This is a proposal to study the molecular mechanisms of human Beta-interferon (IFN) gene regulation using recombinant DNA techniques and mammalian cell transformation procedures. A cloned human Beta-IFN gene will be stably introduced into a mouse fibroblast cell line using a bovine papillomavirus (BPV) expression vector which replicates as an extrachromosomal DNA element. The DNA sequences required for poly(I)-poly(C) induction of the extrachromosomal Beta-IFN gene will be identified by introducing deletion and single base mutations into the 5 feet flanking region of the gene and examining the effect of these mutations on the inducibility of the gene in cells in culture. Once the cis-acting regulatory sequences have been identified, nuclease and dimethyl sulfate protection experiments will be carried out to analyze possible protein-DNA interactions in the regulatory region and to determine whether structural changes can be detected upon poly(I)-poly(C) activation of the gene. Attempts will also be made to identify and analyze regulatory proteins which interact with the Beta-IFN regulatory region using filter binding assays or a bioassay which involves the injection of the cloned Beta-IFN gene into frog oocyte nuclei followed by the injection of extracts from poly(I)-poly(C) treated human cells. Finally, experiments will be carried out to identify mutations in cellular genes that control Beta-IFN gene expression. This will be accomplished by developing an in situ replica plating assay for IFN gene expression. If successful, the proposed experiments should lead to a better understanding of the mechanisms by which human cells resist viral infections.
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