title: Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli.
abstract: 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 phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & 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 ispA 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.
authors.affiliation: Department of Chemistry, Faculty of General Education, Gifu University.
authors.forename: S
authors.initials: S
authors.lastname: Fujisaki
authors.forename: H
authors.initials: H
authors.lastname: Hara
authors.forename: Y
authors.initials: Y
authors.lastname: Nishimura
authors.forename: K
authors.initials: K
authors.lastname: Horiuchi
authors.forename: T
authors.initials: T
authors.lastname: Nishino
mesh_terms: D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D051228:Geranyltranstransferase; D045782:Hemiterpenes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D009943:Organophosphorus Compounds / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D014166:Transferases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D003062:Codon; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C004809:isopentenyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase