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

作为内共生真细菌的后代，氢小体--线粒体的厌氧形式--代表了真核细胞中细胞内生命的一种高度减少的形式。通过比较在壶菌、纤毛虫和滴虫中独立进化的氢小体的蛋白质组成，我们想要研究这些细胞器之间功能相似的程度，并确定它们是否都是由平行的选择性力量(适应厌氧栖息地)塑造的。我们建议从菊属真菌Neocallimastix Frontalis中提取5000个EST，作为鉴定氢酶体蛋白的基础，该蛋白通过双向电泳法分离，并通过质谱仪进行测序。结合来自滴虫的类似项目的数据和厌氧纤毛虫的EST数据，我们希望能够深入了解不同真核生物组的氢小体之间的关系。此外，我们建议探索厌氧纤毛虫中氢小体与内共生产甲烷古细菌的异常联系，以确定内共生菌对宿主细胞基因表达的影响。为此，我们将建立没有内共生体的纤毛虫培养物，并使用来自有和没有内共生菌甲烷菌的培养物的信使-RNA来检测从EST数据中选择的斜纹藻cDNA阵列上的表达水平。总而言之，这些实验应该会在理解厌氧真核细胞的细胞内生命方面取得实质性进展。

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.