CONSTRUCTION AND ANALYSIS OF TRP/PAB HYBRID GENES

TRP/PAB杂合基因的构建与分析

• 批准号: 3128078
• 负责人: BRIAN P NICHOLS
• 金额: $ 12.13万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-01-01 至 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3128078
• 关键词: Escherichia coli; enzymes; fluorescence spectrometry; gene expression; genetic manipulation; genetic mapping; genetic recombination; mutant; nucleic acid sequence; o aminobenzoate; p aminobenzoate; plasmids; radiotracer; Escherichia coli; enzymes; fluorescence spectrometry; gene expression; genetic manipulation; genetic mapping; genetic recombination; mutant; nucleic acid sequence; o aminobenzoate; p aminobenzoate; plasmids; radiotracer

E. coli p-aminobenzoate synthetase and anthranilate synthetase are structurally and genetically related enzymes that catalyze similar, but slightly different, reactions. Each enzyme is composed of two dissimilar subunits, one responsible for glutamine amidotransferase (GAT) activity, and the other responsible for the aromatization of chorismate. The difference in the products is the disposition of amino- and carboxy- substituents on a benzene ring. The subunits of PABS are encoded by pabA and pabB and the subunits of anthranilate synthetase are encoded by trpE and trpG. The nucleotide and amino acid sequences of these four genes have been determined, and clearly indicate divergence from a common ancestor. Similarity is approximately 30% between the large subunits and 45% between the small subunits of the enzymes. The GAT subunits perform identical functions, and differ only in the specific interactions with the appropriate large subunit. The nucleotide sequence of a GAT subunit that functions in both anthranilate and PABA synthesis in A. calcoaceticus has also been determined, and is very similar to the E. coli pabA sequence. The experiments in this proposal focus on the determination of specific differences between the pab and trp genes that are responsible for divergence of function. The experimental approaches include construction of hybrid genes by exploiting in vivo recombination and in vitro DNA replication techniques, in vitro inter- and intrageneric subunit exchange, in vitro mutagenesis, and analysis of second site reversions. The results of the proposed studies will include information on the ways in which genes evolve from a common ancestor after a gene duplication event. The extent of total nucleotide and amino acid sequence divergence has been documented, and my aim is to determine the fraction of the divergence that is responsible for alteration of function. We will also gain insight into a mechanism in which biochemical specificity has been maintained while function has diverged. The approach and methodology are unique in that a functional determination of the consequences of sequence divergence will accompany a descriptive approach on the extent of sequence divergence.

大肠杆菌P-氨基苯甲酸酯合成酶和蒽合成酶是 结构和遗传相关的酶催化相似，但 略有不同的反应。 每个酶由两个不同的酶组成 亚基，负责谷氨酰胺酰胺转移酶（GAT）活性的亚基， 另一个负责假酸的芳香化。 这 产品的差异是氨基和羧基的处理 苯环上的取代基。 PAB的亚基由PABA编码 PABB和炭疽菌合成酶的亚基由TRPE编码 和TRPG。 这四个基因的核苷酸和氨基酸序列具有 已确定，清楚地表明与共同祖先的差异。 大型亚基之间的相似性约为30％，在45％之间 酶的小亚基。 GAT亚基执行相同 功能，仅在与特定的相互作用中不同 适当的大亚基。 GAT亚基的核苷酸序列 炭疽菌和PABA合成的功能在曲霉中的功能 也已确定，与大肠杆菌paba序列非常相似。 本提案中的实验重点是确定特定 负责的PAB和TRP基因之间的差异 功能发散。 实验方法包括施工 通过利用体内重组和体外DNA来利用杂化基因的 复制技术，体外间和内部亚基交换， 体外诱变和第二站点逆转的分析。 拟议研究的结果将包括有关方式的信息 基因在基因重复事件后的共同祖先演变。 总核苷酸和氨基酸序列差异的程度已经 记载的，我的目的是确定差异的比例 负责改变功能。 我们还将深入了解 维持生化特异性的机制 功能有所不同。 方法和方法是独一无二的 序列差异后果的功能确定将 伴随描述性方法在序列差异的程度上。