This laboratory has been involved in studying the interferon (IFN) receptors and the early events in IFN action. Receptors recognized by IFN-alpha and IFN-beta (type I IFN) and IFN- gamma (type II IFN) have been identified and some of their characteristics have been studied. Studies are planned to determine whether the receptors for human IFN (HuIFN)- alpha/beta consist of subunits (or other factors) associated through non-covalent bonds, a possibility suggested by some of our results, by their chromatographic behavior and radiation inactivation analysis to determine their target size, and if indicated, to determine their subunit composition. Role of IFN receptors in differential induction of cellular gene expression by type I and type II IFNs will be studied. We have isolated, from a cDNA library, two cDNA clones that are complementary to mRNAs induced by HuIFN-gamma. One of the cDNA clones is complementary to a 2.2 kb mRNA that is induced very strongly by HuIFN-gamma (- 100-fold) but less so by HuIFN-alpha 2 ( -10- fold). Whereas the induction by HuIFN-gamma is sustained for days, the induction by HuIFN-alpha 2 is transient and is followed by a shut-off, indicating differential regulation by the two types of IFNs. The isolated cDNA clones will be further characterized by restriction mapping and by Southern blotting analysis of genomic DNA to determine whether the corresponding gene exists as a single copy gene or in multiple copies in the genome. The cognate mRNA will be isolated by hybridization selection and translated in vitro to identify the protein product. Nuclear run- off transcription experiments will be carried out to determine whether this induction is a transcriptional effect or a post- transcriptional effect. Such cDNA clones will be used as probes to study whether the differential induction of cellular genes by type I and type II IFNs is due to events at the receptor level or at post-receptor level. For example, we will determine whether the transient induction by HuIFN-alpha 2 and sustained induction by HuIFN-gamma is due to receptor down regulation and recycling, respectively, or due to the appearance of a negative control mechanism in the case of HuIFN-alpha 2 and not with HuIFN- gamma, whether inhibitors of receptor recycling (e.g. monensin) would affect the persistant nature of induction in the case of HuIFN-gamma, and test whether amino acid analogs would prevent the shut-off observed with HuIFN-alpha 2. Finally, we will make attempts to develop a cell-free system which may allow an investigation of the nature of the signal (mediators) generated upon IFN receptor interaction leading to an activation of specific cellular genes.
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