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研究生:呂秋錦
研究生(外文):Chiu_chin Lu
論文名稱:確認小鼠神經元細胞中adenosinetoinosine之mRNA目標編輯作用
論文名稱(外文):Identify A-to-I editing targets on mRNA of mouse neuron cells
指導教授:黃弘文黃弘文引用關係
指導教授(外文):Hurng-wern Huang
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物醫學科學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:69
中文關鍵詞:RNA編輯作用microarray分析
外文關鍵詞:inosine-specific cleavagemicroarray analysisRNA editing
相關次數:
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將RNA上adenosine脫氨 (deamination) 的編輯作用 (editing) 是由
ADAR family進行催化,ADAR可以改變RNA結構,利用錯誤配對打開
二級結構 (secondary structure) ,把原先A-U鹼基配對 (base-pair) 改變
成G-U鹼基配對,此種修飾作用是具有頻率差異性,以不同編輯頻率調
整蛋白質 (protein) 的編碼,而編輯作用也會依照不同組織有不同頻
率,藉此調節細胞或回應細胞因外在環境改變而改變蛋白質編輯頻
率,在哺乳動物,神經傳導作用 (neurotransmission) 中接受蛋白
(receptor protein) 的麩胺酸接受蛋白 (glutamate receptor) 及血清素接
受蛋白 (serotonin receptor) 受到編輯作用調節其選擇性,將adenosine
轉變成inosine , 目前受限於使用方法無法大量篩選候選者
(candidates) ,因此設計一系列方法建立篩選編輯候選者系統,包括利
用RNase T1作用、純化mRNA、以帶有poly T的核酸序列作為引子
(primer),合成cDNA以及探針 (probe) 以微矩陣 (microarray) 分析結
果,組合上述方法找出有多少mRNA是具有inosine,結合二個微矩陣找
出有意義差異訊號的基因約有100個,確認這些基因後即可更進一步研
究mRNA編輯作用對生理功能的影響。
RNA editing by adenosine deamination is catalyzed by members of an enzyme family
known as adenosine deaminases that act on RNA (ADARs). ADARs can change the
structure of RNA by changing an AU base-pair to an IU mismatch. This frequently
modifies the function of the encoded protein, and an emerging theme associated with
A-to-I mRNA editing is that tissues often regulate the ratio of proteins expressed from
edited and unedited mRNAs to fine-tune cellular responses and functions. In mammals,
pre-mRNA of receptor proteins involved in neurotransmission, including serotonin
receptors and glutamate receptors, are edited. Currently, only a limited number of
human ADAR substrates are known, whereas indirect evidence suggests a substantial
fraction of all pre-mRNAs being affected. To identify RNAs containing inosine residues,
this study used a multi step approach; including (1) inosine-specific base cleavage and
RNase T1 digestion, (2) purification of polyA-tailed mRNA, (3) RT w/ T7-polydT
primer, (4) probe synthesis and microarray analysis. Using this method it is possible to
identify novel targets of A to I editing. Approximately 100 genes showed a significant
decrease in two arrays. Future analysis of these targets should reveal the biomedical
significance of A-to-I editing.
致謝.......................................................................................................... Ⅰ
英文摘要.................................................................................................. Ⅱ
中文摘要.................................................................................................. Ⅲ
縮寫表...................................................................................................... Ⅴ
主要目的.................................................................................................... 1
重要性........................................................................................................ 1
前言............................................................................................................ 2
ADAR1 與胚胎發育.............................................................................................8
ADAR2 與神經系統的發育.................................................................................8
其他A-to-I 編輯作用的例子...............................................................................8
先前研究.............................................................................................................10
實驗設計.................................................................................................. 14
材料與方法.............................................................................................. 17
結果.......................................................................................................... 24
討論.......................................................................................................... 28
結論.......................................................................................................... 32
文獻............................................................................................................. i
附錄..........................................................................................................-1-
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