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論文中文名稱:對鏈黴菌端粒複製所必需的Tac蛋白質會辨識結合SCP1質體端粒的單股DNA。 [以論文名稱查詢館藏系統]
論文英文名稱:A terminal patching required protein, Tac, recognizes the single-stranded DNA of SCP1 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:98
出版年度:99
中文姓名:潘信佑
英文姓名:Sin-You Pan
研究生學號:97688007
學位類別:碩士
語文別:中文
口試日期:2010-07-26
論文頁數:74
指導教授中文名:黃志宏
口試委員中文名:陳文盛;楊千金;陳月茸
中文關鍵詞:鏈黴菌末端蛋白末端伴隨蛋白SCP1
英文關鍵詞:StreptomycesSCP1terminal patchingTacTpc
論文中文摘要:鏈黴菌具有線型的染色體及質體,其共同的特徴之一都是端粒片段都是由廻紋序列組成的,並且在端粒DNA的5’端上有末端蛋白(TPs)以共價鍵的方式結合於其上。在多數鏈黴菌中端粒系統分為兩類,第一類的典型端粒系統的末端序列相似,及端粒上所鍵結的TP,以及將TP帶到端粒作用的Tap蛋白序列是相似的,第二類是非典型端粒系統,其末端序列則是不相似的,且對應的Tap及Tpg序列也是不相似的。先前的研究中指出非典型的SCP1端粒DNA序列,其對應的TP及Tap在酵母雙雜交的結果顯示SCP1的TP及Tap無交互作用,與其它已知鏈黴菌端粒複製的機制有所不同。為了進一步研究SCP1的末端複製機轉,所以先對Tpc(SCP1的TP)及Tac(SCP1上可能等同於Tap的蛋白質)做生化活性分析,我們首先分別重新建構在N端及C端帶有組胺酸標記的Tac及Tpc之融合蛋白,並且證實這兩個融合蛋白質於TK64中具有複製SCP1端粒複製的功能。相同的融合蛋白質轉到E.coli內大量表現並純化後,部份拿去產生抗體,其它的則以EMSA的方法檢測其對SCP1端粒的結合活性。目前實驗結果顯示Tac及Tpc都可分別與單股及雙股的SCP1端粒DNA結合,但Tac對單股SCP1端粒DNA是否具專一性結合能力仍然未知,而Tpc則可能因為PI值高,所以與DNA結合的能力強,因此不具有專一性的結合能力。
論文英文摘要:The linear replicons, including chromosomes and plasmids, of Streptomyces share common features that the palindrome-rich telomeric DNAs are covalently-bound with terminal proteins (TPs) at DNA’s 5’ ends. These telomeres show a replication intermediate with single-stranded 3’ overhang, and a telomere associated protein (Tap) was shown specifically binding on this single-stranded DNA and to recruited TP to here for further terminal patching. Comparison the telomeric DNA and their related TP and Tap, the telomeric DNA shows either the similar sequences and arranged palindromes with similar sequences of TP and Tap, or diverse sequences with unique TP and Tap. SCP1 owns unique telomeric DNA, capped Tac (TP of SCP1), and Tac which may work as Tap. Like the TP and Tap, Tac and Tpc are essential for terminal patching of SCP1. Remarkably, our previous studies showed no interaction between Tac (TP of SCP1) and Tpc (telomere associate protein of SCP1) by yeast two-hybrid system. For further studying the terminal patching of SCP1, the alternative strategy is to use biochemical assay of these two proteins. First, the his-tagged Tac and Tap were constructed and shown normal function in Streptomyces. These His tagged protein of Tpc and Tac purified from Escherichia coli displayed DNA binding activity with either single- or double-stranded DNA by gel mobility shift assay (EMSA). However, it is unknown whether Tac has the binding specificity on the single-stranded DNA of SCP1 telomere. The interaction of Tac and Tpc will be tested by immunoprecipitation.
論文目次:目 錄

中文摘要 …i
英文摘要 .….iii
誌謝 .….v
目錄 …..vi
圖目錄 ....viii
第一章 序論 ...…1
1.1 鏈黴菌簡介 ...…1
1.2 鏈黴菌的端粒DNA複製機制 ...…2
1.3 鏈黴菌端粒序列的差異 ...4
1.4 Tap及Tpg的生化功能……………………………………….. ...6
1.5 本論文的目的 .…10
第二章 材料與方法 ….12
2.1 菌種及質體 .…12
2.2 論文所需的引子 ….14
2.3 藥品、酵素………………………………………………………15
2.4 培養基及緩衝容液 ….15
2.5 菌種儲存…………………………………………………………15
2.6 大腸桿菌之轉型…..………………………………………………………16
2.7 大腸桿菌質體之分離與純化………………...…………………………16
2.8 鏈黴菌原生質體(protoplast)的製備與轉型…………...……..……16
2.9 鏈黴菌基因組DNA的分離與純化……………………………….……16
2.10 限制酶、T4系列酵素及各式Kit的處理與使用……………...…….16
2.11 聚合酶連鎖反應PCR(polymerase chain reaction)…………………16
2.12 誘導蛋白質表現……………………….…………………………………17
2.13 陽離子交換管柱純化方法……………..………………………………17
2.14 Refolding的測試……………….…………………………………………17
2.15 鎳離子交換管柱純化方法………………………………………17
2.16 Bradford assay……………………………………………………18
2.17 單股DNA的製備………………….……………………………18
2.18 Binding assay………………….…………………………………18
第三章 實驗結果…………………………………………………………19
3.1 測試Tac、Tpc的交互作用…………………………………...……19
3.2 Tac及Tpc的純化…………………………………………………31
3.3 Tac、Tpc與末端序列的交互作用……………..…………………48
第四章 討論………………………………………………………………55
參考文獻……………………………………………………………………59
附錄
Appendix A Media and buffer .63
Appendix B E.Coli Competent cell preparation and transformation ….67
Appendix C Plasmid isolation from E. coli ….70
Appendix D Preparation of Streptomyces protoplast and transformation……71
Appendix E. Isolation the total DNA of Streptomyces……………………73
Appendix F. Polymerase Chain Reaction……………………………….74
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論文全文使用權限:同意授權於2010-08-27起公開