Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12.
Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12.
复制标题
编码大肠杆菌 K-12 谷氨酸和天冬氨酸载体的基因的克隆和测序。
DOI：
10.1128/jb.172.6.3214-3220.1990
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发表时间：
1990
影响因子：
3.2
通讯作者：
Lum,D
中科院分区：
文献类型：
--
作者：
Wallace,B;Yang,YJ;Hong,JS;Lum,D
通过使用高拷贝数质粒pBR 322将编码谷氨酸和天冬氨酸载体蛋白的基因克隆到大肠杆菌K-12菌株BK 9 MDG中。该基因（命名为gltP）可能与最近从大肠杆菌中克隆的一个基因相同。coli B（Y.出口岛Yamato和Y. Anraku，J. Bacteriol. 171：1314-1319）。将含有gltP的1.6-腺苷酸酶DNA片段亚克隆到表达质粒pT 7 -5和pT 7 -6中，用噬菌体T7 RNA聚合酶-T7启动子偶联系统（S.塔博尔和C. C. Richardson，Proc. Natl. Acad. Sci. USA 82：1074-1078）作为具有38千道尔顿的表观质量的多肽。gltP多肽的一部分与细胞质膜相关。测定了1.6-腺苷酸酶片段的核苷酸序列。它含有一个开放的阅读框架，能够编码一个高度疏水的多肽的395个氨基酸，含有四个可能的跨膜段。谷氨酸和天冬氨酸的摄取增加5.5倍和4.5倍，分别在含有gltP质粒的菌株。谷氨酸的摄取是不敏感的Na+的浓度，并抑制L-半胱氨酸和β-羟基天冬氨酸。这些结果表明，gltP是一个结构基因的载体蛋白的Na（+）-非依赖性，结合蛋白-非依赖性谷氨酸-天冬氨酸转运系统。
A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322. The gene (designated gltP) is probably identical to a gene recently cloned from E. coli B (Y. Deguchi, I. Yamato, and Y. Anraku, J. Bacteriol. 171:1314-1319). A 1.6-kilobase DNA fragment containing gltP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter coupled system (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078) as a polypeptide with an apparent mass of 38 kilodaltons. A portion of the gltP polypeptide was associated with the cytoplasmic membrane. The nucleotide sequence of the 1.6-kilobase fragment was determined. It contained an open reading frame capable of encoding a highly hydrophobic polypeptide of 395 amino acids, containing four possible transmembrane segments. Uptake of glutamate and aspartate was increased 5.5- and 4.5-fold, respectively, in strains containing gltP plasmids. Glutamate uptake was insensitive to the concentration of Na+ and was inhibited by L-cysteate and beta-hydroxyaspartate. These results suggest that gltP is a structural gene for a carrier protein of the Na(+)-independent, binding-protein-independent glutamate-aspartate transport system.