EVIDENCE FOR A SINGLE ROLLING CIRCLE IN THE REPLICATION OF POTATO SPINDLE TUBER VIROID
EVIDENCE FOR A SINGLE ROLLING CIRCLE IN THE REPLICATION OF POTATO SPINDLE TUBER VIROID
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DOI：
10.1073/pnas.85.23.9128
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发表时间：
1988-12-01
影响因子：
11.1
通讯作者：
ROBERTSON, HD
中科院分区：
文献类型：
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作者：
BRANCH, AD;BENENFELD, BJ;ROBERTSON, HD
我们对体内标记的RNA进行了分析，以确定所提出的两种滚圆模型中哪一种更可能描述马铃薯纺锤型块茎类病毒的复制周期。区别这两种模型的一个关键特征是其中一种模型中存在环状单体负链，而另一种模型中没有。用CF11纤维素柱层析从单链后代中分离出含有复制中间体的组份。用热变性和凝胶电泳寻找可能的环状模板。进行滚环复制所需的物种。加热后，32P标记的RNA被释放。限制性核酸酶消化(“缺口”)显示，这是一个单位长度的环状RNA。指纹鉴定它是一条正链。在这个群体中或在含有复制复合体的RNaseT1抗性核心的核酸酶处理的样本中，没有检测到环状负链。因此，马铃薯纺锤型块茎类病毒似乎使用了一种不对称的途径，在这种途径中，负链通过滚圈复制合成，而正链则不是。需要了解各种类病毒RNA所使用的复制途径的更多细节，这将有助于建立这些病原体之间的进化关系。
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.sbd.species required for rolling-circle replication to take place. Upon heating, a 32P-labeled RNA was released. Limited nuclease digestion ("nicking") revealed that that was a unit-length 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 relationship among these infectious agents.