Human type I interferons offer a relevant system to examine cell-specific inducible gene expression. Interferon genes are transcriptionally activated in a variety of cell types following induction by synthetic double-stranded RNA (poly I:C) and viruses. A transient expression system was developed which permits regulated expression of different IFN-CAT (chloramphenicol acetyltransferase) hybrid genes in human cells. Using this system in vivo competition assays identified positive and negative cellular factors interacting with the IFN-$ beta$ promoter. A factor that recognizes negative upstream regulatory sequences was also identified in uninduced myeloid cell extracts. Complementary studies demonstrated that transcription of the IFN-$ beta$ gene in vitro could be inhibited by a 44 bp synthetic oligonucleotide corresponding to the interferon regulatory element (IRE). This element is comprised of two genetically distinct positive regulatory domains, PRDI and PRDII, that are essential for maximal induction by virus or poly I:C. Binding and competition analysis showed that the PRDI element interacts with a factor(s) present in uninduced and virus-induced Hela extracts. The DNA-binding specificity of the PRDI factor(s) is characteristic of proteins recently shown to be involved in IFN-stimulated gene transcription. The PRDII domain is unrelated to PRDI, but shares 80% nucleotide homology with the NF-$ kappa$B binding site in the immunoglobulin kappa enhancer. PRDII was found to interact with an NF-$ kappa$B-like activity in both lymphoid and non-lymphoid cell extracts. An HIV-1 enhancer oligonucleotide containing two repeated $ kappa$B elements was interchangeable with PRDII in the gel retardation assay. UV-crosslinking analysis further revealed that several distinct inducible and constitutive proteins bind specifically to PRDII in different cell types, and suggests that multiple factors may interact with this element to regulate IFN-$ beta$ transcription. Taken togeth
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.74353 |
| Date | January 1990 |
| Creators | Xanthoudakis, Steven |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001232622, proquestno: AAINN63760, Theses scanned by UMI/ProQuest. |
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