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从感染TMV的普通烟叶中分离到含有TMV-RNA复制酶的无细胞提取物(20,000×g沉淀),它能在四种三磷酸核苷(其中三磷酸尿苷用氚标记)、镁离子和放线菌素D的存在下离体合成双链RNA,经酚-SDS提取、Serva纤维素柱层析获得的~3H-双链RNA,用聚丙烯酰胺-琼脂糖凝胶电泳、核糖核酸酶处理、热变性、自退火等手段,证实~3H-双链RNA中含有TMV-RNA的复制型(RF)和复制中间体(RI),分子量分别为4.0×10~6和5.0×10~6,分子杂交竞争试验表明~3H-双链RNA中新生链的60—70％为TMV-RNA正链。
A cell-free extract containing TMV-RNA replicase (20,000 xg pellet) was isolated from normal tobacco infected TMV and was isolated on four types of nucleoside triphosphates (where tritiated triphosphate was labeled), magnesium ions and Double stranded RNA was synthesized in vitro in the presence of actinomycin D, and the 3H-double stranded RNA obtained by phenol-SDS extraction and Serva cellulose column chromatography was electrophoresed on polyacrylamide-agarose gel, and ribonuclease (RF) and replication intermediates (RI) of TMV-RNA in ~ 3H-double stranded RNA were confirmed by the methods of thermostability, thermal denaturation and self-annealing. The molecular weights of TMV-RNA were 4.0 × 10-6 and 5.0 × 10-6 Molecular hybridization competition assays show that 60-70% of nascent strands of ~ 3H-double stranded RNA are TMV-RNA positive strands.