Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21822
標題: Stenotrophomonas maltophilia 染色體外DNA 之探討
Investigation of extra-chromosomal DNA from Stenotrophomonas maltophilia
作者: 黃証鴻
Huang, Cheng-Hang
關鍵字: phage;噬菌體;plasmid;repA;repB;ori;southern blotting;transformation efficiency;stenotrophomonas maltophilia;質體;複製蛋白質;複製起始區;南方轉漬法;轉型效率
出版社: 分子生物學研究所
引用: 參考文獻 張曉娟, 2004. Stenotrophomonas maltophilia 噬菌體 ΦSMA5 與 ΦSMT13 之分離與分析。國立中興大學分子生物學研究所碩士論文。 林晉玄, 2004. 十字花科蔬菜黑腐病菌小熱休克蛋白質 HspA 之功能探討及 Stenotrophomonas maltophilia 質體 pSM76 之定序與分析。國立中興大學分子生物學研究所碩士論文。 張昕淳, 2002. 十字花科蔬菜黑腐病菌的β-lactam類抗生素敏感性突變株 XKBLA 之分析。國立中興大學分子生物學研究所碩士論文。 Briani, F., Deho, G., Forti, F., and Ghisotti, D. 2001. The plasmid status of satellite bacteriophage P4. Plasmid 45:1-17. Bjerve, K. and Lindgvist, B. H. 1996. The N-Terminal Part of Bacteriophage P2 Capsid Protein Is Essential for Postassembly Maturation of P2 and P4 Capsids. Virology:224:568–572. Boethling R. S. 1975. Purification and properties of a serine protease from Pseudomonas matophilia. J Bacteriol. 121:933-941. Calendar, R. 1988. The bacteriophages. Vol. 2. Plenum, New York, USA. Chattoraj, D. K. 2000. Control of plasmid DNA replication by iterons:no longer paradoxical. Mol. Microbiol. 37:467-476. del Solar, G. and Espinosa, M. 2000. Plasmid copy number control:an ever-growing story. Mol. 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摘要: 
由培養 Stenotrophomonas maltophilia T13 的上清液中,分離出一株溫和型噬菌體 ΦSMT13。 電子顯微鏡觀察顯示, ΦSMT13具有一個外型是二十面體結構的頭部,直徑約 55 ~ 65 nm,並具有可伸縮的尾部構造。 ΦSMT13具有雙股 DNA,定序結果顯示,基因體全長為 33,525 bp,推測有 43個 ORFs。 為了了解 ΦSMT13 噬菌體 DNA 在噬菌體顆粒內、及在宿主體內存在的型式,利用含有噬菌體 cos site 區域的片段為探針,進行南方轉漬分析。結果顯示噬菌體DNA,在噬菌體顆粒內以線狀形式存在,而在菌體內可能以環狀形式存在。
pSM76 是由 S. maltophilia T76 分離獲得的質體。 序列分析後,推測帶有四個 ORF,其中 ORF1 與 ColE2-type 質體之 RepA protein 具有 54 % 的相似度, ORF2 與 Neisseria plasmid pJD1 之 RepB protein 具有 55 ~ 65 % 的相似度。 本實驗中確認此質體之 ori 位於 141 bp ( 935~1,075 bp) 區域內。
ORF1 與 ORF2 是質體複製所需,兩個 ORF 必須同時存在,質體才能進行複製。 此外,又將先前已構築好分別以Gmr 基因破壞ORF3 及ORF4 之質體,pSM76PG 及 pSM76SG,以電穿孔方式送入 S. maltophilia 10737及Xc17中,測試其轉形效率。 發現以 S. maltophilia 10737 為宿主的轉形效率略高於 Xc17,而pSM76SG 的轉形效率也略高於 pSM76PG。 ORF4產物是否與質體 copy number 的調控有關尚不清楚。 pSM76 的衍生質體應可應用於選殖、轉殖與表現基因於 S. maltophilia 菌株中。

ΦSMT13 is a temperate phage isolated from the supernants of S. maltophilia T13 culture media. The structure of phage ΦSMT13 under electron microscope revealed an icosahedron structure head approximately 55~65 nm in diameter and a contractable tail. ΦSMT13 genome is double stranded DNA and the size of the whole genome is 33,525 bp containing 43 putative open reading frames. The DNA fragment containing phage cos site region was used as a probe for southern blotting analysis to identify the phage DNA conformation in phage particle and host. The results indicated that phage DNA is a liner form in phage particle, and maybe circular form in the host.
Plasmid pSM76 is isolated from S. maltophilia T76 with four predicted open reading frames. The sequence of ORF1 is 54 % similar to RepA protein of ColE2-type plasmid, and ORF2 is 55 ~ 65 % similar to RepB protein of Neisseria plasmid pJD1. The pSM76 ori is located in nt 935 ~ 1,075 within the intergenic region between ORF4 and ORF1. ORF1 and ORF2 are essential for plasmid replication. Previously we have constructed plasmids pSM76PG and pSM76SG with the ORF3 and ORF4 sequence interrupted by Gm resistance gene, respectively. To test the transformation efficiency, pSM76PG and pSM76SG were transformed into S. maltophilia 10737 and Xc17 by electroporation. The efficiency of the host cell S. maltophilia 10737 is higher than that of Xc17, and the transfromation efficiency of pSM76SG is higher than pSM76PG. However, whether the gene product of ORF4 is related to the regulation of plasmid copy number was not clear. The results showed that the derivative of plasmids pSM76 can be used for possibly selection, transfromation, and gene expression for S. maltophilia.
URI: http://hdl.handle.net/11455/21822
其他識別: U0005-0906200711375700
Appears in Collections:分子生物學研究所

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