核受体辅助抑制因子中ETO结合结构域的确定

Determination of ETO interaction domain within nuclear receptor co-repressor

目的 为消除急性髓系白血病标志基因AML1/ETO的活性 ,确定核受体辅助抑制因子(nuclearreceptorco repressor,N CoR)与ETO基因相互作用的活性部位。方法 用PCR扩增不同区段的N CoR(N CoRY) ,克隆入酵母表达质粒pGADGL中 ,构建获得pGADGL/N CoRY。应用酵母双杂交技术及X gal染色确定pGADGL/N CoRY 10个区段的N CoR是否与ETO结合。结果 N CoR的 988～ 112 6氨基酸残基与 15 5 1～ 180 1氨基酸残基共存时 ,才能与ETO发生结合作用 ,是与ETO相互作用的结构域。结论 N CoR两个结构域能与ETO的两个锌指结构作用 ,保持两种蛋白之间的稳定结合。

Objective To determine the ETO interaction domain of nuclear receptor co repressor (N CoR) for abolishing the biological function of AML1 ETO. Methods Ten different regions of N CoR (N CoRYs) were generated by means of polymerase chain reaction (PCR), and cloned into yeast expression plasmid pGADGL to construct pGADGL/N CoRYs. The yeast two hybrid technique and X gal staining were used to determine the binding between the 10 different regions of N CoR and ETO. Results It was shown that the co existance of 988～1?126 and 1?551～1?803 amino acid residues of N CoRY was the ETO interaction domains required for the binding with ETO. Conclusion Two domains of N CoR that interact with two zinc fingers of ETO, and keep stable binding between the two proteins were identified.