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J. Biol. Chem., Vol. 275, Issue 38, 29672-29684, September 22, 2000
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From the We have used nylon membranes spotted in duplicate
with full-length polymerase chain reaction-generated products of each
of the 4,290 predicted Escherichia coli K12 open reading
frames (ORFs) to measure the gene expression profiles in otherwise
isogenic integration host factor IHF+ and IHF
Global Gene Expression Profiling in Escherichia coli
K12
THE EFFECTS OF INTEGRATION HOST FACTOR*,
,
, and Departments of Biological Chemistry and
¶ Microbiology and Molecular Genetics, College of Medicine,
§ Department of Ecology and Evolutionary Biology, School of
Biological Sciences, and Department of Chemical Engineering and
Material Sciences, School of Engineering, University of California,
Irvine, California, 92697
strains. Our results demonstrate that random hexamer rather than 3'
ORF-specific priming of cDNA probe synthesis is required for accurate measurement of gene expression levels in bacteria. This is
explained by the fact that the currently available set of 4,290 unique
3' ORF-specific primers do not hybridize to each ORF with equal
efficiency and by the fact that widely differing degradation rates
(steady-state levels) are observed for the 25-base pair region of each
message complementary to each ORF-specific primer. To evaluate the DNA
microarray data reported here, we used a linear analysis of variance
(ANOVA) model appropriate for our experimental design. These
statistical methods allowed us to identify and appropriately correct
for experimental variables that affect the reproducibility and accuracy
of DNA microarray measurements and allowed us to determine the
statistical significance of gene expression differences between our
IHF+ and IHF strains. Our results demonstrate
that small differences in gene expression levels can be accurately
measured and that the significance of differential gene expression
measurements cannot be assessed simply by the magnitude of the fold
difference. Our statistical criteria, supported by excellent agreement
between previously determined effects of IHF on gene expression and the
results reported here, have allowed us to identify new genes regulated
by IHF with a high degree of confidence.
*
This work was supported in part by National Institutes of
Health Grant GM55073 (to G. W. H.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a supplemental figure.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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