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研究生(外文):Imelda Margaretha Aritonang
論文名稱(外文):Differential Effects of Heat Stress and UVB Radiation on Nucleotide Excision Repair in Zebrafish (Danio rerio) Embryos
指導教授(外文):Todd Hsu
口試委員(外文):Todd HsuMing-Wei ChaoLing-Huei Yih
外文關鍵詞:UVBnucleotide excision repairheat stressin vitro transcription
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魚類在早期胚胎階段對環境壓力比其他發育階段較為敏感,因此需要DNA修復系統來維持基因完整性。在快速發育的生物中,若基因修復效率受到干擾,則會增加致命突變的發生機率。本論文主要藉以DNA轉錄為基礎的修復實驗及電泳遷移實驗(EMSA)觀察暴露於+8.5 ᵒC的水中及非致死劑量UV-B下,對斑馬魚胚胎的核酸切割修復(NER)反應之影響。在轉錄修復實驗中,使用UV-C照射表現質體為修復標的,隨著UV-C劑量的上升,愈抑制其轉錄成cDNA的效率。受精後10小時(10 hpf)斑馬魚胚胎的粗蛋白質萃取液與受UV-C照射受損的質體同時培養,能夠提升質體轉錄cDNA的效率,另外,經過急性熱休克(37ᵒC 30分鐘)的10hpf斑馬魚胚胎更能提升轉錄效率,表示熱休克可以提升NER的功能。雖然使用200 J/m2 的UV-B照射胚胎能微量提升NER活性,但是在400 和 800 J/m2 卻有抑制效果。在電泳遷移實驗中,使用6-4光產物(6-4 PP)或環丁烷嘧啶雙體(CPD)的寡核苷酸做為辨識的目標,UV-B照射的胚胎會抑制NER辨識的步驟。因此,UV-B會干擾損傷識別階段來抑制NER的修復效率。
Fish at early embryonic stages are more sensitive to environmental stressors than fish at other developmental stages. DNA repair systems are critical to the maintenance of genetic integrity and lethal mutations may arise in fast-growing organisms if the efficiencies of DNA repair systems are disturbed. The objective of this study was to explore the responses of nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos to a +8.5ᵒC water temperature elevation and sublethal UV-B exposure using a transcription-based DNA repair assay and gel shift assay. Irradiation of an expression plasmid with increasing UV-C dose caused a dose-dependent inhibition of cDNA transcription. Incubation of the plasmid with 10 hpf zebrafish extracts promoted a significant up-regulation of UV-C suppressed cDNA transcription and heat-stressed (37ᵒC for 30 min) 10 hpf zebrafish extracts displayed a better NER capacity than non-stressed zebrafish extracts. UVB at 200 J/m2 induced a slight increase of NER activity, but NER activities were inhibited in embryos exposed to UVB at 400 and 800 J/m2. Gel shift assay using an oligonucleotide carrying a UVC-induced (6-4) photoproducts or cyclobutane pyrimidine dimer (CPD) as the binding target demonstrated that UVB irradiation on 10 hpf embryos generally imposed inhibitory effects on the damage recognition step of NER. Hence, UVB was believed to inhibit NER by disturbing its damage recognition stage.
摘要 ii
1-1 Thermal Pollution and UVB Exposure Effect 1
1-2 Heat Shock Protein (HSPs) 2
1-3 DNA Damage and Repair 3
1-4 Nucleotide Excision Repair 4
1-5 Research objectives................. 5
2-1 Materials................................ 6
2-2 Experimental Equipment 8
2-3 Experimental Method 9
2-3.1 Collection of zebrafish embryos and developing 9
2-3.2 Zebrafish embryo heat treatment 9
2-3.3 Zebrafish embryo UV-B irradiation treatment 10
2-3.4 Preparation of the extracts of zebrafish embryos 10
2-3.5 Protein Quantification use BCA Protein Assay 10
2-3.6 DNA Plasmid Extraction 11
2-3.7 Quantification and charaterization of plasmid 11
2-3.8 Linearization of plasmid 11
2-3.9 DNA Electrophoresis Gel 12
2-3.10 UVC-irradiation on plasmid 12
2-3.11 In vitro transcription based DNA repair assay 12
2-3.12 RNA Electrophoresis Gel of product in vitro Transcription 13
2-3.13 Preparation of 6,4 PP probe 14
2-3.14 Electrophoretic mobility shift assay (EMSA) 14
3.1 UVC-dose dependent of plasmid DNA detected by in vitro transcription assay 16
3.2 Heat stress stimulates NER activities in 10 hpf zebrafish embryo 16
3.3 Low doses of UVB-irradiation in zebrafish embryo induced NER activities 17
3.4 Differential effect of UVB-damaged binding activities on 17
6,4 PP lesion at different UVB doses 17
3.5 Differential effect of UVB-damaged binding activities on 18
6,4 PP lesion at different UVB doses 18
REFERENCES........................ 22
APPENDIX......................... 32
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