Understanding Complex Gene Editing Systems in Protists

描述(申请人提供)：这项研究的目标是研究原生动物中不寻常的生物信息处理方式，重点是在纤毛虫中的基因解读。这项拟议的研究结合了解决这个问题的不同方法来解决问题，并在基因组、进化和发育水平以及时间尺度上检验假设。全球目标是使用这个模型系统来探索微生物真核基因组中复杂重写系统的潜在机制。具体目标包括：a)探索我们在基因组规模上研究的模式系统Oxytricha trifalax中杂乱基因的全球分布，以及早期分化的螺旋藻物种中杂乱同源基因的存在，以便在进化时间尺度上进行比较。B)在发育时间尺度上探索基因解读过程中的中间体种群。C)将外源DNA或RNA引入发育中的大核的功能实验。D)测试小RNA在棘毛虫大核发育中的作用。E)鉴定和测试与棘毛虫大核发育有关的候选基因。对分子进化和直接实验数据的计算分析--例如扰乱基因重排的时间进程--将提供一个窗口，了解此类生物计算的复杂性和规则，例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读的深入理解提供了一个独特的机会--这一现象仍然让研究界感到困惑。

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.