Using budding yeast to study LINE (L1) retrotransposition

描述（由申请人提供）：我们对理解基因组寄生虫和宿主生物体之间正在进行的进化斗争以及为应对这些冲突而进化的细胞程序有着长期的兴趣。我们研究长散布核元素（LINES或L1），因为它们在人类基因组中取得了惊人的成功。L1是逆转录转座子，通过RNA中间体复制并整合回染色体的遗传元件。L1负责产生超过三分之一的哺乳动物基因组序列，并且L1反转录转座导致人类个体之间的基因组结构变异。此外，L1通常在生殖细胞中表达，L1调控的丧失与哺乳动物的不育有关。这可能是由于L1反转录转座的遗传毒性作用。由于大多数人类不育病例在分子水平上还不清楚，这对生育研究具有潜在的意义。我们想知道L1是如何复制的，以及L1是如何被调节的。我们正在使用一个芽殖酵母（酿酒酵母）模型来研究未知方面的L1反转录转座。S.酿酒酵母非常适合这一目的;它是研究基本真核生物过程的遗传学和细胞生物学的杰出生物体，并且通常是分子生物学中最新技术的试验场。S.酿酒酵母染色体易于操作，提供了很大的实验灵活性，简化的基因组和蛋白质组简化了大型数据集的分析（相对于高等真核生物）。我们在芽殖酵母模型中特别使用的元件是来自白色念珠菌的L1同源物。我们将克隆并分析反转录转座插入。我们将探讨环状反转录转座产物形成的机制。我们将研究L1蛋白如何识别和结合L1 RNA。我们将研究L1核糖核蛋白（RNP）的位置和动力学。我们还将进行遗传筛选，以确定参与L1复制周期的宿主因素，并询问减数分裂是否代表一个特定的许可“状态”，这是L1 RNP行动的最佳状态。最后，我们将模拟一个家庭的L1元素在以前的L1-naove主机（芽殖酵母）的引入和扩展。总的来说，这些研究将深入了解L1复制的机制以及L1如何与宿主细胞相互作用。 公共卫生相关性：我们的研究涉及L1元件的复制和调节，L1元件是产生超过三分之一的哺乳动物基因组的遗传元件。这些因素导致DNA损伤和基因组重排，当不受控制时，与哺乳动物的不育有关。我们的研究将帮助我们了解这些L1元件如何改变我们的基因组的分子细节，它们如何与我们的细胞相互作用，以及它们对我们的细胞有多危险。

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. PUBLIC HEALTH RELEVANCE: Our studies concern the replication and regulation of L1 elements, which are genetic elements that have produced over one third of mammalian genomes. These elements cause DNA damage and genome rearrangements, and when uncontrolled are associated with infertility in mammals. Our studies will help us understand the molecular details of how these L1 elements change our genome, how they interact with our cells, and how dangerous they are to our cells.