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乳酸乳球菌乳酸亚种胸苷酸合成酶基因的克隆与特性分析

Cloning and characterization of the thymidylate synthase gene from Lactococcus lactis subsp. lactis.

作者信息

Ross P, O'Gara F, Condon S

机构信息

Department of Food Microbiology, University College, Cork, Ireland.

出版信息

Appl Environ Microbiol. 1990 Jul;56(7):2156-63. doi: 10.1128/aem.56.7.2156-2163.1990.

Abstract

The thymidylate synthase (thyA) gene has been isolated from Lactococcus lactis subsp. lactis. The cloned gene was strongly expressed in Escherichia coli both in vivo and in vitro (maxicells and cell-free transcription and translation systems) and complemented E. coli thyA mutants. DNA-DNA hybridizations demonstrated that the thyA gene is encoded by the chromosome of L. lactis subsp. lactis. By sequential deletion of DNA outside the complementing region, the thyA gene was localized to a 1.1-kilobase DNA fragment. The nucleotide sequence of the lactococcal thyA gene was determined by the dideoxy-chain termination technique. The derived amino acid sequence indicated a protein size of 32,580 daltons, which is in good agreement with results obtained from maxicell and in vitro transcription and translation experiments. The primary sequence is homologous to 12 other thyA proteins from a variety of other organisms. Upstream from the structural gene, -10 and -35 promoter sequences which were almost canonical sigma-70 promoter sequences were identified, which may explain the strong expression of the thyA gene observed in E. coli. An A-T-rich sequence characteristic of gram-positive promoters was also noted adjacent to the -35 region. The thyA gene has potential as a marker for plasmid maintenance and selection in food systems.

摘要

胸苷酸合成酶(thyA)基因已从乳酸乳球菌乳酸亚种中分离出来。克隆的基因在大肠杆菌体内和体外(在最大细胞以及无细胞转录和翻译系统中)均能强烈表达,并能互补大肠杆菌thyA突变体。DNA-DNA杂交表明,thyA基因由乳酸乳球菌乳酸亚种的染色体编码。通过依次缺失互补区域外的DNA,thyA基因被定位到一个1.1千碱基的DNA片段上。利用双脱氧链终止技术测定了乳球菌thyA基因的核苷酸序列。推导的氨基酸序列表明蛋白质大小为32,580道尔顿,这与从最大细胞以及体外转录和翻译实验获得的结果高度一致。其一级序列与来自多种其他生物的12种其他thyA蛋白同源。在结构基因上游,鉴定出了几乎是典型的σ-70启动子序列的-10和-35启动子序列,这可能解释了在大肠杆菌中观察到的thyA基因的强烈表达。在-35区域附近还发现了一个革兰氏阳性启动子特有的富含A-T的序列。thyA基因有潜力作为食品系统中质粒维持和选择的标记。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dfb0/184576/eb5989b34010/aem00088-0187-a.jpg

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