CLONING AND NUCLEOTIDE-SEQUENCE OF THE ISPA GENE RESPONSIBLE FOR FARNESYL DIPHOSPHATE SYNTHASE ACTIVITY IN ESCHERICHIA-COLI
CLONING AND NUCLEOTIDE-SEQUENCE OF THE ISPA GENE RESPONSIBLE FOR FARNESYL DIPHOSPHATE SYNTHASE ACTIVITY IN ESCHERICHIA-COLI
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DOI：
10.1093/oxfordjournals.jbchem.a123327
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发表时间：
1990-12-01
影响因子：
2.7
通讯作者：
NISHINO, T
中科院分区：
文献类型：
--
作者：
FUJISAKI, S;HARA, H;NISHINO, T
本文报道了大肠杆菌法呢基二磷酸（FPP）合酶（EC 2.5.1.1）ispA基因的分子克隆和核苷酸序列测定。E. coli ispA菌株具有温度敏感型FPP合酶，缺陷基因位于染色体上约min 10处。野生型ispA基因是从拟南芥中亚克隆的。从Kohara等人的比对基因组文库中挑选的含有约10分钟的染色体片段的阶段克隆[Kohara，Y.，Akiyama，K.，Isono，K.（1987）Cell 50，495-508]。通过在ispA菌株中恢复和扩增FPP合酶活性，鉴定所克隆的基因为ispA基因。测定了克隆片段的1，452个核苷酸的序列。该序列指定了两个开放阅读框，ORF-1和ORF-2，分别编码预期分子量为8，951和32，158的蛋白质。ORF-2的部分氨基酸序列与真核生物FPP降解酶和光合细菌crtE产物的序列相似。在lac启动子下游携带ORF-2的质粒弥补了ispA突变体的FPP合酶活性的缺陷，表明ORF-2编码的产物是ipsA产物。maxicell分析表明，分子量为36，000的蛋白质是基因产物，与推导的ORF-2编码的蛋白质的分子量大致一致。
The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a .lambda. phase clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ipsA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.