Kuhn Josef, Binder Stefan
Molekulare Botanik, Universität Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany.
Nucleic Acids Res. 2002 Jan 15;30(2):439-46. doi: 10.1093/nar/30.2.439.
Gene expression in plant mitochondria is still inadequately analyzed. To learn more about transcription and RNA processing in plant mitochondria, the 5'- and 3'-RNA extremities and the promoters of the cytochrome oxidase gene (cox2) were analyzed in pea. Both 5' and 3' ends of cox2 transcripts were examined by RT-PCR across the ligation site of circularized mitochondrial RNA as template. This approach identified 5' ends a few nucleotides shorter than three major 5' ends mapped by primer extension analysis. Presumably, only monophosphate 5' ends derived from processing can be ligated. In vitro transcription assays using a homologous mitochondrial protein extract from pea strongly suggest the major 5' ends to derive from transcription initiation. The cDNA analysis of the head-to-tail ligated cox2 mRNA identified 3' ends within a thymidine stretch approximately 300 nt downstream of the reading frame in a sequence segment that was not present in the previous investigation of this gene. Nuclease S1 protection experiments confirmed this newly identified 3' terminus and corroborated the validity of this technique in mRNA end analysis. The general use of the circularized RNA (CR)-RT-PCR approach for the simultaneous analysis of the 5' and 3' extremities of mRNA molecules is discussed.
植物线粒体中的基因表达仍未得到充分分析。为了更多地了解植物线粒体中的转录和RNA加工过程，对豌豆细胞色素氧化酶基因（cox2）的5'-和3'-RNA末端以及启动子进行了分析。以环化线粒体RNA的连接位点为模板，通过RT-PCR检测cox2转录本的5'端和3'端。该方法鉴定出的5'端比通过引物延伸分析确定的三个主要5'端短几个核苷酸。推测只有加工产生的单磷酸5'端才能被连接。使用来自豌豆的同源线粒体蛋白提取物进行的体外转录分析强烈表明，主要的5'端源自转录起始。对头尾连接的cox2 mRNA进行的cDNA分析在一个阅读框下游约300 nt的胸苷延伸区内鉴定出3'端，该序列片段在该基因先前的研究中并不存在。核酸酶S1保护实验证实了这个新鉴定的3'末端，并证实了该技术在mRNA末端分析中的有效性。本文讨论了环化RNA（CR）-RT-PCR方法在同时分析mRNA分子5'端和3'端方面的普遍应用。
