Degradative Plasmids: Comparative Regulatory Sequences

这项建议旨在研究假单胞菌分解代谢和合成代谢基因的调控模式，以确定哪些核苷酸序列对下游基因的转录起关键作用(S)。要研究的系统包括：(I)CLC簇，一组与氯代儿茶酚降解有关的质粒携带的基因；(Ii)CatBC操纵子，与邻苯二酚降解有关的两个染色体基因紧密相连的簇；以及(Iii)alg簇，这是一组参与海藻多糖生物合成的染色体基因。我们建议通过定点突变改变不同位置的特定核苷酸，然后检测这种改变对下游基因表达的影响，从而确定clcABD、catBC和algD基因簇上游区域的关键核苷酸。为了检测其他位置的其他关键核苷酸，将使用非特异性突变来随机改变上游核苷酸，并注意这种改变的效果。由于已知许多以积极方式控制结构基因表达的调控基因与转录起始点上游50至100个核苷酸之间的上游序列相互作用，因此，CLcr和CatR基因将在超产后得到纯化，并将通过足迹和电泳蛋白DNA结合分析来研究它们与-50到-100区域上游序列的相互作用。这些实验应该为我们提供关于假单胞菌基因调控性质的丰富信息，假单胞菌是一种细菌物种，有时是人类病原体，在某些情况下是重要的植物病原体，在其他情况下正被越来越多地用于工业目的。

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.