Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome
Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome
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DOI：
10.1099/mic.0.25834-0
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发表时间：
2003-08-01
期刊：
影响因子：
2.8
通讯作者：
Namba, S
中科院分区：
文献类型：
--
作者：
Miyata, S;Oshima, K;Namba, S
胸苷酸激酶（TMK）在原核生物和真核生物的dTTP合成的从头和补救途径中催化dTMP磷酸化以形成dTDP。洋葱黄化植原体是一种寄生于植物韧皮部和昆虫器官的植物病原体，在洋葱黄化植原体的基因组文库中发现了细菌胸苷酸激酶基因的两个同源物。Southern杂交分析表明，OY基因组中含有一个拷贝的tmk-b基因和多个拷贝的tmk-a基因。通过扩增tmk-a产生的PCR产物的测序使得能够鉴定tmk-a的另外三个拷贝，尽管这些拷贝中的ORF均被点突变中断。由两个完整基因编码的蛋白质TMK-a和TMK-b含有催化活性的保守基序。这两种蛋白在大肠杆菌中过表达为具有多组氨酸标签的融合蛋白并纯化，并且TMK-b显示具有胸苷酸激酶活性。这被认为是植原体蛋白的催化活性的第一个报告，并且OY植原体是第一个被发现在其基因组中具有两个完整同源物的细菌物种。
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.