Thaller M C, Berlutti F, Schippa S, Selan L, Rossolini G M
Dipartimento di Biologia, II Università di Roma "Tor Vergata", 00133 Rome, Italy.
Biotechnol Prog. 1998 Mar-Apr;14(2):241-7. doi: 10.1021/bp980009t.
The Morganella morganii phoC gene, encoding a class A acid phosphatase, was used to generate gene fusions with modified amino-terminal moieties of the Escherichia coli lacZ gene carrying a multiple-cloning site flanked by phage-specific promoters and recognition sites for universal sequencing primers. The corresponding hybrid proteins retained a PhoC-like enzymatic activity which is easily detectable by a plate histochemical assay, rendering similar gene fusions potentially useful as targets for cloning-dependent insertional inactivation. Cloning experiments performed in plasmids carrying similar lacZ-phoC fusions confirmed their usefulness as cloning vectors for direct screening of recombinants. As compared to conventional lacZ alpha-complementation-based vectors, which can only be used in E. coli hosts carrying specific lacZ mutations, the lacZ-phoC fusion-based vectors can be used in combination with any E. coli host and require a less expensive histochemical assay for screening of recombinants, while retaining all the advantageous features that made the former so popular as general purpose cloning vehicles.
摩根氏摩根菌的phoC基因编码一种A类酸性磷酸酶，该基因被用于与大肠杆菌lacZ基因的修饰氨基末端部分产生基因融合，lacZ基因带有一个多克隆位点，两侧是噬菌体特异性启动子和通用测序引物的识别位点。相应的杂合蛋白保留了类似PhoC的酶活性，通过平板组织化学测定法很容易检测到，这使得类似的基因融合有可能作为克隆依赖性插入失活的靶点。在携带类似lacZ-phoC融合体的质粒中进行的克隆实验证实了它们作为直接筛选重组体的克隆载体的有用性。与传统的基于lacZα互补的载体相比，传统载体只能用于携带特定lacZ突变的大肠杆菌宿主，而基于lacZ-phoC融合体的载体可以与任何大肠杆菌宿主结合使用，并且筛选重组体所需的组织化学测定成本较低，同时保留了使前者作为通用克隆载体如此受欢迎的所有有利特征。
