Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23808
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dc.contributor.advisor張功耀zh_TW
dc.contributor.advisorKung-Yao Changen_US
dc.contributor.author蘇美綺zh_TW
dc.contributor.authorSu, Mei-Chien_US
dc.date2004zh_TW
dc.date.accessioned2014-06-06T07:21:12Z-
dc.date.available2014-06-06T07:21:12Z-
dc.identifier.urihttp://hdl.handle.net/11455/23808-
dc.description.abstract中文摘要 嚴重急性呼吸道症候群其致病原為SARS冠狀病毒,由基因序列得知其ORF1a與ORF1b序列之間有一個stop codon可以使轉譯停止,導致ORF1ab無法被轉譯以形成replicase complex proteins,進而使得病毒無法進行複製與轉錄,因此ribosome必需進行-1轉譯框架移轉,才能使ORF1ab的轉譯繼續進行。根據序列比對及RNA二級結構的預測顯示SARS冠狀病毒具有-1轉譯框架移轉所需的slippery sequence及可能的pseudoknot structure信號,且其pseudoknot structure在loop 2的區域可形成一額外的stem loop (stem 3)。 利用in vitro transcription coupled translation的分析,我們證實了SARS冠狀病毒核酸包含了slippery sequence及可能pseudoknot structure的序列能夠誘發-1轉譯框架移轉。若破壞這兩個信號皆會導致-1轉譯框架移轉無法進行,並且也觀察到破壞stem 3之間的鹼基配對會使得-1轉譯框架移轉效率下降,一旦回復stem 3之間的配對其-1轉譯框架移轉效率則又回升,顯示stem 3的鹼基配對能影響-1轉譯框架移轉效率,但非-1轉譯框架移轉所必需的信號。此外我們在slippery sequence的上游SARS13222-13369區域觀察到可能具有調節SARS冠狀病毒-1轉譯框架移轉效率的RNA信號,但由目前的實驗結果還無法確切的瞭解這個RNA信號是如何調節-1轉譯框架移轉的效率。最後已知可以提高及減低一些病毒系統中之-1轉譯框架移轉效率的Sparsomycin及Anisomycin,在我們的藥物測試實驗中似乎不太能影響SARS冠狀病毒-1轉譯框架移轉的效率。zh_TW
dc.description.abstractAbstract The servere acute respiratory syndrome is caused by SARS coronavirus (SARS-CoV). The genomic sequence of SARS-CoV possesses a stop codon between the junction of ORF1a and ORF1b that can cause the translational termination, and thus block ORF1ab fusion protein synthesis crucial for replicase complex formation. Eventhally, it will lead to the inhibition of virus replication. The continuing translation of ORF1ab will need a —1 ribosomal frameshift, and sequence alignment as well as RNA secondary structure prediction indicate the existence of a set of putative frameshifting signal for SARS-CoV. It is formed by a slippery sequence and a downstream atypical pseudoknot structure with an extra stem loop (stem 3) in the loop 2 of the pseudoknot structure. We have shown the putative SARS-CoV —1 frameshifting signal can induce —1 frameshift in vitro. Disruption of either signal can eliminates the —1 frameshift. Furthermore, the disruption of the base pairs forming stem 3 will reduce the efficiency of —1 frameshift while restoring the base pairs also restore the —1 frameshift efficiency. The result indicates the integrity of stem 3 is a factor for the —1 frameshift, but is not essential for —1 frameshift to occur. In addition, we locate an attenuation RNA signal (SARS13222-13369) upstream of the slippery sequence with activity of regulating the SARS-CoV —1 frameshifting efficiency. However, the mechanisms of this attenuation remain unknown. Finally, two antibiotics, sparsomycin and anisomycin, capable of modulating —1 frameshift efficiency in several viral systems fail to modulate the efficiency of —1 frameshift of SARS-CoV in our assay system.en_US
dc.description.tableofcontents目錄 目錄………………………………………………………………………………………………………Ⅰ 圖表目錄 ……………………………………………………………………………………………Ⅲ 中文摘要 ………………………………………………………………………………………………1 英文摘要 Abstract ……………………………………………………………………………2 第一章 緒論 ………………………………………………………………………………………4 1.1 SARS的發現與臨床症狀 ……………………………………………………4 1.2 SARS冠狀病毒生命週期概述 ……………………………………………5 1.3 -1轉譯框架移轉對於SARS-CoV基因表現的重要性 ………7 1.4 -1轉譯框架移轉需要特殊RNA信號的誘導 ……………………8 1.5 —1轉譯框架移轉之機制 …………………………………………………10 1.6 研究動機及目的 …………………………………………………………………13 第二章 實驗材料與方法 …………………………………………………………………16 2.1 試藥來源及分析軟體 …………………………………………………………16 2.2 菌株與質體來源 …………………………………………………………………16 2.3 質體構築 ………………………………………………………………………………17 2.4 Double-strand DNA的定量 ……………………………………………22 2.5 運用In vitro transcription coupled translation探討-1轉譯框架移轉之進行 …………………………………………………22 第三章 實驗結果 ………………………………………………………………………………25 3.1 SARS-CoV—1轉譯框架移轉之分析 …………………………………25 3.2 調節SARS-CoV—1轉譯框架移轉效率之RNA信號分析…34 Ⅰ 3.3 增加slippery sequence及pseudoknot結構之上下游 核苷酸長度可以影響-1轉譯框架移轉效率之分析 ………41 3.4 slippery sequence上游序列對之-1轉譯框架移轉效率之影響程度不因不同的報告基因計算方法而不同 ……………50 3.5 運用Sparsomycin及Anisomycin影響 SARS-CoV—1轉譯框架移轉效率之結果分析 …………………………………………………52 第四章 討論 ………………………………………………………………………………………56 4.1 SARS-CoV -1轉譯框架移轉訊號內之pseudoknot structure與其它冠狀病毒不同 ………………………………………56 4.2 -1轉譯框架移轉之slippery sequence上游可能具有調控移轉效率之attenuation signal…………………………………57 4.3 Sparsomycin與Anisomycin不影響SARS-CoV —1轉譯框架移轉之進行 ………………………………………………………………………59 第五章 附錄 ……………………………………………………………………………………60 第六章 參考文獻 ……………………………………………………………………………65 Ⅱzh_TW
dc.language.isoen_USzh_TW
dc.publisher生物化學研究所zh_TW
dc.subject1 frameshiften_US
dc.subject1轉譯框架移轉zh_TW
dc.titleSARS冠狀病毒-1轉譯框架移轉機制之研究zh_TW
dc.titleStudy of the mechanism of translational -1 frameshift of SARS-CoVen_US
dc.typeThesis and Dissertationzh_TW
Appears in Collections:生物化學研究所
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