Chazenbalk G D, Wadsworth H L, Foti D, Rapoport B
Thyroid Molecular Biology Laboratory, Veterans Administration Medical Center, San Francisco, California 94121.
Mol Endocrinol. 1990 Aug;4(8):1117-24. doi: 10.1210/mend-4-8-1117.
Fragments of the rat ferritin-H 5'-flanking region up to 1 kilobase in length were generated by the polymerase chain reaction using FRTL5 rat thyroid cell genomic DNA as template. Ferritin-H 5'-flanking region fragments of 219, 351, 666, and 1046 basepairs (bp), ligated up-stream to the reporter gene luciferase, were transiently transfected into FRTL5 thyroid cells and NIH-3T3 mouse fibroblasts. In both cell types, constitutive (nonstimulated) ferritin-H promoter activity increased progressively with constructs containing increasing lengths of 5'-flanking region. TSH or (Bu)2cAMP (dBcAMP) stimulation of FRTL5 cells transfected with the shorter (219 and 351 bp) ferritin-H 5'-flanking region fragments increased promoter activity 2- to 3-fold. However, with the longer DNA segments (666 and 1046 bp), the extent of TSH stimulation was less. Exposure of transfected NIH-3T3 cells to dBcAMP mimicked in all respects the effects of TSH and dBcAMP on ferritin-H promoter activity in FRTL5 cells. Transcription initiation sites in the luciferase reporter gene were unaffected by the length of the ferritin-H 5'-flanking region included in the construct or by dBcAMP stimulation. Plasmid constructs with 45 bp of the ferritin-H 5'-flanking region containing a potential cAMP response element did not reveal any promoter activity or dBcAMP responsiveness in this region. Gel shift mobility assays with the -219 bp ferritin-H 5'-flanking region fragment and NIH-3T3 nuclear proteins revealed specific protein-DNA interaction. Reduced DNA mobility was inhibited by excess unlabeled probe DNA, but not by DNA fragments corresponding to the recognition sites for a variety of known trans-activating factors.(ABSTRACT TRUNCATED AT 250 WORDS)
以FRTL5大鼠甲状腺细胞基因组DNA为模板，通过聚合酶链反应生成了长度达1千碱基的大鼠铁蛋白-H 5'-侧翼区片段。将长度分别为219、351、666和1046碱基对（bp）的铁蛋白-H 5'-侧翼区片段连接到报告基因荧光素酶的上游，然后瞬时转染到FRTL5甲状腺细胞和NIH-3T3小鼠成纤维细胞中。在这两种细胞类型中，组成型（未刺激）铁蛋白-H启动子活性随着包含5'-侧翼区长度增加的构建体而逐渐增加。用较短（219和351 bp）铁蛋白-H 5'-侧翼区片段转染的FRTL5细胞经促甲状腺激素（TSH）或双丁酰环磷腺苷（dBcAMP）刺激后，启动子活性增加2至3倍。然而，对于较长的DNA片段（666和1046 bp），TSH刺激的程度较小。将转染的NIH-3T3细胞暴露于dBcAMP在各方面都模拟了TSH和dBcAMP对FRTL5细胞中铁蛋白-H启动子活性的影响。荧光素酶报告基因中的转录起始位点不受构建体中包含的铁蛋白-H 5'-侧翼区长度或dBcAMP刺激的影响。含有潜在环磷腺苷反应元件的45 bp铁蛋白-H 5'-侧翼区的质粒构建体在该区域未显示任何启动子活性或dBcAMP反应性。用-219 bp铁蛋白-H 5'-侧翼区片段和NIH-3T3核蛋白进行凝胶迁移率变动分析，揭示了特异性的蛋白质-DNA相互作用。未标记的过量探针DNA可抑制DNA迁移率降低，但对应于多种已知反式激活因子识别位点的DNA片段则不能。（摘要截短于250字）
