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RNA expression analysis using a 30 base pair resolution Escherichia coli genome array

Nature Biotechnology volume 18, pages 1262–1268 (2000)Cite this article

Abstract

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.

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Figure 1: False-color images of scanned E. coli genome array hybridized with a sample derived from a stationary-phase culture growing in LB.
Figure 2: Comparison of 2max (blue circle), median (red square), and average difference (green triangle) abundance metrics using Bacillus subtilis control RNAs.
Figure 3: The E. coli array can detect strand-specific transcription and can be used to identify (A) small untranslated RNAs, such as csrB, and (B) detection of a previously unidentified antisense RNA in the Rac prophage.
Figure 4: Determination of transcription starts of (A) lpp and (B) rpsO.

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Acknowledgements

We thank J. Edwards for improvements to the labeling protocol, D. Janse for sharing unpublished data, A. Derti and A. Petti for bioinformatics contributions, F. Lam for help with the fermentor, M. Mittmann for array design, P. Juels for impeccable computer tech support, W. Rindone and J. Aach for expression database support, B. Cohen, R. Mitra, M. Bulyk, P. Estep, M. Steffen, and the rest of the Church lab for the many helpful discussions and encouragement that made this work possible. We also thank the reviewers for significant improvements to the manuscript. This work was supported by grants from Aventis Pharma, Lipper Foundation, DOE, and NSF.

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Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, 02115, MA

    Douglas W. Selinger & George M. Church

  2. Harvard College, Cambridge, 02138, MA

    Kevin J. Cheung

  3. Affymetrix Inc., 3380 Central Expressway, Santa Clara, CA

    Rui Mei, Erik M. Johansson & David J. Lockhart

  4. Genomics Institute of the Novartis Research Foundation, 3115 Merryfield Row, San Diego, 92121, CA

    David J. Lockhart

  5. Laboratory of Genetics, University of Wisconsin, Madison, 53706, WI

    Craig S. Richmond & Frederick R. Blattner

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Correspondence to George M. Church.

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Selinger, D., Cheung, K., Mei, R. et al. RNA expression analysis using a 30 base pair resolution Escherichia coli genome array. Nat Biotechnol 18, 1262–1268 (2000). https://doi.org/10.1038/82367

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