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中文論文名稱 黑腹果蠅端粒TART反轉子ORF2p內切酶之表現、純化及活性測試
英文論文名稱 Expression,purification and activity of the ORF2p endonuclease from TART retrotransposon of Drosophila telomere
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 96
學期 2
出版年 97
研究生中文姓名 曾奕之
研究生英文姓名 Yi-Chih Tzeng
電子信箱 491170105@s91.tku.edu.tw
學號 695180611
學位類別 碩士
語文別 中文
口試日期 2008-07-17
論文頁數 98頁
口試委員 指導教授-陳銘凱
委員-鄧述諄
委員-林賜恩
中文關鍵字 黑腹果蠅  內切酶 
英文關鍵字 Drosophila melanogaster  TART  endonuclease 
學科別分類 學科別醫學與生命科學生物學
中文摘要 黑腹果蠅(Drosophila melanogaster)易於培養和繁殖。在基因研究方面,果蠅是最常見的研究對象,它有四對染色體,且可以顯示很多變異,所以我們經常利用它作基礎的研究。端粒的長短與生物老化的過程息息相關,希望能藉由黑腹果蠅端粒的研究模型,了解端粒之演化基礎及物種老化的過程。
黑腹果蠅其端粒是由三種的non-LTR的retrotransposons組成,其分別為HeT-A、TART和TAHRE的重複序列,而其中TART可以轉譯出兩種蛋白質,ORF1p和ORF2p。TART ORF1p已證明是當共表現HeT-A ORF1p時,用來移動HeT-A ORF1p到telomere-associated structures上的蛋白質。而ORF2p則可能具有endonuclease(EN)和reverse transcriptase(RT)的活性。
本實驗是研究TART-EN的部分,目前已將endonuclease、點突變蛋白和二種的截短蛋白大量的表現在最適合的E.coli宿主上,並且能夠在具有活性的前提之下把endonuclease純化出來並做有效的保存。已確定此種endonuclease確實具有活性,可以將supercoiled form DNA的受質切成open circular form DNA。在活性確認之後,便可進而研究此endonuclease之DNA切位專一性以及辨認序列,以瞭解TART之作用方式。
英文摘要 The Drosophila melanogaster is easy to culture and propagation. In gene studies, the Drosophila melanogaster is the most common experimental animal. It has four pairs of chromosome, and have much genetic variation, so it is often used in basic research. The length of the telomere is closely correlated with aging process. We hope that by studying the Drosophila melanogaster's telomere we can understand the evolution of telomere and the aging process in general.
Drosophila melanogaster telomere is composed of three non-LTR retrotransposons, HeT-A, TART and TAHRE, which are repeated sequences, and TART can translate two kinds of proteins, ORF1p and ORF2p. TART ORF1p has already proved its function in shifting the telomere-associated structures only when coexpressed with HeT-A ORF1p. And ORF2p might have endonuclease (EN) and reverse transcriptase (RT) activity.
In this study, we investigate TART-EN expression and purification. We have already over-expressed Endop, point-mutant Endop, and two truncated mutant Endop in suitable E.coli. We Confirmed Endop have endonuclease activity by cutting supercoiled form DNA into open circular form DNA. We can also keep endonuclease activity of purified Endop effectively by storing it in 30% glycerol and -20℃. After the enzyme activity affirmation, we can study the cutting hot spots of endonuclease and identify their sequences further in order to understand the function of TART.
論文目次 致謝……………………………………………………………………………………I
中文、英文摘要………………………………………………………………………II
中英對照表…………………………………………………………………………III
目錄……………………………………………………………………………………IV
圖、表目錄……………………………………………………………………………V
1. 序論 …………………………………………………………………………………1
1.1. 端粒…………………………………………………………………………5
1.2. 端粒酶(telomerase)…………………………………………………………7
1.3. 黑腹果蠅端粒………………………………………………………………8
1.4. 轉位子(transposon…………………………………………………………9
1.4.1. 逆轉位子(Retrotransposon)…………………………………………10
1.4.2. LTR Retrotransposons …………………………………………………10
1.4.3. Non-LTR Retrotransposons ……………………………………………11
1.5. Non-LTR Retrotransposition Model………………………………………12
1.6. TART Retrotransposon………………………………………………………15
1.6.1. The Structure of TART…………………………………………………15
1.6.2. Targeting of HeT-A and TART to chromosome ends……………………15
1.6.3. Gag Proteins from HeT-A and……………………………………………16
TART Have Specific Nuclear Localizations
2. 材料………………………………………………………………………………17
2.1. E.coli strains…………………………………………………………………17
2.2. Plasmid………………………………………………………………………18
2.3. Primers………………………………………………………………………19
2.4. Media and reagents …………………………………………………………20
3. 實驗步驟…………………………………………………………………………22
3.1. 小量質體DNA製備…………………………………………………………23
3.1.1. 以化學法抽取plasmid…………………………………………………23
3.1.2. 以High-Speed Plasmid MiniKit抽取plasmid…………………………24
3.1.3. 以BamHI限制酶處理確認plasmid ……………………………………25
3.1.4. 以NotI限制酶處理確認plasmid………………………………………26
3.2. 定點突變……………………………………………………………………27
3.2.1. QuikChange Site-Directed Mutagenesis Kit…………………………28
3.2.2. 以DpnI限制酶處理親代模版…………………………………………29
3.2.3. 以PCR方式簡易確認突變……………………………………………30
3.2.4. 以定序方式確認突變…………………………………………………31
3.3. 勝任細胞(Competent Cell)製備……………………………………………36
3.4. 轉形至表現載體(Transformation)…………………………………………37
3.5. 蛋白質表現…………………………………………………………………38
3.5.1. 小量菌落培養…………………………………………………………40
3.5.2. Large-scale Protein Expression………………………………………40
3.5.2.1. LB Medium添加D-sorbitol……………………………………40
3.5.2.2. 表現蛋白質在各種溫度下………………………………………41
3.5.3. 生長曲線………………………………………………………………41
3.5.4. 超音波破碎細胞………………………………………………………42
3.5.4.1. 小量超音波破碎細胞……………………………………………42
3.5.4.2. 大量超音波破碎細胞……………………………………………42
3.5.5. 蛋白質電泳(SDS-PAGE……………………………………………43
3.6. 蛋白質純化…………………………………………………………………51
3.6.1. His-tag Fusion System…………………………………………………52
3.6.2. FPLC(Fast Performance Liquid Chromatography)……………………57
3.6.3. Mono QTM 5/50 GL column……………………………………………58
3.6.4. Q Sepharose Fast Flow column………………………………………69
3.7. 活性檢測……………………………………………………………………77
3.7.1. CsCl-Hoechst Density Gradient Centrifugation………………………78
3.7.2. Ultrapure plasmid extraction…………………………………………81
3.7.3. AP-DNA………………………………………………………………82
3.7.4. Nicking Assay…………………………………………………………82
4. 結果與討論………………………………………………………………………86
4.1. Plamid確認結果 ……………………………………………………………86
4.2. 點突變結果…………………………………………………………………87
4.3. 大量表現結果………………………………………………………………89
4.4. 純化結果……………………………………………………………………89
4.4.1. His-tag純化結果………………………………………………………89
4.4.2. Mono QTM 5/50 GL column純化結果…………………………………89
4.4.3. Q Sepharose Fast Flow column純化結果……………………………90
4.5. 活性測試結果………………………………………………………………91
4.6. 討論…………………………………………………………………………92
5. 參考文獻…………………………………………………………………………93
6. 附錄………………………………………………………………………………95
6.1. H1定序表……………………………………………………………………95
6.2. H4定序表……………………………………………………………………97


Figure 1 黑腹果蠅……………………………………………………2
Figure 2 基因轉殖示意圖……………………………………………3
Figure 3 細菌移位子構造……………………………………………10
Figure 4 LTR Retrotransposons構造………………………………10
Figure 5 L1 retrotransposition的步驟……………………………13
Figure 6 Target primed reverse transcription(TPRT)………14
Figure 7 Overview of the QuikChange II site-directedmutagenesis method………………………………………27
Figure 8 DNAssist V2.2程式比對H1…………………………………32
Figure 9 DNAssist V2.2程式比對H4…………………………………34
Figure 10 LB BL21(DE3) Endop表現…………………………………47
Figure 11 LB RosettaTM (DE3) Endop、Endo3tp、Endo5tp表現…48
Figure 12 D-sorbitol tRNA380-pET28c H230A表現………………49
Figure 13 LB RosettaTM(DE3)-pGH H230A表現……………………49
Figure 14 TALON™ Metal Affinity Resins………………………52
Figure 15 TALON™ Metal Affinity Resins實驗流程圖…………53
Figure 16 萃取條件資料圖……………………………………………54
Figure 17 BL21 Endop purification………………………………55
Figure 18 Rosetta Endop purification……………………………56
Figure 19 Rosetta En3tp purification……………………………56
Figure 20 Rosetta En5tp purification……………………………56
Figure 21 tRNA380 pET28C H230A purification…………………57
Figure 22 BL21 Endop first purification………………………59
Figure 23 BL21 Endop second purification………………………60
Figure 24 Rosetta Endop first purification……………………61
Figure 25 Rosetta Endop second purification……………62
Figure 26 Rosetta Endo3tp first purification…………………63
Figure 27 Rosetta Endo3tp second purification………………64
Figure 28 Rosetta Endo5tp first purification…………………65
Figure 29 Rosetta Endo5tp second purification………………66
Figure 30 tRNA380 pET28C-H230A first purification…………67
Figure 31tRNA380 pET28C-H230A second purification…………68
Figure 32 BL21 Endop purification………………………………70
Figure 33 Rosetta Endop purification……………………………71
Figure 34 Rosetta Endo3tp purification…………………………72
Figure 35 Rosetta Endo5tp purification…………………………73
Figure 36 tRNA380 pET28C-H230A purification…………………74
Figure 37 Rosetta pGH-H230A purification………………………75
Figure 38 各蛋白質純化圖……………………………………………76
Figure 39 活性檢測圖各時間點反應結果…………………………82
Figure 40 Endop和各突變種所做的對照圖(AP的pB)……………83
Figure 41 Endop和各突變種所做的對照圖(一般的pBS)…………84
Figure 42 超量蛋白質活性檢測………………………………………85
Figure 43 Plamid確認結果……………………………………………86
Figure 44 點突變結果………………………………………………88

表目錄
表一 宿主………………………………………………………………17
表二 Plasmid……………………………………………………………18
表三 Primers……………………………………………………………19
表四 Media and reagents……………………………………………20
表五 BamHI限制酶處理確認plasmid試劑……………………………25
表六 NotI限制酶處理確認plasmid試劑………………………………26
表七 點突變PCR反應條件………………………………………………28
表八 點突變PCR檢測……………………………………………………29
表九 DpnI限制酶處理親代模版試劑…………………………………29
表十 點突變PCR快速檢測試劑…………………………………………30
表十一 點突變PCR快速檢測條件………………………………………31
表十二 分離凝膠的反應試劑…………………………………………43
表十三 焦集凝膠的反應試劑…………………………………………43
表十四 表現比較表……………………………………………………46
表十五 生長曲線表……………………………………………………50
表十六 Nicking Assay測試試劑………………………………………82
參考文獻 Richard M Cawthon, Ken R Smith, Elizabeth O'Brien, Anna Sivatchenko and Richard A Kerber (2003) Association between telomere length in blood and mortality in people aged 60 years or older. The Lancet, Volume 361, Issue 9355, 1 February 2003, Pages 393-395.

McClintock, B. (1939) The Behavior in Successive Nuclear Divisions of a Chromosome Broken at Meiosis. Proc Natl Acad Sci U SA,25,405-416.

Rashkova, S., Karam, S.E., Kellum, R. and Pardue, M.L. (2002a) Gag proteins of the two Drosophila telomeric retrotransposons are targeted to chromosome ends. J Cell Biol, 159, 397-402.

Rashkova, S., Karam, S.E. and Pardue, M.L. (2002b) Element-specific localization of Drosophila retrotransposon Gag proteins occurs in both nucleus and cytoplasm. Proc Natl Acad Sci U S A, 99, 3621-3626.

Blackburn, E.H. (1992) Telomerases. Annu Rev Biochem, 61, 113-129. Cost, G.J., Feng, Q., Jacquier, A. and Boeke, J.D. (2002) Human L1 element target-primed reverse transcription in vitro. Embo J, 21, 5899-5910.

Biessmann, H., Carter, S.B. and Mason, J.M. (1990) Chromosome ends in Drosophila without telomeric DNA sequences. Proc Natl Acad Sci U S A, 87, 1758-1761.

Biessmann, H. and Mason, J.M. (1988) Progressive loss of DNA sequences from terminal chromosome deficiencies in Drosophila melanogaster. Embo J, 7, 1081-1086.

Blackburn E.H. and Gall J.G. (1978) A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. J Mol Biol. 1978 Mar 25;120(1):33-53.

Moyzis RK. (1991) The human telomere.Sci Am. 1991 Aug;265(2):48-55.

Luan, D.D., Korman, M.H., Jakubczak, J.L. and Eickbush, T.H. (1993) Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell, 72, 595-605.
Cost, G.J., Feng, Q., Jacquier, A. and Boeke, J.D. (2002) Human L1 element target-primed reverse transcription in vitro. Embo J, 21, 5899-5910.

Malik, H.S., Burke, W.D. and Eickbush, T.H. (1999) The age and evolution of non-LTR retrotransposable elements. Mol Biol Evol, 16, 793-805.

Danilevskaya, O.N., Traverse, K.L., Hogan, N.C., DeBaryshe, P.G. and Pardue, M.L.(1999) The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol Cell Biol, 19, 873-881.

Levis, R.W., Ganesan, R., Houtchens, K., Tolar, L.A. and Sheen, F.M. (1993) Transposons in place of telomeric repeats at a Drosophila telomere. Cell, 75, 1083-1093.

Sheen, F.M. and Levis, R.W. (1994) Transposition of the LINE-like retrotransposon TART to Drosophila chromosome termini. Proc Natl Acad Sci U S A, 91, 12510-12514.

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