GENET & MOLEC ANALYSIS OF HUMAN LINE1 RETROTRANSPOSITION

转座因子是可以移动（即转座）到不同基因组位置的DNA序列，它们存在于几乎所有被研究的生物体的基因组中。转座子有两大类：转座子和反转座子。逆转录转座子通过RNA中间体动员并通过称为逆转录转座的复制机制转座。长穿插核元件（LINES或L1s）是人类基因组中最丰富的反转录转座子，它们约占DNA的15%。估计有30-60个人类L1因子具有逆转录转位能力，生殖系和体细胞L1逆转录转位事件都可引起疾病。还有一种假设是，由具有逆转录转位能力的L1s编码的蛋白质调动了某些sine（例如，Alu元件）和加工过的假基因，这些假基因构成了另外10%的人类DNA。Alu逆转录也具有诱变性，在过去7年中发现了11个新的Alu插入引起不同的疾病。因此，无论是直接的还是通过细胞rna的混杂动员，l1都是人类基因组中强有力的诱变剂。尽管L1具有致突变的潜力，但关于L1逆转录的机制方面的研究还处于起步阶段。最近开发的一种检测方法用于监测培养的人类细胞中的L1逆转录转位，分子生物学和生物化学方法将用于在分子水平上确定L1逆转录转位的机制。具体而言，实验将进行：1)鉴定L1 RNA中的顺式作用序列和L1编码蛋白中逆转录所需的功能域；2)确定控制l1编码蛋白选择模板的因素；3)鉴定L1逆转录途径的体内中间体。该项目的长期目标是获得对L1逆转录如何促进人类疾病和遗传多样性的基本理解。对L1逆转录转位的基本机制的理解也将使工程L1作为小鼠基因组的转座子诱变剂和人类研究的潜在基因传递载体的实际发展成为可能。

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.