Churikov N A, Chistiakova L G, Zavil'gel'skiĭ G B, Manukhov I V
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
Genetika. 2000 Jan;36(1):23-7.
To study the effect of RNA interference (RNAi) on the activity of gene lon in Escherichia coli, genetic constructs were used that could express RNA molecules complementary to the 5' region of lon mRNA in the same direction. These RNAs were termed parallel RNAs (pRNAs). Two approaches were used to control expression. In one approach, lon gene activity was estimated genetically, based on the effect of the Lon protease on bioluminescence determined by the Vibrio fischeri lux regulon. The other approach was direct testing of ATP-dependent proteolysis in vitro. It was found that pRNA considerably suppressed lon expression. The antiparallel RNA (apRNA) was a less effective suppressor of this gene. The specific RNAi was found to decay gradually by the 40th generation. The data obtained indicate that Eubacterium cells have mechanisms for specific regulation of gene activity that are sensitive to the formation of both parallel and antiparallel RNA duplexes involving mRNA of the given gene.
为研究RNA干扰(RNAi)对大肠杆菌中lon基因活性的影响,使用了能够同向表达与lon mRNA 5'区域互补的RNA分子的基因构建体。这些RNA被称为平行RNA(pRNA)。采用了两种方法来控制表达。在一种方法中,基于Lon蛋白酶对费氏弧菌lux操纵子所决定的生物发光的影响,从遗传学角度估计lon基因活性。另一种方法是直接在体外测试ATP依赖性蛋白水解。发现pRNA能显著抑制lon表达。反平行RNA(apRNA)对该基因的抑制作用较弱。发现特异性RNAi在第40代时逐渐衰减。所获得的数据表明,真细菌细胞具有对基因活性进行特异性调控的机制,该机制对涉及给定基因mRNA的平行和反平行RNA双链体的形成敏感。