Understanding Complex Gene Editing Systems in Protists

原生生物拥有一系列前所未有的遗传组织，令分子生物学家感到惊讶， 自剪接RNA和端粒酶的发现，到基因大小的片段，程序性DNA缺失， 纤毛虫的DNA重排。因此，这些生物体是自然的地方， 结合机械，进化和计算程序DNA重排的研究。在 特别是纤毛虫中的“乱序基因”，为研究其起源提供了一个独特的实验系统。 以及复杂的遗传和计算机制的功能。混乱的基因存在于许多其他 从原生生物到人类。然而，基因片段很少真的被“缝”回去 在DNA水平上结合在一起，从所有片段中创建一个连续的基因。DNA剪接的复杂性 在纤毛虫中发生的事件使它们成为最有趣和最具挑战性的研究案例之一。 此外，频繁的DNA缺失和其他广泛的基因组重排事件越来越多地被发现。 与几种癌症有关，强调了研究这些生物事件的重要性。 最近发现，通常被认为是基因表达管道的RNA， 在纤毛原生动物中的作用激发了这项建议的研究。目标是研究基因组 纤毛类Oxytricha的基因重排，将一个接近完成的基因组计划与最近的发现相结合， 和工具使尖毛藻成为一个强大的模型系统。例如，RNA模板可以提供DNA和DNA的双重功能。 DNA重排的组织指南和可以传递自发体细胞突变的模板 传给下一代这种RNA引导的DNA修复的机会在尖毛藻中意义深远，因为 有机体通过全球重排破坏了95%的生殖系DNA，然后对它们进行分类和重新排序。 剩余部分。本研究的具体目标都集中在有关加工的问题， 在基因组、进化或发育的尺度上扰乱基因。全球的目标是利用这种模式 系统来探索微生物真核基因组中复杂的重写机制。 具体目标包括： 1. RNA模板要能够编程DNA重排的最低要求是什么？ 2.了解RNA模板核苷酸取代的表观遗传。 3.尖毛虫属及其近缘纤毛虫柱甲属的小分子RNA及其序列分布。 4.尖毛藻大核发育相关候选基因的鉴定和检测。 5.随机基因在O. trifallax和数据库的重大更新。 6.高度杂乱生殖系核糖体RNA基因座的比较研究。

Protists have surprised molecular biologists with an unprecedented array of genetic organization, from the discoveries of self-splicing RNA and telomerase, to gene-sized pieces, programmed DNA deletion, and scrambled DNA rearrangements in ciliates. Therefore these organisms are the natural place to focus a combined mechanistic, evolutionary, and computational study of programmed DNA rearrangements. In particular, the "scrambled genes" in ciliates offer a unique experimental system in which to study the origin and function of a complex genetic and computational mechanism. Scrambled genes exist in many other systems, ranging from protists to humans. Rarely, however, are the gene fragments actually 'sewn' back together at the level of DNA, to create a contiguous gene from all pieces. The complexity of DNA splicing events that take place in ciliates makes them one of the most interesting and challenging cases to study. Moreover, frequent DNA deletions and other extensive genome rearrangement events are increasingly associated with several cancers, underscoring the importance of studying such biological events. The recent discovery that RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa motivates the research in this proposal. The goal is to study genome rearrangements in the ciliate Oxytricha, pairing a nearly-complete genome project with recent discoveries and tools that make Oxytricha a powerful model system. For example, RNA templates can provide both an organizing guide for DNA rearrangements and a template that can transmit spontaneous somatic mutations to the next generation. This opportunity for RNA-guided DNA repair is profound in Oxytricha, because this organism destroys 95% of its germline DNA through global rearrangements, and then sorts and reorders the remaining segments. The specific aims of this research all focus on questions about the processing of scrambled genes at the genomic, evolutionary, or developmental scale. The global aim is to use this model system to explore the complex rewriting mechanisms in microbial eukaryotic genomes. Specific aims include: 1. What are the minimal requirements for an RNA template to be able to program DNA rearrangement? 2. Understanding the epigenetic inheritance of nucleotide substitutions from an RNA template. 3. Small RNAs and their sequence distribution in Oxytricha and the related ciliate Stylonychia. 4. Identification and testing of candidate genes involved in macronuclear development in Oxytricha. 5. The genomic distribution of scrambled genes in O. trifallax, and significant updates to a database. 6. Comparative studies of the highly scrambled germline ribosomal RNA locus.