McKendry, Janet Roslyn; (1992) Mutations in interferon signalling pathways. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The thesis describes the successful use of a genetic system set up to analyse early steps in the cellular response to interferon (IFN)-α/β. The human cell line 2fTGH is an HPRT cell line containing the selectable marker guanine phosphoribosyltransferase regulated by IFN-. Mutagenised 2fTGH cells were selected in 6-thioguanine plus IFN-α to yield cell lines unresponsive to IFN-α, or in hypoxanthine, aminopterin, thymidine (HAT) medium minus IFN-α to yield cell lines expressing IFN-α-inducible genes constitutively. Mutant cell lines defining four complementation groups of unresponsive mutants have been selected (U1-U4). Complementation group U1 has already been described. Cell lines from complementation group U2 show no response to IFN-α/p and some genes have lost their response to IFN-γ, i.e. 6-16, 9-27, and 1-8U. This indicates that there is a connection between the IFN-γ response of these genes and the IFN-α/β response. A major defect appears to be in the synthesis or activation of E, the transcription factor mediating the primary response to IFN-α/β. Band shift complementation assays reveal that IFN-α-treated U2 cells contain the functional Ea subunit of E but that untreated or IFN-γ-treated U2 cells lack the functional Eα subunit. Complementation groups U3 and U4 have no response to IFNs-α/β, or -γ, and are partially defective in their response to double-stranded RNA. This indicates that the response pathways to these three effectors are likely to share two common components or their function is dependent on a common enzyme or transcription factor. A major defect appears to be in the synthesis or activation of the Eα subunit of E. Three cell lines which define three complementation groups of constitutive mutants have been selected (C1-C3). All three express IFN constitutively. C1 is a recessive mutant which produces IFN-α indicating the presence of a trans-acting negative regulator of IFN-α expression. C2 is a dominant mutant which produces at least two sub-types of IFN-βα indicating the presence of a trans-acting positive regulator of IFN-α expression. C3 is a dominant mutant which produces predominantly IFN-β indicating the presence of a trans-acting positive regulator of IFN-β expression.

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