Chen Li-ling, Wu Ning-hua, Shen Yu-fei, Guan Xin-min
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2002 Jun;24(3):285-8.
To investigate the effect of p53 binding site (+31/+60) of hsp90 beta gene on its transcriptional regulation.
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 hsp90 beta gene fragment containing mutated p53 binding site and Jurkat cell nuclear extract transfected by p53 expression vector.
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.
The core sequence of p53 binding site plays a key role in the trans binding of p53 to hsp90 beta gene.
