Fujii N
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Gene. 1997 Nov 12;201(1-2):55-62. doi: 10.1016/s0378-1119(97)00427-7.
Carrot cells cultured in vitro in a medium supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) proliferate as unorganized cell clusters. Upon removal of 2,4-D from the culture medium, these cells undergo somatic embryo formation through globular, heart, and torpedo stages. Since the proximal -255 bp upstream region of the rolC gene of the Ri plasmid confers somatic embryogenesis-related activation on the uidA gene in transgenic carrot cell culture, we investigated the interaction of nuclear proteins with the proximal -255bp upstream sequences to characterize the mechanism of somatic embryogenesis-related activation. Gel retardation experiments revealed that there were several different profiles of the relative levels of DNA binding activities in nuclear protein extracts from calli, PEMs (proembryogenic masses), globular embryos, and heart/torpedo embryos. The binding activity associated with a fragment (-203 bp to -92 bp) of one protein (BI) was most abundant in globular embryos. Another DNA binding protein (AII) showed the highest DNA binding activity in calli, but had low binding activities in PEMs and globular embryos. Such an altered pattern of DNA binding activities of nuclear proteins may contribute to somatic embryogenesis-related activation of the rolC promoter. Competition experiments with oligonucleotides revealed that the BI protein interacts with AT-rich sequences.
在添加了2,4 - D（2,4 - 二氯苯氧乙酸）的培养基中体外培养的胡萝卜细胞以无组织的细胞团块形式增殖。从培养基中去除2,4 - D后，这些细胞会经历球形、心形和鱼雷形阶段的体细胞胚胎形成过程。由于发根农杆菌Ri质粒rolC基因上游近端- 255 bp区域在转基因胡萝卜细胞培养中赋予了与体细胞胚胎发生相关的uidA基因激活作用，我们研究了核蛋白与近端- 255 bp上游序列的相互作用，以表征与体细胞胚胎发生相关的激活机制。凝胶阻滞实验表明，来自愈伤组织、原胚性细胞团（PEMs）、球形胚和心形/鱼雷形胚的核蛋白提取物中DNA结合活性的相对水平存在几种不同的模式。与一种蛋白（BI）的片段（- 203 bp至- 92 bp）相关的结合活性在球形胚中最为丰富。另一种DNA结合蛋白（AII）在愈伤组织中显示出最高的DNA结合活性，但在PEMs和球形胚中具有较低的结合活性。核蛋白DNA结合活性的这种改变模式可能有助于rolC启动子与体细胞胚胎发生相关的激活。用寡核苷酸进行的竞争实验表明，BI蛋白与富含AT的序列相互作用。
