Insights into the mechanism of L1 retrotransposition

L1 retrotransposons have had a tremendous impact on mammalian genomes through a variety of mechanisms including retrotransposition, transduction of flanking sequence, and mobilization of non-autonomous retrotransposons in trans. The significance of L1 elements is becoming increasingly apparent, but the mechanism of L1 retrotransposition remains poorly understood. In this thesis, I describe the creation of an Enhanced Green Fluorescent Protein (EGFP) based retrotransposition cassette and demonstrate that is can be used to detect near real-time retrotransposition in a single cell. I use the cassette to tag human L1 elements and test the elements in a new cultured cell assay. I next describe the creation of a mouse model of human L1 retrotransposition using tagged human L1 elements. Retrotransposition in the male germ line of transgenic mice occurs at frequency as high as 1 in 100 sperm. The L1 promoter is germ line-specific and retrotransposition often occurs in late meiosis and post-meiosis. The mouse model is valuable as a system to study L1 retrotransposition in vivo and may pave the way to an eventual random mutagenesis system in mouse.; In a subsequent chapter, I describe a bioinformatics project that has provided insight into several phenomena of L1 retrotransposition. First, I show that L1-mediated transduction is a common process and that it may have created up to 0.6% of the mass of the genome. L1-mediated transduction may be a source of genetic diversity as it may move exons or regulatory regions to new areas of the genome. Second, I demonstrate that L1 elements in the genome frequently show inversion and truncation of their 5′ ends. I hypothesize that L1 inversion is a consequence of its mechanism of insertion, a coupled process of reverse transcription and integration called target primed reverse transcription. I propose a model called “twin priming” to explain the inversions and provide evidence in support of the model. In the last chapter, I describe the discovery of an active SVA retrotransposon. I demonstrate that SVA elements are active non-autonomous human retrotransposons and that they are mobilized by using the L1 retrotransposition machinery in trans.