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    Please use this identifier to cite or link to this item: http://tkuir.lib.tku.edu.tw:8080/dspace/handle/987654321/74152

    Title: (I) 啤酒酵母菌YNR029Cp的表現和純化及甲基化活性之研究 (II) 過度表現啤酒酵母菌ADH3p對其粒線體形態影響
    Other Titles: (i) Study of saccharomyces cerevisiae YNR029Cp : experssion, purification, and methylation activity. (ii) Effect of overpressing saccharomyces cerevisiae ADH3p on mitochondrial morphology
    Study of saccharomyces cerevisiae YNR029Cp : experssion, purification, and methylation activity
    Effect of overpressing saccharomyces cerevisiae ADH3p on mitochondrial morphology
    Authors: 陳宏仁;Chern, Hang-Jen
    Contributors: 淡江大學化學學系碩士班
    陳銘凱;Chern, Ming-Kai
    Keywords: 甲基轉移酶;甲基化;粒線體形態;酒精去氫酶III;methyltransferase;methylation;mitochondrial morphology;ADH3
    Date: 2011
    Issue Date: 2011-12-28 18:10:57 (UTC+8)
    Abstract: 表觀遺傳調控(epigenetic regulation),泛指在沒有改變DNA序列的情況下,透過DNA外的分子修飾,造成DNA的結構上的改變,進而影響基因活化或表現的修飾作用。然而這類的修飾作用包含了甲基化(methylation)、去甲基化(demethylation)、磷酸化(phosphorylation)、去磷酸化(dephosphorylation)、乙醯化(acetylation)、去乙醯化(deacetylation)、泛素化(ubiquitination)等。這其中甲基化更是常見的修飾作用,因此本研究第一部分即是利用啤酒酵母菌進行甲基轉移酶的探討。

    根據Steven Clarke所提出一份經由序列及數學演算分析可能具有甲基轉移酶活性名單,我們挑選了感興趣的啤酒酵母菌基因YNR029C進行深入探討。我們利用了重組蛋白技術及His-tag純化,純化出YNR029Cp並與去除YNR029C基因之啤酒酵母菌(統稱△YNR029C)受質進行甲基化活性分析。結果表示啤酒酵母菌基因YNR029C目前尚無法利用本研究的方法證實其具有蛋白質甲基轉移酶的活性。

    本研究第二個部份觀察基因表現與胞器形態之間的關係。我們所著重的是啤酒酵母菌中ADH3p對粒線體形態所造成的影響。我們將建構之ADH3-pYES2、ADH3G211A- pYES2;ADH3-GFP- pYES2、ADH3G211A-GFP- pYES2轉形至酵母菌(BY4742、BJ2168、△YLL001W),並進行蛋白質表現誘導及利用螢光顯微鏡觀察過度表現ADH3p對粒線體形態的影響。結果發現當過度表現ADH3p確實會造成粒線體形態發生改變,形態變成較小且數目較多的顆粒狀粒線體。且我們也誘導了ADH3-GFP- pYES2,將粒線體使用紅螢光染色與ADH3-GFP- pYES2所帶之綠螢光蛋白進行對照分析,試著了解ADH3-GFP蛋白於粒線體中的分佈位置與分布量,結果發現粒線體與ADH3-GFP螢光顯示部分重疊,這說明粒線體形態的改變可能與ADH3p的活性有關。同時我們使用流式細胞儀對ADH3-GFP及ADH3G211A-GFP進行蛋白質定量分析。根據流式細胞儀的結果表示,過度表現ADH3p造成粒線體形態改變是受蛋白質活性影響。
    Epigenetic regulation is causing DNA structural changes by modifier which leaves DNA sequence unchanged. Further epigenetic regulation affects gene activation or expression. Therefore, this modification includes methylation), demethylation, phosphorylation, dephosphorylation, acetylation, deacetylation, and ubiquitination. The methylation is the most common of modification. Consequently, my first part of research is using Saccharomyces cerevisiae to proceed the study of methyltransferase.

    According to Steven Clarke who provided a list which shows potential methyltransferase activities by mathematical calculation and analysis of sequences.
    We were interested in the Saccharomyces cerevisiae gene YNR029C which was selected for in-depth exploration. We use recombinant protein technology and His-tag purification to purify YNR029Cp. We take the YNR029C-knockout strain Saccharomyces cerevisiae (△YNR029C) as substrate. We take YNR029Cp and the protein extract of △YNR029C for reaction which analyses the methylation activity. The result indicates YNR029Cp does not exhibit protein methyltransferase activity by the methodology employed in this research.

    My second part of research is observing relationship of gene expression with organelle morphology. We stress on Saccharomyces cerevisiae ADH3p affecting mitochondrial morphology. We transform yeast strains (BY4742, BJ2168 and △YLL001W) with ADH3-pYES2, ADH3G211A- pYES2, ADH3-GFP- pYES2 and ADH3G211A-GFP- pYES2, and induce protein expression. We use fluorescence microscope to observe mitochondrial morphology upon overexperessing ADH3p. The overexpression of ADH3p indeed effects mitochondrial morphology change. This change is in the size of mitochondria which become smaller and the number increases. Then we also induce ADH3-GFP- pYES2 expression, and use red fluorescent to stain mitochondria superimposed with ADH3-GFPp image, trying to understand the spatial distribution of ADH3-GFP protein relative to the mitochondrial localization. We found that mitochondria and ADH3-GFP fluorescence overlap partially, indicating that mitochondrial morphological changes may be due to ADH3p of activity. We use flow cytometery to analyze the ADH3-GFP and ADH3G211A-GFP protein quantitatively. The results of flow cytometery support that overexpression ADH3p causing changes in mitochondrial morphology is effected by activity of the protein.
    Appears in Collections:[Graduate Institute & Department of Chemistry] Thesis

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