Accepted Manuscript

Evolution of circadian rhythms: from bacteria to human

Utpal Bhadra, Nirav Thakkar, Paromita Das, Manika Pal-Bhadra

PII: S1389-9457(17)30190-9
DOI: 10.1016/j.sleep.2017.04.008
Reference: SLEEP 3383

To appear in: Sleep Medicine

Received Date: 12 January 2017

Revised Date: 7 April 2017
Accepted Date: 18 April 2017

Please cite this article as: Bhadra U, Thakkar N, Das P, Pal-Bhadra M, Evolution of circadian rhythms:
from bacteria to human, Sleep Medicine (2017), doi: 10.1016/j.sleep.2017.04.008.

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Review

Evolution of circadian rhythms: from bacteria to human

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Utpal Bhadra a,*, Nirav Thakkar a, Paromita Das b, Manika Pal-Bhadra b
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Functional Genomics and Gene Silencing Group, Centre for Cellular and Molecular
Biology, Hyderabad-500007, India

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b
Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad-500007,
India

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Running Title: Evolutionary shift of Circadian rhythm

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* Corresponding author. Functional Genomics and Gene Silencing Group, Centre for Cellular

and Molecular Biology, Uppal Road, Hyderabad, India-50007. Tel.: +91 40 2719 2513; fax:
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+91 40 2716 0591.

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E-mail address: utpal@ccmb.res.in (U. Bhadra).

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ABSTRACT

The human body persists in its rhythm as per its initial time zone, and transition always
occurs as per solar movement around the earth over 24 h. But during transition, while

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traveling, exceeding that of the earth the movements supercedes, which is far beyond the
toleration of the body’s biological clock. This poses an alteration in our physiological
activities of sleep–wake pattern, mental alertness, organ movement, and eating habits,

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causing them to temporarily lose the track of time. This is further re-synchronized with the
physiological cues of the destination over time. The mechanism of resetting of the clocks

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with varying time zones and cues occur in organisms from bacteria to humans. It is the result
of the evolution of different pathways and molecular mechanisms over the time. There has
been evolution of numerous comprehensive mechanisms using various research tools to get

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a deeper insight into the rapid turnover of molecular mechanisms in various species. This
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review reports insights into the evolution of the circadian mechanism and its evolutionary
shift which is vital and plays a major role in assisting different organisms to adapt in different
zones and controls their internal biological clocks with changing external cues.
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Keywords:
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Feedback loop
Bacteria
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Human evolutionary divergence

Transcriptional and post-transcriptional regulation
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Introduction

The last universal common ancestor (LUCA) and all the subsequent life forms that evolved

from it, including humans, have eternally experienced the diurnal changes in the form of day

and night [1,2]. Evolutionary pressures and natural selection have favored the formation of
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genetic circuits that keep track of time and alter the physiology and behavior of life forms so

as to adapt to the daily changes in light and temperature [3]. The day–night cycle resulting

from the earth’s rotation provides cues to these genetic circuits, which act as internal

timekeepers. This allows the life forms to adapt not only to the diurnal changes but also to

the annual changes reflected in the duration of the day versus night [4,5]. This timekeeper

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genetic circuit or the organism’s internal clock inform it about when to venture out for

hunting, when to hide, when to store food for the adverse season, when to prepare for

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hibernation/estivation, and even when to reproduce. These behavioral responses are often

critical to the survival of an organism. Experiments in cyanobacteria, show that in

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competition experiments, strains that exhibit an internal clock periodicity, which is similar to

external environmental periodicity, have a better Darwinian fitness compared to mutant

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strains whose internal periodicity is not synchronous with external environmental periodicity.
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Drosophila lines with a mutation in genes involved in the maintenance of internal periodicity

exhibit different rates of development as well as differences in their lifespan. In higher

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animals such as humans, the periodicity of circadian rhythms is most prominently expressed
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as the sleep–wake cycle. Continued long-term disturbances in a sleep–wake cycle of higher

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animals have been linked to several disorders ranging from reduced cognitive ability to a

higher incidence of cancers.

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The genetic circuits that regulate the internal clock or pacemaker are conserved from

bacteria to plants and humans, though the individual components or the cogs of these
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genetic circuits differ [6]. These circadian pacemakers can make an approximate
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measurement of time and so their phase needs to be set daily in order to keep the internal

clock in synchrony with the environmental clock. Thus, it is not surprising to know that light is

the leading entrainment cue for all circadian circuits encompassing organisms from

unicellular ones to mammals. In animals, like Zebrafish and Drosophila, light–dark cycles

can entrain peripheral oscillators also, which obviously is only possible if at least some

photons reach all internal organs. The evolution of larger and opaque organisms
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necessitated the development of different non-photic entrainment pathways, comprising a

highly specialized master pacemaker in the suprachiasmatic nucleus (SCN) in mammals [7–

9]. In this review, we discuss the development of circadian clocks in different organisms from

prokaryotes to plants and from plants to animals, its molecular pathways and the similarities

and variation during the evolutionary time.

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Circadian rhythms in cyanobacteria

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Circadian rhythms are not restricted to eukaryotes, but also exist in the most primitive

organisms like cyanobacteria. The organism Synechococcus elongates PCC 7942 is a

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preferable genetic model for understanding the molecular details pertaining to clock gene

regulation. Pioneer workers like Susan Golden, Carl Johnson, Masahiro Ishiura, and Takao

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Kondo have incorporated luciferase to monitor rhythmic expression of the gene causing first
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clock mutants in cyanobacteria to evolve. Cyanobacteria are the simplest known organisms

known to exhibit circadian rhythms (Fig. 1). Several species of Cyanobacteria are capable of
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photosynthesis and nitrogen fixation. Oxygen is a by-product of photosynthesis. However,

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the enzymes involved in nitrogen fixation are inhibited in the presence of oxygen. Unlike

heterocystous filamentous cyanobacteria, non-heterocystous and unicellular cyanobacteria

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cannot spatially separate photosynthesis and nitrogen fixation; hence, they resort to
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temporal separation of these two processes. Photosynthesis takes place during the daylight,

while nitrogen fixation peaks during night/dark. Interestingly, this rhythmicity is maintained
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even when they are shifted to constant light (LL), albeit with temperature rhythmicity.
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Reporter gene (luciferase) insertions throughout the chromosome of unicellular

cyanobacteria, Synechococcus elongates revealed that all the 800 insertion lines, which

displayed quantifiable fluorescence, exhibited circadian rhythmicity. Ethylmethane sulfonate

(EMS) mutagenesis and rescue experiments showed that the kai gene cluster, composed of

kaiA, kaiB and kaiC genes, is crucial for the maintenance of circadian rhythmicity. kaiA is

transcribed as a monocistronic mRNA from the kaiA promoter while kaiB and kaiC are

transcribed as a di-cistronic mRNA from the kaiBC promoter, which is then translated into
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two distinct proteins, such as KaiB and KaiC. The process of phosphorylation of the KaiC is

regulated by two proteins, namely KaiA and KaiB. KaiA protein positively regulates the KaiC

phosphorylation and activates accumulation of phosphorylated KaiC, while the KaiB protein

is the negative regulator and promotes the accumulation of non-phosphorylated KaiC protein

[10–12]. Phosphorylated KaiC protein is suggested to stop expression of the kaiBC gene,

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thus forming the negative feedback regulation of the kaiBC gene expression [13]. It has been

discovered [14] that rhythmic phosphorylation of KaiC protein persists in cyanobacteria in

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constant dark conditions (DD). In DD conditions, transcription, translation, as well as

degradation processes, are inhibited [15]. It was further studied [16] that the KaiC

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phosphorylation rhythm in an in vitro system occurs only if proper amounts of KaiA, KaiB and

KaiC proteins as well as ATP exist [16–18]. Research until now reveals the biochemical

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interactions among KaiA, KaiB, and KaiC proteins and their role in driving circadian
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oscillations, but still a regulatory mechanism of phosphorylation of KaiC protein needs to be

elucidated.
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The loci, crm (circadian rhythm modulator) and rpaB (regulator of phycobilisome association
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B) comprise the principal clock. crm comprises the gene encoding a 62 peptide is situated
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upstream of rpaA presumed earlier to affect the expression of RpaA [19]. Earlier, the genes

kai or rpaA were only thought to be responsible for causing arrhythmia, which was later
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disproved [19].

The Crm peptide acts as an allosteric modulator of RpaA, acting as codon initiating
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translation complement to a crm1 mutant. It may be that the altered peptide of transposon
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insertion recessively exerts an effect adverse to the clock. The RpaB response regulator

also plays a part in regulating transcription of the circadian genes. Being highly conserved

[20] it conjugates with the histidine kinase NblS, to regulate genes related to cold, light, and

osmotic and oxidative stress [21–23]. When phosphorylated, RpaB binds to HLR1 (highlight

regulatory 1) regulating the gene expression [21,23]. RpaB activates gene expression, but

usually it inhibits expression of the promoters downstream [21,23,24].

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An HLR1 element in the PkaiBC promoter shows peak binding at dawn when the light is

constant [24], which is 12 h apart from the peak stage of RpaA getting bound to PkaiBC.

Thus, RpaA and RpaB cooperatively perform regulation of the expression of the circadian

target, by displacing RpaB. Genes like kaiBC (class 1) and purF (class 2), that regulate

RpaA have been found to undergo negative regulation by RpaB (87). A phosphorylated

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RpaA shows circadian oscillation, while RpaB phosphorylation occurs when cells are grown

under 12 h of light followed by 12 h of dark [25]. The degree of phosphorylation decreased

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with an overexpressed RpaB receiver domain, which suggests that besides RpaA, PraB too

competes for target binding and function as RpaA modulator via regulating RpaB

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phosphorylation [25]. This clearly unfolds the cross-talk occurring between the circadian

pathway and the environmentally governed pathway and the synchronizations occurring to

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generate rhythmic expression of the genes. To regulate a number of peroxide levels [26], an
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antioxidant named peroxiredoxin is found to be beneficial; circadian rhythms are therefore

better modified using a conserved marker of peroxiredoxin oxidation [2,27,28]. The 24-h
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redox cycles are entrainable and also temperature compensated when checked for the
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oxidized active state. In the eukaryotic cells, however, the rhythms generated via the
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peroxiredoxin oxidation function independently of the feedback loops regulating transcription

and translation [28]. Organisms like flies, mice, Neurospora, S. elongates and the archaeon
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Halobacterium salinarum sp. NRC-1 show rhythms of peroxiredoxin oxidation [2], which

suggest that there may be circadian rhythm in archaeal organisms too. S. elongate rhythms
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of peroxiredoxin oxidation show that besides the canonical clock mechanisms, rhythms are
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driven via an oscillator still unknown to researchers, which might use more light on the

ancient clock driven by oxidative cycles. In this way, a model previously thought of as linear

with input, oscillator, and output, has emerged to be an intricately integrated network,

comprising elements with multiple functions. Hence, it has made huge leaps in expanding

our knowledge of the cyanobacterial circadian clock with future prospects of unfolding

several more interesting aspects in the system.

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Internal ‘clock’ of Neurospora

Over several decades, Neurospora crassa, the filamentous fungus, has served as a brilliant

model for the study of biological, regulatory and circadian rhythms. It typically thrives on

woody biomass in the laboratory on agar-containing medium. Recent studies have

demonstrated the disruption of the natural circadian clock in the dark with a consistent

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metabolism, extending the possibility of removal of the negative feedback loop operating in

case of the circadian oscillator. The core clock component, frq[29] gets activated by the

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White Collar Complex (WCC), which is made of two PAS domains having White Collar-1

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(WC-1) and White Collar-2(WC-2) [30–32]. WC-1 is the photoreceptor for blue light while

WC-2 helps in the light signaling pathway. The circadian late night and early morning cause

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promotion of the transcription of frq in the presence of WCC. FRQ shows a peak during the
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circadian midday 4, where FRQ operates through a negative feedback loop, which causes

inhibition of transcription of frq gene. Once inhibition occurs, frq levels show a decline in the
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circadian in the afternoon and in the early evening. FRQ also upregulates the transcription of

WC-1 and wc-2. The gene, vivid (vvd) is found to be controlled by WCC and shows photo-
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adaptation [33]. VVD shows a competitive interaction with WC-1 to inhibit the formation of
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WCC and transcription of vvd [34].

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Thus, the circadian rhythms get stabilized by the interlocking of the negative and positive

feedback loops [35,36]. FRQ undergoes progressive phosphorylation, making it susceptible

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to ubiquitination and degradation that change its conformation [34,35,37]. Once degraded,
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FRQ generates a fresh cycle of activation of transcription by WCC.

Most studies on Neurospora crassa pertain to circadian rhythms, since it reproduces both

asexually as well as sexually during the day/light, while at night/dark, aerial hyphae are

produced perpendicularly to form the asexual spores, macroconidia. The conidia formation

process is regulated in a daily manner and peaks can be seen just before the dawn. The

rhythm of growth and conidia formation is monitored using ‘race tubes’. At 25°C, the growth
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front advances with a periodicity of about 21.6 h [38]. Many mutations that resulted in

changes in this periodicity were mapped to the frq gene. FRQ protein regulates its own

transcription in a classical negative feedback loop. As the FRQ protein accumulates it is

progressively phosphorylated, by calcium/calmodulin-dependent kinase (CaMK1) and casein

kinase II (CKII), which marks it for degradation. Inhibition of FRQ phosphorylation reduces

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FRQ turnover and interestingly also increases the frq mRNA levels, suggesting that

phosphorylation is not only important for FRQ turnover but also its ability to negatively

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regulate its own transcription. Two PAS domain containing transcription factor proteins –

WC1 and WC2 – form a complex and induce the transcription of frq. WC1 protein acts as a

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photoreceptor and its transcription is induced by light, while WC2 is constitutively expressed.

Phosphorylated FRQ inhibits the transcription-promoting activity of theWC1–WC2 complex,

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thus inhibiting its own transcription. Turnover of phosphorylated FRQ at night permits the
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WC1–WC2 complex to activate transcription of freq. Another PAS domain containing protein,

VVD controls the light response of internal clock to definitive periods of time, such that even
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in constant light conditions, the light-induced processes occur only transiently. VVD also
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regulates its own transcription in a negative feedback loop [39–42]. Rhythmicity of the
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internal clock is also responsive to temperature. At higher temperatures, the FRQ oscillates

at a higher level, such that the trough level of FRQ at 28°C is higher than the peak level at
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21°C. Thus, shifting of Neurospora culture growing at 21–28°C would result in phase s hifting

of the internal clock, which would now correspond to the time of night when the FRQ is
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lowest. Response to temperature may be an intrinsic property of FRQ protein, as some

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mutants of FRQ are not responsive to temperature change. Normal environmental conditions

assume a higher temperature during the day/light phase and a lower temperature

corresponding to the dark/night phase. Interestingly, when Neurospora is placed in

conditions where it is competing for temperature–light, entrainment happens with

temperature cycle, such that conidiation is observed during the light/daytime (lower

temperature). This suggests that light may not be the dominant signal for entrainment of

Neurospora clock [43–46].

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There is an intricate communication between the cell cycle and circadian clock that regulates

cell division cycles, whose alterations cause diseases like cancer. A highly conserved

coupling has been found between the circadian clock and cell cycle via the serine/threonine

protein kinase-29, which currently proposes that circadian oscillations of cell cycle partners

help to synchronize nuclear divisions. It experimentally demonstrates G1 and G2 cyclins,

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CLN-1 and CLB-1 to show oscillation with bioluminescence reporters. Such oscillations are

found to be abolished in the frq knockout mutant. Thus, it shows that the molecular dynamics

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of cell cycle that modulate the circadian clock synchronize nuclear divisions in Neurospora.

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Circadian Rhythm in Plants

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Plants were found to show a variety of movement that was driven by rhythms, like the leaf

movement, growth, germination, stomatal movement, enzyme activity, gas exchange, flower
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opening, photosynthetic activity, and fragrance emission [47]. Kloppstech [48] had elucidated

circadian rhythm in the pea in the abundance of three nuclear transcripts that code for the
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light-harvesting chlorophyll a/b binding protein (LHCB), the small subunit of ribulose-1,5-
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bisphosphate carboxylase/oxygenase and an early light-induced protein. In wheat, the rate

of Cab-1 transcription was found to be controlled by circadian rhythms [49]. Amongst, pea
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and wheat, Arabidopsis thaliana was preferable to combine forward genetic analysis with

molecular gene cloning [50]. Later, the rate of transcription and accumulation of LHCB
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transcript in Arabidopsis [51] were found to regulated by circadian factors.

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Arabidopsis show a rhythmic movement of the cotyledon and leaf, which is mostly based on

differential growth. The elongation rate of the abaxial and adaxial cells of the petiole has a

circadian rhythm that helps in oscillating the positions of cotyledons and leaves. Besides,

there is a change in the rate of elongation of hypocotyl [52] and stem inflorescence [53].The

Arabidopsis genome sequencing has [54] identified novel genes having interlocked feedback

loops that are conserved. The Arabidopsis genome sequencing has led to the identification
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of different orthologs of the recognized clock proteins. The Arabidopsis clock is unique

bearing conserved feedback loops consisting of three interlocked feedback loops, with two

single Myb motif transcription factors, LATE ELONGATED HYPOCOTYL (LHY) and

CIRCADIAN AND CLOCK ASSOCIATED1 (CCA1). CCA1 and LHY are responsible for

repression of the expression of TOC1, the evening-phased protein; this is carried out by the

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evening elements binding in its promoter region [55, 56].The family of PSEUDO-RESPONSE

REGULATOR (PRR) protein has a PSEUDO-RECEIVER (PR) domain and a CONSTANS

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(CO) domain, CO-like domain, TOC1 (CCT) domain and these proteins act as a general

repressor of transcription with DNA binding activity. TOC1 is a member of this protein family

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[57]. TOC1 mediates DNA binding and transcriptional activities through the C-terminus CCT

motif and the N-terminus PR motif, respectively [57–59]. Other experiments revealed that in

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the evening, the expression of TOC1 inhibits the accumulation of CCA1/LHY. These factors
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control the positive regulation of three TOC1 relatives, PRR5, PRR7, andPRR9. TOC1 is a

clock component that controls the oscillation of the mRNA and the protein produced thereof.
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CCA1/LHY is negatively regulated by PRR5/7/9. CCA1 and LHY are transcriptionally

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repressed post-degradation by ZTL to regulate the titer of TOC1, which primarily determines
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the circadian period [60]. Other experiments revealed that in the evening, the expression of

TOC1 inhibits the accumulation of CCA1/LHY. In the morning, mRNA abundance of

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PSEUDORESPONSE REGULATOR 7 (PRR7) and PRR9 is caused by CCA1/LHY

[4,61,62]. In the evening, the circadian loop comprises GIGANTEA (GI), TOC1, EARLY
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FLOWERING 3 and 4(ELF3 and ELF4), and LUX ARRHYTHMO (LUX) [63,64] (Fig. 2).
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There is a formation of complex like evening complex (EC). It is made of ELF3, ELF4 and

LUX suppress the LUX expression, ELF4, GI, TOC1 and PRR9 (Fig. 1) [65–67]. Evening

loop and morning loop are involved in up the fundamental model of the circadian clock in

Arabidopsis [55,68–71].

A novel family of proteins comprising ZEITLUPE (ZTL), LOV KELCH PROTEIN2 (LKP2),

and FLAVIN binding KELCH REPEAT F-BOX (FKF), with PAS/LOV domains, Kelch repeats,
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and F-boxes [72–74], has revealed interesting aspects of the role of protein degradation in

the circadian clock of Arabidopsis. The LOV domains are photoactive, while FKF is solely

concerned with photoperiodism, while ZTL and LKP2 alter the clock. Mutations in ztl-1 and

ztl-2 cause alterations in the length of the period of various rhythm [73,75,76]. The

abundance of ZTL mRNA is not regulated by the clock, the protein is in its peak at dusk, and

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troughs at dawn. The proteasomal degradation pathways are modulated by F-box proteins

that specifically undergo poly-ubiquitination of targets for degradation. Thus, the activity of

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TOC1 is regulated through repression of transcription by CCA1 and LHY, which determines

the circadian period.

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In Arabidopsis, a positive correlation exists between the period length of a set of natural

factors and the length of the day encountered at latitude of origin of the factors, which

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determine whether the altered clock function gets selected under differing environmental
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conditions. The length of the period depends upon carbon fixation, biomass, and survival too

[77] by altering the timing of flowering [78]. The lengthened period delay of CO mRNA
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accumulation, which increases the critical day length essential for CO protein accumulation.
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This late flowering is, therefore, advantageous in the high altitudes before the freezing
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weather sets in.

Circadian rhythms in Drosophila

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Drosophila melanogaster is a holometabolous insect having defined stages of development

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from egg, to larva, then pupa, and finally to an adult. Larva, which emerges from the egg,
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passes through three instar stages and then forms an immobile pupa. Inside the pupa,

metamorphosis transforms it into a fly, which on development, emerges from the pupa in a

process called eclosion [79]. It was observed that eclosion is ‘gated’ by the circadian clock,

such that most flies eclose 1–2 h before dawn [80–82]. Further, the locomotor behavior

assays show that wild-type flies are active during the day/light and inactive during the

night/dark in an LD (light–dark) cycle. Even in constant darkness, flies continue to maintain

the rhythm showing a high locomotor active phase and an inactive (resting) phase
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corresponding to subjective day and night [83]. In addition to locomotion and eclosion, other

behaviors controlled by the clock are egg laying, courtship, gustatory sensitivity, olfactory

sensitivity and learning and memory. Caffeine suppresses the resting phase in flies, which is

similar to its effect on sleep in humans. Similarly, hydroxyzine, a sedative drug used for the

treatment of anxiety and tension in humans, enhances the resting phase in Drosophila [84–

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89] (Fig. 3).

The Drosophila clock

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In a mutagenesis screen, the mutants that displayed short (pers), long (perl) and arrhythmic

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(per0) periodicities mapped to the gene ‘period’ (per) on X chromosome of Drosophila

melanogaster, encoding a PAS domain protein. Different periodicities were observed in

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eclosion as well as locomotor activity [90]. Timeless (tim) was identified in a screen for
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recessive mutations that affect the Drosophila clock. As with per gene, short (tims), long

(timl) and arrhythmic (tim0) mutations of tim have also been identified. dClock (dClk) and
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cycle (cyc; also known as dBmal1) encode bHLH/PAS domain transcription factors that form
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an integral part of the internal clock in flies. Drosophila doubletime (dbt) gene encodes

casein kinase 1, which is directly or indirectly involved in phosphorylation of PER and its
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clearance. SHAGGY (SGG) phosphorylates TIM and controls its entry into the nucleus and
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thereby role in a feedback loop. Clearance of TIM is regulated by light, although light directly

does not affect TIM protein in vitro. TIM degradation is important in resetting the internal
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clock. Mutant flies that lack eyes or the visual transduction pathways can be entrained to
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light and exhibit degradation of TIM in response to light, indicating the presence of a

photoreceptor, which functions independently of vision. CRYPTOCHROME (CRY) was

found to perform the function of the circadian photoreceptor. In cryb mutants, TIM is not

degraded in response to light and both per and time expressed constitutively at most places,

where they are expressed with the exception of lateral neurons. Their expression in lateral

neurons continues to be rhythmic in cryb mutant flies and locomotor rhythms are maintained

[91–99] (Table 1).

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Transcriptional and post-transcriptional feedback models

Biological timekeeping is carried out by the circadian clock. The periodical oscillation of the

circadian clock is known as circadian rhythm. In the case of Drosophila melanogaster, the

period (per) and timeless (tim) core genes are responsible for the circadian rhythm. Gene

expression of per is regulated at transcriptional and post-transcriptional levels. Per genes

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undergo changes in the circadian rhythm at the transcriptional level. The rise of per and tim

mRNA in the evening/dark phase leads to accumulation of PER and TIM in the cytoplasm.

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But Tim protein is light sensitive and accumulates only after dark and stabilizes when it

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forms a dimer along with PERIOD. Again here, PER alone is unstable and dimerization with

TIM stabilizes it. In the absence of TIM, PER is phosphorylated by Double time (Dbt), which

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has kinase activity, and afterwards it is ubiquitinated by Slimb the F-Box protein and
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degraded in the proteasome. Later, the TIM and PER heterodimers enter the nucleus and

accumulate. Further, this dimer binds to Clk/Cyc dimers via a Per-Clk interaction. This
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interaction causes hyperphosphorylation of the clock and thus prevents Clk/Cyc dimers from

binding to the DNA. This negatively regulates transcription of per and tim genes. This
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creates a first negative feedback loop where Period inhibits its own transcription. Further Clk
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mRNA is transcribed in an opposite way to tim/per mRNA, achieving a peak in the early

morning. Clk/Cyc dimers are formed, which activate transcription of two proteins, namely
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Vrille and Pdp1e. Both proteins bind to the V/P boxes in the promoter region of Clk. Vrille

inhibits the transcription of Clk while Pdp1e activates Clk transcription. This forms the
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second negative feedback loop integrated within the first [3,82,100–105] (Fig. 4).
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Evolutionary divergence in clock principles

Ever since life dawned on this planet, there has been a sea change in the environment to

produce the amiable and pleasant conditions that exist at present. With very insignificant

changes in the revolution of the earth around its orbit being approximately 365 days, there

have been appreciable changes in the period of rotation of the earth on its axis, and also in
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the period of revolution of the moon around the earth. This has brought about significant

variations in the circadian clock over the evolutionary time scales. Over the past 4 billion

years, the distance between the earth and the moon has changed greatly, owing to the

effects of the tidal drag, and this has revealed that initially, the earth completed one rotation

cycle in a period of 4 h which extended to approx. 20 h during the Cambrian period [106].

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Organisms evolved and persisted in the ever-changing environment, by virtue of their

Darwinian fitness which resonated between the endogenous periodicity of the organism and

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the period changes of the organism’s environment, which was very beautifully depicted by

the cyanobacteria, Synechococcus [107]. This organism simultaneously evolved a circadian

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mechanism that caused the organism to delay its mechanism over a millennium about 1.9

billion years ago, using KaiB for 0.25–0.5 billion years ago followed by KaiA until the

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geophysical period extended to 24 h as in the present. The cryptochromes present now
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perform functions in both animals as well as plant circadian clocks. The Cry genes have

undergone extensive duplication, as a result of which, there is a modulation of functions. In

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mammals, the CRYs play a role of negative regulation of the circadian clock [108], with no
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significant role in light signaling [109–111].

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The six Cry genes in zebrafish negatively regulate light signaling while in Drosophila, the

single cry gene helps in blue-light signaling the central pacemaker neurons [112].The two
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Cry genes in Arabidopsis help in transmitting blue-light signaling to the clock [113]. In plants,

the photolyase domain may have undergone fusion with the C-terminal DAS domain
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400 million years ago since this domain concurrently occurs in both mosses and angiosperm
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[114]. The C-terminal domains have undergone extensive divergence in animals, such as

Arabidopsis [115], but in Drosophila [97] and Xenopus [116], they play a role in regulation.

Cryptochromes, therefore, act as molecules that exist as homologs/paralogs to the clock,

playing similar roles in plants and animals. The divergence between the two phyla occurred

about 1.6 million years ago, in the pre-Cambrian age. The cryptochromes not only act as

part of the cellular response to ultraviolet (UV) damage but also evolve for executing

functions related to the clock. p53 undergoes phosphorylation by casein kinase 2 when
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subjected to UV damage, thus they activate the tumor suppressor [117]. Besides the yeast

casein kinase 1, HRR25 function in transcriptional response to UV damage. These proteins

are important for the phosphorylation of main plant and mammalian [118] clock proteins [70],

Neurospora [115], flies [93,119]. But these proteins are also crucial for the Wnt signaling,

being important players. Post-Cambrian, the evolution of clock genes is mainly due to gene

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duplication and functional divergence. Per genes show maximum divergence and are

believed to have duplicated first in the vertebrate lineages.

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Duplication in higher eukaryotes exists, where a copy sustains the original functions while

the other undergoes changes in their amino acids to become functionally divergent [120].The

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same genetic trend is set by the circadian system, where the gene related to the clock such

as the per, Cry, cycle, and Clock exists in single copies in insects but as duplicate copies in

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vertebrates. The duplication of a clock gene in a lineage causes it to shuffle to another
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chromosome, with the creation of variation with respect to the ancestral copy. The two

copies show divergence with overlapping occurring very frequently, as in the case of the
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three mPer genes in the murine model that have rhythmic responses in the SCN but have
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variable responses to light [121].

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The mper2 presumptively looks like the positive regulator of the gene, Bmal1 [108] but does

not modulate the rhythm although it regulates the circadian rhythm. The very same per gene
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in quails shows a functional divergence only with qPer2, but not with qPer1 mRNA when

induced by light [122]. The Drosophila timeless (tim) genes have two paralogs, tim and tim2
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(also called timeout), but a single copy of Tim exists in mammals [123–125]. The Tim in
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mammals apparently is orthologous to tim2 in the fly, which means that tim2/mTim is the

ancestor and has undergone a recent duplication in the insects, which is evident in Dipterans

and Lepidopterans only, but is missing from the genome of the honeybee. Thus, we can

conclude that the duplication of tim may have preceded the above two orders more than

200 million years ago. mTim has been reported to regulate the clock in mammals [126] but

Drosophila does not provide any such evidence for tim2.

 ACCEPTED MANUSCRIPT
Gene duplication is exemplified by another example of the silkmoth, Antheraea pernyi per

gene has two paralogs on different sex chromosomes perZand perW [127]. Several other

sequences of the per encode an antisense transcript in the female sex chromosome W but

are missing in the homogametic males (chromosomes ZZ), who are rhythmic, which proves

that the perW is not essential for clock function. The genes perW and perZ show a great

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degree of identical sequence, which suggests that perW may evolve by translocating very

recently from the Z chromosome [127].

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There are certain situations when paralogs show divergence and in doing so lose their

complete function, turning out to be pseudogenes, as in the case of the hPer4 in human and

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rmPer4 in Rhesus monkey [124,128], in that neither gets transcribed nor codes for

contiguous segments that produce the PER-like protein. Per4 may have its origin from Per3

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via retrotransposition, which perhaps might have to move the gene to its current position
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[128]. After such findings shedding light on the evolution of the clock gene per, there is much

waiting to be discovered about the evolution of the other genes such as cry.
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Protein regulation of circadian rhythms

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CLOCK is Circadian Locomotor output cycles kaput. The clock is responsible for the

generation of the circadian rhythms. In the case of most of the clock genes Per-Arnt-Sim
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(PAS) domain mediates protein interaction and helps in the regulation of the circadian

rhythm. PAS is also related to the transcription factor and also works as a heterodimer.
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There are numerous numbers of Rof clock genes being cloned in different genera of
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organisms. In the case of neuro POR white collar 1 and white collar 2 and frequency are

present, in the case of humans period (per) and timeless (tim) genes are present. In

Drosophila period (per) double time, timeless (tim), and clock genes are present. In the case

of mammals period genes per 1, per 2, per3, bmal1, clock and timeless (tim) genes are

present. In the case of mammals clock genes or bmal 1genes through the help of

transcription binding factor, E-Box, which is located near the period gene, activate per genes

namely per 1, per 2, per 3. After the transcription of per 1, per 2, per 3, mRNAs are moved
 ACCEPTED MANUSCRIPT
and are translated to PER 1 PER 2 and PER 3 proteins. CRY 1 and CRY 2 are

cryptochrome proteins and they act as a negative feedback loop. PER after interaction with

cryptochrome proteins, translocated per proteins from the cytoplasm to the nucleus.

Heterodimers of per and cry translocate back to the nucleus repressing their own

transcription by the complex formation of CLOCK and BMAL1. There is another regulatory

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loop by a heterodimer of CLOCK and BMAL1. The retinoic acid-related orphan nuclear

receptor is activated for transcription. There is competition between REV-ERBα and RORα

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to bind to the retinoic acid related orphan nuclear receptor. Bmal 1 is activated for

transcription by RORs whereas transcription process is repressed by REV-ERBα. ROR and

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REV-ERBα are both responsible for positive and negative regulation of the circadian

oscillation [92,93,119,129–134].

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The mammalian clock environment

Physiological parameters such as temperature, blood pressure, metabolism, retinal

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electroretinogram responses and the circulating titers of hormones in mammals are

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subjected to a 24-h rhythm that persists even in the absence of the cycles of sleep/wake or
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light/dark [56,135–137].The circadian clock in mammals has three main components which

include the input signaling pathways, the main pacemaker or the central oscillator and output
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signaling pathways. The signals or cues received from the environment are known as

zeitgebers and get communicated to the main pacemaker through the input pathway where
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they synchronize the oscillations in the pacemaker with respect to the changes in the solar
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day–night cycle. Zeitgebers includes factors such as temperature, light, noise, food, and

melatonin [138].The pacemaker for the circadian clock includes two clusters of neurons

called the SCN, comprising about ~100,000 neurons in each, present in the hypothalamus

just above the optic chiasma [139]. Light cues are perceived by the retinal cells and get

transmitted to this region via the retinohypothalamic tract where signal transduction occurs

via the influx of calcium after an interaction with the NMDA receptors to activate the IP3 and

ryanodine receptors. The central oscillator then generates and maintains the rhythmicity
 ACCEPTED MANUSCRIPT
upon the necessary input perception. These signals are then diffused consecutively to the

peripheral organs via the output pathway such as the glucocorticoid pathway [140].The core

genes of the mammalian circadian clock comprise 14 genes, including members of the Per,

Cry, Bmal, Clock, Ror and Rev-Erb families, that show an interaction via the positive and

negative feedback loops of the transcription and translation [141].

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The circadian day ensues with the CLOCK/BMAL1 activator heteromeric complex formation,

which binds the E-box sequences at the promoter of the genes, Per, Cry, Ror and Rev-Erb

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[118,138,142]. The two major loops, REV-ERB/Bmal/ROR and PER/CRY loops,

interconnect [142]. The transcription of the Per 1, 2, 3 and Cry 1, 2 genes activates in the

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PER/CRY loop and, in turn, inhibits the transcription of the clock genes via the

CLOCK/BMAL1.The REV-ERB/Bmal/ROR loop helps in driving the oscillations which are

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self-sustained [142]. Both of the loops actually operate independently but need to
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interconnect concomitantly to produce oscillations within a period of 24 h, which is why they

comprise two transcription/translation feedback loops (TTFLs) [143,144].

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As an exception to the transcriptional rhythms, in some cases there also exists a non-
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transcriptional/translational rhythm as in the case of the human red blood cells. These cells
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are non-nucleated where peroxiredoxins undergo oxidization in a circadian rhythm with the

temperature-compensation ability of the oscillators [15,16,145,146]. On the contrary, the

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nucleated cells are influenced by the cytoplasmic peroxiredoxin clock, which takes place via

the transcriptional clock factors [146].

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Resetting the human clock

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The biological clock is an innate mechanism of one’s body. This clock regulates the normal

physiological functioning of the body. There are several genes responsible for the biological

clock. The circadian clock gets reset by an external signal such that these rhythms

generated get synchronized with variations occurring daily in the environment [147]. The

SCN present in the anterior position in the hypothalamus is responsible for the physiological
 ACCEPTED MANUSCRIPT
functioning of our body. The SCN works as a pacemaker of the body. Several genes, namely

per, cry, clock, are responsible for the functioning of the biological clock (Table 2). Although

in today’s world of globalization and fast-moving life, external sources such as mobile

phones laptops in some ways regulate a human biological clock. But among several factors,

light is the major factor in the regulation of circadian rhythm in the mammalian body. The

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mper1 is a gene sequence present in mammals which is homologous to the per gene in

Drosophila and for genes in Neurospora. The per and freq are clock genes in Drosophila and

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Neurospora. For the determination expression profile of mper1 and its potential role, light

treatment was used. The light treatment helped to reset the circadian clock. The reaction of

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circadian rhythm towards light is used as a mechanistic process to determine the circadian

pacemaker and the organization of the circadian system. The expression profile of mper1

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peaks during daytime and lowers during the night. This expression profile suggests
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dependency of mper1 on the daytime light [29,118,148–155] (Fig. 5).
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The clock gene expression is thought to be a culmination of molecular mechanisms

operating under the influence of light that translate mRNA to a functional regulatory protein.
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Light acts as the chief modulator and cue for the entrainment of the biological clock. The
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ambient light/dark cycle intricately regulates the phases of the SCN. There is a very fast

entrainment in the presence of transient light exposure at night [156]. Light stimulates the
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retinal ganglion cells that express melanopsin and release the neurotransmitter at the

retinohypothalamic tract terminals [156,157], which binds to the postsynaptic receptor. Then
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they activate the second messenger-based signal transduction pathway in the SCN. Light
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cues, when perceived by the central oscillator, cause the stimulation of the mitogen and

stress-activated kinases that activate CREB. CREB is responsible for the expression of

genes with CREB in the promoters such as c-Fos and Per1 [158]. The mRNA translation

occurs mainly at the initiation step [159] with the CREB co-activator, CRTC [160,161].

Translation initiates when the m7GpppN structure present at 5′-end gets recognized by the

eukaryotic translation initiation factor 4E [162], where eIF4E binds the complex of eIF4F
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having eIF4A and eIF4G [159,163]. eIF4E acts as the rate-limiting translation factor whose

presence in abundance occurs due to the presence of mTOR protein. The mTOR obstructs

binding of eIF4E to eIF4G, hence the mRNA translation is obstructed [159,163].The activity

of the eIF4E gets modified by the p38 and p42/44 MAP kinases through phosphorylation at a

single amino acid, Ser209 [164]. The MAPK interacts with the MAPK-interacting

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serine/threonine kinases such as MNK1 and MNK2 [165].

A recent study has exposed that MAPK/MNK phosphorylation of the cap-binding protein,

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eIF4E, that in turn regulates the translational initiation of the mRNA of the clock proteins

PER1 and PER2. This phosphorylation is intricately regulated in the SCN by light and shows

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a constant circadian rhythm. In situations demanding an upregulated PER, eIF4E gets

phosphorylated to trigger the Per protein; but under constant environmental light conditions,

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eIF4E just timely modulates the PER synthesis.
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An unprecedented increase in the PER leads to the disruption of the dynamic balance of the

feedback loops and causes the resetting of the phase of the SCN clock [157]. In this way,
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the activity of eIF4E is highly regulated to ensure a well-timed protein synthesis and normal
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functioning of the circadian clock.

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A common link in the clock control process between plant and animals
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The circadian clock generates certain kinds of oscillations known as circadian rhythm.

Circadian rhythm is found in all kinds of organisms starting from Cyanobacteria, Neurospora,
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plants, mammals, and other animals. In plants, there are three transcriptional loops present
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in the plant circadian clock. PSEUDO-RESPONSE REGULATOR 1 (PRR1) or TOC 1 is the

clock regulatory gene, Circadian Clock associated 1 (CCA1) and LATE ELONGATED

HYPOCOTYL (LHL). CCA1 and LHL bind to TOC 1 and block its transcription. TOC 1 is

hypothesized to activate an unknown compound X. It has been considered that there may be

other evening-faced genes. In the case of animals, SCN is an important component of the

circadian clock. It is considered to be a pacemaker of the circadian clock. The mammalian

circadian oscillation requires both negative and positive elements. The negative elements
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are per 1, per 2 genes and cry 1 and cry 2. The positive-acting proteins are Bmal 1 and clock

genes. In plants, circadian rhythm is essential for maintaining the balance of the whole

system. Circadian rhythm in plants helps plants to get accustomed to seasonal changes,

enzymatic activity, stomatal changes, gas exchange, germination, pollination, leaf

movement, photosynthetic activity, fragrance emission, and growth. It also helps in

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synchronization to the light/dark cycle. In the case of animal circadian genes, per 1, per 2,

cry 1, and cry 2 are homologous to the plant circadian rhythm. The function of both genes is

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to maintain circadian oscillation and thus help in the correct running of the biological clock

[55,77,166–171].

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From bacteria to humans

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In the case of cyanobacteria, kaiA, kaiB, and kaiC are important in the regulation of circadian
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oscillation. These three genes are very important for circadian oscillation. Deletion of any of

the genes will result in arrhythmicity of the system. Kai BC complex and Kai A transcript are
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numerous. In the case of negative feedback regulation, Kai BC is repressed by

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overexpression of KaiC. This results in the elimination of the loss of rhythmicity. On the other

hand, positive feedback regulation is found when Kai A overexpression activates Kai B C
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complex. In the case of mammals, period genes per 1, per 2, per3, bmal1, clock, and
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timeless (tim) genes are present. In the case of mammals, clock genes or bmal 1 genes

through the help of transcription binding factor, E-Box, which is located near the period gene,
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activates per genes namely per 1, per 2, per 3. After the transcription of per 1, per 2, and per
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3, mRNAs are moved and are translated to PER 1, PER 2, and PER 3 proteins. CRY 1 and

CRY 2 are cryptochrome proteins and they act as a negative feedback loop. PER after

interaction with cryptochrome proteins translocate per proteins from the cytoplasm to the

nucleus. Then heterodimers of per and cry translocate back to the nucleus repressing their

own transcription by the complex formation of CLOCK and BMAL1. There is another

regulatory loop by a heterodimer of CLOCK and BMAL1. The retinoic acid-related orphan

nuclear receptor is activated for transcription. There is a competition between REV-ERBα

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and RORα to bind to the retinoic-acid-related orphan nuclear receptor. Bmal 1 is activated

for transcription by RORs whereas the transcription process is repressed by REV-ERBα.

ROR and REV-ERBα are both responsible for positive and negative regulation of the

circadian oscillation. Bacterial circadian rhythm is very important; circadian rhythm plays a

role in cell division and adaption to the environment. Similarly, in the case of humans,

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circadian rhythm carries out physiological work. It helps in creating a balance in the all the

physiological behavior, starting from the sleeping pattern, hormonal balance, feeding pattern,

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cell regeneration and other biological activities. In the case of both cyanobacteria and

humans, genes responsible for the circadian clock are homologous. Both in cyanobacteria

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and humans, circadian rhythm is responsible for maintaining balance in physiological

activities. There are differences in the structural organization of the genes, but they perform

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the same task of maintaining circadian rhythm. The regulation of genes responsible for
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circadian rhythm in cyanobacteria and humans is also similar [12,172–178].
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Bioinformatics outlook in the circadian evolutionary processes

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Bioinformatics studies pertaining to circadian rhythm involve the use of an appropriate

experimental model that helps to organize information and helps to understand the
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hypothesis to be investigated. Such models might include a diagram correlating different

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genes and proteins or may be some mathematical equations. Models can be implemented

as contextual or conceptual; they may be represented as diagrams or in the form of

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computations. Quantitatively they may be discrete, continuous, stochastic or deterministic

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[179]. A model to determine circadian attributes needs to have essentially the characteristics

of rhythmicity with a circadian period and should get compensated with temperature and

should be able to be entrained by external cues with amplitudes that can produce a

functional output and never get dampened under constant conditions. To date, various

models have been created to mimic the circadian clock in diverse species through

mathematical differential equations as in the case of mammals, such as mus musculus,

[142,180,181], for insects such as D. melanogaster [182–185], plants particularly, A. thaliana

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[186–188] Ostreococcus tauri [189,190], fungi (Neurospora crassa) [191–193], and

cyanobacteria [194,195]. The core components of the clock mechanism need to be set to

find out its properties such as the endogenous self-sustained oscillations, robustness,

cellular synchronization and ability to be entrained by external cues. This can be achieved by

setting up models whose other functions may be to understand the alterations in the

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properties of the clock and to correlate the situation with a de-regulated pathological

condition [142,196]. Furthermore, there are certain models that try to simulate the network of

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the circadian components at the level of the system using clock-controlled genes which react

to a circadian rhythm in a tissue-specific manner [197–199], hence they can be an answer to

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studying circadian effects in a specific tissue. It is important to understand the structure of

the gene regulatory network via the identification of various enhancer or repressor binding

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sites at the promoter of the gene using ChIP-seq.
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A simple sequence or position weight matrix (PWM) can help in identifying motifs such as
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the E-, D-boxes, and RREs, for the principal clock genes. Then this matrix can be matched

to the genomic positions and analyzed using statistics of occurrence of the specific gene
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targeting the clock [200].The Goodwin oscillator represents the first biochemical negative-
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feedback oscillator model that interconnects three components with rates of production and

degradation [201]. Coupled oscillators represent the focus on different influences on the
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different clock components [202]. A mathematical description of the circadian system may be

obtained using the conventional ordinary differential equations (ODEs), gene expression
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changes over time [180,181,203], or the delayed differential equations (DDEs) to explicitly
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express time [183,204]. The latter has been found to be useful in overcoming the missing

factor and in simplifying the model [205]. Besides, Boolean models sometimes mimic the

basic properties of the core components of the clock in A. thaliana [206]. For the preparation

of a mathematical model from data, one needs to overlook the experimental inaccuracies via

statistics [207].
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The changes in gene expression should be unfolded via luciferase assays [208,209],

producing data having direct or indirect information about the gene’s circadian phenotype.

Trigonometric functions can be commonly used, such as the one below, which can fit the

expression into a linear regression [210] corresponding to the phase shifts in each of its

peaks.

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The circadian model is primarily the consolidation of the network structure with an additional

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data on time-series. The network structure designs a set of the equation and the time series

determines the values of the model such as the rate of inhibition of the proteins or RNAs or

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any other experimental parameters. Large and complex mathematical models are usually
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optimized using a global optimization algorithm like the Evolutionary Algorithms [211] or

approaches based on the Markov Chain Monte Carlo (MCMC) sampling, like the simulated
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annealing [212]. COSOPT is an algorithm that helps to incorporate the dampening factor and
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provides necessary information about the periods or the amplitude [213]. A database called
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CircaDB [214] has been found to be instrumental in annotating the expression of several

circadian genes with time. Alternately, one can use the maSigPro package meant for
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Bioconductor to understand the differential expression of the genes with time. The online

service provider BioDare is useful for recognizing the period of the circadian time [215].
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Analysis of the bifurcation diagrams identifies key parameters pertaining to a particular

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circadian phenotype that mimics a pathological condition, and hence identifies the genes or

proteins that disrupt the biological clock and hence find out its role in the disease

progression.

Systems biology methods involving analyzing and integrating the different types of large-

scale data such as those of gene co-expression, ChIP-seq data, protein–protein interactions,

GO terms and analysis of the KEGG pathways [216,217] are preferred for understanding the

relation of the clock-controlled genes to a larger network of the similarly functioning genes
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[218–220]. Hence, an integration of such interdisciplinary approaches may furnish newer

strategies in the field of chemotherapeutic treatment and may shed abundant light in

unleashing factors that will restore the circadian rhythm timing in various diseases due to

perturbed clock factors.

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Conclusion

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Taken together, the diversity of hierarchical mechanisms of an individual organism is

impressive, tightly organized, but limited to their normal sleep/wake distribution. The

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machinery can be compared to an orchestra’s conductor. The circadian rhythms combat and

overcome many gestures to set the timing of the sleep/wake cycle of individual musicians

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playing their own instruments. However, to overcome sleeping disorders, the success of the
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circadian symphony not only depends on the role of the conductor but also relies on the

individual organisms as musicians to play their roles.

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Remarkable progress in the field of circadian rhythms or sleep/wake cycles has been made
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in recent decades. The identification of many key genes and their roles in sleep/wake
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balance and the discovery of the roles in peripheral oscillators have advanced our

understanding, but a clear picture of the circadian timing in various organisms is far from
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complete. We speculate that the hierarchical evolution and inventory of clock factors have

just begun. The present report is in support of this, but numerous clock components still
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remain to be identified in the future.

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The mechanism of circadian rhythm in the fly provides an excellent opportunity as a model

organism, and can promote the understanding of a complex mechanism such as the

sleep/wake rhythm in human. In many organisms, sleep disorders should focus on

interconnecting the central oscillation to sleep behaviors and physiology. The cyclic rhythmic

transcripts and their integration with diversified related protein molecule and small molecule
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rhythm might provide cutting edge technological emerge. These complete pictures in the

different organism might show an overall picture of sleep behavior and physiology.

In addition, we pointed out that the relationship or coupling of clock protein and specific

signals cascade illustrated the different molecular mechanisms of circadian rhythm. The

endeavors between physiology and circadian behaviors will require the applicability of many

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genetic tools, genomics, proteomics, physiology, and behavior. In fact, in different

organisms, major attraction lies in multidisciplinary events, and it is speculated that future

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experiments for illustrating circadian rhythm in different organisms will not only bring us

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enormous exciting surprises, but will also reward us with immense adventures.

Conflict of interest

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The authors declare that they have no competing interests.
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Acknowledgements
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We greatly acknowledge the discussion and technical assistance provided by all lab
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members. The work was supported by an HFSP Young Investigator Grant (RGY020),

Wellcome trust fellowship (GP0145) awarded to UB. Wellcome Trust International

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Fellowship was awarded to MPB (GAP0065). We dedicate this manuscript to the memory of
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a renowned physician in Calcutta, Dr Malaya Kr. Pal, who inspired us in Science.

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Fig. 1. Phylogenetic tree showing simplest organisms to higher forms exhibiting circadian

regulation.

Fig. 2. Circadian feedback loops in plants.

Fig. 3. Molecular mechanism of Drosophila clock.

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Fig. 4. Interlocked feedback loops: light–temperature.

Fig. 5. Transcriptional–translational feedback loop in the mammalian clock.

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Table 1. Fly Clock genes and proteins.

Gene Protein

period (per) RNA and protein cycle. Binds to TIM. Inhibits dCLK/CYC function.

RNA and protein cycle. Binds to PER and facilitates PER nuclear

timeless (tim) transport. Inhibits dCLK/CYC function. Degrades in response to

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light.

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Doubletime Constitutively expressed. Ser/Thr kinase (CK1ἐ). Phosphorylates

(dbt) TIM-free PER, promoting its degradation.

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RNA and protein cycle. bHLH–PAS protein. Heterodimerizes with
dClock (dClk)
CYC and promotes transcription from E-box.

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Constitutively expressed. bHLH–PAS protein. Heterodimerizes
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cycle (cyc)
with dCLK and promotes transcription from E-box.

cryptochrome RNA cycles. Circadian photoreceptor. Promotes light-dependent

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(cry) degradation of TIM. May be essential for some peripheral clocks.

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RNA and protein cycle. bZIP transcription factor. May repress per
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Constitutively expressed. Ser/Thr kinase (GSK-3). Promotes TIM

shaggy
phosphorylation and nuclear localization of PER/TIM complex.
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Table 2. Mammalian Clock genes and proteins.

Gene Protein
Period 1 (Per1) RNA and proteins cycle. Physically associates with CRY and among
Period 2 (Per2) PER proteins. Activator of BMAL1 function. (Mutation in Per2 is
Period 3 (Per3) associated with FASPS.)
Constitutively expressed. Closest Drosophila relative is timeout.
Timeless (Tim)
Homozygous null mutant is lethal, making it impossible to
conclusively establish clock function.

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Casein kinase 1ἐ Constitutively expressed. Protein kinase (CK1 ἐ). Phosphorylates
PER and affects PER stability.
Circadian locomotor

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output cycle kaput Constitutively expressed. Heterodimerizes with BMAL1 and binds to
E-box. Promotes transcription of Per and Cry.

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(Clock)

Bmal1/MOP3 RNA cycles. Heterodimerizes with CLOCK and binds to E-box.

Promotes transcription of Per and Cry.

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Cryptochrome 1 AN
(Cry1)
RNA cycles. Mutations alter rhythmicity in mice, implying a central
oscillator function. Physically associates with and stabilizes PER.
Cryptochrome 2
Inhibits transcription of Per and Cry.
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(Cry2)

Nfil3/E4BP4 In chicken, represses cPer expression78. In mouse, suppresses

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mPer1 expression in cell-culture assays79

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Highlights

• The genetic circuits regulating the pacemaker are conserved from bacteria to
humans.

• Light is the leading entrainment cue for all the circadian circuits across all taxa.

• Light entrains through peripheral oscillators in lower taxa and via pacemakers in the

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higher taxa.

• The per and Cry genes have extensively duplicated to show modulation of function

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The hierarchical mechanisms of an organism’s normal sleep/wake distribution are
intricately organized.

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