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研究生:張凱甯
研究生(外文):Kai-Ning Chang
論文名稱:小球藻DNA配對錯誤辨識蛋白親合性分離與ATP調節作用
論文名稱(外文):Isolation of DNA mismatch binding proteins from Chlorella pyrenoidosa by affinity adsorption and ATP-dependent regulation of mismatch binding
指導教授:吳清熊
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:61
中文關鍵詞:小球藻
外文關鍵詞:Chlorella pyrenoidosa2Daffinity adsorptionUV-crosslinkingATP
相關次數:
  • 被引用被引用:1
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本論文使用小球藻( Chlorella pyrenoidosa )做為探討配對錯誤修
復系統的材料,藉小球藻蛋白抽取液與單一GT 配對錯誤的38-mer
雙股核酸反應,以凝膠抗阻試驗(EMSA)來分析錯誤配對辨識蛋白質
之活性。實驗結果顯示小球藻蛋白質在30µg 時可產生明顯之辨識蛋
白複合體。小球藻蛋白在0.3 及1.0 mM ATP 的存在下會另外產生一
些鬆散的辨識複合體,但是原有的結合活性並不受到影響。由EMSA
結果顯示小球藻中某些蛋白質會與正常配對的DNA 產生結合,但是
與GT 配對錯誤的雙股核酸反應時,這些蛋白出現的位置便消失而在
別的位置另外產生新的辨識結合,另外由UV-crosslinking 實驗中也可
證明此種現象。UV-crosslinking 實驗發現添加1 mM ATP 後可使有一
條分子量約48-kDa 蛋白質核酸鍵結物強度減弱。接著親和性的吸附
實驗配合銀染可以知道在62 及48-kDa 左右有兩個蛋白質會辨識單一
GT配對錯誤的雙股核酸。藉由二維電泳的分析也可發現在35~75-kDa
分子量之間有數個會辨識單一GT 配對錯誤的雙股核酸的蛋白質。親
和性吸附實驗配合SYPRO Ruby 螢光染色發現有13-kDa 有個明顯胜
肽以及48-kDa 有一條較弱的胜肽。2-D 分析配合SYPRO Ruby 螢光
染色可發現有兩個13-kDa 胜肽pI5.3 及5.5。上述兩13-kDa 胜肽經質
V
譜儀分析後,以Mascot 資料庫比對其身分。對照何者已知蛋白與其
相似度最高,目前的初步結果為與綠色植物Viridiplantae (Green Plants)
中的阿拉伯芥(Arabidopsis thaliana)及水稻Oryza sativa (rice)有較高的
相似度。pI 值5.3 的胜肽與阿拉伯芥比的Serine/threonine protein
phosphatase PP1 isozyme 有五條peptide 符合。pI 值5.5 的胜肽與一
真菌中之ATP-dependent RNA helicase 有五條peptide 相符合。
但這只是一個初步的比對,未來更須經由質譜儀作更進一步的
MS/MS 分析。
DNA mismatches occur because of incorporation of
noncomplementary, Watson-Crick bases opposite each other in the DNA
helix during replication or recombination. Transition mispairs (G-T or
A-C) are repaired by the mismatch repair process more efficiently than
transversion mispairs (G-G, A-A, G-A, C-C, C-T, and T-T).In this study, a
G-T probe was used to detect mismatch binding proteins.
DNA mismatch binding activities in the extracts of the unicellular
alga Chlorella pyrenoidosa were examined by electrophoretic mobility
shift assay(EMSA). A protein concentration-dependent binding study
revealed the presence of mismatch binding activities having low or no
affinity for homoduplex DNA. Addition of 0.3 and 1.0 mM ATP to EMSA
mixtures induced the formation of some high-shifting complexes
reflecting the regulation of ATP on DNA mismatch recognition.
Two polypeptides about 62 and 48 kDa estimated by SDS-PAGE
were found to bind with high specificity to a biotin-labeled G-T probe
immobilized on streptavidin-conjugated agarose beads and a few
62-kDa G-T binding polypeptides possessing pIs ranging from 5.4 to 5.8
were identified by two-dimensional gel electrophoresis after silver
staining. Staining of affinity-captured proteins on a SDS-polyacrylamide
gel by fluorescent SYPRO Ruby dye, however, detected a 13-kDa and a
weak 48-kDa GT binding polypeptide. Two 13-kDa G-T binding
polypeptides were found by fluorescence staining of 2-D gels. Peptide
mass fingerprinting (PMF) of the 13-kDa polypeptide with pI5.3 matched
best to a arabidopsis thaliana Serine/threonine protein phosphatase. The
other polypeptide with pI5.5 matched best to a yeast ATP-dependent
RNA helicase. The exact identities of the two polypeptides need to be
detected.
總目次

總目次………………………………………………………………….…I
謝辭………………………………………………………………...…....II
英文摘要………………………………………………………….....…III
中文摘要……………………………………………………………IV
名詞縮寫……………………………………………………………..…VI
壹、前言…………………………………………………………………1
貳、材料與方法…………………………………………………………6
一、材料………………………………………………………………6
二、方法……………………………………………………………..18
參、結果………………………………………………………………31
一、 小球藻生長曲線…………………………………………………31
二、 小球藻DNA錯誤配對辨識活性及ATP調節作用…………31
三、 親和性吸附試驗及一維與二維電泳分離錯誤配對辨識蛋白.32
四、 錯誤配對辨識蛋白質之鑑定……………………………33
肆、討論………………………………………………………………..35
伍、實驗圖表…………………………………………………………39
陸、參考文獻…………………………………………………………..56
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