PubMed Health⌕ Search

Biomedical subjects

J C Dunlap

Publications and source records attributed to J C Dunlap.

At least 19 recordsLinked to original sources
PubMed2001

The PAS protein VIVID defines a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting.

vvd, a gene regulating light responses in Neurospora, encodes a novel member of the PAS/LOV protein superfamily. VVD defines a circadian clock-associated autoregulatory feedback loop that influences light resetting, modulates circadian gating of input by connecting output and input, and regulates light adaptation. Rapidly light induced, vvd is an early repressor of light-regulated processes. Further, vvd is clock controlled; the clock gates light induction of vvd and the clock gene frq so identical signals yield greater induction in the morning. Mutation of vvd severely dampens gating, especially of frq, consistent with VVD modulating gating and phasing light-resetting responses. vvd null strains display distinct alterations in the phase-response curve to light. Thus VVD, although not part of the clock, contributes significantly to regulation within the Neurospora circadian system.

Amino Acid Sequence↗
PubMed2001

WC-2 mediates WC-1-FRQ interaction within the PAS protein-linked circadian feedback loop of Neurospora.

Eukaryotic circadian clocks comprise feedback loops where PAS domain-containing transcriptional activators drive gene expression of negative elements. In NEUROSPORA:, clock models posit a White Collar complex (WCC) containing WC-1 and WC-2 that activates expression of the central clock gene frequency (frq); FRQ protein is hypothesized to feed back to block the activity of the WCC. We have characterized the WC-2 protein and its role in this complex: WC-2 is an abundant constitutive nuclear protein, in contrast to rhythmically expressed FRQ and WC-1. WC-2 interacts with WC-1 and FRQ but, significantly, WC-1 and FRQ do not interact in the absence of WC-2. By quantifying the relative numbers of WC-2, FRQ and WC-1 proteins and complexes in cell extracts, both the numbers and types of complexes at different circadian times were estimated, yielding results consistent with the model. Constitutive and abundant WC-2 appears to provide a scaffold allowing for the interaction of two limiting and rhythmically out-of-phase proteins, FRQ and WC-1, and this temporal and physical relationship may be responsible for rhythmic expression of frq.

Biological Clocks↗
PubMed2001

The Neurospora circadian clock regulates a transcription factor that controls rhythmic expression of the output eas(ccg-2) gene.

The circadian clock provides a link between an organism's environment and its behaviour, temporally phasing the expression of genes in anticipation of daily environmental changes. Input pathways sense environmental information and interact with the clock to synchronize it to external cycles, and output pathways read out from the clock to impart temporal control on downstream targets. Very little is known about the regulation of outputs from the clock. In Neurospora crassa, the circadian clock transcriptionally regulates expression of the clock-controlled genes, including the well-characterized eas(ccg-2) gene. Dissection of the eas(ccg-2) gene promoter previously localized a 68 bp sequence containing an activating clock element (ACE) that is both necessary and sufficient for rhythmic activation of transcription by the circadian clock. Using electrophoretic mobility shift assays (EMSAs), we have identified light-regulated nuclear protein factors that bind specifically to the ACE in a time-of-day-dependent fashion, consistent with their role in circadian regulation of expression of eas(ccg-2). Nucleotides in the ACE that interact with the protein factors were determined using interference binding assays, and deletion of the core interacting sequences affected, but did not completely eliminate, rhythmic accumulation of eas(ccg-2) mRNA in vivo, whereas deletion of the entire ACE abolished the rhythm. These data indicate that redundant binding sites for the protein factors that promote eas(ccg-2) rhythms exist within the 68 bp ACE. The ACE binding complexes formed using protein extracts from cells with lesions in central components of the Neurospora circadian clock were identical to those formed with extracts from wild-type cells, indicating that other proteins directly control eas(ccg-2) rhythmic expression. These data suggest that the Neurospora crassa circadian clock regulates an unknown transcription factor, which in turn activates the expression of eas(ccg-2) at specific times of the day.

Base Sequence↗
PubMed2001

Analysis of expressed sequence tags from two starvation, time-of-day-specific libraries of Neurospora crassa reveals novel clock-controlled genes.

In an effort to determine genes that are expressed in mycelial cultures of Neurospora crassa over the course of the circadian day, we have sequenced 13,000 cDNA clones from two time-of-day-specific libraries (morning and evening library) generating approximately 20,000 sequences. Contig analysis allowed the identification of 445 unique expressed sequence tags (ESTs) and 986 ESTs present in multiple cDNA clones. For approximately 50% of the sequences (710 of 1431), significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function) were detected. About 50% of the ESTs (721 of 1431) showed no similarity to previously identified genes. We hybridized Northern blots with probes derived from 26 clones chosen from contigs identified by multiple cDNA clones and EST sequences. Using these sequences, the representation of genes among the morning and evening sequences, respectively, in most cases does not reflect their expression patterns over the course of the day. Nevertheless, we were able to identify four new clock-controlled genes. On the basis of these data we predict that a significant proportion of the expressed Neurospora genes may be regulated by the circadian clock. The mRNA levels of all four genes peak in the subjective morning as is the case with previously identified ccgs.

Blotting, Northern↗
PubMed2001

Circadian clock-specific roles for the light response protein WHITE COLLAR-2.

To understand the role of white collar-2 in the Neurospora circadian clock, we examined alleles of wc-2 thought to encode partially functional proteins. We found that wc-2 allele ER24 contained a conservative mutation in the zinc finger. This mutation results in reduced levels of circadian rhythm-critical clock gene products, frq mRNA and FRQ protein, and in a lengthened period of the circadian clock. In addition, this mutation altered a second canonical property of the clock, temperature compensation: as temperature increased, period length decreased substantially. This temperature compensation defect correlated with a temperature-dependent increase in overall FRQ protein levels, with the relative increase being greater in wc-2 (ER24) than in wild type, while overall frq mRNA levels were largely unaltered by temperature. We suggest that this temperature-dependent increase in FRQ levels partially rescues the lowered levels of FRQ resulting from the wc-2 (ER24) defect, yielding a shorter period at higher temperatures. Thus, normal activity of the essential clock component WC-2, a positive regulator of frq, is critical for establishing period length and temperature compensation in this circadian system.

Amino Acid Sequence↗
PubMed2001

Genetic and molecular analysis of circadian rhythms in Neurospora.

Over the course of the past 40 years Neurospora has become a well-known and uniquely tractable model system for the analysis of the molecular basis of eukaryotic circadian oscillatory systems. Molecular bases for the period length and sustainability of the rhythm, light, and temperature resetting of the circadian system and for gating of light input and light effects are becoming understood, and Neurospora promises to be a suitable system for examining the role of coupled feedback loops in the clock. Many of these insights have shown or foreshadow direct parallels in mammalian systems, including the mechanism of light entrainment, the involvement of PAS:PAS heterodimers as transcriptional activators in essential clock-associated feedback loops, and dual role of FRQ in the loop as an activator and a repressor; similarities extend to the primary sequence level in at least one case, that of WC-1 and BMAL1. Work on circadian output in Neurospora has identified more than a dozen regulated genes and has been at the forefront of studies aimed at understanding clock control of gene expression.

Circadian Rhythm↗
PubMed2000

Interconnected feedback loops in the Neurospora circadian system.

In Neurospora crassa, white collar 1 (WC-1), a transcriptional activator and positive clock element, is rhythmically expressed from a nonrhythmic steady-state pool of wc-1 transcript, consistent with posttranscriptional regulation of rhythmicity. Mutations in frq influence both the level and periodicity of WC-1 expression, and driven FRQ expression not only depresses its own endogenous levels, but positively regulates WC-1 synthesis with a lag of about 8 hours, a delay similar to that seen in the wild-type clock. FRQ thus plays dual roles in the Neurospora clock and thereby, with WC-1, forms a second feedback loop that would promote robustness and stability in this circadian system. The existence also of interlocked loops in Drosophila melanogaster and mouse clocks suggests that such interlocked loops may be a conserved aspect of circadian timing systems.

Amino Acid Sequence↗
PubMed2000

Dimerization and nuclear entry of mPER proteins in mammalian cells.

Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells. Previously, mCRY1 and mCRY2 have been found to complex with PER proteins leading to nuclear import. Here we report that nuclear translocation of mPER1 and mPER2 (1) involves physical interactions with mPER3, (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2 double-deficient cells lacking a functional biological clock. Moreover, nuclear localization of endogenous mPER1 was observed in cultured mCry1/mCry2 double-deficient cells as well as in the liver and the suprachiasmatic nuclei (SCN) of mCry1/mCry2 double-mutant mice. This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions (i.e., in the intact mouse) in the absence of any CRY protein. The mPER3 amino acid sequence predicts the presence of a cytoplasmic localization domain (CLD) and a nuclear localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophila is conserved in mammals, but with the novel twist that mPER3 can act as the dimerizing partner.

Animals↗
PubMed2000

Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock.

Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (>30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock.

Adenine↗
PubMed2000

Microbial circadian oscillatory systems in Neurospora and Synechococcus: models for cellular clocks.

Common regulatory patterns have emerged among the feedback loops lying within circadian systems. Significant progress in dissecting the mechanism of clock resetting by temperature and the role of the WC proteins in the Neurospora light response has accompanied documentation of the importance of nuclear localization and phosphorylation-induced turnover of FRQ to this circadian cycle. The long-awaited molecular description of a transcription/translation loop in the Synechococcus circadian system represents a quantal step forward, followed by the identification of additional important proteins and interactions. Finally, the adaptive significance of rhythms in Synechococcus and by extension in all clocks nicely ties up an extraordinary year.

Biological Clocks↗
PubMed1999

Circadian biology: clocks for the real world.

The circadian system of Neurospora crassa includes a molecular feedback loop that is entrainable by light. A recent study has shown that a second, elusive oscillator interacts with the feedback loop to drive output rhythms.

Animals↗
PubMed1999

Regulation of clock genes.

A recent explosion in the identification of new clock components in cyanobacteria, fungi, insects, mammals as well as potential candidates in plants has uncovered common themes among the structure, function and regulation of these components. Positive and negative interactions that are organized in negative feedback loops have been found crucial for clock function. Both transcriptional and posttranscriptional mechanisms appear to be important for circadian rhythm generation in all of these organisms.

Animals↗
PubMed1999

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. Post-transcriptional control governs the amount and type of FRQ and makes the clock responsive to temperature.

Animals↗
PubMed1998

Nuclear localization is required for function of the essential clock protein FRQ.

The frequency (frq) gene in Neurospora encodes central components of a circadian oscillator, a negative feedback loop involving frq mRNA and two forms of FRQ protein. Here we report that FRQ is a nuclear protein and nuclear localization is essential for its function. Deletion of the nuclear localization signal (NLS) renders FRQ unable to enter into the nucleus and abolishes overt circadian rhythmicity, while reinsertion of the NLS at a novel site near the N-terminus of FRQ restores its function. Each form of FRQ enters the nucleus soon after its synthesis in the early subjective day; there is no evidence for regulated sequestration in the cytoplasm prior to nuclear entry. The kinetics of the nuclear entry are consistent with previous data showing rapid depression of frq transcript levels following the synthesis of FRQ, and suggest that early in each circadian cycle, when FRQ is synthesized, it enters the nucleus and depresses the level of its own transcript.

Biological Clocks↗
PubMed1998

How temperature changes reset a circadian oscillator.

Circadian rhythms control many physiological activities. The environmental entrainment of rhythms involves the immediate responses of clock components. Levels of the clock protein FRQ were measured in Neurospora at various temperatures; at higher temperatures, the amount of FRQ oscillated around higher levels. Absolute FRQ amounts thus identified different times at different temperatures, so temperature shifts corresponded to shifts in clock time without immediate synthesis or turnover of components. Moderate temperature changes could dominate light-to-dark shifts in the influence of circadian timing. Temperature regulation of clock components could explain temperature resetting of rhythms and how single transitions can initiate rhythmicity from characteristic circadian phases.

Biological Clocks↗
PubMed1998

Glyceraldehyde-3-phosphate dehydrogenase is regulated on a daily basis by the circadian clock.

Circadian clocks function to govern a wide range of rhythmic activities in organisms. An integral part of rhythmicity is the daily control of target genes by the clock. Here we describe the sequence and analysis of a novel clock-controlled gene, ccg-7, showing similarity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme widely used as a constitutive control in a variety of systems. That ccg-7 encodes GAPDH was confirmed by demonstrating that in vitro synthesized CCG-7 possesses GAPDH activity. Rhythms in both ccg-7 mRNA accumulation and CCG-7 (GAPDH) activity are observed in a clock wild-type strain where the peak in GAPDH activity lags several hours behind the peak in ccg-7 mRNA accumulation in the late night. Together with our previous observation that ccg-7 mRNA is not developmentally regulated, we show that ccg-7 is not induced by environmental stresses such as glucose or nitrogen deprivation (which also trigger development), heat shock, or osmotic stress. Thus, the finding that GAPDH is clock-regulated points to a specific role for the circadian clock in controlling aspects of general metabolism and provides evidence for circadian regulation of a gene found in most living organisms.

Amino Acid Sequence↗