GENETICS AND BIOCHEMISTRY OF A MURINE RETROPOSON

LINE-1(长散布重复序列1，或L1)是主要的 哺乳动物基因组中的动态力。逆行转位存放于 L1的后代遍及整个基因组，有时导致基因 干扰、修饰相邻基因的表达和/或转导 邻近的DNA。此外，L1作为散布的、重复的DNA， 为错配子的同源重组提供底物 序列，导致基因复制、缺失、染色体 易位和潜在的外显子洗牌。所有这些动态 事件可以导致疾病；事实上，第1行插入突变已经 被发现与血友病和肌肉营养不良有关，因为 以及人类的乳腺癌和结肠癌。因此，它是极其重要的 重要的是要了解涉及的中间产品的详细信息 逆转录转座及其调控表达的机制 以及在体内的运动。如果L1表达的正常控制机制 和逆转位变得疯狂，在发育过程中 (配子发生或早期胚胎发生)或在体细胞中反应 对环境的侮辱、L1序列的移动和重排 可能有助于遗传性疾病的产生，生育 缺陷和癌症。LINE-1逆转录转座始于转录 全长有义链L1 RNA，需要两个L1编码的 多肽。这些蛋白质可能也会催化反转。 正弦的转录和整合(短、散布、重复 序列)和经过处理的假基因，从而放大了 哺乳动物基因组动力学中的LINE-1。我们的长期目标是 详细了解反转位过程，包括 涉及的生化中间体及其在遗传和免疫调节中的作用 进化的时间。具体地说，这里提出的研究是设计的 目的：1)阐明L1编码的ORF1蛋白在 通过鉴定其核酸和蛋白质的逆转座- 详细的蛋白质相互作用活动，以及测试这一点 蛋白质能够促进互补链的退火和 链交换；2)分离小鼠基因组DNA前体 小鼠新品系-1在进化中获得的启动子 时间，以及；3)使用我们新开发的转座子托盘试验来 描述发生的插入类型，以及确定 内源性L1和L1介导的逆转座事件的频率 在有和没有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.