An ordered Arabidopsis thaliana mitochondrial cDNA library on high-density filters allows rapid systematic analysis of plant gene expression:: a pilot study
An ordered Arabidopsis thaliana mitochondrial cDNA library on high-density filters allows rapid systematic analysis of plant gene expression:: a pilot study
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DOI：
10.1046/j.1365-313x.1998.00242.x
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发表时间：
1998-09-01
期刊：
影响因子：
7.2
通讯作者：
Brennicke, A
中科院分区：
文献类型：
--
作者：
Giegé, P;Konthur, Z;Brennicke, A
基因组完整序列的可获得性使系统分析其表达成为可能。转录水平的特定于基因的变异以及转录加工和RNA编辑等现象需要检查大量的克隆。对于完全测序的拟南芥线粒体基因组，我们采用了机器人技术来识别和鉴定表达的基因。构建了一个包含约5万个克隆的cDNA文库，机器人将其排列成384孔的微滴定板，并将其斑点到高密度滤膜上。这些过滤器允许在单个杂交步骤中分离大量特定的cdna克隆。用cox1、cox2和cox3基因评价该方法的可行性和有效性。在cox3编码区之外观察到的一簇RNA编辑位点确定了高等植物中一个新的阅读框架orf95，与呼吸链复合体II的一个亚单位有很大的相似性。
The availability of the complete sequence of a genome allows a systematic analysis of its expression. Gene-specific variations of transcription levels and phenomena such as transcript processing and RNA editing require large numbers of clones to be examined. For the completely sequenced mitochondrial genome of Arabidopsis thaliana we adapted robot technology to identify and characterize expressed genes. A cDNA library of about 50 000 clones was constructed, robot-ordered into 384-well microtitre plates and spotted onto high-density filter membranes. These filters permit the isolation of large numbers of specific cDNA clones in a single hybridization step. The cox1, cox2 and cox3 genes were used to evaluate the feasibility and efficiency of this approach. A cluster of RNA editing sites observed outside the cox3 coding region identifies a novel reading frame orf95 in higher plants with significant similarity to a subunit of respiratory chain complex II.