純化及鑑定負向調節AGP基因表現的轉錄因數

Abstract

近幾年來本實驗室研究老鼠肝臟的α l－Acid Glycoprotein ( AGP )基因表現之調控機制，此基因啟動子 ( promoter )的鹼基序列 －180/+10 之間，包括五個主要序列，分別為A，B，C，D及E。同時也鑑定出幾 個轉錄因數，其中之一AGP/EBP可以結合到A，C，D，E四個序列，加強AGP基因的表現，而另一方面B序列經 特異位置突變( site-directed mutagenesis )及功能性分析 ( functional assays )已確定其為一負向序 列( negative element )。 本篇論文主要就是應用傳統的生物化學方法，將肝臟細胞的核萃取液通過離子交換層析管柱及DNA親和性層 析管柱，藉以純化和B序列相結合，且具有負向調節效果的轉錄因數AGP/NTF。此因數對熱大致穩定，經 south–western blot的分析，發現至少有六個多勝?，其中分子量45一66kDa之間，具有主要結合B序列的活性。

α 1-acid glycoprotein (AGP) is an acute-responsive, 1iver-specific gene. Its expression is regulated by both positive and negative trans-acting factors. At least five cis-e1ements have been identified within the 180 bp region of AGP promoter. Four of these sites, A, C, D and E are recognized by AGP/EBP or AGP/EBP-like factors in liver nuclear extracts. Site-directed mutagenesis and trasfection analysis indicate that A, C, D and E regions are positive regulatory elements enhancing the expression of AGP gene while B motif is a negative regu1atory element repressing the expression of AGP gene. We have purified several proteins that bind to the B region in the AGP gene promoter. These proteins were purified from rat liver nuclear extracts by chromatography on heparin-agarose and oligonucleotide affinity columns. Analysis of purified protein fractions by southwestern blot reveals more than six polypeptides having binding activity to B motif. The purified protein from each fraction was recovered from SDS-PAGE, and those of the molecular size ranging from 45 to 66 kDa were demonstrated to have major binding activity toward B region of the AGP promoter.