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DNA regions of the filamentous phage φLf required for autonomous replication and integration into the chromosome of Xanthomonas campestris pv. campestris
No full text摘 要
本實驗室前曾發現, 線狀噬菌體 φLf 的 RF DNA 之不同段落皆能
integrate 於宿主 Xanthomonas campestris pv. campestris
之染色體。 本實驗為釐清其 integration 機制, 將各個不同
的段落予以選殖之後, 分別送入 Xc17 之 recA 與 himA 突變株。
結果顯示 himA 基因非 integration 所需; recA 非 site-specific
integration 所需, 但卻為 homologous recombination 不可或
缺。 為了瞭解 φLf 的嵌入點, 本實驗在 Xc17 的染色體上
選殖到一段長達 4,445-bp 的 φLf-homologous 片段。 將之定序後,
發現有一部份序列與 φLf 相同。 其中包括 gene VIII, 與 attP 相同
的 attB 之 core region, 及疑似 IHF binding site 之
sequence 在內。 將該 φLf-homologous DNA 片段刪除之後,
會引起菌體之 deletion induced filamentation, 使菌體外表呈
細長線形。 此情形與 E. coli 之 dif 突變株類似。 經比對發現, Xc17
的 attB 之 core region 與 E. coli 之 dif site 序列具高
度同質性。 該 φLf-homologous 段落 經刪除後, 亦使 φLf
無法再嵌入, 顯示此 4,445-bp 片段是 homologous recombination
所需。 但若將 attB 之 core region 連同附近一小段 DNA (共 51 bp)
嵌回到 4,445-bp 被刪除之菌株後, 發現菌體可回復成
wild type 之表型, 且可供只含有 attP 之 φLf 段落
(72 bp) 嵌入。 顯示後者為 site-specific integration, 且所需之
integrase 基因不在 attB 附近。 而將 φLf 縮減到 72-bp 之後, 仍能
進行 integration 一節, 顯示 integrase 基因亦不
在噬菌體之基因體上。 此種情形與一般噬 菌體或質體嵌入宿主
染色體之情彷不同。
本實驗將 φLf 之 RF DNA 進行刪除, 得知 1,013-bp (nt 1386-2398)
長之片段即能 自主複製。 以 in trans 提供 gene II 功能之
互補試驗得知, φLf 的複製起始點係位於 gene II 的主導區內,
由 nt 1,591 至 1,711 的 121-bp 中。 實驗中並發現, φLf 之
gene II 能提供其功能, 使來自於 Tn903 的 kanamycin resistance
gene 能在 X. campestris 存活。 最後, 利用 φLf 之自
主複製片段 (nt 1,35-2,400) 構築了穿梭載體 p2GP, 供選殖基
因於 E. coli 及 X. campestris。Abstract
Different regions of the filamentous bacteriophage φLf were
previously found to integrate into the host Xanthomonas
campestris pv. campestris chromosome. In this study,
efforts were made to investigate the mechanisms involved
in the integration of φLf. The results indicated that himA,
the gene encoding the α subunit of integration host
factor (IHF), was not required for integration. The
recA gene was required for homologous
recombination, but not for site-specific integration. In order
to localize the sites for φLf integration, a 4,445-bp φ
Lf-homologous region was cloned from the host chromosome.
Sequence comparison showed high homology between the
fragment and φLf, including the gene encoding the major coat
protein, the attB carrying a 15-bp AT-rich core for φLf
integration, and the putative IHF binding site. Deletion of
this 4,445-bp region caused filamentation of the cells, a
consequence similar to dif deletion in E. coli which loses
normal partitioning of chromosome. In addition, after deletion
of the 4,445-bp fragment, φLf could no longer
integrate, indicating the φLf-homologous region
to be required for homologous recombination of φLf.
Replacement of the 4,445-bp fragment with a 59-bp fragment,
containing the attB core sequence of Xc17, restore to the
deletion mutant, a 72-bp segment including the attP core
sequence simultaneously the wild-type phenotype and the
ability to facilitate site-specific integration. These data
indicate that the integrase required for site-specific
integration of φLf is not located in the 4,445-bp
fragment of Xc17 chromosome or included in the φLf
genome. This situation is different from the site-specific
integration systems for phages and plasmids which
have integrase genes located near attP.
A 1,013-bp fragment (nt 1386-2398) of the φLf RF DNA containing
the gene II coding region was found to be maintained
autonomously as a minireplicon, and the replication origin
(ori) was localized in a segment of 121-bp (nt 1,591 to
1,711) within the gene II coding region, as assayed by providing
gene II function in trans. Gene II was able to provide
function for the kanamycin resistance gene from Tn903 to
replicate in Xc17. Finally, by cloning the autonomous
replicating fragment (nt 1,235-2,400) of φLf into pOK12, a
shuttle cloning vector, p2GP, was constructed which could
maintain in X. campestris and E. coli.封面
目錄
英文摘要
中文摘要
縮寫表
Ⅰ.前言
Ⅱ.結果與討論
第一部份: ΦLf嵌入Xc17染色體的模式之探討
一、ΦLf之不同片段均能嵌入於寄主染色體
二、以recA突變株(Xc17NT3)測試,ΦLf證明小 之片段大多係理由同質嵌入染色體
三、疑似IHF binding site之存在與否不影響ΦLf之定點嵌入
四、E.coli之IHF蛋白似無法與ΦLf的疑似IHF binding site結合
五、himA基因突變對ΦLf的site-specific integration及homelogous recombination均垂影響
六、himA基因突變降低ΦLf的複製效率
七、Xc17染色體上的ΦLf同質片段RV61之選殖
八、RV61片段之核酸定序與分析
九、ΦLf嵌入點之選殖
十、刪除Xc17染色體上與ΦLf同質之片段後,ΦLf片段即無法再嵌入
十一、叫除Xcl7染色體上與ΦLf同質之片段後造成茵體之線形化( filamentation )
十二、刪除attB區域後並不影響homologousre
十三、將attB序列嵌回染色體原來位置後可使菌體恢復正常外表型
十四、將attB序列在染色體上之位置更換後封菌體線形化之影響
十五、Site-spceific integration所需之integrase基因並不在ΦLfattP或染色體attB附近
第二部份: ΦLf複製區之研究
一、ΦLf之intergenic region( IR )
二、orfII2似與噬菌體之成熟(maturation)與釋放有關
三、ΦLfRFDNA之自主複製片段
四、ΦLf之複製起始點被包含在geneII的主導區內
五、ΦLf複製起始區之定位
六、ΦLffgeneII 蛋白之表現與功能
七、ΦLf之geneII可提供複製功能使pOK12及Km cartridge都能存活於X.campestris
八、穿梭載體p2GP之構築及應用
Ⅲ.材料與方法
一、茵種,噬茵體及質體
二、藥品、酵素及放射性同位素
三、培養基及緩衝溶液
四、抗生素濃度
五、DNA之製備
六、電泳分析
七、質體之構築(cloning)
八、細茵的轉形作用(transformation)
九、聚合脢連銷反應(polymerase chainreaction.PCR)
十、DNA-DNA雜配(DNA-DNA hybridyzation )
十一、IHF蛋白粗萃取液(crude extract )之製備
十二、蛋白質含量之測定
十三、膠體阻滯分析(gel rettardation assat)
十四、核甘酸定序(DNA sequencing )
十五、線狀噬菌體ΦLf之增殖(amplification)
十六、線狀噬菌體ΦLf濃度(titer) 之測定
十七、點測試法(spot test)
十八、活體外之轉錄及轉釋作用(in vitro transcription/translation coupled reaction)
Ⅳ.參考文獻
Ⅴ.已發表之論文
1. Lm. Nien-Tsung Bih-Yuh You, Chang-Yi Huang, Chin-Hsing Lin, Chung-Wen Kno, Fu-Shyan Wen and Yi-Hshmg Tseng. (1994). Characterization of two novel filamentous phages of Xanthomonas. J. Gen. ViroL 71:1881
2. Lin, Cheng-Sheng, Nien-Tsung Lin. Bih-Ying Yang, Shu-Fen Weng, and Yi-Hshing Tseng. (1995). Nucleotide sequence and expression of UDP-ghicose dehydrogenase gene required for the synthesis ofxanthan gum in Xanthomonas campestris. Biochem. Biophys. Res. Comm. 207:223
3. Lm. Nien-Tsung. Fu-Shyan Wen, and Yi-Hsiung Tseng- (1996) A region of the filamentous phage ΦLf genome that can support autonomous replication and mmiphage production. Biochem. Biophys. Res. Comm. 218:12-16
4. Lee, Tze-Ching, Nien-Tsung Lm and Yi-Hsiung Tseng. (1996) Isolation and characterization of the recA gene of Xanthomonas campestris pv. campestris. Biochem Biophys. Res. Comm. (In press)
5. Weng, Shu-Fen, Nien-Tsung Ln4 Yu-Fen Fan, Juey-Wen Lin, and Yi-Hsiung Tseng. (1996) Characterization of the 1.8-kb plasarid pXV64 from Xanthomonas campestris pv. vesicatoria. Bot. Bull Acad. Sin. (In press)
6. Lin. Nien-Tsung and Yi-Hshmg Tseng. (1996) A phagemid shuttle vector based on filamentous phage ΦLf and pOK12 for use in Xanthomonas campestris. Gene (accept after minor revision
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