Sakurai H, Izumi S, Tomino S
Department of Biology, Tokyo Metropolitan University, Japan.
Biochim Biophys Acta. 1990 Sep 10;1087(1):18-24. doi: 10.1016/0167-4781(90)90115-i.
An efficient cell-free transcription system was developed from the extract of BmN cells established from an ovarian tissue of the silkworm, Bombyx mori. The cloned genes coding for major plasma proteins of B. mori including SP 1, SP 2 and 30K protein, were faithfully and efficiently transcribed in the extract prepared from BmN cells. The S1 nuclease mapping and primer extension analyses demonstrated that the transcription initiation site recognized in vitro is identical to that which functions in vivo. The transcription assay reconstituted from the fractionated BmN cell extract revealed that at least four protein factors are required for accurate transcription of the SP 1 and adenovirus major late genes. The results of in vitro transcription experiments employing a series of the 5' deleted mutant templates of the SP 1 gene indicated that partial deletion of the TATA box results in considerable loss of faithful transcript, while complete removal of the TATA-sequence totally abolishes the transcript. These observations suggest that the promoter element essential for transcription in cell-free systems is located in a region between nucleotide positions -44 and +16 of the SP 1 gene.
利用家蚕（Bombyx mori）卵巢组织建立的BmN细胞提取物，开发了一种高效的无细胞转录系统。编码家蚕主要血浆蛋白（包括SP 1、SP 2和30K蛋白）的克隆基因，在从BmN细胞制备的提取物中得到了准确且高效的转录。S1核酸酶图谱分析和引物延伸分析表明，体外识别的转录起始位点与体内发挥作用的位点相同。由分级分离的BmN细胞提取物重建的转录分析显示，SP 1和腺病毒主要晚期基因的准确转录至少需要四种蛋白质因子。使用一系列SP 1基因5'端缺失突变模板进行的体外转录实验结果表明，TATA框的部分缺失会导致忠实转录本的大量丢失，而TATA序列的完全去除则会完全消除转录本。这些观察结果表明，无细胞系统中转录所必需的启动子元件位于SP 1基因核苷酸位置-44至+16之间的区域。
