Ishisaki A, Murayama T, Ballagi A E, Funa K
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.
Eur J Biochem. 1997 May 15;246(1):142-6. doi: 10.1111/j.1432-1033.1997.t01-2-00142.x.
To determine the regulatory mechanism of the expression of the mouse platelet-derived growth factor (PDGF) beta-receptor gene, a 1.9-kb 5' flanking genomic fragment was cloned and analyzed. Site-directed mutagenesis of a CCAAT motif, located 60 bp upstream of the transcriptional-start site, completely abolished the promoter activity [Ballagi, A. E., Ishisaki, A., Nelin, J.-O. & Funa, K. (1995) Biochem. Biophys. Res. Commun. 210, 165-1751. The sequence around the intact CCAAT motif was protected by in vitro DNase-I-footprinting analysis. Electrophoresis-mobility-shift assays with anti-[nuclear factor Y(NF-Y)]Ig revealed binding of the NF-Y complex to the CCAAT box. Furthermore, the double-stranded oligonucleotides corresponding to the sequence around the CCAAT motif were conjugated with DNA-affinity magnetic beads. The binding proteins were affinity purified and identified as the NF-Y transcription factor by western blotting. Our results indicate that NF-Y controls the basal transcription activity of the mouse PDGF beta-receptor gene.
为确定小鼠血小板衍生生长因子（PDGF）β受体基因表达的调控机制，克隆并分析了一个1.9 kb的5'侧翼基因组片段。对位于转录起始位点上游60 bp处的CCAAT基序进行定点诱变，完全消除了启动子活性[巴拉吉，A. E.，石崎，A.，内林，J.-O. & 富纳，K.（1995年）《生物化学与生物物理研究通讯》210，165 - 175]。通过体外DNase - I足迹分析，完整的CCAAT基序周围的序列受到保护。用抗[核因子Y（NF - Y）]Ig进行的电泳迁移率变动分析显示NF - Y复合物与CCAAT框结合。此外，将与CCAAT基序周围序列对应的双链寡核苷酸与DNA亲和磁珠偶联。通过蛋白质印迹法对结合蛋白进行亲和纯化并鉴定为NF - Y转录因子。我们的结果表明NF - Y控制小鼠PDGFβ受体基因的基础转录活性。
