Borneman J, Tritz R, Hampel A, Altschuler M
Plant Molecular Biology Center, Northern Illinois University, DeKalb 60115, USA.
Gene. 1995 Jul 4;159(2):137-42. doi: 10.1016/0378-1119(95)00173-4.
In order to examine ribozyme (Rz) activity in vivo, we have adapted a virus to deliver Rz to plants. DNA fragments that code for both active and mutant cis-hairpin Rz were cloned into the double-stranded DNA plant virus, cauliflower mosaic virus (CaMV). These Rz constructs successfully infected Brassica campestris rapa (turnip). The plants that were infected with the active-Rz construct showed, on average, a one-week delay in the appearance of viral symptoms, when compared to the mutant-Rz control. Since CaMV replicates through reverse transcription of a full-length RNA intermediate, Rz cloned into the CaMV DNA should be transcribed within this viral RNA. If these Rz constructs cleave, the amount of intact virus RNA should be reduced, resulting in attenuated viral symptoms. In addition, lysate RNase protection assays showed fragments corresponding to the sizes of both the 5' and 3' cis cleavage products in the active Rz tissue. No cleavage products were observed from plant tissue infected with the mutant Rz. Both the attenuated systemic viral symptoms and the cleavage products from the protection assay strongly support in vivo transcription and cleavage of this hairpin Rz. This is the first report of an in vivo transcribed Rz showing cleaved products by direct RNA analysis (non-PCR) in plants or animals.
为了在体内检测核酶（Rz）活性，我们改造了一种病毒，以便将Rz递送至植物。编码活性和顺式发夹突变体Rz的DNA片段被克隆到双链DNA植物病毒花椰菜花叶病毒（CaMV）中。这些Rz构建体成功感染了芜菁（Brassica campestris rapa）。与突变体Rz对照相比，感染活性Rz构建体的植物平均病毒症状出现延迟一周。由于CaMV通过全长RNA中间体的逆转录进行复制，克隆到CaMV DNA中的Rz应在这种病毒RNA内转录。如果这些Rz构建体发生切割，完整病毒RNA的量应减少，从而导致病毒症状减轻。此外，裂解物核糖核酸酶保护分析显示，活性Rz组织中存在与5'和3'顺式切割产物大小对应的片段。感染突变体Rz的植物组织未观察到切割产物。病毒症状减轻和保护分析中的切割产物都有力地支持了这种发夹Rz在体内的转录和切割。这是首次报道通过直接RNA分析（非PCR）在植物或动物体内转录的Rz显示出切割产物。
