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: costa@civ.bio.unipd.it .‘Department of Genetics, University of Leicester, Adrian Building, University Road, Leicester, LEl 7RH, UK; e-mail: cpk@leicester.ac.uk

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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