| الوصف: |
Circadian rhythms are self-autonomous rhythms generated at cellular level with a period close to 24 hours in constant conditions. They are present in diverse organism, from bacteria to mammals. Although they appear independently throughout evolution, the molecular bases governing them are conserved. The central circadian oscillator is composed of a transcriptional-translational feedback loop (TTFL), where the negative element inhibits directly the positive element, which promotes its transcription. In Neurospora crassa the negative element is the protein FREQUENCY, encoded by the gene frq, and the positive element the White Collar Complex (WCC), composed by White Chollar-1 (WC-1) and White Chollar-2 (WC-2). WC-1 is also a photoreceptor, allowing the incorporation of the light input to the clock. The rhythmic information is transmitted to the output pathways causing the oscillation of different biological processes, as metabolism and conidiation. This is mediated, in part, by a hierarchical arrangement of transcription factors; which allows the rhythmic expression of genes controlled by the clock (ccgs). To improve our knowledge in the genetic topological plasticity of the clock, in this thesis we used transcriptional rewiring technics, a synthetic biology approach, to generate new circuitry topologies of the central clock. We generate Hybrid Oscillators (HOs) changing the promoter of frq with the promoter of a ccg and evaluated the capacity of the system to generate and sustain rhythms even the TTFL circuit architecture is severely modified. Using this approach, we demonstrated the ability of the core oscillator of Neurospora to sustain rhythms, even when the evolutionary conserved core oscillator is dramatically modified. The HO with better performance was denominated HO-10. HO-10 displays circadian oscillations which are subjected to FRQ posttranslational dynamics and is temperature compensated. Interestingly, in HO-10 the light response and phase determination are different. We confirmed that HO-10 has an ... |
