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Nucleic Acids Research, 1994, Vol. 22, No. 11 1948-1953
© 1994

MOLECULAR BIOLOGY

Sequence-specific interactions of a nuclear protein factor with the promoter region of a rice gene for {alpha}-amylase, RAmy3D

Shin-ichiro Mitsunaga*, Raymond L. Rodriguez1 and Junji Yamaguchi

Nagoya University, BioScience Center Chikusa-ku, Nagoya 464-01, Japan 1Department of Genetics, University of California, Davis CA 95616, USA

*To whom correspondence should be addressed at: Department of Life and Health Science, Joetsu University of Education, Yamayashiki 1, Joetsu, Niigata 943, Japan

Received May 2, 1994. Accepted May 6, 1994.

The expression of a rice gene for {alpha}-amylase, RAmy3D, in suspension-cultured cells is induced at the transcriptional level by the deprivation of sugars. Binding of a nuclear protein from suspension-cultured rice cells to the promoter region of the RAmy3D gene was studied by gel-retardation and DNase I footprinting assays. Gel-retardation assays indicated that a 358-bp fragment of the promoter region interacted specifically with a protein factor from suspension-cultured cells. DNase I footprinting analysis allowed us to define three protein-binding regions. Each of these protein-binding sequences contained the GCCG G/C CG motif, which is specifically present in the promoter region of the sugar-regulated gene, RAmy3D, for rice {alpha}-amylase and not in that of the gibberellin-regulated RAmy1A gene. Subsequent cross-competition experiments using gel-retardation assay and synthetic oligonucleotides showed that the GCCG G/C CG motifs directly mediated the binding of a nuclear protein. These observations are discussed in relation to expression of the gene for {alpha}-amylase in suspension-cultured cells.


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