Generation of rodent malaria parasites with a high mutation rate by destructing proofreading activity of DNA polymerase δ.
Generation of rodent malaria parasites with a high mutation rate by destructing proofreading activity of DNA polymerase δ.
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DOI：
10.1093/dnares/dsu009
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发表时间：
2014-08
期刊：
影响因子：
--
通讯作者：
Tanabe K
中科院分区：
文献类型：
--
作者：
Honma H;Hirai M;Nakamura S;Hakimi H;Kawazu S;Palacpac NM;Hisaeda H;Matsuoka H;Kawai S;Endo H;Yasunaga T;Ohashi J;Mita T;Horii T;Furusawa M;Tanabe K
恶性疟原虫疟疾在整个热带地区造成严重的公共卫生问题。虽然遗传工具对于充分调查疟疾寄生虫极为重要，但目前可用的正向和反向工具相当有限。预计具有高突变率的寄生虫很容易获得新的表型/性状；然而，它们仍然是疟疾生物学中尚未开发的工具。在这里，我们使用定点诱变和基因转染技术产生了一个突变疟原虫（以下称为“疟疾突变体”）。DNA聚合酶δ (PbMut) 3′→5′外切酶活性有缺陷的突变株和具有野生型DNA聚合酶δ (PbCtl)的对照株通过每周传代在小鼠体内维持了122周。高通量基因组测序分析显示，两个PbMut系有175-178个突变，突变率比PbCtl系高86- 90倍。PbMut、PbCtl及其亲本菌株PbWT表现出相似的感染过程。有趣的是，PbMut在连续传代过程中失去了形成配子体的能力。我们相信疟疾突变体系统可以为研究疟疾生物学提供一种新颖而有用的工具。
Plasmodium falciparum malaria imposes a serious public health concern throughout the tropics. Although genetic tools are principally important to fully investigate malaria parasites, currently available forward and reverse tools are fairly limited. It is expected that parasites with a high mutation rate can readily acquire novel phenotypes/traits; however, they remain an untapped tool for malaria biology. Here, we generated a mutator malaria parasite (hereinafter called a ‘malaria mutator’), using site-directed mutagenesis and gene transfection techniques. A mutator Plasmodium berghei line with a defective proofreading 3′ → 5′ exonuclease activity in DNA polymerase δ (referred to as PbMut) and a control P. berghei line with wild-type DNA polymerase δ (referred to as PbCtl) were maintained by weekly passage in ddY mice for 122 weeks. High-throughput genome sequencing analysis revealed that two PbMut lines had 175–178 mutations and a 86- to 90-fold higher mutation rate than that of a PbCtl line. PbMut, PbCtl, and their parent strain, PbWT, showed similar course of infection. Interestingly, PbMut lost the ability to form gametocytes during serial passages. We believe that the malaria mutator system could provide a novel and useful tool to investigate malaria biology.
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