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Characterization of diphtheria-toxin-resistant mutants lacking receptor function or containing nonribosylatable
Abstract:
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.
Insights
Researchers identified two types of diphtheria toxin (DT) resistant mutants in Chinese hamster ovary cells: toxin-entry and translational mutants affecting elongation factor 2 (EF-2). These findings suggest two functional copies of the EF-2 gene exist in these cells.
Area of Science:
- Cell Biology
- Molecular Genetics
- Toxicology
Background:
- Diphtheria toxin (DT) exerts its cytotoxic effect by inhibiting protein synthesis.
- Elongation factor 2 (EF-2) is the target of DT's ADP-ribosylating activity.
- Chinese hamster ovary (CHO-K1) cells provide a model for studying toxin resistance mechanisms.
Purpose of the Study:
- To isolate and characterize stable mutants of CHO-K1 cells resistant to diphtheria toxin.
- To elucidate the molecular mechanisms underlying DT resistance.
- To investigate the gene dosage of elongation factor 2 (EF-2) in CHO-K1 cells.
Main Methods:
- Induction of stable mutants resistant to diphtheria toxin using various mutagens.
- Classification of mutants into toxin-entry (DTrI) and translational (DTRII) groups.
- Quantification of [125I]DT uptake and analysis of EF-2 ADP-ribosylation status via two-dimensional gel electrophoresis.
Main Results:
- DTrI mutants showed no DT binding and possessed only ADP-ribosylatable EF-2.
- DTRIIb mutants exhibited partial resistance and contained both ADP-ribosylatable and nonribosylatable EF-2.
- A fully resistant mutant (KEE1) was derived, displaying resistance to both DT and Pseudomonas exotoxin A (PA), with only nonribosylatable EF-2.
- Somatic cell hybridization data indicated codominant inheritance of the DTRII phenotype.
Conclusions:
- The study identified distinct mechanisms of DT resistance related to toxin entry and EF-2 modification.
- The results strongly support the hypothesis of two functional copies of the EF-2 gene in CHO-K1 cells.
- This provides insights into gene regulation and toxin resistance in mammalian cells.