摘要
人類轉譯後修飾性蛋白質SUMO (small ubiquitin-like modifier protein)，包含三種蛋白質SUMO-1/2/3，它的胺基酸序列與ubiquitin僅有18%相同，但與ubiquitin的立體結構卻十分相似。它可參與細胞內許多蛋白質的相互作用，故SUMO掌握了許多細胞內的重要功能，包括蛋白質在細胞中分佈的位置、蛋白質的活性與穩定度、細胞凋零以及細胞週期的調節等，SUMO在轉譯後修飾作用中扮演相當重要的角色。先前利用data-mining方式找出人類SUMO-1/2/3分別座落於染色體上的位置2q33、17q25.1及21q22.3，SUMO-1/2分別有八、23個偽基因，而SUMO-3則沒有偽基因。除此之外在基因序列上發現SUMO-1有四個外子，SUMO-2/3則有三個外子。本研究之目的在探討SUMO-1/2/3在細胞內分佈的情形，我們的結果顯示大部分的SUMO- 1/2/3分別位於細胞核膜、細胞核體及細胞質內，當切除N端胺基酸序列的SUMO-1，其位於細胞核膜和細胞質內；而突變型的SUMO-2/3則表現於細胞質中。只未活化的SUMO-3會表現於細胞質內，當給予（1 μM）actinomycin D作用後，使得SUMO-3改變了它原本在細胞中的位置，被活化的SUMO-3由細胞質轉移到細胞核內。然而給予nocodazole和arsenic trioxide作用後，發現減少了未活化SUMO-3，推論這些減少的SUMO-3已經被活化，進而去參與細胞間的作用。

Abstract
Human ubiquitin-like SUMO-1/2/3 proteins have been identified. The 3-D structure of the SUMO-1 has been shown to be very similar to that of ubiquitin, although their sequences share only 18 % identity. Unlike ubiquitination targets proteins for degradation, sumoylation appears to regulate a number of cellular processes such as protein-protein interaction, subcellular localization, protein stability, apoptosis, cell cycle and so on . Our laboratory has cloned cDNAs encoding human SUMO-2, mouse SUMO-2 and SUMO-3, as well as a single SUMO gene from nematode and Drosophila. Recently (Su & Li, Gene 296:65-73,2002), Su & Li have performed data-mining on current human genomic sequence and found the presence of only three SUMO-1/2/3 functional genes located at chromosome no. 2q33, 17q25.1 and 21q22.3, respectively, as well as eight SUMO-1 pseudogenes and 23 SUMO-2 pseudogenes. The protein-coding sequence of SUMO-1 gene is interrupted by four introns , while those of SUMO-2/3 genes are interrupted by only three introns. In this study , most of SUMO-1/2/3 proteins were show to be localized on nuclear membrane, nuclear bodies and cytoplasm, respectively. The N-terminus-deleted SUMO-1 proteins was further shown to be localized on nuclear membrane and in cytosol, while the mutant SUMO-2/3 proteins were localized only in the cytosol. The inactive precursor form of SUMO-3 was exclusively localized in the cytosol. The activation of SUMO-3 in HeLa cells was triggered by actinomycin D and its location was shifted from cytosol to nucleus. Further, the inactive precursor of SUMO-3 was reduced in HeLa cells treated with nocodazole and arsenic trioxide.

目錄
誌謝
中文摘要………………………………………………….………………I
英文摘要…………………………………………………………….…..Ⅲ
英文縮寫表……………………………………………………………..Ⅴ
壹、序言…………………………………………………………….….…1
一、Ubiquitin蛋白質的介紹……………………………………….…1
二、SUMO蛋白質的介紹……………………………………………2
三、SUMO與ubquitin胺基酸結構的相關性……………………….3
四、SUMO與ubquitin共價結合的相關性………………………….4
五、SUMO-1已知的生理功能……………………………………….6
六、內質網壓力……………………………………………………….8
貳、研究目的……………………………………………………….…..12
參、材料與方法…………………………………………………..……14
一、建構不同型式的SUMO-1/2/3質體的DNAs………….………14
二、細胞株的處理…………………………………………….……..19
三、從HeLa細胞株抽取RNA……………………………….……..22
四、寡核甘酸序列的製作…………………………………….……..23
五、反轉錄聚合酶連鎖反應………………………………….……..24
六、質體的建構……………………………………………….……..25
七、質體DNA少量置備…………………………………….………26
八、基因選殖……………………………………………….………..27
九、轉殖…………………………………………………………..…29
十、蛋白質的萃取…………………………………………………..30
十一、西方漬點法…………………………………………………..31
十二、免疫螢光染色……………………………………….……….32
十三、免疫沈澱法……………….……………………….…………33
十四、蛋白質的表現………………………………………………..33
十五、SDS-PAGE的配置…………………………………………..34
十六、蛋白質純化…………………………………………………..35
十七、蛋白質的濃縮和保存………………………………………..36
十八、蛋白質定量………………………………………………......37
肆、結果………………………………………………...……………...38
一、SUMO-1/2/3蛋白最原始的生理特性........................................38
二、不同型式的SUMO-1/2/3在細胞中的位置……………..…….40
三、未活化的SUMO-3在Hela細胞內受異常緊迫之變化……...43
四、HUbc9蛋白的建構以及純化………………………..….…..…45
伍、討論………………………….……………………………….........46
陸、結論………………………….……………………………….........51
柒、參考文獻…………………………………………………….....…53
圖表……………………………………………………………….……...58
附錄……………………………………………………………….……...84

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