Chen L J, Rogers S A, Bennett D C, Hu M C, Orozco E M
Department of Agronomy, University of Illinois.
Curr Genet. 1990 Jan;17(1):55-64. doi: 10.1007/BF00313249.
Promoters for spinach chloroplast genes were cloned 5' to a strong factor-independent transcription terminator from E. coli. These "minigene" constructions were transcribed in vitro by a transcriptionally active extract of spinach chloroplasts. Transcription of supercoiled DNA templates resulted in synthesis of discretely-sized RNAs that were readily quantifiable. The efficiency of transcription was up to 3.5 RNAs per template. The transcription termination system described in this report was used to identify the primary transcripts for the plastid atpB gene. Four in vivo transcripts for the atpB gene have been previously identified with 5' untranslated leaders of approximately 455, 275, 180 and 100 nucleotides, respectively. In this report we show that the "-455", "-275" and "-180" regions function as chloroplast promoters in vitro. In addition, a fourth promoter was found that yields a primary transcript totally lacking an untranslated leader.
菠菜叶绿体基因的启动子被克隆到来自大肠杆菌的一个强的不依赖因子的转录终止子的5'端。这些“小基因”构建体在体外由菠菜叶绿体的转录活性提取物进行转录。超螺旋DNA模板的转录导致了大小离散的RNA的合成，这些RNA易于定量。转录效率高达每个模板3.5个RNA。本报告中描述的转录终止系统被用于鉴定质体atpB基因的初级转录本。先前已鉴定出atpB基因的四种体内转录本，其5'非翻译前导序列分别约为455、275、180和100个核苷酸。在本报告中，我们表明“-455”、“-275”和“-180”区域在体外作为叶绿体启动子起作用。此外，还发现了第四个启动子，它产生的初级转录本完全没有非翻译前导序列。
