In recent years it has become clear that insertions of retrotransposons, including L1 and Alu elements, account for about 1 in 300-600 human mutations, a small, yet biologically important fraction. Most of these insertions are truncated L1 elements. L1 elements are the only known human elements capable of encoding proteins, including reverse transcriptase (RT), required for their retrotransposition. We have now tracked down precursors of two of these recent insertions, one into the factor VIII gene producing hemophilia A and the other into the dystrophin gene causing muscular dystrophy. These two precursor elements, one of which maps to chromosome 22 and the other to chromosome 1, are the first two active human transposable elements isolated to date. In addition, we have demonstrated that active L1 elements encode an RT activity demonstrable in vivo in a yeast assay system. However, a myriad of questions remain as to the number of transposable L1 elements in the human genome, how frequently they are """"""""copied and pasted"""""""" into a new genomic location, and the mechanisms by which these retrotransposition events occur. In this application we continue the effort to answer these questions through a variety of new and innovative experiments, some of which depend upon assays of transposition In yeast and mammalian cells. Success of our Specific Aims will allow us to learn the following: 1) unusual aspects of the expression of the second active element isolated, 2) characteristics of the yeast assay system for the detection of RT activity encoded by active L1 elements, 3) structural features of the RT through semiquantitative analysis of a large number of active and potentially active elements, 4) an estimate of the number of active human L1 elements and the isolation of the large majority of these elements, 5) regions of L1 important in retrotransposition in human teratocarcinoma cells in culture, and 5) an estimate of the number of spontaneous L1 insertions into the human genome and the isolation of some of these new events. This information will greatly increase our knowledge of retrotransposable elements and the retrotransposition process. Furthermore, it may aid us in creating a usable vector system for gene delivery based on the L1 element, similar to the P element vector now in use in Drosophila.
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