大鼠4S多环芳香烃结合蛋白对细胞色素P450IAl基因体外转录的激活作用

STIMULATION OF IN VITRO TRANSCRIPTION OF THE RAT CYTOCHROME P450IA1 GENE BY 4S PAH-BINDING PROTEIN

作者以Hela细胞液体系,研究大鼠细胞色素P450IA-1(P 450 C)基因在4S多环芳香烃(PAH)结合蛋白存在下的体外转录作用。用含大鼠细胞色素P 450 IA1的-880至外显子的第1000碱基对((bp)的质粒pA9[长约1.94千碱基对(kb)],经内切酶切割成不同大小的片段为模板。以插有腺病毒-2大部晚期启动子(Ad-2)的质粒PHML-1作为阳性对照。所得结果显示Hela细胞体系能按模板准确地转录出预期的产物。加入4S PAH-结合蛋白使pA9模板的转录增加2～3倍,而对PHML-1的转录无增强作用,说明4S PAH-结合蛋白是专一加强细胞色素P450IA1基因转录的正调节因素。推测4S PAH-结合蛋白也可能诱导大鼠体内细胞色素P 450 IA1基因的表达。

In this investigation, we have used a HeLa cell lysate system to study the in vitro transcription of the rat cytochrome P450IAl(P450) gene in the presence and absence of rat 4S polycyclic aromatic hydrocarbon (PAH)-binding protein. The HeLa cell lysate system has been shown to contain an active RNA polymerase II and the necessary factors which can accurately transcribe exogenous DNA templotes. For this investigation, the plasmid pA9, which contains about 1.9kb of cloned rat oytoohrome P450IA1 sequences spanning -880 through exon 1 to+ 1 000 bp, was used to prepare several endonuclease digestion fragments which were used as oytoohrome P450IA1 DNA templates. The plasmid PHML-1, which contains the adenovirus-2 major late promoter insert of approximately 456bp,was used as a positive control. Our results indicate that the Hela cell lysate system produced transcripts corresponding to the expected size with both the positive control PHML-1 and the pA9 templates. The addition of 4S PAH-binding protein resulted in a 2-3 fold stimulation of the in vitro transcription of pA9 templates, but not of the control template PHML-1. These results indicate that in vitro, the 4S PAH-binding protein may act as a positive transcription factor to specifically enhance the expression of the oytoohrome P450IA1 gene. The 4S PAH-binding protein may play a role in vivo in the PAH-induoed expression of the cytochrome P450IA1 gene in the rat.