Forward and reverse genetic approaches to unveil novel regulators of the Neurospora crassa circadian clock
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2019
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Abstract
All organisms in which circadian clocks have been characterized exhibit a common molecular circuit, which is based on a positive element able to activate the expression of a negative one that then represses the action of the former, inhibiting its own expression. This system is capable of sustaining oscillations under several external perturbations, but the compensation mechanisms remain unknown. Studies in mammals, Drosophila and Arabidopsis have revealed some new transcriptional inputs, maintaining the central clock circuitry, by modulating the expression of several of the core-clock components. In Neurospora crassa the core-clock central circuit has been well characterized but the participation of other associated transcriptional components remain unknown. In this chapter, we are trying to identify these novel transcriptional networks controlling the clock in N. crassa through a reverse genetic screen based on luminescent circadian reporters and a Neurospora Knockout collection of transcription factors. Thus, we have defined a set of 8 transcriptional regulators that modulate circadian features in our experimental conditions, which fall in a broad range of functions or processes. Finally, we selected the KO strain for the transcription factor for kpr-16 to defying its circadian behavior to environmental changes; this strain shown a remarkable period lengthening of ~2 hours and by literature are associated to temperature sensibility. We demonstrated that in absence of this transcription factor the Neurospora clock was unable to respond to temperature changes over the compensation temperature range, showing the further usefulness of this kind of approach.
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Tesis (Doctor en Ciencias Biológicas, mención Genética Molecular y Microbiología)--Pontificia Universidad Católica de Chile, 2019