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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃楓婷(Feng-Ting Huang) | |
| dc.contributor.author | Yu-Pu Kao | en |
| dc.contributor.author | 高瑜璞 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:11:57Z | - |
| dc.date.available | 2011-08-20 | |
| dc.date.copyright | 2011-08-20 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-17 | |
| dc.identifier.citation | Allen RC, Armitage RJ, Conley ME, Rosenblatt H, Jenkins NA, Copeland NG, Bedell MA, Edelhoff S, Disteche CM, Simoneaux DK, et al. (1993) CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome. Science 259: 990-993
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Mol Biol Evol 10: 60-72 Luby TM, Schrader CE, Stavnezer J, Selsing E (2001) The mu switch region tandem repeats are important, but not required, for antibody class switch recombination. J Exp Med 193: 159-168 Maizels N (2005) Immunoglobulin gene diversification. Annu Rev Genet 39: 23-46 Manis JP, Gu Y, Lansford R, Sonoda E, Ferrini R, Davidson L, Rajewsky K, Alt FW (1998) Ku70 is required for late B cell development and immunoglobulin heavy chain class switching. J Exp Med 187: 2081-2089 Martin A, Scharff MD (2002a) AID and mismatch repair in antibody diversification. Nat Rev Immunol 2: 605-614 Martin A, Scharff MD (2002b) Somatic hypermutation of the AID transgene in B and non-B cells. Proc Natl Acad Sci U S A 99: 12304-12308 Martomo SA, Gearhart PJ (2006) Somatic hypermutation: subverted DNA repair. Curr Opin Immunol 18: 243-248 Mizuta R, Iwai K, Shigeno M, Mizuta M, Uemura T, Ushiki T, Kitamura D (2003) Molecular visualization of immunoglobulin switch region RNA/DNA complex by atomic force microscope. J Biol Chem 278: 4431-4434 Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T (2000) Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102: 553-563 Mussmann R, Courtet M, Schwager J, Du Pasquier L (1997) Microsites for immunoglobulin switch recombination breakpoints from Xenopus to mammals. Eur J Immunol 27: 2610-2619 Nakamura M, Kondo S, Sugai M, Nazarea M, Imamura S, Honjo T (1996) High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells. Int Immunol 8: 193-201 Nickoloff JA (1992) Transcription enhances intrachromosomal homologous recombination in mammalian cells. Mol Cell Biol 12: 5311-5318 Odegard VH, Schatz DG (2006) Targeting of somatic hypermutation. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45275 | - |
| dc.description.abstract | After stimulation with external antigens and coupling with activation of T cell , naïve B cells can differentiate into plasma cells that produce antibodies . External antigens are processed and bound to B cells. That activates the appropriate immune response in B cells and antibody production to protect the body. Antibody can be divided into five main isotypies according to constant regions, namely IgM, IgG, IgA, IgD and IgE; Different antigens stimulate B cells to undergo class switch recombination (CSR) for production of different types of antibodies. Different isotypies of antibodies trigger different immune response so as to enhance the ability of removing different kinds of antigens. class switch recombination is initiated by activation-induced cytidine deaminase (AID), which in two switch (S) regions of the antibody genes so that S region can further generate double-stranded breaks and then two S region are combined by non-homologous end joining (NHEJ) and other mechanisms.
When RNA polymerase transcribes DNA in the S region, RNA is produced and base paired with the template strand DNA, so that another strand of DNA exists in single-stranded form, which is called the R-loop. The target of AID is single-stranded DNA. Therefore, it’s assumed that R-loop structures may provide AID’s substrates, and which makes AID trigger CSR. However, this hypothesis needs to be confirmed with more experiments. Previous research indicates that R-loop structures exist at the Sμ, Sγ2b and Sγ3 in primary B cells of mice, and which makes it more possible that R-loops structure provide substrates for AID. In order to better apprehend the function of R-loop structures, we test whether R-loop structures existat the Sα in CH12F3-2A cells (a murine B cell line). The CH12F3-2A cell would switch to IgA positive cells through CSR after stimulation. If R-loops exist at the Sα, where are initiation sites and the endpoints of R-loops? In addition, whether the frequency of R-loops is directly proportional to the frequency of CSR? If so, it can further determine that R-loops structure is important to CSR. In the thesis, results show that R-loop structures also exist at the Sα, and it supports that R-loops are the common features of S regions. Surprisingly, the initiation sites of some R-loops are located in the region upstream of the Sα; this phenomenon is never observed in other acceptor switch regions, and this particular R-loop has a recurring phenomenon, which may be correlated with that fact that the G density of Sα is lower than the other S regions. However, it needs to be confirmed by more experiments. In addition, R-loop with initiation sites in the Sα region are detected in stimulated cells but not in unstimulated cells. With increasing of stimulation time, there is no significant difference of the pattern of R-loops. Moreover, since CSR breakpoints often appear in the core region of Sα, we also detect and analyze R-loops in the core region of the Sα. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:11:57Z (GMT). No. of bitstreams: 1 ntu-100-R98b47209-1.pdf: 19656247 bytes, checksum: fd03a3373195445b0f423cfee51bbedb (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 口試委員會審定書 #
中文摘要 i 英文摘要 ii 縮寫表 iv 目錄 v 第一章 緒論 1 概述 1 產生更有效率的抗體 2 抗體 (Antibody) 2 抗體多樣性 (Antibody Diversification) 3 CD40 (TNF receptor superfamily member 5) 3 抗體類型轉換重組相關機制 4 抗體類型轉換重組 (Class Switch Recombination, CSR) 4 抗體基因的變換區 (Switch region, S region) 5 Germ-line transcription 6 R –loops 6 R-loops的生理意義 7 誘發活化性胞嘧啶核苷脫氨酶 8 BER和MMR對CSR的重要性 (Role of BER and MMR in CSR) 9 非同源黏合 (Non-homologous End Joining) 對CSR的重要性 9 第二章 研究動機及目的 11 第三章 實驗材料與方法 13 實驗藥品與試劑 13 實驗方法 15 細胞培養與刺激 15 流式細胞儀分析 (FACS analysis) 15 核酸萃取 (DNA extraction) 15 BamHⅠ限制酶切 16 RNase H處理 16 亞硫酸鹽處理DNA (Sodium bisulfite treatment) 17 聚合酶連鎖反應 (Polymerase Chain Reaction，PCR) 17 洋菜膠電泳 (Agarose gel electrophoresis) 20 TA cloning (TOPO TA Cloning® Kits, Invitrogen) 20 大腸桿菌培養基製作 21 大腸桿菌勝任細胞 (Competent cell) 製備 21 電轉型作用(Electroporation Transformation) 21 質體DNA小量萃取 (Mini-preparation) 22 DNA 定序分析 22 R-loops訊號頻率分析 22 第四章 實驗結果 24 抗體基因之Sα受刺激兩天後有長鏈單股DNA結構存在 24 利用RNase H確認Sα中觀察之單股DNA結構為R-loops 24 R-loops在Sα中的起始位置 25 R-loops在Sα中的終點位置 26 在Sα中偵測R-loops訊號的頻率 26 R-loops在Sα之core region分布情形 27 第五章 討論 29 第六章 實驗圖表 34 圖一、受刺激兩天細胞Sα中非模板之單股DNA偵測結果 34 圖二、未受刺激細胞之R-loops起始位置 35 圖三、受刺激一天細胞之R-loops起始位置 36 圖四、受刺激兩天細胞之R-loops起始位置 37 圖五、受刺激三天細胞之R-loops起始位置 38 圖六、R-loops在Sα中的終點位置 40 圖七、刺激時間不同影響R-loops偵測頻率 41 圖八、R-loops在Sα之core region分布情形 42 表一、RNase H處理刺激兩天細胞對R-loops形成之影響 43 表二、刺激時間不同影響R-loops起始位置偵測頻率 43 附圖一、Sα (YPK2-FH9) G density示意圖 44 附圖二、本論文所使用引子的相對位置示意圖 45 附圖三、不同acceptor S region之R-loops起始位置比較 47 附圖四、不同acceptor S region之R-loops終點位置比較 49 附圖五、Sα breakpoints 示意圖 50 第七章 參考文獻 51 | |
| dc.language.iso | zh-TW | |
| dc.subject | 抗體 | zh_TW |
| dc.subject | 抗體類型轉換重組 | zh_TW |
| dc.subject | 抗體基因的變換區 | zh_TW |
| dc.subject | R-環 | zh_TW |
| dc.subject | 誘發活化性胞嘧啶核苷 | zh_TW |
| dc.subject | 脫氨酶 | zh_TW |
| dc.subject | Class switch recombination | en |
| dc.subject | Immunoglobulin | en |
| dc.subject | Activation induced cytidine deaminase | en |
| dc.subject | R-loops | en |
| dc.subject | Switch region | en |
| dc.title | 在抗體基因Sα區域的R-loops之特徵分析 | zh_TW |
| dc.title | Detection and characterization of R-loops
at the immunoglobulin switch alpha (Sα) region | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 常怡雍,陳彥榮 | |
| dc.subject.keyword | 抗體類型轉換重組,抗體基因的變換區,R-環,誘發活化性胞嘧啶核苷,脫氨酶,抗體, | zh_TW |
| dc.subject.keyword | Class switch recombination,Switch region,R-loops,Activation induced cytidine deaminase,Immunoglobulin, | en |
| dc.relation.page | 64 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-08-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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