GENETICS AND FUNCTION OF THE ARABIDOPSIS CIRCADIAN CLOC

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Circadian rhythms influence many physiological functions in humans and affect the efficacy, side effects, and toxicity of drug treatments. Disruption of daily rhythms can have significant deleterious effects, causing disorders such as chronic insomnia. A better understanding of circadian clocks will allow further optimization of many pharmacological treatments and improved therapies for sleep disorders. Studies in several model organisms have led to the identification of a number of genes involved in circadian timekeeping. However, no unequivocal clock genes have yet been cloned in higher plants. The aims of my research proposal are to: 1) identify new clock mutants in the model plant Arabidopsis, and 2) determine the function of several Myb-like transcription factors that have been implicated in circadian regulation in plants. Since overexpression of clock components perturbs circadian timekeeping, I will insertionally mutagenize plants using an 'activation tag' construct to drive gene overexpression. Flowering time is disrupted in Arabidopsis circadian mutants, allowing us to first screen for plants that flower either earlier or later than wild type plants (an easier assay than looking directly for circadian mutants). Plants with altered circadian rhythms will then be identified by examining the expression pattern of a clock-controlled luciferase reporter gene. After eliminating plants with altered photoreceptor-signalling pathways, circadian mutants will be cloned using the activation tag. These genes will be introduced into plants as transgenes to confirm their ability to perturb the clock when overexpressed. The functional activity of a family of seven Myb-like transcription factors implicated in the functioning of the plant clock will also be examined. Although mammalian Myb protein use two Myb domains to bind to DNA, these plant proteins each contain a single DNA binding domain, suggesting that they bind to DNA as homo- or heterodimers. The ability of these proteins to bind to themselves and to each other will be assessed by yeast two-hybrid analysis, and their consensus DNA-binding specificity determined by a PCR-based random oligomer-binding assay. The ability of these Myb proteins to transactivate putative target genes will also be assessed.