臺灣博碩士論文加值系統

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

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