T7启动子具有真核生物聚合酶Ⅱ顺式作用因子的功能  

The Function of T7 Promoter as cis-Acting Elements for Polymerase Ⅱ in Eukaryotic Cell

作　　者：李月琴[1] 朱嘉明[1] 李弘剑[1] 谢卫兵[1] 周天鸿[1] 王彤歌[2] 

机构地区：[1]暨南大学生物工程系,广州5106325 [2]暨南大学附属医院,广州510630

出　　处：《Acta Genetica Sinica》2000年第5期455-461,共7页

基　　金：国家自然科学基金!(39770411)

摘　　要：根据ａ－鹅膏蕈碱（ａ－ａｍａｎｉｔｉｎ）对真核生物ＲＮＡ聚合酶的选择性抑制，以氯霉素乙酰转 移酶基因（ＣＡＴ）作为报道基因进行体内表达实验，证明Ｔ７噬菌体启动子可为真核生物ＲＮＡ 聚合酶Ⅱ所启动。应用建立的竞争性ＤＮＡ－蛋白质凝胶泳动技术，分别以ＴＡＴＡ框、ＣＡＡＴ 框、ＧＣ框和八核苷酸序列（ｏｃｔａｍｅｒ）为竞争性寡核苷酸分子，发现人工合成的Ｔ７启动子可能 与 ＴＦⅡＤ起始转录因子结合，形成 ＤＮＡ－核蛋白质结合物。将 ＴＡＴＡ框和八核苷酸序列分别 接入Ｔ７启动子上游，ＣＡＴ实验显示八核苷酸序列可以增强ＲＮＡ聚合酶Ⅱ对Ｔ７启动子的转录 起始作用。实验结果表明Ｔ７启动子可作为ＲＮＡ聚合酶Ⅱ的顺式作用因子。Using the chlorampheniol acetyltransterase gene as reporter, the function of phage T7 promoter in mammalian cells was studied by inhibition of transcription with a-amanitin. The experiment proved that the reporter under T7 promoter was transcribed by RNA polymerase Ⅱ. Competitive electropho retic mobility shift assay (CEMSA) with TATA box, CAAT box, GC box and octamer showed that the TATA box was competitive molecular for synthetic T7 promoter. It is possible that T7 promoter is bound with TF ⅡD transcription factor. The TATA box and octamer were inserted into Pvu Ⅱ site upstream from the T7 promoter of pT7CAT. Two recombinant plasmids, pT7TATACAT and pT7OCTCAT, were constructed and transfected into CHO cells. CAT-activity test showed that T7 promoter strength was increased by octamer factor, not by TATA box. These results suggested that T7 promoter functions as cis-acting elements of RNA polymerase Ⅱ transcriptional system in eucaryotic cells.

关 键 词：T7启动子 顺式作用因子 转录起始 真核生物POLⅡ 

分 类 号：Q754[生物学—分子生物学]