Systematic analyses of biological functions of helicase-like proteins in eukaryote

本研究旨在鉴定真核生物中解旋酶样蛋白的所有成员并阐明其生物学功能。我着重于下面所述的两个科学主题。生物信息学新发现解旋酶样蛋白的功能研究。通过表征RNAi突变体研究线虫解旋酶样蛋白的功能。在整个研究过程中，我新获得了以下几点观察结果。在出芽酵母中发现的20个新的orf中，有6个被确定为基因破坏所必需的。通过对43个解旋酶样orf的Northern分析，确定了几个在特定条件下高表达的基因。研究还表明，超过70%的解旋酶样基因的转录本水平在热休克后显著增加。Ydl084wp是新发现的rna依赖性atpase。利用10种新的非必需蛋白，用酵母双杂交法检测了基因产物Yhl218wp和YklO 17cp与几种细胞蛋白的相互作用。一些遗传学证据表明，Ynl218wp可能在DNA重组中起作用。已经明确了三个新的基因产物（即Ydr332wp， Ygl064cp和Yol095cp）在线粒体功能中起重要作用。线虫解旋酶样基因RNAi突变体的系统表征揭示了该基因在真核生物中的进化保守性及其功能。由于超过40%的RNAi突变体在细胞中表现出各种类型的异常。这表明解旋酶样蛋白在高等真核生物中的生物学重要性。

This research has aimed to identify all members of helicase-like proteins in eukaryote and clarify their biological functions. I have focused on two scientific subjects described below.1. Functional study of novel helicase-like proteins that were found by bioinformatics.2. Functional study of nematode helicase-like proteins by characterizing RNAi mutants.I have newly obtained the following observations throughout this research.1. Six out of 20 novel ORFs that were found in budding yeast, were identified to be essential by gene disruptions.2. Several genes that were highly expressed under specific conditions were identified by Northern analyses of 43 helicase-like ORFs. It also clarified that the levels of transcripts from more than 70% of examined helicase-like genes significantly increased upon heat-shock.3. Ydl084wp has been newly found to be RNA-dependent ATPase.4. Interactions of gene products such as Yhl218wp and YklO 17cp with several cellular proteins were detected by yeast two-hybrid assays using 10 novel nonessenfial proteins. Some genetic evidences suggested that Ynl218wp might act in DNA recombination.5. It has been clarified that three novel gene products (i.e., Ydr332wp, Ygl064cp, and Yol095cp) play important roles in mitochondria functions.6. Systematic characterization of nematode RNAi mutants of helicase-like genes revealed the evolutional conservation of the genes and their functions in eukaryote. Since more than 40% of examined RNAi mutants exhibited various kinds of abnormalities in cell. proliferation and development, suggesting biological importance of helicase-like proteins in higher eukaryote.