Donald R G, Schindler U, Batschauer A, Cashmore A R
Department of Biology, University of Pennsylvania, Philadelphia 19104.
EMBO J. 1990 Jun;9(6):1727-35. doi: 10.1002/j.1460-2075.1990.tb08296.x.
G box and I box sequences of the Arabidopsis thaliana ribulose-bisphosphate-1,5-carboxylase small subunit (RBCS) promoter are required for expression mediated by the Arabidopsis rbcS-1A promoter in transgenic tobacco plants and are bound in vitro by factors from plant nuclear extracts termed GBF and GA-1, respectively. We show here that a -390 to -60 rbcS-1A promoter fragment containing the G box and two I boxes activates transcription from a truncated iso-1-cytochrome c (CYC1) gene promoter in Saccharomyces cerevisiae. Mutagenesis of either the rbcS-1A G box or both I box sequences eliminated the expression mediated by this fragment. When polymerized, I box oligonucleotides were also capable of enhancing expression from the truncated CYC1 promoter. Single-copy G box sequences from the Arabidopsis rbcS-1A, Arabidopsis Adh and tomato rbcS-3A promoters were more potent activators and were used in mobility shift assays to identify a DNA binding activity in yeast functionally similar to GBF. In methylation interference experiments, the binding specificity of the yeast protein was indistinguishable from that obtained with plant nuclear extracts.
拟南芥1,5 - 二磷酸核酮糖羧化酶小亚基(RBCS)启动子的G盒和I盒序列,对于转基因烟草植株中由拟南芥rbcS - 1A启动子介导的表达是必需的,并且在体外分别与来自植物核提取物的称为GBF和GA - 1的因子结合。我们在此表明,一个包含G盒和两个I盒的-390至-60 rbcS - 1A启动子片段,可激活酿酒酵母中截短的异-1-细胞色素c(CYC1)基因启动子的转录。对rbcS - 1A G盒或两个I盒序列进行诱变,可消除该片段介导的表达。当I盒寡核苷酸聚合时,也能够增强截短的CYC1启动子的表达。来自拟南芥rbcS - 1A、拟南芥Adh和番茄rbcS - 3A启动子的单拷贝G盒序列是更强的激活剂,并用于迁移率变动分析,以鉴定酵母中与GBF功能相似的DNA结合活性。在甲基化干扰实验中,酵母蛋白的结合特异性与植物核提取物的无异。