Alt-Moerbe J, Rak B, Schröder J
Institut für Biologie II, Universität Freiburg, Schänzlestrasse 1, D-7800, Freiburg, FRG.
EMBO J. 1986 Jun;5(6):1129-35. doi: 10.1002/j.1460-2075.1986.tb04337.x.
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 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.
Ti质粒的vir区域负责将T区域从农杆菌转移至植物细胞；先前的实验表明，独立T区域DNA环的形成是这一过程中的一个步骤。为了在大肠杆菌中研究这一步骤，我们开发了一种二元载体系统。一种质粒（=底物）含有来自章鱼碱型质粒pTiAch5的正确定向的右边界和左边界。在这些边界之间克隆了一个具有反向选择功能的基因（galK）。galK基因受tac启动子-操纵子以及lac阻遏物的控制，laci基因也在选择盒中。这种构建方式能够确定失去了可选择的galK功能的底物质粒突变体。该系统的第二个组分是一组兼容质粒中的一种，这些兼容质粒含有来自胭脂碱型质粒pTiC58的vir区域的各种克隆片段。结果表明，vir区域的一个3.6-kbp片段对于产生底物质粒突变体是必要且充分的，这些突变体相当于含有完整左边界的T区域。从这个vir区域片段中，有四种离散的蛋白质在小细胞中表达。编码区域被定位到在胭脂碱型和章鱼碱型质粒中保守的一部分；在章鱼碱型质粒中，它似乎对应于virC/D。
