Validation of a modified method for Bxb1 mycobacteriophage integrase-mediated recombination in Plasmodium falciparum by localization of the H-protein of the glycine cleavage complex to the mitochondrion.
Validation of a modified method for Bxb1 mycobacteriophage integrase-mediated recombination in Plasmodium falciparum by localization of the H-protein of the glycine cleavage complex to the mitochondrion.
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DOI：
10.1016/j.molbiopara.2010.04.005
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发表时间：
2010-08
影响因子：
1.5
通讯作者：
Prigge, Sean T.
中科院分区：
文献类型：
--
作者：
Spalding, Maroya D.;Allary, Marina;Gallagher, John R.;Prigge, Sean T.
甘氨酸裂解复合物（GCV）是恶性疟原虫中单碳供体5，10-亚甲基-四氢叶酸（5，10-CH 2-THF）的潜在来源。单碳（C1）供体单位是氨基酸和核苷酸生物合成以及线粒体和质体翻译起始所必需的。在其他生物体中，GCV活性与丝氨酸羟甲基转移酶（SHMT）的活性密切协调。恶性疟原虫含有胞质和线粒体SHMT亚型，因此，GCV的亚细胞位置是其在疟疾代谢中作用的重要指标。为了确定GCV的亚细胞定位，我们使用了已发表的用于在恶性疟原虫中进行分枝杆菌噬菌体整合酶介导的重组的方法的修改版本，以产生含有GCV的组分蛋白之一的H蛋白与GFP融合的细胞系。在这里，我们表明，这种修改的结果在染色体整合的转基因寄生虫的快速生成，我们表明，H-蛋白定位到peption。Bxb 1整合酶系统被用来定位恶性疟原虫H-蛋白的p53。对转染方法的修改导致选择时间减少。
The glycine cleavage complex (GCV) is a potential source of the one carbon donor 5,10-methylene-tetrahydrofolate (5,10-CH2-THF) in the malaria parasite Plasmodium falciparum. One carbon (C1) donor units are necessary for amino acid and nucleotide biosynthesis, and for the initiation of mitochondrial and plastid translation. In other organisms, GCV activity is closely coordinated with the activity of serine hydroxymethyltransferase (SHMT) enzymes. P. falciparum contains cytosolic and mitochondrial SHMT isoforms, and thus, the subcellular location of the GCV is an important indicator of its role in malaria metabolism. To determine the subcellular localization of the GCV, we used a modified version of the published method for mycobacteriophage integrase-mediated recombination in P. falciparum to generate cell lines containing one of the component proteins of the GCV, the H-protein, fused to GFP. Here, we demonstrate that this modification results in rapid generation of chromosomally integrated transgenic parasites, and we show that the H-protein localizes to the mitochondrion. The Bxb1 integrase system was used to localize the Plasmodium falciparum H-protein to the mitochondrion. Modifications to the transfection method resulted in decreased selection times.
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