Light-Dependent and Circadian Transcription Dynamics In Vivo Recorded with a Destabilized Luciferase Reporter in Neurospora

Abstract

We show that firefly luciferase is a stable protein when expressed at 25°C in Neurospora, which limits its use as transcription reporter. We created a short-lived luciferase by fusing a PEST signal to its C-terminus (LUC-PEST) and applied the LUC-PEST reporter system to record in vivo transcription dynamics associated with the Neurospora circadian clock and its blue-light photosensory system over the course of several days. We show that the tool is suitable to faithfully monitor rapid, but also subtle changes in transcription in a medium to high throughput format.

Transcriptional refractoriness is dependent on core promoter architecture

“…Construction of the vector containing frq-vvdLRE-luc PEST was performed in three steps. A pFH62-luc PEST plasmid 35 was used as parental plasmid. An AscI and a XbaI sites were introduced by site-directed mutagenesis up-and downstream, respectively, of the frq LRE.…”

“…5b). To facilitate quantification of the LP-induced transcription waves we chose a stable luciferase (t ½ B6-8 h) 35 . Luciferase levels accumulated for B1 h after the LP and then decreased rather slowly ( Supplementary Fig.…”

“…5c). Since the turnover of luc RNA is much faster than the turnover of luciferase 35 , the maximal luciferase levels (B1-2 h after the LP) are approximately proportional to a temporal integration of the number of mRNA molecules produced in response to the LP irrespective of the shape (peak heights versus width) of the transcription wave. As expected, vvd-luc was expressed at higher levels than frq-luc ( Supplementary Fig.…”

Tet-on, or Tet-off, that is the question: Advanced conditional gene expression in Aspergillus

“…In order to conduct gene oscillatory studies in Aspergillus spp., we decided to test two modified luciferase reporters, including a fungal codon optimized version luc (Gooch et al, 2008) and a reporter encoding a proline, glutamic acid, serine, and threonine (PEST) protein degradation sequence luc-PEST (Cesbron et al, 2013). We reasoned that these modifications would increase fluorescent intensity and decrease luciferase half-life, which is essential for accurate measurement of gene expression during oscillations.…”

“…A luc-PEST reporter has previously been used to quantify circadian rhythms in Neurospora crassa, where improved sensitivity enabled accurate reporting of oscillatory transcriptional patterns (Cesbron et al, 2013). In order to test if luc-PEST in A. niger was similarly sensitive, we simulated circadian oscillations by addition of Dox to growth medium, which was followed by a growth period of 5h and subsequent removal of Dox by washing cultures with fresh media.…”