Molecular analysis of zebrafish photolyase/cryptochrome family: two types of cryptochromes present...

Molecular analysis of zebra®sh photolyase/cryptochromefamily: two types of cryptochromes present in zebra®sh

Yuri Kobayashi1, Tomoko Ishikawa1, Jun Hirayama1, Hiromi Daiyasu2, Satoru Kanai2,Hiroyuki Toh2, Itsuki Fukuda1, Tohru Tsujimura3, Nobuyuki Terada3, Yasuhiro Kamei4,Shunsuke Yuba4, Shigenori Iwai2 and Takeshi Todo1,*1Radiation Biology Center, Kyoto University, Yoshidakonoe-cho, Sakyoku, Kyoto 606-8501 Japan2Biomolecular Engineering Research Institute, Furuedai 6-2-3, Suita, Osaka 565-0874 Japan3The First Department of Pathology, Hyogo College of Medicine, Mukogawa 1-1, Nishinomiya, Hyogo 663-8501 Japan4Institute for Molecular and Cellular Biology, Osaka University, Yamada-oka 1-3, Suita, Osaka 565-0871 Japan

Abstract

Background: Cryptochromes (CRY), members of the

DNA photolyase/cryptochrome protein family,

regulate the circadian clock in animals and plants.

Two types of animal CRYs are known, mammalian

CRY and Drosophila CRY. Both CRYs participate in

the regulation of circadian rhythm, but they have

different light dependencies for their reactions and

have different effects on the negative feedback loop

which generates a circadian oscillation of gene

expression. Mammalian CRYs act as a potent inhibi-

tor of transcriptional activator whose reactions do

not depend on light, but Drosophila CRY functions

as a light-dependent suppressor of transcriptional

inhibitor.

Results: We cloned seven zebra®sh genes that carry

members of the DNA photolyase/cryptochrome

protein family; one (6-4)photolyase and six cry

genes. A sequence analysis and determination of

their in vitro functions showed that these zebra®sh cry

genes constitute two groups. One has a high

sequence similarity to mammalian cry genes and

inhibits CLOCK:BMAL1 mediated transcription.

The other, which has a higher sequence similarity to

the Drosophila cry gene rather than the mammalian

cry genes, does not carry transcription inhibitor

activity. The expressions of these cry genes oscillate

in a circadian manner, but their patterns differ.

Conclusions: These ®ndings suggest that functionally

diverse cry genes are present in zebra®sh and each

gene has different role in the molecular clock.

Introduction

Cryptochromes are members of the DNA photolyase/cryptochrome ¯avoprotein family (Todo et al. 1996;Cashmore et al. 1999; Todo 1999) which are widelydistributed across both plants and animals. Crypto-chromes ®rst were characterized in Arabidopsis as bluelight photoreceptors which mediate a variety of lightresponses in the plant, including the regulation ofseedling growth, ¯owering, phototropism and theentrainment of circadian rhythms (Ahmad & Cashmore1993; Ahmad et al. 1998; Guo et al. 1998; Lin et al.1998; Somers et al. 1998). They are also present in ferns

and algae and are apparently ubiquitous in the plantkingdom. Plant cryptochromes are similar in sequenceto CPD (cyclobutane pyrimidine dimer) photolyase(Ahmad & Cashmore 1993), a type of DNA photolyasewhich mediates repair of the cyclobutane pyrimidinedimer (CPD), a type of UV-induced DNA damage, in alight-dependent manner (Sancar 1994). CPD photo-lyases, ubiquitous in both prokaryotes and eukaryotes,are thought to be the presumptive evolutionary pre-cursor of cryptochromes (Kanai et al. 1997). Crypto-chromes are also present in animals. Their primarystructures are similar to, but distinct from, those of plantcryptochromes, forming an animal cryptochromegroup. Initially, animal cryptochromes were detected astwo kinds of proteins encoded by human genes related toanother type of DNA photolyase (6-4)photolyase, which

q Blackwell Science Limited Genes to Cells (2000) 5, 725±738 725

Communicated by: Shu Narumiya* Correspondence: E-mail: [email protected]

mediates the repair of (6-4)photoproducts, another typeof UV-induced DNA damage (Todo et al. 1993; Todoet al. 1996). In spite of their high degree of similarity to(6-4)photolyase, the two human proteins lack DNArepair activity (Hsu et al. 1996; van der Spek et al. 1996;Todo et al. 1997a). Their functions had not beenidenti®ed, but were predicted to be similar to those ofplant cryptochromes, and so the human proteins havebeen designated human cryptochromes, hCRY1 andhCRY2 (Hsu et al. 1996). A type of protein with aprimary structure similar to that of (6-4)photolyase,but lacking DNA photolyase activity, is also found inmice (mCRY1 and mCRY2) (Kobayashi et al. 1998;Miyamoto & Sancar 1998) and Drosophila (DCRY)(Emery et al. 1998; Stanewsky et al. 1998; Ishikawa et al.1999), indicating that such proteins are ubiquitous inthe animal kingdom and have therefore been classi®edas animal cryptochromes. All animal cryptochromeshave been designated as cryptochromes from theirprimary structure prior to an identi®cation of thefunction of each protein. Therefore the de®nition of acry gene is not clear at present. A simple de®nition isthat it is a gene that encodes a putative protein thatdoes not have DNA photolyase activity but does havehomology to the DNA photolyase/cryptochromeprotein family (Hsu et al. 1996; Cashmore et al. 1999;Todo 1999).

Participation of the animal cryptochromes in thecircadian clock (oscillator) recently was shown inDrosophila (Emery et al. 1998; Stanewsky et al. 1998;Ishikawa et al. 1999) and mice (Thresher et al. 1998; vander Horst et al. 1999; Vitaterna et al. 1999). Circadianrhythms are oscillations that have daily periodicities inthe physiological and behavioural functions of organ-isms. These rhythms are generated by a cell-autono-mous circadian oscillator that is synchronized with theenvironmental light (Hall 1995; Dunlap 1999). Becauseanimal cryptochromes were initially predicted to be ablue-light photoreceptor (Hsu et al. 1996; Todo et al.1996), it was speculated that they mediate circadianphotoreception (Miyamoto & Sancar 1998). This turnedout to be the case in Drosophila. The circadian clock ischaracterized by an intracellular feedback loop, inwhich the expression of a group of genes results inthe production of proteins that then switch off theexpression of those genes. The circadian feedback loopin Drosophila is controlled by a set of transcriptionalactivators (dCLK and CYC/dBMAL1) and inhibitorsthat block these activators (PER and TIM) (Darlingtonet al. 1998). dCRY binds TIM and represses theinhibitory action of PER/TIM in a light-dependentmanner (Ceriani et al. 1999). The feedback loop

maintains synchrony with the environment's light-dark cycles by shifting phase in response to light (Hall1995; Dunlap 1999). The Drosophila cryb mutant, whichhas a mutation at the dcry gene, shows no response tobrief light pulses used to induce a phase shift. These®ndings indicate that CRYs mediate circadian photo-reception in Drosophila (Stanewsky et al. 1998). Unlikethe light-dependent function of dCRY, mice CRYsfunction at the feedback loop in a light-independentmanner. In the mouse circadian feedback loop, mPER1,2 and 3 and mTIM inhibit the activity of the tran-scriptional activators (CLOCK and BMAL1), but theinhibition is weak (Jin et al. 1999). In contrast, themCRY protein ef®ciently inhibits CLOCK:BMAL1-mediated transcription. The inhibition by the mCRYprotein is greater than that of any of the mPER proteinsor mTIM, and neither the ability for nor degree ofinhibition is dependent on light (Grif®n et al. 1999;Kume et al. 1999). In mice de®cient in both mCRY1and 2, who show arrhythmic behaviour in the free-running condition (van der Horst et al. 1999; Vitaternaet al. 1999), mPer1 and mPer2 mRNA levels are high inthe suprachiasmatic nucleus (SCN, the site of the centralcircadian oscillator), consistent with these in vitro results.Furthermore, a light pulse induces mPer2 in mice thatare de®cient in both CRYs, as well as in wild-type mice(Okamura et al. 1999; Vitaterna et al. 1999). Theseresults show that mCRYs act as transcriptional inhibitorswithin the circadian feedback loop but not as circadianphotoreceptors. Both insect and mammalian CRYsoriginated from DNA photolyase, which suggests thatthey have a common basic mechanism (Kanai et al.1997; Cashmore et al. 1999; Todo 1999), but theircorresponding reactions seem to differ. To account forthis discrepancy and to investigate the basic mechanismcommon to these CRYs, we worked on isolating genesof this protein family from a lower vertebrate thezebra®sh.

Zebra®sh provide an attractive model for the study ofthe vertebrate biological clock (Whitmore et al. 1998).We isolated seven cDNA clones of this protein family,one (6-4)photolyase and six cry genes. Four of the sixzcry genes, which are more similar to mouse/humanCRY in primary structure than the other two, havetranscription inhibitor activity as do mouse/humanCRY. The other two zcry genes do not, however, showany transcription inhibitor activity. The topology of thephylogenetic tree of this protein family shows that thelatter two genes diverged earlier than the others in the(6-4)photolyase/animal CRY cluster, indicative thatthey have a novel function in the circadian clock whichhas not been identi®ed in mammals.

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726 Genes to Cells (2000) 5, 725±738 q Blackwell Science Limited

Results

Cloning of zebra®sh (6-4)photolyase/CRY

family genes

Two strategies were devised to identify and isolate(6-4)photolyase/CRY family homologues in zebra®sh;PCR cloning and cDNA library screening. For thePCR cloning, degenerate PCR primers were designedfrom amino acid sequences highly conserved in(6-4)photolyase and human/mouse CRY and used toamplify partial zebra®sh photolyase/cry cDNAsequences. The distinct 203 bp products of thesePCRs were subcloned and subjected to nucleotidesequence analysis. Three types of DNA fragmentshighly homologous to the known (6-4)photolyase/crygenes were obtained. Primers were designed from thesequences of these fragments to obtain longer cDNA bythe further extension of each fragment by RACE.Three cDNA clones with full length coding sequenceswere obtained. Determination of their putative aminoacid sequences showed that all are members of the

(6-4)photolyase/CRY family. These cDNAs were usedas DNA probes to further screen the genes of this familyin the zebra®sh adult cDNA library. Phage plaques thatgave weak signals when hybridized with all threecDNAs were picked up, and the sequences of thecDNA inserts of each phage clone determined. Fournew cDNA clones were isolated. In total, seven cDNAclones that have high amino acid sequence identitieswith the known (6-4)photolyase and CRY wereidenti®ed (Fig. 1).

The amino acid sequences of these putative proteinswere aligned with the known (6-4)photolyase andCRYs, after which a phylogenetic tree was constructed(Fig. 2). It was similar in topology to a tree constructedpreviously (Kanai et al. 1997) and one drawn by anothergroup (Cashmore et al. 1999). One clone has a higherdegree of conservation with Xenopus (6-4)photolyase(72.9% identity) than with hCRY1 and 2 (58.6% and58.1%, respectively) and clusters with Xenopus andDrosophila (6-4)photolyase in the tree (subcluster 2).This clone was named z6-4phr. The other ®ve cloneshave higher degrees of conservation with hCRY1and 2 (68±88.3%) than with Xenopus (6-4)photolyase(55.9±59.2%). Of these ®ve clones, two pairs areclustered in the tree. The clones which showed highersimilarity to hCRY1 were designated zCry1a andzCry1b, the other two zCry2a and zCry2b, and theremaining clone zCry3. The last clone, which hasthe same relatively low conservation with Xenopus(6-4)photolyase and hCRY1 and 2 (51.6% and 51%,respectively), was named zCry4.

Detection of (6-4)photolyase activity

Members of the (6-4)photolyase/CRY protein familyare divided into two types according to function; DNArepair enzymes and proteins with no DNA repairactivity. DNA repair activities of all the gene productswere tested in Escherichia coli. E. coli SY32/pRT2, whichdoes not have (6-4)photolyase activity, was transformedwith pGEX-z6-4phr, pGEX-zcry1b, pGEX-zcry2a,pGEX-zcry3 or pGEX-zcry4, after which light-dependent DNA repair activity was rated by the survivalof transformed cells after UV-irradiation followed byillumination of photoreactivating light. The positivecontrol was pGEX-Xl(6-4)phr which has the Xenopus(6-4)photolyase gene placed in the same vector. pGEX-z6-4phr conferred light-dependent UV resistance on E.coli SY32/pRT2, as did pGEX-Xl6-4phr, whereas theother plasmids had no effect on the UV sensitivity of E.coli cells after transformation (Fig. 3A). To con®rm thatthe z6-4phr gene product has the (6-4)photolyase

Zebra®sh cry gene family


Figure 1 Structures of cloned zebra®sh (6-4)photolyase/cryp-

tochrome. Proteins in this family have two regions, a conserved

N-terminal and a C-terminal extension. Amino acid sequences

of the conserved N-terminal region are well conserved in all

members of this protein family, whereas the sequence and length

of the C-terminal extension varies with the protein. The bold,

solid line indicates the conserved N-terminal region of each

clone, the broken line the C-terminal extension, and the

numerals the amino acid sequences. DDBJ accession numbers

are as follows: zCry1a ABO42248, zCry1b ABO42249, zCry2a

ABO42250, zCry2b ABO42251, zCry3 ABO42252, zCry4

ABO42253, z6-4phr ABO42254.

activity, the recombinant z6-4phr protein expressed inE. coli was partially puri®ed and its enzymatic activitytested in vitro. It speci®cally bound (Fig. 3B) and repaired(Fig. 3C) (6-4)photoproducts in a light-dependentmanner.

zCry1a, 1b, 2a and 2b block

CLOCK:BMAL1-induced transcription

Except for z6-4phr, the clones do not have DNAphotolyase activity, indicative that these are cry genesthat function as clock components. The critical role ofmammalian CRY in the circadian feedback loop isinhibition of CLOCK:BMAL1-mediated transcription(Kume et al. 1999). We therefore used a luciferasereporter gene assay to examine whether zCry inhibits

this transcription (Fig. 4). The reporter construct utilizesthe promoter region of the mouse arginine vasopressingene that carries a CACGTG E box enhancer (Gekakiset al. 1998; Jin et al. 1999; Kume et al. 1999). CLOCKand BMAL1 act together on the enhancer and activateexpression of the reporter gene. zCry1a, 1b, 2a and 2binhibited CLOCK:BMAL1-induced transcription by>90%, whereas zCry3, 4 and z6-4phr did not causeinhibition.

zcry family genes have differential oscillation

patterns of expression in adult tissues

A property shared by many clock genes is that the levelsof their transcripts undergo circadian oscillation(Dunlap 1999). Northern blotting (Fig. 5) and RNase

Y Kobayashi et al.


Figure 2 Phylogenetic analysis of the

photolyase/cryptochrome family. The

tree was constructed by the Neighbour

Joining (NJ) method. Type I CPD photo-

lyase, Type II CPD photolyase and

(6-4)photolyase/CRYs were included.

Sequences analysed were retrieved from

public electronic databases. The accession

number and registered name are given,

together with the species name. NJ analysis

used 100 bootstrap replicates. The boot-

strap probability for each node is shown. A

bootstrap probability $ 90.0% was the

criterion for statistical signi®cance.

Animal CRYs form a cluster with the

(6-4)photolyases of animals and plants.

This cluster, which includes a homologue

from Synechocystis sp., is here called the

animal CRY subfamily. This subfamily

divides roughly into two subclusters; one

has mammalian CRYs and the homolo-

gues derived from zebra®sh, the other the

(6-4)photolyases from Drosophila melano-

gaster, Xenopus laevis and zebra®sh. The

former is here denoted subcluster 1, the

latter subcluster 2. A CRY from D.

melanogaster, a Cry homologue 4 from

zebra®sh, a (6-4)photolyase from Arabi-

dopsis thaliana, and an ORF product from

Synechocystis sp., however, are not included

in either NJ tree subcluster. A cluster of

CRYs derived from various plants is a

neighbour to the animal CRY subfamily.

This cluster is here referred to as the plant

CRY subfamily.

protection analyses (Fig. 6) of RNAs isolated fromvarious tissues after entrainment under a standard light-dark condition (LD 14 : 10) were used to assay zcrymRNAs for circadian oscillation. Both assays gaveidentical results. All zcry gene expressions show differ-ences in abundance between Zeitgeber time (ZT) 3 and15 (ZT0 corresponding to light on and ZT14 to lightoff) (Fig. 5), differences being clearer in the eye andbrain than in the body. More precise oscillation patternswere determined for the whole body (Fig. 6A) and eye(Fig. 6B). In the eye all zcry mRNAs manifest 5±40-foldamplitude cycling, but the oscillation pattern differs foreach cry gene (Fig. 6B). Expressions of zcry1a and b arehigh during the day, reaching a trough during the night

(ZT15-19), and increasing again at dawn (ZT23). zcry2a and 2b mRNA expressions peak at dusk (ZT13 or15) and reach a trough at ZT23-1. The oscillationpattern of zcry 4 is similar to the patterns of zcry2a and2b, but it has an earlier peak at ZT9. The oscillationpattern of zcry 3 differs from the patterns of zcry2a, 2band 4. It shows a trough at dusk (ZT9-15) and a peak atdawn (ZT 23-1).

The eye and pineal gland are two de®ned pacemakerstructures in zebra®sh (Cahill 1996). In situ hybridiza-tion analysis of the eye and brain showed that all zcrys areexpressed in the ganglionic cell layer of the retina. In thebrain they are expressed throughout it, including thepineal gland (data not shown).



Figure 3 Photoreactivating activity detec-

ted in z(6-4)phr but not in zCRYs.

(A) Effects of photoreactivation on the

survival of UV-irradiated E. coli SY32/

pRT2 cells carrying the pGEX4T-2 vector

(W), pGEXXl6-4phr (K), pGEXz6-

4phr(A), pGEXzcry1b(L), pGEXz-

cry2a(X), pGEXzcry3(O), or pGEXz-

cry4(B). After UV irradiation, E. coli

cells plated on LB plates were illuminated

with a ¯uorescent lamp for 1 h then

incubated overnight in the dark. Survival

rate was calculated from the number of

colonies formed. Each point is the mean of

three independent experiments. (B) Gel

shift analysis showing (6-4)photoproduct-

speci®c binding of z6-4phr protein. The

49 bp duplex oligonucleotides (1 nM)

undamaged (Lane 1) or having either of

CPD (Lane 2) (6-4)photoproduct (Lane

3), or the DEWAR isomer of (6-4)photo-

product (Lane 4) was mixed with 3 mg of

partially puri®ed GST-z6-4phr protein

then electrophoresed in a nondenaturing

gel. (C) Photolyase assay showing the

(6-4)photoproduct speci®c repair activity

of z6-4phr. The 49 bp duplex oligo-

nucleotides (10 nM) undamaged (Lane 1)

or having either CPD (Lane 2) or the

(6-4)photoproduct (Lane 3), or DEWAR

isomer of the (6-4)photoproduct (Lane 4)

were mixed with 37 mg of partially

puri®ed GST-z6-4phr protein, illuminated

with a ¯uorescent lamp for 1 h on ice, then

treated with phenol/chloroform. Samples

were treated with MseI then separated in a

10% polyacrylamide gel.

We also measured zcry mRNA cycling under con-ditions of constant darkness (DD; Fig. 7A) and constantlight (LL; Fig. 7B). Cycling of zcry2a, 2b and 3expressions persist under these conditions. Under DD ithas a lower, approximately twofold amplitude, thanunder constant light, where the cycling of higheramplitude persists. Similar results were obtained withthe zcry1a, 1b and 4 transcripts (data not shown).

Discussion

We isolated seven cDNA clones from zebra®sh thatcorrespond to (6-4) photolyase/cry family genes. Oneclone, z6-4phr, has (6-4)photolyase-like activity, whereasthe other six do not. A de®nition of a cry gene is that it isa gene that encodes a putative protein that does not haveDNA photolyase activity but does have homology tothe DNA photolyase/cryptochrome protein family(Hsu et al. 1996; Cashmore et al. 1999; Todo 1999).On the basis of this de®nition, six of the zebra®sh cloneswere classi®ed as cry genes; zcry1a, 1b, 2a, 2b, 3 and 4. Acomparison of the primary structures of their putativegene products showed that zCry3 and 4 have an

evolutionary history that differs from that of zCry1a, 1b,2a and 2b. Figure 2 shows the NJ tree of the photolyase/cryptochrome family, in which subcluster 1 consists ofthe mammalian CRY1s and 2s from humans and miceand the zebra®sh CRY homologues zCry1a, 1b, 2a, 2band 3. The bootstrap probability for the clustering ofsubcluster 1 was 100%. Mammalian CRY1s and zCry1aand 1b clustered together; a bootstrap probability of99.9%. zCry1a and 1b therefore are thought to be thezebra®sh counterparts of mammalian CRY1. zCry2aand 2b were closer to mammalian CRY1 than tomammalian CRY2; a 100% bootstrap probability of thenode being associated with the cluster of mammalian

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Figure 4 zCRY1a, 1b, 2a and 2b potentially inhibit CLOCK:

BMAL1-mediated transcription. Inhibition of CLOCK:BMAL1-

mediated transcription from the vasopressin (AVP) promoter by

zCRY1a, 1b, 2a and 2b (250 ng each). Each value is the mean

6 SEM of three independent experiments. In each experiment

the luciferase activity of the CLOCK:BMAL1-containing sample

was adjusted to 100.

Figure 5 Expression of zcry genes in several tissues. The mRNA

level of zcry in each tissue was measured at ZT3 and ZT15 by

Northern blotting. Transcript sizes are zcry1a (2.7 kb), zcry1b

(3.5 kb), zcry2a (6.5 and 3.7 kb), zcry2b (3.2 kb), zcry3 (4.3 kb),

zcry4 (2.6 kb), and z6-4phr (2.3 kb).

CRY1s and zCry1a, 1b, 2a and 2b. zCry3 was notincluded in this set of mammalian CRY1s and zebra®shrelatives, suggesting that zCry3 diverged ®rst insubcluster 1. The tree also suggests that zCry4 divergedfrom the lineage of subcluster 1 immediately after thedivergence of subclusters 1 and 2, although the nodecorresponding to the divergence of zCry4 had the lowbootstrap probability of 28.4%. On the basis of thesesequence analyses, zcry genes could be classi®ed in oneof three groups. Two groups have only a single member;the ®rst zcry4, and the second zcry3. The third groupconsists of zcry1a, 1b, 2a and 2b. Members of this groupare closer to mammalian CRYs, in particular mouse/human CRY1.

The ®rst two groups (zCry3 and 4) and the thirdgroup (zCry 1a, 1b, 2a and 2b) are functionallydifferent. Animal CRYs can be divided into two types

based on function. One is mammalian CRY, which actsas a potent inhibitor of CLOCK:BMAL1-mediatedtranscription (Kume et al. 1999). The other is DrosophilaCRY, which does not inhibit CLOCK:BMAL1(CYC)-mediated transcription, functioning instead asa light-dependent suppressor of PER/TIM-mediatedinhibition of transcription (Ceriani et al. 1999). Thethird group of zebra®sh Crys (zCry1a, 1b, 2a and 2b),which are evolutionarily closer to mammalian CRYsthan the other groups, has the same function asmammalian CRYs. These CRYs ef®ciently inhibitCLOCK:BMAL1-mediated transcription (Fig. 4), andare therefore classi®ed as mammalian type CRY. Incontrast, the other groups (zCry3 and 4) do not inhibitCLOCK:BMAL1-mediated transcription (Fig. 4), nordoes dCRY. Therefore they can be classi®ed asDrosophila type CRY although their function has not



Figure 6 Oscillation of zcry gene expression. zcry mRNA cycling in the whole body and the eye were measured by Northern blotting

(A) or the RNase protection assay (B). Times of sample collection (ZT) are indicated. Open bars correspond to day, and ®lled bars to

night. The protected probes are indicated in (B). Quanti®cation of three RNase protection assays (each point the mean of three

independent experiments with standard deviation) is shown in the bottom panels. zcry mRNA signals were normalized to the bactin

signal, and the peak reading for each experiment was adjusted to 100. zcry1a (X), zcry1b (A), zcry2a (B), zcry2b (W), zcry3 (K) and zcry4

(O).

been identi®ed yet. Whether they are functionallysimilar to Drosophila CRY or constitute a new type ofCRY family which differs functionally from bothDrosophila and mammalian CRYs is therefore a matterof interest. Identi®cation of their in vitro functionsshould provide answers to these questions.

The expression of zcrys oscillates in a circadianmanner (Figs 5, 6 and 7), as do expressions of the crygenes of Drosophila (Emery et al. 1998; Stanewsky et al.1998; Ishikawa et al. 1999) and mice (Miyamoto &Sancar 1998). The oscillation pattern, however, differfor each gene. The expressions of zcry1a and 1b showdistinct but similar patterns of oscillation. Both have atrough at ZT15±19. The oscillation patterns of zcry2aand 2b are the same, both mRNA peaking atZT13±15. The oscillation patterns of zcry3 and 4differ from those of zcry1s and 2s. zcry3 mRNA hasits peak at ZT23±1, and zcry4 mRNA at ZT9. Tissuespeci®c expression pattern also differs for each gene(Fig. 5). zcry2a is highly expressed only in the eye,

whereas zcry2b is expressed in all the tissues. In thesetissues, both mRNA expressions show cycling withhigh amplitude. zcry1a, 1b and 3 expressions are highin the eye and brain but low in the body. Theamplitude of oscillation in the body also is low. Theseresults suggest that each cry gene has a distinct role onthe zebra®sh circadian clock and their activity differin each tissue. The in vivo function of the mammaliantype zcry (zcry 1a, b, 2a and b) genes might beanalogous to that of mCry genes. They function as thenegative regulator in the CLOCK:BMAL1-mediatedtranscriptional feedback loop and their inhibitoryeffect is mediated by the direct interaction withCLOCK/BMAL1 heterodimer (Grif®n et al. 1999).Several Clock and Bmal1 genes are present in zebra®sh(unpublished data). Whether individual zCry1a, b, 2aand b interact with different CLOCK and BMAL1and how the expression of each genes are regulatedare the matters of interest. Detailed analysis of theinteraction of zCrys with CLOCK and BMAL1 and

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Figure 7 Oscillating zcry2a, 2b and 3

expression in zebra®sh eye in constant

darkness (A) and constant light (B).

Bottom panels: quanti®cation of an

RNase protection assay. Gray and black

bars in (A) represent constant darkness

(DD), grey being subjective day and black

subjective night. Grey and white bars in

(B) represent constant light (LL), white

being subjective day and grey subjective

night. zcry mRNA signals were normal-

ized to the bactin signal. The peak reading

for each experiment was adjusted to 100.

zcry2a (B), zcry2b (W) and zcry3 (K).

of the regulation of the expression of zCrys shouldprovide clearer understanding of the role of CRY inthe circadian clock.

In Fig. 2, the node connecting subclusters 1 and 2 hada low bootstrap probability of 53.6%. A CRY from D.melanogaster, a CRY homologue from zebra®sh (zCry4),a (6-4)photolyase from A. thaliana and an ORF productfrom Synechocystis sp. are not included in either sub-cluster in the NJ tree. The presence of the(6-4)photolyase from A. thaliana and an ORF productfrom Synechocystis sp. in the animal CRY subfamilystrongly suggests that divergence of the animal CRYandplant CRY subfamilies preceded that of animals andplants. The bootstrap probabilities associated with thedivergence of the (6-4)photolyase from A. thaliana(65.0%) and of the ORF product of Synechocystis(66.2%), however, were not particularly high. Incontrast, a CRY from D. melanogaster formed a clusterwith other animal CRYs and their relatives, and the

node corresponding to the divergence of this CRYfrom D. melanogaster had the high probability of 100.0%.In addition to the above examples, the NJ tree includesuncertainty about several protein relationships that arecritical for investigating the evolution of animal CRYsand their relatives. To con®rm the tree topologiesobtained by the NJ method, the evolutionary relation-ships within the animal CRY subfamily were investi-gated by ML analysis. The ML tree is shown in Fig. 8.The topology of this tree is basically the same as that ofthe NJ tree. Most nodes that corresponded to nodeswith low bootstrap probabilities in the NJ tree also havelow bootstrap probabilities in the ML tree. A majordifference between the NJ and ML trees is the locationsof zCry4 and Drosophila CRY. In the ML tree, zCry4and Drosophila CRY form a subcluster with the highbootstrap probability of 96.0%. This suggests thefunctional similarity of dCRYand zCry4 more stronglythan that do NJ tree ®ndings.



Figure 7 Continued

Our NJ and ML analyses suggest that: (i)divergence of the animal CRY and plant CRYsubfamilies preceded that of plants and animals, (ii)(6-4)photolyases evolved from the animal CRY, (iii)several gene duplications occurred, increasing thecopy number of animal CRYs before the divergenceof insects and vertebrates or of mammals and ®sh, and(iv) increases in copy number have recently occurredin zebra®sh. The relationship between zCry1a and 1bis one example, and that between zCry2a and 2banother. These recent increases would have beencaused by chromosomal duplication; often observed in®sh.

Of the ®ndings obtained from the phylogenetic treethe most interesting is that the (6-4)photolyasesevolved from the animal CRY. This indicates thatthe ancestor of animal CRY had the potential to act

functionally as a DNA photolyase. In other words, itretained the ability to recover DNA repair activity,even though it had already obtained the CRYfunction. Together with our previous report that theancestral gene of the DNA photolyase/CRY proteinfamily encoded CPD photolyase (Kanai et al. 1997),this suggests that a common mechanism operates inthis protein family despite the diversity of function. Afurther analysis of the zCry function should clarifythis.

Another issue of interest is why there are so many crygenes in zebra®sh. As described above, chromosomalduplication may be one reason. The pairs of zCry1aand b, zCry2a and b are examples. They may havefunctions which are analogous to those of mammalianCRYs. In addition to mammalian type CRYs,zebra®sh have two extra Crys (zCry3 and 4). They

Y Kobayashi et al.


Figure 8 Phylogenetic tree obtained by

the maximum likelihood (ML) method.

Numbers at the nodes indicate the boot-

strap probabilities. It was dif®cult to

construct an ML tree for the 44 sequences

used in the NJ analysis (Fig. 2) because of

the large amount of computational time

required for calculation. The following

restrictions therefore were introduced into

the ML analysis: The number of sequence

data were reduced. All members of the

animal CRY subfamily, except the ORF

product of Synechosystis sp., were used in

the ML analysis. Only four sequences from

the plant CRY subfamily were left for

analysis as an outgroup against the animal

CRY subfamily. In addition, the topo-

logies of several subclusters were ®xed to

reduce computation time. Selection of

subclusters with ®xed topologies is

described in the methods section. Nodes

related to the ®xed topology are indicated

by closed circles. The difference in AIC

between the ML and the second best tree

was greater than 1.0.

might be lost in the course of evolution to mammalsand have a function not found in mammals. Recentresearch on several organisms has clearly shown thatcircadian gene expression oscillates both in the de®nedclock structure and in peripheral tissues (Tonishi &Menaker 1996; Plautz et al. 1997; Balsalobre et al.1998; Whitmore et al. 1998; Zylka et al. 1998;Miyamoto & Sancar 1999). The mechanism of lightentrainment of these oscillators is different in mammalsand in Drosophila and zebra®sh. In mammals, lightadjusts the circadian pacemaker in SCN throughsignals from eyes, and this master pacemaker thensynchronizes peripheral clocks using chemical signals(Sakamoto et al. 1998; Brown & Schibler 1999). InDrosophila, on the other hand, peripheral tissues havecell autonomous circadian oscillators which can bedirectly light-entrained by a photoreceptor (Plautz et al.1997). dCRY clearly participates in the light entrain-ment (Stanewsky et al. 1998). This fact is indicativethat dCRY is a circadian photoreceptor in theperipheral tissues as well as in the master pacemaker.Recently it was shown that the zebra®sh peripheraloscillator can also be set directly by an environmentallight-dark cycle (Whitmore et al. 2000). The nature ofthe light receptor therefore is a matter of interest.zCry4 is a likely candidate for the zebra®sh circadianphotoreceptor because of the six zCrys found in it, it isstructurally closest to dCRY. Identi®cation of the invitro function of zCry4 should provide a clearerunderstanding of its in vivo role in light perception inthe zebra®sh clock.

Experimental procedures

Fish

Zebra®sh were maintained at 26±29 8C under a 14 h light/10 h

dark cycle. Adult ®sh were killed at each Zeitgeber (ZT) time

(ZT0 corresponding to light on and ZT14 to light off) by rapid

immersion in chilled water followed by decapitation. After

dissection, the eyes and brain were immediately frozen on dry ice

and used in the RNA analysis.

cDNA cloning

A combination of a PCR-based cloning strategies and low-

stringency hybridization screening was used to isolate the

zebra®sh (6-4)photolyase/cry gene family.

PCR-based cloning strategies were used in the initial step.

Primers speci®c for sequences conserved in the (6-4)photolyase

subgroup of the photolyase/CRY family were designed and used

in a PCR to amplify a 200-bp DNA fragment from cDNA

obtained from adult zebra®sh RNA. First-strand cDNA was

prepared from 5 mg of total RNA using reverse transcriptase (RT)

(BRL, MD, USA) according to the manufacturer's directions.

The PCR used two degenerated oligonucleotides as primers: f50-

TGG(A/C)G(A/G/C/T)GA(A/G)TT(C/T)T(A/T)(C/T)TA

(C/T)AC-30 and r50-TG(A/G/C/T)C(G/T)GGC(A/G/C/

T)AG(A/G)TG(A/G)TG(A/G/C/T)ATCCA-30. After sample

denaturation at 94 8C for 3 min, the PCR was run for 30 cycles

that consisted of denaturation at 94 8C for 1 min, annealing at

42 8C for 1 min, and extension at 72 8C for 2 min. Ampli®cation

was terminated by a 10-min ®nal extension step at 72 8C. After

RT-PCR product size was veri®ed by 1% agarose gel electro-

phoresis, the products were ligated to the pGEM-T vector

(Promega), and the sequence of the cloned fragment was

determined with an Applied Biosystems DNA sequencer.

Sequence determination classi®ed the PCR ampli®ed fragments

in one of three groups. Full length cDNA of the coding sequence

of each group was obtained by the RACE (rapid ampli®cation

of cDNA ends) method (Frohman et al. 1988). Sequences of

the three independent PCR clones for each full length

cDNA were determined. In further screening of the cDNA of

the (6-4)photolyase/cry gene family, the clones obtained by

PCR-based cloning strategies were used as probes to screen the

adult cDNA library (purchased from Clontech). The phage

library was screened as described elsewhere (Sambrook et al.

1989). Phage clones that showed weak signals with all the cDNA

probes after being hybridized and washed under mild conditions

(1 ´ SSC at 62 8C) were isolated, and the sequences of the

inserted cDNA determined. Four new cDNA clones were

identi®ed.

Detection of DNA photolyase activity

The full-length coding regions of z6-4phr, zcry1b, zcry2a, zcry3 and

zcry4 were ampli®ed with the KOD polymerase (Toyobo) from

plasmid DNA or cDNA (zcry4) then ligated into the pGEX4T

expression vector (Pharmacia). The resulting plasmids, designated

pGEX-z6-4phr, pGEX-zcry1b, pGEX-zcry2a, pGEX-zcry3 and

pGEXzcry4, were sequenced and shown to be correct.

In the photoreactivation of UV-irradiated E. coli cells, E. coli

SY32 (uvrAÿ, recAÿ, phrÿ) cells that carry plasmid pRT2 were

used as the hosts for the expression of zebra®sh 6-4photolyase

or cry genes. Plasmid pRT2 bears the E. coli phr� gene. UV-

irradiated E. coli was photoreactivated as described elsewhere

(Todo et al. 1997b).

For the gel shift and in vitro repair assays, glutathione

S-transferase (GST)-z6-4phr fusion protein was expressed in

E. coli SY2 (uvrAÿ, recAÿ, phrÿ). The GST fusion protein was

puri®ed as previously described (Hitomi et al. 1997). 49 bp

DNAs, which have a single UV photoproduct (either a CPD,

(6-4)photoproduct or DEWAR isomer) at the MseI site, were

prepared as described elsewhere (Hitomi et al. 1997) and used as

the substrate in the gel shift and in vitro repair assays. For the gel

shift assay, 3 mg of partially puri®ed GST-z6-4phr fusion protein

was added to the 32P-labelled DNA substrate (1 nM), and the

mixture electrophoresed on a nondenaturing acrylamide gel as

previously described (Hitomi et al. 1997). The in vitro repair assay

was done as described elsewhere (Hitomi et al. 1997); 37 mg of



GST-z6-4phr fusion protein was mixed with 32P-labelled DNA

substrate (10 nM) then illuminated with photoreactivating light.

After treatment of the mixture with phenol, the DNA substrate

was recovered by ethanol precipitation, digested with MseI then

electrophoresed on a denaturing acrylamide gel.

RNA analysis

RNA was extracted by the acid-phenol method (Sambrook et al.

1989). For the Northern blot analysis, 20 mg of total RNA

was treated with glyoxal then fractionated by electrophoresis

through a 1.2% agarose gel. The fractionated RNAs then were

transferred to nitrocellulose and hybridized as previously

described (Sambrook et al. 1989). Whole coding sequences of

each gene were the probes.

Ribonuclease (RNase) protection analysis was done with

20 mg total RNA obtained at the designated times. Riboprobe

templates were prepared by subcloning a fragment from each zcry

cDNA into pBSIISK-(Stratagene). Each subcloned fragment

carried the zcry cDNA as follows: nucleotides 1490±1574 of

zcry1a; 1536±1700 of zcry1b; 1727±1968 of zcry2a; 1609±1752 of

zcry2b and 1490±1707 of zcry4. Two kinds of bactin probe were

used as the control for normalizing the amount of applied RNA.

DNA fragments bearing nucleotides 957±1070 or 957±1009

of the bactin gene were ampli®ed from zebra®sh adult RNA by

RT-PCR then subcloned into pBSIISKÿ. They next were

linearized with EcoRI (zcry1a, 1b and 4) or Asp718 (zcry2a, 2b

and 3 and bactin) and transcribed with T7 RNA polymerase to

generate the anti-sense riboprobes. Hybridization and RNase

digestion were done with an RPA II kit (Ambion) according to

the manufacturer's recommendations. Protected fragments were

separated in 5% acrylamide/8M Urea gels and made visible by

autoradiography. Quanti®cation of the protected fragments was

done in a Bio Image Analyser (BAS1000, Fuji, Tokyo), the levels

of each zcry mRNA being normalized to that of bactin at each

time point.

Transcription assay

Luciferase reporter gene assays were carried out on NIH3T3 cells

as previously described (Jin et al. 1999; Kume et al. 1999). Cells

(3 ´ 105) were seeded in six-well plates and transfected the next

day with Lipofectamine-Plus (Gibco-BRL). Reporter constructs

were made as described elsewhere (Jin et al. 1999). A 200-bp

segment of the 50 ¯anking region of mouse vasopressin gene was

ampli®ed from the genomic DNA by PCR and subcloned into

the pGLC-Basic vector (Promega). Each transfection had the

reporter plasmid (10 ng) and pRL vector (Promega) (25 ng).

Mouse Clock and BMAL1 cDNAs were obtained from mouse

brain RNA by RT-PCR. Mouse Clock, mouse BMAL1, and

zebra®sh cry1a, 1b, 2a, 2b, 3 and 4 were each subcloned into

individual pcDNA3.1 vectors (Invitrogen) at 250 ng per trans-

fection. The total amount of DNA per well was adjusted to 1 mg

by adding pcDNA3.1 vector as carrier. Forty-eight hours after

transfection, cells were harvested and ®re¯y and Renilla luciferase

activities determined by luminometry. The reporter luciferase

activity was normalized for each sample by determining the

®re¯y:Renilla luciferase activity ratios. All experiments were

carried out three times.

Computational studies

A multiple sequence alignment of the photolyase-blue-light

photoreceptor family was constructed with the alignment

software, CLUSTAL W 1.7 (Thompson et al. 1994). The align-

ment obtained was modi®ed by visual inspection to adjust the gap

positions so that no gaps were included in secondary structure

elements. We then examined the evolutionary relationships within

the protein family by the neighbour-joining (NJ) (Saitou & Nei

1987) and maximum likelihood (ML) methods (Felsenstein

1981). In the molecular phylogenetic analysis, sites that included

gaps were excluded from multiple alignment. To construct an NJ

tree, the genetic distance between each pair of aligned sequences

was calculated from the JTT model (Jones et al. 1992) as an ML

estimate (Felsenstein 1996) for the amino acid substitutions.

Based on these distances, a tree was constructed by the NJ

method. The statistical signi®cance of the NJ tree topology was

evaluated by bootstrap analysis (Felsenstein 1985) with 1000

iterative constructions of the tree. Alignment also was subjected

to molecular phylogenetic analysis by the ML method. The

number of sequences, however, was too large for analysis by that

method. Therefore, we reduced the number of sequence data

used in the ML analysis and ®xed the topology of the subcluster

for that analysis when the bootstrap probability of the subcluster

was greater than 80.0% in the NJ tree. The remaining branching

pattern was examined by the ML method. The statistical

signi®cance of the topology of the ML tree was also evaluated

by bootstrap analysis. The JTT model was used as the amino acid

substitution model in ML analysis. Two software packages, PHYLIP

3.5c (Felsenstein 1993) and MOLPHY 2.3b3 (Adachi et al. 1996),

were used in the molecular phylogenetic analyses. The trees

obtained were drawn by TREEVIEW (Page 1996).

Acknowledgements

This work was supported by Grants-in-Aid from the ministry

of Education, Science, Sports and Culture of Japan (09308020,

11146206, 11480140 and 11878093) and by the REIMEI

Research Resources of Japan Atomic Energy Research

Institute.

ReferencesAdachi, J. & Hasegawa, M. (1996) MOLPHY (programs for

molecular phylogenetics), 2.3b3. Tokyo: Institute of StatisticalMathematics.

Ahmad, M. & Cashmore, A.R. (1993) HY4 gene of A. thalianaencodes a protein with characteristics of a blue-light photo-receptor. Nature 366, 162±166.

Ahmad, M., Jarillo, J.A., Smirnova, O. & Cashmore, A.R. (1998)Cryptochrome blue-light photoreceptors of Arabidopsisimplicated in phototropism. Nature 392, 720±723.

Y Kobayashi et al.


Balsalobre, A., Damiola, F. & Schibler, U. (1998) A serum shockinduces circadian gene expression in mammalian tissue culturecells. Cell 93, 929±937.

Brown, S.A. & Schibler, U. (1999) The ins and outs of circadiantime keeping. Curr. Opin. Genet. Dev. 9, 588±594.

Cahill, G.M. (1996) Circadian regulation of melatonin produc-tion in cultured zebra®sh pineal and retina. Brain Res. 708,177±181.

Cashmore, A.R., Jarillo, J.A., Wu, Y.-J. & Liu, D. (1999) Crypto-chrome: blue light receptors for plants and animals. Science284, 760±765.

Ceriani, M.F., Darlington, T.K., Staknis, D., et al. (1999) Light-dependent sequestration of TIMELESS by cryptochrome.Science 285, 553±556.

Darlington, T.K., Wager-Smith, K., Ceriani, M.F., et al. (1998)Closing the circadian loop: CLOCK-induced transcription ofits own inhibitors per and tim. Science 280, 1599±1603.

Dunlap, J.C. (1999) Molecular bases for circadian clocks. Cell 96,271±290.

Emery, P., So, W.V., Kaneko, M., Hall, J.C. & Roshbash, M.(1998) CRY, a Drosophila clock and light-regulated crypto-chrome, is a major contributor to circadian rhythm resettingand photosensitivity. Cell 95, 669±679.

Felsenstein, J. (1981) Evolutionary trees from DNA sequences: amaximum likelihood approach. J. Mol. Evol. 17, 368±376.

Felsenstein, J. (1985) Con®dence limits on phylogenies: anapproach using the bootstrap. Evolution 39, 783±791.

Felsenstein, J. (1993) PHYLIP (phylogeny inference package),Version 3.5c. Seattle: Department of Genetics, University ofWashington.

Felsenstein, J. (1996) Inferring phylogenies from proteinsequences by parsimony, distance, and likelihood method.Meth. Enzymol. 266, 418±427.

Frohman, M.A., Dush, M.K. & Martin, G.R. (1988) Rapidproduction of full-length cDNAs from rare transcripts:ampli®cation using a single gene-speci®c ologonucleotideprimer. Proc. Natl. Acad. Sci. USA 85, 8998±9002.

Gekakis, N., Staknis, D., Nguyen, H.B., et al. (1998) Role of theCLOCK protein in the mammalian circadian mechanism.Science 280, 1564±1569.

Grif®n, E.A. Jr, Staknis, D. & Weitz, C.J. (1999) Light-independent role of CRY1 and CRY2 in the mammaliancircadian clock. Science 286, 768±777.

Guo, H., Yang, H., Mockler, T.C. & Lin, C. (1998) Regulationof ¯owering time by Arabidopsis photoreceptors. Science 279,1360±1363.

Hall, J.C. (1995) Tripping along the trail to the molecularmechanisms of biological clocks. Trends Neurosci. 18,230±240.

Hitomi, K., Kim, S.-T., Iwai, S., et al. (1997) Binding andcatalytic properties of Xenopus (6±4) photolyase. J. Biol. Chem.272, 32591±32598.

van der Horst, G.T.J., Muijtjens, M., Kobayashi, K., et al. (1999)Mammalian Cry1 and Cry2 are essential for maintenance ofcircadian rhythms. Nature 398, 627±630.

Hsu, D.S., Zhao, X., Zhao, S., et al. (1996) Putative human blue-light photoreceptors hCRY1 and hCRY2 are ¯avoproteins.Biochemistry 35, 13871±13877.

Ishikawa, T., Matsumoto, A., Kato, T. Jr, et al. (1999) DCRY is aDrosophila photoreceptor protein implicated in light entrain-ment for circadian rhythm. Genes Cells 4, 57±66.

Jin, X., Shearman, L.P., Weaver, D.R., et al. (1999) A molecular

mechanism regulating rhythmic output from the suprachias-matic circadian clock. Cell 96, 57±68.

Jones, D.T., Taylor, W.R. & Thornton, J.M. (1992) The rapidgeneration of mutation data matrices from protein sequences.Comput. Appl. Biosci. 8, 275±282.

Kanai, S., Kikuno, R., Toh, H., Ryo, H. & Todo, T. (1997)Molecular evolution of the photolyase-blue-light photo-receptor family. J. Mol. Evol. 45, 535±548.

Kobayashi, K., Kanno, S., Smit, B., et al. (1998) Characterizationof photolyase/blue-light receptor homologs in mouse andhuman cells. Nucl. Acids Res. 26, 5086±5092.

Kume, K., Zylka, M.J., Sriram, S., et al. (1999) mCRY1 andmCRY2 are essential components of the negative limb of thecircadian clock feedback loop. Cell 98, 193±205.

Lin, C., Yang, H., Guo, H., et al. (1998) Enhancement ofblue-light sensitivity of Arabidopsis seedlings by a blue lightreceptor cryptochrome 2. Proc. Natl. Acad. Sci. USA 95,2686±2690.

Miyamoto, Y. & Sancar, A. (1998) Vitamin B2-based blue-lightphotoreceptors in the retinohypothalamic tract as the photo-reactive pigments for setting the circadian clock in mammals.Proc. Natl. Acad. Sci. USA 95, 6097±6102.

Miyamoto, Y. & Sancar, A. (1999) Circadian regulation ofcryptochrome genes in the mouse. Mol. Brain Res. 71,238±243.

Okamura, H., Miyake, S., Sumi, Y., et al. (1999) Photic induc-tion of mPer1 and mPer2 in Cry-de®cient mice lacking abiological clock. Science 286, 2531±2534.

Page, R.D. (1996) TreeView: an application to display phylo-genetic trees on personal computers. Comp. Appl. Biosci. 12,357±358.

Plautz, J.D., Kaneko, M., Hall, J.C. & Kay, S. (1997) Independentphotoreceptive circadian clocks throughout Drosophila. Science278, 1632±1635.

Saitou, N. & Nei, M. (1987) The neighbor-joining method: anew method for reconstructing phylogenetic trees. Mol. Biol.Evol. 4, 406±425.

Sakamoto, K., Nagase, T., Fukui, H., et al. (1998) Multi tissuecircadian expression of rat period homolog (rPer2) mRNA isgoverned by the mammalian circadian clock, the supra-chiasmatic nucleus in the brain. J. Biol. Chem. 273,27039±27042.

Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) MolecularCloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor,NY: Cold spring Harbor Laboratory Press.

Sancar, A. (1994) Structure and function of DNA photolyase.Biochemistry 33, 2±9.

Somers, D.E., Devlin, P.F. & Kay, S.A. (1998) Phytochromes andcrypthochromes in the entrainment of the Arabidopsiscircadian clock. Science 282, 1488±1490.

van der Spek, P.J., Kobayashi, K., Bootsma, D., et al. (1996)Cloning, tissue expression, and mapping of a human photo-lyase homolog with similarity to plant blue-light receptors.Genomics 87, 177±182.

Stanewsky, R., Kaneko, M., Emery, P., et al. (1998) The crybmutation identi®es cryptochrome as a circadian photoreceptorin Drosophila. Cell 95, 681±692.

Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994)CLUSTALW: improving the sensitivity of progressive multiplealignment through sequence weighting, position-speci®c gappenalties, and weight matrix choice. Nucl. Acids Res. 22,4673±4680.



Thresher, R.J., Vitaterna, M.H., Miyamoto, et al. (1998) Role ofmouse cryptochrome blue-light photoreceptor in circadianphotoresponses. Science 282, 1490±1494.

Todo, T. (1999) Functional diversity of the DNA photolyase/blue light receptor family. Mutat Res. 434, 89±97.

Todo, T., Kim, S.-T., Hitomi, K., et al. (1997b) Flavin adeninedinucleotide as a chromophore of the Xenopus (6±4) photo-lyase. Nucl. Acids Res. 25, 764±768.

Todo, T., Ryo, H., Yamamoto, K., et al. (1996) Similarity amongthe Drosophila (6±4) photolyase, a human photolyase homo-log, and the DNA photolyase-blue-light photoreceptor family.Science 272, 109±112.

Todo, T., Takemori, H., Ryo, H., et al. (1993) A new photo-reactivating enzyme that speci®cally repairs ultraviolet light-induced (6±4) photoproducts. Nature 361, 371±374.

Todo, T., Tsuji, H., Otoshi, E., et al. (1997a) Characterization ofa human homolog of (6±4) photolyase. Mutat. Res. 384,195±200.

Tonishi, G. & Menaker, M. (1996) Circadian rhythms in culturedmammalian retina. Science 272, 419±421.

Vitaterna, M.H., Selby, C.P., Todo, T., et al. (1999) Differentialregulation of mammalian Period genes and circadian rhyth-micity by cryptochromes 1 and 2. Proc. Natl. Acad. Sci. USA96, 12114±12119.

Whitmore, D., Foulkes, N.S. & Sassone-Corsi, P. (2000) Lightacts directly on organs and cells in culture to set the vertebratecircadian clock. Nature 404, 87±91.

Whitmore, D., Foulkes, N.S., Strahle, U. & Sassone-Corsi, P.(1998) Zebra®sh Clock rhythmic expression reveals inde-pendent peripheral circadian oscillators. Nat. Neurosci. 1,701±707.

Zylka, M., Shearman, L.P., Weaver, D.R. & Reppert, S.M.(1998) Three period homologs in mammals: differential lightresponses in the suprachiasmatic circadian clock and oscillatingtranscripts outside of brain. Neuron 20, 1103±1110.

Received: 19 April 2000

Accepted: 5 June 2000

Y Kobayashi et al.


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