REGULATION OF AROMATIC HYDROCARBON DEGRADATION GENES

该项目的目标是进一步表征 NAH 7萘的nah和sal操纵子的调节 来自恶臭假单胞菌的降解质粒 这两个操纵子是 由一个调控基因nahR协调控制，该基因编码一个 诱导物依赖性转录激活因子。 具体实验将 包括分析nahR基因之间的蛋白质-DNA相互作用 产物以及NAH和SAL操纵子的启动子。 这将是 通过以下步骤完成：（1）体外诱变sal启动子- galK融合 质粒和随后的分析，以确定涉及的关键核苷酸 在nahR蛋白和诱导物的转录激活中， 水杨酸盐 (2)nahR启动子galK的体外诱变和分析 融合质粒，以确定启动子结构和可能的调控。 (3)纯化大肠杆菌nahR基因产物。大肠杆菌细胞， 利用放射化学示踪法过表达克隆的nahR基因 涉及放射化学纯的nahR蛋白作为测定。 (4)分析 和nahR基因产物的生物学性质的表征 及其与NAH 7 DNA的分子相互作用（例如，特异性结合 nah和sal启动子，体外转录刺激，水杨酸盐 绑定等）。 (5)nahR基因DNA序列分析 确定与其他阳性细胞中已知功能结构域的同源性 调节蛋白 (6)nah启动子- galK融合质粒的分析 为了证实NAH和SAL操纵子在相同的 在分子水平上。 这些实验的结果将 提供了一个详细的了解分子间的相互作用， 这个非E。coli中，正调控基因系统。 这些信息将 对了解细菌性疾病过程很重要， 治疗，并将证明许多革兰氏阴性病原菌， 可以进行详细的分子遗传分析 此外，基本 了解独特的遗传调控系统和机制将有助于 了解人类遗传系统中的类似遗传系统和情况 疾病状态。

The goal of the project is to further characterize the molecular basis of the regulation of the nah and sal operons of the NAH7 napthalene degradation plasmid from Pseudomonas putids. These two operons are coordinately controlled by a regulatory gene, nahR, which encodes an inducer-dependent activator of transcription. Specific experiments will involve analysis of the protein-DNA interactions between the nahR gene product and the promoters of the nah and sal operons. This will be accomplished by: (1) In vitro mutagenesis of sal promoter - galK fusion plasmids and subsequent analysis to determine critical nucleotides involved in transcription activation by the nahR protein and the inducer, salicylate. (2) In vitro mutagenesis and analysis of nahR promoter - galK fusion plasmids to determine promoter structure and possible regulation. (3) Purification of the nahR gene product from E. coli cells which overexpress the cloned nahR gene utilizing a radiochemical tracer method involving the radiochemically pure nahR protein as an assay. (4) Analysis and characterization of the biological properties of the nahR gene product and its molecular interactions with NAH7 DNA (e.g., specific binding to the nah and sal promoters, stimulation of transcription in vitro, salicylate binding, etc.). (5) Analysis of the DNA sequence of the nahR gene to determine homologies with known functional domains in other positive regulatory proteins. (6) Analysis of nah promoter - galK fusion plasmids to confirm that the nah and sal operons are controlled in an identical manner at the molecular level. The results of these experiments will provide a detailed understanding of the molecular interactions involved in this non-E. coli, positively regulated gene system. This information will be important to an understanding of bacterial disease processes and treatments and will demonstrate that many gram negative pathogenic bacteria are amenable to detailed molecular genetic analysis. In addition basic knowledge of unique genetic regulatory systems and mechanisms will help in understanding analogous genetic systems and situations involved in human disease states.