Isolation of an RNA-Directed RNA polymerase-specific cDNA clone from tomato
Isolation of an RNA-Directed RNA polymerase-specific cDNA clone from tomato
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DOI：
10.1105/tpc.10.12.2087
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发表时间：
1998-12-01
期刊：
影响因子：
11.6
通讯作者：
Wassenegger, M
中科院分区：
文献类型：
--
作者：
Schiebel, W;Pélissier, T;Wassenegger, M
从番茄中分离到一个3600 bp的RNA定向RNA聚合酶（RdRP）特异性cDNA，该cDNA包含一个1114个氨基酸的开放阅读框架（ORF）。由该ORF编码的推定蛋白质与任何特征蛋白质不具有同源性。针对其序列已从ORF推导的合成肽产生的抗体显示出特异性检测127-kD番茄RdRP蛋白。对抗体的免疫应答与在Q-、poly（A）-和paly（U）-Sepharose、羟基磷灰石和Sephadex G-200柱上层析后RdRP的酶活性谱相关。DNA凝胶印迹分析表明，RdRP基因在番茄中的单拷贝。通过聚合酶链反应鉴定了矮牵牛、拟南芥、烟草和小麦中的RdRP同源物。序列比较表明，同源序列RdRP也存在于酵母裂殖酵母粟酒裂殖酵母和线虫秀丽隐杆线虫。先前描述的类病毒感染后RdRP活性的诱导显示与相应mRNA的稳态水平增加相关。这迄今为止功能难以捉摸的植物RNA聚合酶在同源依赖的基因沉默的可能参与进行了讨论。
A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and paly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed.