現在位置首頁 > 博碩士論文 > 詳目
  • 同意授權
論文中文名稱:找尋複製中鏈黴菌DNA端粒的引子酶辨認序列之研究 [以論文名稱查詢館藏系統]
論文英文名稱:To find the primase recognition site at the telomere of Streptomyces [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
畢業學年度:100
出版年度:101
中文姓名:林蕙盈
英文姓名:Huei-ying Lin
研究生學號:99688010
學位類別:碩士
語文別:中文
口試日期:2012-07-30
論文頁數:80
指導教授中文名:黃志宏
口試委員中文名:楊千金;陳月茸
中文關鍵詞:鏈黴菌延遲股引子酶
英文關鍵詞:Streptomyceslagging strandprimase
論文中文摘要:鏈黴菌線狀染色體與線狀質體的複製,由中間朝向兩側複製,當延遲股(lagging strand)最後一個岡崎片段(Okazaki fragment)複製完,在模板股3’端會突出裸露一段單股DNA稱之為單股間隙。在不同的端粒DNA上顯示為250~320 bp的間隙,然後以末端蛋白(TP)為將其補齊。
黃譯慧的研究中,使用聚合連鎖反應(Polymerase Chain Reaction)能夠確認單股間隙與雙股DNA交接的位置,發現帶有S. lividans端粒DNA的線狀質體複製,其單股間隙為300 nt。由上述研究結果推測端粒DNA應有一段序列能夠由引子酶(primase)所辨識,如能找出此序列,可知道鏈黴菌在複製時引子酶辨識的序列。
本論文中使用線狀質體實驗,分別帶有S. liviidans、S. coelicolor以及SLP2三種菌株的端粒DNA。實驗發現當端粒DNA過短時,無法發現觀察到複製中間產物,猜測可能是因為單股間隙過短而無法得到複製中間產物。另外本論文中藉由突變距離S. liviidans端粒DNA的末端223~297 nt序列,是否會影響到單股間隙的長度,來得知引子酶所辨識序列的精確位置。所以經由上述實驗結果發現帶有S. lividans端粒DNA的線形質體,距離末端約281~285 -nt中間,應有引子酶所辨識的序列。
論文英文摘要:Streptomyces chromosomes and some of plasmids are linear, the linear replicon initiate replication from the middle to both sides. When the lagging strand to the last Okazaki fragment and the primer was removed, it will leave a 3’ strands overhang at end. Rich palindromes synthesis proceeded in this single-strand make this sequence form a stable secondary structure that could be identified by Tap with TP protein is then working as primer and single-strand gap as a template to complete the end of the replication via DNA polymerase.
Previous studies, showed that the linear plasmids carried the telomeres of S. rochei pSLA2, S. coelicolor and SLP2 generate single-stranded gaps about 280 nt, 320 and 250 nt in length, respectively. Huang I-Huei’s studies, using polymerase chain reaction, found that for linear plasmid replication with S. lividans telomeres, the single-stranded gap is about 300 nt long.
We set lead a hypothesis based on all the previous research results that those gap structures should be caused by a specific DNA sequence recognized by primase of the last Okazaki fragment. In our study we focus on identifying the primase recognition site of Streptomyces linear replicons.
In this study, we used the linear plasmids with different telomeres from S. liviidans, S. coelicolor and SLP2. We found that when DNA telomeres were too short, the replication intermediates couldn’t be observed. It suggested that to the length of single strand part could be too short to be isolated. But the previous studies did showed the possible location. We mutated the sequence, to see how it affects the length of the single strand gap to get the exact location of the primase recognition site and results suggested that the linear plasmid with the telomeres of S. lividans has its primase recognition site located approximately 275 to 285 nt from the end.
論文目次:目錄

摘 要..................................................i
ABSTRACT..............................................ii
誌 謝..................................................iv
目錄...................................................v
表目錄.................................................vii
圖目錄.................................................viii
第一章 緒論...............................................1
1-1 鏈黴菌簡介............................................1
1-2 細菌、腺病毒與鏈黴菌的DNA複製機制.........................2
1-3 噬菌體與其他細菌的引子酶辨認序列..........................3
1-4 本論文的內容...........................................5
第二章 材料與方法...........................................8
2-1 菌種與質體.............................................8
2-2 本篇論文所使用的引子序列..................................9
2-3 藥品及酵素............................................10
2-4 培養基及緩衝液.........................................10
2-5 菌種之貯存............................................10
2-6 大腸桿菌的轉形.........................................11
2-7 大腸桿菌質體之分離與純化.................................11
2-8 鏈黴菌原生質體之製備與轉形...............................11
2-9 鏈黴菌total DNA的純化.................................11
2-10 檢測鏈黴菌之生長曲線...................................12
2-11 鏈黴菌之對數期中期複製DNA的純化..........................12
2-12 南方點墨法............................................13
2-13 聚合酶連鎖反應.........................................13
2-14 End gap junction位置的檢測實驗設計流程..................13
2-15 DNA長度檢量法.........................................14
第三章 實驗結果............................................15
3-1 改良染色體端粒DNA的複製中間產物之純化......................15
3-2 末端端粒DNA序列的長短會影響到複製中間產物出現...............18
3-3 RNaseH能有效移除單股間隙上的RNA primer...................28
3-4 目前S. lividans染色體端粒DNA最後一個引子酶辨認序列位在261~297 –nt間...........................................31
3.5 使用突變來縮小S. lividans色體端粒DNA最後一個引子酶辨認序列範圍是在276~297 -nt序列區間中............................39
3-6 S. coelicolor和S. lividans端粒DNA的最後一個引子酶辨認序列可能是5’-d(CGC)-3’或是5’-d(GGC)-3’....................47
第四章 討論................................................55
文獻參考...................................................59
附錄......................................................63


表目錄

表 1-3 噬菌體與其他細菌的引子酶辨認序列整理表....................12


圖目錄

圖1-4-1 鑑定單股間隙的方法之策略..............................15
圖2-11-1 純化5g菌絲體的total DNA之流程......................22
圖3-1-1 TK64[P3]複製中間產物純化之偵測.......................24
圖3-2-1 TK64[pHY17L]複製中間產物之偵測......................28
圖3-2-2 TK64[pKH04L]複製中間產物之偵測......................30
圖3-2-3 TK64[pKH04L]複製中間產物移除單股DNA之偵測............32
圖3-2-4 TK64[pLUS881L]複製中間產物之偵測....................34
圖3-3-1 TK64[pLUS970L]複製中間產物處理RNaseH之偵測.........37
圖3-4-1 TK64[P250]複製中間產物的單股間隙之偵測..............41
圖3-4-2 TK64[P3]複製中間產物的單股間隙之偵測................43
圖3-4-3 TK64[P280]複製中間產物的單股間隙之偵測...............45
圖3-5-1 構築質體pBLU2KS(-)+S. l tsr end P297及限制酶切割電泳分析圖...................................49
圖3-5-2 構築質體 pHL01(pLUS970-tsr end 281~297 deletion)及限制酶切割電泳分析圖...............................50
圖3-5-3 構築質體pBLU2KS(-)+S. l tsr end P276及限制酶切割電泳分析圖......................................51
圖3-5-4 構築質體 pHL02(pLUS970-tsr end 276 mutation)及限制酶切割電泳分析圖..................................52
圖3-5-5 構築質體pBLU2KS(-)+S. l tsr end P285及限制酶切割電泳分析圖..............................................53
圖3-5-6 構築質體pHL03(pLUS970-tsr end 285 insertion)及限制酶切割電泳分析圖.....................................54
圖3-6-1 TK64[pHL01L]複製中間產物的單股間隙之偵測.............57
圖3-6-2 TK64[pHL02L]複製中間產物的單股間隙之偵測.............59
圖3-6-3 TK64[pHL03L]複製中間產物的單股間隙之偵測.............61
論文參考文獻:1. Angert ER: Alternatives to binary fission in bacteria. Nat Rev Microbiol 2005, 3(3):214-224.
2. Bao K, Cohen SN: 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 2003, 17(6):774-785.
3. Bao K, Cohen SN: Reverse transcriptase activity innate to DNA polymerase I and DNA topoisomerase I proteins of Streptomyces telomere complex. Proc Natl Acad Sci U S A 2004, 101(40):14361-14366.
4. Bao K, Cohen SN: Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces. Genes Dev 2001, 15(12):1518-1527.
5. Bentley SD, Brown S, Murphy LD, Harris DE, Quail MA, Parkhill J, Barrell BG, McCormick JR, Santamaria RI, Losick R et al: SCP1, a 356,023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2). Mol Microbiol 2004, 51(6):1615-1628.
6. Bentley SD, Chater KF, Cerdeno-Tarraga AM, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D et al: Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 2002, 417(6885):141-147.
7. Bhattacharyya, S., and M. A. Griep. DnaB helicase affects the initiation specificity ofEscherichia coli primase on single-stranded DNA templates. Biochemistry 2000. 39:745-752.
8. Bhattacharyya, S., and M. A. Griep. 2000. DnaB helicase affects the initiation specificity of Escherichia coli primase on single-stranded DNA templates. Biochemistry 39:745-752.
9. Cha, T. A., and B. M. Alberts. Studies of the DNA helicase-RNA primase unit from bacteriophage T4. A trinucleotide sequence on the DNA template starts RNA primer synthesis. J. Biol. Chem. 1986. 261:7001-7010.
10. Chang PC, Cohen SN: Bidirectional replication from an internal origin in a linear streptomyces plasmid. Science 1994, 265(5174):952-954.
11. Frick, D. N., and C. C. Richardson. 2001. DNA primases. Annu. Rev. Biochem. 70:39-80.
12. Hopwood DA, Kieser T, Wright HM, Bibb MJ: Plasmids, recombination and chromosome mapping in Streptomyces lividans 66. J Gen Microbiol 1983, 129(7):2257-2269.
13. Huang CH, Chen CY, Tsai HH, Chen C, Lin YS, Chen CW: Linear plasmid SLP2 of Streptomyces lividans is a composite replicon. Mol Microbiol 2003, 47(6):1563-1576.
14. Huang CH, Tsai HH, Tsay YG, Chien YN, Wang SL, Cheng MY, Ke CH, Chen CW: The telomere system of the Streptomyces linear plasmid SCP1 represents a novel class. Mol Microbiol 2007, 63(6):1710-1718.
15. Johnson, S. K., S. Bhattacharyya, and M. A. Griep. 2000. DnaB helicase stimulates primer synthesis activity on short oligonucleotide templates. Biochemistry 39:736-744.
16. Khopde, S., E. E. Biswas, and S. B. Biswas. 2002. Affinity and sequence specificity of DNA binding and site selection of primer synthesis by Escherichia coli primase. Biochemistry 41:14820-14830.
17. Kinashi H, Shimaji-Murayama M, Hanafusa T: Nucleotide sequence analysis of the unusually long terminal inverted repeats of a giant linear plasmid, SCP1. Plasmid 1991, 26(2):123-130.
18. Koepsell SA, Larson MA, Griep MA, Hinrichs SH: Staphylococcus aureus helicase but not Escherichia coli helicase stimulates S. aureus primase activity and maintains initiation specificity. J Bacteriol 2006, 188(13):4673-4680.
19. Koepsell, S. A., M. A. Larson, M. A. Griep, and S. H. Hinrichs. Staphylococcus aureus helicase but not Escherichia coli helicase stimulates S. aureus primase activity and maintains initiation specificity. J. Bacteriol. 2006. 188:4673-4680.
20. Larson, M. A., R. Bressani, K. Sayood, J. E. Corn, J. M. Berger, M. A. Griep, and S. H. Hinrichs. Hyperthermophilic Aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines. Nucleic Acids Res. 2008 36:5260-5269.
21. Lin YS, Kieser HM, Hopwood DA, Chen CW: The chromosomal DNA of Streptomyces lividans 66 is linear. Mol Microbiol 1993, 10(5):923-933.
22. Musialowski MS, Flett F, Scott GB, Hobbs G, Smith CP, Oliver SG: Functional evidence that the principal DNA replication origin of the Streptomyces coelicolor chromosome is close to the dnaA-gyrB region. J Bacteriol 1994, 176(16):5123-5125.
23. Salas M: Protein-primed DNA replication. Annu Rev Biochem 1991, 60:37-91.
24. Tabor, S., and C. C. Richardson. : Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7. Proc. Natl. Acad. Sci. U. S. A. 1981. 78:205-209.
25. Thirlway, J., and P. Soultanas.: In the Bacillus stearothermophilus DnaB-DnaG complex, the activities of the two proteins are modulated by distinct but overlapping networks of residues. J. Bacteriol. 2006. 188:1534-1539.
26. Thompson CJ, Fink D, Nguyen LD: Principles of microbial alchemy: insights from the Streptomyces coelicolor genome sequence. Genome Biol 2002, 3(7):REVIEWS1020.
27. Tseng, T. Y., D. N. Frick, and C. C. Richardson. : Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6. Biochemistry 2000. 39:1643-1654.
28. Volff JN, Viell P, Altenbuchner J: Artificial circularization of the chromosome with concomitant deletion of its terminal inverted repeats enhances genetic instability and genome rearrangement in Streptomyces lividans. Mol Gen Genet 1997, 253(6):753-760.
29. Yang CC, Huang CH, Li CY, Tsay YG, Lee SC, Chen CW: The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins. Mol Microbiol 2002, 43(2):297-305.
30. Yoda K., and T. Okazaki.: Specificity of recognition sequence of Escherichia coli primase.Mol. Gen. Genet. 1991 227:1-8.
31. Zhang R, Yang Y, Fang P, Jiang C, Xu L, Zhu Y, Shen M, Xia H, Zhao J, Chen T et al: Diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids. Appl Environ Microbiol 2006, 72(9):5728-5733.
論文全文使用權限:同意授權於2014-09-03起公開