INITIAL CONDITIONS FOR PARASITISM IN TRYPANOSOMES: APOSSIBLE ROLE OF ANAEROBIC PYRIMIDINE BIOSYNTHESIS

在这个项目中，我们的目的是验证锥虫体内从头合成嘧啶的生物合成途径的进化意义，该途径包括六个顺序的酶反应。该途径的第四和第六种酶的基因，分别是二氢罗酸脱氢酶(DHOD)和鸟苷-5‘-单磷酸脱羧酶(OMPDC)，先前已被证明起源于侧向基因转移(LGT)，这突显了该途径在锥虫中的独特性质。我们证明了查加斯病的病原体克氏锥虫的遗传多样性和DHOD的动力学特性。克氏锥虫吐拉湖株的3个DHOD基因全长942个碱基，有26个核苷酸的差异，导致8个氨基酸残基的替换。重组DHOD具有相似的酶学性质，包括底物和电子受体的最适pH、最适温度、V&lt；max&gt；和Km。这些结果表明，尽管存在遗传变异，但这三种…的动力学性质OMPDC基因作为OMPDC-OPRT融合基因，定位于锥虫的糖体中，而在动植物中则以OPRT-OMPDC融合的形式存在。基因克隆和系统发育分析表明，OMPDC-OPRT融合是通过OPRT-OMPDC的裂解和再融合，出现在由锥虫和类锥虫组成的动质体的共同祖先中。OMPDC-OPRT融合在另一个真核生物群中也被发现。系统发育重建从统计学上拒绝了稻草型和动质型OMPDC-OPRT的OPRT结构域的单一性，表明尽管这些基因具有相同的基因序列，但它们并不具有共同的进化起源。因此，OMPDC-OPRT融合很可能是在这些真核生物中独立出现的。我们的结论是，基因融合事件发生的频率比以前认为的更高，LGT对真核细胞中OPRT和OMPDC基因重排结构的建立做出了显着贡献。较少

In this project, we aimed to verify evolutionary implications of de novo pyrimidine biosynthetic pathway, which includes six sequential enzymatic reactions, in trypanosomes. Genes for the fourth and sixth enzymes of the pathway, dihydroorotate dehydrogenase (DHOD) and orotidine-5'-monophosphate decarboxylase (OMPDC), respectively, have been previously shown to have a lateral-gene-transfer (LGT) origin, highlighting the distinct nature of the pathway in trypanosomes. We demonstrated that genetic diversity and the kinetic properties of DHOD in Trypanosoma cruzi, the agent of Chagas' disease. Three DHOD genes in T. cruzi Tulahuen strain were comprised of 942 bp and differed by 26 nucleotides, resulting in replacement of 8 amino acid residues. Recombinant DHODs showed similar enzymatic properties, including optimal pH, optimal temperature, V_<max>, and K_m for the substrate and electron acceptor. These results suggest that, despite their genetic variations, kinetic properties of the three … More T. cruzi DHODs are conserved.OMPDC gene is fused with the fifth enzyme, orotate phosphoribosyltransferase (OPRT) as OMPDC-OPRT fusion and localized in glycosomes in trypanosomes, while they occur as OPRT-OMPDC fusion in animals and plants. Gene cloning and phylogenetic analyses suggested that OMPDC-OPRT fusion emerged in a common ancestor of kinetoplastids that comprises trypanosomatids and bodonids, via split of OPRT-OMPDC and re-fusion. OMPDC-OPRT fusion was also found in another eukaryotic group, stramenopiles. Phylogenetic reconstruction rejected statistically the monophyly of the OPRT domains of stramenopile and kinetoplastid OMPDC-OPRT, demonstrating that these gene fusions do not share a common evolutionary origin, despite the identical gene order. Thus, the OMPDC-OPRT fusion is likely to have emerged independently in these eukaryotic groups. We conclude that gene fusion events occur more frequently than previously thought and that LGT has made a marked contribution to establishment of the rearranged structure of OPRT and OMPDC genes in eukaryotes. Less