Understanding Complex Gene Editing Systems in Protists

了解原生生物中复杂的基因编辑系统

• 批准号: 7736832
• 负责人: LAURA F LANDWEBER
• 金额: $ 39.17万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7736832
• 关键词: Address; Architecture; Back; Bioinformatics; Biological; Biological Models; Candidate Disease Gene; Chromosomes; Comparative Study; Complementary DNA; Complex; Computational Science; DNA; DNA Repair; DNA Sequence Rearrangement; Data; Data Set; Databases; Development; Disease; Epigenetic Process; Event; Evolution; Fostering; Functional RNA; Gene Expression; Gene Rearrangement; Genes; Genetic; Genetic Recombination; Genome; Genomic Instability; Genomics; Goals; Growth; Guide RNA; Human; Inherited; Investigation; Lead; Light; Logic; Malignant Neoplasms; Maps; Molecular; Molecular Biology; Mutation; Nucleotides; Organism; Orthologous Gene; Oxytricha; Point Mutation; Process; Property; Protozoa; Public Health; RNA; RNA Interference; RNA Splicing; Research; Ribosomal DNA; Ribosomal RNA; Role; Series; Small RNA; Somatic Mutation; Sorting - Cell Movement; System; Telomerase RNA Component; Testing; Transposase; Tumor Suppressor Genes; Update; Validation; biological information processing; cancer genome; chimeric gene; computer studies; genome wide association study; genome-wide; human disease; in vivo; interest; microbial; multidisciplinary; next generation; novel; programs; public health relevance; scale up; tool; tumor

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: DNA rearrangements (deletions, inversions, and duplications) are a major factor contributing to genome instability associated with many human diseases, including cancer, with DNA translocations and gross deletions responsible for a significant portion of cancers and inherited diseases. Recombination often occurs between "hotspots", which may be short identical repeats that resemble "pointers" at DNA recombination junctions in Oxytricha, and such events can result in either deletion of tumor-suppressing genes, the formation of chimeric genes, or duplication and subsequent over-expression of genes that promote tumor stability. Because of its magnitude of DNA rearrangements and the recent demonstration that RNA templates can reprogram DNA rearrangements in vivo, Oxytricha is unparalleled as a model system to shed light on the complex events during DNA rearrangement and similar mechanism(s) responsible for cancer and genome instability in humans.

描述（由申请人提供）：原生生物以前所未有的遗传组织令分子生物学家感到惊讶，从自我剪接RNA和端粒酶的发现，到基因大小的片段，程序性DNA缺失和纤毛虫中的乱序DNA重排。因此，这些生物体是关注程序化DNA重排的组合机制，进化和计算研究的自然场所。特别是，纤毛虫中的“乱序基因”提供了一个独特的实验系统，可以研究复杂的遗传和计算机制的起源和功能。乱序基因存在于许多其他系统中，从原生生物到人类。然而，基因片段很少在DNA水平上真正“缝合”在一起，从所有片段中创建一个连续的基因。纤毛虫中发生的DNA剪接事件的复杂性使其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与几种癌症相关，强调了研究此类生物事件的重要性。最近的发现，RNA，通常被认为是基因表达的管道，在纤毛原生动物中有一种新的作用模式，激发了这项建议的研究。目标是研究纤毛类Oxytricha的基因组重排，将一个几乎完整的基因组计划与最近的发现和工具配对，使Oxytricha成为一个强大的模型系统。例如，RNA模板既可以为DNA重排提供组织指导，也可以将自发的体细胞突变传递给下一代。这种RNA引导的DNA修复的机会在尖毛藻中意义深远，因为这种生物通过全局重排破坏了95%的种系DNA，然后对剩余的片段进行排序和重新排序。这项研究的具体目标都集中在基因组，进化或发育尺度上的混乱基因的处理问题。全球的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模板中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。 公共卫生关系：DNA重排（缺失、倒位和重复）是导致与许多人类疾病（包括癌症）相关的基因组不稳定性的一个主要因素，其中DNA易位和总体缺失是很大一部分癌症和遗传性疾病的原因。扩增通常发生在“热点”之间，所述热点可以是类似于尖毛霉属中DNA重组连接处的“指针”的短的相同重复序列，并且这样的事件可以导致肿瘤抑制基因的缺失、嵌合基因的形成或促进肿瘤稳定性的基因的复制和随后的过表达。由于其DNA重排的幅度和最近的证明，RNA模板可以在体内重新编程DNA重排，Oxytricha是无与伦比的模型系统，以阐明DNA重排过程中的复杂事件和导致人类癌症和基因组不稳定性的类似机制。