GENETICS AND BIOCHEMISTRY OF A MURINE RETROPOSON

LINE-1（长散布重复序列1，或L1）是一种主要的 哺乳动物基因组中的动力。 逆行转位使 整个基因组中L1的后代，有时导致基因 相邻基因的破坏、修饰表达和/或转导 相邻的DNA。 此外，L1作为散布的重复DNA， 为错配的同源重组提供了底物， 序列，导致基因复制，缺失，染色体 易位和潜在的外显子改组。 所有这些动态 事件可能导致疾病;事实上，LINE-1插入突变 被发现是血友病和肌肉萎缩症的原因， 以及乳腺癌和结肠癌。 因此， 重要的是要了解中间体的细节， 逆转录转座和用于控制其表达的机制 和体内运动。 如果L1表达的正常控制机制 和反转录转座变得异常 （配子发生或早期胚胎发生）或体细胞中的反应 环境损伤、L1序列的移动和重排 可能有助于遗传疾病的产生， 缺陷和癌症。 LINE-1逆转录转座始于转录 一个全长的，有义链的L1 RNA，需要两个L1编码的 多肽。 这些蛋白质也可能催化逆转 西内斯（短间隔重复序列）的转录和整合 序列）和加工的假基因，从而放大 哺乳动物基因组动力学中的LINE-1。 我们的长远目标是 详细了解逆转录过程，包括 参与的生化中间体及其在遗传和 进化时间 具体而言，这里提出的研究旨在 1）阐明L1编码的ORF 1蛋白在 逆转录转座通过表征其核酸和蛋白质- 蛋白质相互作用的活动，以及测试这一点， 蛋白质促进互补链退火的能力， 2）分离小鼠基因组DNA祖细胞，其中一个是 小鼠LINE-1最近在进化中获得的启动子 时间，和; 3）采用我们新开发的转座子托盘测定， 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 在有无外部代理人的情况下。

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.