Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21860
標題: Stenotrophomonas maltophilia 噬菌體Smp14的基因體與蛋白體之探討
Investigation of genome and proteome of Stenotrophomonas maltophilia phage Smp14
作者: 陳芝融
Chen, Chiy-Rong
關鍵字: 噬菌體;phage;基因體;蛋白體;genome;proteome
出版社: 分子生物學研究所
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摘要: 
中文摘要
我們從不同醫院之廢液樣品中分離到14株可以感染Stenotrophomonas maltophilia之噬菌體,分別命名為 ΦSMA1 ~ ΦSMA8、Smp14、Smp36、Smp54、Smp104、ΦSMC14和 ΦSMC36,其中以溶裂型噬菌體 Smp14殺菌力最強。於電子顯微鏡下觀察Smp14外型,為屬於噬菌體分類中的Myoviridae。利用PFGE電泳分析Smp14基因體,得知其基因體大小約160 kb。Smp14 DNA除了可被限制酶MseI切割外,對其他多種限制酶具抗性。利用HPLC分析 Smp14 DNA上所包含鹼基種類,發現除了A、T、C、G和m4C外,另含有2種未知形式修飾之鹼基(非m5C或m6A)。將純化之噬菌體DNA以shotgun cloning搭配primer walking的定序策略,完成 Smp14基因體之定序。噬菌體 Smp14基因體大小為159,910 bp,基因體GC比為54%,推測 Smp14基因體具有20 tRNA基因,230 ORFs,其中70個ORFs與已知功能之蛋白質有同源性,40個ORFs與hypothetical proteins具同源性,其他120 ORFs為Smp14基因體上unique hypothetical genes。Smp14與T4-type噬菌體之結構蛋白相關基因和複製重組相關基因相似,根據 GP23構築之演化樹分析顯示Smp14屬於T4-type噬菌體之新亞群。Smp14基因體具有與核酸生成有關之基因,但未發現與核酸修飾相關之基因。此外, Smp14基因體上推測具有32個由T4-type σ55因子所辨識的晚期啟動子,藉由RT-PCR實驗證明gp4和gp48上游晚期啟動子之功能。於Smp14蛋白質體分析上,N端定序結果顯示 Smp14 GP23經post-translational processing後,產生不同程度修飾之蛋白質。配合蛋白質二維電泳與MALDI-TOF質譜分析,顯示 Smp14 GP23除了經post-translational processing外,亦可能藉由其他修飾作用產生不同分子量及不同pI值之蛋白質群。

Abstract
Fortheen Stenotrophomonas maltophilia lytic phages (named ΦSMA1 ~ ΦSMA8, Smp14, Smp36, Smp54, Smp104, ΦSMC14 and ΦSMC36) from sewage samples of varions hospitals were isolated. One of them, Smp14, carrying the best effect to lyse various strain of S. maltophilia cells was characterized. The results of electron microscopy showed that the morphology of Smp14 is similar to the members of family Myoviridae. We estimated that Smp14 has a genome size of about 160 kb by using PFGE analysis. Smp14 DNA is refractory to digestion by many restriction endonucleases except MseI. HPLC analysis results showed that phage Smp14 DNA contained A, T, C, G, m4C and two types of unknown modified deoxynucleosides otherthan m5C nor m6A. Purified phage DNA was used for genome sequencing via shotgun cloning strategy and primer walking. Phage Smp14 genome consists of 159,910 bp with a G + C content of 54%. It encodes 230 putative protein-encoding open reading frames (ORFs). Of the total 230 ORFs, 70 were similar to known proteins, 40 were similar to hypothetical proteins. The analyzed results showed that Smp14 genome contains 20 tRNA genes. The structural gene cluster and replication-recombination gene cluster of phage Smp14 showed the same organization with that of T4, and phylogentic analysis based on GP23 classified Smp14 into a novel single-membered T4-type subgroup. Smp14 genome has nucleotide metabolism-related genes but not nucleotide-modification gene. In addition, Smp14 genome contains 32 putative late promoters which are possibly recognized by phage-encoded T4-type σ factor. By RT-PCR analysis, we identified two late promoters which are located upstream of gp4 and gp48. Studying on Smp14 proteome, the results of GP23 N-terminal sequencing showed that Smp14 GP23 has different post-translational processed products. The MALDI-TOF data also showed that the protein modification mechanism maybe exist, causing the formation of various GP23 isoforms.
URI: http://hdl.handle.net/11455/21860
其他識別: U0005-2408200714280100
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