Transfection with thymidine kinase permits bromodeoxyuridine labelling of DNA replication in the human malaria parasite Plasmodium falciparum.
Transfection with thymidine kinase permits bromodeoxyuridine labelling of DNA replication in the human malaria parasite Plasmodium falciparum.
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DOI：
10.1186/s12936-015-1014-7
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发表时间：
2015-12-02
期刊：
影响因子：
3
通讯作者：
Merrick CJ
中科院分区：
文献类型：
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作者：
Merrick CJ
恶性疟原虫是人类严重疟疾的病原体，是一种早期分化的原生动物，其生命周期具有许多不寻常的特征，包括其复制模式。疟原虫细胞周期的研究主要通过单细胞生殖而不是典型的二分裂发生，由于缺乏可以从模式生物细胞周期研究中转移的工具和标记而受到阻碍。用于研究人类细胞中DNA复制和细胞周期的常用工具是用修饰的核苷酸溴脱氧尿苷（BrdU）标记新复制的DNA，然后进行免疫荧光检测。然而，疟原虫寄生虫不掺入BrdU，因为它们仅依赖于嘧啶的从头合成，并且不抢救胸苷类似物如BrdU以转化为核苷酸。疟原虫中的生化途径的分析表明，酶胸苷激酶（TK）的情况下，可能是唯一的障碍，BrdU在这种生物体的掺入。因此，从单纯疱疹病毒的TK基因，引入恶性疟原虫3D7株和BrdU标记的效果进行了评估，通过酶联免疫吸附试验和免疫荧光显微镜。引入TK基因产生的寄生虫确实可以合并BrdU。这形成了DNA复制的敏感指标，可以通过群体水平或单细胞水平上的定量和定性测定来检测。恶性疟原虫在表达TK时，对BrdU毒性变得异常敏感。BrdU标记是一个重要的新工具，调查DNA复制和疟原虫的细胞周期。本文的在线版本（doi：10.1186/s12936 - 015 - 1014 - 7）包含补充材料，可供授权用户使用。
Plasmodium falciparum, the causative agent of severe human malaria, is an early-diverging protozoan whose lifecycle has many unusual features, including its modes of replication. Research on the Plasmodium cell cycle, which occurs primarily via schizogony instead of canonical binary fission, has been hampered by a lack of tools and markers that can be transferred from cell cycle studies in model organisms. A common tool used to study DNA replication and the cell cycle in human cells is the labelling of newly-replicated DNA with the modified nucleotide bromodeoxyuridine (BrdU), followed by immunofluorescent detection. Plasmodium parasites, however, do not incorporate BrdU because they rely only on de novo synthesis of pyrimidines and do not salvage thymidine analogues like BrdU for conversion into nucleotides. Analysis of biochemical pathways in Plasmodium indicated that the absence of the enzyme thymidine kinase (TK) may be the only impediment to BrdU incorporation in this organism. A TK gene from Herpes simplex was, therefore, introduced into the Plasmodium falciparum 3D7 strain and the effect on BrdU labelling was assessed by enzyme-linked immunosorbent assay and immunofluorescence microscopy. Introduction of a TK gene produces parasites that can indeed incorporate BrdU. This forms a sensitive indicator of DNA replication, which can be detected by both quantitative and qualitative assays on either a population level or a single-cell level. Plasmodium falciparum, when expressing TK, becomes unusually sensitive to BrdU toxicity. BrdU labelling represents a significant new tool for investigating DNA replication and the cell cycle in Plasmodium. The online version of this article (doi:10.1186/s12936-015-1014-7) contains supplementary material, which is available to authorized users.