LINES-1 elements (L1) are the prototype for LTR-less transposons (retroposons). Retroposons are distributed throughout many eukaryotes including protozoa, insects, plants, and mammals. These elements are very abundant and in mammals make up somewhere between 10 and 30% of the sequence in the genome! The replication mechanism employed by these elements is not yet elucidated although it is clear that an RNA intermediate is involved. Ll elements have been shown to be an ongoing source of insertional mutagenesis and in species with an active Ll family Ll can be a major source of RFLP's in the genome. In Drosophila it has also been shown that the bulk of spontaneous mutations involves recombination between sequences repetitive in the genome at nonhomologous locations. If this is also true for mammals, LINES-1 elements would be expected to be a major contributor to the genetic load and hence to be determinative in which species survive and which do not. As a consequence we are very interested in the factors which give rise to Ll replication rates and copy number.
Our specific aims are organized in terms of this issue but of course the experiments should also be thought of as an assault on the population dynamics and replication strategy of this new class of transposon. We will continue our exploration of the sequences which control Ll transcription. We have identified three protein binding sites in murine Ll 5' sequences and will determine the sequence specificity of this binding. We will identify the Ll promoter and determine the transcriptional start site. We will determine the tissue and temporal specificity of Ll regulatory sequence controlled transcription in transgenic mice. We will construct a system wherein Ll elements are replicated at a rate sufficient to study the biochemical composition of replication intermediates. This will be done by replacing the normal Ll transcriptional control sequences with a strong promoter/ enhancer. If this does not give adequate rates of Ll transposition we will explore the rat chloroleukaemic cell line in which Ll has been shown to be inducible (300,000 new elements in 24 hours). We will try to replicate this induction effect in mouse cell lines to capitalize on our previous information about mouse Lls. If this is not successful we will study Ll replication in the rat cell line.
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