TRANSLOCATION OF DNA ACROSS THE AGROBACTERIUM ENVELOPE

核酸在生物膜上的转移是 原核生物中不同细胞过程的基本重要性 真核生物。一个引人注目的DNA易位例子，有着深刻的 在根癌农杆菌研究中发现基因组进化 植物细胞的感染。响应特定植物信号 分子，这种原核病原体合成具有易位能力的 DNA/蛋白质复合体，不仅跨越细菌的包膜，而且还 植物膜通向细胞核。被转移者的表达 基因最终会破坏植物内源激素的平衡，导致 在失去细胞分裂控制和肿瘤形成的过程中。因为 这种通过DNA刺激植物肿瘤形成的独特能力 转移，根癌农杆菌植株转化系统提供了一个理想的 检验与宿主-病原体信号有关的基本过程的模型 交换、大分子运输与真核细胞分裂调控 和肿瘤的发生。这项研究计划的重点是阐明 介绍了该装置的结构和功能特点。 向植物输出大分子所需的根癌细胞膜 细胞。 根据几个遗传和生化标准，大约9.5千碱基(Kb) VIRB操纵子代码，用于这个跨王国的部分或全部组件 运输系统。拟议的研究将评估这些贡献 两个假定的ATP结合/水解蛋白，VirB4和VirB11，以 这个DNA的运输过程。这两种蛋白质都含有保守结构域。 发现于原核生物和真核单核苷酸超家族中 结合/水解性蛋白质。初步研究表明，提纯的 VirB11蛋白与ATP结合，具有ATPase活性， 体外自磷酸化。经典与分子的结合 基因技术和蛋白质生物化学将被用来研究 这些蛋白质的结构和功能。 病毒B11是DNA运输所必需的，但这一点仍有待确定 为VirB4建立的。病毒B4蛋白在DNA转运中的重要性 将通过构建非极性VirB4零突变和 评估相应突变体运输DNA的能力。 这两种蛋白质的生化活性将由蛋白质来表征。 三磷酸腺苷结合、三磷酸腺苷水解酶和三磷酸腺苷的纯化和体外检测 磷酸化。对病毒B4和病毒B11重要的特定残基 生化活性将通过定点定向来识别和突变 诱变。将引入随机突变来识别其他 对结构和/或功能重要的蛋白质区域。突变型 将检查蛋白质的酶活性变化和对 根癌农杆菌的毒力和DNA转运。VirB4和VirB11膜 拓扑、亚细胞定位和相互作用的可能性 与其他细胞成分的关系将得到评估。对应 突变蛋白质的分析将有助于识别结构域或 对蛋白质构型至关重要的残基。 本实验室建立了一种新的DNA转移检测方法。 根癌农杆菌转移含内含子报告基因的能力 植物细胞和原生质体的基因。化验的灵敏度将会 通过评估DNA转移在整个拟议的研究中进行测试 野生型菌株及virB4和virB11突变体的效率。这些 研究将测试该检测作为遗传筛查的实用性。 鉴定DNA转移缺陷突变体。这项化验将形成 为今后根癌农杆菌感染过程的遗传学研究奠定基础。

Translocation of nucleic acids across biological membranes is of fundamental importance for diverse cellular processes in prokaryotes and eukaryotes. A striking example of DNA translocation with profound implications for genome evolution occurs during Agrobacterium tumefaciens infection of plant cells. In response to specific plant signal molecules, this prokaryotic pathogen synthesizes translocation-competent DNA/protein complex that cross not only the bacterial envelope but also plant membranes enroute to the nucleus. Expression of the transferred genes ultimately disrupts endogenous plant hormone balances, resulting in loss of cell division control and the formation of tumors. Because of this unique ability to incite plant tumor formation through DNA transfer, the A. tumefaciens plant transformation system offers an ideal model for examining fundamental processes related to host-pathogen signal exchange, macromolecular transport, and eukaryotic cell division control and tumorigenesis. The focus of this research program is to elucidate the structural and functional features of the apparatus at the A. tumefaciens membrane required for exporting macromolecules to plant cells. By several genetic and biochemical criteria, the about 9.5 kilobase (kb) virB operon codes for some or all of the components of this interkingdom transport system. The proposed research will evaluate the contributions of two putative ATP-binding/hydrolysis proteins, VirB4 and VirB11, to this DNA transport process. Both proteins contain conserved domains found in a superfamily of prokaryotic and eukaryotic mononucleotide binding/hydrolysis proteins. An initial study showed that purified VirB11 protein binds ATP, possesses ATPase activity, and autophosphorylates in vitro. A combination of classical and molecular genetic techniques, and protein biochemistry, will be used to examine the structures and functions of these proteins. VirB11 is required for DNA transport, but this remains to be definitively established for VirB4. The importance of VirB4 protein for DNA transport will be examined by constructing a nonpolar virB4 null mutation and assessing the ability of the corresponding mutant to transport DNA. Biochemical activities of both proteins will be characterized by protein purification and in vitro assays for ATP binding, ATP hydrolysis, and phosphorylation. Specific residues important for VirB4 and VirB11 biochemical activities will be identified and mutated by site-directed mutagenesis. Random mutations will be introduced to identify other regions of the proteins important for structure and/or function. Mutant proteins will be examined for altered enzymatic activity and effects on A. tumefaciens virulence and DNA transport. VirB4 and VirB11 membrane topologies, subcellular localization, and the potential for interacting with other cellular constituents will be evaluated. Corresponding analyses of mutant proteins will facilitate identification of domains or residues that are critical for protein configuration. A novel DNA transfer assay has been developed in this laboratory based on the ability of A. tumefaciens to transfer intron-containing reporter genes to plant cells and protoplasts. The sensitivity of the assay will be tested throughout the proposed studies by evaluating DNA-transfer efficiencies of wild-type strains and virB4 and virB11 mutants. These studies will test the utility of the assay as a genetic screen for identifying DNA-transfer deficient mutants. This assay will form the basis of future genetic studies of the A. tumefaciens infection process.