Abstract:
My thesis research had two goals. One was to identify physiological signals that induce the transcription factor NF-kappaB in pre-B and T cells. The second was to understand the biological basis of the paradox that NF-kappaB regulated inducible genes, such as IL-2 receptor alpha, are not constitutively active in B cells where NF-kappaB is constitutively present. In most cells, NF-kappaB exists in the cytoplasm in association with its inhibitor, IkappaB. Only in B cells is it constitutively present in the nucleus. Artificial inducing agents such as phorbol esters were known to induce NF-kappaB translocation to the nucleus on non-B cells. We were interested in determining key physiological signals that induce NF-kappaB. We showed that NF-kappaB is induced via the T cell receptor in T cells activated by antigen. To determine whether the inducible NF-kappaB had a physiological role, we were interested in finding NF-kappaB regulated genes in non-B cells. We identified a functional, NF-kappaB binding sequence, TCR-B, in the vicinity of previously identified, T cell-specific DNAaseI hypersensitive sites within the T cell receptor beta2 locus. Many NF-kappaB-regulated genes are expressed when others are not. We investigated the contribution of different kappaB-like DNA sequences to NF-kappaB function. The sequences from constitutive genes had a higher affinity for B cell NF-kappaB and were transcriptionally more active than sites from inducible genes. To study the NF-kappaB protein itself and protein interactions in vivo I developed a peptide rabbit antiserum to the p50 sub-unit C-terminus. This antibody discriminates between p50/p65 and p50/c-rel heterodimers in an electrophoretic mobility shift assay (EMSA), indicating a difference in p50 C-terminus accessibility. In super-shift EMSA I found that p50 participated in several NF-kappaB complexes including TCR induced complexes. Immunoprecipitation from B and T cells revealed a novel protein, p28, that co-precipitates with nuclear p50. Infection with human T cell leukemia virus type 1 (HTLV-1) results in adult T cell leukemia. HTLV-1 can infect a wide variety of cell types but transformation is restricted to T cells. HTLV-1 tax does not bind DNA itself yet somehow induces NF-kappaB to activate transcription. In transient transfection assays HTLV-1 tax transactivated NF-kappaB dependent transcription more potently in T cells than nonlymphoid cells and therefore could be a factor contributing to the disease specificity of HTLV-1. (Full text available from University Microfilms International, Ann Arbor, MI, as Order No. AAD93-22575)
Keywords: B-Lymphocytes/CHEMISTRY HTLV-I/GENETICS NF-kappa B/*GENETICS/PHYSIOLOGY Receptors, Antigen, T-Cell/GENETICS Sequence Analysis, DNA *Signal Transduction T-Lymphocytes/CHEMISTRY Transcription, Genetic/GENETICS THESIS 940530
M9450896
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