Autoradiographic detection of mutation to exotoxin-A resistance in mouse fibroblasts treated with ethyl methanesulfonate, X-rays and ultraviolet light.
P. aeruginosa exotoxin-A (PE) blocks protein synthesis in mammalian cells by inactivating elongation factor 2 (EF-2). Toxin-resistant mutant cells can be detected autoradiographically, in cultures grown on microscope coverslips in the presence of PE, and then exposed to [3H]leucine. The frequency of PE-resistant cells detected by the autoradiographic assay in non-mutagenized cells of the established mouse cell line LTKA is 9.7 +/- 0.6 X 10(-5). Upon treatment with ethyl methanesulfonate (EMS), X-rays or ultraviolet (UV) light it increases in a dose-dependent fashion. The mutational nature of the resistance detected by the assay is indicated by its clonal inheritance, and by the dose-dependent increase in the frequency of resistant cells after mutagenesis. On the basis of the high frequency of PE-resistant cells detected by the autoradiographic assay, and their cross-resistance to diphtheria toxin (DT), we suggest that the PE-resistant mutants detected by the autoradiographic assay are of class II, i.e., they are altered in the structural gene for EF-2. The autoradiographic assay for PE resistance is similar to that for DT resistance, but is applicable also to mouse cells, which are naturally resistant to DT. Being independent of colony formation, the autoradiographic assay for PE resistance can be used with non-dividing cells, either in vitro or in vivo.
Tiah
M
M
Department of Genetics, Hebrew University of Jerusalem, Israel.
Ronen
A
A
D036002:ADP Ribose Transferases; D000818:Animals; D001345:Autoradiography; D001427:Bacterial Toxins; D002470:Cell Survival / Q000187:drug effects; D005020:Ethyl Methanesulfonate / Q000633:toxicity*; D005098:Exotoxins / Q000633:toxicity*; D005347:Fibroblasts / Q000187:drug effects / Q000528:radiation effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D014466:Ultraviolet Rays; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005020:Ethyl Methanesulfonate; D005098:Exotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A