The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes.
The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes.
复制标题
大肠杆菌K12 aspC 和tyrB 基因的克隆及序列分析。
作者：
Ian G. Fotheringham;Sandra A. Dacey;Paul P. Taylor;Terry J. Smith;Michael G. Hunter;Mark E. Finlay;Sandy B. Primrose;David M. PARKERt;R. Mark;Edwardsi
本文介绍了大肠杆菌K12中编码芳香族转氨酶和天冬氨酸转氨酶的tyrB和aspC基因的克隆和序列分析。TyrB基因是从携带附近dna B基因的粘粒中分离出来的，通过其补充dna B损伤的能力进行鉴定。缺失和连接子插入分析将tyrB基因定位于1.7kb碱基的NruI-HindIII-酶切片段。序列分析显示，该基因编码43000Da的多肽。该基因以GTG密码子开始，紧随其后的是类似于Rho独立终止子的结构。从原营养型大肠杆菌K12菌株制备的基因库中筛选出能够补充氨基转移酶缺陷菌株HW225中ASPC tyrB损伤的质粒，从而克隆了AsPC基因。亚克隆和缺失分析将aspC基因定位在1.8kb的HincII-StuI-Digest片段上。序列分析表明，存在一个编码43000Da蛋白的基因，其序列与以前从大肠杆菌B中获得的天冬氨酸氨基转移酶的序列完全相同，这两种酶的产量都相当高。我们将推导的蛋白质序列与猪线粒体和细胞质天冬氨酸氨基转移酶的序列进行了比较。根据观察到的广泛的同源性，我们能够提出这两种大肠杆菌酶具有亚基结构、亚基相互作用以及辅酶结合和底物结合部位，它们彼此非常相似，并且与哺乳动物的那些非常相似，因此也必然具有非常相似的催化机制。对aspc和tyrB基因序列的比较表明，在功能和密码子使用的限制下，它们似乎已经尽可能地分化。
In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage.