GENETICS AND BIOCHEMISTRY OF A MURINE RETROPOSON

鼠逆转录子的遗传学和生物化学

• 批准号: 6385730
• 负责人: SANDRA L MARTIN
• 金额: $ 26.12万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385730
• 关键词: RNase protection assay; SDS polyacrylamide gel electrophoresis; biochemical evolution; developmental genetics; fluorescent in situ hybridization; gene expression; genetically modified animals; genome; image processing; laboratory mouse; nucleic acid repetitive sequence; nucleic acid sequence; polymerase chain reaction; protein biosynthesis; protein protein interaction; protein structure function; ribonucleoproteins; transposon /insertion element

LINE-1 (long interspersed repeated sequence one, or L1) is a major dynamic force in the mammalian genome. Retrotransposition deposits the progeny of L1 throughout the genome, sometimes leading to gene disruption, modified expression of adjacent genes, and/or transduction of neighboring DNA. In addition, L1, as interspersed, repetitive DNA, provides a substrate for homologous recombination of mispaired sequences, leading to gene duplication, deletion, chromosome translocation and, potentially, exon shuffling. All of these dynamic events can lead to disease; in fact, LINE-1 insertional mutagenesis has been found to be responsible for hemophilia and muscular dystrophy, as well as breast and colon cancer in humans. Thus, it is extremely important to understand the details of the intermediates involved in retrotransposition and the mechanisms used to control their expression and movement in vivo. If the normal control mechanisms of L1 expression and retrotransposition become deranged and during development (gametogenesis or early embryogenesis) or in somatic cells in response to environmental insults, movement and rearrangement of L1 sequences could be instrumental in the generation of genetic diseases, birth defects and cancer. LINE-1 retrotransposition begins with transcription of a full-length, sense-strand L1 RNA and requires two L1-encoded polypeptides. These proteins probably also catalyze the reverse transcription and integration of SINEs (short interspersed repeated sequences) and processed pseudogenes, thereby amplifying the effects of LINE-1 in mammalian genome dynamics. Our long-range goal is to understand the retrotransposition process in detail, including the biochemical intermediates involved as well as its control in genetic and evolutionary time. Specifically, the studies proposed here are designed to: 1) Elucidate the role of the L1-encoded ORF1 protein during retrotransposition by characterizing its nucleic acid and protein- protein interaction activities in detail, as well as to test this protein for its ability to promote complementary strand annealing and strand-exchange; 2) Isolate the mouse genomic DNA progenitor of one of the promoters that was acquired by mouse LINE-1 recently in evolutionary time, and; 3) Employ our newly developed transposon tray assay to characterize the types of insertions that occur, as well as determine the frequency of endogenous L1 and L1-mediated retrotransposition events in the presence and absence of external agents.

LINE-1（长散布重复序列一，或 L1）是一个主要的 哺乳动物基因组中的动力。 逆转录转座沉积 L1 的后代遍布整个基因组，有时会导致基因 破坏、修改邻近基因的表达和/或转导 邻近的 DNA。 此外，L1作为散布的重复DNA， 为错配的同源重组提供底物 序列，导致基因复制、缺失、染色体 易位和可能的外显子改组。 所有这些动态 事件可能导致疾病；事实上，LINE-1插入突变 已被发现与血友病和肌营养不良症有关， 以及人类乳腺癌和结肠癌。 因此，这是极其 了解所涉及的中间体的详细信息很重要 逆转录转座及其表达控制机制 以及体内的运动。 如果 L1 表达的正常控制机制 和逆转录转座变得混乱并且在发育过程中 （配子发生或早期胚胎发生）或体细胞中的反应 环境损害、L1 序列的移动和重新排列 可能有助于遗传疾病的产生、出生 缺陷和癌症。 LINE-1 逆转录转座从转录开始 全长、有义链 L1 RNA 并需要两个 L1 编码 多肽。 这些蛋白质可能也会催化相反的过程 SINE 的转录和整合（短散布重复 序列）和处理的假基因，从而放大的效果 哺乳动物基因组动力学中的 LINE-1。 我们的长期目标是 详细了解逆转录转座过程，包括 涉及的生化中间体及其对遗传和遗传的控制 进化时间。 具体来说，这里提出的研究旨在 1) 阐明 L1 编码的 ORF1 蛋白在 通过表征其核酸和蛋白质进行逆转录转座 详细的蛋白质相互作用活性，以及​​测试这一点 蛋白质具有促进互补链退火的能力， 链交换； 2) 分离小鼠基因组 DNA 祖细胞之一 小鼠 LINE-1 最近在进化过程中获得的启动子 时间，以及； 3) 采用我们新开发的转座子托盘检测 描述发生的插入类型，并确定 内源性 L1 和 L1 介导的逆转录转座事件的频率 在有或没有外部代理的情况下。