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首页> 外文期刊>Molecular and Cellular Biology >Purification and characterization of chicken ovalbumin gene upstream promoter transcription factor from homologous oviduct cells.
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Purification and characterization of chicken ovalbumin gene upstream promoter transcription factor from homologous oviduct cells.

机译：从同源输卵管细胞中纯化和鉴定鸡卵清蛋白基因上游启动子转录因子。

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Previous studies established that the chicken ovalbumin gene upstream promoter (COUP) sequence, which lies between -70 and -90 base pairs upstream from the cap site, is essential for the efficient transcription of the ovalbumin gene. A transcription factor which binds to this sequence has been purified from the homologous chicken oviduct cells. The purification scheme starting from oviduct nuclear extract involved a combination of conventional column and sequence-specific DNA affinity chromatography steps. Using gel retardation and DNase I footprinting techniques to assay COUP-binding activity, we achieved extensive purification of this factor. Binding competition studies with the purified factor indicated that it bound specifically to the COUP sequence and that the binding could be competed for only by the promoter DNA fragments or synthetic oligonucleotides containing the COUP sequence. The purified protein preparation showed multiple polypeptide bands on polyacrylamide gel electrophoresis. Renaturation of separated polypeptides after extraction from the gel matrix was carried out. The majority of renatured polypeptides exhibited specific binding to the COUP sequence.
机译：先前的研究表明，鸡卵清蛋白基因上游启动子（COUP）序列位于帽位点上游-70至-90个碱基对之间，对卵清蛋白基因的有效转录至关重要。已经从同源鸡输卵管细胞中纯化了与该序列结合的转录因子。从输卵管核提取物开始的纯化方案涉及常规色谱柱和序列特异性DNA亲和色谱步骤的组合。使用凝胶阻滞和DNase I足迹技术分析COUP结合活性，我们实现了该因子的广泛纯化。与纯化因子的结合竞争研究表明，它与COUP序列特异性结合，并且该结合只能由启动子DNA片段或含有COUP序列的合成寡核苷酸竞争。纯化的蛋白制品在聚丙烯酰胺凝胶电泳上显示出多个多肽条带。从凝胶基质中提取后分离的多肽进行复性。多数变性多肽表现出与COUP序列的特异性结合。

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