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研究生:楊善倍
研究生(外文):Shan-Bei Yang
論文名稱:RNA結構和接收模板的濃度對D型肝炎病毒RNA重組的影響
論文名稱(外文):RNA Recombination of Hepatitis Delta Virus:Role of RNA Structure and Acceptor Concentration
指導教授:趙玫趙玫引用關係
指導教授(外文):Mei Chao
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:107
中文關鍵詞:D型肝炎RNA重組
外文關鍵詞:HDVRNA recombination
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D型肝炎病毒 (HDV) 為唯一以細胞RNA聚合脢 (polymerase) 催化RNA基因體複製之動物病毒,且複製過程中也有RNA 重組的現象發生。本文以template switch機制為出發點探討可能影響HDV RNA重組的因素-RNA二級結構及接收模板 (acceptor template) 的濃度。HDV RNA雖為桿狀不分枝結構,但在複製過程中桿狀結構有機會在RNA聚合酶附近鬆解而形成二級結構,進而造成RNA 聚合酶與新合成之RNA一起掉落並跳至接收模板。首先,我分析重組頻率特別高的區域附近可能形成之二級結構,找到一個可能影響重組率的stem-loop,將其單點突變,增加stem-loop的穩定度,發現此區域的重組率提升;再用雙點突變降低此處之穩定度,發現重組分佈回復如野生型,證實重組頻率會受RNA二級結構的影響;在分析過程中觀察到另ㄧ有趣之現象,即HDV RNA重組存在aberrant homologous recombination。再者,我利用不同比例的兩株HDV分析接收模板 (acceptor template) 濃度的影響,從初步結果看來,重組的產生多來自於基因股合成方向,且接收模板的濃度也是影響重組發生頻率高低的重要因素。這些結果皆支持HDV RNA重組是經由template switch的機制進行的,如此不僅能幫助我們更了解HDV重組的分子機制,也可擴展寄主RNA聚合酶功能的多樣性。
Hepatitis delta virus (HDV) is the only animal virus that replicated it’s RNA genomes by host RNA polymerase. RNA recombination has been found in a large number of RNA viruses including HDV. Although the molecular mechanism of RNA recombination is not fully understood, the replication-dependent template-switching mechanism has been proposed to contribute to most of the RNA recombination events. RNA secondary structure and the concentration of acceptor template have been suggested to determine recombination frequency. The aim of my thesis is to investigate the role of these factors in HDV RNA recombination. Although the HDV RNA is a rod-like structure, it could undergo dynamic conformational changes around the polymerase binding site during genome replication. These changes may contribute to the formation of a local RNA secondary structure which induce polymerase pausing and template switching. It has been published that nt 1159-1207 on the HDV genome is a RNA recombination hot spot. Interestingly, I found a G-C rich stem-loop structure at the 5’-end of this hot spot. To investigate the potential role of this structure in HDV RNA recombination, point mutations that affect the stability of the stem-loop were constructed. The results indicated that the recombination frequency around the stem-loop was increased significantly when a point mutation was introduced to strengthen the stem-loop. In contrast, this effect was reversed when a compensatory mutation of this point mutation was constructed to reduce the stem-loop stability. Further analyses of the recombination junctions of the recombinants, aberrant homologous recombination was observed in HDV. To investigate the role of the acceptor concentration in recombination frequency, I examined the recombination patterns between two HDV genotypes with different ratios. The results suggested that HDV recombination occurs mainly during genomic RNA synthesis, and the acceptor concentration is an important factor determining the recombination frequency. Taken together, HDV RNA recombination could be carried out by template- switching mechanism. These results not only contribute to the elucidation of, but also expand the capacity of host RNA polymerase.
指導教授推薦書…………………………………………………………… i
口試委員會審定書………………………………………………………… ii
授權書……………………………………………………………………… iii
誌謝………………………………………………………………………… iv
中文摘要…………………………………………………………………… v
英文摘要…………………………………………………………………… vi
目錄………………………………………………………………………… vii
圖表目錄………………………………………………………...……… viii
第一章 緒論……………………………………………………………… 1
第二章 材料與方法……………………………………………………… 23
第三章 實驗結果………………………………………………………… 33
第四章 討論……………………………………………………………… 48
參考文獻…………………………………………………………………… 59
圖表………………………………………………………………………… 72
附錄………………………………………………………………………… 102
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