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Mol Cell Biol 2001 Apr;21(8):2619-28

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

Collett MA, Dunlap JC, Loros JJ. Department of Genetics, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.

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.

Cell 2001 Feb 9;104(3):453-64

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

Heintzen C, Loros JJ, Dunlap JC. Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA.

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.

Genetics 2001 Mar;157(3):1057-65

Analysis of Expressed Sequence Tags From Two Starvation, Time-of-Day-Specific Libraries of Neurospora crassa Reveals Novel Clock-Controlled Genes.

Zhu H, Nowrousian M, Kupfer D, Colot HV, Berrocal-Tito G, Lai H, Bell-Pedersen D, Roe BA, Loros JJ, Dunlap JC. Department of Chemistry and Biochemistry, Advanced Center for Genome Technology, University of Oklahoma, Norman, Oklahoma 73019.

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.

EMBO J 2001 Jan 15;20(1-2):109-17

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

Denault DL, Loros JJ, Dunlap JC. Departments of Biochemistry and Genetics, Dartmouth Medical School, Hanover, NH 03755, USA.

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.

Genes Dev 2000 Jun 1;14(11):1353-1363

Yagita K, Yamaguchi S, Tamanini F, van Der Horst GT, Hoeijmakers JH, Yasui A, Loros JJ, Dunlap JC, Okamura H
Department of Anatomy and Brain Science, Kobe University School of Medicine, Chuoku, Kobe 650-0017, Japan.

            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.

Proc Natl Acad Sci U S A 2000 Jan 4;97(1):234-9

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

Liu Y, Loros J, Dunlap JC

Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA.

            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.

Science 1998 Aug 7;281(5378):825-9

Liu Y, Merrow M, Loros JJ, Dunlap JC

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844, USA.

            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.

EMBO J 1998 Aug 10;17(5):1228-35

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

Luo C, Loros JJ, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

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.

PMID: 9482720, UI: 98151347

Cell 1997 May 2;89(3):469-76

Alternative initiation of translation and time-specific phosphorylation yield multiple forms of the essential clock protein FREQUENCY.

Garceau NY, Liu Y, Loros JJ, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

The frequency (frq) gene encodes central components of the
transcription/translation-based negative-feedback loop comprising the core of
the Neurospora circadian oscillator; posttranscriptional regulation associated
with FRQ is surprisingly complex. Alternative use of translation initiation
sites gives rise to two forms of FRQ whose levels peak 4-6 hr following the
peak of frq transcript. Each form of FRQ is progressively phosphorylated over
the course of the day, thus providing a number of temporally distinct FRQ
products. The kinetics of these regulatory processes suggest a view of the
clock where relatively rapid events involving translational regulation in the
synthesis of FRQ and negative feedback of FRQ on frq transcript levels are
followed by slower posttranslational regulation, ultimately driving the
turnover of FRQ and reactivation of the frq gene.

PMID: 9150146, UI: 97294390

Science 1997 May 2;276(5313):763-9

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

Crosthwaite SK, Dunlap JC, Loros JJ
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

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. Data shown here suggest that wc-1 is a clock-associated gene and
wc-2 is a clock component; both play essential roles in the assembly or
operation of the Neurospora circadian oscillator. Thus DNA binding and
transcriptional activation can now be associated with a clock gene that may
provide a positive element in the feedback loop. In addition, similarities
between the PAS-domain regions of molecules involved in light perception and
circadian rhythmicity in several organisms suggest an evolutionary link between
ancient photoreceptor proteins and more modern proteins required for circadian
oscillation.

Comments:

Comment in: Science 1997 May 2;276(5313):753-4

PMID: 9115195, UI: 97277213

Proc Natl Acad Sci U S A 1997 Apr 15;94(8):3877-82

Dissection of a circadian oscillation into discrete domains.

Merrow MW, Garceau NY, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

The circadian oscillator in Neurospora is a negative feedback loop involving as
principal players the products of the frequency (frq) locus. frq encodes
multiple forms of its protein product FRQ, which act to depress the amounts of
frq transcript. In this scheme there are two discrete and separable steps to
the circadian cycle, negative feedback itself (repression) in which FRQ acts to
decrease the levels of its own transcript, and recovery from repression
(derepression) in which frq transcript levels return to peak amounts. By
introducing an exogenously regulatable frq transgene into a frq
loss-of-function strain (frq9), we created an artificial system in which the
two separate steps in the circadian cycle can be initiated and followed
separately for purposes of observing their kinetics. Under these conditions the
frq-FRQ cycle occupies the time scale of a full circadian cycle. During this
time, the process of negative feedback of FRQ on frq transcript levels is rapid
and efficient; it requires only 3 to 6 h and can be mediated by on the order of
10 molecules of FRQ per nucleus, a level even less than that seen in the normal
oscillation. In contrast, recovery from negative feedback requires 14 to 18 h,
most of the circadian cycle, during which time de novo FRQ synthesis has
stopped, and existing FRQ is progressively posttranslationally modified.
Altogether the time required to complete both of these steps is in good
agreement with the 22-h observed period length of the normal circadian cycle.

PMID: 9108072, UI: 97268664

Prog Brain Res 1996;111:11-27

The genetic and molecular dissection of a prototypic circadian system.

Dunlap JC, Loros JJ, Merrow M, Crosthwaite S, Bell-Pedersen D, Garceau N, Shinohara M, Cho H, Luo C
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

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.g. Loros et al., 1989; Loros
and Dunlap, 1991; Aronson et al., 1994a) that will help to pave the way for
proving the identity of clock components in more complex systems, for
understanding how clocks are regulated by entraining factors, and for showing
how time information eventually is used to regulate the behaviors of clock
cells, and of whole organisms.

Publication Types:

Review
Review, tutorial

PMID: 8990904, UI: 97145245

Annu Rev Genet 1996;30:579-601

Genetics and molecular analysis of circadian rhythms.

Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

The first part of this review summarizes the two best understood aspects of the
two best understood circadian systems, the feedback oscillators of Neurospora
and Drosophila, concentrating on what we know about the frequency (frq), period
(per) and timeless (tim) genes. In the second part, the general circadian
genetic and molecular literature is surveyed, with an eye to describing what is
known from a variety of systems about input to the oscillator (entrainment),
and how the oscillator might work and be temperature compensated, in emerging
systems including Synechococcus, Gonyaulax, Arabidopsis, hamsters, and mice.
Finally, the conversation of the molecular components of clocks is analyzed:
both frq and per are widely conserved in their respective phylogenetic classes.
Pharmacological data suggests that most other organisms use a day-phased
oscillator of the type seen in Neurospora rather than a night-phased oscillator
such as in Drosophila.

Publication Types:

Review
Review, academic

PMID: 8982466, UI: 97137089

Ciba Found Symp 1995;183:3-17; discussion 17-25

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

Dunlap JC, Loros JJ, Aronson BD, Merrow M, Crosthwaite S, Bell-Pedersen D, Johnson K, Lindgren K, Garceau NY

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

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. The timing of peak expression is
distinct in the two genes, frq expression peaking after dawn and per expression
peaking near midnight. Also, although expression of per from a constitutive
promoter can rescue rhythmicity in a fly lacking the gene, constitutive
expression of frq will not rescue rhythmicity in Neurospora frq-null strains,
and in fact causes arrhythmicity when expressed in a wild-type strain. These
data suggest that frq is and/or encodes a state variable of the circadian
oscillator. Recent molecular genetic analyses of frq have shed light on the
origin of temperature compensation and strongly suggest that this property is
built into the oscillatory feedback loop rather than appended to it. It seems
plausible that clocks are adjusted and reset through adjustments in central
clock components such as frq, and, by extension, per.

Publication Types:

Review
Review, tutorial

PMID: 7656691, UI: 95385481

Proc Natl Acad Sci U S A 1994 Aug 2;91(16):7683-7

Circadian clock locus frequency: protein encoded by a single open reading frame defines period length and temperature compensation.

Aronson BD, Johnson KA, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

The frequency (frq) locus encodes a key component, a state variable, in a
cellular oscillator generating circadian rhythmicity. Two transcripts have been
mapped to this region, and data presented here are consistent with the
existence of a third transcript. Analysis of cDNA clones and clock mutants from
this region focuses attention on one transcript encoding a protein. FRQ, which
is a central clock component: (i) mutations in all of the semidominant frq
alleles are the result of single amino acid substitutions and map to the open
reading frame (ORF) encoding FRQ; (ii) deletion of this ORF, or a frameshift
mutation within it, results in a strain with a recessive clock phenotype
characterized by the loss of rhythm stability and compensation. Single amino
acid substitutions within, or disruption of, this single ORF are thus
sufficient to drive major alterations in both period length and temperature
compensation, two canonical characteristics of circadian systems. The 989-amino
acid FRQ protein species the circadian function of frq in the assembly of the
Neurospora biological clock.

PMID: 8052643, UI: 94329580

EMBO J 1994 May 15;13(10):2257-66

Intergeneric complementation of a circadian rhythmicity defect: phylogenetic conservation of structure and function of the clock gene frequency.

Merrow MW, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

The Neurospora crassa frequency locus encodes a 989 amino acid protein that is
a central component, a state variable, of the circadian biological clock. We
have determined the sequence of all or part of this protein and surrounding
regulatory regions from additional fungi representing three genera and report
that there is distinct, preferential conservation of the frequency open reading
frame (ORF) as compared with non-coding sequences. Within the coding region,
many of the domain hallmarks of the N. crassa protein are highly conserved,
especially an internal region bearing the causative mutations in frq1 and frq7,
the most extreme alleles in the frequency allelic series. Despite considerable
diversity among the strains analyzed in terms of morphology, growth, circadian
clock output and frq sequence, the ORF from the most distantly related fungus
included in this study (Sordaria fimicola) rescues rhythmicity in a N.crassa
frequency null strain. Both sequence conservation, and the ability of frequency
from a genus displaying one developmental program to complement circadian
defects in a separate genus with a distinct, clock-regulated developmental
program, are consistent with a central role of the frequency gene product in a
general circadian oscillator capable of controlling diverse outputs in a
variety of systems.

PMID: 8194516, UI: 94252311

Science 1994 Mar 18;263(5153):1578-84

Negative feedback defining a circadian clock: autoregulation of the clock gene
frequency.

Aronson BD, Johnson KA, Loros JJ, Dunlap JC
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook NY 11794.

The frequency (frq) locus of Neurospora crassa was originally identified in
searches for loci encoding components of the circadian clock. The frq gene is
now shown to encode a central component in a molecular feedback loop in which
the product of frq negatively regulated its own transcript, which resulted in a
daily oscillation in the amount of frq transcript. Rhythmic messenger RNA
expression was essential for overt rhythmicity in the organism and no amount of
constitutive expression rescued normal rhythmicity in frq loss-of-function
mutants. Step reductions in the amount of FRQ-encoding transcript set the clock
to a specific and predicted phase. These results establish frq as encoding a
central component in a circadian oscillator.

Comments:

Comment in: Science 1994 Mar 18;263(5153):1570-2

PMID: 8128244, UI: 94174300

Brain Res Brain Res Rev 1993 Sep-Dec;18(3):315-33

Circadian rhythms.

Aronson BD, Bell-Pedersen D, Block GD, Bos NP, Dunlap JC, Eskin A, Garceau NY, Geusz ME, Johnson KA, Khalsa SB, et al
Section on Biochemical Pharmacology, National Institute of Mental Health, Bethesda, MD 20892.

Circadian rhythms are a ubiquitous adaptation of eukaryotic organisms to the
most reliable and predictable of environmental changes, the daily cycles of
light and temperature. Prominent daily rhythms in behavior, physiology, hormone
levels and biochemistry (including gene expression) are not merely responses to
these environmental cycles, however, but embody the organism's ability to keep
and tell time. At the core of circadian systems is a mysterious mechanism,
located in the brain (actually the suprachiasmatic nucleus of the hypothalamus)
of mammals, but present even in unicellular organisms, that functions as a
clock. This clock drives circadian rhythms. It is independent of, but remains
responsive to, environmental cycles (especially light). The interest in
temporal regulation--its organization, mechanism and consequences--unites
investigators in diverse disciplines studying otherwise disparate systems. This
diversity is reflected in the brief reviews that summarize the presentations at
a meeting on circadian rhythms held in New York City on October 31, 1992. The
meeting was sponsored by the Fondation pour l'Etude du Systeme Nerveux (FESN)
and followed a larger meeting held 18 months earlier in Geneva, whose
proceedings have been published (M. Zatz (Ed.), Report of the Ninth FESN Study
Group on 'Circadian Rhythms', Discussions in Neuroscience, Vol. VIII, Nos. 2 +
3, Elsevier, Amsterdam, 1992). Some speakers described progress made in the
interim, while others addressed aspects of the field not previously covered.

Publication Types:

Review
Review, tutorial

PMID: 8401597, UI: 94004978

Annu Rev Physiol 1993;55:683-728

Genetic analysis of circadian clocks.

Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

Publication Types:

Review
Review, academic

PMID: 8466189, UI: 93221124

Chronobiol Int 1992 Jun;9(3):231-9

Molecular analysis of the Neurospora clock: cloning and characterization of the frequency and period-4 genes.

Aronson BD, Johnson KA, Liu Q, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844.

Genetic analysis of Neurospora crassa has identified many mutants that affect
the biological clock. In this article we review the cloning of two of these
genes, frq and prd-4. Both genes were isolated using a chromosome walk
technique. Subcloning experiments and subsequent Northern analysis of frq
implicate the importance of two transcripts that emanate from this locus. In
preliminary data, no protein-coding region is evident in the smaller
transcript; the larger transcript contains a 962-amino acid open reading frame.
The open reading frame shows limited homology to per, a clock gene identified
in Drosophila. Sequence analysis of all existing frq alleles suggests that the
defect in each case lies within the open reading frame. Successful cloning of
the prd-4 gene required walking a distance of greater than 40 kb. A physical
map of this region has been constructed using restriction analysis. The
dominance-recessive relationship of prd-4 and prd-4+ was established by
examining the period lengths of strains harboring a wide range of prd-4/prd-4+
nuclear ratios.

Publication Types:

Review
Review, tutorial

PMID: 1535290, UI: 92306196

Trends Genet 1990 May;6(5):159-65

Closely watched clocks: molecular analysis of circadian rhythms in Neurospora and Drosophila.

Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03756.

Circadian rhythms represent a type of cellular regulation common to most
eukaryotes. Analysis of the genetic basis of this phenomenon is beginning to
provide information about how clocks function at the molecular level.
Surprisingly, the first two cloned 'clock genes', one from a fruit fly and one
from a fungus, share some common characteristics both genetically and in the
nature of the proteins they encode. In related work, the recent identification
and molecular analysis of clock-controlled genes is revealing how biological
clocks control gene expression, and may pave the way for the isolation of novel
'clock genes' in the future.

Publication Types:

Review
Review, tutorial

PMID: 2142347, UI: 90312655

Nature 1989 Jun 15;339(6225):558-62

The Neurospora clock gene frequency shares a sequence element with the
Drosophila clock gene period.

McClung CR, Fox BA, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03756.

The isolation and characterization of single gene mutations affecting the
circadian biological clocks of several organisms has left little doubt that
circadian rhythms can be subjected to classical genetical analysis. Many of
these mutations occur at the same few genetic loci (frequency (frq) in the
fungus Neurospora, and period (per) in fruit fly Drosophila); these loci
represent the best studied clock-affecting genes known. Mutant strains are
usually affected in more than one basic clock property, suggesting an
inter-relatedness at the molecular level among these basic properties that
would not have been predicted a priori. The extensive background information
available concerning the frq locus provides a basis for the molecular
dissection of the Neurospora circadian clock--the most minimal circadian system
thus far described. We report here the cloning and analysis of the frq locus
and show it to be larger and more complex than would have been predicted from
the available genetic data. Complete rescue of all of the pleiotropic mutant
phenotypes of the recessive frq allele requires transformation with a
7.7-kilobase (kb) region of DNA encoding at least two transcripts. Sequence
analysis of this region has allowed the identification of a common element
between frq and per which, given the background similarities in their classical
genetic characteristics, suggests the possibility of a common element in the
clock mechanisms of these two organisms.

PMID: 2525233, UI: 89281721

Science 1989 Jan 20;243(4889):385-8

Molecular cloning of genes under control of the circadian clock in Neurospora.

Loros JJ, Denome SA, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03756.

To investigate the regulation of messenger RNA abundance by circadian clocks,
genomic and complementary DNA libraries were screened with complementary DNA
probes enriched, by means of sequential rounds of subtractive hybridization,
for sequences complementary to transcripts specific to either early morning or
early evening cultures of Neurospora. Only two morning-specific genes were
identified through this protocol. RNA blot analysis verified that the abundance
of the transcripts arising from these genes oscillates with a period of 21.5
hours in a clock wild-type strain and 29 hours in the long-period clock mutant
strain frq7. Genetic mapping through the use of restriction fragment length
polymorphisms shows the two genes, ccg-1 and ccg-2, to be unlinked. These data
provide a view of the extent of clock control of gene expression.

PMID: 2563175, UI: 89100287

Proc Natl Acad Sci U S A 1988 Feb;85(4):1096-100

On the role of protein synthesis in the circadian clock of Neurospora crassa.

Dunlap JC, Feldman JF
Department of Biology, University of California, Santa Cruz CA 95064.

Inhibitors of protein synthesis reset the biological clocks of many organisms.
This has been interpreted to mean either that the synthesis per se of proteins
is a step in the oscillatory feedback loop or merely that certain unstable
protein(s) are required at certain times of the cycle to complete the feedback
loop. We report here that Neurospora strains bearing the clock mutation frq-7
are relatively insensitive to the resetting action of the
protein-synthesis-inhibitor cycloheximide. Protein synthesis itself in this
mutant is inhibited by the drug to the same extent as in wild type. Since the
clock of frq-7 continues to run relatively unimpeded even in the virtual
absence of protein synthesis, it is unlikely that synthesis per se can be a
part of the feedback cycle. Rather, we suggest that for normal operation of the
Neurospora clock, certain protein(s) with a high turnover rate are required
daily and, thus, must be resynthesized each day (at least) during discrete
times in the cycle. The frq-7 mutation simultaneously alters several distinct
clock characteristics--period length, temperature compensation, and resetting
by cycloheximide. A model is presented to unify these observations.

PMID: 2963337, UI: 88124982

Cell Mol Life Sci 1999 Aug 15;55(10):1195-205

Liu Y, Heintzen C, Loros J, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH
03755, USA. Yi.Liu@dartmouth.edu

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.

Publication Types:

Review
Review, academic

PMID: 10487202, UI: 99415124

Genes Cells 1999 Jan;4(1):1-10

Eukaryotic circadian systems: cycles in common.

Dunlap JC, Loros JJ, Liu Y, Crosthwaite SK
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
jay.c.dunlap@dartmouth.edu

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.

Publication Types:

Review
Review, tutorial

PMID: 10231388, UI: 99248422

Cell 1999 Jan 22;96(2):271-90

Molecular bases for circadian clocks.

Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

Publication Types:

Review
Review, tutorial

PMID: 9988221, UI: 99140720