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研究生:阮婷儀
論文名稱:單胞藻細胞萃取液中錯誤配對辨識蛋白之偵測與鑑定
論文名稱(外文):Detection and characterization of mismatch repair activities in cell-free extracts of unicellular alga(chlorella pyrenoidosa)
指導教授:許濤許濤引用關係
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:81
中文關鍵詞:錯誤配對辨識
相關次數:
  • 被引用被引用:1
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摘要
生物細胞中的核酸配對錯誤會造成蛋白質層次錯亂,當這些核酸錯誤配修復蛋白蛋白產生突變時,便會造成易突變之表現型 (mutator phenotype)。然而對植物界錯誤配對辨識系統進行探討的報告是非常少的,本實驗使用單胞藻當探討配對錯誤修復系統的研究材料,藉小球單胞藻抽取液與不同單一配對錯誤的38-mer雙股核酸反應,進行electrophoretic mobility shift assay (EMSA),發現小球藻中錯誤辨識蛋白結合能力最強的錯誤配對為G-T、C-T,其次是G-G,而G-A、C-C最弱。此種結合即使在 1M KCl下亦不會受到影響。由EMSA的結果顯示, 在ATP的存在下( 0、0.75、1.5 mM )會產生一個鬆散的錯誤辨識複合體,但原有的結合活性並不受影響。而在競爭性實驗中,小球單胞藻抽取液對G-T之結合完全不會被帶有紫外光照射過之CPD(cyclobutane pyrimidine dimmer)或 (6-4) photoproduct核酸探針所競爭,由此結果顯示,單胞藻中對配對錯誤辨識結合的蛋白質是非常專一性的,可看出此種錯誤辨識結合蛋白不會辨識因紫外光造成之核酸扭曲結構。
在蛋白質抽取試驗方面,由凝膠上將錯誤辨識結合基團(band)取出, 經過SDS-PAGE、銀染分析,發現在15 ~ 40 KDa的範圍有數個蛋白質對G-T 比正常配對核酸探針有明顯較強的親和力,故取其中一18Kda之蛋白質去進行MALDI-TOF質譜分析,所得質譜分析結果與現已知之資料庫比對發現並無相似者,表示此蛋白質之結構可能具有小球藻專一性。
本實驗中藉著試管中修復實驗(In vitro repair assay)檢測小球藻對錯誤配對修補的能力;利用 GT、CC之f1PM突變噬菌體(在其5`或3`end 具有一個nick),來進行試管中修復實驗。但由於單胞藻cell-free extracts 中核酸切割酶活性過高,僅能觀察到微弱之修復結果,故此這部分沒有辦法得到明確的結論,但由EMSA的實驗結果的確可看出algal cell-free extracts具有明確的辨識活性,但未來必須開發小球藻抽取液適用之修補基質,以克服非專一性核酸切割活性。

Abtract
Mismatch repair (MMR) is a DNA repair pathway that removes DNA mispairs or insertion-deletion loops generated during replication, recombination or spontaneous deamination of 5-methylcytosine. The mechanisms MMR in plants are still unclear and the unicellular alga (Chlorella pyrenoidosa) was used in this research as a model system to study MMR proteins in plants. The extracts of C. pyrenoidosa showed a strong preferential binding to a hetroduplex probe carrying a G-T or a C-T mispair as determined by electrophoretic mobility shift assays (EMSA). Algal extract proteins bound weakly to C-C mispair and intermediate binding efficiencies were observed for G-A and G-G mispairs. Addition of KCl up to 1M to EMSA mixtures did not affect the binding of C.pyrenoidosa extracts to mismatched DNA. High-shifting binding complexes with loose structures were formed after incubating the algal extracts and the heteroduplex probes in the presence of 0.75 and 1.5mM ATP. A UV-damaged duplex probe carrying either a cyclobutane pyrimidine dimer or a 6-4 photoproduct was unable to compete the binding of the G-T probe to C. pyrenoidosa extracts, indicating the specificity of mismatch recognition proteins in C.pyrenoidosa to their binding targets.
SDS-PAGE analysis and silver staining of the proteins eluted from G-T and homoduplex DNA binding complexes revealed the presence of several mismatch-binding polypeptides with molecular weights between 15 and 40KDa. One 18KDa G-T binding polypeptide with very low affinity for the homoduplex probe was excised from the gel for MALDI-TOF (Matrix Assisted Laser Directed Ionization-Time of Flight), and the mass spectrum of this polypeptide showed no homology with that of any protein stored in the Mascot data bank, suggesting that the 18KDa polypeptide was a novel mismatch binding protein.
The algal extracts were found to contain a weak repair activity as shown by in vitro repair assay after incubation with bacteriophages inserted with a heteroduplex fragment containing a nick at the 5`end of DNA mismatches. The repair efficiency was very limited because of nonspecific nucleolytic activities present in the algal extracts. Inclusion of polydI;dC in repair reaction mixtures slightly improved the repair efficiency and repair substrates suitable for algal extract proteins must be developed.

目 錄
英文摘要………………………………………………………………1
中文摘要………………………………………………………………3
前言……………………………………………………………………5
研究目的………………………………………………………………11
材料與方法……………………………………………………………12
一、 材料…………………………………………………………12
二、 方法…………………………………………………………29
A. 藻類細胞之培養…………………………………………29
B. 藻類細胞萃取液之製備…………………………………29
C. 蛋白質濃度之測定………………………………………33
D. 放射性錯誤配對雙股DNA的製備……………………34
D.1 單一錯誤配對雙股DNA探針的黏合……………34
D.2 錯誤配對雙股DNA之5’端放射性磷酸基標定…34
D.3 放射性錯誤配對雙股DNA的純化………………35
E. 活性分析…………………………………………………36
E.1 凝膠阻抗試驗………………………………………36
E.2 試管中之配對錯誤修復反應………………………37
E.3 試管中修復情形之雙重酵素切割…………………38
E.4 錯誤辨識蛋白之MALDI-TOF分析………………39
E.4.1 蛋白質萃取試驗……………………………39
E.4.2 SDS電泳膠體的製備………………………41
E.4.3 SDS-PAGE…………………………………41
E.4.4 銀染…………………………………………42
結果……………………………………………………………………43
一、小球單胞藻中核酸錯誤配對辨識活性之偵測……………43
二、藻類錯誤辨識蛋白之鑑定…………………………………45
三、試管中之修復反應…………………………………………45
討論……………………………………………………………………47
附圖
附圖一………………………………………………………………51
附圖二………………………………………………………………52
附圖三………………………………………………………………53
附圖四………………………………………………………………54
附圖五………………………………………………………………55
附圖六………………………………………………………………56
附圖七………………………………………………………………57
附圖八………………………………………………………………58
附圖九………………………………………………………………59
附圖十………………………………………………………………60
附圖十一……………………………………………………………61
附圖十二……………………………………………………………62
附圖十三……………………………………………………………63
附圖十四……………………………………………………………64
附圖十五……………………………………………………………65
附圖十六……………………………………………………………66
附圖十七……………………………………………………………67
附圖十八……………………………………………………………68
參考文獻………………………………………………………………69

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