Genetic and Molecular Analyses of Human LINE-1 Retrotransposition

人类 LINE-1 逆转录转座的遗传和分子分析

基本信息

批准号：
7902174
负责人：
JOHN V. MORAN
金额：
$ 29.98万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-30 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7902174
关键词：
Affect Alu Elements Baculovirus Expression System Biochemical Biological Biological Assay Cells Cultured Cells Cytidine Deaminase DNA Damage DNA Repair DNA Repair Pathway DNA Sequence DNA Transposable Elements Development Disease Elements Engineering Event Evolution Family Functional RNA Funding Gene Family Genes Genetic Genetic Variation Genome Genomics Germ Lines Hereditary Disease Host Defense Human Human Genome In Vitro Individual Integration Host Factors L1 Elements Laboratories Lead Learning Length Location Long Interspersed Elements Mammalian Cell Mediating Minisatellite Repeats Mobile Genetic Elements Molecular Molecular Genetics Monitor Mutation Nonhomologous DNA End Joining Paste substance Pathway interactions Process Proteins Protocols documentation RNA Reagent Retrotransposition Retrotransposon Reverse Transcription Short Interspersed Nucleotide Elements Small Nucleolar RNA Somatic Cell Structure System U6 small nuclear RNA Variant human DNA human disease insight meetings member public health relevance reconstitution response success tool

项目摘要

DESCRIPTION (provided by applicant): Long Interspersed Element-1 (LINE-1 or L1) is an abundant mobile genetic element that comprises approximately 17% of human DNA. Active human L1s are about 6 kb in length and they encode two proteins (ORF1p and ORF2p) that are critical for retrotransposition. Ongoing LINE-1 retrotransposition events contribute to inter-individual genetic variation, and, on occasion, LINE-1 insertions into genes can result in genetic disease. During the past decade, my laboratory has developed efficient assays to monitor LINE-1 retrotransposition in cultured mammalian cells. Here, we will use these assays in conjunction with genetic, molecular, and biochemical approaches to further elucidate the mechanism of L1 retrotransposition. We also will identify host proteins that affect LINE-1 retrotransposition in human cells. A basic mechanistic understanding of the process of L1 retrotransposition will lead to greater insight about how transposable element activity contributes to human disease, human genome evolution, and human diversity. PUBLIC HEALTH RELEVANCE: Long INterspersed Element-1 (LINE-1) is an abundant mobile genetic element that comprises 17% of human DNA. Active LINE-1 elements are able to retrotranspose or "jump," inserting themselves into a new genomic location by a copy and paste mechanism. On occasion, new LINE-1 insertions in the germ line, in early development, or in somatic cells can result in disease-producing mutations. Despite the mutagenic potential of LINE-1 elements, little is known about the molecular mechanism of LINE-1 retrotransposition and even less is known about cellular proteins that influence this process. During the past decade, my laboratory has developed a toolbox to study LINE- 1 retrotransposition in a controlled manner in mammalian cultured cells. These tools have allowed significant progress in this field by my lab and others. In this proposal, we will use genetic, molecular biological, and biochemical approaches to further elucidate the molecular mechanism of LINE-1 retrotransposition. We also will explore how mutations in host proteins involved in host defense and/or DNA repair pathways affect LINE-1 jumping. Through these studies, we will learn more about how this intriguing family of repetitive DNA sequences contributes to the structure and function of the human genome.

描述（由申请人提供）：长散布元件-1（LINE-1或L1）是一种丰富的可移动遗传元件，约占人类DNA的17%。活跃的人类 L1 长度约为 6 kb，它们编码两种对于逆转录转座至关重要的蛋白质（ORF1p 和 ORF2p）。正在进行的 LINE-1 逆转录转座事件会导致个体间的遗传变异，有时，LINE-1 插入基因可能会导致遗传病。在过去的十年中，我的实验室开发了有效的检测方法来监测培养的哺乳动物细胞中的 LINE-1 逆转录转座。在这里，我们将使用这些测定结合遗传、分子和生化方法来进一步阐明 L1 逆转录转座的机制。我们还将鉴定影响人类细胞中 LINE-1 逆转录转座的宿主蛋白。对 L1 逆转录转座过程的基本机制了解将有助于更深入地了解转座元件活性如何影响人类疾病、人类基因组进化和人类多样性。公共卫生相关性：长散布元件 1 (LINE-1) 是一种丰富的可移动遗传元件，占人类 DNA 的 17%。活性 LINE-1 元件能够逆转录转座或“跳跃”，通过复制和粘贴机制将自身插入到新的基因组位置。有时，生殖系、早期发育或体细胞中新的 LINE-1 插入可能会导致产生疾病的突变。尽管 LINE-1 元件具有诱变潜力，但人们对 LINE-1 逆转录转座的分子机制知之甚少，对影响这一过程的细胞蛋白更是知之甚少。在过去的十年中，我的实验室开发了一个工具箱，用于在哺乳动物培养细胞中以受控方式研究 LINE-1 逆转录转座。这些工具使我的实验室和其他人在该领域取得了重大进展。在本研究中，我们将利用遗传学、分子生物学和生化方法进一步阐明LINE-1逆转录转座的分子机制。我们还将探讨参与宿主防御和/或 DNA 修复途径的宿主蛋白突变如何影响 LINE-1 跳跃。通过这些研究，我们将更多地了解这个有趣的重复 DNA 序列家族如何对人类基因组的结构和功能做出贡献。