Fabiny J M, Jayakumar A, Chinault A C, Barnes E M
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
J Gen Microbiol. 1991 Apr;137(4):983-9. doi: 10.1099/00221287-137-4-983.
Escherichia coli expresses a concentrative ammonium (methylammonium) transport system which is strongly repressed under conditions of nitrogen excess. We have previously reported the cloning of a structural gene (amtA) for this transporter by complementation. In this study, a 3.4 kb HindIII-BamHI fragment containing amtA was cloned into the pBluescript KS(+) vector, and unidirectional nested deletions from each end of this 3.4 kb fragment were generated by exonuclease III digestion. The deletions were analysed by complementation of the structural gene mutation produced by Tn10 insertion. This allowed amtA to be localized within a 1.4 kb region which spans the site of the mutation. By application of the Sanger dideoxy method, we sequenced the region containing amtA. The gene contains an open reading frame which encodes a protein with a predicted molecular mass of 27 kDa. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by an inverted repeat which might function as a simple transcription terminator. Hydropathic analysis of the inferred amino acid sequence of the gene product predicts that amtA encodes a cytoplasmic component of the ammonium transport system.
大肠杆菌表达一种浓缩铵（甲基铵）转运系统，该系统在氮过量条件下受到强烈抑制。我们之前曾报道通过互补作用克隆了该转运蛋白的一个结构基因（amtA）。在本研究中，将包含amtA的3.4 kb HindIII - BamHI片段克隆到pBluescript KS(+)载体中，并通过核酸外切酶III消化从该3.4 kb片段的两端产生单向嵌套缺失。通过对由Tn10插入产生的结构基因突变进行互补分析来研究这些缺失。这使得amtA能够定位在跨越突变位点的1.4 kb区域内。通过应用桑格双脱氧法，我们对包含amtA的区域进行了测序。该基因包含一个开放阅读框，编码一种预测分子量为27 kDa的蛋白质。开放阅读框之前有一个假定的SD序列，之后有一个反向重复序列，其可能作为一个简单的转录终止子发挥作用。对该基因产物推断的氨基酸序列进行亲水性分析预测，amtA编码铵转运系统的一个细胞质成分。
