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研究生:謝宜静
研究生(外文):Yi-Ching Hsieh
論文名稱:酵母菌蛋白Imp4p生化特性分析
論文名稱(外文):Characterization of Saccharomyces cerevisiae Imp4p
指導教授:林敬哲林敬哲引用關係
指導教授(外文):Jing-Jer Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
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:端粒
外文關鍵詞:telomere
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端粒 (Telomere) 是染色體末端之特殊結構,保護染色體完整防止其被分解。在酵母菌S. cerevisiae中,端粒序列為長約300 bp的雙股 C1-3A/TG1-3重複核酸序列以及會隨著細胞週期變化長度的單股TG1-3核酸序列所組成。Cdc13p為單股TG1-3的端粒結合蛋白,由在in vitro pull down analysis及in vivo yeast two-hybrid及CoIP的實驗結果顯示Cdc13p和Imp4p有交互作用。Imp4p為酵母菌Saccharomyces cerevisiae的蛋白,包含290個胺基酸,在SDS PAGE上觀察到的大小約為42 kDa。Imp4p的序列中包含brix domain以及其內的σ70-like motif,二個可能和RNA或DNA結合有關的區域。Imp4p屬於U3 snoRNP (small nucleolar ribonucleoprotein) 中的一員,也是其中唯一具有古生物analog的蛋白,已知其參與細胞合成成熟的18S rRNA。為了進一步分析Imp4p在端粒所扮演的角色,我們利用純化的6His-Imp4p進行electrophoretic mobility shift assay來分析Imp4p其端粒結合的特性。我們發現Imp4p具有單股TG1-3 DNA結合的能力,由於Imp4p對端粒DNA序列具有專一性,顯示Imp4p可能是端粒結合蛋白之一。為證明σ70-like motif及Brix domain是否影響Imp4p對單股TG1-3 DNA結合的能力,我們分別對此兩區域最conserved的胺基酸imp4E249A及imp4R234A做點突變。由於此兩點突變並不會影響端粒DNA結合及對細胞生長,顯示這兩個胺基酸並不是直接參與在端粒DNA的結合。除此之外,在RMSA實驗中,我們發現Imp4p和端粒酉每RNA分子TLC1也有結合作用,我們並可在Imp4p的免疫沈澱物中偵測到端粒酉每RNA的存在。顯示Imp4p可能與telomerase RNA有結合能力。綜合以上實驗結果,Imp4p除了參與rRNA的processing之外,亦可能經由與Cdc13p、單股TG1-3 DNA、或TLC1 RNA的結合來參與端粒功能的調節。
Telomeres, the DNA-protein complex located at the ends of most eukaryotes, protect chromosome ends from nucleolytic digestion and facilitate complete replication of the genome. Cdc13p is a telomere binding protein of Saccharomyces cerevisiae. Through a screen for proteins that interact with Cdc13p in a two-hybrid system, we identified a protein Imp4p that interact with Cdc13p both in vivo and in vitro. Imp4p is a component of the U3 snoRNPs (small nucleolar ribonucleproteins) that is involving in pre-18S rRNA processing. It encodes 290 amino acids with molecular mass of 32 kDa. Sequence analysis of Imp4p indicated that it has a brix domain and σ70-like motif. The brix domain is considered to be responsible for ribosome synthesis andσ70-like motif with prokaryotic origins has ability to binding single strand RNA homopolymer. These conserved sequences indicate that Imp4p may participate in DNA or RNA binding. Applying an in vitro gel shift assay, we found that Imp4p is capable of binding to single strand telomeric DNA, suggest that Imp4p is a component of telomeric complex. We also found that Imp4p bound to telomerase RNA, TLC1, with high affinity. Since Imp4p was identified in partially purified telomerase fractions and TLC1 RNA was detected in the Imp4p immunoprecipitates together these results indicated that Imp4p is also a component of telomerase complex. We created the site-directed mutation inσ70-like motif and Brix domain to study the function of Imp4p involved in the telomere. Unfortunately, these two mutated Imp4pE249Aand Imp4pR234A does not affect the binding activity of Imp4p to ss telomeric DNA or the cell viability. Besides, the overexpression of Imp4p does not affect the telomere length in vivo. Since Imp4p is capable to interact with both telomeres and telomerase complex. We propose that Imp4p may play a role in telomere function in mediate accessibility of telomerase to telomeres.
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