Characterization of diphtheria-toxin-resistant mutants lacking receptor function or containing nonribosylatable elongation factor 2.
Stable mutants resistant to diphtheria toxin (DT) were isolated from Chinese hamster ovary cells (CHO-K1) by single-step mutations with various mutagens. All the mutants were classified into two major groups as reported by other workers (4-6): toxin-entry mutants (DTrI) and translational mutants (DTRII) at the level of elongation factor 2 (EF-2). These mutants were further characterized by directly measuring the specific uptake of [125I]DT and the content of nonribosylatable EF-2 by two-dimensional gel analysis. DTrI mutants, which showed no cross-resistance to Pseudomonas exotoxin A (PA), had no ability to associate with [125I]DT and contained only ADP-ribosylatable EF-2, like wild-type cells. DTRIIb mutants maintained about 50% of the normal level of cellular protein synthesis in the presence of DT, and two-dimensional gel analysis directly showed that they contained equivalent amounts of ADP-ribosylatable and nonribosylatable EF-2 molecules. Fully toxin-resistant cells, named KEE1 (DTRIIa), were isolated from a DTRIIb mutant (KE1) by two-step mutation. KEE1 cells showed full resistance to DT and PA, the normal level of association with [125I]DT, and produced only nonribosylatable EF-2. Biochemical analysis of somatic cell hybrids indicated that the DT-resistant character of class II behaved codominantly. These results strongly supported the hypothesis that two copies of the gene for EF-2 are functional in CHO-K1 cells.
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004167:Diphtheria Toxin / Q000494:pharmacology*; D004351:Drug Resistance; D005098:Exotoxins / Q000494:pharmacology; D005260:Female; D005796:Genes; D066257:Heparin-binding EGF-like Growth Factor; D006822:Hybrid Cells; D036341:Intercellular Signaling Peptides and Proteins; D009154:Mutation; D010053:Ovary; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D014176:Protein Biosynthesis; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000378:metabolism*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D001427:Bacterial Toxins; D004167:Diphtheria Toxin; D005098:Exotoxins; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A