臺灣博碩士論文加值系統

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

Bai, M. S. 1989. Study on the insertion of filamentous phage DNA into the host chromosomes. Master thesis, Institute of botany, National Taiwan University.
Beck, E., and B. Zink. 1981. Nucleotide sequence and genome organization of filamentous bacteriophages f1 and fd. Gene 16:35—58.
Birnboim, H. C. and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids. Res. 7: 1513-1523.
Blumer, K. J., and D. A. Steege. 1989. Recognition and cleavage signals for mRNA processing lie within local domains of the phage f1 RNA precursors. J. Biol. Chem. 264:20770—20777.
Cashman, J. S., and R. E. Webster. 1979. Bacteriophage f1 infection of Escherichia coli: identification and possible processing of f1-specific mRNAs in vivo. Proc. Natl. Acad. Sci. USA 76:1169—1173.
Cashman, J. S., R. E. Webster, and D. A. Steege. 1980. Transcription of bacteriophage f1. The major in vivo RNAs. J. Biol. Chem. 255:2554—2562.
Chen, W. P., C. M. Cheng, A. H. J. Wang, and T. T. Kuo. 1996. Single-stranded DNA binding protein from bacteriophage cf: characterization, gene localization and protein-ssDNA complex. Biochim. Biophys. Acta 1309:147—155.
Cheng, C. M., H. J. Wang, H. J. Bau, and T.T. Kuo 1999. The primary immunity determinant in modulating the lysogenic immunity of filmentous bacteriophage cf. J. Mol. Biol. 287: 867-876.
Dai, H., K. S. Chiang, and T. T. Kuo. 1980. Characterization of a new filamentous phage cf from Xanthomonas citri. J. Gen. Virol. 46:277—289.
De Feyter, R., Y. Yang, and D. W. Gabriel. 1993. Gene-for-genes interactions between cotton R genes and Xanthomonas campestris pv. malvacearum avr genes. Mol. Plant Microbe Interact. 6: 225-237.
Edens, L., R. N. H. Konings, and J. G. G. Schoenmarkers. 1978. A cascade mechanism of transcription in bacteriophage M13 DNA. Virology 86:354—367.
Fann, J. H., M. H. Lee, and T. T. Kuo. 1989. Identification and nucleotide sequence of attachment site of the Cf1t filamentous phage from Xanthomonas campestris pv. citri. Chinese J. Microbiol. Immunol. 22:151—162.
Feinberg, A. P. and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonucleases fragments to high specific activity. Anal. Biochem. 132: 6-13.
Friedman, D. I., and D. L. Court. 1995. Transcription antitermination: the lambda paradigm updated. Mol. Microbiol. 18 : 191-200.
Ghisotti, D., F. Briani, F. Forti, F. Piassa, S. Polo, P. Sabbattini, T. Sturniolo, S. Terzano, S. Zangrossi, and M. Zappone. 1995. Mutiple regulatory mechanisms controlling phage-plasmid P4 propagation. FEMS Microbiol. Rev. 17, 127-134.
Gussin, G., A. Johnson, C. Pabo, and R. Sauer. 1983. Repressor and Cro protein: structure, function, and role in lysogenization, p. 93—122. In “Lambda II” (R. W. Hendrix, J. W. Roberts, F. W. Stahl, and R. A.Weisberg, Eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Heinrich, J., M. Velleman, and H. Schuster. 1995. The tripartite immunity system of phages P1 and P7. FEMS Microboil. Rev. 17, 121-126.
Horiuchi, K. 1997. Initiation mechanisms in replication of filamentous phage DNA. Genes Cells. 2: 425-32.
Katzen, F., A. Becker, A. Zorreguieta, A.Puhler, and L.Ielpi. 1996. Promoter analysis of the Xanthomonas campestris pv. campestris gum operon directing biosynthesis of the xanthan polysaccharide. J Bacteriol. 178: 4313-8.
Kuo, T. T., Y. S. Chao, Y. H. Lin, B. Y. Lin, L. F. Liu, and T. Y. Feng. 1987a. Integration of the DNA of filamentous bacteriophage Cf1t into the chromosomal DNA of its host. J. Virol. 61: 60-65.
Kuo, T. T., T. C. Huang, and T. Y. Chow. 1969. A filamentous bacteriophage from Xanthomonas orgzae. Virology 39: 548-555.
Kuo, T. T., Y. H. Lin, C. M. Huang, S. F. Chang, H. Dai, and T. Y. Feng.1987b. The lysogenic cycle of the filamentous phage cf1t from Xanthomonas campestris pv. citri. Virology 156: 305-312.
Kuo, T. T., C. C. Chiang, S. Y. Chen, J. H. Lin, and J. L. Kuo. 1994a. A long lytic cycle in filamentous phage Cf1tv infecting Xanthomonas campestris pv. citri. Arch. Virol. 135: 253-264.
Kuo, T. T., G. J. Shieh, J. L. Kuo, W. P. Chen, and H. J. Bau. 1994b. Xanthomonas campestris pv. citri infected with filamentous phage cf1: plaque turbidity, viral DNA integration, and immunity. Bot. Bull. Acad. Sin. 35:1—8.
Kuo, T. T., Y. H. Lin, C. M. Hung, S. F. Chang, H. Dai, and T. Y. Feng. 1987b. The lysogenic cycle of the filamentous phage Cf1t from Xanthomonas campestris pv. citri. Virology 156: 305-312.
Kuo, T. T., M. S. Tan, M. T. Su, and M. K. Yang. 1991. Complete nucleotide sequence of filamentous phage Cf1c from Xanthomonas campestris pv. citri. Nucl. Acids Res. 19:2498.
Lawn, A. M., E. Meynell, G. G. and N. Datta. 1967. Sex pili and the classification of sex factors in the Enterobacteriaccae. Nature 216: 343-346.
Lu, M. C. 1985. A filamentous bacteriophage fLf of Xanthomonas campestris pv. campestris. Master thesis. Institute of Botany, National Chung Hsing University.
Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Marvin, D. A., and B. Hohn. 1969. Filamentous bacterial viruses. Bacteriol. Rev. 33: 172-209.
Masukata, H., and J. I. Tomizawa. 1986. Control of primer formation for ColE1 plasmid replication: conformational change of the primer transcript. Cell 44:125—136.
Minamishima, Y., K. Takeya, Y. Ohnishi, and K. Amako. 1986. Physicochemical and biological properties of fibrous Pseudomonas bacteriophage. J. Virol. 2: 208-213.
Model, P., and M. Russel. 1988. Filamentous bacteriophage, p. 375—456. In “The Bacteriophages” (R. Calendar, Eds.), Vol. 2. Plenum, New York.
Nihimura, A., M. Morita, Y. Nishimura, and Y. Sugino. 1990. A rapid and highly efficient method for preparation of competent Escherichia coli cells. Nucl. Acids Res. 18: 6169.
Novick, R. P. 1998. Contrasting lifestyles of rolling-circle phages and plasmids. TIBS 23 : 434-438.
Oberto, J., and R. A. Weisberg. 1989. Structure and function of the nun gene and the immunity region of the lambdoid phage HK022. J. Mol. Biol. 207:675—693.
Peeters, B. P., R. M. Peter, J. G. G. Schoenmarker, and R. N. H. Konings. 1985. Nucleotide sequence and genetic organization of the N-specific filamentous bacteriophage Ike. J. Mol. Biol. 181: 27-39.
Sabine, B., and E. G. H. Wagner. 2000. Antisense RNA-mediated transcriptional attenuation: an in vitro study of plasmid pT181. Mol. Microbiol. 35: 1469-1482.
Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491.
Sambrook. J., E. F. Fritsch., and T. Maniatis. 1989. Molecular cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.
Shieh, G. J., C. H. Lin, J. L. Kuo, and T. T. Kuo. 1995. Characterization of an open reading frame involved in site-specific integration of filamentous phage Cf1t from Xanthomonas campestris pv. citri. Gene 158:73—76.
Shieh, G. J., Y. C. Charng, B. C. Yang, J. Tu, H. J. Bau, and T. T. Kuo. 1991. Identification and nucleotide sequence analysis of an open reading frame involved in high frequency conversion of turbid to clear plaque mutants of filamentous phage Cf1t. Virology 185:316—322.
Shieh, G. J. 1992. Studies on the plaque turbidity gene and the integration of phage DNA into host chromosome of filamentous phage cf1 from Xanthomonas campestris pv. citri. Doctor thesis, Institute of Botany, National Taiwain University, Taipei, Taiwan.
Simons, R. W. and N. Kleckner. 1988. Biological regulation by antisense RNA in prokaryotes. Ann. Rev. Genet. 22: 576-600.
Southern, E. M. 1975. Detection of a specific sequence among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.
Wang, C. N. 1996. Identification of the 5’ ends of filamentous phage cf transcripts. Master thesis, Institute of Botany, National Taiwan University.
Wang, H. J. 1995. Transcription in bacteriophage cf-infected Xanthomonas campestris pv. citri. Master thesis, Institute of Botany, National Taiwan University.
Wang, H. J., C. M. Cheng, C. N. Wang, and T. T. Kuo. 1999. Transcription of the genome of the filamentous bacteriophage cf from both plus and minus DNA strands. Virology. 256: 228-232.
Weng, S.F., M. Y. Shieh, F. Y. Lai, Y. Y. Shao, J. W. Lin, and Y. H. Tseng. 1996. Construction of a broad-host-rang promoter-probing vector and cloning of promoter fragments of Xanthomonas campestris. Biochem. Biophys. Res. Commun. 228: 386-390.
Wu, W. P. 1998. Regulation of the recA gene expression in Xanthomonas campestris pv. citri. Master thesis, Institute of Biology, Fu-Jen University, Taipei, Taiwan.
Yang, M. K., H. M. Huang, Y. C. Yang, and W. C. Su. 1995. Molecular cloning and expression of the coat protein genes of Cf, a filamentous bacteriophage of Xanthomonas campestris pv. citri. Bot. Bull. Acad. Sin. 36:207—214.
Yang, M. K., W. C. Su, and T. T. Kuo. 1991. High efficient transfection of Xanthomonas campestris by electroporation. Bot Bull. Acad. Sin. Taipei 32: 197-203.
Zaman, G. J.R., A. M. Kaan, J. G. G. Schoenmakers, and R. N. H. Konings. 1992. Gene V protein-mediated translation regulation of the synthesis of gene II protein of the filamentous bacteriophage M13: a dispensable function of the filamentous-phage genome. J. Bacteriol. 174: 595-600
Zinder, M., and K. Horiuchi. 1985. Multiregulation element of filamentous bacteriophage. Microbiol Rev. 49: 101-106
Zuker, M. 1989. On finding all suboptimal folding of an RNA molecule. Science 244: 48-52.