Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway.
We have selected mutations in genes encoding components of the signaling pathway for alpha interferon (IFN-alpha) by using a specially constructed cell line. The upstream region of the IFN-regulated human gene 6-16 was fused to the Escherichia coli guanine phosphoribosyltransferase (gpt) gene and transfected into hypoxanthine-guanine phosphoribosyltransferase-negative human cells. These cells express gpt only in the presence of IFN-alpha. They grow in medium containing hypoxanthine, aminopterin, and thymidine plus IFN and are killed by 6-thioguanine plus IFN. Two different types of mutants were obtained after treating the cells with mutagens. A recessive mutant, selected in 6-thioguanine plus IFN, was completely resistant to IFN-alpha but responded normally to IFN-gamma and, unexpectedly, partially to IFN-beta. A constitutive mutant, selected in hypoxanthine-aminopterin-thymidine alone, was abnormal in expressing endogenous genes in the absence of IFN. Both types revert infrequently, allowing selection for complementation of the defects by transfection.
Pellegrini
S
S
Imperial Cancer Research Fund, London, United Kingdom.
John
J
J
Shearer
M
M
Kerr
I M
IM
Stark
G R
GR
D015152:Blotting, Northern; D002459:Cell Fusion; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D005786:Gene Expression Regulation; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase; D007370:Interferon Type I / Q000378:metabolism / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D009154:Mutation; D010430:Pentosyltransferases / Q000235:genetics; D015398:Signal Transduction; D013866:Thioguanine / Q000494:pharmacology; D014162:Transfection
D007370:Interferon Type I; D007371:Interferon-gamma; D010430:Pentosyltransferases; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine