Dzianott A M, Bujarski J J
Plant Molecular Biology Center, Northern Illinois University, Dekalb 60115.
Proc Natl Acad Sci U S A. 1989 Jul;86(13):4823-7. doi: 10.1073/pnas.86.13.4823.
An in vitro transcription system has been developed that utilizes self-processing to adjust 3' termini in transcribed viral RNAs. Transcription vectors were constructed by ligating a cDNA "cassette" containing self-cleavage sequences from the satellite RNA of tobacco ringspot virus (STobRV RNA) with the 3' end of brome mosaic virus (BMV) cDNA clones. Transcripts were obtained either from templates linearized at positions located downstream from the cassette or from nonlinearized plasmids. In both cases, a spontaneous self-cleavage reaction produced BMV RNAs that contained 19 heterologous nucleotides at their 3' ends. These RNAs were biologically active, indicating that 19 extra nucleotides did not remove the infectivity. The progeny BMV RNA isolated from infected plants did not retain the 19-base heterologous sequence. BMV RNAs containing longer 3' heterologous sequences were not infectious. The STobRV self-cleavage cassette is potentially applicable to a variety of RNA systems.
已开发出一种体外转录系统，该系统利用自我加工来调整转录的病毒RNA的3'末端。通过将包含来自烟草环斑病毒卫星RNA（STobRV RNA）的自我切割序列的cDNA“盒”与雀麦花叶病毒（BMV）cDNA克隆的3'末端连接，构建了转录载体。转录本可从在盒下游位置线性化的模板或从非线性化质粒获得。在这两种情况下，自发的自我切割反应产生了在其3'末端含有19个异源核苷酸的BMV RNA。这些RNA具有生物活性，表明19个额外的核苷酸并未消除其感染性。从受感染植物中分离出的子代BMV RNA没有保留19碱基的异源序列。含有更长3'异源序列的BMV RNA没有感染性。STobRV自我切割盒可能适用于多种RNA系统。
