In the fungus Neurospora crassa, the blue light photoreceptor(s) and signaling pathway(s) have not been identified. We examined light signaling by exploiting the light sensitivity of the Neurospora biological clock, specifically the rapid induction by light of the clock component frequency (frq). Light induction of frq is transcriptionally controlled and requires two cis-acting elements (LREs) in the frq promoter. Both LREs are bound by a White Collar-1 (WC-1)/White Collar-2 (WC-2)-containing complex (WCC), and light causes decreased mobility of the WCC bound to the LREs. The use of in vitro-translated WC-1 and WC-2 confirmed that WC-1, with flavin adenine dinucleotide as a cofactor, is the blue light photoreceptor that mediates light input to the circadian system through direct binding (with WC-2) to the frq promoter.

The biological clock of Neurospora crassa includes interconnected transcriptional and translational feedback loops that cause both the transcript and protein encoded by the frequency gene (frq) to undergo the robust daily oscillations in abundance, which are essential for clock function. To understand better the mechanism generating rhythmic frq transcript, reporter constructs were used to show that the oscillation in frq message is transcriptionally regulated, and a single cis-acting element in the frq promoter, the Clock Box (C box), is both necessary and sufficient for this rhythmic transcription. Nuclear protein extracts used in binding assays revealed that a White Collar (WC)-1-and WC-2-containing complex (WCC) binds to the C box in a time-of-day-specific manner. Overexpression of an ectopic copy of FRQ or addition of in vitrogenerated FRQ resulted in reduced WCC binding to the C box. These data suggest that oscillations in frq transcript result from WCC binding to the frq promoter and activating transcription with subsequent changes in FRQ levels having an inverse effect on WCC binding. In this way rhythmic expression and turnover of FRQ drives the rhythm in its own transcription. Life on earth has evolved under the continual daily fluctuations in light and temperature. Many organisms have evolved the ability to anticipate these external changes in their environment by using endogenous ''biological clocks.'' In recent years, the molecular components that make up these intracellular clocks have begun to be identified, and similarities among a wide range of organisms have emerged (1-5). The clock in a fungus, fruit fly, or mammal contains a negative feedback loop in which two PAS domain-containing proteins [White Collar (WC)-2͞ WC-1, dCLOCK (dCLK)͞CYCLE (CYC), CLOCK͞BMAL1] heterodimerize and act as positive elements to activate the expression of a negative element [FREQUENCY (FRQ), PERIOD (PER)͞TIMELESS (TIM), CRYPTOCHROME 1 (CRY1)͞CRY2͞mPERs]. The negative element(s) in turn feeds back to repress the activity of the positive elements. These transcription͞translation-based negative feedback loops ultimately generate self-sustaining circadian (circa ϭ about; dies ϭ day) oscillations or rhythms in the level(s) of one or more of the elements within the loop. The robustness and stability of these oscillations is enhanced further by interlocking positive feedback loops (6-8) and multiple layers of regulation (2,3,9).In Neurospora, a negative feedback loop comprised of the products of the frq, wc-1, and wc-2 genes is central to clock function. WC-1 and WC-2 are predominately nuclear transcription factors containing trans-activation domains and Zn-finger DNA-binding domains (10, 11). They form a WC complex (WCC) by heterodimerizing via PAS domains (12) and act as positive elements in the expression of frq (13); in a wc-1 KO or wc-2 KO strain, very limited, unregulated transcription of frq occurs (14-16). During the course of a day, FRQ is progressively phosphorylated and degraded (17)(18)(19), but when present FRQ ...

band, an allele enabling clear visualization of circadianly regulated spore formation (conidial banding), has remained an integral tool in the study of circadian rhythms for 40 years. bd was mapped using single-nucleotide polymorphisms (SNPs), cloned, and determined to be a T79I point mutation in ras-1. [Keywords: Circadian; output; band; ras-1; ROS] Supplemental material is available at http://www.genesdev.org. Alterations in light-

BackgroundThe main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.ResultsWe expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase.ConclusionsGene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.

Phytochromes (Phys) comprise a superfamily of red-/far-red-light-sensing proteins. Whereas higher-plant Phys that control numerous growth and developmental processes have been well described, the biochemical characteristics and functions of the microbial forms are largely unknown. Here, we describe analyses of the expression, regulation, and activities of two Phys in the filamentous fungus Neurospora crassa. In addition to containing the signature N-terminal domain predicted to covalently associate with a bilin chromophore, PHY-1 and PHY-2 contain C-terminal histidine kinase and response regulator motifs, implying that they function as hybrid two-component sensor kinases activated by light. A bacterially expressed N-terminal fragment of PHY-2 covalently bound either biliverdin or phycocyanobilin in vitro, with the resulting holoprotein displaying red-/far-red-light photochromic absorption spectra and a photocycle in vitro. cDNA analysis of phy-1 and phy-2 revealed two splice isoforms for each gene. The levels of the phy transcripts are not regulated by light, but the abundance of the phy-1 mRNAs is under the control of the circadian clock. Phosphorylated and unphosphorylated forms of PHY-1 were detected; both species were found exclusively in the cytoplasm, with their relative abundances unaffected by light. Strains containing deletions of phy-1 and phy-2, either singly or in tandem, were not compromised in any known photoresponses in Neurospora, leaving their function(s) unclear.Light is essential for life on earth, serving as a primary energy source for organisms ranging from single-celled bacteria to higher plants. Plants, in particular, contain a complex network of light perception and signal transduction systems that enables them to track and respond to fluctuations in multiple parameters within their light environment, including intensity, directionality, daily duration, and spectral quality. Plants employ at least three photoreceptor types for light perception, the cryptochromes and phototropins, which monitor the blue/UV region of the spectrum, and the phytochromes (Phys), which monitor the red-light (R)/far-red-light (FR) region (52). The generic Phy in higher plants is a soluble homodimer, consisting of two ϳ120-kDa polypeptides, each bearing a single bilin (or linear tetrapyrrole) chromophore. The bilin is bound covalently by an autocatalytic mechanism to an N-terminal pocket that, once assembled, serves as the sensory module (73). Through interactions between the bilin and the apoprotein, Phys reversibly photointerconvert between two stable conformers, an R-absorbing Pr form that is biologically inactive and an FR-absorbing Pfr form that is biologically active. Via interconversion between Pr and Pfr, Phys act as reversible switches in photoperception. The C-terminal half of Phys bears contacts for dimerization and sensory output activities, the nature of which is currently unclear (reviewed in references 58 and 71).The deluge of genomic sequence information has greatly expanded the Phy family, with n...

In plants and animals, cryptochromes function as either photoreceptors or circadian clock components. We have examined the cryptochrome from the filamentous fungus Neurospora crassa and demonstrate that Neurospora cry encodes a DASH-type cryptochrome that appears capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). The cry transcript and CRY protein levels are strongly induced by blue light in a wc-1-dependent manner, and cry transcript is circadianly regulated, with a peak abundance opposite in phase to frq. Neither deletion nor overexpression of cry appears to perturb the free-running circadian clock. However, cry disruption knockout mutants show a small phase delay under circadian entrainment. Using electrophoretic mobility shift assays (EMSA), we show that CRY is capable of binding single-and double-stranded DNA (ssDNA and dsDNA, respectively) and ssRNA and dsRNA. Whole-genome microarray experiments failed to identify substantive transcriptional regulatory activity of cry under our laboratory conditions.

No abstract

Summary The modification of behavior in response to experience is crucial for animals to adapt to environmental changes. Although factors such as neuropeptides and hormones are known to function in the switch between alternative behavioral states, the mechanisms by which these factors transduce, store, retrieve and integrate environmental signals to regulate behavior are poorly understood. The rate of locomotion of the nematode Caenorhabditis elegans depends on both current and past food availability. Specifically, C. elegans slows its locomotion when it encounters food, and animals in a food-deprived state slow even more than animals in a well-fed state. The slowing responses of well-fed and food-deprived animals in the presence of food represent distinct behavioral states, as they are controlled by different sets of genes, neurotransmitters and neurons. Here we describe an evolutionarily conserved C. elegans protein, VPS-50, that is required for animals to assume the well-fed behavioral state. Both VPS-50 and its murine homolog mVPS50 are expressed in neurons, are associated with synaptic and dense-core vesicles and control vesicle acidification and hence synaptic function, likely through regulation of the assembly of the V-ATPase complex. We propose that dense-core vesicle acidification controlled by the evolutionarily conserved protein VPS-50/mVPS50 affects behavioral state by modulating neuropeptide levels and presynaptic neuronal function in both C. elegans and mammals.