Genetic complementation in apicomplexan parasites
Genetic complementation in apicomplexan parasites
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DOI：
10.1073/pnas.092525699
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发表时间：
2002-04-30
影响因子：
11.1
通讯作者：
Abrahamsen, MS
中科院分区：
文献类型：
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作者：
Striepen, B;White, MW;Abrahamsen, MS
一个强大的正向遗传模型顶复门可以大大提高这些重要的原生动物病原体的基因功能分析。我们已经开发并成功地测试了基因互补策略的基础上基因组插入在弓形虫。将重组克隆技术应用于基因组DNA，我们发现互补序列可以在寄生虫基因组和细菌质粒之间穿梭，为候选基因的回收和功能评估提供了一种有效的方法。我们展示了一种缺乏次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖基转移酶和T。弓形虫cDNA文库我们还探讨了这种方法的实用性克隆基因的基础上，从其他顶复门寄生虫弓形虫作为替代品的功能。一个异源文库含有隐孢子虫基因组DNA的产生，我们确定了一个C parvum基因编码肌苷5-单磷酸脱氢酶（IMPDH）。有趣的是，系统发育分析表明，该基因明确的真细菌起源，并强烈建议其从ε-变形菌的横向转移。这种酶的原核起源可能使其成为针对隐孢子虫的有希望的治疗靶点。
A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient too] for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from epsilon-proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.