A 3.6‐kbp segment from the vir region of Ti plasmids contains genes responsible for border sequence‐directed production of T region circles in E. coli
A 3.6‐kbp segment from the vir region of Ti plasmids contains genes responsible for border sequence‐directed production of T region circles in E. coli
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Ti 质粒 vir 区的 3.6 kbp 片段包含负责在大肠杆菌中边界序列指导产生 T 区环的基因
DOI：
10.1002/j.1460-2075.1986.tb04337.x
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
J. Schröder
中科院分区：
文献类型：
--
作者：
J. Alt;B. Rak;J. Schröder
Ti质粒的vir区负责将T区从农杆菌转移到植物细胞;先前的实验表明，形成独立的T区DNA环是该过程中的一个步骤。为了在大肠杆菌中研究这一步骤，我们开发了一个双元载体系统。一个质粒（=底物）含有来自章鱼碱质粒pTiAch 5的正确取向的右边界和左边界。在这些边界之间克隆了具有反选择功能的基因（galK）。galK基因在tac启动子-操纵子和lac阻遏子的控制下，laci基因也在选择盒中。这种构建允许确定已经失去可选择的galK功能的底物质粒突变体。该系统的第二个组成部分是一组相容的质粒之一，这些质粒含有来自胭脂碱质粒pTiC 58的vir区的各种克隆部分。vir区的3.6 kbp片段被证明是产生底物质粒突变体所必需和足够的，其代表了含有完整左边界的T区的等价物。从这个vir区域片段中，四种离散的蛋白质在小细胞中表达。编码区被定位到胭脂碱和章鱼碱质粒中保守的部分;在后者中，它似乎对应于virC/D。
The vir region of Ti plasmids is responsible for the transfer of the T region from Agrobacteria to plant cells; previous experiments suggested that formation of independent T region DNA circles is one step in this process. To study this step in Escherichia coli, we developed a binary vector system. One plasmid (=substrate) contains correctly oriented right and left borders from octopine plasmid pTiAch5. A gene with a counterselectable function (galK) was cloned between these borders. The galK gene is under the control of the tac promoter–operator and the lac repressor with the laci gene also in the selection cassette. This construction allows determination of substrate plasmid mutants which have lost the selectable galK function. The second component of the system is one of a set of compatible plasmids harbouring various cloned parts from the vir region of nopaline plasmid pTiC58. A 3.6‐kbp segment of the vir region turned out to be necessary and sufficient for production of substrate plasmid mutants which represented the equivalent of the T region containing a complete left border. From this vir region fragment four discrete proteins were expressed in minicells. The coding regions were mapped to a part conserved in nopaline and octopine plasmids; in the latter it appears to correspond to virC/D.