Miskimins W K
Department of Biochemistry and Molecular Biology, University of South Dakota, School of Medicine, Vermillion 57069.
J Cell Biochem. 1992 Aug;49(4):349-56. doi: 10.1002/jcb.240490405.
Nuclear factors from HeLa cells were isolated by elution of DNA-cellulose bound proteins with a double stranded synthetic oligonucleotide corresponding to the region from -34 to -79 of the human transferrin receptor (TR) gene promoter. The eluted proteins were further purified and separated from the oligonucleotide by ion exchange chromatography. Proteins within the resulting fraction bound with specificity to the TR promoter. Retardation gel analysis and competition with specific double-stranded oligonucleotides show that multiple factors present in this fraction compete for binding within the same region of the TR promoter. Footprinting experiments demonstrate that these factors contact a GC-rich element that is within the region that is required for enhanced expression of the gene in proliferating cells. One of the factors protects an extended DNA sequence but still contacts the GC-rich element.
通过用与人类转铁蛋白受体（TR）基因启动子-34至-79区域对应的双链合成寡核苷酸洗脱结合在DNA-纤维素上的蛋白质，从HeLa细胞中分离出核因子。洗脱的蛋白质进一步纯化，并通过离子交换色谱与寡核苷酸分离。所得组分中的蛋白质与TR启动子特异性结合。阻滞凝胶分析以及与特异性双链寡核苷酸的竞争表明，该组分中存在的多种因子在TR启动子的同一区域竞争结合。足迹实验表明，这些因子与富含GC的元件接触，该元件位于增殖细胞中基因增强表达所需的区域内。其中一个因子保护一个延伸的DNA序列，但仍与富含GC的元件接触。
