Functional characterization of putative Fe-hydrogenase maturases from the hydrogenosomes of Trichomonas vaginalis

作为内共生真细菌的后代，氢酶体-线粒体的厌氧形式-代表了真核细胞中细胞内生命的高度简化形式。通过比较的蛋白质互补的氢化酶体，独立的壶菌，纤毛虫和滴虫谱系进化，我们要研究这些细胞器之间的功能相似性的程度，并确定它们是否都是由平行的选择力（适应厌氧栖息地）。我们建议产生5000 ESTs的壶菌真菌Neocallimastix frontalis的氢体蛋白的鉴定的基础上分离的二维电泳和质谱测序。再加上从一个类似的项目上的毛滴虫和EST数据从厌氧纤毛虫Plagiopyla frontata的数据，我们希望获得洞察氢酶体从不同的真核生物群体之间的关系。此外，我们建议探索不寻常的协会厌氧纤毛虫与内共生产甲烷古细菌的氢化酶体，以确定在宿主细胞中的基因表达的内共生体的影响。为此目的，我们将建立纤毛虫文化没有内共生体和信使RNA的文化与和没有内共生产甲烷菌测定表达水平的阵列上的斜角虫cDNA从EST数据选择。总之，这些实验应该提供了一个实质性的进步，在厌氧真核细胞的细胞内生命的理解。

As descendants of endosymbiotic eubacteria hydrogenosomes - anaerobic forms of mitochondria - represent a highly reduced form of intracellular life in eukaryotic cells. By comparing the protein complement of hydrogenosomes that evolved independently in chytrid fungi, ciliate, and trichomonad lineages, we want to study the extent of functional similarity between these organelles and determine whether they were all shaped by parallel selective forces (adaptation to anaerobic habitat). We propose to generate 5000 ESTs from the chytrid fungus Neocallimastix frontalis as the basis for the identification of hydrogenosomal proteins isolated by 2D-electrophoresis and sequenced by mass spectrometry. Together with data from a similar project on trichomonads and EST data from the anaerobic ciliate Plagiopyla frontata we expect to obtain insight into the relationships between hydrogenosomes from different eukaryotic groups. In addition, we propose to explore the unusual association of hydrogenosomes in anaerobic ciliates with endosymbiotic methanogenic archaebacteria to determine the influence of the endosymbionts on gene expression in the host cells. For that purpose we will establish ciliate cultures without endosymbionts and use messenger-RNA from cultures with and without endosymbiotic methanogens to assay expression levels on arrays of Plagiopyla cDNAs selected from the EST data. Together, these experiments should provide a substantial advance in the understanding of intracellular life in anaerobic eukaryotic cells.