CONSTRUCTION AND ANALYSIS OF TRP/PAB HYBRID GENES

E.大肠杆菌对氨基苯甲酸合成酶和邻氨基苯甲酸合成酶， 结构和遗传上相关的酶，催化类似的，但 稍微不同的反应。 每种酶都由两种不同的 亚基，一个负责谷氨酰胺转移酶（GAT）活性， 另一个负责分支酸的芳构化。 的 产物的不同之处在于氨基和羧基的处置。 苯环上的取代基。 PABS的亚基由pabA编码 pabB和邻氨基苯甲酸合成酶的亚基由trpE编码 和trpG。 这四个基因的核苷酸和氨基酸序列具有 这一点已经确定，并清楚地表明从共同的祖先分歧。 大亚基之间的相似性约为30%， 酶的小亚基。 GAT子单元的性能相同 功能，不同之处仅在于与 适当的大亚基。 GAT亚基的核苷酸序列， 在A.醋酸钙有 也被确定，并且与E. coli pabA序列。 本提案中的实验侧重于确定特定的 pab和trp基因之间的差异， 功能发散 实验方法包括构建 通过利用体内重组和体外DNA 复制技术，体外属间和属内亚基交换， 体外诱变和第二位点回复的分析。 拟议研究的结果将包括关于如何 哪些基因在基因复制事件后从共同祖先进化而来。 总核苷酸和氨基酸序列差异的程度已经被证实。 我的目标是确定 负责改变功能。 我们还将深入了解 一种机制，其中生化特异性一直保持， 功能已经分化。 方法和方法的独特之处在于， 序列趋异后果的功能性确定将 伴随着对序列分歧程度的描述性方法。

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.