Ogawa T, Shinohara A, Ogawa H, Tomizawa J
Department of Biology, Faculty of Science, Osaka University, Japan.
J Mol Biol. 1992 Aug 5;226(3):651-60. doi: 10.1016/0022-2836(92)90622-q.
We developed a novel genetic method for finding functional regions of a protein by the analysis of chimeras formed between homologous proteins. Sets of chimeric genes were made by intramolecular homologous recombination in a linearized plasmid DNA carrying both recA genes of Escherichia coli and Pseudomonas aeruginosa. A recBCsbcA strain of E. coli was used for isolation of plasmids carrying recombinants between these genes. Examination of properties of E. coli strains deleting the recA gene and carrying a plasmid with a chimeric gene shows that chimera formation at certain positions inactivates a RecA function. Frequently, all chimeras with a junction in a certain region of the protein inactivate a function. Rather than a direct effect of the presence of the junction at a particular position, mismatching of the regions both sides of the junction that are derived from the different species is responsible for the inactivation. For a chimeric protein to be functional, certain pairs of sequences in different regions of the protein must derive from the same parent. Four pairs of such sequences were found: two are involved in activities for genetic recombination and for resistance to ultraviolet light irradiation and the others in formation of active oligomers. Regions defined by these sequences are located in the looped regions of the protein. A pair of regions may co-operate to form a functional folded structure.
我们开发了一种新的遗传方法，通过分析同源蛋白之间形成的嵌合体来寻找蛋白质的功能区域。嵌合基因集是通过在携带大肠杆菌和铜绿假单胞菌recA基因的线性化质粒DNA中进行分子内同源重组而构建的。大肠杆菌的recBCsbcA菌株用于分离携带这些基因之间重组体的质粒。对缺失recA基因并携带嵌合基因质粒的大肠杆菌菌株的特性进行检测，结果表明在蛋白质的某些位置形成嵌合体可使RecA功能失活。通常，在蛋白质特定区域有连接点的所有嵌合体都会使一种功能失活。造成失活的原因不是连接点在特定位置的直接影响，而是连接点两侧源自不同物种的区域不匹配。要使嵌合蛋白具有功能，蛋白质不同区域的某些序列对必须来自同一亲本。发现了四对这样的序列：两对参与基因重组和抗紫外线照射的活性，另外两对参与活性寡聚体的形成。由这些序列定义的区域位于蛋白质的环状区域。一对区域可能协同形成功能性折叠结构。
