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研究生:林書葦
研究生(外文):Sue-Wei Lin
論文名稱:鑑定參與在受Torso調控之抑制複合體中的蛋白質
論文名稱(外文):Identification of proteins in Torso-regulated repression complex
指導教授:廖國楨
指導教授(外文):Gwo-Jen Liaw
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:黃體果蠅抑制複合體
外文關鍵詞:Drosophila melanogastertaillessrepression complex
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在果蠅胚胎頭尾發育的過程中,由tailless (tll)所表現出來的細胞核受體蛋白(nuclear receptor)扮演者很重要的角色。當這個基因產生變異時,果蠅的大腦、後腸、馬氐管、肛門骨板等器官都會有發育不正常的現象。之前的研究顯示,tll是受到Torso (TOR)受體(receptor)所引發的訊息傳遞鏈(signal transduction pathway)所調控。當果蠅胚胎兩端的TOR受體和配體(ligand)結合時,會透過一連串磷酸化的反應,使下游的轉錄阻遏物(transcriptional repressor)失去抑制tll基因表現的能力而導至tll在胚胎兩端表現。許多的阻遏物,包括:Groucho (GRO)、Capicua (CIC)和Tramtrack69 (TTK69)已經被發現,但這些阻遏物都已被證實,不會和tll基因的最小調控區域(minimal regulatory region)結合。且當這些基因的母體效應(maternal effect)被去除時,tll的表現往胚胎中間延伸的現象,比均勻活化整個胚胎中的TOR受器(torso gain-of-function)時要弱得多。這暗示著還有更多未知的阻遏物等待被發現。
為了找到更多的調控蛋白,我使用了三種方法,包括:去氧核糖核酸層析法(DNA chromatography),染色質免疫沉澱法(Chromatin immunoprecipitation)和環化跳離法(loop-out method)。用這些方法所純化出來的蛋白質,則以質譜儀進行鑑定。使用去氧核糖核酸層析法,16個蛋白質,包括TTK69,HSC4和TER94,已被鑑定出參與在能與tll基因的最小調控區域結合的蛋白質複合體中。至於染色質免疫沉澱法和環化跳離法的部分,所需的材料,包括能辨識TTK69的抗體,TOR受器均勻活化的果蠅胚胎,和兩株基因轉殖果蠅:SW11和SW25,都已經準備完成。
In Drosophila melanogaster, tailless (tll) gene encodes a nuclear receptor required for development of brain at anterior, hindgut, Malpighian tubules, anal pad and spiracles at the posterior. Previous studies showed that tll expression was regulated by the Torso (TOR) signaling pathway through relief of the transcriptional repression. Several repressors including Groucho (GRO), Capicua (CIC), and Tramtrack69 (TTK69) are involved in the repression. However, none of these are torso response element (tor-RE) binding protein. When maternal activity of either one of the genes was removed, expansion of tll expression is much less than that in embryos from torso gain-of-function mothers. These suggest that fully functional repression requires more transcription factors.
To find more factors, three methods: DNA chromatography, Chromatin immunoprecipitation (ChIP), and loop-out method were performed. The proteins purified by these methods were identified using mass spectrometer. By DNA chromatography, 16 proteins include TTK69, HSC4, and TER94 have been identified to be involved in the protein complexes that bind to the tailless minimal regulatory region (tll-MMR). The tools for ChIP and loop-out method have been prepared, including anti-TTK69 antibody, torso gain-of-function embryos, and two transgenic fly lines: SW11 and SW25.
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