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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄧述諄(Shu-Chun Teng) | |
| dc.contributor.author | Yu-Chen Wang | en |
| dc.contributor.author | 王郁軫 | zh_TW |
| dc.date.accessioned | 2021-06-13T00:29:39Z | - |
| dc.date.available | 2007-08-08 | |
| dc.date.copyright | 2007-08-08 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-07-25 | |
| dc.identifier.citation | 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. (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. Curcio, M.J. and Garfinkel, D.J. (1991) Single-step selection for Ty1 element retrotransposition. Proc Natl Acad Sci U S A, 88, 936-940. 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. Greider, C.W. (1996) Telomere length regulation. Annu Rev Biochem, 65, 337-365. Henson, J.D., Neumann, A.A., Yeager, T.R. and Reddel, R.R. (2002) Alternative lengthening of telomeres in mammalian cells. Oncogene, 21, 598-610. Hohjoh, H. and Singer, M.F. (1996) Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA. Embo J, 15, 630-639. Kulpa, D.A. and Moran, J.V. (2006) Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles. Nat Struct Mol Biol, 13, 655-660. Levis, R.W. (1989) Viable deletions of a telomere from a Drosophila chromosome. Cell, 58, 791-801. 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. 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. Lundblad, V. (2002) Telomere maintenance without telomerase. Oncogene, 21, 522-531. 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. McClintock, B. (1939) The Behavior in Successive Nuclear Divisions of a Chromosome Broken at Meiosis. Proc Natl Acad Sci U S A, 25, 405-416. Melnikova, L. and Georgiev, P. (2002) Enhancer of terminal gene conversion, a new mutation in Drosophila melanogaster that induces telomere elongation by gene conversion. Genetics, 162, 1301-1312. Mikhailovsky, S., Belenkaya, T. and Georgiev, P. (1999) Broken chromosomal ends can be elongated by conversion in Drosophila melanogaster. Chromosoma, 108, 114-120. Ostertag, E.M. and Kazazian, H.H., Jr. (2001) Biology of mammalian L1 retrotransposons. Annu Rev Genet, 35, 501-538. Pardue, M.L. and DeBaryshe, P.G. (1999) Telomeres and telomerase: more than the end of the line. Chromosoma, 108, 73-82. 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. Ricchetti, M. and Buc, H. (1993) E. coli DNA polymerase I as a reverse transcriptase. Embo J, 12, 387-396. Savitsky, M., Kravchuk, O., Melnikova, L. and Georgiev, P. (2002) Heterochromatin protein 1 is involved in control of telomere elongation in Drosophila melanogaster. Mol Cell Biol, 22, 3204-3218. 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. Siriaco, G.M., Cenci, G., Haoudi, A., Champion, L.E., Zhou, C., Gatti, M. and Mason, J.M. (2002) Telomere elongation (Tel), a new mutation in Drosophila melanogaster that produces long telomeres. Genetics, 160, 235-245. Teng, S.C., Kim, B. and Gabriel, A. (1996) Retrotransposon reverse-transcriptase-mediated repair of chromosomal breaks. Nature, 383, 641-644. Teng, S.C. and Zakian, V.A. (1999) Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae. Mol Cell Biol, 19, 8083-8093. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28921 | - |
| dc.description.abstract | 大部分真核生物的端粒長度是由端粒酶 (telomerase) 所維持，端粒酶以自身的 RNA 作為模板進行反轉錄以延長染色體末端。然而，在已知的所有果蠅品種內皆不具有端粒酶活性，目前研究的結果發現，果蠅末端是由兩種非 LTR 反轉錄子 – HeT-A 及 TART 不間斷的形成。TART 會產生 ORF1p 及 ORF2p 兩個蛋白質。TRAT ORF2p 蛋白質序列與其他研究較廣的非 LTR 反轉錄子，如人類的 LINE 1 的蛋白質序列相比，具有內切酶 (endonucleaase) 與反轉錄酶 (reverse transcriptase) 兩種酵素的蛋白質序列相似性。雖然已有研究報導 TART 會移動到染色體末端，但截至目前為止，都沒有證據顯示這種移動是利用反轉錄移位 (retrotransposition) 還是重組機制 (recombination)。我的研究主要是利用 in vitro 與 in vivo 兩種系統釐清 TART 是否真具有反轉錄酶的活性。在 in vitro 方面，利用大腸桿菌表現 TRAT 反轉錄酶，並純化偵測是否有酵素活性，預測之活性點突變及去除此片段之蛋白質反轉錄酶活性偵測結果皆顯示目前測試過的 TART RT 片段可能不包含完整的反轉錄酶全長。in vivo 方面，在酵母菌系統內將反轉錄子 Ty1 的 TYB 反轉錄酶片段置換為 TART ORF2 ，在 in vivo RT assay 可測到 TART 反轉錄酶活性。南方墨點法顯示因 TART 反轉錄酶作用造成 marker 插入質體的頻率約為 4.5x10-9。將此具有 marker 插入之質體定序，結果發現 TART ORF2 進行反轉錄過程時發生模板轉換現象 (template switch)，且 TART ORF2 mRNA 部分可能有強烈二級結構 (secondary structure) 使得 TRAT ORF2p 進行 cDNA 合成時容易掉落。由我的研究證實果蠅端粒連結反轉錄子 TART 的確具有反轉錄酶活性。 | zh_TW |
| dc.description.abstract | In most eukaryotes, telomeres are maintained by telomerase. Telomerase extends telomeres by reverse transcribing a segment of its internal RNA template onto the end of the chromosome. However, telomeres in all Drosophila species are composed of tandem arrays of repeated sequences formed by two non-LTR retrotransposons, HeT-A, TART. TART encodes two proteins, ORF1p and ORF2p. ORF2 shares homology to two enzyme activities, endonuclease (EN) and reverse transcriptase (RT) to other non-LTR retrotransposons. Sheen and Levis previously found TART can move to chromosome ends. But there is no evidence showing that this movement is through retrotransposition or recombination. My project focuses on identifying TART RT activity and the detail mechanism of TART. In the in vitro approach, I purified TART RT protein to detect its activity. However, both RT assay of point mutations at RT active motif YADD and the truncated protein showed that the construct may not have full length TART RT. In the in vivo assay , I set up a yeast retrotransposition system, as described previously (Teng et al., 1996), and Southern blot analysis was performed to obtain a insertion frequency, which is about 4.5x10-9. The sequencing data showed that the template switch may happen when TART ORF2 synthesized cDNA in VLPs. And TRAT ORF2p may prefer to fall out from mRNA because of the mRNA secondary structure in TART ORF2. My studies demonstrated that TART possesses RT activity. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T00:29:39Z (GMT). No. of bitstreams: 1 ntu-96-R93445102-1.pdf: 1439820 bytes, checksum: 3d304fa305ea9f61c4773b2a156b8ff0 (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract ii Abbreviations iii Table of Contents vi 1. Introduction 1 1.1. Mammal telomere and maintenance 1 1.2. Drosophila telomere 3 1.3. Retrotransposons 4 1.3.1. LTR retrotransposons 4 1.3.2. Non-LTR retrotransposons 5 1.4. Non-LTR retrotransposition model 6 1.5. TART retrotransposon 7 1.5.1. The structure of TART 7 1.5.2. Targeting of HeT-A and TART to chromosome ends 8 1.5.3. Gag proteins from HeT-A and TART have specific nuclear localizations 8 2. Aims 9 3. Materials and Methods 10 3.1. Plasmid construction 10 3.2. E. coli strains 11 3.3. Polymerase chain reaction (PCR) 12 3.3.1. General PCR 12 3.3.2. Site-directed mutagenesis PCR 12 3.4. Agarose gel electrophoresis 12 3.5. Competent cells preparation 13 3.5.1. Heat shock competent cells 13 3.5.2. KCM competent cells 13 3.6. E. coli transformation 14 3.6.1. Heat shock method 14 3.6.2. KCM method 14 3.7. Protein expression 15 3.8. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 16 3.9. Protein purification 16 3.9.1. GST fusion system 16 3.9.2. His-tag fusion system 17 3.10. Western blot analysis 18 3.11. Site-directed mutagenesis 19 3.12. Reverse transcriptase assay (RT assay) 20 3.13. Cell culture 21 3.14. Stable transfection and retrotransposition assay 21 3.14.1. General stable transfection and retrotransposition assay 21 3.14.2. Modified stable transfection-transient assay 22 3.15. Yeast retrotransposition assay 23 3.16. Southern blot analysis 23 4. Results 25 4.1. TART RT activity was undetectable in E. coli system. 25 4.1.1. GST-TART-RT (ORF2 nt 1065 to 2618) 25 4.1.2. 6xHis-TART-RT (ORF2 nt 798 to 2652) and mutants 26 4.1.3. GST-TART-RT (ORF2 nt 823 to 2667) and mutants 28 4.2. TART retrotransposition could not be detected in a cell culture system. 28 4.2.1. Overview of the cell culture retrotransposition system 28 4.2.2. General and modified stable transfection for retrotransposition assay 29 4.3. The RT of Drosophila TART was able to function in vivo in a yeast retrotransposition assay system. 30 4.3.1. Overview of the in vivo yeast retrotransposition assay system 30 4.3.2. Southern blot analysis and HIS3 insertion frequency of His+ prototrophy 31 4.3.3. Southern blot analysis of the rescued plasmids 32 4.3.4. Sequencing analysis of the rescued plasmids 33 5. Discussion 34 6. Figures 37 Figure 1 Non-LTR Retrotransposition Model 37 Figure 2 The hypothesis for the telomere elongation in Drosophila 38 Figure 3 Expression and purification of GST-TART-RT (ORF2 nt 1065 to 2618) 39 Figure 4 RT activity of GST-TART-RT (ORF2 nt 1065 to 2618) 40 Figure 5 Determination of the optimal conditions of TART RT 41 Figure 6 Expression of 6xHis-TART-RT in different strains 42 Figure 7 Expression of 6xHis-TART-RT at different temperatures and induction time 43 Figure 8 Expression and purification of 6xHis-TART-RT and mutants 44 Figure 9 RT activity of the 6xHis-TART RT and mutants 45 Figure 10 Expression of GST-TART-RT (ORF2 nt 823 to 2667) and mutants in different strains and at different temperatures 46 Figure 11 RT activity of GST-TART-RT (ORF2 nt 823 to 2667) 47 Figure 12 A system to detect TART retrotransposition 48 Figure 13 Yeast retrotransposition assay 49 Figure 14 Southern blot analysis of Histidine prototrophy 50 Figure 15 Retrotransposition frequency 51 Figure 16 Southern blot analysis of rescued plasmids 52 Figure 17 The donor plasmid and HIS3 insertion plasmid 53 Figure 18 Mechanism of plasmid event mediated by TART RT. 54 7. Tables 55 Table 1 Oligonucleotide Primers 55 Table 2 Plasmids 57 Table 3 E. coli strains 60 Table 4 Antibodies 61 Table 5 Cell Lines and Plasmids 62 Table 6 Yeast Strains and Plasmids 63 Reference 64 | |
| dc.language.iso | en | |
| dc.subject | 反轉錄酶 | zh_TW |
| dc.subject | 果蠅 | zh_TW |
| dc.subject | Reverse Transcriptase | en |
| dc.subject | Drosophila | en |
| dc.subject | TART | en |
| dc.title | 果蠅端粒連結反轉錄子TART之反轉錄酶活性研究 | zh_TW |
| dc.title | Drosophila Telomere-Associated Retrotransposon TART Encodes a Functional Reverse Transcriptase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李財坤(Tsai-Kun Li),陳銘凱(Ming-Kai Chern) | |
| dc.subject.keyword | 果蠅,反轉錄酶, | zh_TW |
| dc.subject.keyword | Drosophila,TART,Reverse Transcriptase, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-07-26 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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