Leung P S, Preiss J
J Bacteriol. 1987 Sep;169(9):4349-54. doi: 10.1128/jb.169.9.4349-4354.1987.
The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.
鼠伤寒沙门氏菌LT2的ADP葡萄糖焦磷酸化酶（glgC）和糖原合酶（glgA）的结构基因被克隆到pBR322的SalI位点上一个5.8千碱基对的插入片段中。用一个在M13mp8中包含大肠杆菌K-12 glgC基因N端的单链特异性放射性探针，与λ1059中的鼠伤寒沙门氏菌基因组文库进行杂交。从显示阳性杂交信号的噬菌斑中分离出DNA，亚克隆到pBR322中，并转化到大肠杆菌K-12 RR1和大肠杆菌G6MD3（一个缺失glg基因的突变体）中。用碘对转化体进行染色以检测糖原的存在。大肠杆菌K-12 RR1转化体染成深棕色，而G6MD3转化体染成黄绿色，并且它们都被证明在pBR322的SalI位点含有一个5.8千碱基对的插入片段，命名为pPL301。对携带pPL301的大肠杆菌K-12 G6MD3进行酶活性测定，恢复了ADP葡萄糖焦磷酸化酶和糖原合酶的活性。大肠杆菌K-12 RR1（pPL301）中ADP葡萄糖焦磷酸化酶和糖原合酶的比活性分别提高了6至7倍和13至15倍。免疫和动力学研究表明，在转化的大肠杆菌K-12 G6MD3细胞中表达的ADP葡萄糖焦磷酸化酶活性与野生型酶非常相似。
