Roberts Radclyffe L, Metz Matthew, Monks Dave E, Mullaney Matthew Lockwood, Hall Tyler, Nester Eugene W
Department of Microbiology, University of Washington, Seattle 98195-7242, USA.
Proc Natl Acad Sci U S A. 2003 May 27;100(11):6634-9. doi: 10.1073/pnas.1132022100. Epub 2003 May 9.
The bacterium Agrobacterium tumefaciens transforms eukaryotic hosts by transferring DNA to the recipient cell where it is integrated and expressed. Bacterial factors involved in this interkingdom gene transfer have been described, but less is known about host-cell factors. Using the yeast Saccharomyces cerevisiae as a model host, we devised a genetic screen to identify yeast mutants with altered transformation sensitivities. Twenty-four adenine auxotrophs were identified that exhibited supersensitivity to A. tumefaciens-mediated transformation when deprived of adenine. We extended these results to plants by showing that purine synthesis inhibitors cause supersensitivity to A. tumefaciens transformation in three plant species. The magnitude of this effect is large and does not depend on prior genetic manipulations of host cells. These data indicate the utility of yeast as a model for the transformation process and identify purine biosynthesis as a key determinant of transformation efficiency. These findings should increase the utility of A. tumefaciens in genetic engineering.
根癌土壤杆菌通过将DNA转移到受体细胞并在其中整合和表达来转化真核宿主。参与这种跨界基因转移的细菌因子已被描述，但对宿主细胞因子的了解较少。我们以酿酒酵母作为模型宿主，设计了一个遗传筛选来鉴定转化敏感性改变的酵母突变体。我们鉴定出了24个腺嘌呤营养缺陷型突变体，当缺乏腺嘌呤时，它们对根癌土壤杆菌介导的转化表现出超敏感性。我们通过表明嘌呤合成抑制剂在三种植物物种中会导致对根癌土壤杆菌转化的超敏感性，将这些结果扩展到了植物。这种效应的程度很大，并且不依赖于宿主细胞先前的基因操作。这些数据表明酵母作为转化过程模型的实用性，并确定嘌呤生物合成是转化效率的关键决定因素。这些发现应该会增加根癌土壤杆菌在基因工程中的实用性。
