A series of mutants of the ME-180 (human cervical carcinoma) cell line resistant to the cytotoxic effects of interferon-gamma have been isolated. Such mutants have been found to be defective in indoleamine 2,3-dioxygenase (IDO) induction, the first enzyme of the tryptophan catabolic pathway. Second round mutants have also been isolated lacking completely IDO induction and affected also in other interferon-inducible responses, such as HLA and oligo A synthetase induction. The relationship between IDO induction, tryptophan transport and metabolism will be investigated in order to ascertain the physiological significance of this pathway. Using a cloned cDNA we shall investigate whether the IDO-mutants are point mutations or result from mutation in regulatory sequences using gel- retardation assays. Northern blot and Southern analyses will be done with """"""""global"""""""" mutants to investigate whether such mutants result from deletions or alterations in the signalling pathway. Receptor binding studies using I125 interferon will also be done. The structure of interferon responsive sequences in IDO genomic DNA will be investigated. The regulation of this gene by interferon-gamma will be investigated using mutant and wild-type cells. Finally a genetic analysis of the mutants will be undertaken to locate the gene(s) affected to their respective chromosomes, and to ascertain whether the mutations are allelic.
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