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Nucleic Acids Res. 2013 Sep;41(17):e164. doi: 10.1093/nar/gkt655. Epub 2013 Jul 26.

A genome-wide screen for identifying all regulators of a target gene.

Author information

1
Laboratoire Adaptation et Pathogénie des Microorganismes, Université Joseph Fourier, CNRS UMR5163, 38700 La Tronche, France and INRIA Grenoble-Rhône-Alpes, 38334 Saint Ismier Cedex, France.

Abstract

We have developed a new screening methodology for identifying all genes that control the expression of a target gene through genetic or metabolic interactions. The screen combines mutant libraries with luciferase reporter constructs, whose expression can be monitored in vivo and over time in different environmental conditions. We apply the method to identify the genes that control the expression of the gene acs, encoding the acetyl coenzyme A synthetase, in Escherichia coli. We confirm most of the known genetic regulators, including CRP-cAMP, IHF and components of the phosphotransferase system. In addition, we identify new regulatory interactions, many of which involve metabolic intermediates or metabolic sensing, such as the genes pgi, pfkA, sucB and lpdA, encoding enzymes in glycolysis and the TCA cycle. Some of these novel interactions were validated by quantitative reverse transcriptase-polymerase chain reaction. More generally, we observe that a large number of mutants directly or indirectly influence acs expression, an effect confirmed for a second promoter, sdhC. The method is applicable to any promoter fused to a luminescent reporter gene in combination with a deletion mutant library.

PMID:
23892289
PMCID:
PMC3783194
DOI:
10.1093/nar/gkt655
[Indexed for MEDLINE]
Free PMC Article

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