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研究生:楊育明
研究生(外文):Yu-Ming Yang
論文名稱:鯉魚轉譯起始因子eIF2α,eIF2β及eIF4E之選殖以及卵發育過程中蛋白質合成之研究
論文名稱(外文):Molecular Cloning of Translational Initiation Factors eIF2α , eIF2βand eIF4E and The Study of Protein Synthesis During Oogenesis In Carp
指導教授:黃火鍊張月霞張月霞引用關係
指導教授(外文):Fore-Lien HuangYea-Sha Chang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:動物學研究所
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:2000
畢業學年度:88
語文別:中文
論文頁數:65
中文關鍵詞:轉譯起始因子蛋白質合成卵細胞發育鯉魚
外文關鍵詞:translational initiation factorsprotein synthesisoogenesiscarp
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在卵細胞發育過程中, 轉譯活動的調控非常重要。卵細胞中許多特定mRNA只有在特定時期才會表現, 另外, 還有許多mRNA在卵中並不合成蛋白質, 而是受精後的某個胚胎發育時期被轉譯, 參予調控胚胎的發育與組織及器官的形成。
在鯉魚卵細胞的發育過程中也觀察到類似的情形。本實驗室發現,鯉魚卵膜蛋白質ZP2及ZP3在卵發育初期就已經被大量轉錄出來, 但是其蛋白質卻要到卵細胞進行卵黃堆積時才開始合成(Chang et al., 1996, 1997)。這種轉錄與轉譯非同步進行的現象也見於其他生物卵細胞中的母源mRNA (maternal mRNA)。 在這篇論文中, 首先藉由分析細胞中polysome 的分布與含量來了解發育不同時期卵細胞轉譯活動的進行。 結果發現, 不同時期卵細胞中的polysome含量並不相同, 在卵黃堆積前期的卵細胞, polysome的含量遠小於發育成熟的卵細胞。 因此, 我們推論發育晚期卵細胞整體的轉譯活動比早期旺盛。進一步以polysome RNA當作模板進行RT-PCR實驗發現, 在發育成熟的卵細胞中進行轉譯活動的ZP2 mRNA之數量約為卵黃堆積前期卵細胞的5.2倍。這個結果與之前西方轉印法的結果吻合, 表示ZP2 卵膜蛋白質在卵細胞發育晚期才開始大量合成。
為欲了解轉譯起始因子(Translation initiation factors)是否涉及調控鯉魚卵細胞的轉譯活動, 我們選殖鯉魚的eIF2α, eIF2β及eIF4E三個轉譯起始因子, 以它們為工具, 探討卵細胞發育過程中蛋白質合成的可能調控機制。 其中, eIF2α cDNA全長為1279 bps, 所推論之蛋白質由307個胺基酸所組成, 胺基酸序列與其他生物之同源基因十分相似。而eIF2β及eIF4E則只得到部分cDNA序列, 分別為645 bps與336 bps。由此部分序列所推論之胺基酸序列也與其它生物具有相當高程度的保守性。而北方轉印法的結果則顯示, 發育晚期卵細胞的eIF4E mRNA有增加的現象。
但是由於卵細胞發育中晚期卵黃大量堆積的影響, 因此無法利用西方轉印法進一步分析, 轉譯起始因子eIF2α, eIF2β及eIF4E是否參予發育不同階段鯉魚卵細胞轉譯活動的調控。

Translational control is very important for oogenesis. Expression of many messages in oocytes is temporally regulated. These messages may not be translated immediately. Instead, they are utilized to produce proteins that participate in the formation of tissues and organs in a particular phase of embryogenesis.
Our data indicates that a large amount of the mRNA of chorion components, including ZP2 and ZP3, are transcribed during early stage of oogenesis, yet synthesis of ZP2 and ZP3 proteins are delayed until the late stage of oogenesis. Therefore, the transcription and translation of ZP2 and ZP3 are asynchronous. Such a phenomena is also found in many other organisms. Therefore, we intended to investigate the possible mechanisms that may regulate the protein synthesis during oogenesis.
In order to realize the protein synthesis rate in oocyte of different developmental stages, the polysome profile of previtellogenic oocytes and fully-grown oocytes was investigated. The content of polysome of fully-grown oocytes is far exceeding that of previtellogenic oocytes by about 150 folds, suggesting that the fully-grown oocytes have a much higher protein synthesis rate than that of the previtellogenic oocytes. In order to study whether such a lower protein synthesis rate was also happened to the chorion components, the amount of ZP2 mRNA involved in translation in previtellogenic oocytes and fully-grown oocytes was compared. It is found that the amount of ZP2 mRNA present in polysome fraction of fully-grown oocytes is 5 folds higher than that present in previtellogenic oocytes.
Initiation factors of translation are key regulators of protein synthesis. For the sake to understand whether these factors are actually involved in regulation of protein synthesis during oogenesis in carp, I clone three initiation factors of translation, eIF2α, eIF2β, and eIF4E. The eIF2αis 1279 bps in length that encodes a protein of 307 amino acids. The partial sequence of eIF2βand eIF4E were also cloned. All these factors are homologous to the ones of other organisms. Northern blotting reveals that eIF4E expresses more in the fully-grown oocytes than in the previtellogenic oocytes.
In the present study, the recombinant proteins of eIF2α, eIF2β, and eIF4E were expressed and used to induce antibodies. Western blotting data indicated that only the antiserum of eIF2αcould recognize a specific protein of expected molecular weight from the crude extract of previtellogenic oocytes and liver but fail to do so on the crude extract of fully-grown oocytes. Therefore, I can not obtain any information from Western blots as whether eIF2α, eIF2β, and eIF4E may regulate the protein synthesis in carp oocytes.

目錄
中文摘要………………………………………………………………i
英文摘要……………………………………………………………iii
第一章. 前言
簡介真核生物的轉譯活動起始步驟………………………1
真核生物轉譯活動的調控機制……………………………3
轉譯起始因子的調控………………………………………3
mRNA---5’UTR的調控………………………………………5
mRNA---3’UTR的調控………………………………………7
核糖體的調控………………………………………………9
研究主題……………………………………………………10
第二章. 材料與方法
eIF2α, eIF2β, eIF4E基因之選殖………………………11
北方轉印法分析………………………………………………18
重組蛋白質之製備與純化……………………………………20
SDS-PAGE 電泳解析與染色…………………………………22
西方轉印法分析………………………………………………23
polysome之製備與polysomal RNA之分析…………………25
第三章. 結果
不同發育階段卵細胞polysome含量之變化…………………28
不同發育階段的卵細胞之ZP2 mRNA的轉譯…………………29
鯉魚eIF2α, eIF2β及eIF4E基因之選殖……………………30
eIF2α, eIF2β及eIF4E 重組蛋白的製備與純化…………31
發育過程中eIF2α, eIF2β及eIF4E mRNA含量之變化……33
發育過程中eIF2α, eIF2β及eIF4E蛋白質含量之變………33
第四章. 討論……………………………………………………………35
參考文獻…………………………………………………………………40
圖表

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曾煥仁, 黃火鍊 1976 鯉魚卵生長之研究 科學發展 4(4) : 2306-2315

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