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

Abstract

We have isolated temperature-sensitive diphtheria toxins (DT-A(ts)) to develop a method that allows temporal impedement of cellular functions. Four DT-A(ts) genes were isolated in a mutagenesis screen using the yeast, Saccharomyces cerevisiae. When expressed in yeast, these DT-A(ts) arrest growth at 18 degrees C but not at 30 degrees C. Three DT-A(ts) 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-A(ts) mutations are novel. Interestingly, the fourth DT-A(ts) carries the same point mutation as the extensively characterized CRM197, an ADP ribosyltransferase-defective form of diphtheria toxin.