Circadian rhythmicity during prolonged chemostat cultivation of Neurospora crassa

Tralau, Tewes; Lanthaler, Karin; Robson, Geoff; Crosthwaite, Susan K.

doi:10.1016/j.fgb.2006.11.003

Cited by 8 publications

(10 citation statements)

References 62 publications

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“…Additionally, genes that are commonly used as reference genes in other organisms have been shown to be under clock control in N. crassa . In general, the actin gene is frequently used as a normalizer in gene expression studies [9] – [11] , though in most cases there is no mention of stability verification. Therefore, the overall objective of this study was to examine a suite of genes for the potential to serve as appropriate reference genes under a variety of environmental conditions during growth in N. crassa .…”

Section: Introductionmentioning

confidence: 99%

Selection and Evaluation of Reference Genes for Expression Studies with Quantitative PCR in the Model Fungus Neurospora crassa under Different Environmental Conditions in Continuous Culture

et al. 2014

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Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR) is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1) light/dark cycling: btl, asl, and vma1; (2) all-dark growth: btl, tbp, vma1, and vma2; (3) temperature flux: btl, vma1, act, and asl; (4) all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated), expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring gene expression changes during growth in bioreactors.

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Section: Introductionmentioning

confidence: 99%

Selection and Evaluation of Reference Genes for Expression Studies with Quantitative PCR in the Model Fungus Neurospora crassa under Different Environmental Conditions in Continuous Culture

et al. 2014

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“…3a ). The addition of polyacrylic acid (Junlon, average MW ~100,000) to the culture medium 26 31 and a second bioengineering technique termed “dynamic agitation” were required to disperse the fungal mats that formed in the reactor. The dynamic agitation method cycled the propeller periodically from 400 rpm to a high (1000 rpm) and a low (100 rpm) revolution rate ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…While these assays are designed for understanding the fungus in a liquid culture, some factors such as nutrient diffusion, localized pH gradients, and oxygen tension, are not well-controlled and the resulting environmental stressors could impact the circadian clock independently. These stressors should be controlled in order to determine if they are affecting the physiological state of the fungus in a way that influences the circadian clock 26 . This can be accomplished by cultivating N. crassa within bioreactors which allows for the constant removal of medium, cells, and waste material with the input of fresh medium/nutrients (termed “continuous culture”) 27 which minimizes potential changes in the culture medium.…”

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confidence: 99%

Suppressing the Neurospora crassa circadian clock while maintaining light responsiveness in continuous stirred tank reactors

Cockrell

Pirlo

Babson

et al. 2015

Sci Rep

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Neurospora crassa has been utilized as a model organism for studying biological, regulatory, and circadian rhythms for over 50 years. These circadian cycles are driven at the molecular level by gene transcription events to prepare for environmental changes. N. crassa is typically found on woody biomass and is commonly studied on agar-containing medium which mimics its natural environment. We report a novel method for disrupting circadian gene transcription while maintaining light responsiveness in N. crassa when held in a steady metabolic state using bioreactors. The arrhythmic transcription of core circadian genes and downstream clock-controlled genes was observed in constant darkness (DD) as determined by reverse transcription-quantitative PCR (RT-qPCR). Nearly all core circadian clock genes were up-regulated upon exposure to light during 11hr light/dark cycle experiments under identical conditions. Our results demonstrate that the natural timing of the robust circadian clock in N. crassa can be disrupted in the dark when maintained in a consistent metabolic state. Thus, these data lead to a path for the production of industrial scale enzymes in the model system, N. crassa, by removing the endogenous negative feedback regulation by the circadian oscillator.

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“…Clock-controlled genes (ccgs) are related to a myriad of cellular functions, including intermediary metabolism, protein synthesis and processing, chromatin changes, cellular signalling, and transcriptional regulation (Loros & Dunlap 2001;Vitalini et al 2006). Since these ccgs can feed back onto regulation of the clock itself it is not unexpected that specific biochemical and nutritional requirements must be met for a circadian rhythm to be properly revealed (Eisensamer & Roenneberg 2004;Ivleva et al 2005;Vitalini et al 2006;Tralau et al 2007). It is possible, therefore, that the absence of observed conidiation rhythms in T. atroviride and T. viride (Betina & Zajacová 1978;Deitzer et al 1988;Schrü fer & Lysek 1990) was due to a lack of knowledge of the appropriate environmental conditions and that conidiation is under the control of an endogenous rhythm in the genus Trichoderma.…”

Section: Resultsmentioning

confidence: 99%

Rhythmic conidiation in the blue-light fungus Trichoderma pleuroticola

et al. 2010

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Circadian rhythmicity during prolonged chemostat cultivation of Neurospora crassa

Cited by 8 publications

References 62 publications

Selection and Evaluation of Reference Genes for Expression Studies with Quantitative PCR in the Model Fungus Neurospora crassa under Different Environmental Conditions in Continuous Culture

Selection and Evaluation of Reference Genes for Expression Studies with Quantitative PCR in the Model Fungus Neurospora crassa under Different Environmental Conditions in Continuous Culture

Suppressing the Neurospora crassa circadian clock while maintaining light responsiveness in continuous stirred tank reactors

Rhythmic conidiation in the blue-light fungus Trichoderma pleuroticola

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