REGULATION OF AROMATIC HYDROCARBON DEGRADATION GENES

拟议项目的主要目标将是在 NAH和SAL基因表达的分子水平调控机制 恶臭假单胞菌nah质粒的操纵子。这将是 实现方法：(1)利用水杨酸诱导的分子杂交 和含有各种突变型NaH的恶臭假单胞菌细胞的未诱导RNA 用来评估转录调控的质粒。(2)净化和 Nahr基因产物的特性，它介导诱导 不是和萨尔的歌剧。(3)调控基因的克隆和序列分析 NAH和SAL野生型和结构性突变体的区域 歌剧。(4)Nahr蛋白与蛋白质相互作用的物理分析 通过结合研究nah和sal操纵子的调控序列，在 DNA-蛋白质交联的体外转录实验和分析。 此外，nah质粒基因与Very的分子关系 其他碳氢化合物氧化细菌中的类似基因将通过 Southern杂交分析。这些研究应该会得出更好的结果 对基因调控机制和代谢进化的理解 负责有毒致癌污染物生物降解的途径。 这些研究还应该提供关于分子的基本信息 重要属植物基因表达的正调控机制 假单胞菌。

The primary goal of the proposed project will be to elucidate at the molecular level the control mechanism of the expression of the nah and sal operons of the NAH plasmid in Pseudomonas putida. This will be accomplished by: (1) Using molecular hybridization of salicylate-induced and uninduced RNA from P. putida cells containing various mutant NAH plasmids to evaluate transcriptional control. (2) Purification and characterization of the nahR gene product which mediates induction of the nah and sal operons. (3) Cloning and sequence analysis of the regulatory regions from wild-type and constitutive mutants of the nah and sal operons. (4) Physical analysis of the interaction of the nahR protein with the regulatory sequences of the nah and sal operons by binding studies, in vitro transcription experiments and analysis of DNA-protein crosslinking. In addition, the molecular relationship of the NAH plasmid genes to very similar genes in other hydrocarbon oxidizing bacteria will be analyzed by Southern hybridization analysis. These studies should result in a better understanding of the evolution of gene control mechanisms and metabolic pathways responsible for biodegradation of toxic, carcinogenic pollutants. The studies should also provide basic information about molecular mechanisms of positive control of gene expression in the important genus Pseudomonas.