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研究生:王惠貞
研究生(外文):wang, huei-jen
論文名稱:線狀噬菌體cf1t的基因轉錄機制與免疫因子的特性分析
論文名稱(外文):The transcription mechanism and the immunity determinant of the filamentous bacteriophage cf1t
指導教授:郭宗德郭宗德引用關係
指導教授(外文):Kuo, tsong-teh
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:79
中文關鍵詞:線狀噬菌體基因轉錄潛溶菌免疫
外文關鍵詞:cf1ttranscriptionlysogenimmunity
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cf1t 為線狀噬菌體cf的變種,感染柑橘潰瘍病菌後會將DNA插入細菌基因體中,抑制其他cf1t的感染,具有免疫作用。Cftv2為cf1t之突變株,可感染帶有cf1t之潛溶菌,使其失去免疫作用。北方轉印分析發現cf1t感染後,其PstI(0, 7308) - NcoI(1196)區域,在菌體內會產生2種轉錄產物-RNA e1與RNA e2,其中RNA e1是以負股DNA為模版,而RNA e2則以正股DNA為模版。對照cftv2 與cf1t此區域之DNA序列,發現cftv2在核苷酸771-819處有49bp的缺失,cftv2感染後不會轉錄RNA e2,RNA e1雖會產生但其轉錄起始點由G783往前移至G752,故知此區域為決定cf1t無法感染潛溶菌的所在。將此區域分段接入pUFR047質體中,送入柑橘潰瘍病菌中模擬cf1t潛溶菌,尋找潛溶菌中抑制cf1t感染的因子,發現RNA e1為主要的抑制調控因子,RNA e1中有一閱讀框63(ORF63),破壞此ORF63後仍具有抑制噬菌體cf1t的感染,所以ORF63不是cf1t潛溶菌中的抑制因子。RNA e1並不會抑制線狀噬菌體cf1t進入宿主後的一開始的負股DNA複製,而是使噬菌體DNA無法大量複製,它調控著噬菌體後期的DNA複製。將基因II上游cf1t免疫調控區接上luxAB,發現轉錄物e1會影響基因II啟動子的活性,而對於與其5’端重疊的轉錄物e2的啟動子無影響;轉錄物e1對其下游基因轉錄的調控,應是抑制噬菌體二次感染的重要因素。
The filamentous bacteriophage cf1t infects the bacterium Xanthomonas campestris pv. citri and set up a stable lysogenic state with its genome integrated into the host chromosome. Northern blot analysis with probes derived from various restriction fragments of cf1t replicative form DNA(RF DNA)has revealed the presence of five major phage-specific transcripts in infected cells. Four of these transcripts were shown to be derived from the region of the cf genome extending from gene II to gene VIII and are consistent with the cascade model of transcription proposed for Ff coliphage. Unlike the well-characterized Ff phage, in which only the minus strand of viral DNA serves as a transcription template, both strands of the RF DNA of phage cf1t appeared to be transcribed. From the isolation and characterization of a virulent mutant, cftv2, we report the first investigation into the mechanisms of the immunity established by the filamentous bacteriophage. The mutation in cftv2 enables the phage to produce plaques on lawns of cf1t-lysogen. The mutation was defined as a 49-nucleotide deletion located in a 0.59kb NcoI/KpnI fragment of cf1t RF DNA. Two messages, e1 and e2, transcribed from the immunity region of cf1t but in opposite directions, were detected. In cftv2, the 49-nucleotide deletion abolishes e2 transcription. The transcript e1 from the immunity region of cf was detected by RT-PCR in a cf1t-lysogenic host or by Northern blot in Xanthomonas cells that carry the immunity element. In cf1t-infected Xanthomonas carrying plasmids containing the immunity region that could confer immunity, the RF DNA and single-stranded DNA of cf1t were detected at a low level by Southern blot. A promoter reporter plasmid was constructed with the promoter region of phage cf1t fused to the luxAB reporter gene. Assays of luciferase activity show that the cf1t promoter is regulated in cells carrying immunity element which confer immunity. These observations suggest that the transcript e1 expressed in cf1t-lysogen acts as a riboregulator, which regulates the activity of the cf1t promoter and inhibits replication of superinfecting phage.
目錄………………………………………………………………………. I
圖目錄……………………………………………………………………. III
表目錄……………………………………………………………………. V
中文摘要…………………………………………………………………. VI
英文摘要…………………………………………………………………. VII
緒言………………………………………………………………………. 1
實驗材料與方法
一.菌種、噬菌體、質體及引子……………………………… ..11
二.藥品與酵素………………………………………………… ..14
三.培養基……………………………………………………………… ..15
四.方法
1.DNA的抽取、剪接與電泳分析……………………………………… ..15
2.大腸桿菌之轉型作用…………………………………………………..15
3.噬菌體之放大與力價的測定…………………………………………..16
4.噬菌體複製型…………………………………………………………..17
5.經cf感染後菌體的RNA之萃取……………………………………… ..18
6.柑橘潰瘍病菌之轉形作用……………………………………………..19
7.探針的製作……………………………………………………………..20
8.南方雜合反應…………………………………………………………..21
9.北方雜合法……………………………………………………………..23
10.聚合酶鏈反應……………………………………………………… ..24
11.DNA定序法……………………………………………………………..25
12.Primer extension………………………………………………… ..25
13.RT-PCR……………………………………………………………… ..27
14.細胞冷光酵素活性之測定………………………………………… ..28
結果
一.線狀噬菌體cf1t在柑橘潰瘍病菌中的轉錄情形
1.以北方轉印雜合法分析cf1t在柑橘潰瘍病菌中的轉錄……………..29
2.線狀噬菌體cf1t的雙向轉錄…………………………………………..33
3.線狀噬菌體cf1t轉錄機制……………………………………………..39
二.線狀噬菌體cf1t與突變株cftv2的比較
1.cf1t與cftv2 DNA序列的比較……………………………………… ..42
2.cf1t與cftv2 基因轉錄的差異………………………………………..49
三.cf1t潛溶菌的免疫調控
1.抑制cf1t二次感染的DNA片段……………………………………… ..52
2.RNA e1是一個RNA調節因子………………………………………… ..56
3.RNA e1與cf1t的基因體複製…………………………………………..59
4.RNA e1與cf1t基因轉錄………………………………………………..61
討論………………………………………………………………………..65
參考文獻…………………………………………………………………..71
附錄………………………………………………………………………..80
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