ABSTRACT
Mutant cell lines B3 and B10, which are unresponsive to both interferon (IFN)-alfa and IFN-gama, and line B9, which does not respond to IFN-gama stimulation, are described. The mutants were submitted to fluorescence-activated cell sorting (FACS) from a cellular pool, which was obtained from the parental cell line 2C4 after several rounds of mutagenesis. The unresponsiveness to IFN stimulation was observed both in terms of expression of cell surface markers (CD2, class I and II HLAs) and mRNA expression of IFN-stimulated genes (2'-5' oligoadenylate synthetase (OAS), 9-27, and guanylate binding protein (GBP)). Genetic crossing of B3, B9 and B10 with U3 (STAT1-),gama2 (JAK2-) and U4 (JAK1-) mutants, respectively, did not restore IFN responsiveness to the hybrid cell lines. However, when these cell lines were crossed with the same mutants, but using the pairwise crosses B3 x U4, B9 x U3 and B10 x U3, the cell hybrids recovered full IFN responsiveness. The present genetic experiments permitted us to assign the mutant cell lines B3, B9 and B10 to the U3, gama2 and U4 complementation groups, respectively. These conclusions were supported by the analysis of IFN-stimulated genes in the mutants.
Subject(s)
Protein-Tyrosine Kinases/deficiency , Signal Transduction , Cell Line/enzymology , Cell Separation , Flow Cytometry , Interferons , Mutation , Ribonucleases , Transcriptional ActivationABSTRACT
A recessive mutant cell line, B7, which is partially responsive to both interferon (IFN)-(alpha and IFN-gamma is described. B7 was FACS sorted from a cellular pool, which was obtained from the parental cell line 2C4 after several rounds of mutagenesis. The partial responsiveness to IFN was observed both in terms of expression of cell surface markes (CD2, class I and II HLAS) and mRNA expression of IFN-stimulated genes (9-27; 6-16; 2'-5' OAS; GBP and HLA-DRalpha). A genetic cross with the U4 mutant (JAK I -, a member of the Janus family of nonr ceptor tyrosine kinase) did not restore full IFN responsiveness to B7 and JAK1 cDNA transfection into B7 restored the wild phenotype of the cell line, defining B7 as a member of the U4 complementation group. Nevertheless, JAK1 mRNA was not detected in this mutant Transcriptional regulator complexes such as IRF 1/2 (IFN-regulatory factor) and ISGF3gamma (IFN-stimulated gene factor) were constitutively formed in the B7 mutant and co-migrated with the IFN-induce complexes expressed in the parental cell line 2C4. Thus, this cell line seems to be useful for understanding cis-acting elements governing JAK1 mRNA expression.