Genetic Analysis of Flaeellum Biogenesis in Caulobacter

1)在这项资助的过程中，我们计划：检查克隆鞭毛基因在含有各种fla突变的菌株中的表达，作为确定鞭毛基因之间调节相互作用的手段。2)完成包含新发现鞭毛基因突变的菌株的表征，并确定这些新基因如何适应鞭毛基因调控层次。3)利用脉冲场凝胶电泳技术（PFGE）完成月桂草染色体物理图谱的构建，并确定额外营养基因的位置，以便利用脉冲场凝胶电泳技术快速准确地定位新的突变，而不是繁琐的经典遗传学方法。4)通过凝胶阻滞实验检测不同f1bT表达水平菌株中与鞭毛蛋白基因启动子融合的报告基因的表达，证实f1bT编码一种调节鞭毛蛋白基因表达的抑制蛋白。并通过分离对f1bT抑制因子不敏感的操作突变体。5)通过分离和鉴定非连锁抑制突变来研究参与鞭毛蛋白加工的基因的作用

1) During the course of this grant we plan to : examine the expression of cloned flagellar genes in strains containing various fla mutations as a means of determining regulatory interactions among the flagellar genes. 2) Complete the characterization of strains containing mutations in newly identified flagellar genes and to determining how these new genes fit into the flagellar genes regulatory hierarchy. 3) Complete the construction of a physical map of the C. crescentus chromosome using pulsed field gel eletrophoresis (PFGE) and to determine the location of additional nutritional genes so that new mutations can be mapped rapidly and precisely using PEGE rather than the more tedious methods of classical genetics. 4) Confirm that f1bT encodes a repressor protein which regulates flagellin gene expression by examining the expression of reporter genes fused to flagellin gene promoters in strains containing various levels of f1bT expression, by gel retardation assays, and by the isolation of operator mutants which are insensitive to the f1bT repressor. 5) Examine the role of the gene involved in flagellin processing by isolating and characterizing unlinked suppressor mutations.//