Understanding Complex Gene Editing Systems in Protists

描述（由申请人提供）：本研究的目的是研究原生生物中不寻常的生物信息处理形式，重点研究刺毛纤毛虫的基因解码。提出的研究结合了不同的方法来解决这个问题，并在基因组，进化和发育水平和时间尺度上测试假设。总体目标是使用该模型系统来探索微生物真核生物基因组中复杂重写系统的机制。具体目标包括：a)探索三叶草中混乱基因的全球分布，我们在基因组尺度上研究的模型系统，以及在进化时间尺度上比较早期分化的螺旋体物种中混乱同源物的存在。b)在发育时间尺度上探索基因解码过程中的中间群体。c)将外源DNA或RNA引入发育中的巨核的功能实验。d)测试小rna在Oxytricha大核发育中的作用。e)木犀草大核发育相关候选基因的鉴定和检测。分子进化和直接实验数据的计算分析，如基因重组的时间进程，将为了解这种生物计算的复杂性和规则提供一个窗口，如正确基因处理的序列和分子要求。分子生物学、功能基因组学、计算生物学和进化实验的结合为建立对纤毛虫基因解码的深刻理解提供了一个独特的机会——这一现象至今仍令研究界感到困惑。

DESCRIPTION (provided by applicant): The goal of this research is to study unusual forms of biological information processing in protists, with emphasis on gene unscrambling in stichotrichous ciliates. The proposed research combines different approaches to this problem to address questions and test the hypotheses at the genomic, evolutionary, and developmental levels and time scales. The global aim is to use this model system to explore the mechanisms underlying complex rewriting systems in microbial eukaryotic genomes. Specific aims include: a) Probing the global distrbution of scrambled genes in Oxytricha trifallax, our model system for studies on a genomic scale, and the presence of scrambled orthologs in earlier diverging spirotrichous species for comparisons on an evolutionary time scale. b) Probing the populations of intermediates during the process of gene unscrambling on a developmental time scale. c) Functional experiments introducing foreign DNA or RNA into the developing macronucleus. d) Testing the role of small RNAs in macronuclear development in Oxytricha. e) Identification and testing of candidate genes involved in macronuclear development in Oxytricha. Computational analysis of both molecular evolutionary and direct experiment data-such as the time course of scrambled gene rearrangements-will provide a window into the complexity and rules of such biological computations, such as the sequence and molecular requirements for correct gene processing. The combination of molecular biology, functional genomics, computational biology, and evolutionary experiments in this proposal offers a unique opportunity to build a strong understanding of gene unscrambling in ciliates-a phenomenon that still mystifies the research community.