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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17754

Title: 1.熱休克蛋白70在發育中斑馬魚自發性表現與轉譯調節機制 2.斑馬魚胚胎萃取液中DNA傷害切割活性之偵測
1.Translational regulation of heat shock cognate 70 synthesis in developing zebrafish (Danio rerio) 2.Dectection of UV-dependent DNA incision activity in extracts of zebrafish (Danio rerio) early embryos
Authors: You-Hsin Chang
張友信
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 斑馬魚;熱休克蛋白70;胚胎;紫外線傷害DNA;切割試驗;卵黃前質蛋白
zebrafish;HSP70;embryo;UV-damaged DNA;Incision assay;vitellogenin
Date: 2008
Issue Date: 2011-07-04
Abstract: 第一部分 先前研究指出斑馬魚胚胎受精後會產生自發性熱休克蛋白70,此自發性HSC70的產生在轉譯層級所調控。從in vitro translation 實驗中發現hsc70 mRNA在rabbit reticulocyte lysate的轉譯效率很弱,而hsp70 mRNA卻有很強的轉譯活性,因此HSC70與HSP70的轉譯機制完全不同。本論文主要目的探討斑馬魚胚胎發育過程中的自發性熱休克蛋白70之轉譯調節機制,因此進行RT-PCR-PAT assay來偵測hsc70 mRNA在胚胎發育不同時期的poly(A) tail長度變化,結果發現斑馬魚在12 hpf至120 hpf之間,其hsc70 mRNA之poly(A) tail長度沒有明顯變化,故HSC 70的轉譯調控機制並不是由其poly(A) tail進行調控。接著利用RNA mobility shift assay分析hsc70 mRNA 5’與3’-UTR是否被特定RNA結合蛋白辨識,結果顯示斑馬魚萃取液對於sense與anti-sense hsc70 mRNA有著很強的非專一性結合活性,而造成背景值太高,因此無法找到專一性的RNA結合蛋白辨識到hsc70 mRNA 5’或3’-UTR進行轉譯調控。 第二部分 EMSA實驗中發現斑馬魚12 hpf CE對紫外線受損DNA有高度辨識活性,而此辨識蛋白為斑馬魚卵黃前質1類似蛋白 (Vg1-like protein),接著是否會進一步對受損DNA進行切割。而本論文利用biontin標定探針作為斑馬魚切割蛋白之受質,探討斑馬魚胚胎發育時期的切割能力;接著進行質體切割實驗來探討是否ATP會影響其切割能力。在botin標定的探針切割實驗中發現,斑馬魚120 hpf CE在ATP作用下活化很強的外切酶活性,必須加入poly(dI.dC)及poly(lys) 去除非專一性蛋白來減少影響切割實驗的進行,接下來遀著蛋白質濃度增加對受損DNA探針訊號有下降現象,但無看到切割後產物出現,由於此biotin標定探針本身受到UV照射後,照成探針背景值訊號過高而影響本實驗觀察。而在質體切割試驗中發現,斑馬魚12 hpf CE在EMSA buffer中不須ATP作用,對受損質體有專一性切割能力,似乎與EMSA實驗中辨識蛋白的結合才能進行專一性切割作用。反而加入ATP作用產生非專一性的切割活性,接著質體切割實驗中,12 hpf CE在EMSA 環境下加入Anti-Vg1抗體發現切割活性並不受影響,似乎不是Vg1-like蛋白參與切割作用。
PART ONE In our previous studies, we detected a high expression of heat shock cognte 70 (HSC 70) in developing zebrafish and the spontaneous HSC 70 expression was found to be controlled by translational regulation. The purpose of this study was to explore the mechanisms of the translational regulation that produce the spontaneous HSC 70 synthesis. RT-PCR-PAT assay indicated that the poly(A) tail of hsc70 mRNA isolated from 12 to 120 hpf zebrafish carried the same length in developing zebrafish. So translational regulation of HSC 70 synthesis was not conctrolled by changing poly(A) tail length. RNA mobility shift assay showd the sequence of both sense and anti-sense from Hsc70 mRNA 5’UTR and 3’-UTR was shift by specific RNA binding protein. The embryo extracts generated very strong non-specific binding to both sense and anti-sense hsc 70 mRNA, so the mechanism of translational regulation by the binding of specific RNA binding proteins to hsc70 5’ and 3’ -UTR could not be established. PART TWO A strong UV-damaged-DNA binding activity had been detected in the extracts of zebrafish embryos at 12 hrs post-fertilization by gel shift assay. We attempted to study the components of this binding activity and their importance in DNA damage recognition. In incision assay using a biotin-labeled oligonucleotide, we found 12 hpf zebrafish extracts expressed strong UV-independent exonuceloase activities that could be enhanced by ATP, but inhibited by the addition of poly(dI.dC) and poly(lys). When exonuclease activities were blocked, no UV-specific incision fragments were produced along with increasing protein concentration. In incision assay using a supercoiled plasmid in EMSA buffer, a UV-dependent increase in the level of open form plasmid was detected, indicating the association between UV-specific binding and DNA incision. Addition of ATP to 12 hpf zebrafish extracts caused a very intense damage-independent incision. Inclusion of an anti-zebrafish Vg1 antibody in incision mixtures did not inhibit the production of open form plasmid. Taken together, a UV-induced DNA incision activity independent of ATP is expressed in zebrafish early embryos.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M95360037
http://ntour.ntou.edu.tw/ir/handle/987654321/17754
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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