No Evidence That Homologs of Key Circadian Clock Genes Direct Circadian Programs of Development or mRNA Abundance in .

Many organisms harbor circadian clocks that promote their adaptation to the rhythmic environment. While a broad knowledge of the molecular mechanism of circadian clocks has been gained through the fungal model , little is known about circadian clocks in other fungi. belongs to the same class as many important plant pathogens including the vascular wilt fungus .

Mapping the bacterial ecology on the phyllosphere of dry and post soaked grass hay for horses.

Soaking hay fodder to reduce dust and soluble carbohydrate (WSC) contents prior to feeding is common practice among horse owners. Soaking can increase bacteria load in hay but no information exists on how this process alters the bacteria profile, which could pose a health risk or digestive challenge, to horses by introducing foreign bacteria into the gastrointestinal tract and so altering the normal profile. The current objectives were to map the bacterial profile of 3 different hays and determine how soaking alters this with the aim of improving best practice when feeding stabled horses.

Transcriptional interference by antisense RNA is required for circadian clock function.

Eukaryotic circadian oscillators consist of negative feedback loops that generate endogenous rhythmicities. Natural antisense RNAs are found in a wide range of eukaryotic organisms. Nevertheless, the physiological importance and mode of action of most antisense RNAs are not clear.

Natural antisense transcripts and long non-coding RNA in Neurospora crassa.

The prevalence of long non-coding RNAs (lncRNA) and natural antisense transcripts (NATs) has been reported in a variety of organisms. While a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. Here, we use RNA sequencing data from the ABI SOLiD platform to identify lncRNA and NATs obtained from samples of the filamentous fungus Neurospora crassa grown under different light and temperature conditions.

Comprehensive modelling of the Neurospora circadian clock and its temperature compensation.

Circadian clocks provide an internal measure of external time allowing organisms to anticipate and exploit predictable daily changes in the environment. Rhythms driven by circadian clocks have a temperature compensated periodicity of approximately 24 hours that persists in constant conditions and can be reset by environmental time cues. Computational modelling has aided our understanding of the molecular mechanisms of circadian clocks, nevertheless it remains a major challenge to integrate the large number of clock components and their interactions into a single, comprehensive model that is able to account for the full breadth of clock phenotypes.

Circadian timekeeping in Neurospora crassa and Synechococcus elongatus.

At first, the saprophytic eukaryote Neurospora crassa and the photosynthetic prokaryote Synechococcus elongatus may seem to have little in common. However, in both organisms a circadian clock organizes cellular biochemistry, and each organism lends itself to classical and molecular genetic investigations that have revealed a detailed picture of the molecular basis of circadian rhythmicity. In the present chapter, an overview of the molecular clockwork in each organism will be described, highlighting similarities, differences and some as yet unexplained phenomena.

Identification of macrodomain proteins as novel O-acetyl-ADP-ribose deacetylases.

Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD(+) to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways.

Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi.

A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNAs associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus.

Neurospora crassa heat shock factor 1 Is an essential gene; a second heat shock factor-like gene, hsf2, is required for asexual spore formation.

Appropriate responses of organisms to heat stress are essential for their survival. In eukaryotes, adaptation to high temperatures is mediated by heat shock transcription factors (HSFs). HSFs regulate the expression of heat shock proteins, which function as molecular chaperones assisting in protein folding and stability.

The PAS/LOV protein VIVID controls temperature compensation of circadian clock phase and development in Neurospora crassa.

Circadian clocks are cellular timekeepers that regulate aspects of temporal organization on daily and seasonal time scales. To allow accurate time measurement, the period lengths of clocks are conserved in a range of temperatures--a phenomenon known as temperature compensation. Temperature compensation of circadian clock period aids in maintaining a stable "target time" or phase of clock-controlled events.

Northern analysis of sense and antisense frequency RNA in Neurospora crassa.

In Northern analysis the presence of specific RNA transcripts is detected and their quantity can be estimated. RNA is separated using denaturing agarose gel electrophoresis and is subsequently transferred and fixed to a solid support, such as a nitrocellulose filter. When labeled probes are hybridized to these immobilized RNA molecules, their presence can be visualized by autoradiography.

Circadian rhythmicity during prolonged chemostat cultivation of Neurospora crassa.

Following exposure to light and attainment of steady-state in the chemostat, Neurospora was grown in constant conditions of darkness at 25 degrees C for 6 days. Biomass samples were taken every 4h for the extraction of RNA and protein, and the state of the circadian clock was assessed by assaying the levels of three rhythmically expressed mRNAs; frequency (frq), antisense frq (qrf) and clock-controlled gene-14 (ccg-14), and by monitoring the clock-controlled rhythm of sporulation. Our results indicate that the Neurospora clock continued to run in the chemostat.

Post-transcriptional control of nuclear-encoded cytochrome oxidase subunits in Trypanosoma brucei: evidence for genome-wide conservation of life-cycle stage-specific regulatory elements.

Trypanosomes represent an excellent model for the post-transcriptional regulation of gene expression because their genome is organized into polycistronic transcription units. However, few signals governing developmental stage-specific expression have been identified, with there being no compelling evidence for widespread conservation of regulatory motifs. As a tool to search for common regulatory sequences we have used the nuclear-encoded components of the cytochrome oxidase (COX) complex of the trypanosome respiratory chain.

Circadian clocks and natural antisense RNA.

Eukaryotes regulate gene expression in a number of different ways. On a daily and seasonal timescale, the orchestration of gene expression is to a large extent governed by circadian clocks. These endogenous timekeepers enable organisms to prepare for predictable environmental conditions from one day to the next and thus allow adaptation to a given temporal niche.

Role for antisense RNA in regulating circadian clock function in Neurospora crassa.

The prevalence of antisense RNA in eukaryotes is not known and only a few naturally occurring antisense transcripts have been assigned a function. However, the recent identification of a large number of putative antisense transcripts strengthens the view that antisense RNAs might affect a wider variety of processes than previously thought. Here we show that in the model organism Neurospora crassa entrainment of the circadian clock, which is critical for the correct temporal expression of genes and their products, is controlled partly by an antisense RNA arising from a clock component locus.

The Neurospora circadian clock: simple or complex?

The fungus Neurospora crassa is being used by a number of research groups as a model organism to investigate circadian (daily) rhythmicity. In this review we concentrate on recent work relating to the complexity of the circadian system in this organism. We discuss: the advantages of Neurospora as a model system for clock studies; the frequency (frq), white collar-1 and white collar-2 genes and their roles in rhythmicity; the phenomenon of rhythmicity in null frq mutants and its implications for clock mechanisms; the study of output pathways using clock-controlled genes; other rhythms in fungi; mathematical modelling of the Neurospora circadian system; and the application of new technologies to the study of Neurospora rhythmicity.

A brief history of circadian time.

Recent progress in clock research has revealed major molecular components in the mechanisms responsible for circadian time keeping in mammals. The first vertebrate clock mutation (tau) was discovered in the Syrian hamster more than a decade ago and, using the power of comparative genomics, this gene has now been cloned. We now know that tau is the mammalian homologue of a Drosophila circadian clock component (double-time) that plays an important role in regulating clock protein turnover.

Eukaryotic circadian systems: cycles in common.

Common regulatory patterns can now be discerned among eukaryotic circadian systems, extending from fungi through to mammals. Complexes of two distinct PAS domain-containing transcription factors play positive roles in clock-associated feedback loops by turning on classic clock proteins such as FRQ, PER and TIM. These in turn appear to act as negative elements, interfering with their own activation and thus giving rise to an oscillatory negative feedback loop.

Neurospora wc-1 and wc-2: transcription, photoresponses, and the origins of circadian rhythmicity.

Circadian rhythmicity is universally associated with the ability to perceive light, and the oscillators ("clocks") giving rise to these rhythms, which are feedback loops based on transcription and translation, are reset by light. Although such loops must contain elements of positive and negative regulation, the clock genes analyzed to date-frq in Neurospora and per and tim in Drosophila-are associated only with negative feedback and their biochemical functions are largely inferred. The white collar-1 and white collar-2 genes, both global regulators of photoresponses in Neurospora, encode DNA binding proteins that contain PAS domains and are believed to act as transcriptional activators.

The genetic and molecular dissection of a prototypic circadian system.

A great deal is known about this archetypal circadian system, and it is likely that Neurospora will represent the first circadian system in which it will be possible to provide a complete description of the flow of information from the photoreceptor, through the components of oscillator, out to a terminal aspect of regulation. In Neurospora the strongest case has been made for there being a state variable of clock identified (Hall, 1995), it has now been shown that light resetting of the clock is mediated by the rapid light induction of the gene encoding this state variable, and a number of defined clock-regulated output genes have been identified, in two of which the clock-specific parts of the promoters have been localized. In addition to the importance of these factoids themselves, our efforts towards understanding of this system has allowed the development of tools and paradigms (e.

Light-induced resetting of a circadian clock is mediated by a rapid increase in frequency transcript.

To understand how light entrains circadian clocks, we examined the effects of light on a gene known to encode a state variable of a circadian oscillator, the frequency (frq) gene. frq is rapidly induced by short pulses of visible light; clock resetting is correlated with frq induction and is blocked by drugs that block the synthesis of protein or translatable RNA. The speed and magnitude of frq induction suggest that this may be the initial clock-specific event in light resetting.

The genetic basis of the circadian clock: identification of frq and FRQ as clock components in Neurospora.

Genetic approaches to the identification of clock components have succeeded in two model systems, Neurospora and Drosophila. In each organism, genes identified through screens for clock-affecting mutations (frq in Neurospora, per in Drosophila) have subsequently been shown to have characteristics of central clock components: (1) mutations in each gene can affect period length and temperature compensation, two canonical characteristics of circadian systems; (2) each gene regulates the timing of its own transcription in a circadian manner; and (3) in the case of frq, constitutively elevated expression will set the phase of the clock on release into normal conditions. Despite clear genetic and molecular similarities, however, the two genes are neither molecular nor temporal homologues.