本論文係以酵母菌S. cerevisiae為模式來研究SV之複製，利用SV-replicase replicon必須經由RNA複製步驟才能表現外源基因之特性，將其構築於可在酵母菌中複製之載體，並以hygromycin抗藥基因為篩選標的（selectable marker），構築於病毒次基因體RNA啟動子（subgenomic promoter）之後，因此可藉由抗藥性菌落之產生以及RT-PCR偵測負股RNA之形成，做為判斷病毒RNA是否複製之依據。為了確認所構築之重組質體S1/Rep21/hyg的確是經由完整RNA複製之步驟，來提供酵母菌對hygromycin之抗藥性，因此刪除複製中所必需之3’UTR序列及破壞複製所需之replicase，結果發現S1/Rep21/hyg/△3’UTR仍可產生抗藥性菌落且可偵測到負股RNA合成，而S1/Rep21/hyg/△P雖不能產生抗藥性菌落但卻可偵測到負股RNA合成，因此推論在抗hygromycin基因之後有一在載體上之反向DNA啟動子。為解決此干擾，進一步將S1/Rep21/hyg直線化，結果證實以直線化之S1/Rep21/hyg確實可提供酵母菌對hygromycin之抗藥性，因此建立了S1/Rep21/hyg可在酵母菌中複製之系統。此外，並針對已知在哺乳動物細胞中較不毒之nsP2（P726L）突變與野生型nsP2比較，發現S1/Rep21/hyg比S1/Rep21/hyg_nsP2(wt)者菌落生長較大且其生長速率在進入log phase後變更快，這可能是因P726L突變造成S1/Rep21/hyg複製時對酵母菌比較不毒之故。故本論文建立以酵母菌為良好的研究模式來探討SV之複製，未來更可進一步研究宿主因子與SV複製間之交互作用，藉此對SV在細胞中複製之分子機轉能有深入之瞭解。

In this study, we use yeast S. cerevisiae as a model to study the replication of sindbis virus (SV), which is a positive sense, single-stranded animal RNA virus belonging to alphavirus. To serve as a viral expression vector, foreign genes are often constructed under control by SV subgenomic RNA promoter, so that the expression of the given foreign genes must be the result of SV replication process. Characteristically, it is sufficient for SV to complete viral RNA replication only through its replicase complex derived from virus non-structural genes. In order to study SV replication in yeast system, we constructed a series of SV-replicase replicon in a yeast expression plasmid (pYES-X1) and used the hygromycin resistant gene as a selectable marker that was under the subgenomic RNA promoter. The generation of hygromycin-resistant colonies and the detection of virus negative strand RNA by RT-PCR were the two major assessments to evaluate SV replication in yeast system. Our results show that S1/Rep21/hyg could result in hygromycin resistant yeast colonies. To examine whether this was the outcome of SV RNA replication, we deleted the 3’UTR sequence which is required for replication, and we found S1/Rep21/hyg/△3’UTR was unexpectedly able to generate resistant colonies and negative strand of virus RNA. In contrast, S1/Rep21/hyg/△P, whose replicase was partially deleted, could not generate resistant colonies but was still able to synthesize the negative strand of virus RNA. These results suggest that there may be a reverse DNA promoter behind hygromycin resistant gene on vector that initiates the synthesis of negative strand of virus RNA. To overcome this interference, we linearized the plasmid of S1/Rep21/hyg and found that the linear form of S1/Rep21/hyg was indeed able to form resistant colonies, clearly suggesting that S1/Rep21/hyg is capable of replication in yeast. In conclusion, this study establishes a SV replication system in yeast, and hopefully this system can be extended to study the interactions between host factors and SV. So we can further realize the molecular mechanism of SV replication in the cellular levels.

目 錄 ………………………………………………………… 1
中文摘要 …………………………………………………… 2
英文摘要 ……………………………………………………… 3
第一章　前言 ………………………………………………… 5
第二章 材料與方法 ………………………………………… 11
第三章 結果 ………………………………………………… 25
第四章 討論 ………………………………………………… 32
第五章 參考文獻 ………………………………………… 39
第六章 圖表 ………………………………………………… 43
第七章 附錄 ………………………………………………… 51

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