The Laboratory of Genetics, The Rockefeller University, New York, NY 10021.
Proc Natl Acad Sci U S A. 1988 Dec;85(23):9128-32. doi: 10.1073/pnas.85.23.9128.
We analyzed in vivo-labeled RNA to determine which of the two proposed rolling-circle models is more likely to depict the replication cycle of potato spindle tuber viroid. A key feature distinguishing the two models is the presence of a circular monomeric minus strand in one and not the other. Chromatography on cellulose CF11 was used to purify a fraction containing the replication intermediates free from single-stranded progeny. Heat denaturation followed by gel electrophoresis was used to seek possible circular templates-species required for rolling-circle replication to take place. Upon heating, a (32)P-labeled RNA was released. Limited nuclease digestion ("nicking") revealed that this was a unitlength circular RNA. Fingerprinting identified it as a plus strand. No circular minus strands were detected in this population or in nuclease-treated samples containing RNase T1-resistant cores of the replication complex. Thus, potato spindle tuber viroid appears to use an asymmetric pathway in which minus strands are synthesized by rolling-circle copying, but plus strands are not. More details of the replication pathways used by various viroid-like RNAs are needed and will help to establish the evolutionary relationships among these infectious agents.
我们分析了体内标记的 RNA，以确定这两种提出的滚环模型中哪一种更有可能描述马铃薯纺锤块茎类病毒的复制周期。区分这两个模型的一个关键特征是一个模型中存在环状单体负链，而另一个模型中则没有。纤维素 CF11 上的色谱法用于纯化不含单链产物的复制中间体的馏分。热变性随后进行凝胶电泳，以寻找可能的环状模板-发生滚环复制所需的物种。加热后，释放出 (32)P 标记的 RNA。有限的核酸酶消化（“切口”）表明这是一个单元长度的环状 RNA。指纹图谱将其鉴定为正链。在这个群体或在含有复制复合物 RNase T1 抗性核心的核酸酶处理样品中，均未检测到环状负链。因此，马铃薯纺锤块茎类病毒似乎采用不对称途径，其中负链通过滚环复制合成，但正链则不是。需要了解各种类病毒样 RNA 所使用的复制途径的更多细节，这将有助于确定这些感染因子之间的进化关系。
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