CELL INTERACTION-REGULATED GENE EXPRESSION IN M XANTHUS

拟议研究的长期目标是了解如何 细胞间的相互作用控制着发育基因的表达。公克- 阴性细菌，黄色粘球菌，提供了一种非常有吸引力的 这些研究的实验系统，因为它经历了一个简单的 多细胞发育需要细胞间的信号，而且它是 对生化和遗传分析都是适用的。建议进行的研究 寻求阐明连接信号的分子机制(S) 与基因激活的相互作用。这些研究具有广泛的意义。 因为细胞间的通讯控制着两个生物体的发育 以及成熟的多细胞生物体内的许多过程，以及 寄主/病原菌和寄主/共生菌的相互作用。此外，它是 越来越清楚的是微生物之间的相互作用会影响 许多重要的生物现象。 至少有四种信号相互作用控制着粘球菌的发育和 其中最具特点的是由CsgA介导的，它编码 一种作为C-信号分子的17kda蛋白质。CsgA突变体 不能正常表达许多发育调节的粘球菌 基因，最初是由转座子产生的LacZ识别的 融合。这些依赖于C的基因为 拟议的调查，旨在探索分子 连接C信号和基因激活的机制(S)。这个 几个依赖C的基因的调节区将被克隆和 其特点是在体内检测启动子活性，然后 启动子区域到缺失分析，转录起始点 测绘和DNA测序。与之相互作用的调节蛋白 C依赖基因的调节区将使用一种 序列特异性DNA结合蛋白的迁移率改变分析 重建C依赖基因的体外转录。监管机构 蛋白质将被提纯，相应的基因将被克隆和 测序，并将开发基因和抗体探针供使用 在旨在了解调控蛋白如何对 C-信号转导和调控C-依赖基因表达。

The long-term objective of the proposed research is to understand how cell-cell interactions control developmental gene expression. The gram- negative bacterium, Myxococcus xanthus, provides a very attractive experimental system for these studies because it undergoes a simple multicellular development requiring cell-to-cell signaling, and it is amenable to both biochemical and genetic analyses. The proposed studies seek to elucidate the molecular mechanism(s) that connects a signaling interaction to gene activation. These studies are of broad significance because cell-to-cell communication controls both organismal development and many processes within mature multicellular organisms, as well as host/pathogen and host/symbiont interactions. In addition, it is becoming increasingly clear that interactions between microbes influence many important biological phenomena. At least four signaling interactions control Myxococcus development and the best-characterized of these is that mediated by csgA, which encodes a 17 Kda protein that serves as the C-signal molecule. A csgA mutant fails to normally express many developmentally regulated Myxococcus genes, which were originally identified by transposon-generated lacZ fusions. These C-dependent genes provide a starting point for the proposed investigation which is aimed at exploring the molecular mechanism(s) that connects C-signaling to gene activation. The regulatory regions of several C-dependent genes will be cloned and characterized by testing for promoter activity in vivo, then subjecting promoter regions to deletion analyses, transcriptional start site mapping, and DNA sequencing. Regulatory proteins that interact wit the regulatory regions of C-dependent genes will be identified using a mobility shift assay for sequence-specific DNA-binding proteins and by reconstructing C-dependent gene transcription in vitro. The regulatory proteins will be purified, the corresponding genes will be cloned and sequenced, and both gene and antibody probes will be developed for use in studies aimed at understanding how the regulatory proteins respond to C-signaling and control C-dependent gene expression.