Functional and evolutionary implications clock genes
659
of natural variation in
Rodolfo Costa* and Charalambos P Kyriacout
Nearly all studies of natural variation within clock genes involve
the period (per) locus, which was originally isolated in the
fruit-fly. Intra- and interspecific work on per has focused mostly
on a region of Thr-Gly or Ser-Gly repeats, which show rapid
length and sequence evolution. The functional implications of
nucleotide variation in this repetitive array have been
characterised using behavioural, molecular, ecological,
structural and statistical analyses. A population genetics
approach to variation in per has also been useful in defining
species histories within Drosophilids and, in some cases, in
implicating selective processes in the evolution of the per
gene. Interspecific analysis of per expression patterns reveals
evolutionary alterations in this clock gene’s regulation.
Addresses “Dipartimento dl Biologia, Universita di Padova, Via Ugo Bassi 58/B, Italy; e-mail: [email protected] .‘Department of Genetics, University of Leicester, Adrian Building, University Road, Leicester, LEl 7RH, UK; e-mail: [email protected]
Current Opinion in Neurobiology 1998, 8:659-664
http://biomednet.com/elecref/0959438800800659
G Current Biology Ltd ISSN 0959-4388
Abbreviations bHLH basic helix-loop-helix
frq frequency
LD light/dark
per period
tim timeless
WC2 white collar-2
Introduction Endogenous circadian clocks represent the result of an
ancient adaptation to the rotation of the earth; they make
it possible for organisms from bacteria to humans to antic-
ipate the relentless 24 h cycles of light and dark. Most of
the knowledge available on the molecular mechanisms
underlying circadian timing stems from studies in
;V~I~IV.S~III.N (‘I‘USSN and Ih.suphilc/ mlhogmfe~~ [ 1 -I]. A n um-
ber of genes have been identified that appear to encode
l/o//N,fide clock components involved in the genesis of bio-
logical rhythmicity; these genes include pe/&’ (paj and
timhss ftitu) in II. mhmg~.stu~ [5], and frequetlcy (fiq) and
u/rite ml/h-2 ~~2) in XWIU.S~OI.N [h]. Both per and W-L’
encode versions of the PAS dimerization domains [7.8].
hlore recently, another gene, C/O/# - which was originall)
identified as a circadian rhythm mutation in the mouse [C,]
- has been isolated at the molecular Ic\~l and found to
encode a novel bHI,ll-PAS protein [lO,ll]. hloreovcr, the
rcccnt identification and characrerization of mammalian
pe!‘ gent homologues (12-l 41 suggests that the same clock
molecules are mediaring and controlling circadian rhyth-
micity in highi!- diverged organisms. l:or pn; tim and frq,
the genes’ Uanscripts cycle in abundance during circadian
periods, as do their products, with a lag between the
mRNA and protein rhythms [ 141. ‘I-his time delay allows
the effects of the proteins to feed back and suppress their
own transcription (see [1,4] for further discussions and
reviews of experiments demonstrating this negative feed-
back). Negative autoregulation is believed to lie at the
heart of the circadian pacemaker mechanism.
(liven the recent demonstrations of the conservation of
clock molecules between different species - such as
mouse and fly pn- - one COLII~ justifiably ask whether
there is any relevant genetic variation within a species in
any clock gene thar is worth studying. \Ve shall show in this
review how natural variation, both intra- and interspecific,
has led to a wider appreciation of the functional and evoiu-
&nary constraints in particular regions within clock genes.
Clocks and natural selection - intraspecific variation It seems reasonable to assume that having a good 24 h clock
must confer selective advantages to an organism. A recent
experimental test of this article of faith comes from
cyanobacrcria, in which mutant strains expressing normal
(24 h). long or short periods, were competed against each
other in different light/dark (13) cycles [1.5”]. Long-period
mutants \f’ere at a selective advantage during long I,D
cycics, and short-period mutants during short LD cycles.
‘I’he relative fitnesses of the mutant genotypes were high
only in tight cycles that resonated with their own endoge-
nous periods [15”]. ‘I-his striking example reveals the
adaptive value of the clock’s 24 h circadian period; however,
to demonstrate natural selection for more subtle, naturally
occurring clock variants, particularly in organisms that do not
have c)anobacteria’s obvious advantages for multi-genera-
tion competition studies, is problematic. For example, in
Dros~phifu, how would one detect whether one clock geno-
type MTIS at a sclcctive advantage in comparison to another,
when a basic rule of population genetics states that ielative
fitness increments as little as l/n, (where ne is the effective
population size - about 10” in D. IWP/NNO~OSS~~) are visible to
natural selection [ lh]? The answer is. ‘with difficulty’.
The Thr-Gly repeat in per \Vithin prr, considerdbic length and nucleotide polymor-
phism exists both in laboratory and natural populations of
11. r&N/qnster; particularly in a repetitive stretch that
encodes alternating threonine-glycine (‘i’hr-Gly) and scr-
ine-glycine (Ser-Giy) pairs [17.1X]. ‘I-his extensive
\.ariation has of itself pro\idcd a useful model for studying
the \,arious mutational mechanisms at \+,ork in such tan-
dcm arrays [19,20’]. i>oes this genetic variation have any
phenotypic consequences? &ographical analysis of the
structure of this length polymorphism has rcvcaled the
660 Molecular clocks
existence of a robust latitudinal cline, with the two major
allelic variants encoding 17 and 20 Thr-Gly pairs, predom-
inating in southern and northern Europe, respectively [Zl]
(Figure 1). This striking spatial pattern suggests that either
a historical accident, perhaps resulting from the northern
migration of this species from its ancestral home in Africa,
or, alternatively, natural selection, may be maintaining the
polymorphism. Behavioural experiments under different
temperature regimes have suggested that the period of the
clock is subtly different in the major Thr-Gly variants,
with the more common southern variant having a period
very close to 24 h under hotter conditions, and the more
common northern variant having a period that is better
buffered against temperature swings (i.e. better ‘tempera-
ture compensated’) [2X?*]. Each variant is, therefore, well
adapted to its own climatic environment.
Fiaure 1
a) Chimeric per genes (b) Locomotor activity
D. pseudoobscura 100
I I
0
I 1
D. melanogaster per gene
ti O
a
c) Temperature compensation (d) Conformation
I I I I
14 17 20 23
Thr-Gly length
There is more to this, however, than a familiar ‘just-so’ story,
which is often found in loose adaptionist thinking.
Conformational and theoretical analyses of (Thr-Gly), pep-
tides show that a (Thr-Gly)3 hexamer generates a stable
beta-turn, and is probably the structural ‘monomer’ [23]
(Figure 1). The major Thr-Gly variants found in nature
have 14, 17, 20 and 23 Thr-Gly pairs, conspicuously jump-
ing from 14 to 23 by the addition of the (Thr-Gly),3
conformational monomer [18]. Very rare variants that fall out
of step with this (Thr-Gly), interval, such as (Thr-Gly),s
and (Thr-Gly)21, have significantly poorer responses to
thermal challenges (as measured by larger swings in their
periods) than their more common relatives, which show a
linear pattern of temperature compensation [ZZ”]
( Figure 1). Thus, there appears to be a correlation between
behaviour (temperature stability of the period), the length
An orgy of Thr-Gly biology. (a) The per gene
in D. melanogaster is represented in the figure
at the bottom, with the Thr-Gly region shown
as a clear segment labelled TG. Above this
are two chimeric genes in which the
D. melanogasfer 5’coding regions (in black)
are ligated to the 3’ D. pseudoobscura
regions (in grey), either at the beginning of
pseudoobscura’s longer Thr-Gly repeat
(longer clear segment) or at a junction 60
amino acids upstream of the longer repeat.
(b) The two locomotor activity histograms on
the right reveal that the first chlmeric gene
barely restores circadian cycles to arrhythmic
pep transformants, whereas the second,
carrying more D. pseudoobscura material,
restores the cycle perfectly (redrawn from
[27**]). This shows how the immediate
5’-flanking region of the repeat co-evolves
(shown by horizontal curved arrows above the
chimeric genes) with repeat length, as
predicted in [25,26]. (c) The graph below the
D. melanogaster gene illustrates the
temperature compensation properties of the
major D. melanogaster Thr-Gly length
variants. The Y-axis represents the period at
29°C minus the period at 16°C in hours. Note
how the (Thr-Gly),o variant is extremely well
compensated (redrawn from [22”]). The
linear relationship between temperature
compensation and Thr-Gly length, and the
interval of (Thr-Gly), between the major
variants reveals the functional properties of
the (Thr-Gly), conformational monomer,
(d) illustrated by the ball-and-stick figure,
which shows the p-turn structure (redrawn
from [23]). (e) Finally, the European cline is
shown at the bottom, with pie diagrams
representing the frequency of the (Thr-Gly),,
variant (in black), the (Thr-Gly),,, (in white),
and all the other variants (grey) (redrawn from
[21]). Thus, the better temperature
compensated (Thr-Gly),O allele predominates
in the generally colder regions of northern
Europe.
e) European cline
hrrent Optnion in Neurobdogy
Natural variation in clock genes Costa and Kyriacou 661
of the Thr-Gly region, its conformational structure, and
gent frequencies in populations on a continental scale.
Rosato UT N/. [20’,24] have reported evidence consistent
with the operation of natural selection on this region of
per in both 12. simtdans and D. rnelonogostrr; using various
models designed to test for present and past selectivc
events using patterns of natural polymorphism linked to
the ‘l‘hr-Gly repeat. Finally, to bludgeon the point
home, interspecific sequence analyses of many
Drosophilid species have revealed that the length and
amino acid composition of this repetitive region differs
between species, but its predicted secondary structure
appears to be conserved [18,25]. [Ising simple statistical
analyses of the relelrant sequences, the interspecific
length of the repeat has been suggested to have co-
evolved with the immediately flanking 60-amino-acid
amino-terminal and IS-amino-acid carboxy-terminal
regions [25,263. This was tested experimentally by gen-
erating chimeric per genes in which the repeat of a
‘long-‘l’hr-Gly’ species \vas ligated to the flanking region
of a ‘short-‘l’hr-Gly’ species [27”] (Figure I). The result
\vas that the hybrid per gene in such a transformant bare-
ly functions at all, in that locomotor activity cycles are
not restored (I;igure 1 ), thereby confirming the idea that,
as the repeat lengthens during evolution, the flanking
amino acids must compensate for any changes in PER
protein structure. Other interspecific constructs involv-
ing chimeric junctions in this region lead to dramatically
temperature-sensitive circadian periods, consistent with
the effects discussed above regarding intraspecific
Thr-Gly length variants [ZZ”].
Thus, as the repeat length evolves, if compensatory muta-
tions are not fixed within the flanking regions, then
temperature compensation of the clock is altered, subtly in
the case of intraspecific length variation [Z?“], or dramati-
cally with interspecific changes. Consequently, a number of
independent lines of evidence converge to suggest that the
intra- and interspecific variation observed in and around the
repetitive region ofper is under natural selection.
Interspecific studies of per and tim in Diptera ‘l’he first comparative analyses of clock genes involved per
and revealed that, within Drosophih, pw is one of the more
variable genes that has been studied to date [28].
Interspecific transformation of the per genes of
11. pse&oo&zr~z and D. simzrl~ns in D. rmel’anogaster revealed
that both the species-specific patterns of locomotor behav-
iour [29] and the ultradian male lovesong cycles that can be
attered by mutations in per [30,31”] can be transferred to
the hosts in an all-or-none fashion [32]. l’hese remarkable
results show that interspecific coding sequence changes
within pet rather than regulatory changes, determine the
characteristics of these two biological rhythms. The rele-
vance of lovesong cycles to sexual selection and isolation is
obvious, but different patterns of circadian locomotor
rhythms could also contribute to sexual isolation; for
example, if one species is active while another is not.
Therefore, both per-determined phenotypes could play a
role in the speciation process itself. If so, per- sequences
might be expected to show the signature of natural selection.
In fact. for the song cycle, the key species-specific
sequences are those surrounding the ‘I’hr-Gly repeat [Xl,
which shows the hallmarks of selection in both
D. mpla)logas~erand 11. slmulat~s ([20’,24]; as discussed above).
Various statistical tests have been used to analyse
nucleotide diversity in a number of fly species, either to
investigate whether other regions ofper show evidence for
selection or simply to use per as a useful marker to track
speciation history [33,34]. For example, Kliman and Hey
[35] analysed a region of per located upstream to the
Thr-Gly-encoding repeat in species of the D. melanogaster
complex, but they did not find any significant departure
from neutrality in the distribution of the levels of variabil-
ity. In two more recent papers by Hey and colleagues
(36,371, the speciation history of the D. virihs group [36]
and of D. psetdookwn and its close relatives [37] were
investigated using per sequence variation in the 3’ part of
the gene. I;ive closely related taxa were analysed in the
11. vh-ilis group, and the authors were unable to reject the
neutral hypothesis except in the case of D. novon~exirccna
[36]. Even for this latter species, alternative explanations
other than natural selection could explain the nucleotide
divergence patterns. In the second study, the significant
reduction in the nucleotide variation detected in
D. psezdoohwa hogofam may reflect the action of natural
selection, either on per or a sequence nearby [37].
One way of marrying the behavioural/functional analyses
ofpp;r- and the population genetics based studies would be
to narrow down the sequences that control species-specif-
ic behaviour and study these with the neutrality tests, as
was done with song rhythms. In this particular case, how-
ever, because the repetitive Thr-Gly region is the culprit,
the assumptions on which these tests are based are violat-
ed. and so alternative approaches had to be used [20’,24].
Nevertheless, if the species-specific pattern of locomotor
behaviour could be dissected down to a narrow nonrepeti-
tive region of the per coding sequence - using, for
example, the D. p.seudoobsmralD. ndanogaster comparison
mentioned earlier - then it would be interesting to deter-
mine what the neutrality tests would conclude about the
relevant sequences.
Natural variation in timeless The per partner molecule, tim, whose product dimerises
with PER and escorts it into the nucleus, mediates the
negative feedback of the two proteins on their transcrip-
tion [Z-4]. The sequence of tiln in D. melanogaste~ D. vidis
and the partial sequence from 11. &dei [38,39,40”], plus
sequences corresponding to amino-terminal fragments in
other Drosophila species [41’], have been reported. These
data suggest that the TIM protein is better conserved than
PER. There is a 76% overall identity for TIM between
662 Molecular clocks
I!. mefumgmter and D. virih compared with 54% overall
identity for PER [40”]. Interestingly, the PER and TIhI
interaction domains have very similar levels of conserva-
tion (SO--85%) between these two species, suggesting that
selective forces are acting to conserve these functionally
important regions [40”].
‘I’he amino terminus of TIhl reveals that in all species
examined so far [39,40”,41’], apart from D. melmoguster
[.3X,41’], translation is putatively initiated from a methion-
ine codon situated 23 amino acids downstream from the
site originally suggested to represent the start codon in
I). mehmgu.ster: A quick survey of D. tnehnogaster strains
revealed that, in fact, this species is polymorphic for a
mutation that generates a stop codon between the two
methionines, and so flies can potentially generate either a
long and a short, or just a short, TIRI product [41’]. This
particular polymorphism is reminiscent of Neurosportl,
because the clock gene ,frq also generates by alternative
translational initiation a short and long FRQ product
[42”]. At different temperatures, the ratios of the two
products changes dramatically, and each FRQ product res-
cues the arrhythmic phenotype of a frq null mutant,
particularly at its favoured temperature [43”]. Thus, the
two forms of FRQ extend the physiological temperature
range at which it will work. One might therefore wonder
whether the two forms of TIhI (assuming the long ‘I’lhl
variant is translated) have functions analogous to the fun-
gal phenomena. Preliminary results from our laboratories,
involving a large-scale survey of natural European popula-
tions of I). tr~e/~)~ogz.s~e/; suggest that this TIIU length
polymorphism is ubiquitous, and that. intriguingly, it
shows a latitudinal cline in its geographical distribution
(hl Zordan et (I/., unpublished data).
Interspecific comparison of per outside the Diptera ‘l’he per gene has now been isolated in mammals (Inper)
[l&14], silkmoths [44], cockroaches [44] and bees
(DP ‘I’oma, GE Robinson, personal communication).
‘I’hree mammalian per genes have been sequenced (mperl,
mper2 and mpeL?) [ 12-14,45”], and four regions of homol-
ogy have been identified between the mammalian and fly
genes; these regions include the PAS domain and the
repetitive ‘Thr-Gly/Ser-Gly region. However, mperl and
mpen? are more similar to each other than they are to N~PPIIJ
[35”], and this appears to reflect itself in the responses of
the three m/x/- transcripts to light pulses. All three tran-
scripts oscillate in various brain and peripheral tissues,
including the suprachiasmatic nucleus (SCN) and the
eyes, but only /riper2 and /per2 are acutely light-responsive
during the subjective night phase in the SCN [12-14,45”].
‘I’he mRNA oscillations of these three mper genes
[12-14.45”.46,47] is reported to be similar in all tissues
where they are expressed [45”], and the presence of mper oscillations in peripheral tissues such as skeletal muscle,
liver and testis, is reminiscent of the situation in Dro.wphi/cr [4X], where light-sensitive dper oscillations are found in
many tissues. ‘l’he clear implication is that autonomous
clocks are found in the periphery.
Silkmoth per k>oses some interesting anomalies, not least of
which is the existence of an anti-sense per mRNA that
cycles in antiphase to the sense molecule, and the apparent
absence of nuclear localisation of PRR in the small number
of adult brain neurons in which it is expressed [44,49]. Note
that the negative feedback model whereby I’ER and ‘I’lhl
negatively regulate their own transcription requires nuclear
localisation of their products [Z-4]. ‘I’hese difficulties
notwithstanding, studying the interspecific differences in
per and [i/n should provide answers to the altered expres-
sion patterns of clock genes that will inevitably be seen as
we move across species borders. This is not to say that peg
and ti~l are the only loci of interest to the evolutionary
chronobiologist, because a number of newly identified
clock molecules have recentlv hit the headlines.
Briefly, as mentioned in the introduction, one of these
novel clock molecules is the C%& gene, which was initial-
ly identified in a screen for circadian mutants in the mouse,
and subsequently isolated by positional cloning [9-l 11. Its
Dru@i/cl homologue was identified in another screen for
behavioral mutants, and also by low-stringency hybridisa-
tion [SO,Sl]. CLOCK (CLK) is a bHI,H-PAS transcription
factor that dimerises with the product of the /:)v (/?I(/e) gene
[51,X?]: the latter was also identified by a forward genetic
screen of behavioral mutants in Drasophh [S3]. *l-he I:YI’
gene is, in turn, the Drus~phih homologue of the
bHLH-PAS-encoding mammalian llm~I/ gene. (:\r’(:/(X,K
heterodimers act as positive elements in the feedback
loop, binding to E boxes that are present inpel-and till/ pro-
moters and driving their expression [51,52]. PER and
‘IX1 can interact with these positive factors, and repress
their own transcription, presumably via hcterotypic PAS
interactions, thereby closing the loop [Sl]. Finally, the c//~/r
(~&/e-tjr~/p) locus was identified in another screen for fly
circadian variants, and encodes a protein similar to human
casein kinase 1~ [54.55]. The kinase phosphorylates PER
and regulates its accumulation, giving rise to the delay
between transcription of per and the high levels of PER
required for nuclear translocation [S-l..%]. Comparative
studies of these new clock genes remain to be initiated.
The frequency gene in Neurospora Finally, and perhaps of little relevance to neurobiologists.
but for the sake of completeness, we should point out the
interspecific comparisons offrq in different fungdl genera.
revealing a similarity of 86% at the DNA level between.fig
in .V. ,-rt.s.sn and S’ord~trk ,fiml/nh [56]. l’he .frq gene in
,Veuro.spor~ regulates the circadian conidiation cycle [ 11. :I developmental prograrnme that is absent in ~Ynrhriu.
Nevertheless. the .fiq homologue from ,Sorhrh rescues
reasonably well the conidiation rhythm (i.e. production of
asexual spores) when transformed into a :V. uxs.scr,frq null
mutant host. These results suggest that,fi-q may play a cen-
tral role in the pacemakers of the two orKdnisms, rather
Natural variation in clock genes Costa and Kyriacou 663
than ;I peripheral role related to the output of the clock sig-
nal co a specific phenotype. However, two recent studies,
one a formalistic analysis based on a new model of the cir-
cadian system, and the other a study of genetic interactions
between .fiy and metabolic mutants, both hint that frq could also be a component of the input system [57.58].
Conclusions Natural variants of clock genes, whether intra- or interspe-
cific, have thus been extremely useful, not only in the
study of circadian phenomena, but also in defining mech-
anisms of mutations in repeated sequences, sorting out
species histories, and understanding how molecular evolu-
tion can produce behavioural and reproductive isolation in
the fly. The pa- gene has been particularly prominent
because it was c-he first clock gene to be isolated. It has pro-
vided a rare example of a case where studies of natural
variation have encompassed almost all levels of biology,
from the structural to the ecological. The impact of the
recent flurry of new clock genes that have been isolated in
the fly and the mouse means that LI new reservoir of natur-
al clock gene variation remains to be tapped.
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