RHIZOBIUM NODULATION GENE EXPRESSION AND GENE PRODUCTS

我们研究细菌细胞与特定真核宿主的相互作用， 在一个既涉及多细胞生物的发育事件， 微生物，以及细菌的致病机理。 苜蓿根瘤菌入侵， 在包括苜蓿在内的特定宿主植物中诱导根瘤形成 （Medicago sativa）. 当它们入侵时，细菌改变了宿主的细胞壁 合成，诱导正常非分裂细胞恢复细胞分裂，和 刺激新宿主基因表达。 细菌也会分化 并表达新的基因。 我们已经识别并克隆了 用于入侵和结节诱导的细菌基因。 自由生活 细菌几乎没有或没有与nod基因片段同源的RNA， 这表明它们可能不会在自由生活的细胞中转录。 没有蛋白质 在野生型细菌和转座子Tn5之间观察到差异 诱导Nod突变体。 一个框内翻译的基因融合体， lacZ已被构建，并用于分析nod基因 在各种生长条件下和感染期间表达。 的dna 已经确定了该区域大部分的序列，并且在体外， 对于开放式阅读，已经实现了微型单元和大型单元的表达 nod基因片段中的框架。 正在产生抗CD4A的抗体， 在E.大肠杆菌，和两个合成肽预测 已经构建并偶联到 载体 我们将继续对nod基因表达和nod基因的研究 通过构建进一步的操纵子和nod基因之间的基因融合来产生 以及指示基因lacZ、CAT和lux。 体外融合将基于 这些共生基因的完整DNA序列 随机操纵子融合 将利用Tn5衍生物，其将lacZ或CAT指示剂 在插入位点受启动子控制的基因。 我们会利用基因 融合物作为定位和突变调节转录基因的测定。 nod基因蛋白将在E.大肠杆菌，并纯化， 产生抗体，用于确认患者的身份 基因，以跟踪基因表达在自由生活的增长， 感染，并确定基因产物的细胞定位 in vivo. 这些基因表达的时间和细胞定位 产品将提供洞察他们在入侵期间可能的功能 和根瘤诱导。

We study the interaction of bacterial cells with specific eukaryotic hosts, in a system which relates both to developmental events in multicellular organisms, and to bacterial pathogenesis. Rhizobium meliloti invades and induces nodule formation in specific host plants including alfalfa (Medicago sativa). As they invade, the bacteria alter the host's cell wall synthesis, induce normally non-dividing cells to resume cell division, and stimulate new host gene expression. The bacteria also differentiate structurally and express new genes. We have identified and cloned the bacterial genes used in invasion and nodule induction. Free living bacteria have little or no RNA homologous to the nod gene fragments, suggesting they may not be transcribed in free-living cells. No protein differences were observed between wild type bacteria and transposon Tn5 induced Nod- mutants. An in-frame translational gene fusion of nodC and lacZ has been constructed and this is being used to analyze nod gene expression in various growth conditions and during infection. The DNA sequence has been determined for most of this region, and in vitro, mini-cell and maxi-cell expression has been achieved for open reading frames in the nod gene segment. Antibody is being generated to nodA which has been overexpressed in E. coli, and two synthetic peptides predicted from the DNA sequence of nodC have been constructed and coupled to carrier. We will continue our studies of nod gene expression and nod gene products by constructing further operon and gene fusions between nod genes and indicator genes lacZ, CAT, and lux. In vitro fusions will be based on the complete DNA sequence for these symbiotic genes. Random operon fusions will take advantage of Tn5 derivatives which place lacZ or CAT indicator genes under control of promoters at the insertion site. We will use gene fusions as assays for locating and mutating regulatory nodulation genes. The nod gene proteins will be produced in E. coli and purified for generating antibody, which will be used to confirm the identity of the genes, to follow gene expression in free-living growth and during infection, and to determine the cellular localization of the gene products in vivo. The timing of expression and cellular location of these gene products will provide insight into their possible function during invasion of and nodule induction in their host.