A study will be made of plant nuclear genes encoding the chloroplast protei s ribulose-1, 5-biphosphate carboxylase (RBCS) and the polypeptide components of the light-harvesting chlorophyll a/b (CAB) complex. Cis-acting promoter elements mediating the transcriptional regulation of these photoregulated genes will be characterized in detail. The genes in question will include the rbcs-3A gene from Lycopersicon esculentum (tomato), the rbcS-1A gene fr m Arabidopsis thaliana, and the cab-E gene from Nicotiana plumbaginifolia (tobacco). These studies will be carried out by constructing chimeric gene containing RBCS and CAB promoter elements fused to reporter genes. These promoter elements will be manipulated in vitro by deletion and site-directe mutation. The expression of the chimeric genes will be examined in transgenic tobacco plants generated by Agrobacterium-mediated transformatio . Trans-acting factors that bind to these promoter elements will be further characterized. This will be achieved by band-shift assays, footprinting, a d protein purification. A previously-demonstrated role for protein phosphorylation in modulating binding of factors to promoter elements will be characterized. The nature of the DNA sequences and protein factors that mediate diurnal and circadian expression rhythms will be examined. Photoresponse mutants in tomato and Arabidopsis will be examined in order t determine if they lack expression of any individual RBCS or CAB genes. A transient expression assay will be developed in protoplassts so as to facilitate a rapid analysis of cis-acting promoter elements. The described studies will both contribute to a better understanding of the complexities of transcriptional regulation of gene expression and will assist in the development of novel methods by which the nutritional quality of food crops will be improved.
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