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博碩士論文 etd-0908109-103513 詳細資訊
Title page for etd-0908109-103513
論文名稱
Title
Senseless 蛋白相撲化位置之研究
Investigation of the sumoylation sites of senseless
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
113
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2009-06-23
繳交日期
Date of Submission
2009-09-08
關鍵字
Keywords
相撲蛋白、果蠅
Senseless, Drosophila, SUMO
統計
Statistics
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中文摘要
Senseless為含有四個鋅手指的轉錄因子,它能和bHLH (basic helix-loop-helix) 神經前驅性蛋白 (proneural protein) 作用,調控果蠅神經前驅細胞 (sensory organ precusors) 的發展。我們利用生物資訊比對 Senseless 蛋白中相撲化 (SUMO蛋白結合位置) 的保留序列 後,發現許多相撲化的序列位置。本研究中我們透過試管中相撲化實驗 (in vitro sumoylation),發現這些片段可以產生相撲化複合物,且Senseless蛋白和相撲蛋白一起共同出現於核體中;因此,我們認為Senseless能被相撲化。
Abstract
The zinc-finger transcription factor Senseless is co-expressed with basic helix-loop -helix (bHLH) proneural protein in Drosophila sensory organ precusors and is required for their normal development. Recently, we analysed Senseless protein sequence with bioimformatics and found many SUMOs consensus sites. In this study, we successfully performed in vitro sumoylation of Senseless proteins and observed the colocalisation between Senseless and SUMOs protein.
目次 Table of Contents
中文摘要…………………………………………………………… 1
英文摘要…………………………………………………………… 3
縮寫表……………………………………………………………… 4
壹、緒論…………………………………………………………… 7
1. Senseless………………………….……………………………… 7
1.1 Senseless和其同源蛋白質結構……………………….......... 7
1.2 GPS蛋白質功能………………………................................... 8
1.3 GPS蛋白質能防止細胞凋亡……………..……………….... 9
1.4 GPS蛋白質對於細胞增殖的影響…………………………... 10
1.5 GPS蛋白質和bHLH蛋白質間的作用……………………… 11
1.6 GPS蛋白質在細胞發育命運及細胞分化中扮演重要角色. 14
2. 相撲蛋白 (Small ubiquitin-related modifiers, SUMOs)……….. 15
2.1 相撲蛋白……………………………………………………... 15
2.2 SUMO-1與泛激素(Ubiquitin)………………..……………... 16
2.3 相撲化 (Sumolyation)………………………..…………........ 16
2.4 SUMO-1在細胞中的位置……..………………………......... 17
2.5 SUMO-1在細胞中的功能…………………………………… 18
2.5.1 調控核體的組成性……………………………………….. 18
2.5.2 調控蛋白質在細胞中的位置……………………………….. 19
2.5.3 調控蛋白質活性和穩定性………………………………….. 21

2.6 SUMO-interacting Motifs….……………………………........ 24
貳、研究目的……………………………………………………… 25
参、實驗方法與材料……………………………………………… 27
一、E. Coli expression plasmids的製備……….…………………. 27
二、PCR反應……………………………………………………… 28
三、DNA Cloning…………………………………………………… 30
四、Site-Directed Mutagenesis…………………………………….. 35
五、蛋白質表現、純化和保存方法………………………………. 39
六、In Vitro Sumoylation assay……………….................................. 42
七、蛋白質電泳與西方點墨法 (Western blots, WB)……………. 43
八、細胞培養………………………………………………………. 48
九、轉殖plasmid進入細胞中 (transfection) …………………….. 50
十、螢光顯微鏡分析(fluorescence microscope analysis)………….. 51
肆、結果……………………………………………………………. 52
一、試管中Senseless胜肽片段的相撲化反應 (In vitro sumoylation of senseless peptide fragment)…………………...
52
二、細胞中Senseless蛋白與相撲蛋白的相互作用 (In vivo
interaction between Senseless and SUMOs)…………………..
58
三、Senseless蛋白相撲化位置的辨認 (Identification of the senseless sumoylation site)…………………………………..
60

(一) Senseless蛋白主要相樸化位置的單點突變 (Single mutation on the major sumoylation sites of Senseless)………………….

60
(二) Senseless蛋白主要相樸化位置的多點突變 (Multiple mutations on the major sumoylation sites of Senseless)……..
64
(三) Senseless蛋白主要及次要相樸化位置的多點突變 (Multiple mutations on the major and minor sumoylation sites of Senseless)……...........................................................................

70
伍、討論……………………………………………………………. 81
陸、未來工作…………………………………………………….. 86
柒、參考文獻…………………………………………………….. 87
捌、 附錄…………………………………………………………… 94
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