Montandon P E, Wagner R, Stutz E
EMBO J. 1986 Dec 20;5(13):3705-8. doi: 10.1002/j.1460-2075.1986.tb04703.x.
Resistance to streptomycin (Sm) of Euglena gracilis chloroplasts can be due to a single C to T transition of the 16S rRNA gene in an invariant position which is equivalent to C912 of the Escherichia coli 16S rRNA. Since Euglena chloroplasts cannot be transformed we introduced, by site-directed mutagenesis, a C912 to T transition in the cloned rrnB operon (pKK3535) of E. coli and used this new construct (pEM109) in transformation experiments. Transformed E. coli cells were selected for Sm resistance by colony plating and stepwise increase of Sm up to 25 micrograms/ml of culture medium. Several Sm-resistant colonies were obtained. Ribosomes were isolated from pEM109-transformed Sm-resistant and pKK3535-transformed Sm-sensitive cells. The ribosomes were assayed in vitro for Sm-induced misreading of poly(U) mRNA. We isolated 16S rRNA and sequenced the crucial RNA region by reverse transcription. The results clearly show that ribosomes from Sm-resistant cells correctly read the poly(U) mRNA in the presence of 25 micrograms Sm/ml of reaction mixture and the 16S rRNA contains the C912 to U transition. We conclude that C912 is involved in a translation step(s) which is (are) sensitive to streptomycin.
纤细裸藻叶绿体对链霉素（Sm）的抗性可能是由于16S rRNA基因在一个不变位置上发生了单个C到T的转变，该位置相当于大肠杆菌16S rRNA的C912。由于纤细裸藻叶绿体无法进行转化，我们通过定点诱变在大肠杆菌克隆的rrnB操纵子（pKK3535）中引入了C912到T的转变，并在转化实验中使用了这个新构建体（pEM109）。通过菌落平板培养并逐步将链霉素浓度提高到每毫升培养基25微克来选择对链霉素具有抗性的转化大肠杆菌细胞。获得了几个抗链霉素的菌落。从pEM109转化的抗链霉素细胞和pKK3535转化的链霉素敏感细胞中分离核糖体。在体外检测核糖体对链霉素诱导的聚（U）mRNA错读情况。我们分离出16S rRNA，并通过逆转录对关键RNA区域进行测序。结果清楚地表明，在每毫升反应混合物中存在25微克链霉素的情况下，抗链霉素细胞的核糖体能够正确读取聚（U）mRNA，并且16S rRNA包含从C912到U的转变。我们得出结论，C912参与了对链霉素敏感的一个或多个翻译步骤。
