Fluorescent protein tagging in Toxoplasma gondii:: identification of a novel inner membrane complex component conserved among Apicomplexa
Fluorescent protein tagging in Toxoplasma gondii:: identification of a novel inner membrane complex component conserved among Apicomplexa
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DOI：
10.1016/j.molbiopara.2004.05.007
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发表时间：
2004-09-01
影响因子：
1.5
通讯作者：
Striepen, B
中科院分区：
文献类型：
--
作者：
Gubbels, MJ;Wieffer, M;Striepen, B
弓形虫是一种专性细胞内寄生虫，其亚细胞组织显示出明显的适应这种生活方式。除了所有真核生物共有的细胞器外，生物体还具有许多在宿主细胞入侵和细胞内存活中发挥重要作用的专门隔室。寄生虫生物学的这些独特方面也反映在其基因组中。目前正在进行的弓形虫和相关顶复门的基因组测序工作预测，该门特有的基因比例很高，在其他模式生物中缺乏同源物。了解这些基因产物的亚细胞定位将是对其功能表征的重要的第一步。我们使用了一种文库方法，其中寄生虫基因组DNA融合到黄色荧光蛋白（YFP）基因。通过流式细胞术和荧光显微镜筛选用该文库转化的寄生虫。分离在多种亚细胞区室（细胞核、线粒体、ER、致密颗粒（分泌的）、剪接体、质膜、顶质体、内膜复合体）中标记的克隆，并使用区室特异性标记物进行确认。使用体外重组系统对具有寄生虫特异性定位的标签的克隆进行插入拯救和表型验证。在所鉴定的基因中，有一个新的内膜复合体基因（IMC 3）在顶复门中是保守的。(C)2004 Elsevier B.V.保留所有权利。
Toxoplasma gondii is an obligate intracellular parasite, and its sub-cellular organization shows clear adaptations to this life-style. In addition to organelles shared among all eukaryotes, the organism possesses a number of specialized compartments with important roles in host cell invasion and intra-cellular survival. These unique aspects of the parasite's biology are also reflected in its genome. The ongoing genome sequencing efforts for T gondii and related apicomplexans predict a high proportion of genes unique to the phylum, which lack homologs in other model organisms. Knowing the sub-cellular localization of these gene products will be an important first step towards their functional characterization. We used a library approach wherein parasite genomic DNA was fused to the yellow fluorescent protein (YFP) gene. Parasites transformed with this library were screened by flow cytometry and fluorescence microscopy. Clones tagged in a wide variety of sub-cellular compartments (nucleus, mitochondria, ER, dense granules (secreted), spliceosome, plasma membrane, apicoplast, inner membrane complex) were isolated and confirmed using compartment specific markers. Clones with tags in parasite-specific localizations were subjected to insert rescue and phenotypic verification using an in vitro recombination system. Among the genes identified is a novel inner membrane complex gene (IMC3) conserved among Apicomplexa. (C) 2004 Elsevier B.V. All rights reserved.