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At least 1,117 records · Page 62Linked to original sources
PubMed2003

Periodic notch inhibition by lunatic fringe underlies the chick segmentation clock.

The segmented aspect of the vertebrate body plan first arises through the sequential formation of somites. The periodicity of somitogenesis is thought to be regulated by a molecular oscillator, the segmentation clock, which functions in presomitic mesoderm cells. This oscillator controls the periodic expression of 'cyclic genes', which are all related to the Notch pathway. The mechanism underlying this oscillator is not understood. Here we show that the protein product of the cyclic gene lunatic fringe (Lfng), which encodes a glycosyltransferase that can modify Notch activity, oscillates in the chick presomitic mesoderm. Overexpressing Lfng in the paraxial mesoderm abolishes the expression of cyclic genes including endogenous Lfng and leads to defects in segmentation. This effect on cyclic genes phenocopies inhibition of Notch signalling in the presomitic mesoderm. We therefore propose that Lfng establishes a negative feedback loop that implements periodic inhibition of Notch, which in turn controls the rhythmic expression of cyclic genes in the chick presomitic mesoderm. This feedback loop provides a molecular basis for the oscillator underlying the avian segmentation clock.

Animals↗
PubMed2004

The clock gene Per2 influences the glutamatergic system and modulates alcohol consumption.

Period (Per) genes are involved in regulation of the circadian clock and are thought to modulate several brain functions. We demonstrate that Per2(Brdm1) mutant mice, which have a deletion in the PAS domain of the Per2 protein, show alterations in the glutamatergic system. Lowered expression of the glutamate transporter Eaat1 is observed in these animals, leading to reduced uptake of glutamate by astrocytes. As a consequence, glutamate levels increase in the extracellular space of Per2(Brdm1) mutant mouse brains. This is accompanied by increased alcohol intake in these animals. In humans, variations of the PER2 gene are associated with regulation of alcohol consumption. Acamprosate, a drug used to prevent craving and relapse in alcoholic patients is thought to act by dampening a hyper-glutamatergic state. This drug reduced augmented glutamate levels and normalized increased alcohol consumption in Per2(Brdm1) mutant mice. Collectively, these data establish glutamate as a link between dysfunction of the circadian clock gene Per2 and enhanced alcohol intake.

Acamprosate↗
PubMed2006

Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods.

The cyanobacterial circadian clock can be reconstituted in vitro by mixing recombinant KaiA, KaiB and KaiC proteins with ATP, producing KaiC phosphorylation and dephosphorylation cycles that have a regular rhythm with a ca. 24-h period and are temperature-compensated. KaiA and KaiB are modulators of KaiC phosphorylation, whereby KaiB antagonizes KaiA's action. Here, we present a complete crystallographic model of the Synechococcus elongatus KaiC hexamer that includes previously unresolved portions of the C-terminal regions, and a negative-stain electron microscopy study of S. elongatus and Thermosynechococcus elongatus BP-1 KaiA-KaiC complexes. Site-directed mutagenesis in combination with EM reveals that KaiA binds exclusively to the CII half of the KaiC hexamer. The EM-based model of the KaiA-KaiC complex reveals protein-protein interactions at two sites: the known interaction of the flexible C-terminal KaiC peptide with KaiA, and a second postulated interaction between the apical region of KaiA and the ATP binding cleft on KaiC. This model brings KaiA mutation sites that alter clock period or abolish rhythmicity into contact with KaiC and suggests how KaiA might regulate KaiC phosphorylation.

Amino Acid Sequence↗
PubMed2006

Transcriptional regulation of the Neurospora circadian clock gene wc-1 affects the phase of circadian output.

WHITE COLLAR-1 (WC-1) is the limiting component of the White Collar Complex (WCC) controlling expression of the Neurospora circadian clock protein Frequency (FRQ). Accumulation of WC-1 is supported by FRQ on a post-transcriptional level. Here, we show that transcription of wc-1 is organized in a complex way. Three promoters drive transcription of wc-1. Pdist is dependent on WCC. Pprox is independent of WCC in darkness, but inducible by light in a WCC-dependent manner. A third promoter, Pint, is located in the wc-1 open reading frame and promotes expression of an amino-terminally truncated WC-1 isoform of unknown function. Expression of wc-1 by Pdist or Pprox alone, or by a heterologous promoter, affects the entrained phase of circadian conidiation and the response of Neurospora to light. Our results indicate that transcriptional regulation of wc-1 is required to modulate the circadian phase of clock output.

Amino Acid Sequence↗
PubMed1999

Chronobiology of the hair follicle: hunting the " hair cycle clock".

The hair follicle (HF) is the only mammalian organ that undergoes life-long, cyclic transformations from long stages of growth (anagen), via rapid, apoptosis-driven organ involution (catagen) to a stage of relative "resting" (telogen). The controls that underlie these transformations clearly reside in and/or around the HF itself, and are likely to reflect - essentially autonomous, yet highly manipulable - changes in the local signalling milieu of e.g., hair growth-modulatory growth factors, cytokines, hormones and adhesion molecules. Yet the molecular nature and organization of the "hair cycle clock" (HCC) that drives these cyclic switches in the local signalling milieu remain obscure, and there is not even a fully satisfactory theory of hair cycle control. Since deciphering of the HCC is of paramount clinical importance, and since corresponding working hypotheses are badly needed to guide the design of more incisive experiments that identify the elusive central "oscillator" mechanism behind the HCC, we discuss basic requirements any convincing HCC theory should meet. After arguing that at least four distinct timing devices underlie HF chronobiology ("morphogenesis clock", "cycling inducer", "desynchronizer", and the actual HCC), previously proposed HCC theories are briefly and critically reviewed. In the light of intriguing regulatory similarities between the HCC and the cell cycle machinery, we suggest here that the HCC may be driven by autonomous, cell cycle-coupled secretory activities of the HF mesenchyme, namely by changes in the G0/G1-associated secretion of "papilla morphogens" by dermal papilla fibroblasts. Hopefully, this provocative hypothesis will encourage the proposition of novel, comprehensive HCC theories.

Animals↗
PubMed2001

Clock gene protein mPER1 is rhythmically synthesized and under cAMP control in the mouse pineal organ.

The mammalian clock gene Per1 is an important element of endogenous oscillators that control daily rhythms in central and peripheral tissues. Although such autonomous clock function is lost in the mammalian pineal gland during evolution, mPer1 mRNA and mPER1 protein were found to be strongly elevated in the mouse pineal organ during the dark period compared to daytime values. In vitro studies showed that mPer1 mRNA and mPER1 protein in mouse pineal gland are induced following the activation of a signalling pathway of fundamental importance for pineal physiology, the norepinephrine/cAMP/phosphoCREB cascade. mPER1 may function in the mouse pineal gland as a time-measuring molecule to participate in regulating rhythmic cellular responses in vivo.

Animals↗
PubMed2002

The Neurospora crassa pheromone precursor genes are regulated by the mating type locus and the circadian clock.

Pheromones play important roles in female and male behaviour in the filamentous ascomycete fungi. To begin to explore the role of pheromones in mating, we have identified the genes encoding the sex pheromones of the heterothallic species Neurospora crassa. One gene, expressed exclusively in mat A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by Kex2 processing sites. Strains of the opposite mating type, mat a, express a pheromone precursor gene whose polypeptide contains a C-terminal CAAX motif predicted to produce a mature pheromone with a C-terminal carboxy-methyl isoprenylated cysteine. The predicted sequences of the pheromones are remarkably similar to those encoded by other filamentous ascomycetes. The expression of the pheromone precursor genes is mating type specific and is under the control of the mating type locus. Furthermore, the genes are highly expressed in conidia and under conditions that favour sexual development. Both pheromone precursor genes are also regulated by the endogenous circadian clock in a time-of-day-specific fashion, supporting a role for the clock in mating.

Amino Acid Sequence↗
PubMed2003

The circadian clock regulated RNA-binding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre-mRNA.

The clock-regulated RNA-binding protein AtGRP7 is part of a negative feedback circuit through which the protein influences circadian oscillations of its own transcript. Constitutive overexpression of AtGRP7 in transgenic plants leads to the appearance of a low amount of an alternatively spliced Atgrp7 transcript with a premature stop codon. It is generated by the use of a 5' cryptic splice site in the middle of the intron at the expense of the fully spliced mRNA, indicating a role for AtGRP7 in splice site selection. Accelerated decay of this transcript accounts for its low steady state abundance. This implicates a mechanism for the AtGRP7 feedback loop: Atgrp7 expression is downregulated, as AtGRP7 protein accumulates over the circadian cycle, partly by the generation of an alternate transcript that due to its instability does not accumulate to high levels and does not produce a functional protein. Recombinant AtGRP7 protein specifically interacts with the 3' untranslated region and the intron of its transcript, suggesting that the shift in splice site selection and downregulation involves binding of AtGRP7 to its pre-mRNA. AtGRP7 also influences the choice of splice sites in the Atgrp8 transcript encoding a related RNA-binding protein, favoring the production of an alternatively spliced, unstable Atgrp8 transcript. This conservation points to the importance of this regulatory mechanism to control the level of the clock-regulated glycine-rich RNA-binding proteins and shows how AtGRP7 can control abundance of target transcripts.

Alternative Splicing↗
PubMed2001

Calcium and pituitary adenylate cyclase-activating polypeptide induced expression of circadian clock gene mPer1 in the mouse cerebellar granule cell culture.

Mammalian circadian clock genes Per1 and Per2 are rhythmically expressed not only in the suprachiasmatic nucleus where the mammalian circadian clock exists, but also in other brain regions and peripheral tissues. The induced circadian oscillation of Per genes after treatment with high concentrations of serum or various drugs in cultured cells suggests the ubiquitous existence of the oscillatory mechanism. These treatments also result in a rapid surge of expression of Per1. It has been shown that multiple signaling pathways are involved in Per1 gene induction in culture cells. We used a dispersed primary cell culture made up of mouse cerebellar granule cells to examine the stimuli inducing the mPer genes and their signaling pathways in neuronal tissues expressing mPer genes. We demonstrated that mPer1, but not mPer2, mRNA expression was dependent on the depolarization state controlled by extracellular KCl concentration in the granule cell culture. Nifedipine treatment reduced mPer1 induction, suggesting that mPer1 mRNA expression depends on intracellular calcium concentration regulated through a voltage-dependent Ca2+ channel. Transient mPer1 mRNA induction was observed after elevating KCl concentration in the medium from 5 mM to 25 mM. This increased expression was suppressed by a calmodulin antagonist, or CaMKII/IV inhibitor, but not by MEK inhibitors. Addition of pituitary adenylate cyclase-activating polypeptide-38 to the medium also induced transient Per1 gene expression. This induction was mimicked by dibutyryl-cAMP and suppressed by a protein kinase A (PKA) inhibitor, but not by MEK inhibitors. These results suggest that Ca2+/calmodulin-dependent protein kinase II/IV- and PKA-dependent pathways are involved in high-KCl and PACAP-induced mPer1 induction, respectively, and neural tissues use multiple signaling pathways for mPer1 induction similar to culture cells.

Animals↗
PubMed2003

Facilitation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor transmission in the suprachiasmatic nucleus by aniracetam enhances photic responses of the biological clock in rodents.

This study was designed to test whether the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-facilitating drug, aniracetam, could potentiate photic responses of the biological clock in the suprachiasmatic nucleus (SCN) of rodents. Using the whole-cell patch technique, we first demonstrated that AMPA currents elicited by either local AMPA application or optic chiasm stimulation were augmented by aniracetam in the neurons of the SCN. The AMPA application-elicited increase of intracellular Ca2+ concentration in SCN slices was also enhanced by aniracetam treatment. The systemic injection of aniracetam dose-dependently (10-100 mg/kg) potentiated the phase delay in behavioral rhythm induced by brief light exposure of low intensity (3 lux) but not high intensity (10 or 60 lux) during early subjective night. Under the blockade of NMDA receptors by (+) MK801, aniracetam failed to potentiate a light (3 lux)-induced phase delay in behavioral rhythm. Aniracetam increased the photic induction of c-Fos protein in the SCN that was elicited by low intensity light exposure (3 lux). These results suggest that AMPA receptor-mediated responses facilitated by aniracetam can explain enhanced photic responses of the biological clock in the SCN of rodents.

Animals↗
PubMed2000

Biological clocks and the digestive system.

Circadian rhythms play a major role in regulating the digestive systems of many organisms. Cell proliferation, migration, differentiation, and even structure vary as a function of time of day in many different digestive organs (i.e., stomach, gut, liver, and pancreas) and cell types, resulting in regionally specific temporal variations in protein and gene expression. Feeding and light set the hands of the digestive clock(s). However, the clockwork has a genetic basis. During the last 10 years, new developments have emerged in our understanding of how cells keep time. Surprisingly, clock genes in mammals are expressed not only in specialized time keepers in the brain, but also in peripheral organs, suggesting that the ability to keep time may also belong to cells within the digestive system. This article reviews several classic examples of circadian variation in the digestive system, with an emphasis on rhythms in cell proliferation, function, and structure. It also briefly summarizes several new ideas about how cells in the brain and possibly the digestive system keep time.

Animals↗
PubMed2004

Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses.

In cyanobacteria, KaiC is an essential hexameric clock protein that forms the core of a circadian protein complex. KaiC can be phosphorylated, and the ratio of phospho-KaiC to non-phospho-KaiC is correlated with circadian period. Structural analyses of KaiC crystals identify three potential phosphorylation sites within a 10-A radius of the ATP binding regions that are at the T432, S431, and T426 residues in the KaiCII domains. When these residues are mutated by alanine substitution singly or in combination, KaiC phosphorylation is altered, and circadian rhythmicity is abolished. These alanine substitutions do not prevent KaiC from hexamerizing. Intriguingly, the ability of KaiC overexpression to repress its own promoter is also not prevented by alanine substitutions at these sites, implying that the capability of KaiC to repress its promoter is not sufficient to allow the clockwork to oscillate. The KaiC structure and the mutational analysis suggest that S431 and T426 may share a phosphate that can shuttle between these two residues. Because the phosphorylation status of KaiC oscillates over the daily cycle, and KaiC phosphorylation is essential for clock function as shown here, daily modulations of KaiC activity by phosphorylation at T432 and S431/T426 seem to be key components of the circadian clockwork in cyanobacteria.

Amino Acid Sequence↗
PubMed2005

Demasking biological oscillators: properties and principles of entrainment exemplified by the Neurospora circadian clock.

Oscillations are found throughout the physical and biological worlds. Their interactions can result in a systematic process of synchronization called entrainment, which is distinct from a simple stimulus-response pattern. Oscillators respond to stimuli at some times in their cycle and may not respond at others. Oscillators can also be driven if the stimulus is strong (or if the oscillator is weak); i.e., they restart their cycle every time they receive a stimulus. Stimuli can also directly affect rhythms without entraining the underlying oscillator (masking): Drivenness and masking are often difficult to distinguish. Here we use the circadian biological clock to explore properties of entrainment. We confirm previous results showing that the residual circadian system in Neurospora can be entrained in a mutant of the clock gene frequency (frq(9), a strain deficient in producing a functional FRQ protein). This finding has implications for understanding the evolution of circadian programs. By comparing data sets from independent studies, we develop a template for analyzing, modeling, and dissecting the interactions of entrained and masked components. These insights can be applied to oscillators of all periodicities.

Air Movements↗
PubMed2003

Toward a detailed computational model for the mammalian circadian clock.

We present a computational model for the mammalian circadian clock based on the intertwined positive and negative regulatory loops involving the Per, Cry, Bmal1, Clock, and Rev-Erb alpha genes. In agreement with experimental observations, the model can give rise to sustained circadian oscillations in continuous darkness, characterized by an antiphase relationship between Per/Cry/Rev-Erbalpha and Bmal1 mRNAs. Sustained oscillations correspond to the rhythms autonomously generated by suprachiasmatic nuclei. For other parameter values, damped oscillations can also be obtained in the model. These oscillations, which transform into sustained oscillations when coupled to a periodic signal, correspond to rhythms produced by peripheral tissues. When incorporating the light-induced expression of the Per gene, the model accounts for entrainment of the oscillations by light-dark cycles. Simulations show that the phase of the oscillations can then vary by several hours with relatively minor changes in parameter values. Such a lability of the phase could account for physiological disorders related to circadian rhythms in humans, such as advanced or delayed sleep phase syndrome, whereas the lack of entrainment by light-dark cycles can be related to the non-24h sleep-wake syndrome. The model uncovers the possible existence of multiple sources of oscillatory behavior. Thus, in conditions where the indirect negative autoregulation of Per and Cry expression is inoperative, the model indicates the possibility that sustained oscillations might still arise from the negative autoregulation of Bmal1 expression.

ARNTL Transcription Factors↗
PubMed2001

Circadian rhythms of female mating activity governed by clock genes in Drosophila.

The physiological and behavioral activities of many animals are restricted to specific times of the day. The daily fluctuation in the mating activity of some insects is controlled by an endogenous clock, but the genetic mechanism that controls it remains unknown. Here we demonstrate that wild-type Drosophila melanogaster display a robust circadian rhythm in the mating activity, and that these rhythms are abolished in period- or timeless-null mutant flies (per(01) and tim(01)). Circadian rhythms were lost when rhythm mutant females were paired with wild-type males, demonstrating that female mating activity is governed by clock genes. Furthermore, we detected an antiphasic relationship in the circadian rhythms of mating activity between D. melanogaster and its sibling species Drosophila simulans. Female- and species-specific circadian rhythms in the mating activity of Drosophila seem to cause reproductive isolation.

Animals↗
PubMed2001

Spatiotemporal variation of metabolism in a plant circadian rhythm: the biological clock as an assembly of coupled individual oscillators.

The complex dynamic properties of biological timing in organisms remain a central enigma in biology despite the increasingly precise genetic characterization of oscillating units and their components. Although attempts to obtain the time constants from oscillations of gene activity and biochemical units have led to substantial progress, we are still far from a full molecular understanding of endogenous rhythmicity and the physiological manifestations of biological clocks. Applications of nonlinear dynamics have revolutionized thinking in physics and in biomedical and life sciences research, and spatiotemporal considerations are now advancing our understanding of development and rhythmicity. Here we show that the well known circadian rhythm of a metabolic cycle in a higher plant, namely the crassulacean acid metabolism mode of photosynthesis, is expressed as dynamic patterns of independently initiated variations in photosynthetic efficiency (phi(PSII)) over a single leaf. Noninvasive highly sensitive chlorophyll fluorescence imaging reveals randomly initiated patches of varying phi(PSII) that are propagated within minutes to hours in wave fronts, forming dynamically expanding and contracting clusters and clearly dephased regions of phi(PSII). Thus, this biological clock is a spatiotemporal product of many weakly coupled individual oscillators, defined by the metabolic constraints of crassulacean acid metabolism. The oscillators operate independently in space and time as a consequence of the dynamics of metabolic pools and limitations of CO(2) diffusion between tightly packed cells.

Biological Clocks↗
PubMed2024

Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Photosynthesis↗
PubMed2002

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Adenosine Triphosphate↗