Using budding yeast to study LINE (L1) retrotransposition

描述(由申请人提供)：我们对了解基因组寄生虫和宿主有机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具有长期兴趣。我们研究长时间散布的核元素(线，或L1)，因为它们在人类基因组的定植上取得了惊人的成功。L1是反转录转座子，是通过RNA中间体复制并整合回染色体的遗传元件。L1s负责产生超过三分之一的哺乳动物基因组序列，而L1逆转录转座导致人类个体之间的基因组结构差异。此外，L1在生殖细胞中普遍表达，在哺乳动物中，L1调控的缺失与不育有关。这可能是由于L1逆转录转座的遗传毒性效应所致。由于大多数人类不孕症还没有在分子水平上被理解，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，L1是如何被调控的。我们正在使用一个萌芽酵母(酿酒酵母)模型来研究L1逆转录转座的未知方面。酿酒酵母非常适合于这一目的；它是研究真核生物基本过程的遗传学和细胞生物学的卓越生物，通常也是分子生物学最新技术的试验场。酿酒酵母的染色体易于操作，提供了极大的实验灵活性，简化的基因组和蛋白质组简化了大数据集的分析(相对于高等真核生物)。我们在发芽酵母模型中专门使用的元素是白色念珠菌的L1同源物。我们将克隆和分析逆转位插入。我们将探索环状逆转录转座产物的形成机制。我们将研究L1蛋白是如何识别和结合L1RNA的。我们将研究L1核糖核蛋白(RNPs)的位置和动力学。我们还将进行遗传筛选，以确定L1复制周期中涉及的宿主因素，并询问减数分裂是否代表一种特定的允许“状态”，该状态对L1 RNP的作用是最佳的。最后，我们将模拟L1元件家族在以前的L1-NAOVE宿主(萌芽酵母)中的引入和扩展。总体而言，这些研究将为L1复制的机制以及L1如何与宿主细胞相互作用提供深入的见解。

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.