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The AGAMOUS gene of Arabidopsis thaliana is a homeotic gene involved in the development of stamens and carpels. This gene encodes a putative DNA-binding protein sharing a homologous region with the DNA-binding domains, MADS boxes, of yeast MCM1 and mammalian SRF. To examine the DNA-binding activity of the AGAMOUS protein, double-stranded oligonucleotides with random sequences of 40 bp in the central region were synthesized and mixed with the AGAMOUS MADS domain overproduced in Escherichia coli. Oligonucleotides which bound to the MADS domain were recovered by repeated immunoprecipitation with an antibody which recognizes the overproduced protein. From a comparison of the recovered DNA sequences, the consensus sequence of the high-affinity binding-sites for the AGAMOUS MADS domain was determined to be 5′-TT(A/T/G) CC(A/T)6GG(A/T/C)AA-3′. DNase I footprinting and methylation interference experiments showed that the MADS domain binds to this motif. Comparisons with the binding-site sequences of other MADS-box proteins revealed that the MCM1 binding-sites in a-mating type-specific promoters of Saccharomyces cerevisiae show similarities with the binding-site sequence of the AGAMOUS MADS domain. A synthetic MCM1 binding-site in the upstream region of the STE2 gene is recognized by the AGAMOUS MADS domain.