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Title: | EB病毒早期基因BBLF2/3的啟動子分析 Functional Analysis of the Promoter of Epstein-Barr Virus BBLF2/3 |
Authors: | Yi-Ting Lin 林怡廷 |
Advisor: | 張麗冠 |
Keyword: | EB病毒,Rta,BBLF2/3,MCAF1,ATF2,AP-1結合序列, EBV,Rta,BBLF2/3,MCAF1,ATF2,AP-1 binding site, |
Publication Year : | 2011 |
Degree: | 碩士 |
Abstract: | Rta與Zta蛋白質為Epstein-Barr virus (EBV)溶裂期的極早期蛋白質,為轉錄活化子,可以活化早期與晚期基因表現。Rta蛋白質可以直接結合到Rta response element (RRE)上,活化BMRF1、BHRF1、BHLF1與BMLF1等病毒早期基因的表現。另外,Rta也可以形成Sp1-MCAF1-Rta複合體,藉由Sp1結合序列活化自身的表現。Rta蛋白質亦可與Zta蛋白質形成Zta-MCAF1-Rta複合體,結合到啟動子上的Zta response element (ZRE)序列造成協同作用,大量地活化BHLF1、BMRF1與BRLF1等基因的表現。BBLF2/3為EB病毒溶裂期早期基因,可以轉譯出引子酶結合因子 (primase-associated factor),參與EB病毒溶裂期DNA複製。然而,目前對於Rta與Zta活化早期基因BBLF2/3的機制尚不清楚,因此本研究的目的在於探討Rta與Zta蛋白質對於BBLF2/3啟動子的調控。首先利用冷光酵素分析發現BBLF2/3啟動子可以被Rta活化,同時轉染Rta與Zta蛋白質時會產生協同作用,大量活化BBLF2/3的表現,而Zta蛋白質則無法單獨活化BBLF2/3啟動子。利用TESS網站分析BBLF2/3啟動子,發現該啟動子-74到-80的序列具有可能的AP-1結合序列。將該位置進行點突變後進行冷光酵素分析,發現Rta對BBLF2/3啟動子的活化大幅降低。將BBLF2/3可能的AP-1結合序列接到具有TATA box的pTATA質體進行冷光酵素分析,發現細胞內Rta或ATF2蛋白質的含量越多時,對於該質體的活化能力就越高。另外,以DNA親和沉澱分析證實BBLF2/3啟動子-74到-80的序列可以與ATF2蛋白質結合,而透過凝膠電泳位移實驗也發現Rta蛋白質可以與該序列結合。因此,本篇研究發現BBLF2/3啟動子上具有AP-1結合序列,而Rta蛋白質可以藉由此序列促進BBLF2/3的轉錄。 Rta and Zta, two transcription factors expressed during the immediate-early stage of the Epstein-Barr virus (EBV) lytic cycle, are required for lytic activation. Rta activates many EBV early genes, including BMRF1, BHRF1, BHLF1 and BMLF1, through direct binding to Rta response elements (RRE). Rta also enhances SP1-mediated transcription through the interaction with MBD1-containing chromatin-associated factor 1 (MCAF1) and Sp1. In addition, the binding of Rta-MCAF1-Zta complex to Zta response elements (ZRE) activates BHLF1, BMRF1 and BRLF1 synergistically. The EBV BBLF2/3 gene encodes a primase-associated factor that is indispensable for EBV lytic DNA replication. However, the mechanism that activates the transcription of BBLF2/3 remains unclear. The purpose of this study is to elucidate how Rta and Zta activate BBLF2/3 transcription. This study finds that the BBLF2/3 promoter is activated by Rta, but not by Zta. Moreover, Rta and Zta activated the BBLF2/3 promoter synergistically. Sequence analysis from the TESS website predicts that BBLF2/3 promoter contains a putative AP-1-binding sequence, TGACACG, which is located at -74 to -80 in the BBLF2/3 promoter. Reporter assay showed that mutating the site lowers the transcription activity. Meanwhile, Rta and ATF2 activate a promoter in a reporter plasmid, pBBLF2/3-3AP1, which contains three copies of AP-1 site from the BBLF2/3 promoter, in a dose-dependent manner. In addition, ATF2 binds to the AP-1 site as demonstrated by DNA-affinity precipitation assay. Rta also interacts with the AP-1 site as shown by electrophoretic mobility shift assay. Taken together, this study demonstrates that the BBLF2/3 promoter contains an AP-1 binding site from -74 to -80. Rta activates the BBLF2/3 transcription via an indirect binding to the AP-1 site. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27641 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生化科技學系 |
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