Genetics & Biochemistry of a Murine Retroposon

描述（由申请人提供）：LINE-1（长穿插重复序列1，或L1）是哺乳动物基因组中的主要动力。逆转录将L1的后代沉积在整个基因组中，有时会导致基因破坏，修饰邻近基因的表达，和/或邻近DNA的转导。此外，L1作为分散的、重复的DNA，为错配序列的同源重组提供了底物，导致基因重复、缺失、染色体易位和潜在的外显子洗牌。所有这些动态事件都可能导致疾病；事实上，LINE-1插入突变已被发现是导致人类血友病、肌肉萎缩症以及乳腺癌和结肠癌的原因。因此，了解参与反转录转位的中间体的细节以及用于控制其体内表达和运动的机制是非常重要的。如果L1表达和逆转录的正常控制机制在发育过程中（配子体发生或早期胚胎发生）或在体细胞中响应环境损害而变得紊乱，那么L1序列的移动和重排可能有助于遗传疾病、出生缺陷和癌症的产生。LINE-1反转录转位开始于全长、义链L1 RNA的转录，需要两个L1编码的多肽。这些蛋白可能还催化了SINEs（短穿插重复序列）和加工假基因的反转录和整合，从而放大了LINE-1在哺乳动物基因组动力学中的作用。我们的长期目标是详细了解逆转录过程，包括所涉及的生化中间体及其在遗传和进化时间上的控制。具体而言，本研究旨在：1)通过详细研究野生型和突变型蛋白的核酸和蛋白-蛋白相互作用活性，阐明l1编码的ORF1蛋白在逆转录转位中的作用；2)鉴定与ORF1p相互作用的细胞蛋白，然后确定它们是否促进和/或抑制L1反转录转位；3)确定ORF2和/或ORF1的翻译是否涉及与基因帽无关的启动机制，从而有助于控制L1逆转录转位。

DESCRIPTION (provided by applicant): 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 either 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 investigating the nucleic acid and protein-protein interaction activities of wild-type and mutant proteins in detail; 2) identify cellular proteins that interact with ORF1p then determine whether they facilitate and/or inhibit L1 retrotransposition, and; 3) determine whether translation of ORF2 and/or ORF1 involves cap-independent mechanisms for initiation that contribute to the control of L1 retrotransposition.