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Genome Scale Reverse Genetics Approaches for Novel Gene Discovery in Arabidopsis thaliana Circadian Clock Transcriptional Networks /

  • Author(s): Nelson, Jeffrey Douglas
  • et al.
Abstract

The rotation of the earth on its axis results in alternating light and temperature conditions that have guided the evolution of nearly all forms of life. The resultant biological timekeepers are known as circadian clocks. This internal 24-hour oscillator orchestrates critical aspects of growth, development, and metabolism to synchronize with the appropriate time of day and impart adaptive advantages. In the plant model organism Arabidopsis thaliana, a number of the molecular components of the circadian clock have been identified. However, critical nodes of this complex transcriptional network remain undiscovered. To address this deficiency, we created a collection of nearly every transcription factor encoded in the Arabidopsis genome. This transcription factor library platform allows us to conduct unbiased genome-wide screens for new genes functioning within the circadian clock. Using heterologous expression systems such as yeast one-hybrid and yeast two-hybrid, we have detected many protein-DNA and protein-protein interactions that indicate heretofore unrecognized regulatory connections. Initial investigations using an abridged transcription factor collection led to the discovery of CCA1 HIKING EXPEDITION (CHE) as a component of the Arabidopsis circadian clock. Further experiments with this platform are enabling the identification of a number of new circadian clock components, thereby greatly expanding our understanding of this genetic network and informing in -silico models of the clock. In addition, we have used the transcription factor library collection to conduct a systematic in-planta overexpression screen for novel regulators of the circadian clock. This approach has helped overcome many of the barriers to gene discovery associated with forward genetic screens. Proving the effectiveness of this method, we were able to identify members of the B-Box Zinc Finger protein family that display previously undiscovered robust circadian phenotypes when overexpressed. Functional genomic toolsets such as this transcription factor library are becoming essential to gaining a systems level understanding of complex biological processes

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