Sutton C A, Ares M, Hallberg R L
Proc Natl Acad Sci U S A. 1978 Jul;75(7):3158-62. doi: 10.1073/pnas.75.7.3158.
Two cycloheximide-resistant mutants of Tetrahymena thermophila were analyzed to determine the site of their cycloheximide resistance. The mutations in both strains had been previously shown to be genetically dominant and located at separate loci (denoted Chx-A and Chx-B). Strains carrying these mutations were readily distinguished by the extent to which they were resistant to the drug. The homozygous double mutant was more resistant than either single mutant. Cell-free extracts of wild type and of the three mutant strains, assayed for protein synthetic activity by both runoff of natural mRNA and poly(U)-dependent phenylalanine polymerization, demonstrated that in vitro the mutants were all more resistant than the wild type. Further fractionation of the cell-free systems into ribosomes and supernates localized cycloheximide resistance to the ribosome for both Chx-A and Chx-B homozygotes. Ribosome dissociation and pairwise subunit mixing in the in vitro system indicated that ribosome resistance was conferred by the 60S subunit from one strain whereas resistance in the other strain was mediated through the 40S subunit. This was further confirmed by reconstruction of all four cycloheximide-resistance "phenotypes" by mixing ribosomal subunits from appropriate strains. This finding suggests that the mechanisms by which these mutations confer resistance to cycloheximide are different.
对嗜热四膜虫的两个抗放线菌酮突变体进行了分析，以确定其抗放线菌酮的位点。先前已表明这两个菌株中的突变在遗传上是显性的，且位于不同的位点（分别表示为Chx - A和Chx - B）。携带这些突变的菌株很容易通过它们对该药物的耐受程度来区分。纯合双突变体比任何一个单突变体都更具抗性。通过天然mRNA的连续转录和聚（U）依赖性苯丙氨酸聚合反应对野生型和三个突变菌株的无细胞提取物进行蛋白质合成活性测定，结果表明在体外突变体都比野生型更具抗性。将无细胞系统进一步分离为核糖体和上清液，结果表明Chx - A和Chx - B纯合子的抗放线菌酮能力都定位于核糖体。体外系统中的核糖体解离和亚基两两混合表明，一个菌株的核糖体抗性由60S亚基赋予，而另一个菌株的抗性则通过40S亚基介导。通过混合来自合适菌株的核糖体亚基重建所有四种抗放线菌酮“表型”，进一步证实了这一点。这一发现表明这些突变赋予抗放线菌酮能力的机制是不同的。
