This proposal addresses a novel form of gene regulation in eukaryotes by asking how a circadian clock regulates the transcription of specific plant genes. The study of clock-regulated transcription will provide an important insight into molecular mechanism of the clock itself, which currently remains a puzzle. Arabidopsis thaliana has been chosen as our model system, because this plant offers numerous advantages for molecular genetic studies. Transcription of the three Adrabidopsis Cab genes is differentially regulated by a circadian clock. The basis for this difference will be investigated by analyzing the cis- and trans-acting factors that mediate the circadian response. This will entail detailed in vitro analysis of specific DNA-protein interactions using nuclear protein extracts prepared from different time-points during the cycle. The in vivo relevance of these interactions will be assessed by analysis of sites-specific constructs in transgenic plants. In addition, novel circadian- regulated marker genes will be identified and characterized by differential and subtractive hybridization. Knowledge of how transcription is regulated by cellular clocks will provide insight into a novel form of gene regulation in plants and will, perhaps, provide information which can be extrapolated to other organisms.
