This proposal concerns the mechanism by which gene expression is regulated by light. The experimental organisms is the plant Arabidopsis thaliana, well known for its merits concerning molecular genetic analyses. The proposed studies concern both the analyses of light- response-elements (LREs) that characterize light-regulated genes and the characterization of a family of protein molecules (the G-box-binding factors or GBFs) that bind to a common element of these LREs. In addition, the mechanism by which irradiation with light modifies the GBFs will be examined. Higher eukaryotes are characterized by genetic systems that respond in complex ways to developmental and environmental stimuli. One component of this complexity is the common occurrence of gene families, the members of which may perform related but distinct functions. An example of such complexity is the Fos/Jun family of transcription factors and the role that this bZIP family of proteins plays in mammalian development and oncogenesis. The GBF family are also bZIp proteins and GBF4, as with Fos, forms heterodimers with the other family members but fails to homodimerize. Both families are characterized by intrafamily genetic regulatory systems. It is proposed to use the power of molecular genetic analyses in Arabidopsis to explore the properties of the GBF family of proteins. The essential features of LREs will be examined by studying the expression of promoter-reporter constructs in both transgenic plants and transient assay systems. The expression of these constructs will be compared with the expression of endogenous Arabidopsis genes. A large fraction of the GBF proteins are found in the cytoplasm. The nature of the GBF sequences that affect the intracellular distribution of these proteins will be determined. The DNA binding properties of GBF are influenced by light. The nature of this modification, apparently involving phosphorylation, will be examined: this study will include a determination of the kinetics of this reaction, the nature of the light requirements, and the GBF sequences that are modified. For these studies, as with other proposed studies, monoclonal antibodies will be obtained and used to distinguish the individual GBF family members. Transcription systems are characterized by very specific protein-protein interactions. In order to further understand the role of the GBF family of proteins in light-regulated gene expression, proteins that interact with this family of bZIP proteins will be selected via the use of a genetic screen in yeast. Two approaches will be used to evaluate the function of the individual GBF family members. A direct PCR-based screen will be used to search for Arabidopsis plants containing GBF genes tagged by T-DNA insertion. In addition, the use of transgenic plants expressing GBF-antisense constructs is being explored. Preliminary results from this latter approach indicate a complex intrafamily regulatory network. Based on the results from these antisense experiments, a novel complementation system is proposed whereby it may be possible to explore the distinguishing features of the GBF family members.
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