DEVELO~1 - TECHNICAL ADVANCE Development of Arabidopsis...

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Unformatted text preview: TECHNICAL ADVANCE Development of Arabidopsis whole-genome microarrays and their application to the discovery of binding sites for the TGA2 transcription factor in salicylic acid-treated plants Franc oise Thibaud-Nissen 1, *, Hank Wu 1 , Todd Richmond 2 , Julia C. Redman 1 , Christopher Johnson 3, , Roland Green 2 , Jonathan Arias 3, and Christopher D. Town 1 1 The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA, 2 NimbleGen Systems Inc., Madison, WI 53711, USA, and 3 University of Maryland, Baltimore County, Baltimore, MD 21250, USA Received 5 January 2006; revised 8 March 2006; accepted 15 March 2006. *For correspondence (fax 1 301 838 0208; e-mail Present address: Cellular Neurophysiology Section, Cellular Neurobiology Research Branch, IPR/NIDA/NIH/DHHS, Baltimore, MD 21224, USA. Present address: Center for Scientific Review, National Institutes of Health, Bethesda, MD 20892, USA. Summary We have developed two long-oligonucleotide microarrays for the analysis of genome features in Arabidopsis thaliana , in particular for the high-throughput identification of transcription factor-binding sites. The first platform contains 190 000 probes representing the 2-kb regions upstream of all annotated genes at a density of seven probes per promoter. The second platform is divided into three chips, each of over 390 000 features, and represents the entire Arabidopsis genome at a density of one probe per 90 bases. ProteinDNA complexes resulting from the formaldehyde fixation of leaves of plants 2 h after exposure to 1 m M salicylic acid (SA) were immunoprecipitated using antibodies against the TGA2 transcription factor. After reversal of the cross-links and amplification, the resulting ChIP sample was hybridized to both platforms. High signal ratios of the ChIP sample versus raw chromatin for clusters of neighboring probes provided evidence for 51 putative binding sites for TGA2, including the only previously confirmed site in the promoter of PR-1 (At2g14610). Enrichment of several regions was confirmed by quantitative real-time PCR. Motif search revealed that the palindromic octamer TGACGTCA was found in 55% of the enriched regions. Interestingly, 15 of the putative binding sites for TGA2 lie outside the presumptive promoter regions. The effect of the 2-h SA treatment on gene expression was measured using Affymetrix ATH1 arrays, and SA-induced genes were found to be significantly over-represented among genes neighboring putative TGA2-binding sites. Keywords: ChIP-chip, immunoprecipitation, microarray, TGA, transcription factor, Arabidopsis thaliana . Introduction Microarrays of complementary DNA, oligonucleotides or amplicons have been developed for expression analysis in Arabidopsis thaliana by many entities, including the Ara- bidopsis Functional Genomics Consortium (Wisman and Ohlrogge, 2000); Affymetrix (Redman et al. , 2004; Zhu and Wang, 2000); Operon (Zanetti et al. , 2005); a European con-...
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This note was uploaded on 08/01/2009 for the course 2231 22444 taught by Professor Park during the Spring '09 term at A.T. Still University.

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DEVELO~1 - TECHNICAL ADVANCE Development of Arabidopsis...

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