Isolation of temperature-sensitive diphtheria toxins in yeast and their effects on Drosophila cells

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Abstract
We have isolated temperature-sensitive diphtheria toxins (DT-At5) to develop a method that allows temporal impedement of cellular functions. Four DT-Ats genes were isolated in a mutagenesis screen using the yeast, Saccharomyces cerevisiae. When expressed in yeast, these DT-Ats arrest growth at 18°C but not at 30°C. Three DT-At5 were subsequently tested in the R1-R6 photoreceptor cells of transgenic fruit flies, Drosophila melanogaster. The toxins show similar temperature dependence in both organisms, suggesting that they may be useful in a wide range of non-homeothermic species. DNA sequence analysis revealed that three of the four DT-At5 mutations are novel. Interestingly, the fourth DT-Ats carries the same point mutation as the extensively characterized CRM197, an ADP ribosyltransferase-defective form of diphtheria toxin.