GENET & MOLEC ANALYSIS OF HUMAN LINE1 RETROTRANSPOSITION

遗传基因

• 批准号: 6182261
• 负责人: JOHN V. MORAN
• 金额: $ 26万
• 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 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182261
• 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.

转座因子是可以移动的DNA序列（即，转置）到不同的基因组位置，并且它们存在于几乎所有研究的生物体的基因组中。 转座因子有两大类：转座子和反转录转座子。 反转录转座子通过RNA中间体移动并通过称为反转录转座的复制机制转座。 长散布核元件（LINES或L1）是人类基因组中最丰富的逆转录转座子，它们占DNA的约15%。 估计有30-60个人类L1元件具有反转录转座能力，并且生殖系和体细胞L1反转录转座事件都引起疾病。 还假设由具有逆转录转座能力的L1编码的蛋白质动员某些西内斯（例如，Alu元件）和加工过的假基因，后者占人类DNA的另外10%。 Alu逆转录转座也具有致突变性，在过去的七年中，已经发现了11种Alu从头插入导致不同的疾病。 因此，无论是直接还是通过细胞RNA的混杂动员，L1都是人类基因组中的强效诱变剂。尽管L1具有致突变潜力，但关于L1反转录转座机制方面的研究仍处于起步阶段。 最近开发的检测方法，以监测L1逆转录转座在培养的人类细胞和分子生物学和生物化学的方法将被用来确定L1逆转录转座的机制在分子水平上。具体而言，将进行实验以：1）鉴定L1 RNA中的顺式作用序列和逆转录转座所需的L1编码蛋白中的功能α 1结构域; 2）鉴定控制L1编码蛋白的模板选择的因素; 3）鉴定L1逆转录转座途径中的体内中间体。 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反转录转座的基本机理的理解也将允许工程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递送载体的实际开发。