This research proposal is aimed at elucidating the mechanism of lymphomagenesis by the slow transforming murine retroviruses. It focuses on the understanding of the tissue-specificity of the diseases caused by these retroviruses. Included in these studies are experiments to further our understanding of the role that the long terminal repeat (LTR) sequences play in this specificity and to identify the cell population(s) which is involved in the genesis of thymic lymphomas in mice. The LTR which will be characterized is that of the MCF13 murine leukemia virus, which causes thymic lymphomas. Analysis of the contribution of MCF13 LTR sequences to this tissue specificity will depend on the generation of a fine deletion map. Small deletions involving only a few nucleotides will be created by the method of oligonucleotide site-directed mutagenesis. These deletions will include nucleotide sequences which are involved in protein binding in the enhancer element and regions upstream and downstream of this element. Protein binding sites will be identified by the electrophoretic mobility shift assay in conjunction with footprinting analysis by DNAaseI nuclease and copper-phenanthroline chemical cleavages. Once sequence motifs important for tissue-specific transcription are identified, their combinatorial interactions with each other and with repressor elements will be examined. The identification of tissues and cell-types which have MCF13 LTR activity will be identified with the use of transgenic mice. The expression of different marker genes whose transcription is dependent on MCF13 LTR activity will be monitored in both prenatal and postnatal animals by RNA analysis. This analysis will be performed by primer extension assays. The phenotype of the cells which express MCF 13 LTR activity will be identified by using fluorescent antibodies specific for T cell antigens, such as Thy1, Lyt-2, L3T4, and MEL-14, and analysis by flow cytometry.
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