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論文中文名稱:利用突變分析的方法探討Streptomyces lividans染色體端粒的複製機制 [以論文名稱查詢館藏系統]
論文英文名稱:Mutational analysis of terminal patching of Streptomyces lividans chromosome telomeres [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
中文姓名:林澤庠
英文姓名:Tse-Hsiang Lin
研究生學號:94688005
學位類別:碩士
語文別:中文
口試日期:2007-07-19
論文頁數:81
指導教授中文名:黃志宏
指導教授英文名:Chih-Hung Huang
口試委員中文名:陳文盛;楊千金
口試委員英文名:Carton W. Chen;Chien-Chin Yang
中文關鍵詞:鏈黴菌端粒線型染色體端粒
英文關鍵詞:Streptomycestelomerelinear chromosome telomere
論文中文摘要:鏈黴菌,是存在於土壤中的格蘭式陽性菌且含量非常豐富,具有線型的染色體以及質體。先前的研究發現當鏈黴菌進行線型DNA複製時 ,由於在lagging strand的非連續性複製導致在3’端留下一缺口。而3’端突出的單股可能會往內自我摺疊形成二級結構後會被一個末端相關蛋白(terminal associate protein,Tap)所辨認。Tap會與末端蛋白(terminal protein,TP)彼此交互作用後將TP 帶往端粒,接著利用TP當作引子來進行末端缺口的修復工作。
在比較了其他鏈黴菌物種的末端序列後發現它們具有高度的保守性。第一,含有緊密相連的迴文結構。第二,迴文結構比序列還要具有保守性。第三,最末端13 bp(palindrome I)的DNA序列是完全相同的。為了探討末端端粒序列在DNA複製時所扮演的功能,我們主要針對Streptomyces lividans染色體的端粒來進行研究。我利用定位突變(site direct mutagenesis)來產生許多不同的突變端粒並且在活體中測試這些線型質體在DNA複製的功能。假設突變端粒可以支持線型DNA複製,則突變的區域對於末端修復則是不必要的;若是突變區域對於末端修復的必要區域,鏈黴菌無法進行線型DNA複製就會改成環型DNA來進行複製。利用這策略我得到了以下結果:(1)末端端粒僅需90 bp就足夠支持線型DNA複製。(2) palindrome II對於線型DNA是必須的,而非palindrome III。(3)最末端第一個鹼基C是重要的,而非第十三個鹼基G。這些結果指出了末端端粒對於末端缺口修補的必要區域。
論文英文摘要:The Gram-positive Streptomyces, which are abundant in soil, carried linear chromosomes and plasmids. In previous studies, during replication of the Streptomyces linear replicons the discontinuous synthesis of the lagging strands will leave a gap on the 3’ end of the DNA. The expose 3’ overhang may fold back and form a secondary structure which is recognized by the terminal associated protein (Tap). Tap will recruit terminal protein (TP) to the telomere, where the TP acts a primer for patching synthesis that fill up the single-strand gaps.
Comparison of terminal sequence of Streptomyces shows a high degree of conservation. Firstly, they are composed by tightly spaced palindromes. Secondly, the palindromic structure is more conserved than primary sequence. Thirdly, the terminal 13 bp(palindrome I)DNA are conserved in all. To study the function of the telomere sequences during replication, we focused on the telomere of Streptomyces lividans. By different site direct mutagenesis methods, we generated a series of mutant telomeres and examined their function on linear plasmids in vivo. If the mutated telomeres can support the replication of the linear plasmids in Streptomyces, the mutated nucleotide(s) is not essential for end patching. Using this strategy, we found that (1) only the first 90 bp of the telomere was sufficient; (2) the palindrome II region was required for linear DNA replication, but not palindrome III; and (3) the first nucleotide (C) was essential but not the thirteenth nucleotide (G). These results define the essential telomere structure for end patching.
論文目次:中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
表目錄 viii
圖目錄 ix
第一章 緒論 1
第二章 材料與方法 7
第三章 實驗結果
3.1執行複製功能的最小單位端粒DNA為多長 15
3.2探討Tap蛋白質結合到末端端粒區域中哪些區域是重要的 26
3.3末端端粒palindrome I前後兩端鹼基突變株 33
3.4 Palindrome I單一鹼基的突變或刪除對維持DNA複製的影響 44
3.5 Palindrome I序列及結構對末端序列DNA複製的影響 51
3.6 Palindrome I序列及結構對末端序列DNA複製的影響 54
第四章 討論 59
參考文獻 65
附錄
A Media and buffer 67
B PCR 69
C TA載體 71
D E.coli勝任細胞的製備及轉殖的方法 72
E E.coli質體的純化 74
F Streptomyces原生質體(protoplast)的製備及轉殖目標質體進入原生質體的方法 75
G 純化鏈黴菌質體 77
H 複殖末端DNA序列 78
I M-fold 80
J其他實驗注意事項 81
論文參考文獻:[1] Bao, K. and S. N. Cohen. "Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces." Genes Dev, vol 15, no. 12, 2001, pp 1518-27.

[2] Bao, K. and S. N. Cohen. "Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication." Genes Dev, vol 17, no. 6, 2003, pp 774-85.

[3] Huang, C. H., C. Y. Chen, et al. "Linear plasmid SLP2 of Streptomyces lividans is a composite replicon." Mol Microbiol, vol 47, no 6, 2003, pp 1563-76.

[4] Huang, C. H., Y. S. Lin, et al. "The telomeres of Streptomyces chromosomes contain conserved palindromic sequences with potential to form complex secondary structures." Mol Microbiol, vol 28, no 5, 1998, pp 905-16.

[5] Mendez, J., L. Blanco, et al. "Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication." Proc Natl Acad Sci U S A, vol 89,no. 20,1992, pp 9579-83.

[6] Qin, Z. and S. N. Cohen "Long palindromes formed in Streptomyces by nonrecombinational intra-strand annealing." Genes Dev, vol 14, no. 14, 2000, pp 1789-96.

[7] Qin, Z. and S. N. Cohen "Survival mechanisms for Streptomyces linear replicons after telomere damage." Mol Microbiol, vol 45, no. 3, 2002, pp 785-94.

[8] Weisblum, B. and V. Demohn "Thiostrepton, an inhibitor of 5OS ribosome subunit function." J Bacteriol,vol 101, no. 3, 1970, pp 1073-5.

[9] Yang, C. C., C. H. Huang, et al. "The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins." Mol Microbiol,vol 43, no. 2, 2002, pp 297-305.


[10] Yang, C. C., C. H. Huang, et al. "The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins." Mol Microbiol, vol 43, no. 2, 2002, pp 297-305.

[11] Zuker, M. "Mfold web server for nucleic acid folding and hybridization prediction." Nucleic Acids Res, vol 31, no. 13, 2003, pp 3406-15.
論文全文使用權限:同意授權於2008-08-21起公開