人Ha-ras基因中蛋白质特异结合DNA位点的鉴定

Identification of Novel Sites of Specific DNA-Protein Interaction in Human Ha-ras Gene

利用迁移率改变法和DNaseⅠ足纹法观察了Ha-ras癌基因5’端上游序列与特异结合蛋白的相互作用。用限制性内切酶XmaⅠ消化6.6kb Ha-ras基因得到约10个片段,进行3’-末端标记,与T24细胞核提取液反应,经低离子强度聚丙烯酰胺凝胶电泳,发现与蛋白质特异结合的416bp片段,位于Ha-ras基因5’-端上游1230—1646区域内,靠近转录起始点1660bp处(CAP位置)。另一个与蛋白质特异结合的389bp片段,位于转录起始点上游162—551处。

By using mobility shift assay and DNase I footprinting analysis the interaction between specific proteins and specific sequences of Ha-ras oncogene was investigated.The 6.6Kb Ha-ras gene was digested with Xma I and the DNA fragments were incubated with 0.4mol/L KCI extracted nuclear proteins from T24 cells. The reaction mixture was subjected to electrophoresis on a low ionic strength polyacrylamide gel. A fragment of 416bp exhibiting specific binding was detected, which was located upstream at 1230 to 1646bp in the 5'-flanking region adjacent to the cap site at lG60bp. Another fragment of 389bp exhibing specific binding was detected, which was located upstream at 162 to 551 bP in the transcriptional initiation site region. Both specific DNA-binding proteins may play important roles in the activation and expression of Ha-ras gene in T24 cells.