GENETIC COMPETENCE APPARATUS OF BACILLUS SUBTILIS

枯草芽孢杆菌能被外源DNA转化 称为能力的生理状态。有能力的细胞能够结合 双链DNA，细胞表面的DNA碎片，并运输 穿过细胞被膜层的单链，伴随着释放 另一条链上的酸溶产物。我们的长期目标 就是在分子水平上理解这些过程。以前的工作 已经确定了十几种基因产品，这些产品似乎是 转化DNA的结合、加工和摄取。这些基因 产品已被克隆、测序和转录图谱绘制，以及 他们的规则被研究过。大多数被预测为膜蛋白，来自 它们的序列。有几种能力蛋白已经被证明是 使用抗体试剂定位的膜，以及它们的性质 对膜附着进行了表征。Comg操纵子编码七个 DNA与细胞结合所需的蛋白质 浮出水面。Come和Comf操纵子编码至少两种蛋白质，它们是 是摄取DNA所必需的，但不是结合DNA所必需的。Comfi蛋白 类似于已知的DNA转位酶和解旋酶，并可能利用 驱动DNA摄取的ATP水解能。ComE3蛋白是 推测是一种致孔蛋白。我们将使用组合基因， 生化、免疫、电生理和高分辨率 用电子显微镜方法研究这些分子的排列 细胞表面的蛋白质及其相互关系和相互作用 转型过程中的职能角色。我们将使用交叉链接来 检测在转化过程中接触DNA的蛋白质。我们会 研究DNA摄取的极性，DNA摄取的作用 竞争力。特定的核酸酶活性，我们将寻求更多 能力基因产物。最后，我们将尝试建立一个In 体外DNA转运系统。

Bacillus subtilis can be transformed by exogenous DNA when in a physiological state known as competence. Competent cells are able to bind double-stranded DNA, fragment the DNA on the cell surface, and transport a single strand across the cell envelope layers, with concomitant release of acid soluble products from the other strand. Our long term objective is to understand these processes at the molecular level. Previous work has identified a dozen gene products that appear to be necessary for the binding, processing and uptake of transforming DNA. The genes for these products have been cloned, sequenced and transcriptionally mapped, and their regulation studied. Most are predicted to be membrane proteins from their sequences. Several competence proteins have been shown to be membrane-localized using antibody reagents, and the nature of their membrane attachment has been characterized. The comG operon encodes seven proteins which appear to be required for the binding of DNA to the cell surface. The comE and comF operons encode at least two proteins which are required for the uptake but not the binding of DNA. The ComFI protein resembles known DNA translocases and helicases, and may utilize the energy of ATP hydrolysis to drive DNA uptake. The ComE3 protein is suggested to be a pore-forming protein. We will use combined genetic, biochemical, immunological, electrophysiological and high resolution electron microscopic approaches to explore the arrangements of these proteins on the cell surface, their interrelationships and their functional roles during transformation. We will use cross-linking to detect proteins that contact DNA during transformation. We will investigate the polarity of DNA uptake, the role of the Competence.specific nuclease activity, and we will seek additional competence gene products. Finally we will attempt to establish an in vitro DNA transport system.