This project explores the molecular biology of LINE-1 (L1) elements and their impact on the mammalian genome. L1 is the prototype for transposable elements which encode reverse transcriptase but do not contain the LTR sequences characteristic of retroviruses. L1 makes up 10% or more of the mammalian genome. Related elements are found throughout eukaryotes including insects and plants. L1 is a significant source of mutations in mammals, including humans. L1 transposition can knock out genes and has been implicated in tumorigenesis. Recombination between L1 elements is also a potential source of mutations. Our studies focus only in the mouse. We intend to characterize L1 transposition intermediates. Since transcription of L1 is the first step in its transposition one study will focus on the regulation of L1 transcription. Analysis of the mouse L1 A- type promoter by saturation mutagenesis will be completed with the intent of defining the transcription binding sites. Gel shift analysis of mutants with altered transcriptional properties will be used to investigate the correspondence between sequences required for promoter activity and protein binding sites. Transgenic mice with an L1 A-type driven reporter gene will be used to study the developmental regulation of the promoter. Studies comparing the transcriptional regulation of a reconstructed ancestral F- type promoter with the modern A-type in tissue culture cells and in gel shift experiments will be extended. Transgenic mice with an F-type driven reporter gene will be constructed and used to are its regulation with the modern A-type. These experiments model evolution of L1 over the last 5-10 million years in modern mouse cells. Another study will focus on the L1 particle as a likely transposition intermediate. We will also construct cell lines overexpressing L1 and use them to produce L1 particles. The structural and enzymatic properties of these part ides will be characterized in detail. Is the presence of L1 useful for its mammalian host? Two approaches will be investigated. 1) Somatic tissue in which L1 is expressed will be studied. Initial observations finding L1 expression in tissues of the immune system will be extended. Possible functions for L1 in the immune system are a) involvement of L1 reverse transcriptase in somatic hypermutation of immunoglobulins b) activation of tissue specific genes by L1 hyper-transposition in antigen presenting cells in the thymus. 2) Anti- L1 ribozymes will be constructed to attempt to create a phenotype in cell lines and transgenic animals.
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