現在位置首頁 > 博碩士論文 > 詳目
  • 同意授權
論文中文名稱:利用突變分析的方法來探討鏈黴菌SCP1末端端粒的複製機制 [以論文名稱查詢館藏系統]
論文英文名稱:Mutational analysis of terminal patching of Streptomyces SCP1 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:區健宇
英文姓名:Kin-U Ao
研究生學號:94688004
學位類別:碩士
語文別:中文
口試日期:2007-07-30
論文頁數:78
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:Wen-Sheng Chen;Chien-Chin Yang
中文關鍵詞:鏈黴菌SCP1端粒迴紋序列末端蛋白
英文關鍵詞:Streptomyces SCP1telomerepalindrometerminal protein
論文中文摘要:鏈黴菌中普遍含有線型染色體及質體。而線型端粒的鏈黴菌複製子也擁有著很多共通的特性,例如豐富的迴紋結構以及5’端共價連結著末端蛋白 (terminal protein)。這些線型複製子在進行複製期間會從內部的複製起始點往兩端及雙向進行複製。在複製的後期會在延緩股3’端的地方留下一段單股的突出序列,最後再由末端蛋白引導複製完成。目前的研究認為3’端的突出單股會自我摺疊成穩定的二級結構,並在末端位置裡存在著大量且保守性甚高的迴紋序列,被認為與末端的填補與複製有關。在已知的鏈黴菌端粒系統中可依據其序列與迴紋結構分成兩大類:典型的由一般的鏈黴菌染色體與質體所組成,具有由緊密的迴紋序列以及彼此間具有完全保守的最末端序列所構成。非典型的則像是SCP1的端粒,它們的迴紋序列相對地並不緊密並且最末端並非迴紋結構,而且非典型的序列結構與典型的鏈黴菌的序列也有很大的不同。而我們將透過不同的點突變方法來集中研究非典型的SCP1端粒的序列以及功能性,並探討突變的端粒其在線型質體內的功能與特性。如果突變的端粒能夠支持線型的質體在鏈黴菌中的正常複製,那麼該突變序列便對於末端複製影響不大。而利用此方法,我們得到以下結論:(1) 只要最末端的84個鹼基對就足以支持端粒的功能!(2) 第一個及第二個的迴紋序列對於DNA的複製是必要的,而非第四個迴紋序列。(3)五個連續的鳥嘌呤便足以支持端粒的功能。(4)第一個迴紋結構內的序列或長短並不是太重要。這些結果將有助於讓我們去拼湊或更了解SCP1端粒的細步複製機轉。
論文英文摘要:The linear chromosomes and plasmids are common in Streptomyces. The telomeres of these linear replicons share the same characters, like palindrome rich sequences and 5’capped terminal proteins (TP). The replications of these linear replicons are initiated from an internal origin then bi-directionally toward to the ends. Finally, the lagging strands will leave a single-stranded gap at the 3’ end, which was supposedly patching up by TP-primed replication. A working model suggests that the protruding 3’ end folds back and forms stably secondary structure, by abundantly and persistently palindromic sequences in the terminal regions, as the intermediate for terminal patching. The known telomeres of Streptomyces are separate into two groups by their DNA sequences and palindromic structures. The typical group, coming from most Streptomyces chromosomes and plasmids, is composed by tightly palindromic sequences and the very end sequences are within a conserved palindrome. The atypical group, which those are composed by loosely palindromic sequences and their very end sequences are not within a palindrome, their sequences can’t alignment well with each other or any one of typical group. To study the function of the telomere sequences in the atypical group during replication, we focused on the best studied SCP1 which has showed 4 to 6 continued “G” on its very end sequence with the following sequence composed by loosely palindromic structures by forcing cloning. 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 84 bp of the telomere was essential; (2) the palindrome I and II regions were required for linear DNA replication, but not palindrome IV; (3) five continued nucleotides (G) was sufficient; and (4) the sequence and length of palindrome I was not important. These results define the essential telomere sequence for end patching of SCP1.
論文目次:目 錄

中文摘要 ii
英文摘要 iv
致謝 vi
目錄 vii
表目錄 ix
圖目錄 x

第一章 緒論 1

第二章 材料與方法 9

第三章 實驗結果
3.1探討執行線型複製功能的最小單位端粒DNA的長度............................................ 18
3.2探討末端端粒序列中哪些區域或迴紋序列是重要的.............................................34
3.3探討最末端端粒到底需要幾個連續鳥嘌呤 (Guanine)才是必要的.............................................44
3.4探討Palindrome I內部單一鹼基的突變或刪除對維持DNA進行線型複製的影響...................................50

第四章 討論...................................58

參考文獻.......................................65

第五章 附錄
1. Media and buffer........................66
2. PCR (Polymerase Chain Reaction).........68
3. T & A Cloning Vector ....................69
4. E.Coli Competent cell preparation and transformation.................................70
5. Plasmid isolation of E.coli.............72
6. Preparation of Streptomyces protoplast and transformation with plasmid DNA................73
7. Isolation Streptomyces total DNA........76
8. End-Cloing..............................77
論文參考文獻:1. Bao, K. and S. N. Cohen (2001). "Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces." Genes Dev 15(12): 1518-27.

2. Bao, K. and S. N. Cohen (2003). "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 17(6): 774-85.

3. Bentley, S. D., K. F. Chater, et al. (2002). "Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2)." Nature 417(6885): 141-7.

4. Chang, H. M., M. Y. Chen, et al. (1996). "The cutRS signal transduction system of Streptomyces lividans represses the biosynthesis of the polyketide antibiotic actinorhodin." Mol Microbiol 21(5): 1075-85.

5. Chen, C. W., T. W. Yu, et al. (1993). "The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule." Mol Microbiol 7(6): 925-32.

6. Hayakawa, T., N. Otake, et al. (1979). "Isolation and characterization of plasmids from Streptomyces." J Antibiot (Tokyo) 32(12): 1348-50.

7. Hinnebusch, J. and K. Tilly (1993). "Linear plasmids and chromosomes in bacteria." Mol Microbiol 10(5): 917-22.

8. Huang, C. H., Y. S. Lin, et al. (1998). "The telomeres of Streptomyces chromosomes contain conserved palindromic sequences with potential to form complex secondary structures." Mol Microbiol 28(5): 905-16.

9. Huang, C. H., H. H. Tsai, et al. (2007). "The telomere system of the Streptomyces linear plasmid SCP1 represents a novel class." Mol Microbiol 63(6): 1710-8.

10. Ikeda, H., J. Ishikawa, et al. (2003). "Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis." Nat Biotechnol 21(5): 526-31.

11. Kinashi, H., E. Mori, et al. (1994). "Isolation and characterization of linear plasmids from lankacidin-producing Streptomyces species." J Antibiot (Tokyo) 47(12): 1447-55.

12. Kinashi, H. and M. Shimaji-Murayama (1991). "Physical characterization of SCP1, a giant linear plasmid from Streptomyces coelicolor." J Bacteriol 173(4): 1523-9.

13. Kinashi, H., M. Shimaji-Murayama, et al. (1991). "Nucleotide sequence analysis of the unusually long terminal inverted repeats of a giant linear plasmid, SCP1." Plasmid 26(2): 123-30.

14. Qin, Z. and S. N. Cohen (1998). "Replication at the telomeres of the Streptomyces linear plasmid pSLA2." Mol Microbiol 28(5): 893-903.

15. Qin, Z. and S. N. Cohen (2000). "Long palindromes formed in Streptomyces by nonrecombinational intra-strand annealing." Genes Dev 14(14): 1789-96.

16. Qin, Z. and S. N. Cohen (2002). "Survival mechanisms for Streptomyces linear replicons after telomere damage." Mol Microbiol 45(3): 785-94.

17. Qin, Z., M. Shen, et al. (2003). "Identification and characterization of a pSLA2 plasmid locus required for linear DNA replication and circular plasmid stable inheritance in Streptomyces lividans." J Bacteriol 185(22): 6575-82.

18. Rekosh, D. M., W. C. Russell, et al. (1977). "Identification of a protein linked to the ends of adenovirus DNA." Cell 11(2): 283-95.

19. Sakaguchi, K., H. Hirochika, et al. (1985). "Linear plasmids with terminal inverted repeats obtained from Streptomyces rochei and Kluyveromyces lactis." Basic Life Sci 30: 433-51.

20. Salas, M. (1991). "Protein-priming of DNA replication." Annu Rev Biochem 60: 39-71.

21. Weaver, D., N. Karoonuthaisiri, et al. (2004). "Genome plasticity in Streptomyces: identification of 1 Mb TIRs in the S. coelicolor A3(2) chromosome." Mol Microbiol 51(6): 1535-50.
論文全文使用權限:同意授權於2007-08-31起公開