摘要

ArsA蛋白是大腸桿菌經由質體R773所產生抗砷系統中的催化次單元位，此抗砷系統利用ArsA蛋白水解ATP的能量，將進入細胞的砷化物或是銻化物排除至細胞外，而造成大腸桿菌對砷化物、銻化物產生抗性。目前，在已知的真核生物基因庫中，當以大腸桿菌的arsA基因進行搜尋與比對時，在人類、小白鼠、原蟲及酵母菌….等細胞均可找到arsA基因的同源基因。但目前除了ArsA蛋白在大腸桿菌可以確定有抗砷功能外，其他生物中的ArsA同源蛋白真正功能仍不明。由先前研究結果顯示，酵母菌的ArsA同源蛋白Ydl100cp缺失並不影響arsA同源基因被破壞的knock-out（KO）菌株對葡萄糖、乙醇或甘油的利用，即Ydl100cp並不直接參與酵母菌在有氧或厭氧條件下對不同碳源的代謝。而YDL100c缺失也不影響KO菌株在30℃時對砷化物或銻化物的抗性，因此YDL100c與抗砷機制無直接相關。但KO菌株在30℃與37℃時，對多種金屬離子具敏感性，而當溫度逐漸上升時WT與KO菌株出現明顯生長差異，且高溫時（40℃）KO菌出現大量死亡。
本實驗使用YEp352載體攜帶YDL100c基因與YDL100c基因自身promoter為材料，以了解在何種情形下能誘發Ydl100c蛋白的表現。實驗結果顯示菌株在37℃時，Ydl100c蛋白明顯較在30℃時增加約30﹪，證明Ydl100c蛋白會隨溫度上升而增加表現。另外在低劑量毒性金屬存在下，Zn2+、Ni2+與Sb3+也能誘發Ydl100c蛋白的表現增加，而在其他環境壓力下發現，pH值偏中鹼性時也能些微誘發Ydl100c蛋白表現，但整體來說以溫度對Ydl100c蛋白的表現影響最為顯著。
針對YDL100c基因進行網路資料庫搜尋(yeast two hybrid 、promoter sequence analysis)，配合二維電泳膠分析、流式細胞儀分析、顯微鏡觀察等實驗中發現，在二維電泳膠分析結果方面，顯示高溫下，WT及KO菌株明顯有一些蛋白質表現的分佈差異；流式細胞儀分析顯示，YDL100c基因缺乏的KO菌株，細胞週期明顯停滯於G2/M phase；顯微鏡下顯示，酵母菌並未完全完成其出芽動作，即經細胞壁染色後發現子細胞與母細胞間細胞壁仍相連接，未分離完全。以致不能形成單一個子細胞個體，造成顯微鏡下呈現細胞致密叢聚及大量巨大多核細胞情形。因此推論Ydl100c蛋白表現受環境中nonpermissive temperature誘發，以參與酵母菌生長環境中當遭遇nonpermissive temperature時的細胞週期，其扮演角色可能類似late mitosis cyclin-like protein或cyclin dependent kinase inhibitor，並同時參與調控下游相關於細胞壁結構、合成基因。所以當遭遇nonpermissive temperature時，KO菌株呈現出隨溫度上升而死亡的現象，即是因為KO菌株缺乏可被溫度誘發產生的Ydl100c蛋白，致使其與WT菌株產生生長差異。
綜合以上實驗結果證實，酵母菌在nonpermissive temperature下所被誘發表現的Ydl100c蛋白，作用可能為大幅降低巨大多核細胞產生，並調控下游相關於late mitosis phase子母細胞間細胞壁合成、結構維持的多種基因表現。所以當細胞處於nonpermissive temperature下時才能安然度過，不至於大量死亡。而先前研究結果顯示，因KO菌株相較於WT菌株會表現出對多種金屬具敏感性，故推斷YDL100c基因可能跟general stress responses相關。但根據本實驗結果顯示，由於YDL100c基因與nonpermissive temperature下的細胞壁結構相關，所以當KO菌株培養於高溫下，KO菌株本身細胞壁結構產生缺失，很可能使位於細胞壁上負責ion transport的位置產生缺陷，以致當毒性金屬，例如鋅、鈷、鎳、銻化物、砷化物存在於環境中時，並無法及由酵母菌體內排除，所以毒性金屬反而更能利用其細胞壁結構缺陷進入細胞中，造成KO菌株細胞死亡而呈現相較於WT菌株的金屬敏感性，以及造成在KO菌株在高溫下細胞去活性大量死亡之後續結果。因此推論如果Ydl100c蛋白在nonpermissive temperature能正常表現，Ydl100c蛋白即能參與高溫時保護細胞的角色以及反應當遭受環境壓力下抗壓作用，例如排除有毒金屬及回復原先偏酸pH值之機制。

Abstract

ArsA protein is the catalytic component of the bacteria plasmid R773-encoded ArsAB pump that is in involved in As3+ detoxification. Homologues of the ArsA protein are found in nearly all organisms but the biological functions of these homolog proteins are still unclear. The ArsA homologue in S. cerevisiae is encoded by the ORF YDL100c.
Initial studies show that deletion of YDL100c in S. cerevisiae was not lethal and had no effect on As3+ sensitivity at 30℃. However, the disrupted strain (KO strain) is unable to grow at 40℃ and shows increased sensitivity to Co2+,Zn2+,As3+ and Sb3+ at 37℃ by spotting assay. In this study, a plasmid (YEp352) carrying the YDL100c under the control of its endogenous promoter was used to study the induction of YDL100c under various stress conditions. The data show that the expression of Ydl100cp increased 30 % at 37℃ compared to that at 30℃, and the expression can be induced by low dosage of Zn2+, Ni2+, Sb3+ and neutral to alkaline pH. Overall, temperature is the best inducer for Ydl100cp expression.
Besides, searching Ydl100cp in Internet yeast two hybrid database and YDL100c promoter sequence analysis database suggest the following experiments and results:（1）2D gel electrophoresis assay to demonstrate different protein patterns between WT and KO strain under nonpermissive temperature. （2）Flow cytometry data indicate most of KO strain cells growth arrest at G2/M phase in nonpermissive temperature. （3）Microscopic data reveal KO stain cells grew very densely and showed cluster phenotype at nonpermissive temperature. When Congo red was used to stain cell wall，it was found that these cluster cells is actually one cell. Although the cell wall between mother and daughter cell can form cleavage furrow, the formation is not complete and cell can’t separate into two individuals. Consequently, the cells grow densely with cluster form and mega-polynuclear cells. It suggests Ydl100cp is induced and plays a role in cell cycle under nonpermissive temperature. The function of Ydl100cp may be a late mitosis cyclin-like protein or cyclin dependent kinase inhibitor that controls several downstream genes related to cell wall formation, maintenance, and structure. Because KO strain does not have Ydl100cp, it shows different growth patterns compared to WT strain when grow at nonpermissive temperature.
Initial studies suggest that YDL100c is involved in general responses because KO strain shows sensitivity to a broad range of metals. However, based on the results have, it is possible that Ydl100cp is involved in cell wall structure, formation and maintenance. Under nonpermission temperature cell wall of KO strain had defect that led to defect in ion transport structure. Therefore cell can remove not only can not poison metals especially Zn2+, Ni2+, Co2+, arsenite and antimonite metals right away but these metals can also pass cell wall into cytoplasm that causes KO strain reveals sensitivity to metals.
To sum up the results, the expression of Ydl100cp can be induced under nonpremssive temperature to decrease mega-polynuclear cells formation and control downstream genes for cell wall formation, maintenance and structure. Therefore yeast cells can survive at nonpermissive temperature instead to be killed.

英文摘要………………………………1
中文摘要………………………………4
前言……………………………………7
材料方法………………………………12
結果……………………………………20
討論……………………………………25
參考文獻………………………………35
圖………………………………………40
表………………………………………56

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