Rhythmic conidiation in Neurospora crassa.

In the filamentous fungus Neurospora crassa the production of asexual spores (conidia) is regulated by its circadian clock. When the fungus is grown on a thin layer of agar medium in long growth tubes (so-called "race tubes"), restricting its growth to one direction only, bright orange bands are clearly visible. This banding pattern persists with a periodicity of approx 24 h in the absence of any environmental stimuli. The bands are formed by alternating zones of nonsporulating mycelium and mycelium laden with orange conidia. Assaying Neurospora conidiation on race tubes is a simple yet powerful and versatile tool for studying the circadian clock of this model organism.

Isolation of total RNA from Neurospora mycelium.

In filamentous fungi, including the model organism Neurospora crassa, plentiful biological tissue from which RNA can be extracted may be obtained by allowing fungal spores to germinate and form a mycelium in liquid culture. The mycelium constitutes a mosaic of multinuclear, tubular filaments known as hyphae or mycelia. In general, when exposed to air, fungal hyphae quickly start to develop spores, which are often colorful. However, when submerged in liquid under rapid agitation large amounts of vegetatively growing mycelium can be obtained, which can be easily harvested by means of filtration. To preserve the physiological state of the culture, the mycelium is snap-frozen, and then to free its contents, the mycelium is ground under liquid nitrogen to break all hyphal structures. Here a method to extract high-quality total RNA from Neurospora mycelium using TRIzol reagent is described.

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. Here we describe Northern hybridization using radioactively labeled riboprobes to show circadian expression of endogenous sense and antisense frequency RNA in the filamentous fungus Neurospora crassa.

Annotation of environmental OMICS data: application to the transcriptomics domain.

Researchers working on environmentally relevant organisms, populations, and communities are increasingly turning to the application of OMICS technologies to answer fundamental questions about the natural world, how it changes over time, and how it is influenced by anthropogenic factors. In doing so, the need to capture meta-data that accurately describes the biological "source" material used in such experiments is growing in importance. Here, we provide an overview of the formation of the "Env" community of environmental OMICS researchers and its efforts at considering the meta-data capture needs of those working in environmental OMICS. Specifically, we discuss the development to date of the Env specification, an informal specification including descriptors related to geographic location, environment, organism relationship, and phenotype. We then describe its application to the description of environmental transcriptomic experiments and how we have used it to extend the Minimum Information About a Microarray Experiment (MIAME) data standard to create a domain-specific extension that we have termed MIAME/Env. Finally, we make an open call to the community for participation in the Env Community and its future activities.

Tidal, daily, and lunar-day activity cycles in the marine polychaete Nereis virens.

The burrow emergence activity of the wild caught ragworm Nereis virens Sars associated with food prospecting was investigated under various photoperiodic (LD) and simulated tidal cycles (STC) using a laboratory based actograph. Just over half (57%) of the animals under LD with STC displayed significant tidal (approximately 12.4 h) and/or lunar-day (approximately 24.8 h) activity patterns. Under constant light (LL) plus a STC, 25% of all animals were tidal, while one animal responded with a circadian (24.2 h) activity rhythm suggestive of cross-modal entrainment where the environmental stimulus of one period entrains rhythmic behavior of a different period. All peaks of activity under a STC, apart from that of the individual cross-modal entrainment case, coincided with the period of tank flooding. Under only LD without a STC, 49% of the animals showed nocturnal (approximately 24 h) activity. When animals were maintained under free-running LL conditions, 15% displayed significant rhythmicity with circatidal and circadian/circalunidian periodicities. Although activity cycles in N. virens at the population level are robust, at the individual level they are particularly labile, suggesting complex biological clock-control with multiple clock output pathways.

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. In a wild-type strain, levels of antisense frequency (frq) transcripts cycle in antiphase to sense frq transcripts in the dark, and are inducible by light. In mutant strains in which the induction of antisense frq RNA by light is abolished, the time of the internal clock is delayed relative to the wild-type strain, and resetting of the clock by light is altered. These data provide an unexpected link between antisense RNA and circadian timing and provide a new example of a eukaryotic cellular process regulated by naturally occurring antisense RNA.