Acta Academiae Medicinae Sinicae 2002;24(3):285-288

The role of p53 binding site on the trans binding of p53 to Hsp90 beta gene.

Li-ling CHEN 1 ; Ning-hua WU ; Yu-fei SHEN ; Xin-min GUAN

Affiliations

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Country

China

Language

Chinese

Abstract

OBJECTIVETo investigate the effect of p53 binding site (+31/+60) of hsp90 beta gene on its transcriptional regulation.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONSThe core sequence of p53 binding site plays a key role in the trans binding of p53 to hsp90 beta gene.