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THE PLANT CELL, Vol 5, Issue 8 831-841, Copyright © 1993 by American Society of Plant Biologists
Dominant Negative Mutants of Opaque2 Suppress Transactivation of a 22-kD Zein Promoter by Opaque2 in Maize Endosperm Cells
E. Unger, R. L. Parsons, R. J. Schmidt, B. Bowen and B. A. Roth
Pioneer Hi-Bred International, Inc., Department of Biotechnology Research, Box 38, Johnston, Iowa, 50131
In maize endosperm, genes encoding the 22-kD zein class of storage proteins
are regulated by the OPAQUE2 locus. The Opaque2 (O2) protein shares
homology with the basic domain/leucine zipper class of transcriptional
activators. Using microprojectile bombardment, we have shown that O2 is
capable of transactivating a 22-kD zein promoter in maize endosperm
suspension cultures and in longitudinal sections of intact endosperm. Two
mutant forms of the O2 gene were constructed by deleting regions that
encode either the basic domain or the first 175 N-terminal residues of the
O2 protein. When either of these mutant O2 genes was coexpressed with
wild-type O2 in a maize endosperm expression system, O2-mediated
transactivation of the 22-kD zein promoter was inhibited specifically and
in a dose-dependent manner. Electrophoretic mobility shift assays and
immunoprecipitation studies indicated that the mutant O2 proteins form
heterodimers with wild-type O2 in vitro. The mutant lacking the basic
domain forms heterodimers with wild-type O2, which can no longer bind DNA.
In contrast, the product of the N-terminal truncation allele forms
homodimers and heterodimers with wild-type O2, both of which can still bind
DNA. Because the N-terminal region contains an activation domain, it is
likely that these latter complexes are deficient in transactivation.
Dominant negative inhibitors of gene expression, such as those constructed
here, provide an alternative to antisense RNA approaches for inactivation
of gene function in plants.
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