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Chaperoning Effects of Heat Shock Protein 70 on Dna Mismatch Repair Factors in Fish Embryos
|Contributors: ||NTOU:Institute of Bioscience and Biotechnology|
|Issue Date: ||2012-04-13T01:06:34Z
|Abstract: ||摘要:熱休克蛋白70 之自發性表現型(Hsc70)與緊迫誘導型(Hsp70)均具分子監護活性可摺 疊變性蛋白質或協助蛋白質跨膜輸送至各胞器內。DNA 錯誤配對修補作用(MMR)辨識 並移除DNA 複製時產生之鹼基錯誤配對與核酸環。本實驗室已發現Hsc70 會在早幼苗 期之斑馬魚中大量表現，此Hsc70 表現主要出現於魚體泳膘並於轉譯層次調控，另外 高溫處理可誘發Hsp70 藉轉錄調控表現在各發育時期之斑馬魚中。我們亦已選殖斑馬 魚MMR 辨識蛋白MSH2 與MSH6 並証明重組蛋白之辨識活性。文獻指出熱處理會刺 激人類細胞之Hsp70 由細胞質移轉至核內且與DNA 單股斷裂修補酵素相互接觸，顯示 Hsp70 監護DNA 修補蛋白之可能性。另外DNA 傷害也會促使MSH2 與MSH6 內往核 內移動。由於魚類胚胎對水污染物之敏感度與MMR 對胚胎發育之重要性，本計畫將 探討Hsc70 與Hsp70 對斑馬魚與點帶石斑魚胚胎中MMR 之分子監護效應與其生理意 義。熱休克蛋白70 與MSH 於核內之交互作用將以雷射共軛顯微鏡觀察並以免疫沉澱、 蛋白質交互連接與再切割配合錯誤配對親合性吸附取得生化證據。功能性分析將利用 一帶有雙約束限制序列之質體檢測核蛋白中斷股指引錯誤配對修補活性變化。熱休克 對MSH 基因轉錄轉譯之影響將以定量PCR、全覆式原位雜交與免疫組織化學來研究 並將解析MSH 基因啟動子構造。顯微注射熱休克因子1 反意寡核酸抑制Hsp70 合成將 探討Hsc70 對MSH 蛋白之一般與泳膘發育時之組織專一性監護效應。|
Abstract:Heat shock protein 70 (Hsp70) and heat shock cognate 70 (Hsc70) are molecular chaperones that refold denatured proteins and facilitate protein transport across organelle membranes. Transcriptional activation of hsp70/hsc70 is mediated by the binding of heat shock factors (HSFs) to the promoter regions. Studies on cultured cells revealed the translocation of Hsp70 from cytosols to nuclei after heat stress and the interactions between Hsp70 and enzymes repairing DNA single strand breaks have been identified. DNA mismatch repair (MMR) removes mutation-prone DNA mismatches arising in DNA replication. MSH2 and MSH6 are mismatch binding proteins associated with eukaryotic MMR and translocation of MSH2/MSH6 into nuclei was also observed in cells exposed to DNA-damaging agents. Our previous studies indicated the presence of a translation-dependent high expression of Hsc70 in zebrafish (Danio rerio) at embryos-larvae stages, while transcription-regulated Hsp70/Hsc70 production could be induced in heat-stressed zebrafish at all developmental stages. Molecular cloning of both MSH2 and MSH6 and expression of recombinant proteins with mismatch binding activities have also been accomplished and a coordinated high expression of msh2/msh6 transcripts was found to arise in zebrafish early embryos. Since fish embryos are sensitive to aquatic pollutants and maintenance of MMR is critical to embryonic development, this study intends to examine the chaperoning effects of Hsp70/Hsc70 on MMR in the embryos of zebrafish and grouper (Epinephelus coioides) at the levels of protein-protein interactions and transcriptional regulation. Confocal microscopy will be employed to reveal if coordinated translocation of Hsp70/Hsc70 and MSH molecules into nuclei occur in heat-stressed embryos of both fish species and if DNA damage-induced MSH translocation pairs with Hsp70/Hsc70 movement into nuclei. Interactions between Hsp70/Hsc70, co-chaperones and MSH2/MSH6 will be monitored by immunoprecipitation, protein cross-linking and release cleavage, and affinity adsorption using an immobilized oligonucleotide carrying a G-T or a G-G mispair. General effects of Hsp70/Hsc70 on msh2/msh6 expression will be examined by QPCR, whole mount in situ hybridization and whole mount immunohistochemistry. A plasmid containing reciprocal recognition sequences for two restriction endonucleases is selected as a repair substrate to test nick-directed MMR activity in nuclear extracts of embryos. If changes in msh2/msh6 transcription or MMR activities are evident, identification of potential binding sites for Hsp70/Hsc70 or HSFs in the regulatory sequences will be undertaken based on bioinformatic analysis and band shift assay following the identification of promoter structures. Microinjection of an antisense morpholino targeting hsf-1 mRNA into fertilized embryos will be conducted to suppress stress-induced Hsp70 production downstream of HSF-1 activation in order to determine the chaperoning effect contributed by Hsc70. Because the spontaneous high expression of Hsc70 appears primarily in the swim bladders of zebrafish early larvae, the final goal of this study is to examine if the spontaneous Hsc70 expression supports tissue-specific synthesis of MSH2/MSH6 during the development of swim bladders.
|Appears in Collections:||[生命科學暨生物科技學系] 研究計畫|
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