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

本研究旨在鉴定真核生物中所有解旋酶样蛋白成员，并阐明其生物学功能。我集中讨论了下面描述的两个科学主题。生物信息学发现的新型解旋酶样蛋白的功能研究.通过RNAi突变体对线虫类解旋酶蛋白的功能研究。在整个研究过程中，我新获得了以下观察结果。在芽殖酵母中发现的20个新的ORF中，有6个是通过基因破坏确定的。通过对43个解旋酶样ORF的北方分析，鉴定了几个在特定条件下高表达的基因。这也说明了70%以上的解旋酶样基因的转录水平在热激后显著增加。Ydl084wp是新发现的RNA依赖性ATP酶.利用酵母双杂交技术，用10个新的非必需蛋白质检测了基因产物Yhl218wp和YklO 17cp与几种细胞蛋白质的相互作用。一些遗传学证据表明Ynl218wp可能在DNA重组中起作用.已经阐明了三种新的基因产物（即，Ydr332wp、Ygl064cp和Yol095cp）在线粒体功能中起重要作用。对线虫类解旋酶基因RNAi突变体的系统研究揭示了该类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各样的异常。增殖和发育，提示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.