Ferretti L, Mottes M, De Fazio G, Sgaramella V
Gene. 1984 Jul-Aug;29(1-2):11-9. doi: 10.1016/0378-1119(84)90160-4.
A 5300-bp DNA segment containing the promoter, the attenuator and the first gene (hisG) of the Escherichia coli his operon has been inserted into an interspecific E. coli-Bacillus subtilis plasmid vector, pHV14. The resulting plasmid pPV48 restores the His+ phenotype to an E. coli hisG mutant, but fails to do so to a corresponding B. subtilis mutant. Experiments aimed at localizing the block to this heterologous expression in B. subtilis have shown that the enzymatic activity of the hisG+ gene product is neither detectable nor inhibited in crude extracts of B. subtilis cells harboring pPV48. Furthermore, electron microscopic, Southern blot and S1 mapping analysis of the transcripts produced in vitro and in vivo by B. subtilis RNA polymerase indicate that the hisG+ region is transcribed, but that the transcripts initiate at sites different from the his promoter, converge towards, and terminate in the vicinity of the attenuator.
一段含有大肠杆菌组氨酸操纵子启动子、弱化子和第一个基因(hisG)的5300碱基对DNA片段已被插入到种间大肠杆菌-枯草芽孢杆菌质粒载体pHV14中。所得质粒pPV48可使大肠杆菌hisG突变体恢复His+表型,但对相应的枯草芽孢杆菌突变体则无此作用。旨在确定枯草芽孢杆菌中这种异源表达受阻位置的实验表明,在携带pPV48的枯草芽孢杆菌细胞粗提物中,hisG+基因产物的酶活性既无法检测到,也未受到抑制。此外,对枯草芽孢杆菌RNA聚合酶在体外和体内产生的转录本进行的电子显微镜、Southern印迹和S1图谱分析表明,hisG+区域被转录,但转录本起始于与组氨酸启动子不同的位点,朝着弱化子汇聚并在其附近终止。