Browsing by Author "Dunlap, Jay C."

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    High-resolution spatiotemporal analysis of gene expression in real time: In vivo analysis of circadian rhythms in Neurospora crassa using a FREQUENCY-luciferase translational reporter
    (2012) Larrondo, Luis F.; Loros, Jennifer J.; Dunlap, Jay C.
    The pacemaker of the Neurospora circadian clock is composed of a transcriptional-translational feedback loop that has been intensively studied during the last two decades. Invaluable information has been derived from measuring the expression of the central clock component frequency (frq) under liquid culture conditions. Direct analyses of frq mRNA and protein levels on solid media - where overt circadian rhythms are normally visualized - have not been trivial due to technical issues. Nevertheless, a frq promoter-luciferase reporter has recently allowed the study of frq transcription under these conditions. It is known that FRQ undergoes extensive posttranslational modifications, and changes in its levels provide important information regarding the clockworks. Here we describe a FRQ-luciferase translational fusion reporter that directly tracks FRQ levels, granting access to a better understanding and analysis of FRQ dynamics in vivo. More generally the method, which allows the investigator to follow continuous gene expression in real time in a spatially and temporally unrestricted manner, should be widely applicable to analyses of environmentally and developmentally regulated gene expression in ascomycete filamentous fungi as well as in basidiomycetes. (C) 2012 Elsevier Inc. All rights reserved.
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    Transcriptional rewiring of an evolutionarily conserved circadian clock
    (2024) Goity, Alejandra; Dovzhenok, Andrey; Lim, Sookkyung; Hong, Christian; Loros, Jennifer; Dunlap, Jay C.; Larrondo, Luis F.
    Circadian clocks temporally coordinate daily organismal biology over the 24-h cycle. Their molecular design, preserved between fungi and animals, is based on a core-oscillator composed of a one-step transcriptional-translational-negative-feedback-loop (TTFL). To test whether this evolutionarily conserved TTFL architecture is the only plausible way for achieving a functional circadian clock, we adopted a transcriptional rewiring approach, artificially co-opting regulators of the circadian output pathways into the core-oscillator. Herein we describe one of these semi-synthetic clocks which maintains all basic circadian features but, notably, it also exhibits new attributes such as a "lights-on timer" logic, where clock phase is fixed at the end of the night. Our findings indicate that fundamental circadian properties such as period, phase and temperature compensation are differentially regulated by transcriptional and posttranslational aspects of the clockworks.