Hughes D
Department of Molecular Biology, Biomedical Center, Uppsala, Sweden.
J Mol Biol. 1990 Sep 5;215(1):41-51. doi: 10.1016/S0022-2836(05)80093-2.
Each of the two genes encoding EF-Tu in Salmonella typhimurium has been inactivated using a mini-Mu MudJ insertion. Eleven independently isolated insertions are described, six in tufA and five in tufB. Transduction analysis shows that the inserted MudJ is 100% linked to the appropriate tuf gene. A mutant strain with electrophoretically distinguishable EF-TuA and EF-TuB was used to show, on two-dimensional gels, that the MudJ insertions result in the loss of the appropriate EF-Tu protein. Southern blotting, using cloned Escherichia coli tuf sequences as probes, shows that each MudJ insertion results in the physical breakage of the appropriate tuf gene. The degree of growth-rate impairment associated with each tuf inactivation is independent of which tuf gene is inactivated. The viability of S. typhimurium strains with either tuf gene inactive contrasts strongly with data suggesting that in the closely related bacterium E. coli, an active tufA gene is essential for growth. Finally the strains described here facilitate the analysis of phenotypes associated with individual mutant or wild-type Tus both in vivo and in vitro.
利用mini-Mu MudJ插入技术使鼠伤寒沙门氏菌中编码EF-Tu的两个基因中的每一个都失活。描述了11个独立分离的插入情况,6个插入到tufA中,5个插入到tufB中。转导分析表明,插入的MudJ与相应的tuf基因100%连锁。使用具有电泳可区分的EF-TuA和EF-TuB的突变菌株,在二维凝胶上显示MudJ插入导致相应的EF-Tu蛋白缺失。以克隆的大肠杆菌tuf序列为探针进行Southern印迹分析表明,每个MudJ插入都会导致相应tuf基因的物理断裂。与每个tuf失活相关的生长速率受损程度与失活的是哪个tuf基因无关。任一tuf基因失活的鼠伤寒沙门氏菌菌株的活力与表明在密切相关的细菌大肠杆菌中,活跃的tufA基因对生长至关重要的数据形成强烈对比。最后,这里描述的菌株便于在体内和体外分析与单个突变型或野生型Tus相关的表型。