Characterization of diphtheria-toxin-resistant mutants lacking receptor function or containing nonribosylatable...

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.