GENET & MOLEC ANALYSIS OF HUMAN LINE1 RETROTRANSPOSITION

转座元件是可以移动(即转座)到不同基因组位置的DNA序列，它们几乎存在于所有被研究的生物的基因组中。一般有两类转座子：转座子和反转录转座子。反转录转座子通过一种称为反转录转座的复制机制，通过一种RNA中间体进行移动和转座。长穿插核素(L1s)是人类基因组中含量最丰富的反转录转座子，约占DNA的15%。据估计，有30-60个人类L1元件具有逆转录转座能力，生殖系和体细胞L1逆转录转座事件都会导致疾病。它还假设，由逆转录转座能力L1编码的蛋白质可以动员某些Sine(例如Alu元件)和加工的假基因，这些假基因占人类DNA的另外10%。Alu逆转座也是诱变的，在过去的七年里，已经发现了11个从头插入的Alu导致不同的疾病。因此，无论是直接还是通过细胞RNA的混杂动员，L1在人类基因组中都是有效的诱变剂。尽管L1s具有诱变潜力，但关于L1逆转座的机制方面的研究仍处于起步阶段。最近开发的一种监测培养的人类细胞中L1逆转录转座的方法和分子生物学和生化方法将被用来在分子水平上确定L1逆转座的机制。具体来说，将进行以下实验：1)确定L1 RNA中的顺式作用序列和逆转录转座所需的L1编码蛋白中的功能结构域；2)确定控制L1编码蛋白选择模板的因素；以及3)在体内识别L1逆转录转座途径中的中间产物。该项目的长期目标是从根本上了解L1逆转座子如何导致人类疾病和遗传多样性。对L1逆转座的基本机制的理解也将使工程L1S作为小鼠基因组的转座子诱变剂和作为人类研究的潜在基因载体的实际开发成为可能。

Transposable elements are DNA sequences that can move (i.e., transpose) to different genomic locations and they are present in the genomes of virtually all organisms studied. There are two general classes of transposable elements: transposons and retrotransposons. Retrotransposons mobilize via an RNA intermediate and transpose by a replicative mechanism termed retrotransposition. Long Interspersed Nuclear Elements (LINES or L1s) are the most abundant retrotransposons in the human genome and they comprise approximately 15 percent of DNA. An estimated 30-60 human L1 elements are retrotransposition-competent, and both germ line and somatic L1 retrotransposition events have caused disease. It also is hypothesized that the proteins encoded by retrotransposition-competent L1s mobilize certain SINES (e.g., Alu elements) and processed pseudogenes, which comprise another 10 percent of human DNA. Alu retrotransposition also is mutagenic and eleven de novo Alu insertions have been found to cause different diseases in the past seven years. Thus, either directly or through the promiscuous mobilization of cellular RNAs, L1s are potent mutagens in the human genome. Despite the mutagenic potential of L1s, studies concerning the mechanistic aspects of L1 retrotransposition are in their infancy. A recently developed assay to monitor L1 retrotransposition in cultured human cells and molecular biological and biochemical approaches will be used to determine the mechanism of L1 retrotransposition at the molecular level. Specifically, experiments will be performed to: 1) identify cis-acting sequences in L1 RNA and function al domains in the L1-encoded proteins required for retrotransposition; 2) identify factors that govern template choice by the L1-encoded proteins; and 3) identify in vivo intermediates in the L1 retrotransposition pathway. The long-term goal of this project is to gain a fundamental understanding of how L1 retrotransposition contributes to human disease and genetic diversity. A fundamental mechanistic understanding of L1 retrotransposition also will allow the practical development of engineered L1s as a transposon mutagen for the mouse genome and as a potential gene delivery vehicle for human studies.