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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳蕙芬(Whei-Fen Wu) | |
| dc.contributor.author | Hsuan-He Chen | en |
| dc.contributor.author | 陳宣合 | zh_TW |
| dc.date.accessioned | 2021-05-20T20:08:04Z | - |
| dc.date.available | 2015-08-03 | |
| dc.date.available | 2021-05-20T20:08:04Z | - |
| dc.date.copyright | 2009-08-06 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-04 | |
| dc.identifier.citation | Ariyanayagam, M. R., and Fairlamb, A. H., (2001) Ovothiol and trypanothione as antioxidants in trypanosomatids. Mol Biochem Parasitol 115: 189-198.
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L., and Clayton, C., (2000) Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress. Mol Microbiol 35: 542-552. Kwon, D. S., Lin, C. H., Chen, S., Coward, J. K., Walsh, C. T., and Bollinger, J. M., Jr., (1997) Dissection of glutathionylspermidine synthetase/amidase from Escherichia coli into autonomously folding and functional synthetase and amidase domains. J Biol Chem 272: 2429-2436. Makino, K., Shinagawa, H., Amemura, M., and Nakata, A., (1986) Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12. J Mol Biol 190: 37-44. Meister, A., and Anderson, M. E., (1983) Glutathione. Annu Rev Biochem 52: 711-760. Nagasawa, S., Ishige, K., and Mizuno, T., (1993) Novel members of the two-component signal transduction genes in Escherichia coli. J Biochem 114: 350-357. Neylon, C., Kralicek, A. V., Hill, T. M., and Dixon, N. E., (2005) Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex. Microbiol Mol Biol Rev 69: 501-526. Nishino, K., Honda, T., and Yamaguchi, A., (2005) Genome-wide analyses of Escherichia coli gene expression responsive to the BaeSR two-component regulatory system. J Bacteriol 187: 1763-1772. Nonaka, G., Blankschien, M., Herman, C., Gross, C. A., and Rhodius, V. A., (2006) Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress. Genes Dev 20: 1776-1789. Oza, S. L., Ariyanayagam, M. R., and Fairlamb, A. H., (2002a) Characterization of recombinant glutathionylspermidine synthetase/amidase from Crithidia fasciculata. Biochem J 364: 679-686. Oza, S. L., Shaw, M. P., Wyllie, S., and Fairlamb, A. H., (2005) Trypanothione biosynthesis in Leishmania major. Mol Biochem Parasitol 139: 107-116. Oza, S. L., Tetaud, E., Ariyanayagam, M. R., Warnon, S. S., and Fairlamb, A. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9063 | - |
| dc.description.abstract | Glutathionylspermidine synthetase (GspS) 是一種具有雙重功能的酵素，具有合成酶 (synthetase) 活性，可催化glutathione與spermidine間醯胺鍵的形成，產生glutathionylspermidine (Gsp)，也有醯胺酶 (amidase) 活性，可使Gsp的醯胺鍵水解成glutathione及spermidine。由於Gsp為原生動物病原蟲與大腸桿菌等生物中所特有，與其相關之酵素遂成為研究對抗此類病原蟲藥物的目標。目前GspS的蛋白質之結晶結構已被解出，對蛋白質活性區域以及反應機制有更進一步的認識，但是GspS在大腸桿菌中扮演何種角色、gspS基因表現受到哪些因子調控，皆尚未清楚。
為了瞭解gspS的調控方式，首先以5’RACE (Rapid-Amplification of cDNA Ends) 確認轉錄起始點 (transcription start site)，並進一步分析gspS上游的promoter區域及cis-binding site位置，包含預測之BaeR binding site。利用不同長度及BaeR binding site突變之gspS-lacZ operon fusion進行β-galactosidase 活性分析，發現在大腸桿菌中BaeR大量表現可誘導gspS表現量上升，顯示BaeR為一可能之轉錄因子，而且即使BaeR大量表現，預測之BaeR binding site突變仍然無法使gspS表現量增加，推測BaeR跟gspS上游的cis-binding site應為直接結合造成gspS表現量上升。 利用不同濃度H2O2對gspS−及一系列對抗氧化壓力 (oxidative stress) 相關突變株 (trxA−、trxB−、grxA−、grxB−) 的影響，發現gspS−grxA−與gspS−grxB−之雙重突變株對H2O2造成之氧化壓力耐受程度較wild type和gspS−、grxA−、grxB−單一突變株低，推測GspS和 GrxA、GrxB在功能上可能可以互相替代。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-05-20T20:08:04Z (GMT). No. of bitstreams: 1 ntu-98-R96623008-1.pdf: 1740948 bytes, checksum: e7a1c721d89a397dd357def1a971fed9 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
摘要 ii Abstract iii 目錄 v 表目錄 viii 圖目錄 ix 第一章、前人研究 1 1.1 Trypanothione 1 1.2 Glutathione 3 1.3 Spermidine 3 1.4 Glutathionyl spermidine, Gsp 4 1.5 Glutathionyl spermidine synthetase, GspS 5 1.6 雙成分控制系統 (two component system) 5 1.7 氧化壓力 (Oxidative stress) 與抗氧化系統 7 1.8 研究動機與目的 9 第二章、材料與方法 10 2.1 一般研究材料 10 2.1.1 菌株及質體 10 2.1.2 藥品及試劑 10 2.2 一般實驗方法 11 2.2.1 質體製備及轉殖 12 2.2.2 勝任細胞 (competent cell) 製備 13 2.2.3 轉形作用 (transformation) 13 2.3 選殖基因表現系統之建立 15 2.3.1 載體 (vector)製備 15 2.3.2 選質基因片段之製備 16 2.3.3 選殖片段之處理 17 2.3.4 接合反應 (ligation) 18 2.3.5 gspS promoter點突變之質體建構 18 2.4 嗜菌體一般實驗方法 19 2.4.1 製備嗜菌體 19 2.4.2 Titer the lysate 20 2.4.3 P1轉導作用 (P1 transduction) 20 2.4.4 λ特定轉導作用 (specialize transduction) 22 2.5 建構gspS−突變株 23 2.6 β-galactosidase 活性分析 25 2.7 cDNA末端快速增幅 (rapid amplification of cDNA ends, RACE) 26 2.8 西方墨點分析 (Western Blotting) 27 2.8.1 SDS蛋白質膠體電泳 (SDS-PAGE) 27 2.8.2 轉印 (Transfer) 29 2.8.3 免疫雜交 (Hybridization) 30 第三章、結果 34 3.1 gspS上游啟動子 (promoter) 區域及cis-element位置之分析 34 3.2 gspS受到BaeR轉錄因子 (transcription factor) 的調控 35 3.3 以gspS 突變株探討gspS之表現型 (phenotype) 37 第四章、討論 40 4.1 gspS promoter 40 4.2 BaeR對gspS的調控 40 4.3 其他調控gspS之轉錄因子 42 4.4 gspS之表現型 42 第五章、參考文獻 44 表目錄 表一、本論文所使用的菌株與嗜菌體 49 表二、本論文所使用的質體 50 表三、本論文所使用之聚合酶連鎖反應引子 51 圖目錄 圖一、E. coli MG1655 gspS基因上游序列圖譜 52 圖二、不同長度的gspS-lacZ fusion (242、195、161、130 bp) 建構 53 圖三、不同長度的gspS (286、236、116、20) 建構及蛋白質表現情形 54 圖四、轉錄因子BaeR對不同長度及突變的gspS-lacZ fusion基因表現量的影響 55 圖五、gspS之BaeR binding site 突變設計 56 圖六、indole處理下對wild type及BaeR−突變株之gspS-lacZ fusion基因表現量的影響 57 圖七、gspS::bla突變株之確認 58 圖八、H2O2對wild type及各突變株生長之抑制 59 圖九、不同濃度之H2O2對wild type及各突變株在不同菌液稀釋度下生長的影響 60 | |
| dc.language.iso | zh-TW | |
| dc.title | 大腸桿菌gspS基因表現之調控 | zh_TW |
| dc.title | Regulation of gspS gene expression
in Escherichia coli | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林俊宏(Chun-Hung Lin),陳建德(Chien Teh Chen),劉俊民 | |
| dc.subject.keyword | GspS,Gsp,BaeR,氧化壓力,GrxA,GrxB, | zh_TW |
| dc.subject.keyword | GspS,Gsp,BaeR,Oxidative stress,GrxA,GrxB, | en |
| dc.relation.page | 60 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2009-08-04 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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