In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus.
In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus.
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烟草花叶病毒全长 cDNA 的感染性 RNA 的体外转录。
DOI：
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发表时间：
1986
影响因子：
11.1
通讯作者：
Y. Okada
中科院分区：
文献类型：
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作者：
T. Meshi;M. Ishikawa;F. Motoyoshi;K. Semba;Y. Okada
我们将烟草花叶病毒（TMV）番茄株系L RNA的全长双链cDNA克隆到转录载体pPM 1中，该载体能从插入的双链cDNA的第一个核苷酸（对应于TMV RNA的5'端）开始正确转录。当质粒DNA在双链cDNA插入片段下游引入的独特限制性位点（Mlu I）线性化，并在m（7）GpppG存在下用作大肠杆菌RNA聚合酶体外转录的模板时，转录的RNA对烟草植物具有感染性。一个简单的重建程序增加感染性>100倍。出乎意料的是，未加帽的转录物和来自未切割的质粒DNA的转录物也是感染性的，尽管它们的感染性非常低。在烟草植物中繁殖的子代病毒准确地反映了克隆的序列。通过同样的方法，我们成功地在体外转录的弱毒株L（11）A，这是表型上区别于野生型TMV在烟草和番茄植物的感染性RNA。
We have cloned full-length double-stranded cDNAs of tobacco mosaic virus (TMV) (tomato strain L) RNA into a transcription vector, pPM1, which facilitates the correct transcription initiation from the first nucleotide of the inserted double-stranded cDNA, corresponding to the 5' end of TMV RNA. When plasmid DNA is linearized at a unique restriction site (Mlu I) introduced just downstream of the double-stranded cDNA insert and used as a template for in vitro transcription by Escherichia coli RNA polymerase in the presence of m(7)GpppG, the transcribed RNAs are infectious for tobacco plants. A simple reconstitution procedure increases the infectivity >100 times. Unexpectedly, both the uncapped transcript and the transcript from the uncut plasmid DNA are also infectious, although their infectivities are very low. The progeny viruses multiplying in tobacco plants accurately reflect the cloned sequence. By the same method, we succeeded in the in vitro transcription of infectious RNA of attenuated strain L(11)A, which is phenotypically distinguishable from wild-type TMV on both tobacco and tomato plants.