Genome-wide expression profiling in Escherichia coli K-12
Genome-wide expression profiling in Escherichia coli K-12
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DOI：
10.1093/nar/27.19.3821
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发表时间：
1999-10-01
影响因子：
14.9
通讯作者：
Blattner, FR
中科院分区：
文献类型：
--
作者：
Richmond, CS;Glasner, JD;Blattner, FR
我们已经建立了高分辨率的方法，用于全球监测基因表达的大肠杆菌，杂交的放射性标记的cDNA斑点杂交尼龙膜上的荧光标记的cDNA杂交的效率和重复性进行了比较。从大肠杆菌K-12（MG 1655）的全基因组序列中为所有4290个注释的开放阅读框（ORF）创建一套完整的PCR引物，制备基于玻璃和尼龙的PCR产物阵列并用于评估基因表达的总体变化，通过两步PCR扩增产生用于阵列打印的全长编码序列。在这项研究中，我们测量了暴露于热休克后和用异丙基-β-D-硫代半乳糖苷（IPTG）处理后RNA水平的变化，放射性和基于荧光的方法都显示出相当的结果。用IPTG处理导致lacZYA和melAB操纵子的高水平诱导。在热休克处理后，119个基因显示出具有显著改变的表达水平，包括35个先前未表征的ORF和热休克刺激子的大多数基因。从杂交到重复阵列的斑点强度的分析鉴定出具有一致高于背景的信号的基因组，表明在丰富培养基中生长期间至少25%的基因以可检测的水平表达。
We have established high resolution methods for global monitoring of gene expression in Escherichia coli, Hybridization of radiolabeled cDNA to spot blots on nylon membranes was compared to hybridization of fluorescently-labeled cDNA to glass microarrays for efficiency and reproducibility. A complete set of PCR primers was created for all 4290 annotated open reading frames (ORFs) from the complete genome sequence of E.coli K-12 (MG1655), Glass- and nylon-based arrays of PCR products were prepared and used to assess global changes in gene expression, Full-length coding sequences for array printing were generated by two-step PCR amplification. In this study we measured changes in RNA levels after exposure to heat shock and following treatment with isopropyl-beta-D-thiogalactopyranoside (IPTG), Both radioactive and fluorescence-based methods showed comparable results. Treatment with IPTG resulted in high level induction of the lacZYA and melAB operons. Following heat shock treatment 119 genes were shown to have significantly altered expression levels, including 35 previously uncharacterized ORFs and most genes of the heat shock stimulon, Analysis of spot intensities from hybridization to replicate arrays identified sets of genes with signals consistently above background suggesting that at least 25% of genes were expressed at detectable levels during growth in rich media.