Biochemical and Molecular Characterization of The Gliadin Multigene Family: Endosperm-Specific Nuclear Binding Factors

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The gliadins, the prolamines of wheat, are the predominant proteins of endosperm tissue. Two gliadin genomic clones from common and diploid wheat have been isolated and their structures determined. Present studies now show that there are endosperm-specific nuclear factors that bind specifically to a gliadin promoter element conserved in several endosperm genes which Dr. Okita has termed the "CACA" box. In this grant he proposes to study the exact DNA sequence binding domain of the "CACA" and other gliadin-promoter binding activities by gel retardation, Southwestrn blotting, and DNase I footprinting assays. The effect of these DNA binding activities on transcription will be evaluated in vivo by electroporation of gliadin gene constructs complexed with the DNA binding activities into protoplasts of monocot suspension cultures or endosperm cells and/or by employing a heterologous in vitro transcription assay. The "CACA" binding factor will be extensively purified utilizing FPLC and DNA affinity chromatography. With the purified binding factor, antibody probes can be obtained to evaluate the tissue-specificity and developmental pattern of accumulation of the factor and eventually to mediated the isolation and molecular characterization of its gene. These studies will contribute to an understanding of how specific plant genes are controlled during plant development, and a determination of the molecular events which are responsible for this control. The gliadin genes are a highly polymorphic family of proteins, which as the major endosperm storage protein of wheat, are largely responsible for the nutritional quality of this crop.