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First published online May 8, 2003; 10.1105/tpc.009548

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The Plant Cell, Vol. 15, 1296-1309, June 2003, Copyright © 2003,
American Society of Plant Biologists

Regulatory Elements of the Floral Homeotic Gene AGAMOUS Identified by Phylogenetic Footprinting and Shadowing

Ray L. Hong1,a,b, Lynn Hamaguchia, Maximilian A. Busch2,a and Detlef Weigel3,a,c

a Plant Biology Laboratory, The Salk Institute for Biological Sciences, La Jolla, California 92037
b Department of Biology, University of California, San Diego, La Jolla, California 92093
c Department of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany

3 To whom correspondence should be addressed. E-mail weigel{at}weigelworld.org; fax 49-7071-601-1412

In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3-kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae species, several other motifs, but not the LFY and WUS binding sites identified previously, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally important for the activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection but also demonstrate that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.




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