Mullineaux P M, Guerineau F, Accotto G P
John Innes Institute, John Innes Centre for Plant Science Research, Norwich, UK.
Nucleic Acids Res. 1990 Dec 25;18(24):7259-65. doi: 10.1093/nar/18.24.7259.
We have used a polymerase chain reaction (PCR) procedure to analyse low abundance complementary sense RNAs of Digitaria streak virus (DSV) from infected leaves of Digitaria setigera. This study has confirmed that both spliced and unspliced RNAs are synthesised by the same transcription unit. The position of the intron has been proven from sequencing cDNAs corresponding to the spliced RNA. Although the majority of cDNAs have 3' ends at coordinate 1063, downstream from a consensus polyadenylation sequence, a minor population of RNAs with heterogeneous 3' ends has also been identified. Two major RNA species with alternative splice sites or 3' ends, previously identified by nuclease S1 protection assays, could not be detected, but a cDNA species was observed with an apparent 90bp insertion at the 5' end of the intron. In transgenic tobacco containing integrated dimers of DSV DNA, the major unspliced RNA could readily be detected, but no spliced RNA was present. This may be a reason why DSV DNA did not replicate in tobacco. In addition, neither the minor population of heterogeneous RNAs nor the cDNA species with the insertion could be detected. The failure of the intron to be spliced in tobacco and its low activity in Digitaria is discussed in relation to recent studies on RNA splicing in plants and has led us to the conclusion that the geminivirus introns may be intrinsically inefficient.
我们采用聚合酶链反应（PCR）方法，对感染了指状虎尾草条纹病毒（DSV）的指状虎尾草叶片中的低丰度互补链RNA进行了分析。本研究证实，剪接和未剪接的RNA均由同一转录单元合成。通过对与剪接RNA对应的cDNA进行测序，已确定了内含子的位置。尽管大多数cDNA的3'末端位于一致的多聚腺苷酸化序列下游的坐标1063处，但也鉴定出了一小部分具有异质3'末端的RNA。先前通过核酸酶S1保护试验鉴定出的具有可变剪接位点或3'末端的两种主要RNA种类未被检测到，但观察到一种cDNA种类在其内含子的5'末端有明显的90bp插入。在含有整合的DSV DNA二聚体的转基因烟草中，主要的未剪接RNA很容易被检测到，但不存在剪接RNA。这可能是DSV DNA在烟草中不复制的原因之一。此外，既未检测到异质RNA的少数群体，也未检测到带有插入片段的cDNA种类。结合近期关于植物RNA剪接的研究，讨论了内含子在烟草中未能剪接及其在指状虎尾草中活性较低的情况，这使我们得出结论，双生病毒内含子可能本质上效率低下。
