Kooij Taco W A, Franke-Fayard Blandine, Renz Jasper, Kroeze Hans, van Dooren Maaike W, Ramesar Jai, Augustijn Kevin D, Janse Chris J, Waters Andrew P
Department of Parasitology, Malaria Group, Leiden University Medical Centre, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
Mol Biochem Parasitol. 2005 Nov;144(1):16-26. doi: 10.1016/j.molbiopara.2005.07.003.
Plasmodium falciparum contains two genes encoding different isotypes of alpha-tubulin, alpha-tubulin I and alpha-tubulin II. alpha-Tubulin II is highly expressed in male gametocytes and forms part of the microtubules of the axoneme of male gametes. Here we present the characterization of Plasmodium berghei alpha-tubulin I and alpha-tubulin II that encode proteins of 453 and 450 amino acids, respectively. alpha-Tubulin II lacks the well-conserved three amino acid C-terminal extension including a terminal tyrosine residue present in alpha-tubulin I. Investigation of transcription by Northern analysis and RT-PCR and analysis of promoter activity by GFP tagging showed that alpha-tubulin I is expressed in all blood and mosquito stages. As expected, alpha-tubulin II was highly expressed in the male gametocytes, but transcription was also observed in the asexual blood stages, female gametocytes, ookinetes and oocysts. Gene disruption experiments using standard transfection technologies did not produce viable parasites indicating that both alpha-tubulin isotypes are essential for the asexual blood stages. Targeted modification of alpha-tubulin II by the addition of the three C-terminal amino acids of alpha-tubulin I did not affect either blood stage development nor male gamete formation. Attempts to modify the C-terminal region by adding a TAP tag to the endogenous alpha-tubulin II gene were not successful. Introduction of a transgene, expressing TAP-tagged alpha-tubulin II, next to the endogenous alpha-tubulin II gene, had no effect on the asexual blood stages but strongly impaired formation of male gametes. These results show that alpha-tubulin II not only plays an important role in the male gamete but is also expressed in and essential for asexual blood stage development.
恶性疟原虫含有两个编码不同α-微管蛋白同种型的基因，即α-微管蛋白I和α-微管蛋白II。α-微管蛋白II在雄配子体中高度表达，并构成雄配子轴丝微管的一部分。在此，我们展示了伯氏疟原虫α-微管蛋白I和α-微管蛋白II的特征，它们分别编码453和450个氨基酸的蛋白质。α-微管蛋白II缺乏α-微管蛋白I中存在的保守的三个氨基酸C末端延伸，包括一个末端酪氨酸残基。通过Northern分析和RT-PCR研究转录，并通过GFP标记分析启动子活性，结果表明α-微管蛋白I在所有血液和蚊子阶段均有表达。正如预期的那样，α-微管蛋白II在雄配子体中高度表达，但在无性血液阶段、雌配子体、动合子和卵囊中也观察到转录。使用标准转染技术进行的基因破坏实验未产生可存活的寄生虫，这表明两种α-微管蛋白同种型对于无性血液阶段都是必不可少的。通过添加α-微管蛋白I的三个C末端氨基酸对α-微管蛋白II进行靶向修饰，既不影响血液阶段的发育，也不影响雄配子的形成。试图通过向内源α-微管蛋白II基因添加TAP标签来修饰C末端区域未成功。在内源α-微管蛋白II基因旁边引入表达TAP标签的α-微管蛋白II的转基因，对无性血液阶段没有影响，但严重损害了雄配子的形成。这些结果表明，α-微管蛋白II不仅在雄配子中起重要作用，而且在无性血液阶段发育中也有表达且必不可少。
