片段被用作DNA来源，以便在多拷贝质粒pBR322上克隆spoT和pyrE基因。获得了该质粒中的插入和缺失。这些质粒被用于转化一个产生微小细胞的菌株，并对合成的基因产物进行了测定。我们的实验表明，spoT和pyrE基因相距约4兆道尔顿，并提示spoT基因产物是一种分子量为80,000的蛋白质。当在粗提取物中进行测定时，携带spoT⁺等位基因的质粒的菌株产生的spoT基因活性比正常spoT⁺菌株高9倍。该菌株被用于制备部分纯化的基因产物——鸟苷5'-二磷酸-3'-二磷酸焦磷酸酶。该酶具有以下特性。（i）它从鸟苷5'-二磷酸-3'-二磷酸的5'-焦磷酸中水解焦磷酸，产生GDP和焦磷酸。（ii）其活性受到Mn²⁺的强烈刺激，并受到盐的轻微刺激。（iii）其活性受到不带电荷的tRNA的抑制。细胞提取物中还有另外两种活性，它们在体外可降解鸟苷5'-二磷酸-3'-二磷酸中的鸟苷，但不是由spoT基因所编码的。
