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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 孫錦虹 | |
dc.contributor.author | Yu-Chang Huang | en |
dc.contributor.author | 黃昱璋 | zh_TW |
dc.date.accessioned | 2021-06-13T15:41:46Z | - |
dc.date.available | 2010-08-13 | |
dc.date.copyright | 2008-08-13 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-07-07 | |
dc.identifier.citation | 1. Alfred L. Goldberg, Protein degradation and protection against misfolded or damaged proteins, Nature 426(2003),pp895-899.
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Bandara and Nicholas B. La Thangue, Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor, Nature 351(1991), pp. 494–497. 25. Li-Hsin Su, Gilbert A. Lee, Yu-Chang Huang, Yi-Hsiu Chen and Chin-Hung Sun, Neomycin and puromycin affect gene expression in Giardia lamblia stable transfection, Mol Biochem Parasitol 156(2007), pp. 124–135. 26. M.L. Sogin, J.H. Gunderson, H.J. Elwood, R.A. Alonso and D.A. Peattie, Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia, Science 243 (1989), pp. 75–77. 27. Marie-Edith Chabouté, Bernadette Clément, Masami Sekine, Gabriel Philipps, and Nicole Chaubet-Gigot, Cell Cycle Regulation of the Tobacco Ribonucleotide Reductase Small Subunit Gene Is Mediated by E2F-like Elements, plant cell 12(2000), pp. 1987–2000. 28. Nicholas Dyson, The regulation of E2F by pRB-family proteins, Genes Dev. 12(1998), pp. 2245–2262. 29. Nicola Logan, Laurent Delavaine, Anne Graham, Carmel Reilly, Jon Wilson, Thijn R Brummelkamp, E Marielle Hijmans, René Bernards and Nicholas B La Thangue, E2F-7: a distinctive E2F family member with an unusual organization of DNA-binding domains, Oncogene 23(2004), pp. 5138–5150. 30. Ning Zheng, Ernest Fraenkel, Carl O. Pabo, and Nikola P. Pavletich, Structural basis of DNA recognition by the heterodimeric cell cycle transcription factor E2F-DP, Genet Dev. 13(1999), pp. 666–674. 31. Patrizia Lavia , Pidder Jansen-Dürr, E2F target genes and cell-cycle checkpoint control, BioEssays 21(1999), pp. 221–230. 32. R. Bernander, J.E. Palm and S.G. Svard, Genome ploidy in different stages of the Giardia lamblia life cycle, Cell Microbiol 1 (2001), pp. 55–62. 33. R.D. Adam, Biology of Giardia lamblia, Clin Microbiol Rev 14 (2001), pp. 447–475. 34. Raluca Verona, Ken Moberg, Scott Estes, Michelle Starz, Juan P. Vernon and Jacqueline A. Lees, E2F activity is regulated by cell cycle-dependent changes in subcellular localization, Mol Cell Biol. 17(1997), pp. 7268–7282. 35. Rebecca Stevens, Luisa Mariconti, Pascale Rossignol, Claudette Perennes, Rino Cella and Catherine Bergounioux, Two E2F Sites in the Arabidopsis MCM3 Promoter Have Different Roles in Cell Cycle Activation and Meristematic Expression, J. Biol. Chem. 277(2002), pp. 32978–32984. 36. Roberto Sitia and Ineke Braakman, quality control in the endoplasmic reticulum protein factory, Nature 426(2003),pp891-894. 37. Shunichi Kosugi and Yuko Ohashi, E2Ls, E2F-like Repressors of Arabidopsis That Bind to E2F Sites in a Monomeric Form, J. Biol. Chem. 277(2002), pp. 16553–16558. 38. Srilata Bagchi, Roberto Weinmann and Pradip Raychaudhuri, The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F, Cell 65(1991), pp. 1063–1072. 39. Zhang Y and Chellappan SP, Cloning and characterization of human DP2, a novel dimerization partner of E2F, Oncogene 10(1995), pp. 2085–2093. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37748 | - |
dc.description.abstract | 當真核細胞的細胞週期由G1 phase進入S phase時,許多與細胞生長以及DNA複製相關的基因會在這個時期被活化,這一時期的基因調控主要是由E2F家族轉錄因子所掌控。梨形鞭毛蟲在演化的分類上,為一個非常原始的真核生物。我們在梨形鞭毛蟲的基因組資料庫中進行搜尋,發現一個帶有E2F DNA結合區域的基因,稱之為Giardia e2f1 (ge2f1)。由胺基酸序列分析發現gE2F1帶有兩個E2F DNA結合區域,並且在兩個DNA binding domain中都具有重要的RRXYD motif,與近幾年發現人類的 E2F-7以及E2F-8相似。我發現ge2f1基因的mRNA量在囊體化時期比在滋養體時期下降。將HA標記接到gE2F1轉染梨形鞭毛蟲,利用免疫螢光染色法可發現在囊體化時期及滋養體時期gE2F1都位在於細胞核中。由EMSA實驗發現gE2F1蛋白質可和thymidine kinase的啟動子結合。經由突變序列分析,也發現gE2F1蛋白質的結合序列為TTTCCGCG,與人類的E2F家族蛋白質的結合特性類似,並且gE2F1與DNA結合能力,是不需要DP蛋白質的協助,符合帶有2個DNA 結合區域之E2F蛋白質特色。我也利用螢光酵素基因接上thymidine kinase基因啟動子,再利用cotransfection assay,可發現gE2F1大量表現時會使螢光酵素和訊息RNA表現量明顯上升,顯示gE2F1可以增加thymidine kinase 啟動子活性,因此對於thymidine kinase基因調控上可能為一個轉錄活化子。另外利用ChIP assay發現到gE2F1於in vivo下可以結合到thymidine kinase基因啟動子,也可以結合到cyst wall protein 1 基因啟動子,但是大量表現gE2F1卻讓cwp1的mRNA以及蛋白質表現量降低,並且讓cyst形成受阻,因此在cwp1基因調控當中gE2F1可能為一個轉錄抑制子。由實驗結果我們認為gE2F1蛋白質在梨形鞭毛蟲中,除了會造成DNA合成基因的活化,使之可以順利進入S-phase之外,另一方面也會抑制形成cyst相關的基因。 | zh_TW |
dc.description.abstract | The E2F family transcription factors regulate the expression of genes involved in the progression of G1/S transition and DNA replication in higher eukaryotic cells. To understand whether E2F transcription factor is present in the primitive protozoan Giardia lamblia, we searched the Giardia lamblia genome database and found a gene harboring E2F DNA-binding domain (ge2f1). The gE2F1 contains two E2F DNA binding domains and both domains have the RRXYD motif which is conserved in all E2F family proteins. Recently the E2F proteins with two DNA binding-domains were found in human, mouse and plants. gE2F1 may belong to this new E2F subfamily.
Epitope-tagged gE2F1 was found to localize to nuclei. During encystation, the levels of the ge2f1 mRNA and protein decreased significantly. Recombinant gE2F1 specifically bound to DNA replication-related thymidine kinase gene promoter in vitro and in vivo. Mutation analysis revealed that TTTCCGCG sequence was required for the binding of gE2F1 and an A-rich sequence can enhance the binding of gE2F1. gE2F1 contains the important RRXYD motif for DNA binding and its binding sequences are similar to those of the known E2F family factors. Like Two DNA binding domain E2Fs in other eukaryotic systems, gE2F1 binds to DNA without the requirement of DP proteins. To further analyze the role of both DNA binding domains in gE2F1, we mutated each of the RRXYD motif in DNA binding domain. Each mutation resulted in a decrease of gE2F1 DNA binding ability, suggesting that both DNA binding domains in gE2F1 are important for its DNA binding. Using cotransfection assay, we found that overexpession of gE2F1 can increase thymidine kinase promoter activity about 30 fold in the luciferase assay and mutation of the TTTCCGCG sequence in the thymidine kinase promoter abolishs the activation, suggesting that gE2F1 acts as an activator in thymidine kinase gene regulation by binding to the positive cis-acting element TTTCCGCG. Using ChIP assay, we also found that gE2F1 was assciated with cwp1 promoter in vivo. In the gE2F1 constitutively expressed cell line, the levels of the cwp1 mRNA and protein gene decreased significantly. In cotransfection assay, overexpressed gE2F1 decreased cwp1 promoter activity. These results suggest that gE2F1 functions as a repressor in cwp1 gene transcriptional regulation. We also compared the cyst number between the gE2F1 overexpressing cell line and the vector only control. We found that overexpression of gE2F1 decreased the cyst formation in both vegetative growth and encysting culture, suggesting that gE2F1 may decrease cwp1 transcription and influence the cyst formation process. Accordingly, our data suggest that DNA binding ability of E2F protein family has been conserved during evolution and gE2F1 is an important transcriptional activator in regulation of Giardia thymidine kinase gene that is involved in DNA replication. In addition, gE2F1 may also play a role in repressing cwp1 gene that is not relative to DNA replication or cell cycle. It is possible that E2F family transcription factor is also involved in Giardia differentiation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T15:41:46Z (GMT). No. of bitstreams: 1 ntu-97-R95445204-1.pdf: 1573298 bytes, checksum: a88d6f70fb7ffa979bea9d7a7e3dbd20 (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | 第一章 前言
1.1 梨形鞭毛蟲 Giardia lamblia……………….………... 1 1.1.1梨形鞭毛蟲簡介……………………….………....... 1 1.1.2梨形鞭毛蟲生活史……………………….……....... 1 1.2 梨形鞭毛蟲基因體及基因調控特性………………........ 2 1.3 E2F家族蛋白質簡介…………………………………… 2 1.4 本篇論文重點………………… ………….……………... 4 第二章 材料與方法 2.1 梨形鞭毛蟲細胞株的培養…………….……………..…… 6 2.2 梨形鞭毛蟲囊體化培養方式………….……………..…… 6 2.3 轉殖質體的建構……………………….……………..…… 6 2.3.1 5’△5N-Pac…………………….………………..…. 6 2.3.2 pPTE2F1………………………….……………..…. 6 2.3.3 pNTK5/pNTK5m………………….……………..… 7 2.3.4 pNW1………………………………………….....… 7 2.3.5 pPTE2F1m1 ………………………….…..………… 7 2.3.6 pPTE2F1m2……………………………………...…. 7 2.3.7 pPTE2F1DD……………………….……………...… 8 2.4 轉殖質體的轉型與萃取………………………………...…. 9 2.4.1 質體的轉型………………………….………………9 2.4.2 質體的萃取…………………………….……………9 2.5梨形鞭毛蟲轉染與選殖……………………………………9 2.6北方墨點法 (Northern blot)………………………………10 2.7反轉錄聚合酶鏈式反應 (RT-PCR)………………………11 2.8即時定量反轉錄聚合酶鏈式反應 (Real-time RT-PCR)…11 2.9西方墨點法 (Western blot)…………………………………12 2.10免疫螢光染色 (Immunofluorescence assay)………………13 2.11螢光酵素分析 (Luciferase assay)…………………………13 2.12重組gE2F1表現與純化……………………………………14 2.13電泳遷移率分析 (Electrophoretic Mobility Shift Assay).. 15 2.14染色質免疫沉澱法(Chromatin Immunoprecipitation Assay) 16 2.15cDNA 5’末端快速擴增技術 (5’RACE assay)……………... 17 2.16囊體計數 (Cyst count)…………………………………….... 17 第三章 結果 3.1梨形鞭毛蟲ge2f1基因的鑑別............................................... 19 3.2梨形鞭毛蟲ge2f1基因的表現情況和細胞內位置............... 20 3.3鑑定gE2F1的DNA結合位置............................................... 21 3.4分析2個E2F DNA binding domain對gE2F1與DNA 結合的關係…………………………………...……………… 23 3.5gE2F1對於thymidine kinase基因啟動子轉作之影響......... 23 3.6探討gE2F1對cyst wall protein 1基因調控之角色.............. 24 3.7鑑定gE2F1之nuclear localization signal (NLS)................. 26 3.8分析各轉染gE2F1突變質體之梨行鞭毛蟲蟲株對 CWP1蛋白質調控之影響.................................................... 27 第四章 討論 4.1 E2F家族轉錄因子存在於梨形鞭毛蟲基因體…………… 30 4.2 gE2F1對DNA結合特性...................................................... 30 4.3gE2F1可被細胞週期調控..................................................... 32 4.4gE2F1在基因調控的功能.............................................. 32 4.5分析gE2F1m1及gE2F1DD此兩個gE2F1突變蛋白質….. 34 圖與附表................................................. 36 參考文獻................................................ 49 | |
dc.language.iso | zh-TW | |
dc.title | 梨形鞭毛蟲的E2F同源蛋白質對於Cyst Wall Protein 1和Tymidine kinase基因的轉錄調控之影響 | zh_TW |
dc.title | A Novel E2F-related Protein Involved in Transcriptional Regulation of Cyst Wall Protein 1and thymidine kinase Genes in Giardia lamblia | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李財坤,李建國,詹迺立,蕭信宏 | |
dc.subject.keyword | 梨形鞭毛蟲,轉錄調控, | zh_TW |
dc.subject.keyword | Giardia lamblia,transcriptional regulation,E2F, | en |
dc.relation.page | 53 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-07-07 | |
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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