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研究生:楊淳淳
研究生(外文):Yang, Chun-Chun
論文名稱:醱酵黴漿菌轉位子IS1550之轉位研究
論文名稱(外文):Transposition studies of IS1550 from mycoplasma fermentans
指導教授:胡文熙
指導教授(外文):Hu, Wen-Si
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:61
中文關鍵詞:生物技術醱酵黴漿菌
外文關鍵詞:BIOTECHNOLOGY
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先前在撥酵黴漿菌incognitus菌株中已找到一個IS-like因子(Hu,et al.,1990)。利用此一IS-like因子部分左端重覆序列及部分右端重覆序列作為引子(primer,一臨床分離到的撥酵黴漿菌M64菌株基因組DNA為模版(template),以PCR的方法找出與IS相關的因子,命名為IS1550。從找到的因子經序列分析可分為四型,其中一型可能為一個具有活性的拷貝(copy),因為它具有以下特性:(1)二個ORFs,ORF1和ORF2,且分別在0與-1 phase;(2)ORF2的氨基酸序列具細菌轉位月每(bacterial transposase)及反轉錄病毒插入月每(retrovirus integrase)兩者所持有的典型 DDEmotif;(3)具典型A6G訊息,並且在A6G下游可能有stem-and-loop的二級結構,可把ORF1和ORF2結合而為一大ORF。將具活性的IS1550嵌入質體 pSK+dLacK(ampicillin resistant,kanamycin resistant)的抗ampicillin基因位置以破壞抗ampicillin,將此一新構築的質體pSK|+IS1550轉形至大腸桿菌ISM 612菌株(Minion,et al.,1995)來探討轉位現象。此菌落能長在含ampicillin的LB盤子中,顯示IS1550從抗ampicillin基因轉出並且恢復其抗藥性。從南方轉漬法(southern blot)分析發現IS1550可在本身的質體中發生分子內的轉移(intramolecular transfer)並且轉移至抗kanamycin基因附近,導致刪除部分抗kanamycin基因。因此,本實驗所得到的轉仕現象可證明IS1550是一可移動的因子。
Until now, insertion sequence (IS) is present in differernt kind of bacteria even though the smallest genome size, mycoplasma. An IS-like element was previously identified from the incognitus strain of Mycoplasma fermentans (Hu, et al., 1990).Using the partial sequence of left terminal repeat and right terminal repeat of IS-like element, a primer set of oligonucleotides were synthesized to amplify IS-like variants from the clinical isolate M64 strain of Mycoplasma fermentans and now designated IS1550. From sequence analyses of IS1550, four types of IS- like elements were classified. One type named as consensus IS1550 is possible an active copy because of the following characteristics (1) two potential open reading frames (ORFs), ORF 1 and ORF 2 in phase 0 and -1 respectively; (2) ORF 2 containing DDE motif similar to the conserved domain which found in bacterial transposases and retroviral integrases and (3) a potential -1 frameshift signal A6G with downstream secondary structure to fuse ORF 1 and ORF 2. A copy of consensus IS1550 was inserted into ampicillin resistant gene in a plasmid pSK+dLacK with kanamycin resistant gene. This newly constructed plasmid pSK+IS1550 was transformed into Escherichia coli ISM 612 (Minion et al., 1995) to study transpositional activity. The bacterial colonies which able to grow in LB plate containing ampicillin indicated IS1550 was excised from ampicillin gene to recover this antibiotic resistance. Southern blot analysis showed the excised IS1550 has intramolecular transfer to the neighbor of kanamycin resistant gene and possible making deletion of this gene. Thus, these transposition events in this study prove that IS1550 is a mobile element.



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