Introducing mutations into a chromosomal rRNA gene using a genetically modified eubacterial host with a single rRNA operon
Introducing mutations into a chromosomal rRNA gene using a genetically modified eubacterial host with a single rRNA operon
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DOI：
10.1046/j.1365-2958.1996.01532.x
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发表时间：
1996-12
影响因子：
3.6
通讯作者：
P. Sander;T. Prammananan;Erik C. Bo¨ttger
中科院分区：
文献类型：
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作者：
P. Sander;T. Prammananan;Erik C. Bo¨ttger
基因失活技术被用来构建含有单一功能rRNA操纵子的真细菌有机体。污垢分枝杆菌的这个突变体允许用来自载体携带的基因的同源片段来替换单个剩余的rRNA操纵子。通过与染色体的同源重组，带有抗性标记的质粒携带的rDNA片段取代了染色体rRNA操纵子的相应区域，导致细胞中突变核糖体的同质性群体。作为第一个结果，我们证明了单等位基因敲除菌株允许分离具有耐药表型的rRNA突变体，绕过了隐藏性的问题，该问题禁止在具有多个rRNA操纵子的生物体中分离此类突变体。随后，通过等位基因交换实验证明，所发现的rRNA突变确实在体内产生了耐药性。该系统为研究核糖体RNA的结构和功能提供了诱人的潜力。
Gene‐inactivation techniques were employed to construct a eubacterial organism harbouring a single functional rRNA operon. This mutant of Mycobacterium smegmatis permits replacement of the single remaining rRNA operon with a homologous fragment from a vector‐borne gene. By homologous recombination with the chromosome a plasmid‐borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, resulting in a homogeneous population of mutated ribosomes in the cell. As a first result we demonstrate that the single allelic knock‐out strain allows for isolation of rRNA mutants with a drug‐resistant phenotype, circumventing the problem of recessivity which prohibits the isolation of such mutants in organisms with multiple rRNA operons. Subsequently, by allelic exchange experiments, it was demonstrated that the rRNA mutation found indeed confers drug resistance in vivo. This system provides intriguing potential for the study of the structure and function of ribosomal RNAs.