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1.
J Comput Biol ; 8(1): 37-52, 2001.
Article in English | MEDLINE | ID: mdl-11339905

ABSTRACT

We consider the problem of inferring fold changes in gene expression from cDNA microarray data. Standard procedures focus on the ratio of measured fluorescent intensities at each spot on the microarray, but to do so is to ignore the fact that the variation of such ratios is not constant. Estimates of gene expression changes are derived within a simple hierarchical model that accounts for measurement error and fluctuations in absolute gene expression levels. Significant gene expression changes are identified by deriving the posterior odds of change within a similar model. The methods are tested via simulation and are applied to a panel of Escherichia coli microarrays.


Subject(s)
Gene Expression , Models, Theoretical , Oligonucleotide Array Sequence Analysis/methods , Algorithms , Bayes Theorem , Escherichia coli/genetics , Gene Expression Profiling/methods , Models, Statistical
2.
Nat Biotechnol ; 18(12): 1262-8, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11101804

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.


Subject(s)
Escherichia coli/genetics , Gene Expression Profiling/methods , Genome, Bacterial , Oligonucleotide Array Sequence Analysis/methods , RNA, Messenger/metabolism , Escherichia coli/growth & development , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Open Reading Frames/genetics , RNA, Antisense/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/genetics , Transcription, Genetic
3.
Nucleic Acids Res ; 27(19): 3821-35, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10481021

ABSTRACT

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.


Subject(s)
Escherichia coli/genetics , Gene Expression Profiling , Genome, Bacterial , Oligonucleotide Array Sequence Analysis , Escherichia coli/drug effects , Genes, Bacterial , Heat-Shock Response , Isopropyl Thiogalactoside/pharmacology , Transcription, Genetic
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