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Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae
Abstract:
We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.
Insights
Researchers identified yeast mutants resistant to diphtheria toxin. These mutants are defective in modifying elongation factor 2, a key step for toxin action, offering insights into toxin resistance mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Diphtheria toxin inhibits protein synthesis by ADP-ribosylating elongation factor 2 (EF-2).
- Saccharomyces cerevisiae spheroplasts are sensitive to diphtheria toxin.
- Understanding toxin resistance mechanisms is crucial for biological and therapeutic applications.
Purpose of the Study:
- To develop a selection method for isolating yeast mutants resistant to diphtheria toxin.
- To identify the genetic basis of diphtheria toxin resistance in Saccharomyces cerevisiae.
- To elucidate the role of elongation factor 2 modification in toxin sensitivity.
Main Methods:
- A selection procedure using mutagenized Saccharomyces cerevisiae spheroplasts in the presence of diphtheria toxin.
- In vitro screening of transformed survivors for resistance to diphtheria toxin-mediated elongation factor 2 modification.
- Genetic analysis including complementation tests, heterozygous diploid formation, and tetrad analysis.
Main Results:
- Isolation of 31 haploid ADP ribosylation-negative mutants exhibiting resistance to diphtheria toxin.
- Mutants were categorized into five complementation groups, indicating defects in distinct genes.
- Genetic analysis confirmed the recessive nature of the resistant phenotype and its segregation as a single Mendelian character.
Conclusions:
- The isolated mutants are defective in enzymatic steps responsible for the posttranslational modification of elongation factor 2.
- This modification is essential for the recognition and action of diphtheria toxin.
- The study provides a genetic tool for investigating diphtheria toxin mechanisms and EF-2 modification.