GENET & MOLEC ANALYSIS OF HUMAN LINE1 RETROTRANSPOSITION

遗传基因

• 批准号: 6921057
• 负责人: JOHN V. MORAN
• 金额: $ 9.56万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6921057
• 关键词: RNA; RNA directed DNA polymerase; endonuclease; enzyme activity; gene mutation; genetic regulatory element; human genetic material tag; human tissue; immunocytochemistry; messenger RNA; molecular genetics; nucleic acid repetitive sequence; protein structure function; ribonucleoproteins; tissue /cell culture; transposon /insertion element

Transposable elements are DNA sequences that can move (i.e., transpose) to different genomic locations and they are present in the genomes of virtually all organisms studied. There are two general classes of transposable elements: transposons and retrotransposons. Retrotransposons mobilize via an RNA intermediate and transpose by a replicative mechanism termed retrotransposition. Long Interspersed Nuclear Elements (LINES or L1s) are the most abundant retrotransposons in the human genome and they comprise approximately 15 percent of DNA. An estimated 30-60 human L1 elements are retrotransposition-competent, and both germ line and somatic L1 retrotransposition events have caused disease. It also is hypothesized that the proteins encoded by retrotransposition-competent L1s mobilize certain SINES (e.g., Alu elements) and processed pseudogenes, which comprise another 10 percent of human DNA. Alu retrotransposition also is mutagenic and eleven de novo Alu insertions have been found to cause different diseases in the past seven years. Thus, either directly or through the promiscuous mobilization of cellular RNAs, L1s are potent mutagens in the human genome. Despite the mutagenic potential of L1s, studies concerning the mechanistic aspects of L1 retrotransposition are in their infancy. A recently developed assay to monitor L1 retrotransposition in cultured human cells and molecular biological and biochemical approaches will be used to determine the mechanism of L1 retrotransposition at the molecular level. Specifically, experiments will be performed to: 1) identify cis-acting sequences in L1 RNA and function al domains in the L1-encoded proteins required for retrotransposition; 2) identify factors that govern template choice by the L1-encoded proteins; and 3) identify in vivo intermediates in the L1 retrotransposition pathway. The long-term goal of this project is to gain a fundamental understanding of how L1 retrotransposition contributes to human disease and genetic diversity. A fundamental mechanistic understanding of L1 retrotransposition also will allow the practical development of engineered L1s as a transposon mutagen for the mouse genome and as a potential gene delivery vehicle for human studies.

转座元件是可以移动（即转座）到不同基因组位置的 DNA 序列，它们几乎存在于所有研究的生物体的基因组中。 转座因子有两类：转座子和反转录转座子。 逆转录转座子通过 RNA 中间体动员并通过称为逆转录转座的复制机制进行转座。 长散布核元件（LINES 或 L1）是人类基因组中最丰富的反转录转座子，约占 DNA 的 15%。 估计有 30-60 个人类 L1 元件具有逆转录转座能力，种系和体细胞 L1 逆转录事件都会引起疾病。 还假设具有逆转录转座能力的 L1 编码的蛋白质会调动某些 SINES（例如 Alu 元件）和经过加工的假基因，其中包含另外 10% 的人类 DNA。 Alu 逆转录转座也具有诱变性，在过去七年中已发现 11 个从头 Alu 插入会导致不同的疾病。 因此，无论是直接的还是通过细胞 RNA 的混杂动员，L1 都是人类基因组中有效的诱变剂。尽管 L1 具有诱变潜力，但有关 L1 逆转录转座机制方面的研究仍处于起步阶段。 最近开发的一种监测培养人类细胞中 L1 逆转录转座的检测方法以及分子生物学和生化方法将用于在分子水平上确定 L1 逆转录转座的机制。具体地，将进行实验以：1)鉴定逆转座所需的L1 RNA中的顺式作用序列和L1编码的蛋白质中的功能域； 2) 确定 L1 编码蛋白控制模板选择的因素； 3) 鉴定 L1 逆转录转座途径的体内中间体。 该项目的长期目标是从根本上了解 L1 逆转录转座如何影响人类疾病和遗传多样性。 对 L1 逆转录转座的基本机制的了解也将允许实际开发工程 L1 作为小鼠基因组的转座子诱变剂和作为人类研究的潜在基因传递工具。