(3.238.186.43) 您好!臺灣時間:2021/02/25 02:10
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:李汶玲
研究生(外文):Lee, Wen-Ling
論文名稱:大腸桿菌中影響DNA複製的基因及其基因產物之特性分析
論文名稱(外文):The Characterization of the Gene Encoding a Protein that Affects the DNA Replication in Escherichia coli
指導教授:張敏政張敏政引用關係
指導教授(外文):Chang, Ming-Chung
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:75
中文關鍵詞:大腸桿菌DNA 複製priB 基因
外文關鍵詞:DNA replicationpriBEscherichia coli
相關次數:
  • 被引用被引用:0
  • 點閱點閱:229
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要

當人類開始對環境中的生物資源加以利用及改進,以謀人類之福祉,生物技術便逐漸發展開來。而其中,大腸桿菌 (Escherichia coli) 一直是生物技術廣泛使用的寄主細菌;由於它構造簡單,容易培養且繁殖迅速,因此在體外將改造後的基因在大腸桿菌內表現,或直接改造大腸桿菌的基因即成了了解生物體內分子機制的一項基本途徑。
之前,本實驗室發現一株E. coli XL1-Blue MRF¢的點突變株?m4(-),其突變地方是發生在基因priB上,並且造成轉錄出來的蛋白質PriB中第77個氨基酸由原本的Phe殘基突變成Val殘基。PriB (又稱為蛋白質n),最早是從引發體 (primosome) 中純化出來的,所謂引發體,是一個由七種蛋白質所組成,具有合成引發子 (primer) 功能的複合體。然而由於無法取得priB基因的突變株,因此其蛋白質產物PriB在生物體內的功能一直是不清楚的;因為m4(-)突變是發生在基因priB上,因此可讓我們直接觀察到此基因對大腸桿菌的影響。
在我們先前的研究發現:基因priB的突變會使得大腸桿菌無法有效地表現外來基因的活性;同時對於一些Drop的ColE1類型質體的複製數目也有大大降低的影響。由於在一般ColE1類型質體的DNA複製,是受RNAI與RNAII的調控,其中RNAII是複製所需的RNA引發子之前趨物,而RNAI則藉由其序列與RNAII的5'端互補,進而阻止RNA引發子的形成,如此調控質體的複製數目。若此類質體中又帶有蛋白質Rop,則Rop會穩定RNAI與RNAII的結合,使得RNAI的抑制效果更顯著。因此,Rop的存在是否會直接影響PriB的機能是目前研究的課題之一。
另外,我們也發現:(1)此一突變的PriB會抑制大腸桿菌m4(-)在44oC的生長,(2) PriB上另外的二個Phe殘基其突變對於大腸桿菌表現外來基因活性之能力,亦有相同於原本發現之突變所造成的情形。根據一些文獻指出:44oC是許多與RNA結合之蛋白質突變株的nonpermissive溫度;而與DNA或RNA結合有關的蛋白質其Phe殘基常扮演重要的角色。綜合上述的研究現象,我們推測PriB可能和參與DNA複製的RNA有調節上的關係。為了進一步了解PriB在菌體生長及質體複製中所扮演的角色,我們首先藉由ColE1類型質體的複製型態來作探討。
在我們的實驗中發現到,PriB可與合成ColE1類型質體的引發子之前驅物RNAII結合,同時,PriB也可與調控子RNAI結合,但並非扮演RNAI的代謝酵素角色。另外,當PriB上第42或者第77位置的Phe殘基發生了突變之後,則其對RNAI或RNAII的結合能力就有明顯減弱的趨勢。這些結果顯示著PriB確實具有結合RNA的能力,而其Phe殘基可能也扮演了重要的角色。根據這些現象我們推測:PriB可能藉著穩定RNA的構形進而維持適當的複製流程。所以更進一步,我們想藉由PriB結合RNA的功能加上PriB參與引發體的特性,以及Rop的存在對於PriB的影響效應,作一個完整的分析,以了解PriB對於體內複製型態的影響。

Abstract
Escherichia coli is widely used as a host-vector system to produce foreign protein in various biotechnological applications. In order to study the molecular mechanism of living cells, the recombinant genes may be transformed into the host, or the genes may be directly modified in vivo.

By UV irradiation mutagen, we obtained a mutant m4(-) derived from E. coli strain XL1-Blue MRF¢ with a mutation in gene priB. The mutated priB gene contains a point mutation and leads to a single amino acid substitution: a Phe to Val at position 77. The product of priB, initially called the n protein, was identified biochemically as one of the proteins which are necessary for the assembly of the DNA primosomal protein complex. The assembly of the primosome requires seven proteins to provide the priming activity for complementary strand DNA synthesis in initiation of fX174 bacteriphage DNA replication. However, no direct evidence for the role of PriB in replication in vivo has been described.

The priB mutation in the strain m4(-) significantly reduces both the copy number of the Δrop ColE1-type plasmids and the chitinase activity even though the mutant strain contains the ch structure gene. The copy number of E. coli plasmids of the ColE1 family is regulated by the formation of an RNA molecule that serves as primer for the enzyme DNA polymerase I. Negative regulation is attained by the interaction between the precursor of the primer and RNAI, a plasmid-encoded small RNA. The RNA-RNA interaction between the two regulating molecules is modulated by the protein Rop. Based on the mechanism, we hypothesize that PriB participates in the replication system, but is affected by Rop protein.

In our study, the mutant strain m4(-) does not grow well at 44℃, which is the nonpermissive temperature for some temperature-sensitive RNA binding proteins. Meanwhile, we find that the other Phe mutation in PriB protein cannot improve the low copy number of Δrop ColE1-type plasmids. There are several precedents in which phenylalanine residues have been implicated in both RNA and DNA binding. To study PriB function, we test the binding activity of PriB in the replication mechanism of ColE1-type plasmid.

Here, we find that the PriB protein can bind some RNAs transcribed from ColE1-type plasmid. However, the mutation of Phe-42 and Phe-77 in PriB protein causes defects in RNA binding activity. We also find that the RNA binding defects may not result from structur perturbation, but rather reflect those residues are in direct or indirect contact with the RNA. Further biochemical research is necessary to gain a more detailed understanding of the binding role of Phe residues in PriB protein during the control of plasmid replication in vivo.

目錄
頁碼
中文摘要 I
英文摘要 III
目錄 V
重要名詞縮寫 VII
第一章 priB的基因產物於大腸桿菌內功能上的探索 1
緒論 1
材料與方法 4
使用之菌株、載體及培養基 4
膠狀幾丁質之配製 5
製備少量的質體DNA 5
以鹼性去磷酸酵素處理載體 6
接合反應 7
大腸桿菌之形質轉換 7
聚合酵素連鎖反應擴增priB基因 8
SDS-PAGE 膠體電泳 9
北方點墨法 10
菌體外模擬轉錄實驗 15
結果 17
討論 21
第二章 Phe殘基在PriB蛋白質中影響其RNA結合能力
的探討 24
緒論 24
材料與方法 26
頁碼
使用之菌株、載體及培養基 26
以二次聚合酵素連鎖反應擴增點突變priB(-)基因 27
利用pET System純化蛋白質PriB 28
PriB抗體的製備 30
西方點墨法 30
結果 33
討論 39
第三章 正常的PriB蛋白質與其各個Phe殘基發生突變之
蛋白質結構上的探討 42
緒論 42
材料與方法 43
利用旋光光譜儀偵測蛋白質的二級結構 43
結果 44
討論 45
參考文獻 47
附表 51
附圖 54
誌謝
著作權聲明

參考文獻
1. Allen, G. C., Jr., and Kornberg, A. (1991). The PriB gene encoding the primosomal replication n protein of Escherichia coli. J. Biol. Chem. 266, 11610-11613.
2. Allen, G. C., Jr., and Kornberg, A. (1993). Assembly of the primosome of DNA replication in Escherichia coli. J. Biol. Chem. 268, 19204-19209.
3. Berges, H., Oreglia, J., Joseph-Liauzun, E., and Fayet, O. (1997). Isolation and characterization of a priB mutant of Escherichia coli influencing plasmid copy number of Drop ColE1-type plasmids. J. Bacteriol. 179, 956-958.
4. Burd, C. G., and Dreyfuss, G. (1994). Conserved structures and diversity of function of RNA-binding proteins. Science 265, 615-621.
5. Castagnoli, L., Scarpa, M., Kokkinids, M., Banner, D. W., Tsernoglou, D., and Cesareni, G. (1989). Genetic and structural analysis of the ColE1 Rop (Rom) protein. EMBO J. 8, 621-629.
6. Cesareni, G., and Banner, D. W. (1985). Regulation of plasmid copy number by complementary RNAs. Trends Biochem. Sci. 10, 303-306.
7. Cesareni, G., Muesing, M. A., and Polisky, B. (1982). Control of ColE1 DNA replication: the rop gene product negatively affects transcription from the replication primer promotor. Proc. Natl. Acad. Sci. USA 79, 6313-6317.
8. Chang, A. C. Y., and Cohen, S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134, 1141-1156.
9. Dnahan, O. (1983). Studies on transformation of E. coli with plasmid. J. Mol. Biol. 166, 557-580.
10. Eguchi, Y., and Tomizawa, J.-I. (1990). Complex formed by complementary RNA stem-loops and its stabilization by a protein: function of ColE1 Rom protein. Cell 60, 199-209.
11. Itoh, T., and Tomizawa, J.-I. (1980). Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc. Natl. Acad. Sci. USA 77, 2450-2454.
12. He, L., Soderbom, F., Wagner, E. G. H., Binnie, U., Binns, N., and Masters, M. (1993). PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids. Mol. Micro. 9, 1131-1142.
13. Lacatena, R. M., Banner, D. W., Castagnoli, L., and Cesareni, G. (1984). Control of initiation of pMB1 replication: purified Rop protein and RNAI affect primer formation in vitro. Cell 37, 1009-1014.
14. Lin-Chao, S., and Bremer, H. (1986). Effect of the bacterial growth rate on replication control of plasmid pBR322 in Escherichia coli. Mol. Gen. Genet. 203, 143-149.
15. Lin-Chao, S., and Cohen, S. N. (1991). The rate of processing and degradation of antisense RNAI regulates the replicaation of ColE1-type plasmids in vivo. Cell 65, 1233-1242.
16. Liu, J., and Parkinson, J. S. (1989). Genetics and sequence annnalysis of the pcnB locus, an Escherichia coli gene involved in plasmid copy number control. J. Bacteriol. 171, 1254-1261.
17. Liu, J., Nurcse, P., and Marians, K. J. (1996). The ordered assembly of the fX-174-type primosome. J. Biol. Chem. 271, 15656-15661.
18. Low, R. L., Shlomai, J., and Kornberg, A. (1982). Protein n, a primosomal DNA replication protein of E. coli. Purification and characterization. J. Biol. Chem. 257, 6242-6250.
19. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989). Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.
20. Masukata, H., and Tomizawa, J. (1984). Effects of point mutations on formation and structure of the RNA primer for ColE1 DNA replication. Cell 36, 513-522.
21. Masukata, H., and Tomizawa, J. (1986). Control of primer formation for ColE1 plasmid replication: conformational change of the primer transcript. Cell 44, 125-136.
22. Masukata, H., and Tomizawa, J. (1990). A mechanism of formation of a persistent hybrid between elongating RNA and template DNA. Cell 62, 331-338.
23. Merril, B. M., Stone, K. L., Cobianchi, F., Wilson, S. H., and Williams, K. R. (1988). Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotrin. J. Biol. Chem. 263, 3307-3313.
24. Morita, M., and Oka, A. (1979). The structure of a transcriptional unit of ColE1 plasmid. Eur. J. Biochem. 97, 435-443.
25. Ng, J. Y., and Marians, K. J. (1996a). The ordered assembly of the fX-174-type primosome. J. Biol. Chem. 271, 15642-15648.
26. Ng, J. Y., and Marians, K. J. (1996b). The ordered assembly of the fX-174-type primosome. J. Biol. Chem. 271, 15649-15655.
27. Predki, P. F., Nayak, L. M., Gottlieb, M. B. C. (1995). Dissecting RNA-Protein interactions: RNA-RNA recognition by Rop. Cell 80, 41-50.
28. Prigodich, R. V., Shamoo, Y., Williams, K. R., Chase, J. W., Konigsberg, W. H., and Coleman, J. E. (1986). 1H-NMR (500MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis. Biochemistry 25, 3666-3672.
29. Raumann, B. E., Rould, M. A., Pabo, C. O., and sauer, R. t. (1994). DNA recognition by b-sheets in the Arc repressor-operator crystal structure. Nature 367, 754-758.
30. Schekman, R., Weiner, J., Weiner, A., and Kornberg, A. (1975). Ten proteins required for conversion of fX174 single-stranded DNA to duplex form in vitro: resolution and reconstitution. J. Biol. Chem. 250, 5859-5865.
31. Schnier, J., Kitakawa, M., and Isono, K. (1986). The nucleotide sequence of an Escherichia coli chromosomal region containing the genes for ribosomal proteins S6, S18, L9 and an open reading frame. Mol. Gen. Genet. 204, 126-132.
32. Shimahara, K., and Takiauchi, Y. (1988). Preparation of crustacean chitin. Methods. Enzymol. 161, 417-423.
33. Sozhamannan, S., and Stitt, B. L. (1997). Effects on mRNA degradation by Escherichia coli transcription termination factor Rho and pBR322 copy number control protein Rop. J. Mol. Biol. 268, 689-703.
34. Tomizawa, J.-I. (1984). Control of ColE1 plasmid replication: the process of binding of RNA I to the primer transcript. Cell 38, 861-870.
35. Tomizawa, J.-I., and Itoh, T. (1981). The importance of RNA secondary structure in ColE1 primer formation. Cell 31, 575-583.
36. Tomizawa, J.-I., Ohmori, H., and Bird, R. E. (1977). Origin of replication of colicin E1 plasmid DNA. Proc. Nat. Acad. Sci. USA 74, 1865-1869.
37. Tomizawa, J.-I., and Som, T. (1984). Control of ColE1 plasmid replication: enhancement of binding of RNA I to the primer transcript by the Rom protein. Cell 38, 871-878.
38. Zavitz, K. H., Digate, R. J., and Marians, K. J. (1991). The PriB and PriC replication proteins of Escherichia coli. J. Biol. Chem. 266, 13988-13995.
39. Zavitz, K. H., and Marians, K. J. (1991). Dissecting the functional role of PriA protein-catalysed primosome assembly in Escherichia coli DNA. Mol. Microbiol. 5, 2869-2873.
40. 洪琬瑜 (1997) 大腸桿菌中影響質體複製的基因之選殖與特性分析,國立成功大學醫學院生物化學研究所碩士論文。

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔