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dc.contributor.advisorMing-Te Yangen_US
dc.contributor.authorLai, Jing-Yingen_US
dc.description.abstractPresence of PstI-like restriction-modification (R-M) system in Xanthomonas campestris pv. phaseoli strain 73 (Xcp73) was implied by the observation that DNA isolated from this organism was insensitive to PstI endonuclease digestion. Two recombinant plasmids, pEXPP3-2 and pEXPP3-15, that resistant to PstI cleavage were isolated from Xcp genomic library. The nucleotide sequence of 6000-bp insert of plasmid pEXPP3-15 was determined on both strands. Sequence analysis revealed five open reading frames (ORFs) that are transcribed in the same direction. The deduced amino acid sequence of the first ORF (ORF596) contained a conserved sequence motif NPP(F/Y) which is characteristic of N6-adenine methyltransferase. The deduced amino acid sequences of the secondary ORF (ORF364) encoding the putative endonuclease. Thus, the first ORF and the secondary ORF were designated as M. xphI and R. xphI, respectively. The in vivo and in vitro activities of M. XphI and R. XphI were demonstrated in this study. Results showed that both enzymes recognized the same 5'-CTGCAG-3' target sequence as PstI and the purified R. XphI cleaved at the 3'-end of the A nucleotide of the target sequence. At 684 bp downstream to the R. xphI, two ORFs (ORF88 and ORF275) flanked by inverted repeats (IR) were identified. The IRs were imperfect, 38 bp for IRL and 37 bp for IRR, with one gap and six mismatches of nucleotides. The initiating site of orf275 had an overlap of seven nt with the 3'-terminus of orf88 and was in frame —1 with respect to ORF88. Protein distance matrix analyses indicated that ORF88 and ORF275 belong to IS407 subgroup of the IS3 family and designated ORF88 as ORFA of ISXpI and ORF279 as ORFB of ISXpI, respectively. An orfA-orfB fusion protein generated by —1 translational frameshifting between the two reading frame was identified in vitro. The copy number of ISXpI in Xcp73 and the distribution of ISXpI-like sequence in X. campestris strains were also determined by Southern blot hybridization. tnpA and tnpR genes were found to locate on the 6 kb upstream DNA fragment from M. xphI gene and that are transcribed in the different direction from xphI restriction-modification system genes. Since the genes involved in transposition were found to locate at the flanking region of xphI R-M system, it was thus of interest to determine if the xphI R-M system could be translocated by the flanking transposase genes. This speculation was confirmed by conjugation and Southern blot analyses. During the course of this study, a highly homologous PstI-like R-M system (XveI) was isolated from X. campestris pv. vesicatoria#7-1. In order to explore the possibility of horizontal transfer of the R-M system between these two strains, Southern blot analyses with tnpA-tnpR, ISXpI, and xphI R-M system genes as probes were performed. Results showed that tnpA-tnpR and ISXpI were not simultaneously existed with the xphI R-M system genes in the detected X. campestris pv. vesicatoria strains. This indicated that transposition of xphI R-M system genes by its flanking mobile elements might only conduct in X. campestris pv. phaseoli but not in pv. vesicatoria.en_US
dc.description.abstract利用 PstI 切割作用, 進一步從 Xcp73 基因庫中篩選出 pEXPP3-2 與 pEXPP3-15 二個含有 PstI-like 限制修飾系統基因。經由 DNA 定序結果顯示, 在所篩選具有 6 kb DNA 嵌入片段的 pEXPP3-15 重組質體中, 包含了 5 個 ORF, 且轉錄方向均相同。根據胺基酸序列進行比對分析後發現,其中第一個 ORF (ORF596) 為一甲基轉移蛋白, 且經由 BglII 切割作用及胺基酸序列比對分析, 推測此一 ORF596 應屬於 N-MTase。 第二個 ORF (ORF364) 經比對分析後顯示其為一內切限制基因, 並將此二個 ORF 之基因分別命名為 M. xphI 與 R. xphI。 進一步經由生體外及生體內之實驗結果顯示, M. xphI 與 R. xphI 均可辨識 ?’- CTGCAG —3’" DNA 序列, 且 R. xphI 並可在 AG 的位置進行切割作用。 此結果與 PstI 甲基轉移及內切限制的特異性相同。 ORF88 與 ORF275 位於 R. xphI 基因停止密碼下游第 684 bp 的位置後, 且在 ORF88 基因之 5’ 端與 ORF275 基因之 3’ 端分別具有 38-bp 及 37-bp 之反對稱序列 (IRL 與 IRR), 且 IRR 與 IRL 間含有六個不同的鹼基對。 ORF275 之轉譯胺基酸序列則為相對於 ORF88 之 —1轉譯位移序列。進一步經由 DNA 序列分析及蛋白序列之演化分析顯示, ORF88 與 ORF275 應屬於 IS3 家族中之 IS407 次族群, 因此將 ORF88 命名為 ISXp1 之 ORFA, 而 ORF275 則命名為 ISXp1 之 ORFB。 同時經由蛋白表現偵測結果顯示, ISXp1 轉位子的確可經由此一轉譯序列位移, 產生 ORFA-ORFB 融合蛋白。 此外, 利用南方墨點法分析 ISXp1 之分佈情形, 結果顯示除了 X. campestris pv. phaseoli 菌株外, ISXp1 在大部分之 Xanthomonas 菌體內部並無大量分佈。 進一步分析 xphI 限制修飾系統基因上游之區域, 發現此限制修飾系統基因上游約 6 kb 左右之 DNA 片段發現包含有 轉位 (tnpA) 與 解離 (tnpR) 基因。 其轉錄方向與 xphI 限制修飾系統基因方向相反。 由於 xphI 限制修飾系統基因之上下游 DNA 片段中顯示均具有與轉位作用相關之轉位子基因, 因此推測 xphI 限制修飾系統基因可藉由轉位子之轉位作用, 而與轉位子同時轉位至其他 DNA 片段上, 而此推論也進一步經由南方墨點法分析得到驗證。 由於本實驗室在 X. campestris pv. vesicatoria 菌株中, 亦發現一與 XphI 限制修飾系統同質性非常高的 PstI 同功 (XveI), 為探討此二菌株之間的相關性, 利用 tnpA-tnpR 部分基因、 xphI 限制修飾系統部分基因與 ISXp1 轉位子基因當成探針, 針對 X. campestris pv. phaseoli (Xcp) 與 X. campestris pv. vesicatoris (Xcv) 菌體進行南方墨點法分析, 藉以偵測此二種不同小種間之差異。 其結果顯示在所偵測的 Xcv 菌株中, 其 xveI 限制修飾系統基因、 ISXp1 轉位子基因與 tnpA-tnpR 基因並不同時存在, 根據此實驗結果推測可能只在 Xcp 菌體內, xphI 限制修飾系統基因才可藉由其上游之轉位與解離之轉位作用, 將 xphI 限制修飾系統基因轉位至不同的 DNA 位置上。zh_TW
dc.description.tableofcontents封面 縮寫字對照表 中文摘要 英文摘要 前言 第一部份、限制修飾系統 第二部份、轉位因子(Tn;IS) 材料與方法 Ⅰ.材料 Ⅱ.實驗方法 第一部份、xphI限制修飾系統基因的篩選與特性之分析 第二部份、xphI限制修飾系統基因下游區域之分析 第三部份、xphI限制修飾系統基因上游區域之分析 結果與討論 第一部份、xphI限制修飾系統基因的篩選與特性之分析 第二部份、xphI限制修飾系統基因下游片段分析及特性之研究 第三部份、xphI限制修飾系統基因上游DNA片段分析及特性之研究 參考文獻 圖表 附錄zh_TW
dc.subjectrestriction-modification systemen_US
dc.subjectinsertion elementen_US
dc.titleMolecular Cloning and Characterization of xphI Restriction-Modification Genes and Its Flanking Sequences from Xanthomonas campestris pv. phaseolien_US
dc.titleXanthomonas campestris pv. phaseoli xphI 限制修飾系統基因及其上下游片段之選殖及特性研究zh_TW
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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