Chetverin A B, Chetverina H V, Munishkin A V
Institute of Protein Research, Academy of Sciences of the U.S.S.R., Moscow.
J Mol Biol. 1991 Nov 5;222(1):3-9. doi: 10.1016/0022-2836(91)90729-p.
Numerous RNA species of different length and nucleotide sequence grow spontaneously in vitro in Q beta replicase reactions where no RNA templates are added deliberately. Here, we show that this spontaneous RNA synthesis by Q beta replicase is template directed. The immediate source of template RNA can be the laboratory air, but there are ways to eliminate, or at least substantially reduce, the harmful effects of spontaneous synthesis. Solitary RNA molecules were detected in a thin layer of agarose gel containing Q beta replicase, where they grew to form colonies that became visible upon staining with ethidium bromide. This result provides a powerful tool for RNA cloning and selection in vitro. We also show that replicating RNAs similar to those growing spontaneously are incorporated into Q beta phage particles and can propagate in vivo for a number of phage generations. These RNAs are the smallest known molecular parasites, and in many aspects they resemble both the defective interfering genomes of animal and plant viruses and plant virus satellite RNAs.
在不特意添加RNA模板的Qβ复制酶反应中，许多不同长度和核苷酸序列的RNA种类会在体外自发产生。在此，我们表明Qβ复制酶的这种自发RNA合成是由模板指导的。模板RNA的直接来源可能是实验室空气，但有方法可以消除或至少大幅降低自发合成的有害影响。在含有Qβ复制酶的琼脂糖凝胶薄层中检测到了单个RNA分子，它们在其中生长形成菌落，用溴化乙锭染色后可见。这一结果为体外RNA克隆和筛选提供了一个强大的工具。我们还表明，与自发生长的RNA相似的复制RNA会被整合到Qβ噬菌体颗粒中，并能在体内传播多个噬菌体世代。这些RNA是已知最小的分子寄生物，在许多方面它们类似于动植物病毒的缺陷干扰基因组和植物病毒卫星RNA。
