Brunel F, Ochoa A, Schaeffer E, Boissier F, Guillou Y, Cereghini S, Cohen G N, Zakin M M
Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
J Biol Chem. 1988 Jul 25;263(21):10180-5.
We have established by transient expression experiments that the 620 base pairs upstream of the cap site of the human transferrin gene contain the information necessary for efficient expression of the gene in hepatoma cells HepG2 or Hep3B but not in HeLa cells. DNase I footprint analysis reveals that at least five distinct factors present in human or rat liver nuclear extracts interact with different sites of this region. One of these factors, binding to nucleotides -193 to -162, is closely related to or identical with the eukaryotic factor CCAAT-binding transcription factor/nuclear factor I; another one, binding to nucleotides -103 to -83 seems to be related to the CCAAT-binding protein. The binding sites of two other factors, not recognized by HeLa nuclear proteins, each contain an identical 10-nucleotide-long sequence (5' TCTTTGACCT 3') in reverse orientation, separated by 400 base pairs. Results of gel retardation assays, cross-competition experiments, and heat inactivation strongly suggest that the proteins binding to these sites are different. One of these sequences and the binding site of the CCAAT-binding protein related factor are located in the region between nucleotides -119 and -45. We have shown by transient expression experiments with 3' deleted vectors that this region is functionally essential for human transferrin gene expression.
我们通过瞬时表达实验确定，人转铁蛋白基因帽位点上游620个碱基对包含该基因在肝癌细胞HepG2或Hep3B中高效表达所需的信息，但在HeLa细胞中则不然。DNA酶I足迹分析表明，人或大鼠肝核提取物中至少有五种不同的因子与该区域的不同位点相互作用。其中一种因子与核苷酸-193至-162结合，与真核因子CCAAT结合转录因子/核因子I密切相关或相同；另一种与核苷酸-103至-83结合的因子似乎与CCAAT结合蛋白有关。另外两种因子的结合位点，HeLa核蛋白无法识别，每个位点都含有一个反向的相同的10个核苷酸长的序列（5'TCTTTGACCT 3'），相隔400个碱基对。凝胶阻滞试验、交叉竞争实验和热失活的结果强烈表明，与这些位点结合的蛋白质是不同的。这些序列之一和CCAAT结合蛋白相关因子的结合位点位于核苷酸-119至-45之间的区域。我们通过使用3'缺失载体的瞬时表达实验表明，该区域对人转铁蛋白基因表达在功能上至关重要。
