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標題: Analysis of insertion sequences in Xanthomonas campestris pv. campestris
作者: 羅大鈞
Lo, Ta-Chun
關鍵字: Xanthomonas campestris pv. campestris
轉位子 IS476
出版社: 分子生物學研究所
摘要: Xanthomonas campestris pv. campestris (Xcc) is the causing agent of black rot of crucifies. Using a sucrose-sensitive plasmid as a baiting plasmid, our laboratory previously isolated 34 plasmids containing IS insertions. Among these plasmids, thirty were characterized and were found to have IS476, IS1478, IS1479 and IS1480 insertion in the plasmids. All but one IS476 insertion mutant has target duplications of 4-6 bases at the insertion sites. An IS476 insertion mutant does not have any target site duplication. This study demonstrates that pXC11-11 containing IS476 insertion carries a 952-bp duplication at the insertion site. Based on presence of both monomers and multimers of plasmids in Xcc11 from which the pXC11-11 mutant plasmid was originally isolated, a model is prepared herein to explain how this long direct repents are generated. The other four insertion sequence-containing plasmids were found to have IS1477, IS1481 and IS1482. Southern blot hybridization of twenty related bacteria indicates that six IS elements (IS476, IS1477, IS1478, IS1479, IS1480, IS1481) are present in multiple copies in the seven Xcc strains tested and two or three other pathovars of Xanthomonas campestris; IS1482 is present in a single copy in four of the seven Xcc strains tested and two other pathovars of Xanthomonas campestris. A spontaneous avirulent mutant (Xcc11A) of Xcc11 carries an extra 352-bp DNA fragment located upstream from an IS1478 in the chromosome. This study demonstrates that the same 352-bp DNA fragment is located six bases downstream from the same IS1478 in Xcc11. It is speculated that an IS1478 is precise-excised from the Xcc11 chromosome and inserted into a site 352-bp downstream from the excision site, thus generating Xcc11A. An Xcc11 2.6-kb EcoRI-BamHI DNA fragment including the 352-bp fragment was found capable of transforming Xcc11A into a strain pathogenic to turnip seedling. An orf (orfF) encoding a protein of 113 amino acid residues is found in the 2.6-kb sequence, which is interrupted by an IS1478 in Xcc11A. This indicates that the protein is pathogenicity-related. Its amino acid sequence shows low but significant homology with the amino acid sequence of the transcripation intiation factor IIF beta subunit (RAP30) of Xenous laevis.
Xanthomonas campestris pv. campestris (Xcc) 屬於革蘭氏陰性菌, 能引起十字花科植物的黑腐病。 實驗室曾以質體 pUCD800 分離到 34 個質體插入變異株, 其中30株已定性出分別有轉位子 IS476, IS1478, IS1479, 或 IS1480的插入。 30 株中有一株 (pXC11-11) 其插入片段具有 IS476 的逆向重複序列, 但不具 IS476的 4 bp 順向重複序列。 本研究證明 pXC11-11在插入的 IS476 的兩側有 952 bp 的順向重複序列, 其產生的原因為 IS476 轉位至一個 dimer 後又發生 adjacent deletion 的緣故。 本研究另外將 34 株中 4 株尚未分析的質體插入變異株定性, 發現分別為轉位子 IS1477, IS1481,IS1482 的轉位變異株。 以這七種 IS (IS476, IS1477, IS1478,IS1479,IS1480,IS1481,IS1482) DNA 片段做成探針, 對 二十種菌進行南方雜配分析。 發現除了 IS1482外的的六種轉位子, 皆以多套存在於七株測試的 Xcc , 及其他二或三個與 Xcc 同種但不同病原小種的染色體上。 但 IS1482 則僅在七株中的4株 Xcc 及其他兩株病原小種中偵測到有一套的存在。 本實驗先前証實在一株 Xcc 的致病株 (Xcc11) 與其自發性無致病力株 (Xcc11A) 的一套 IS1478 上游部份, Xcc11A 比Xcc11 多一段 352 bp DNA 片段。 本研究以 PCR 選殖二菌株此 IS1478 下游 DNA 片段, 並定序發現 Xcc11A 的下游比Xcc11 少一段 6 bp (5’-TAATAA-3’) 及352 bp 序列。 因此推測 Xcc11A 的 IS1478 為 Xcc11 中的 IS1478 往下游352 bp 轉位所造成的結果。 以 352 bp DNA 片段為探針自 Xcc11 的genomic library 選殖一段 13.5 kb 的 DNA 片段。 將其中的 2.6 kb DNA 片段做次選殖入一泛寄主載體中, 此質體可將 Xcc11A 互補成有病原性。 將此 2.6 kb Xcc11 DNA 片段定序後, 發現有一個 113 個胺基酸序列的開讀窗 (orf) , 而在 Xcc11A 中, 此開讀窗則被 IS1478 插壞。 因此, 此開讀窗轉譯出 113 個胺基酸序列之蛋白質應與 Xcc 之病原性有關。 此蛋白質序列和 Xenopus laevis 的transcription initiation factor IIF (RAP30) 有低但顯著的同質性。
Appears in Collections:分子生物學研究所



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