Miranda A, Janssen G, Hodges L, Peralta E G, Ream W
Department of Biology, Indiana University, Bloomington 47405.
J Bacteriol. 1992 Apr;174(7):2288-97. doi: 10.1128/jb.174.7.2288-2297.1992.
During crown gall tumorigenesis, part of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid, the T-DNA, integrates into plant DNA. Direct repeats define the left and right ends of the T-DNA, but tumorigenesis requires only the right-hand repeat. Virulence (vir) genes act in trans to mobilize the T-DNA into plant cells. Transfer of T-DNA begins when the VirD endonuclease cleaves within the right-hand border repeat. Although the T-DNA right-border repeat promotes T-DNA transmission best in its normal orientation, an inverted right border exhibits reduced but significant activity. Two models may account for this diminished tumorigenesis. The right border may function bidirectionally, with strong activity only in its wild-type orientation, or it may promote T-DNA transfer in a unidirectional manner such that, with an inverted right border, transfer proceeds around the entire Ti plasmid before reaching the T-DNA. To determine whether a substantial portion of the Ti plasmid is transferred to plant cells, as predicted by the unidirectional-transfer hypothesis, we examined T-DNAs in tumors induced by strains containing a Ti plasmid with a right border inverted with respect to the T-DNA oncogenes. These tumors contained extremely long T-DNAs corresponding to most or all of the Ti plasmid. To test whether the right border can function bidirectionally, we inserted T-DNAs with either a properly oriented or an inverted right border into a specific site in the A. tumefaciens chromosome. A border situated to transfer the oncogenes first directed T-DNA transfer even from the bacterial chromosome, whereas a border in the opposite (inverted) orientation did not transfer the oncogenes to plant cells. Our results indicate that the right-border repeat functions in a unidirectional manner.
在冠瘿瘤发生过程中，根癌土壤杆菌的部分致瘤（Ti）质粒，即T-DNA，会整合到植物DNA中。同向重复序列界定了T-DNA的左端和右端，但瘤形成仅需要右手边的重复序列。毒性（vir）基因通过反式作用将T-DNA转运到植物细胞中。当VirD核酸内切酶在右手边边界重复序列内切割时，T-DNA的转移开始。尽管T-DNA右边界重复序列在其正常方向上最有利于T-DNA的传递，但反向的右边界活性虽降低但仍显著。有两种模型可以解释这种瘤形成能力的减弱。右边界可能双向发挥作用，仅在其野生型方向上具有强活性，或者它可能以单向方式促进T-DNA转移，这样，对于反向的右边界，转移在到达T-DNA之前会绕整个Ti质粒进行。为了确定是否如单向转移假说所预测的那样，Ti质粒的大部分会转移到植物细胞中，我们检查了由含有与T-DNA致癌基因方向相反的右边界的Ti质粒菌株诱导形成的肿瘤中的T-DNA。这些肿瘤含有对应于大部分或全部Ti质粒的极长T-DNA。为了测试右边界是否能双向发挥作用，我们将具有正确方向或反向右边界的T-DNA插入到根癌土壤杆菌染色体的特定位点。一个定位为首先转移致癌基因的边界即使从细菌染色体也能引导T-DNA转移，而相反（反向）方向的边界则不会将致癌基因转移到植物细胞中。我们的结果表明右边界重复序列以单向方式发挥作用。
