An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites
An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites
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DOI：
10.1093/nar/29.3.716
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发表时间：
2001-02-01
影响因子：
14.9
通讯作者：
Crabb, BS
中科院分区：
文献类型：
--
作者：
O'Donnell, RA;Preiser, PR;Crabb, BS
人疟疾寄生虫恶性疟原虫的转染目前是用环状质粒进行的，所述环状质粒在药物选择下在寄生虫中以附加体形式维持，但当选择压力被去除时，所述环状质粒迅速丢失。在本文中，我们表明，在基因靶向不受欢迎的情况下，转染的质粒可以改变为稳定复制的形式（SRF），在没有药物选择的情况下维持游离。SRF DNA是亲本质粒的大多联体，其包含以头对尾阵列排列的至少九个质粒。我们还表明，原始的不稳定复制形式（URF）也以头-尾多联体的形式存在，但仅包含三个质粒。有限的消化和γ辐射实验显示，尽管URF多联体如预期的那样主要是环状的，但SRF多联体形成包括大量单链DNA的更复杂的结构。在SRF DNA中没有检测到序列重排或额外序列的证据，包括在设计用于选择更有效复制质粒的瞬时复制实验中。令人惊讶的是，这些实验揭示了细菌质粒单独可以在寄生虫中复制。总之，这些结果意味着转染质粒需要形成头到尾的多联体维持在寄生虫和牵连滚环和重组依赖性机制在其复制。
Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids, Limited digestion and gamma irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA, No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids, Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.