Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo 113, Japan.
Proc Natl Acad Sci U S A. 1986 Jul;83(14):5043-7. doi: 10.1073/pnas.83.14.5043.
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.
我们已经将烟草花叶病毒（TMV）（番茄株系 L）的全长双链 cDNA 克隆到转录载体 pPM1 中，该载体有利于从插入的双链 cDNA 的第一个核苷酸开始正确转录，对应于 TMV RNA 的 5'端。当质粒 DNA 在双链 cDNA 插入物下游的独特限制位点（Mlu I）处线性化，并在存在 m(7)GpppG 的情况下用作大肠杆菌 RNA 聚合酶的体外转录模板时，转录 RNA 对烟草植物具有感染性。一个简单的重组程序可将感染性提高>100 倍。出乎意料的是，未加帽的转录本和未切割质粒 DNA 的转录本也具有感染性，尽管它们的感染性非常低。在烟草植物中增殖的子代病毒准确反映了克隆序列。通过相同的方法，我们成功地进行了减毒 L(11)A 株系的感染性 RNA 的体外转录，该 RNA 在烟草和番茄植物上均表现出与野生型 TMV 不同的表型。
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