Chen J Y, Bodley J W, Livingston D M
Mol Cell Biol. 1985 Dec;5(12):3357-60. doi: 10.1128/mcb.5.12.3357-3360.1985.
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.
我们基于白喉毒素可杀死酿酒酵母原生质球这一观察结果（村上等，《分子与细胞生物学》2:588 - 592，1982年）开发了一种筛选程序；该程序产生了对毒素体外作用具有抗性的突变体。在白喉毒素存在的情况下对诱变后的酿酒酵母原生质球进行转化，并在体外筛选转化后的存活者，以寻找对毒素具有抗性的延伸因子2。通过此程序获得了31个单倍体ADP核糖基化阴性突变体，它们包含五个互补组。这些突变体生长正常，且在长期保存下仍保持稳定。将野生型敏感细胞与这些突变体进行杂交产生的杂合二倍体表明，在每种情况下，抗性表型相对于敏感表型都是隐性的。这些二倍体的孢子形成产生了四分体，其中抗性表型作为单一孟德尔性状分离。基于这些观察结果，我们得出结论，这些突变体在负责延伸因子2翻译后修饰的酶促步骤中存在缺陷，而这种修饰是白喉毒素识别所必需的。
