Genetics and Biochemistry of a Murine Retroposon

描述(申请人提供)：LINE-1(长散布核素-1，或L1)是哺乳动物基因组中的主要动力。逆转录转座使L1的后代在整个基因组中沉积，有时会导致基因中断、相邻基因的表达改变和/或相邻DNA的转导。此外，L1作为散布的重复DNA，为错配序列的同源重组提供了底物，导致基因复制、缺失、染色体易位以及潜在的外显子改组。人类基因组中L1基因的存在和移动引起的任何一种动态事件都可能导致疾病；事实上，LINE-1插入突变被发现与多种疾病有关，包括血友病和肌肉营养不良，以及乳腺癌和结肠癌。因此，了解逆转录转座过程中涉及的中间产物的细节以及在体内控制其表达和运动的机制是极其重要的。如果在发育过程中(配子发生或早期胚胎发生)或在体细胞中对环境伤害或衰老的反应中，L1表达和逆转座的正常控制机制发生紊乱，L1序列的移动和重排可能是导致遗传疾病、出生缺陷和癌症的遗传不稳定性的重要组成部分。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，以及它在遗传和进化时间的控制。L1逆转录转座始于全长正义链L1 RNA的转录，需要2个L1编码的多肽作用于顺式。本文提出的研究旨在：1)通过研究突变对逆转录转座活性以及对分离的ORF1蛋白的核酸结合和伴侣活性的影响，进一步阐明L1编码的ORF1蛋白在逆转录转座过程中的作用；2)确定ORF1和ORF2蛋白之间相互作用的基础，并提供有关ORF2蛋白结构和功能的新的生化信息；3)确定2种L1编码蛋白翻译控制的分子基础，并定义与L1RNA从翻译模板向组装的L1逆转录转座机过渡时与其相关的蛋白质组分。

DESCRIPTION (provided by applicant): LINE-1 (Long Interspersed Nuclear Element-1, 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. Any 1 of the dynamic events caused by the presence and movement of L1 in the human genome can lead to disease; in fact, LINE-1 insertional mutagenesis has been found to be responsible for a wide variety of diseases including hemophilia and muscular dystrophy, as well as breast and colon cancer. 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 or aging, movement and rearrangement of L1 sequences could be an instrumental component of the genetic instability responsible for genetic diseases, birth defects and cancers. 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. L1 retrotransposition begins with transcription of full-length, sense-strand L1 RNA and requires the 2 L1-encoded polypeptides acting in cis. The studies proposed here are specifically designed to: 1) Further elucidate the role of the L1-encoded ORF1 protein during retrotransposition by investigating the effects of mutations on retrotransposition activity, and on the nucleic acid binding and chaperone activities of the isolated ORF1 protein; 2) Determine the basis for the interaction between the ORF1 and ORF2 proteins, and provide new biochemical information about the structure and functions of the ORF2 protein; 3) Determine the molecular basis of translational control of the 2 L1-encoded proteins, and define the protein components associated with the L1RNA as it transitions from its function as the translation template to an assembled L1 retrotransposition machine.