The objective of this proposal is to elucidate the role of two DNA-binding proteins in the development of a multicenter organism. ASF-1 and HSBF are tobacco factors which bind to a hexameric motif present in the upstream region of plant histone genes. This motif, TGACGT, is related to the cAMP-responsive element in mammalian systems. ASF-1 and HSBF have different sequence specificities as demonstrated by crosscompetition studies with different binding site probes. In transgenic tobacco, recent data suggest that the activity of a binding site specific for ASF-1 is induced by a plant hormone and is also developmentally-regulated. Tobacco cDNA clones that encode these two DNA-binding proteins have homologies to transcription factors containing the leucine-zipper motif. Northern analyses show that the expression of these genes is organspecific and developmentally-regulated. Both proteins are encoded by small multigene families. We want to characterize the regulation of the various members of these two gene families. Since each individual member may be regulated differently, we propose to isolate gene-specific probes from either the 5' or 3' non-coding region of each member of these two gene families. These probes will be used for nuclear run-on transcription assays to determine whether the genes are regulated at the transcriptional level, and to localize the mRNA of the particular gene to specific cell types by in situ hybridization. The distribution of the gene products will also be examined at the protein level by immune-detection techniques. The role of these gene products in plant development will be studied by alterations of their concentration in vivo through ectopic expression of the corresponding cDNA in transgenic tobacco.
