title: Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome.
abstract: Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. Two homologues of bacterial thymidylate kinase genes were identified in a genomic library of the onion yellows (OY) phytoplasma, a plant pathogen that inhabits both plant phloem and the organs of insects. Southern blotting analysis suggested that the OY genome contained one copy of the tmk-b gene and multiple copies of the tmk-a gene. Sequencing of PCR products generated by amplification of tmk-a enabled identification of three other copies of tmk-a, although the ORF in each of these was interrupted by point mutations. The proteins, TMK-a and TMK-b, encoded by the two intact genes contained conserved motifs for catalytic activity. Both proteins were overexpressed as fusion proteins with a polyhistidine tag in Escherichia coli and purified, and TMK-b was shown to have thymidylate kinase activity. This is believed to be the first report of the catalytic activity of a phytoplasmal protein, and the OY phytoplasma is the first bacterial species to be found to have two intact homologues of tmk in its genome.
authors.affiliation: Division of Integrated Biosciences, The University of Tokyo, 202 Bioscience Bldg, 5-1-5 Kashiwanoha, Chiba 277-8562, Japan.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Miyata
authors.affiliation: Division of Integrated Biosciences, The University of Tokyo, 202 Bioscience Bldg, 5-1-5 Kashiwanoha, Chiba 277-8562, Japan.
authors.forename: Kenro
authors.initials: K
authors.lastname: Oshima
authors.affiliation: Division of Integrated Biosciences, The University of Tokyo, 202 Bioscience Bldg, 5-1-5 Kashiwanoha, Chiba 277-8562, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Kakizawa
authors.affiliation: Division of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Nishigawa
authors.affiliation: Division of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hee-Young
authors.initials: HY
authors.lastname: Jung
authors.affiliation: Department of Agricultural Sciences, School of Agriculture, Ibaraki University, Chuo 3-21-1, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kuboyama
authors.affiliation: Division of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.|Division of Integrated Biosciences, The University of Tokyo, 202 Bioscience Bldg, 5-1-5 Kashiwanoha, Chiba 277-8562, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Ugaki
authors.affiliation: Division of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.|Division of Integrated Biosciences, The University of Tokyo, 202 Bioscience Bldg, 5-1-5 Kashiwanoha, Chiba 277-8562, Japan.
authors.forename: Shigetou
authors.initials: S
authors.lastname: Namba
mesh_terms: D000129:Acholeplasmataceae / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D007303:Insect Vectors / Q000382:microbiology; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics* / Q000378:metabolism*; D010935:Plant Diseases / Q000382:microbiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase