Kiel J A, Elgersma H S, Beldman G, Vossen J P, Venema G
Department of Genetics, Center of Biological Sciences, Haren, The Netherlands.
Gene. 1989 May 15;78(1):9-17. doi: 10.1016/0378-1119(89)90309-0.
Using the glgB gene from Escherichia coli as a hybridization probe, the gene encoding the branching enzyme of the cyanobacterium Synechococcus sp. PCC7942 has been identified on a 3.9-kb PstI fragment which was cloned into plasmid pUC9. Two types of plasmids have been isolated. Plasmid pKVN1 was expressing the Synechococcus sp. gene as was shown by complementation of the glgB mutation of E. coli KV832. Plasmid pKVN2, which carried the same insert in the opposite orientation was unable to complement E. coli KV832, indicating that the promoter of the cloned gene was either absent or was not recognized in E. coli. Determination of branching activity in extracts of Synechococcus sp. and E. coli KV832[pKVN1] showed that the enzyme was optimally active at approximately 35 degrees C. No significant activity was present at temperatures higher than 55 degrees C, reflecting the mesophilic nature of the cloned enzyme. In a cell-free coupled transcription-translation system the cloned gene specified two proteins of 84 kDa and 72 kDa, respectively, which are probably translated independently from the same gene by initiation at two different start codons.
以大肠杆菌的glgB基因作为杂交探针，已在一个克隆到质粒pUC9中的3.9 kb PstI片段上鉴定出编码蓝藻聚球藻属PCC7942分支酶的基因。已分离出两种类型的质粒。质粒pKVN1可表达聚球藻属基因，这通过对大肠杆菌KV832的glgB突变进行互补得以证明。质粒pKVN2携带相同的插入片段，但方向相反，无法对大肠杆菌KV832进行互补，这表明克隆基因的启动子要么不存在，要么在大肠杆菌中无法被识别。对聚球藻属提取物和大肠杆菌KV832[pKVN1]中的分支活性测定表明，该酶在约35℃时活性最佳。在高于55℃的温度下没有明显活性，这反映了克隆酶的嗜温性质。在无细胞偶联转录-翻译系统中，克隆基因分别指定了两种蛋白质，分子量分别为84 kDa和72 kDa，它们可能是通过在两个不同的起始密码子处起始翻译，从同一基因独立翻译而来。
