Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20904
標題: 線狀噬菌體 φLf 基因體上與嵌入及複製有關之段落
DNA regions of the filamentous phage φLf required for autonomous replication and integration into the chromosome of Xanthomonas campestris pv. campestris
作者: 林念璁
Lin, Nien-Tsung
關鍵字: filamentous phage;線狀噬菌體;Xanthomonas;replication;integration;嵌入;複製
出版社: 植物學系
摘要: 
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.
URI: http://hdl.handle.net/11455/20904
Appears in Collections:生命科學系所

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