English  |  正體中文  |  简体中文  |  Items with full text/Total items : 43312/67235
Visitors : 2077141      Online Users : 3
RC Version 5.0 © Powered By DSPACE, MIT. Enhanced by NTU/NCHU Library IR team.
National Chung Hsing University Institutional Repository - NCHUIR > 獸醫學院 > 獸醫病理生物學所 > 依資料類型分類 > 碩博士論文 >  牛冠狀病毒天然缺陷株RNA的3’末端基因序列蘊含正股及負股病毒基因體合成所需之Cis-Acting Elements

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/155156

標題: 牛冠狀病毒天然缺陷株RNA的3’末端基因序列蘊含正股及負股病毒基因體合成所需之Cis-Acting Elements
The 3’Terminal Sequence of Bovine Coronavirus Defective Interfering RNA Harbors Cis-Acting Elements for Both Negative- and Positive-Strand RNA Synthesis
作者: 廖偉瑜
Liao, Wei-Yu
Contributors: 吳弘毅
Hung-Yi Wu
獸醫病理生物學研究所
關鍵字: 論文;中興大學;獸病所;冠狀病毒;負股;正股
thesis;NCHU;GIVP;coronavirus;negative-strand;positive-strand
日期: 2012
Issue Date: 2013-11-21 11:42:16 (UTC+8)
Publisher: 獸醫病理生物學研究所
摘要: 冠狀病毒 (Coronavirus)為具有封套 (envelope)及長度約30 kilobase (kb)基因體的單股、正向RNA病毒。在病毒的複製過程中,負股基因體RNA的形成為冠狀病毒基因體RNA複製的第一步,根據文獻指出,冠狀病毒基因體3’端非轉譯區 (3’ untranslated region,3’UTR)末端最後55個核苷酸為鼠冠狀病毒 (mouse hepatitis coronavirus,簡稱MHV)負股基因體RNA形成的條件之一。但是在此55個核苷酸當中是哪些序列或是結構對於正股以及負股的形成具有重要性,目前尚無文獻記載。為了降低實驗操作的困難性及避免突變對轉譯 (translation)的功能造成影響,在本研究中我們以牛冠狀病毒 (bovine coronavirus,簡稱BCoV)天然缺陷株RNA (defective interfering RNA,簡稱DI RNA)為實驗模組,並利用即時定量聚合酶連鎖反應 (quantitive real-time PCR,qRT-PCR)以及北方墨漬法 (Northern assay)分別探討牛冠狀病毒3’端末端最後55個核苷酸在負股及正股基因體形成上的重要性,以確定最後55個核苷酸之中基因體RNA複製所需之cis-acting elements,進而瞭解病毒複製的機轉、輔助抗病毒藥物的研發。為了克服偵測負股基因體RNA所出現的偽陽性以及數量過少的困難,我們利用T4 RNA ligase I將負股基因體head-to-tail ligation,並利用qRT-PCR定量新生成的負股基因體RNA。我們發現: (i)冠狀病毒基因體3’UTR最後45個核苷酸對於負股DI RNA的形成具有重要性,其中倒數第5至14 (nt -5至nt -14)以及第35至39 (nt -35至nt -39)的核苷酸可能為負股DI RNA合成所需的cis-acting elements; (ii)冠狀病毒基因體 3’UTR最後55個核苷酸對於正股DI RNA的形成皆很重要,其中倒數第3至34的核苷酸 (nt -3至nt -34)可能為正股DI RNA的形成所需之cis-acting elements。 (iii)冠狀病毒基因體3’UTR最後一個核苷酸種類的改變皆會抑制負股DI RNA之合成能力,但是最後一個核苷酸若為胞嘧啶 (cytosine,C)或尿嘧啶 (uracil,U),亦即嘧啶類的核苷酸,對於正股DI RNA的形成較有效率。 (iv)冠狀病毒基因體3’UTR最後55個核苷酸之中,位於倒數第40至44個核苷酸 (nt -40至nt -44) (與nt -10至nt -14鍵結)所形成的雙股二級結構 (secondary structure),對冠狀病毒正股DI RNA的合成具有顯著的影響。綜合以上結果可以得知,牛冠狀病毒天然缺陷株RNA的3’末端基因序列蘊含正股及負股病毒基因體合成所需之cis-acting elements。
Synthesis of the negative-strand complement of the ~30 kilobase, positive-strand coronaviral genome is a necessary early step for genome replication. It has been shown that for negative-strand RNA synthesis from the mouse hepatitis coronavirus (MHV) genome only the 3’-most 55 nucleotides along with the poly (A) tail in the 3’-untranslated region (UTR) are required. However, map positions for specific cis-acting RNA elements for replication (i.e., both negative and positive-strand RNA synthesis) within this 55-nts region remain unknown. Here, we used a bovine coronavirus defective interfering RNA (DI RNA) with the MHV 3’UTR to characterize the cis-acting RNA requirements in the 3’-terminal 55 nucleotides for viral RNA negative-strand synthesis and replication (interpreted as positive-strand RNA synthesis). The major findings are: (i) Nucleotides from -5 to -14 and -35 to -39 within the 3’-terminal 55 nts are the cis-acting elements responsible for negative-strand BCoV DI RNA synthesis, (ii) Nucleotides from -3 to -34 within the 3’-terminal 55 nts are cis-acting elements required for positive-strand BCoV DI RNA synthesis, (iii) Substitution of 3’-most C residue by U (pyrimidine), but not A and G (purine), is more tolerant for positive-strand synthesis. (iv) Base-paired structure mapping at nt -40 to nt -44 (binding with nt -10 to nt -14) is critical for maintaining efficient positive-strand DI RNA synthesis. The results together demonstrate that the 3’-most 55 nucleotides in the 3’UTR of DI RNA harbor cis-acting elements required not only for negative-strand RNA synthesis but also for positive-strand RNA synthesis.
Appears in Collections:[依資料類型分類] 碩博士論文

Files in This Item:

File SizeFormat
index.html0KbHTML147View/Open


 


學術資源

著作權聲明

本網站為收錄中興大學學術著作及學術產出,已積極向著作權人取得全文授權,並盡力防止侵害著作權人之權益。如仍發現本網站之數位內容有侵害著作權人權益情事者,請權利人通知本網站維護人員,將盡速為您處理。

本網站之數位內容為國立中興大學所收錄之機構典藏,無償提供學術研究與公眾教育等公益性使用。

聯絡網站維護人員:wyhuang@nchu.edu.tw,04-22840290 # 412。

DSpace Software Copyright © 2002-2004  MIT &  Hewlett-Packard  /   Enhanced by   NTU/NCHU Library IR team Copyright ©   - Feedback