Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii.
Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii.
复制标题
DOI：
10.1371/journal.ppat.0040036
复制
发表时间：
2008-02-08
期刊：
影响因子：
6.7
通讯作者：
White MW
中科院分区：
文献类型：
--
作者：
Gubbels MJ;Lehmann M;Muthalagi M;Jerome ME;Brooks CF;Szatanek T;Flynn J;Parrot B;Radke J;Striepen B;White MW
顶复体是专性细胞内病原体，其已经微调其增殖策略以匹配多种宿主细胞。这种适应的一个关键方面是一个灵活的细胞周期，在机械水平上仍然知之甚少。在这里，我们描述了一个正向遗传解剖的apicomplexan细胞周期使用弓形虫模型。通过高通量筛选，我们分离到165个温度敏感的寄生虫生长突变体。这些突变体的表型分析表明，通过寄生虫细胞周期与确定的阶段和检查点的调节进展。这些分析还强调了特殊的核内纺锤体作为细胞周期调节的物理枢纽的至关重要性。为了将这些表型与寄生虫基因联系起来，我们开发了一种基于基因组粘粒文库的强大互补系统。使用这种方法，到目前为止，我们已经补充了22个温度敏感的突变体，并确定了18个候选位点，其中8个是独立确认使用一组测序和排列cosmetics。对于其中三个基因座，我们已经鉴定出突变等位基因。鉴定的基因包括纺锤体形成、核运输和蛋白质降解的调节因子。这里描述的遗传学方法应该广泛适用于寄生虫生物学的许多重要方面。顶复门的寄生虫引起许多重要的疾病，包括疟疾、弓形虫病和隐孢子虫病。通过转染修饰这些寄生虫基因组的能力是在分子水平上解开寄生虫病生物学的技术关键。在这项研究中，我们进一步扩展了实验的可能性，通过适应一个经典的正向遗传学方法弓形虫apicomplexans的研究。我们已经开发了产生大量突变寄生虫的方案和试剂，筛选出特别感兴趣的突变体子集，并开发了识别导致表型的突变基因的工具。使用这种新方法，我们从遗传学上剖析了寄生虫在其宿主细胞内分裂和繁殖的方式。这项工作已经产生了一系列高度信息突变体沿着顶复门细胞周期的进展和20多个参与协调寄生虫细胞分裂的基因。重要的是，这种方法应该允许无偏见的遗传分析的任何部分的寄生虫生物学的屏幕可以设计使用弓形虫模型。
Apicomplexa are obligate intracellular pathogens that have fine-tuned their proliferative strategies to match a large variety of host cells. A critical aspect of this adaptation is a flexible cell cycle that remains poorly understood at the mechanistic level. Here we describe a forward genetic dissection of the apicomplexan cell cycle using the Toxoplasma model. By high-throughput screening, we have isolated 165 temperature sensitive parasite growth mutants. Phenotypic analysis of these mutants suggests regulated progression through the parasite cell cycle with defined phases and checkpoints. These analyses also highlight the critical importance of the peculiar intranuclear spindle as the physical hub of cell cycle regulation. To link these phenotypes to parasite genes, we have developed a robust complementation system based on a genomic cosmid library. Using this approach, we have so far complemented 22 temperature sensitive mutants and identified 18 candidate loci, eight of which were independently confirmed using a set of sequenced and arrayed cosmids. For three of these loci we have identified the mutant allele. The genes identified include regulators of spindle formation, nuclear trafficking, and protein degradation. The genetic approach described here should be widely applicable to numerous essential aspects of parasite biology. Parasites of the phylum Apicomplexa cause numerous important diseases, including malaria, toxoplasmosis, and cryptosporidiosis. The ability to modify the genome of these parasites by transfection has been the technological key to unlock the biology of parasitic diseases at a molecular level. In this study we further extend the experimental possibilities for the study of apicomplexans by adapting a classic forward genetic approach for Toxoplasma gondii. We have developed protocols and reagents to generate large numbers of mutant parasites, screens to hone in on a subset of mutants of particular interest, and tools to identify the mutated genes that are responsible for the phenotype. Using this new approach, we have genetically dissected the way the parasite divides and multiplies within its host cell. This effort has yielded a series of highly informative mutants along the progression of the apicomplexan cell cycle and more than 20 genes involved in orchestrating parasite cell division. Importantly, this approach should allow unbiased genetic analysis of any part of parasite biology for which a screen can be devised using the Toxoplasma model.
登录
查看更多内容
影响因子：
1.5
作者：
Chen, Yueqin;Jirage, Dayadevi;Waters, Norman C.
通讯作者：
Waters, Norman C.
影响因子：
1.5
作者：
Gubbels, MJ;Wieffer, M;Striepen, B
通讯作者：
Striepen, B
DOI：
10.1073/pnas.0701893104
发表时间：
2007-06-12
影响因子：
11.1
作者：
Frankel, Matthew B.;Mordue, Dana G.;Knoll, Laura J.
通讯作者：
Knoll, Laura J.
影响因子：
56.9
作者：
HARTWELL, LH;CULOTTI, J;REID, BJ
通讯作者：
REID, BJ
影响因子：
1.5
作者：
de Felipe, Magnolia M. Conde;Lehmann, Margaret M.;White, Michael W.
通讯作者：
White, Michael W.