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题目:: Characterization of transcription patterns of bacteriophage fRSL1 genes during infection cycle
ID:
状态:: 发布时间July 21, 2010 , 更新时间 March 27, 2012
物种:: Ralstonia phage RSL1, Ralstonia solanacearum
摘要:: fRSL1 is a jumbo myovirus stably and lytically infecting the phytopathogenic bacterium Ralstonia solanacearum. In this study, we investigate the infection cycle of fRSL1 and provide a genomic, proteomic and transcriptomic view of this phage. Its 231-kbp genome sequence showed many genes lacking detectable homologs in the current databases and was vastly different from previously studied phage genomes. In addition to these orphan proteins, fRSL1 was found to encode several enzymes that are unique among known viruses. These include enzymes for the salvage pathway of NAD+ and for the biosynthetic pathways of lipid, carbohydrate and homospermidine. A chitinase-like protein was found to be a potential lysis enzyme. Our proteomics analysis suggests that fRSL1 virions contain at least 25 distinct proteins. We identified six of them including a tail sheath protein and a topoisomerase IB by N-terminal sequencing. Based on a DNA microarray analysis, we identified two transcription patterns. Total RNA was isolated from 3 ml of fRSL1-infected MAFF730138 cells (containing 1 x 108 CFU infected at moi 0.5 ~ 4) at 0, 10, 30, 90 min, and 30 h post infection (p.i.) using an RNAprotect Bacteria Reagent kit (Qiagen K.K., Tokyo, Japan) according to the manufacturer’s protocol. The integrity and concentration of total RNA were measured using a bioanalysis unit (Agilent 2100 Bioanalyser, Aligent Tech. Palo Alto, CA). Fluorescence-labeled antisense RNA was synthesized by direct incorporation of Cy5-UTP (GE Healthcare Bio-Science Corp., Piscataway, NJ), using each RNA sample and an RNA Transcript SureLABEL Core kit (Takara Bio Inc.). The labeled antisense RNAs were hybridized simultaneously to the microarray chip. DNA microarray preparation, hybridization, processing, scanning, and analyses were performed by Filgen Inc. (Nagoya Japan). For each fRSL1 ORF, 1 to 5 coding sequences of 35 to 40 nucleotide-long were synthesized. A total of 1,100 sequences were put in duplicate on a 40 x 55 array chip. The fluorescence images of hybridized microarrays were obtained with a GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA). The Array-Pro Analyzer Ver4.5 (Media Cybernetics, Inc., Silver Spring, MD) was used to determine the signal intensity of each spot and its local background. Scan data images were alalyzed using Microarray Data Analysis Tool Ver3.0 software (Filgen Inc.)
实验种类:: transcription profiling by array
样本量:: 5
实验设计:
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数据号:: E-GEOD-23017, GSE23017
数据状态:: 无法自动分析，您可以尝试手动分析数据。

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