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研究生:施錦毅
研究生(外文):C.Y. Shih
論文名稱:斑馬魚胚胎中DNA結構變異辨識蛋白之檢測與分離
論文名稱(外文):Identification and isolation of damage-recognition protein complexes from zebrafish(Danio rerio) early embryos.
指導教授:許濤許濤引用關係
指導教授(外文):T. Hsu
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:60
中文關鍵詞:辨識蛋白胚胎DNA 損傷發育紫外光斑馬魚
外文關鍵詞:binding proteinembryosDNA damagedevelopmentultraviolet lightzebrafish
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  • 下載下載:28
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兩種因紫外光照射而造成DNA損傷的產物主要為CPDs與6-4PPs,其造成DNA結構的變形,而導致DNA轉錄(transcription)與複製(replication)受阻。而紫外光損傷DNA也是探討細胞內可辨識DNA結構扭曲蛋白之良好探針。本實驗為探討脊椎動物胚胎發育過程中,損傷DNA辨識因子之表現,利用斑馬魚發育中之胚胎為系統模式,並以紫外光損傷DNA為辨識基質進行實驗。以受精後12至84小時的斑馬魚胚胎萃取液進行凝膠阻抗試驗,發現隨著胚胎發育時間的演進,12小時胚胎蛋白產生的高分子量辨識複合體,而84小時胚胎蛋白則產生低分子量辨識複合體。以抗人類修補辨識蛋白XPA、RPA-70、RPA-32抗體進行西方墨點法,發現胚胎發育12小時至60小時中,這些辨識蛋白都不表現,而發育至84小時後XPA、RPA-32始可測得,故胚胎早期所含之辨識蛋白應非與人類DNA損傷辨識蛋白相似者。再將84小時胚胎萃取液通過親和性吸附分析發現,84小時所表現的類XPA具有辨識紫外光損傷DNA之活性,因此XPA應參與此時期的損傷辨識作用。分析12小時胚胎萃取液特性之實驗發現,其對NaCl耐受性較84小時萃取液為低,約0.1 M,而競爭性實驗發現12小時萃取液以相同的辨識蛋白群對CPD或6-4PPs進行辨識。透過雙向電泳的分析得知,12小時萃取液的辨識蛋白群其pI值大約集中於7.5至10之間,屬於略呈鹼性的蛋白群。再將12小時萃取液辨識複合體其中之二個polypeptide進行蛋白質質譜儀析與mascot資料庫比對得知,polypeptides 50.2-kDa與29-kDa分別與斑馬魚fast skeletal muscle troponin T與vitellogenin有高度相似。

The expression of distorted DNA-binding factors was studied in developing zebrafish using UV-damaged DNA as the binding target. A strong and high-shifting binding activity was detected in the extracts of zebrafish embryo (12 h after fertilization), and the expression of the activity dramatically decreased in 60 to 84-h-old zebrafish. Unlike the embryonic extract, the extracts of 84-h-old larvae produced only low-shifting binding complexes when incubated with a CPD-specific probe. The binding of 12-hr-old and 84-hr-old extracts to CPD- and 6-4PPs under various concentration of NaCl supported the possibility that different damage-recognition protein were expressed in zebrafish according to their developmental condition. A competitive binding assay indicated that both CPDs and 6-4PPs were recognized by the same binding activity expressed in 12-h-old zebrafish. The binding activity expressed in embryos was apparently unrelated to human DNA repair damage-recognition proteins XPA、RPA-70 and RPA-32, since those polypeptides recognized by an anti-human XPA、RPA-70 and RPA-32 antibody were detected only in 84-h-old zebrafish extracts. Identification of one 50.2 and one 29-kDa UV-damage-DNA binding factor in 12-hr-old zebrafish extracts by EMSA linked to SDS-PAGE and determined by MALDI TOF analysis, indicated that the 29-kDa and 50.2-kDa polypeptides are high homologous to zebrafish fast skeletal muscle troponin T and vitellogenin. UV-binding factors found in 12-h-old zebrafish embryos may be involved in processing developmental stage-specific DNA structures similar to UV-damage DNA.

目次
中文摘要………………………………………………………………..01
英文摘要………………………………………………………………..02
續論……………………………………………………………………..03
壹、DNA結構變異的形成因子及其影響………………………….03
貳、細胞中各類DNA損傷辨識蛋白特性分析…………………….05
參、研究目的…………………………………………………….…09
材料與方法………………………………………………………….….11
壹、材料……………………………………………………………11
貳、實驗方法………………………………………………………20
結果…………………………………...………………………………..32
討論……………………………………………...……………………..36
參考文獻……………………………………………………...………..42
附圖…………………………………………………...………………..47

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