【摘要】 将编码人c myc羧基端bHLH/LZ结构域的 92个氨基酸的cDNA片段 (c myc c92 )对框插入 pGEX 2T原核表达载体中 ,使之与谷胱甘肽转移酶 (GST)编码基因融合 ,并将重组质粒导入大肠杆菌BL2 1(DE3)中 ,由IPTG诱导融合基因高效表达 ,并用GlutathioneSepharose 4B亲和柱纯化融合蛋白GST c Myc c92 ,凝胶阻滞 (EMSA)分析显示该纯化蛋白能与CACGTG序列特异结合 ,并且只有高浓度的GST c Myc c92才能与探针结合 .结果表明在体外情况下高浓度c Myc羧基端能自身二聚体化 ,此发现丰富了Myc Max Mad的调控网络 ,并为进一步研究c myc的功能和调控提供了一定的线索 .更多还原

【Abstract】 The c myc proto oncogene is involved in the progression of a wide range of neoplasias. Oncogenic activation occurs mainly through its gene product, the short lived nuclear c Myc protein, which contains a carboxy terminal basic helix loop helix leucine zipper motif (bHLH/LZ) known to mediate dimerization and sequence specific DNA binding. In this study, c myc c92, which encodes 92 amino acids within the bHLH/LZ motif, was cloned into the prokaryotic expression vector pGEX 2T at Sma I and Eco R I sites. The recombinant protein, GST c Myc c92, was expressed in E.coli BL21(DE3) induced by IPTG and purified by Glutathione Sepharose 4B affinity chromatography. The electrophoretic mobility shift assay (EMSA) results showed that in vitro the recombinant protein at high concentration could bind with 32 p labeled probe with the core sequence CACGTG. This finding suggests that c Myc protein at high concentration can form homodimers in vitro and bind to the E box (5’ CACGTG ), which sophisticates the regulation network of Myc Max Mad. It is generally accepted that cells have c Myc/Max, Max/Max and Mad/Max complexes. DNA bound c Myc/Max dimers activate transcription and induce cell proliferation or apotosis. In contrast, Max homodimers and Mad/Max heterodimers act as antagonists of c Myc/Max through competition for common DNA targets. Some experiments have revealed that the c Myc protein can bind to its own promoter and suppress c myc transcription. However, the mechanism of this negative autoregulation is still elusive. The results described above lead to this hypothesis: When the c Myc protein is overexpressed in the cell, it may form homodimers and bind to its own promoter, thus autoregulate its expression by negative feedback. And since Max is essential for c Myc dependent transactivation, binding of the homodimerized c Myc to the E box will not promote cellular growth and transformation.更多还原