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1.
Genes Dev. 2006 Jul 1;20(13):1776-89.

Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress.

Author information

1
Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California 94143, USA.

Abstract

The heat-shock response (HSR), a universal cellular response to heat, is crucial for cellular adaptation. In Escherichia coli, the HSR is mediated by the alternative sigma factor, sigma32. To determine its role, we used genome-wide expression analysis and promoter validation to identify genes directly regulated by sigma32 and screened ORF overexpression libraries to identify sigma32 inducers. We triple the number of genes validated to be transcribed by sigma32 and provide new insights into the cellular role of this response. Our work indicates that the response is propagated as the regulon encodes numerous global transcriptional regulators, reveals that sigma70 holoenzyme initiates from 12% of sigma32 promoters, which has important implications for global transcriptional wiring, and identifies a new role for the response in protein homeostasis, that of protecting complex proteins. Finally, this study suggests that the response protects the cell membrane and responds to its status: Fully 25% of sigma32 regulon members reside in the membrane and alter its functionality; moreover, a disproportionate fraction of overexpressed proteins that induce the response are membrane localized. The intimate connection of the response to the membrane rationalizes why a major regulator of the response resides in that cellular compartment.

PMID:
16818608
PMCID:
PMC1522074
DOI:
10.1101/gad.1428206
[Indexed for MEDLINE]
Free PMC Article
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2.
BMC Genomics. 2010 Jul 5;11:414. doi: 10.1186/1471-2164-11-414.

ChIP on Chip: surprising results are often artifacts.

Author information

1
Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital and University of Oslo, 0310 Oslo, Norway.

Abstract

BACKGROUND:

The method of chromatin immunoprecipitation combined with microarrays (ChIP-Chip) is a powerful tool for genome-wide analysis of protein binding. However, a high background signal is a common phenomenon.

RESULTS:

Reinvestigation of the chromatin immunoprecipitation procedure led us to discover four causes of high background: i) non-unique sequences, ii) incomplete reversion of crosslinks, iii) retention of protein in spin-columns and iv) insufficient RNase treatment. The chromatin immunoprecipitation method was modified and applied to analyze genome-wide binding of SeqA and sigma(32) in Escherichia coli.

CONCLUSIONS:

False positive findings originating from these shortcomings of the method could explain surprising and contradictory findings in published ChIP-Chip studies. We present a modified chromatin immunoprecipitation method greatly reducing the background signal.

PMID:
20602746
PMCID:
PMC2996942
DOI:
10.1186/1471-2164-11-414
[Indexed for MEDLINE]
Free PMC Article
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