p53反式结合hsp90β基因

The Role of p53Binding Site on the Trans Binding of p53to Hsp90βGene

目的探讨hsp90β基因中p53结合位点(+31/+60)是否参与p53与hsp90β基因的反式结合,以进一步揭示该位点在p53调节hsp90β基因转录中的作用。方法将含有p53结合位点的hsp90β基因片段插入质粒pBS-SK,然后将它与突变引物和选择引物一起依次退火和延伸,经过两轮细菌转化,酶切筛选出突变阳性质粒后进行序列分析。采用电泳迁移率变更测定(EMSA)检测突变后基因片段与转染p53表达质粒的Jurkat细胞核抽提物的结合。结果序列分析证实p53结合位点第2个半位点核心序列的两个碱基已发生突变,EMSA结果表明,突变后基因片段不能与p53结合。结论hsp90β基因中p53结合位点的核心序列在p53与hsp90β基因的反式结合中起关键作用。

Objective To investigate the effect of p53binding site(+31/+60)of hsp90βgene on its transcriptional regulation.Methods The binding site was first inserted into pBS-SK.After the plasmid annealing and elongation with mutagenic and selective primers,nuclease digestion and bacteria transformation was performed twice to select the positive mutated plasmid.Electrophoretic mobility shift assays(EMSA)was employed to detect the binding of hsp90βgene fragment containing mutated p53binding site and Jurkat cell nuclear extract transfected by p53expression vector.Results The sequence analysis profile confirmed a successful mutation of two bases on the core sequence of the second half binding site.EMSA results showed the specific DNA-protein complex band disappeared after the mutation.Conclusions The core sequence of p53binding site plays a key role in the trans binding of p53to hsp90βgene.