Yukawa Yasushi, Matousek Jaroslav, Grimm Michael, Vrba Lukas, Steger Gerhard, Sugiura Masahiro, Beier Hildburg
Institut für Biochemie, Universität Würzburg, Biozentrum, Germany.
Plant Mol Biol. 2002 Nov;50(4-5):713-23. doi: 10.1023/a:1019905730397.
RNA polymerase III-driven cassettes for the expression of antisense RNAs and ribozymes have recently attracted much attention because (1) pol III genes are transcribed abundantly in all kinds of tissues and (2) the transcripts are very stable by virtue of their small and compact size. We have designed two types of pol III-based expression vehicles. Antisense RNA sequences targeted against conserved structural elements or domains in the RNAs of potato spindle tuber viroid, hop latent viroid and potato virus S were either embedded in the anticodon region of a Nicotiana tRNA(Tyr) gene or near the 3' end of an Arabidopsis 7SL RNA gene. Both classes of chimeric genes were transcribed in vitro in a homologous plant extract. Our studies clearly revealed that the modified tRNA and 7SL RNA genes, carrying insertions of up to 90 and 120 bp, respectively, were expressed efficiently in the tobacco nuclear extract, resulting in high levels of stable chimeric transcripts. 7SL RNA (also termed SRP RNA) represents the RNA component of the signal recognition particle. This is the first report of demonstrating the employment of 7SL RNA genes as potential cassettes for the expression of antisense RNA and ribozyme sequences and might be helpful in future experiments to control their localization in specific sub-cellular compartments.
用于表达反义RNA和核酶的RNA聚合酶III驱动的盒式结构最近备受关注，原因如下：（1）聚合酶III基因在各种组织中大量转录；（2）转录本因其小而紧凑的尺寸而非常稳定。我们设计了两种基于聚合酶III的表达载体。针对马铃薯纺锤块茎类病毒、啤酒花潜隐类病毒和马铃薯S病毒RNA中保守结构元件或结构域的反义RNA序列，要么嵌入烟草tRNA（Tyr）基因的反密码子区域，要么靠近拟南芥7SL RNA基因的3'端。这两类嵌合基因都在同源植物提取物中进行了体外转录。我们的研究清楚地表明，分别携带长达90和120 bp插入片段的修饰tRNA和7SL RNA基因，在烟草核提取物中高效表达，产生高水平的稳定嵌合转录本。7SL RNA（也称为SRP RNA）代表信号识别颗粒的RNA成分。这是首次证明将7SL RNA基因用作表达反义RNA和核酶序列的潜在盒式结构的报告，可能有助于未来控制它们在特定亚细胞区室中定位的实验。
