Degradative Plasmids: Comparative Regulatory Sequences

这项建议旨在研究规管 假单胞菌中的分解代谢和合成代谢基因， 确定哪些核苷酸序列对于 下游基因的转录。 要研究的系统 包含（i）的结构和调节遗传区域， CLC簇是一组质粒携带的基因，参与了 降解氯邻苯二酚;（ii）catBC操纵子，a 紧密连锁的两个染色体基因簇参与了 邻苯二酚的降解;和（iii）alg簇，一组 参与生物合成的染色体基因 藻酸多糖 我们建议描绘出 在clcABD、catBC和 algD基因簇通过改变不同位置的特定核苷酸 通过定点诱变定位，然后检查 这种改变对下游基因表达的影响 基因. 为了检测其他关键核苷酸， 位置，非特异性诱变将用于改变 上游核苷酸的随机和这样的影响， 修改将被记录。 由于许多调控基因 以积极的方式控制结构基因的表达， 已知在50到100之间的上游序列处相互作用 转录起始位点上游的核苷酸， 将algR、clcR和catR基因的产物纯化， 过度生产及其与上游的相互作用 在-50到-100个区域的序列将被研究， 足迹法和电泳蛋白质DNA结合测定。 这些实验应该为我们提供了丰富的 关于假单胞菌基因调控性质的信息， 偶尔是人类病原体的细菌物种， 在某些情况下是一种重要的植物病原体，而在另一些情况下， 越来越多地被开发用于工业 目的

This proposal aims at studying the mode of regulation of catabolic and anabolic genes in Pseudomonas with a view to determining which nucleotide sequences are critical for the transcription of downstream gene(s). The systems to be studied comprise the structural and regulatory genetic regions of (i) the clc cluster, a group of plasmid-borne genes involved in the degradation of Chlorocatechol; (ii) the catBC operon, a tightly-linked cluster of two chromosomal genes involved in the degradation of catechol; and (iii) the alg cluster, a group of chromosomal genes involved in the biosynthesis of a polysaccharide alginate. We propose to delineate the critical nucleotides in the upstream regions of the clcABD, catBC and algD gene clusters by altering specific nucleotides at various locations by site directed mutagenesis and then examining the effects of such alterations on the expression of the downstream genes. In order to detect other critical nucleotides at other locations, nonspecific mutagenesis will be used to alter upstream nucleotides at random and the effects of such alterations will be noted. Since many regulatory genes controlling structural gene expression in a positive manner are known to interact at upstream sequences between 50 and 100 nucleotides upstream of the transcription initiation site, the products of algR, clcR and catR genes will be purified after hyperproduction and their interaction with the upstream sequences at -50 to -100 regions will be studied by footprinting and electrophoretic protein DNA binding assays. These experiments should provide us with a wealth of information on the nature of gene regulation in Pseudomonas, a bacterial species which is occasionally a human pathogen, in some cases an important plant pathogen, and in other cases is being exploited to an increasing degree for industrial purposes.