Using budding yeast to study LINE (L1) retrotransposition

使用芽殖酵母研究 LINE (L1) 逆转录转座

• 批准号: 8499365
• 负责人: Jeffrey S Han
• 金额: $ 4.02万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8499365
• 关键词: Address; Animal Model; Back; Beryllium; Binding; Biology; Candida albicans; Cells; Cellular biology; Chromosomes; Cloning; Collection; Conflict (Psychology); DNA; DNA Damage; DNA Sequence Rearrangement; Data Set; Elements; Environment; Eukaryota; Evaluation; Event; Family; Fertility; Frequencies; Generations; Genetic; Genetic Screening; Genome; Genomics; Germ Cells; Homologous Gene; Human; Human Genome; Immune response; Individual; Infertility; Integration Host Factors; Knock-out; L1 Elements; Location; Mammalian Cell; Mammals; Meiosis; Modeling; Molecular; Molecular Biology; Nuclear; ORF2 protein; Organism; Parasites; Pathway interactions; Process; Proteins; Proteome; RNA; Regulation; Relative (related person); Research; Retrotransposition; Retrotransposon; Ribonucleoproteins; Saccharomyces cerevisiae; Saccharomycetales; Small RNA; Sterility; Structure; System; Technology; Time; Variant; Virus; Yeast Model System; Yeasts; flexibility; genetic element; genome sequencing; insight; interest; mammalian genome; mutant; programs; public health relevance; response; success; tool

DESCRIPTION (provided by applicant): We have long-term interests in understanding the ongoing evolutionary battle between genomic parasites and the host organism, and the cellular programs that have evolved in response to these conflicts. We study Long Interspersed Nuclear Elements (LINEs, or L1) due to their spectacular success in colonizing the human genome. L1s are retrotransposons, genetic elements that replicate through an RNA intermediate and integrate back into the chromosome. L1s are responsible for generating over one third of mammalian genome sequence, and L1 retrotransposition causes genome structural variation between human individuals. In addition, L1 is generally expressed in germ cells, and loss of L1 regulation is associated with sterility in mammals. This is likely due to the genotoxic effects of L1 retrotransposition. Since most cases of human sterility are not understood at the molecular level, this has potential significance for fertility research. We want to know how L1s replicate and how L1s are regulated. We are using a budding yeast (Saccharomyces cerevisiae) model to study unknown aspects of L1 retrotransposition. S. cerevisiae is well suited for this purpose; it is a preeminent organism for the study of genetics and cell biology of basic eukaryotic processes, and often the proving ground for the latest technologies in molecular biology. S. cerevisiae chromosomes are easily manipulated, providing great experimental flexibility, and the streamlined genome and proteome simplify the analysis of large data sets (relative to higher eukaryotes). The element we are specifically using in the budding yeast model is an L1 homolog from Candida albicans. We will clone and analyze retrotransposition insertions. We will explore the mechanism for circular retrotransposition product formation. We will investigate how L1 proteins recognize and bind to L1 RNA. We will examine the location and dynamics of L1 ribonucleoproteins (RNPs). We will also carry out genetic screens to identify host factors involved in the L1 replication cycle and ask whether meiosis represents a particular permissive "state" which is optimal for L1 RNP action. Finally, we will model the introduction and expansion of a family of L1 elements in a previously L1-naove host (budding yeast). Overall, these studies will provide insight into the mechanism of L1 replication and how L1s interact with a host cell.

描述（由申请人提供）：我们在理解基因组寄生虫与宿主有机体之间正在进行的进化战以及响应这些冲突后进化的蜂窝计划方面具有长期利益。我们研究了长期散布的核元素（线或L1），因为它们在殖民人类基因组中取得了惊人的成功。 L1是逆转座子，是通过RNA中间体复制并将其整合到染色体中的遗传元素。 L1负责产生超过三分之一的哺乳动物基因组序列，而L1逆转录置位会导致人个体之间的基因组结构变异。另外，L1通常在生殖细胞中表达，L1调节的损失与哺乳动物中的无菌性有关。这很可能是由于L1逆转录置次置的遗传毒性作用。由于在分子水平上不了解大多数人类无菌病例，因此这对于生育研究具有潜在的意义。我们想知道L1如何复制以及如何调节L1。我们正在使用萌芽的酵母（酿酒酵母）模型来研究L1逆转录的未知方面。 S. cerevisiae非常适合此目的；它是研究基本真核过程的遗传学和细胞生物学的杰出生物，通常是分子生物学最新技术的遗嘱理由。酿酒酵母染色体很容易被操纵，提供了极大的实验柔韧性，而精简的基因组和蛋白质组简化了大型数据集的分析（相对于较高的真核生物）。我们在萌芽酵母模型中专门使用的元素是白色念珠菌的L1同源物。我们将克隆并分析反转录插入。我们将探索循环逆转录产物形成的机制。我们将研究L1蛋白如何识别并结合L1 RNA。我们将检查L1核糖核蛋白（RNP）的位置和动力学。我们还将执行遗传筛选，以识别L1复制周期中涉及的宿主因素，并询问减数分裂是否代表特定的允许“状态”，最适合L1 RNP作用。最后，我们将在先前的L1-Naove宿主（Butding酵母）中对L1元素的引入和扩展进行建模。总体而言，这些研究将洞悉L1复制机理以及L1与宿主细胞的相互作用。