RNA expression analysis using a 30 base pair resolution Escherichia coli genome array
RNA expression analysis using a 30 base pair resolution Escherichia coli genome array
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DOI：
10.1038/82367
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发表时间：
2000-12-01
影响因子：
46.9
通讯作者：
Church, GM
中科院分区：
文献类型：
--
作者：
Selinger, DW;Cheung, KJ;Church, GM
我们已经开发了一种高分辨率的“基因组阵列”，用于研究大肠杆菌中的基因表达和调控。该阵列在整个基因组上平均每30个碱基对含有一个25-mer寡核苷酸探针，基因间区域每6个碱基含有一个，4，290个开放阅读框（ORF）每60个碱基含有一个。在低至每个细胞0.2个信使RNA（mRNA）拷贝的水平下可以检测到两倍浓度差异，并且可以在三个数量级的动态范围内观察到差异。在丰富的培养基中，我们检测到97%和87%的ORF在稳定期和对数期，分别转录。我们发现在这些条件下有1，529个转录本差异表达。正如预期的那样，参与翻译的基因在对数期以较高水平表达，而许多已知参与饥饿反应的基因在稳定期以较高水平表达。许多以前未识别的生长期调控基因被确定，如一个假定的受体（b 0836）和30 S核糖体蛋白亚基（S22），这两个都是高度上调的稳定期。从反义链中观察到3，000至4，000个预测的ORF的转录，表明大部分基因组以可检测的水平转录。实例还介绍了高分辨率阵列分析的转录起始和终止位点和RNA二级结构。
We have developed a high-resolution "genome array" for the study of gene expression and regulation in Escherichia coli. This array contains on average one 25-mer oligonucleotide probe per 30 base pairs over the entire genome, with one every 6 bases for the intergenic regions and every 60 bases for the 4,290 open reading frames (ORFs). Twofold concentration differences can be detected at levels as low as 0.2 messenger RNA (mRNA) copies per cell, and differences can be seen over a dynamic range of three orders of magnitude. In rich medium we detected transcripts for 97% and 87% of the ORFs in stationary and log phases, respectively. We found that 1,529 transcripts were differentially expressed under these conditions. As expected, genes involved in translation were expressed at higher levels in log phase, whereas many genes known to be involved in the starvation response were expressed at higher levels in stationary phase. Many previously unrecognized growth phase-regulated genes were identified, such as a putative receptor (b0836) and a 30S ribosomal protein subunit (S22), both of which are highly upregulated in stationary phase. Transcription of between 3,000 and 4,000 predicted ORFs was observed from the antisense strand, indicating that most of the genome is transcribed at a detectable level. Examples are also presented for high-resolution array analysis of transcript start and stop sites and RNA secondary structure.