University of Groningen
Peroxiredoxins are conserved markers of circadian rhythms
Edgar, Rachel S.; Green, Edward W.; Zhao, Yuwei; van Ooijen, Gerben; Olmedo, Maria; Qin,
Ximing; Xu, Yao; Pan, Min; Valekunja, Utham K.; Feeney, Kevin A.
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Nature
DOI:
10.1038/nature11088
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Edgar, R. S., Green, E. W., Zhao, Y., van Ooijen, G., Olmedo, M., Qin, X., Xu, Y., Pan, M., Valekunja, U.
K., Feeney, K. A., Maywood, E. S., Hastings, M. H., Baliga, N. S., Merrow, M., Millar, A. J., Johnson, C. H.,
Kyriacou, C. P., O'Neill, J. S., Reddy, A. B., & O’Neill, J. S. (2012). Peroxiredoxins are conserved markers
of circadian rhythms.
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ARTICLE
doi:10.1038/nature11088
Peroxiredoxins are conserved markers of
circadian rhythms
Rachel S. Edgar
1
*, Edward W. Green
2
*, Yuwei Zhao
3
*, Gerben van Ooijen
4
*, Maria Olmedo
5
*, Ximing Qin
3
,YaoXu
3
, Min Pan
6
,
Utham K. Valekunja
1
, Kevin A. Feeney
1
, Elizabeth S. Maywood
7
, Michael H. Hastings
7
, Nitin S. Baliga
6
, Martha Merrow
5
,
Andrew J. Millar
4,8
, Carl H. Johnson
3
, Charalambos P. Kyriacou
2
, John S. O’Neill
1
& Akhilesh B. Reddy
1
Cellular life emerged 3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable
daily cycles owing to the Earth’s rotation. The advantage conferred on organisms that anticipate such environmental
cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions.
The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock
genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that
oxidation–reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains
of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the
interconnectivity between these metabolic cycles and transcription–translation feedback loops of the clockwork in
each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic
mechanisms after the Great Oxidation Event
2.5 billion years ago.
Circadian rhythms are considered to be a feature of almost all living
cells. When isolated from external stimuli, organisms exhibit self-
sustained cycles in behaviour, physiology and metabolism, with a
period of approximately 24 h
1
. Circadian clocks afford competitive
selective advantages that have been observed experimentally
2,3
, and
disturbance of circadian timing in humans, as seen in rotational shift
work and jet lag, carries long-term health costs
4
. For all organisms in
which the molecular timing mechanism has been investigated, a com-
mon model has arisen, namely a transcription–translation feedback
loop (TTFL). TTFL components are not, however, shared between
organisms. For example, the cyanobacterial clock is modelled around
three proteins: KaiA, B and C. In the fungus Neurospora crassa, a loop
involving the protein FREQUENCY (FRQ) and the WHITE
COLLAR (WC) complex is thought to drive cellular rhythms, whereas
the plant TTFL involves elements including TOC1 and CCA1 (refs 1,
5). Furthermore, although some multicellular organisms such as
Drosophila and humans possess homologous components (for
example, the Period proteins), their functions seem to differ between
organisms
6–8
. Therefore, across phylogenetic kingdoms, there are
apparently no common ‘clock’ components, suggesting that daily
timekeeping evolved independently within different lineages. The
converse, however, could equally be true, and the primary premise
of this study was therefore to test the hypothesis that circadian clocks
may instead have a common ancestry.
Conservation of peroxiredoxin in circadian systems
Recent studies show that the oxidation state of highly conserved per-
oxiredoxin (PRX) proteins exhibit circadian oscillations in cells from
humans, mice and marine algae
9,10
, probably reflecting an endogenous
rhythm in the generation of reactive oxygen species (ROS)
10
. Because
virtually all living organisms possess peroxiredoxins
11
, we proposed
that this marker for circadian rhythms in metabolism may be
functionally conserved across all three phylogenetic domains:
Archaea, Bacteria and Eukaryota. Peroxiredoxins are peroxidases,
the activity of which is dependent on the oxidation of a key
‘peroxidatic’ cysteine residue (C
p
) in the active site, that is absolutely
conserved, as are neighbouring proline and threonine/serine residues:
conforming to a PXXX(T/S)XXC
p
consensus
12
. Crucially, the catalytic
cysteine can become over- or hyperoxidized (PRX-SO
2/3
), rendering
the peroxiredoxin catalytically inactive, but able to participate in ROS
signalling and chaperone activity
13
. Once overoxidized, peroxiredoxin
can be recycled by sulphiredoxin.
We previously characterized circadian cycles of peroxiredoxin oxida-
tion using antiserum directed against the oxidized active site, which
recognizes both over- (PRX-SO
2
) and hyper- (PRX-SO
3
)oxidized
forms
9,10,14
. To determine whether this antiserum (raised against an
oxidized DFTFVCPTEI peptide) could be used to assay over-/hyper-
oxidation in diverse species, we performed several sequence alignments
to compare peroxiredoxin protein sequences across a variety of cir-
cadian model organisms. This revealed a remarkable degree of conser-
vation across all phylogenetic domains, especially within the active site
(Fig. 1a, Supplementary Fig. 1 and Supplementary Tables 1–7). Even
when we examined the structure of HyrA, the most distantly related
peroxiredoxin orthologue in the archaeon Halobacterium salinarum sp.
NRC-1, we found that amino acid substitutions would not perturb the
geometry of the active site (Fig. 1b). Together, these findings suggested
that the same antiserum could be used to probe oxidation rhythms in
potentially any organism expressing a peroxiredoxin protein. This was
confirmed by gene knockout and peroxide treatment in several repres-
entative organisms (Supplementary Figs 3 and 7–9).
*These authors contributed equally to this work.
1
Department of Clinical Neurosciences, University of Cambridge Metabolic Research Laboratories, NIHR Biomedical Research Centre, Institute of Metabolic Science, University of Cambridge,
Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK.
2
Department of Genetics, University of Leicester, Leicester LE1 7RH, UK.
3
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee
37235-1634, USA.
4
Synthetic and Systems Biology (SynthSys), Mayfield Road, Edinburgh EH9 3JD, UK.
5
Department of Molecular Chronobiology, Center for Life Sciences, University of Groningen, 9700
CC Groningen, The Netherlands.
6
Institute for Systems Biology, 401 Terry Avenue North, Seattle, Washington 98109, USA.
7
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
8
School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK.
24 MAY 2012 | VOL 485 | NATURE | 459
Macmillan Publishers Limited. All rights reserved
©2012
Peroxiredoxin rhythms in eukaryotes
Using the PRX-SO
2/3
antiserum, we first examined circadian time
courses from a range of eukaryotes under constant conditions (that
is, in the absence of external timing cues). In mice, PRX-SO
2/3
and
total PRX1 exhibited a daily cycle in liver tissue and also in the central
pacemaker, the suprachiasmatic nuclei (SCN) of the hypothalamus
(Fig. 2a). Interestingly, between the two tissues, peroxiredoxin oxida-
tion rhythms were in distinct, and different, phase relationships with
respect to total PRX1 and BMAL1 protein, suggesting a difference
between brain and peripheral tissue (Fig. 2a) , as observed previously
for other clock components
15
.
To extend these findings beyond vertebrates, we examined perox-
iredoxin rhythms in the fruitfly Drosophila melanogaster. We pooled
whole heads from insects maintained in constant darkness over two
circadian cycles after they had been stably entrained to 12 h light, 12 h
dark cycles. Again, circadian oscillations in PRX-SO
2/3
immuno-
reactivity were observed, as well as in the clock protein Timeless
(TIM) (Fig. 2b).Similarly,seedlingsfrom the plant Arabidopsis thaliana
exhibited robust PRX-SO
2/3
oscillations in free-running conditions of
constant light, which were also seen in the filamentous fungus
Neurospora crassa—another well-characterized clock model system
(Fig. 2c and Supplementary Fig. 2). Therefore, just as in tissue from
‘complex’ vertebrates, this range of ‘simpler’ eukaryotic systems had
robust peroxiredoxin oxidation cycles, with peaks tending to occur
around anticipated dawn.
Peroxiredoxin rhythms in prokaryotes
Having observed ,24 h peroxiredoxin oxidation rhythms in
organisms with nucleated cells, we next sought to examine represent-
ative prokaryotes from each major domain—Bac teria and Archaea.
Time (h)
0 9 15 3 6 12 18 21
PRX1
PRX-SO
2/3
BMAL1
β-actin
Mouse
a
FRQ
Time (h)
0 12 20 4 8 16 24 28 32 40 36 44 48
Coomassie blue
PRX6-SO
2/3
Fungus
c
Fly
b
Time (h)
0 12 20 4 8 16 24 28 32 40 36 44
β-actin
PRX1
PRX-SO
2/3
BMAL1
β-actin
Time (h)
0 12 20 4 8 16
PRX-SO
2/3
TIM
Liver
SCN
48
0 4 8 12162024283236404448
0
20
40
60
80
100
Time (h)
Normalized
Abundance (%)
TIM
PRX-SO
2/3
0 3 6 9 12 15 18 21 24
0
20
40
60
80
100
Time (h)
Normalized
abundance (%)
PRX-SO
2/3
BMAL1
PRX1
Time (h)
Time (h)
Normalized
abundance (%)
0 4 8 12162024283236404448
0
20
40
60
80
100
PRX6-SO
2/3
FRQ
0 4 8 12162024
0
20
40
60
80
100
Normalized
abundance (%)
PRX-SO
2/3
BMAL
1
PRX1
Figure 2
|
Peroxiredoxin oxidation cycles are conserved in eukaryotic
models of the circadian clock. a–c, Representative immunoblots probed for
oxidized/hyperoxidized 2-Cys peroxiredoxin (PRX-SO
2/3
or PRX6-SO
2/3
) are
shown for mouse (Mus musculus; a), fruitfly (D. melanogaster; b), and fungus
(N. crassa; c). For each model system, the organism was sampled under free-
running conditions. Loading controls show either b-actin immunoblots or
Coomassie blue-stained gels loaded with identical samples used for
immunoblotting. Immunoblot quantification by densitometry is shown below
each panel (mean 6 s.e.m.) for n 5 3 biological replicates. See Supplementary
Fig. 2 for plant rhythms, and Supplementary Table 9 for cycle period estimates
(by harmonic regression) and detailed statistics (by ANOVA).
a
Thr
Pro
Cys
Thr
Pro
Cys
Ser
Pro
Cys
Generic
(PRX-V)
PGAFTPGCSKTH PLDFTFVCPTEI PFDFSPVCATEL
b
Human
(PRDX2)
Archaea
(HyrA)
Ot
Se
At
Hs
Mm
Ce
Se
Dm
Nc
Has
Figure 1
|
The peroxiredoxin active site is highly conserved in all domains of
life. a, Multiple sequence alignment showing peroxiredoxin amino acid
sequences. The highly conserved active site is underlined. Representatives
shown from Eukaryota (At, A. thaliana; Ce, Caenorhabditis elegans; Dm, D.
melanogaster; Hs, Homo sapiens; Mm, M. musculus; Nc, N. crassa; Ot, O. tauri;
Sc, S. cerevisiae), Bacteria (Se, S. elongatus sp. PCC7942) and Archaea (Has, H.
salinarum sp. NRC-1). b, Critical residues in the active site of 2-Cys
peroxiredoxins (in bold) are conserved in all organisms. Structures were
derived from human PRX-V (Protein Data Bank (PDB) accession 1HD2)
47
and
human PRDX2 (PDB accession 1QMV)
48
, and modified with PyMOL to show
the predicted structure for archaeal peroxiredoxin (HyrA, GenBank accession
NP_280562.1).
RESEARCH ARTICLE
460 | NATURE | VOL 485 | 24 MAY 2012
Macmillan Publishers Limited. All rights reserved
©2012
For bacteria, we used the best characterized prokaryotic clock model
system, Synechococcus elongatus sp. PCC7942 (ref. 16). The major
proteins involved in the cyanobacterial clockwork are encoded by
the KaiABC cluster from which, remarkably, circadian oscillations
of phosphorylation can be reconstituted in vitro
17
. However, because
all three Kai proteins are expressed together in only a small number of
bacterial species, and in no known archaea
18
, this system cannot
represent a general prokaryotic clock mechanism. We postulated that,
regardless of the timekeeping mechanism, the peroxiredoxin oxida-
tion cycles we observe reflect an absolutely conserved rhythmic
cellular output. We tested this by assaying PRX-SO
2/3
under free-
running conditions (constant light) for 48 h, and observed cycles of
cyanobacterial peroxiredoxin oxidation, peaking later than phos-
phorylated KaiC (Fig. 3a).
Furthermore, we extended our studies to the third phylogenetic
domain, Archaea, assaying rhythms of peroxiredoxin oxidation in
H. salinarum sp. NRC-1. Although no clock mechanisms have been
identified for any archaeon, diurnal transcriptional rhythms were
recently observed in H. salinarum
19
, and it thus represents an ideal
platform to test whether peroxiredoxin oxidation constitutes a
universal marker for cellular rhythms. After entraining the archaea
for three cycles in 12 h light, 12 h dark cycles, we placed them into
constant light at constant temperature. We observed robust, high
amplitude circadian oscillations of PRX-SO
2/3
for three cycles
(Fig. 3b). Together, our findings in evolutionarily diverse prokaryotes
provide compelling evidence that rhythmic peroxiredoxin oxidation
is a conserved circadian marker across phylogenetic domains.
Relations between peroxiredoxin cycles and TTFLs
In all circadian model systems, the proposed clock mechanism
revolves around a TTFL
5,20,21
. How the metabolic rhythms observable
through peroxiredoxin oxidation relate to, and interact with, the
known transcriptional clockwork in different organisms is unclear.
Again, we used several model organisms, with available ‘clock’
mutants, to address this question.
The Drosophila transcriptional clockwork is structurally similar to
the mammalian TTFL, although there are some important differ-
ences
22
. In flies, Clock and Cycle (orthologous to mammalian
BMAL1, also known as ARNTL) comprise the positive limb, driving
oscillatory expression of Period (PER) and Timeless (TIM). PER and
TIM negatively regulate their own expression, closing the loop
5,6
. This
circuit can be disrupted by many mutations
23
. Two mutants, per
01
and
Clk
Jrk
, are behaviourally arrhythmic, and show non-cycling expres-
sion of circadian components, including PER and TIM
24,25
.To
examine peroxiredoxin oxidation patterns in these mutants, they were
entrained as described above for wild-type (Canton-S) flies. We
observed two circadian cycles of PRX-SO
2/3
oscillation, with an
altered circadian phase relative to wild type. This indicates the pres-
ence of an underlying capacity for circadian timing in both mutant
strains, which was clearly perturbed by the absence of functional
transcriptional feedback circuitry (Fig. 4a).
To establish the wider relevance of these findings, we also examined
similar mutants in the fungus Neurospora crassa. The frequency (frq)
locus encodes a critical element in the TTFL of Neurospora,in
addition to the Per–Arnt–Sim (PAS)-containing WC transcription
factors
26
. In the long-period frq
7
mutant
27
, peroxiredoxin oxidation
rhythms showed a similarly lengthened period, with an altered phase
relative to rhythms in FRQ protein abundance (Fig. 4b). Deletion of
the frq locus characterizes the frq
10
strain, and measurable markers of
clock output, such as its spore-forming (‘conidiation’) rhythm, are
profoundly perturbed in these fungi, although apparently stochastic
oscillations can re-emerge under various growth conditions
27–29
.
Circadian rhythms of peroxiredoxin oxidation were, however, clearly
seen in frq
10
mutants sampled in constant darkness (Fig. 4b), with a
delayed phase relative to wild-type (bd) fungi. This illustrates that
peroxiredoxin rhythms represent an alternative readout for an
oscillator that persists in the absence of a FRQ-dependent clock.
We next examined the phenotypes of mutant circadian transcrip-
tional regulators in photosynthetic eukaryotes and prokaryotes. The
transcriptional clockwork of the plant Arabidopsis thaliana and the
alga Ostreococcus tauri are very similar and rely on circadian oscil-
lation of TOC1. Accordingly, overexpressing TOC1 in either species
disrupts transcriptional rhythms
30,31
. In such strains, under constant
light, we observed persistent oscillations of peroxiredoxin oxidation,
albeit with altered amplitude and phase relative to controls (Sup-
plementary Figs 2 and 3). Furthermore, in cyanobacteria we assayed
peroxiredoxin oxidation in the arrhythmic KaiA deletion strain,
AMC702 (ref. 32). Notably, an approximately 24-h rhythm of
peroxiredoxin oxidation persisted despite a functional Kai-based
oscillator being absent, again in an altered phase relative to wild type
(Fig. 5a). Taken together, these observations indicate that metabolic
rhythms remain closely aligned to transcriptional feedback mechan-
isms when those mechanisms are present. Crucially, however,
metabolic rhythms persist even when cycling clock gene transcription
is abolished (summarized in Supplementary Table 9).
Having determined the TTFL influence on peroxiredoxin oxida-
tion rhythms, we reciprocally tested whether rhythmic peroxiredoxin
oxidation is required for timekeeping, using TTFL components as
markers of the clockwork. We assayed reporter bioluminescence
and delayed fluorescence in mutant S. elongatus and A. thaliana lines,
respectively, that were deficient in 2-CysPRX (Synechococcus D2-
CysPRX, GenBank accession AAP49028; Arabidopsis double mutant:
D2-CysPRXA D2-CysPRXB, GenBank accessions NM_111995 and
NM_120712)
33
. In these mutants, circadian rhythms persisted with
wild-type period, albeit significantly perturbed in either phase or
amplitude, relative to controls (Fig. 5b and Supplementary Fig. 9).
This suggests that peroxiredoxins are not required for oscillator
Time (h)
Archaea
Bacteria
a
b
Coomassie blue
PRX-SO
2/3
Time (h)
KaiC
PRX-SO
2/3
Coomassie blue
0 12 20 4 8 16 24 28 32 40 36 44
48
P
NP
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68
0
20
40
60
80
100
Time (h)
Normalized
abundance (%)
PRX-SO
2/3
0 4 8 12162024283236404448
0
20
40
60
80
100
Time (h)
Normalized
Abundance (%)
PRX-SO
2/3
Phospho-KaiC
0 12 20 4 8 16 24 28 32 40 36 44 48 52 56 60 64 68
Figure 3
|
Peroxiredoxin oxidation cycles are conserved in prokaryotic
models of the circadian clock. a, b, Representative immunoblots probed for
oxidized/hyperoxidized 2-Cys peroxiredoxin (PRX-SO
2/3
) are shown for
bacteria (S. elongatus sp. PCC7942; a), and archaea (H. salinarum sp. NRC-1;
b). Before sampling under free-running conditions (constant light),
cyanobacteria were synchronized with a 12 h dark pulse, whereas archaea were
stably entrained to 12 h light, 12 h dark cycles. Loading controls show
Coomassie blue-stained gels loaded with identical samples used for
immunoblotting. Immunoblot quantification by densitometry is shown below
each panel (mean 6 s.e.m.) for n 5 3 biological replicates. See Supplementary
Table 9 for cycle period estimates and detailed statistics. P, phosphorylated
KaiC; NP, non-phosphorylated KaiC.
ARTICLE RESEARCH
24 MAY 2012 | VOL 485 | NATURE | 461
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©2012
function in systems that possess an alternative timing mechanism,
namely a TTFL. On the other hand, our findings in TTFL mutants
reveal that cellular components that are required for rhythmic outputs
are not essential to rhythms in redox metabolism. Given the informa-
tion that we have about the above model organisms, used commonly
to study clock biology, we suggest that both peroxiredoxin and TTFL
components of the circadian system are important, but potentially
individually dispensable for circadian rhythms at the cellular level.
Moreover, the phenotypes of these mutants suggest that the cellular
ROS balance is important for robust clock function, as was described
recently in Neurospora
34
.
Implications for clock evolution
We have observed ,24 h cycles of peroxiredoxin oxidation–reduction
in all domains of life and consequently, the possibility that cellular
rhythms share a common molecular origin seems increasinglyplausible.
Because the cellular role of peroxiredoxins principally involves the
removal of toxic metabolic by-products (that is, ROS), we proposed that
the ability to survive cycles of oxidative stress may have contributed a
selective advantage from the beginnings of aerobic life.
Approximately 2.5 billion years ago, photosynthetic bacteria
acquired the capacity for photo-dissociation of water, leading to the
geologically rapid accumulation of molecular oxygen during the Great
Oxidation Event (GOE), when anaerobic life underwent a cata-
strophic decline
35
. Evidently, organisms that survived the transition
to an aerobic environment were those that respired and/or evolved
oxygen. As electron transport chains involving oxygen inevitably pro-
duce toxic superoxide anions as by-products
36
, during the GOE, suc-
cessful organisms had to acquire ROS removal systems or were
relegated to anaerobic niches
35
. Superoxide dismutase, which converts
superoxide to hydrogen peroxide, is ubiquitous and, like peroxire-
doxin (the major cellular H
2
O
2
‘sink’), is estimated to have arisen
around the time of the GOE
37
, the same era during which the most
ancient known clock mechanism (the Kai oscillator) evolved.
Importantly, we note that (1) during the GOE, rhythms of O
2
pro-
duction/consumption and ROS generation would have been driven by
the solar cycle, as they are today
38–40
; (2) metabolic/oxidation rhythms
seem to be present in every organism with a circadian clock, all of
which are aerobes, and these rhythms persist in the absence of tran-
scriptional cycles; and (3) circadian timekeeping confers a selective
advantage when it facilitates anticipation of environmental change
(noxious or otherwise).
We believe that the most reasonable interpretation is that cellular
metabolism in the most competitive early aerobes adapted to confer
anticipation of, and resonate with, environmental cycles in energy
supply and oxidative stress. We presume that the echoes of this
ancient evolutionary adaptation are revealed by the conserved
peroxiredoxin oxidation cycles we now observe in disparate
organisms, indicating that during the past 2.5 billion years, ROS
and metabolic pathways must have co-evolved with the cellular clock-
work, and are probably interlinked. On top of this, further cellular
mechanisms seem to have been incorporated, when advantageous, as
they arose over time. For example, in eukaryotes, the timekeeping
contribution made by certain post-translational mechanisms (such
as casein kinase) is conserved across several disparate organisms,
whereas clock gene transcription factors are widely divergent, having
evolved and been introduced more recently (see Fig. 6).
If there was considerable pressure for the co-evolution of meta-
bolic/ROS pathways with cellular timekeeping systems, then evidence
for this should exist in the phylogenetic trees of their component
mechanisms. To substantiate this we used the Mirrortree algorithm
41
to assess the degree of co-evolution between the 2-Cys peroxiredoxin
family, representing metabolism/ROS pathways, with the most
ancient characterized clock mechanism: the three cyanobacterial
Kai proteins. Because the Kai proteins are found exclusively in pro-
karyotes, we focused on these for our analysis
18
. All three components
of the cyanobacterial oscillator seem to have co-evolved with 2-Cys
peroxiredoxins, as shown by the strong correlation between the dis-
tances of respective proteins within each phylogenetic tree (KaiA,
r 5 0.784; KaiB, r 5 0.883; KaiC, r 5 0.865; P , 1 3 10
26
for all)
(Fig. 5c and Supplementary Fig. 4). Notably, when evolution of
KaiC (the most ancient member) was compared with other absolutely
conserved protein families, the three highest correlations observed
were for the other two clock components (KaiA and KaiB) and for
Time (h)
0 12 20 4 8 16 24 28 32 40 36 44 48
Coomassie blue
PRX6-SO
2/3
Fungus
b
Coomassie blue
PRX6-SO
2/3
frq
7
frq
10
0 4 8 12162024283236404448
0
20
40
60
80
100
Normalized
abundance (%)
Time (h)
WT
frq
10
frq
7
Fly
a
PRX-SO
2/3
β-actin
PRX-SO
2/3
β-actin
Clk
Jrk
per
01
0 4 8 12 16 20 24 28 32 36 40 44 48
0
20
40
60
80
100
Normalized
abundance (%)
Time (h)
Canton-S (WT)
Clk
Jrk
per
01
Time (h)
0 12 20 4 8 16 24 28 32 40 36 44 48
Figure 4
|
Peroxiredoxin oxidation cycles in circadian clock mutants.
a, b, Representative immunoblots probed for oxidized/hyperoxidized
peroxiredoxin (PRX-SO
2/3
or PRX6-SO
2/3
) are shown for fruitfly (D.
melanogaster; a) and fungus (N. crassa; b). For each model system, organisms
were sampled under free-running conditions (constant darkness). Loading
controls show either b-actin immunoblots or Coomassie blue-stained gels
loaded with identical samples used for immunoblotting. Immunoblot
quantification by densitometry is shown below each panel (mean 6 s.e.m.) for
n 5 3 biological replicates. See Supplementary Table 9 for cycle period
estimates (by harmonic regression) and detailed statistics (by ANOVA), as well
as Supplementary Fig. 10 for TIM and FRQ immunoblots for fruitfly and
fungus, respectively. WT, wild type.
RESEARCH ARTICLE
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©2012