A brief history of circadian time

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understanding the full functions of all genes in a genome, one needs to be aware of the additional dimension and the scale of the experimental effort that would be required to overcome this. But it may be worth noting that one References 1 Wagner, A. (2000) Robustness against mutations in genetic networks of yeast. Nat. Genet. 24, 355–361 2 Smith V. et al. (1996) Functional analysis of the genes of yeast chromosome V by genetic footprinting. Science 274, 2069–2074 3 Winzeler E.A. et al. (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 4 Tautz, D. (1992) Redundancies, development and the flow of information. BioEssays 14, 263–266 5 Nowak M.A. et al. (1997) Evolution of genetic redundancy. Nature 388, 167–171 6 Wagner A. (2000) The role of population size, pleiotropy and fitness effects of mutations in the evolution of overlapping gene functions. Genetics 154, 1389–1401

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consequence of the formulation of Heisenberg’s uncertainty principle was the development of high-energy physics and the building of supercolliders. Should biologists ask for less?

7 Kimura, M. (1983) The Neutral Theory of Evolution. Cambridge University Press, Cambridge 8 Kimura, M. (1986) DNA and the neutral theory. Philos. Trans. R. Soc. London Ser. B 312, 343–354 9 Kreitman, M. (1996) The neutral theory is dead. Long live the neutral theory. BioEssays 18, 678–683 10 Stephan, W. (1997) Mathematical model of the hitchhiking effect, and its application to DNA polymorphism data. In: Advances in Mathematical Dynamics – Molecules, Cells and Man (Arino, O. et al., eds), pp. 29–45, World Scientific 11 Thatcher, J.W. et al. (1998) Marginal fitness contributions of nonessential genes in yeast. Proc. Natl. Acad. Sci. U. S. A. 95, 253–257 12 Akashi, H. (1994) Synonymous codon usage in Drosophila melanogaster: natural selection and translational accuracy. Genetics 136, 927–935

13 14 15 16 17 18

Akashi, H. (1995) Inferring weak selection from patterns of polymorphism and divergence at ‘silent’ sites in Drosophila DNA. Genetics 139, 1067–1076 Ohta, T. (1992) The nearly neutral theory of molecular evolution. Annu. Rev. Ecol. Syst. 23, 263–286 Hurst, L.D. (1999) The evolution of genomic anatomy. Trends Ecol. Evol. 14, 108–112 Schmid, K.J. and Tautz, D. (1997) A screen for fast evolving genes from Drosophila. Proc. Natl. Acad. Sci. U. S. A. 94, 9746–9750 Tautz, D. and Schmid, K.J. (1998) From genes to individuals: developmental genes and the generation of the phenotype. Philos. Trans. R. Soc. London Ser. B 353, 231–240 Hurst, L.D. and Smith, N.G.C. (1999) Do essential genes evolve slowly? Curr. Biol. 9, 747–750

A brief history of circadian time Recent progress in clock research has revealed major molecular components in the mechanisms responsible for circadian time keeping in mammals. The first vertebrate clock mutation (tau) was discovered in the Syrian hamster more than a decade ago and, using the power of comparative genomics, this gene has now been cloned. We now know that tau is the mammalian homologue of a Drosophila circadian clock component (doubletime) that plays an important role in regulating clock protein turnover. iologists have long recognized that circadian clocks are an integral aspect of the physiology of most eukaryotic organisms. As their name indicates (circa about, dies day), circadian clocks have a period of ~24 h. These endogenous timekeepers regulate an enormous array of physiological systems, altering their activity rhythmically on both the daily and seasonal timescales. Circadian-clock-regulated processes in vertebrates include sleep–wake cycles, many hormone rhythms and seasonal fattening, hibernation and reproductive cycles in wild animals. As a result of recent advances in molecular genetics, clock biologists have now gained some remarkable insights into the molecular elements regulating the central circadian clockwork, which reveal an extraordinary conservation in Drosophila and mammals over a 500-million-year time span (Fig. 1).

B

History Key events in our understanding of the genetic basis of circadian rhythms and of the anatomical hard-wiring of mammalian clock function dates back to the early 1970s. Working with Drosophila, Konopka and Benzer used chemical mutagenesis to produce the first circadian clock mutants1. These mutants had different circadian phenotypes (they showed abnormally long or short rhythms and some were arrhythmic) but all mapped to the same genetic locus, which was termed period (per). At the same time, Zucker and Moore published separate studies2,3 reporting the effects of lesions of the suprachiasmatic nucleus (SCN) of the hypothalamus on the circadian physiology of the rat: destruction of this region eliminated circadian rhythmical behaviour. Together, these studies helped to transform the field of circadian biology by defining the genetic components and anatomical structures associated with rhythm generation. The first hint that a genetic approach to clock biology in mammals might be viable came with the accidental discovery of the circadian tau mutation in the Syrian hamster by Ralph and Menaker in the late 1980s4. This autosomal 0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(00)02122-3

co-dominant mutation has a dramatic shortening effect on the circadian wheel-running activity cycle, with circadian periods of 20 h for homozygous animals, 4 h shorter than the wild type. In a spectacular follow-up publication5, they then showed that transplanting fetal SCN tissue to SCN-lesioned recipients restored circadian wheel-running behaviour, but only with a period appropriate to the genotype of the donor animal. This seminal experiment clearly defined the SCN as a primary site in the mammalian brain for the generation of circadian rhythmicity and also showed that, as in Drosophila, single gene mutations have a powerful effect on the clock. Other studies quickly showed that the mutation also altered circadian patterns of hormone secretion6, including the discovery of an independent circadian oscillator driving melatonin rhythms in the retina7. This mutation was also shown to disrupt the seasonal reproductive and endocrine response of the animal to day-length change8. The tau mutation has thus provided clock biologists with a marvellous opportunity to test predictions about the role of circadian oscillators in regulating temporal physiology.

Identifying the tau gene One obvious difficulty in identifying this gene has been that the Syrian hamster would not be anyone’s model of choice for gene mapping. However, a recent paper presents the results of a positional syntenic approach that has been used to identify the tau mutation and the nature of the gene9. There are no YAC or BAC libraries available for the Syrian hamster, eliminating the option of conventional chromosomal walking. The strategy used was to develop markers linked to tau, with the goal of defining a conserved region of synteny between hamster and well characterized species such as human and mouse, which could then be used to identify a candidate gene for tau (Fig. 2). With such a candidate, it would then be possible to confirm a mutation in the Syrian hamster and subsequently to perform functional studies. TIG November 2000, volume 16, No. 11

Andrew S.I. Loudon andrew.loudon@ man.ac.uk Andrei G. Semikhodskii andrei.semikhodskii@ man.ac.uk Susan K. Crosthwaite susan.k.crosthwaite@ man.ac.uk School of Biological Sciences, University of Manchester, Oxford Road, Manchester, UK M13 9PT. 477

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FIGURE 1. The time line of some major discoveries in circadian clock research

per

dClock cyc cry dbt tim wc1 wc2 vri

frq

Gene cloning

1990

1980

1970

Central pacemaker in lateral neurons

per mutants frq mutants

Clock mutation

Peripheral oscillators

Peripheral oscillators

Csnk1e (Tau)

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Timeless mutants

Bmal1 Per3 Clock Tim Per1 Per2 Cry b mutation Cyc mutants Dbt mutants Jrk mutants

Cry1 Cry2

Phenotypes and physiology

Tau mutation

Central role of SCN

Drosophila

Mammals

Neurospora

Independent retinal clock trends in Genetics

The first clock gene mutation (per ) was discovered in 1971 and mapped and cloned in 198438,39. The discovery of per was followed more than a decade later by the cloning of the Drosophila gene timeless, a partner for per. Since the discovery of the hamster tau mutation, there has been a rapid increase in the number of clock gene components identified, virtually all of which have homologues in mammals and insects. Positive transcription factors CLOCK and BMAL1 were discovered first in mammals, and timeless, per and double-time were originally cloned in Drosophila.

Using genetically directed representational difference analysis (GDRDA), the group was able to isolate two polymorphic products (RDA-750 and RDA-650) that mapped to within 5.6 cM of the tau locus. In order to identify the region of the human and mouse genome harbouring the orthologue of the tau gene, polymorphic hamster GDRDA clones were used to screen a hamster genomic l-phage library. One of the positive genomic clones revealed high homology to the mouse Celsr1 gene on chromosome 15. Using several mouse genes located proximal and distal to Celsr1, a 15 cM region was defined that had conserved synteny with the hamster genome containing tau. This region spans segments of human chromosomes 8, 12 and 22. On human chromosome 22, there was a strong candidate for tau, the recently described human homologue of the Drosophila clock gene doubletime (dbt)10. This gene encodes a casein-kinase Ie (CSNK1E), known to be involved in the phosphorylation of PER in Drosophila, which is a key circadian clock protein. Hamster Csnk1e was cloned and sequenced, and a C→T transition was identified in the mutants. The polymorphism was mapped in the hamster F2 population and shown to co-segregate completely with the circadian phenotype.

Clock mechanism It is now clear that the tau mutation fits perfectly within our current molecular understanding of the mechanism of 478

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the circadian clock. Identification of the general mechanisms and molecular components of the mammalian circadian clock has evolved from studies on Drosophila and Neurospora (Fig. 1, Table 1). They define a transcription– translation autoregulatory feedback loop as a driving force of the clockwork mechanism11,12. The loop is made of several positive and negative regulatory elements that interact with each other to generate a time delay in the cyclical transcription of key components. In Drosophila, positive transcription factors (dCLOCK and dBMAL1 or CYC) activate transcription of per, tim and other clockassociated genes by binding specific regulatory elements (E-boxes) in their promoters13,14. The RNA is then transported to the cytoplasm where, for several hours, protein products are synthesized15. Once synthesized, these proteins undergo post-translational modifications, which control their levels in the cytoplasm and enable them to interact with each other and to be imported back into the nucleus. In the nucleus, the negative elements shut down transcription of their own and other genes16. The exact mechanism of this inhibition is still unclear but it could occur by direct interaction between the negative and positive factors, rendering the latter inactive, or by targeting the regulatory elements of gene promoters and making them inaccessible. When the levels of negative transcription regulators drop, owing to protein degradation, the cycle recommences17. An additional loop has recently been discovered and a trio of papers on Drosophila, Neurospora and mice now

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FIGURE 2. Positional syntenic cloning of the Syrian hamster tau gene

Human 22

MB

Mb Cyp11b2 Cacng2

Mb Cyp11b2 Cacng2

(b)

Human 8 CYP11B2 CACNG2

(d)

Csnk1e (Tau) Csnk1e

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Celsr1 (RDA-650) RDA-750

Celsr1 (RDA-650) RDA-750

CSNK1E CELSR1

(a)

Ela1

ELA1

Ela1 (e)

Hamster?

Mouse 15

Human 12

trends in Genetics

Initially (a), DNA markers were identified that were linked to the tau mutation in Syrian hamster (RDA-650 and RDA-750). A genomic clone containing one of the markers isolated from a Syrian hamster l-phage library showed high homology to a known mouse gene, Celsr1, on mouse chromosome 15 (b). To define a region of conserved synteny between mouse and Syrian hamster, polymorphisms in hamster orthologues of mouse genes located near Celsr1 were identified. These polymorphisms mapped to the same linkage group as the tau mutation and the RDA markers (c). Because no candidate genes for tau were found in the mouse, the corresponding region of the human genome was analysed. This region contained an obvious candidate, CSNK1E, a human orthologue of Drosophila dbt gene (d). Sequencing of the hamster CSNK1E gene revealed allelic variation between wild-type and tau animals. The wild-type and mutant alleles co-segregate completely with the circadian phenotype (e).

add a further fascinating twist to the story18–20. In each case, a negative transcription factor has been shown to play an important role as an activator of positive clock transcription factors (e.g. the negative transcription regulator PER2 activates the positive BMAL1 loop in mice20). Thus, a gene involved in an inhibitory feedback loop is also involved in switching on positive activators and, even though different genes are involved in different organisms, the overall process is clearly highly conserved across a large evolutionary time-span. The Drosophila gene dbt encodes the first gene product implicated in the post-translational modification of clock proteins. DBT phosphorylates the PER protein in the cytoplasm, thus affecting its stability and marking it for enzymatic degradation21. The importance of this process is seen in dbtP mutant embryos, in which the clock stops21. In this mutation, the kinase is inactive and PER protein ceases to cycle. Other mutations of Drosophila dbt have the effect of shortening the circadian cycle, as is also the case with mammalian tau, which has an Arg→Cys substitution at a highly conserved position in the phosphate recognition site9. Lowrey et al. were able to show that the resulting conformational change apparently modifies the ability of the enzyme to phosphorylate the substrate PER protein. It

remains to be determined whether, as in Drosophila, altering the timing of nuclear entry effectively speeds up the clock. Consistent with this, the Per1 mRNA rhythm in the hamster SCN has been shown to exhibit a 20 h periodicity in the tau mutants, in contrast to a 24 h cycle in wild-type animals9.

PAS domains The comparative approach has proved to be a most effective method of defining the molecular ‘cogs’ of the circadian clock, and one of the best examples of this is seen in studies of clock-protein structure. A common feature of many of these proteins are PAS domains (Table 1). This region was defined in the Drosophila PER protein22 and subsequently shown also to be present in the Neurospora clock proteins WHITE COLLAR-1 and WHITE COLLAR-2 (Ref. 23), and in the mouse CLOCK protein24. These domains are known to act as sites for protein–protein interaction and ligand binding, and deletion studies in Drosophila have shown that one function of this region is to enable clock-protein dimerization before nuclear entry13. Knowledge of the role of PAS-containing proteins has proved to be an invaluable tool for clock biologists searching for new clock genes and a combination of redundant PCR-based screening, yeast two-hybrid assays TIG November 2000, volume 16, No. 11

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TABLE 1. A–Z of circadian clock and clock-associated genes and their discovery Genea

Function

Discovery and cloning

Refs

Mammals: Bmal1 (Mop3) Clock Cry1 Cry2 Per1 (RIGUI)

bHLH-PAS transcriptional activator bHLH-PAS transcriptional activator Repressor Repressor PAS-containing repressor PAS-containing repressor/activator PAS-containing protein. Function uncertain Kinase involved in PER1 turnover Uncertain

EST identified by BLAST search with PAS sequence Positional cloning EST identified by BLAST with dCry sequence ESTs identified by BLAST search with Cry1 and CRY1 Per1: IMS-PCR based on PAS domain sequence of Drosophila period gene RIGUI: BLAST search with a human chromosome 17-specific cDNA BLAST search with PER1 and Drosophila PER sequence BLAST search with Per2 sequence Positional syntenic cloningb BLAST search with Drosophila TIM

40,41 42,43 44,45 44,45 46 47 48 49 4,9,72 50

bHLH-PAS transcriptional activator bHLH-PAS transcriptional activator Light signal transduction Kinase involved in PER turnover PAS-containing repressor Repressor Transcription factor

cDNA library screening using ESTs identified by search against bHLH-PAS sequence EST identified by BLAST search against mouse PAS sequence Functional complementation P-element rescue Positional cloning and functional complementation Positional cloning Positional cloning

51 52 44,53 54 1,38 55,56 57,58

MYB-like transcription factor MYB-like transcription factor RNA-binding protein PAS-containing protein, may be involved in light-regulated proteolysis

Transposon tagging, IPCR, complementation South-western screening of expression library Subtractive hydridization Positional cloning

59 60 61–63 25,64

Repressor/Activator PAS-containing Zn finger transcriptional activator PAS-containing Zn finger transcriptional activator

Positional cloning and functional complementation Positional cloning and functional complementation

19,65,66 67,68

Screening of genomic library with products of IPCR

69

Activator Unknown Repressor with ATP and GTP binding sites

Functional complementation Functional complementation Functional complementation

70,71 70,71 70,71

Per2 Per3 Tau (Csnk1e) Timeless (Tim) Drosophila : Cycle (Cyc, dBma1) dClock (Jrk) dCry double-time (dbt) period (per) timeless (tim) vrille (vri) Arabidopsis : LHY CCA1 CCR2 ZEITLUPE Neurospora : frequency (frq) white collar-1 (wc-1) white collar-2 (wc-2) Synechococcus: KaiA KaiB KaiC a

Shaded genes: discovered as a result of induced or spontaneous mutations. Other genes discovered by homology to known genes or protein domains or by reverse genetic approaches. CSNK1E was originally cloned in 1995 (Ref. 72); however its role in mammalian circadian clock was clarified by Lowrey et al.9

b

and bioinformatic analysis of genome databases have revealed new clock genes in many different organisms (Table 1). The ubiquitous nature of PAS-containing proteins in the clock processes is illustrated in a recent report from Arabidopsis researchers, showing that mutations of PAS-containing proteins are associated with altered circadian phenotypes25,26. It is now clear that PAS-containing proteins are widespread and are, in many cases, involved in environmental sensing and signal transduction27. It is thus perhaps not surprising that some have been commandeered to function in a system in which a diversity of external temporal cues converge and synchronize internal cellular events.

Genes in different organisms There are subtle differences in the way in which clock genes operate in different organisms. Several clock gene copies are known to be present in mammals and zebrafish, apparently owing to the polyploid nature of the vertebrate genome. For example, there are three mammalian orthologues of the Drosophila gene per, which have adopted different roles to their Drosophila counterpart20,28. Another example are the cryptochromes, which were isolated from Arabidopsis during studies of light signal transduction29. In Drosophila, the dCry gene is now known to be a core component in light signal transduction directly to the circadian clock30. A more central role for cryptochromes in circadian clock function is seen in mammals. Here, a search for blue480

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light photoreceptors led to the discovery of two homologues of plant cryptochromes (Cry1 and Cry2), which lead to complete circadian behavioural arrhythmicity when combined in a double knockout31. These genes play a major role in the mammalian clock: CRY1 and CRY2 dimerize with PER proteins, and the resulting complex regulates their own expression and that of other clock genes20. In contrast to Drosophila, however, cryptochromes from mammals seem not to be involved in light-signal transduction32.

Conclusion We have had to wait 12 years since the discovery of the tau mutant hamster for the biochemical identity of this first vertebrate clock mutation to be revealed. The stunning achievement of Lowrey and colleagues now brings us full circle back to per. The role of tau in the molecular physiology of the per feedback loop provides the clearest possible indication that the major molecular components of the central circadian oscillator might now have been defined. By using the obvious parallels between clocks in different organisms, the team were able to search across two genomes for syntenic regions, which contained a clock gene candidate. This study shows that comparative genomics can release biologists from earlier constraints, and that geneticists can now tackle poorly characterized genomes with increased confidence. Many in the field are now focused on defining the ways in which clock-driven processes engage physiological systems. In the past three years, we have become aware of the

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existence of cycling clock gene components in peripheral tissues, an observation first made in the Drosophila Malpighian tubule33 but that we now suspect extends to many mammalian tissues34 and even well established cell lines35. How are these millions of separate clocks synchro-

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nized by output from central oscillators in the brain and what is their function? These and other questions will keep attention focused on clocks for at least another 12 years and build on a foundation laid in this field by Pittendrigh36 and Aschoff37.

associated PAS protein from Arabidopsis. Cell 101, 319–329 26 Nelson, D.C. et al. (2000) FKF1, a clock-controlled gene that regulates the transition to flowering in Arabidopsis. Cell 101, 331–340 27 Taylor, B.L. and Zhulin, I.B. (1999) PAS domains: internal sensors of oxygen, redox and light. Microbiol. Mol. Biol. Rev. 63, 479–506 28 Field, M.D. et al. (2000) Analysis of clock proteins in mouse SCN demonstrates phylogenetic divergence of the circadian clockwork and resetting mechanisms. Neuron 25, 437–447 29 Ahmad, M. (1999) Seeing the world in red and blue: insight into plant vision and photoreceptors. Curr. Opin. Plant Biol. 2, 230–235 30 Ceriani, M.F. et al. (1999) Light-dependent sequestration of TIMELESS by CRYPTOCHROME. Science 285, 553–556 31 Horst van der, G.T. et al. (1999) Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature 398, 627–630 32 Griffin, E. et al. (1999) Light-independent role of CRY1 and CRY2 in the mammalian circadian clock. Science 286, 768–771 33 Giebultowicz, J.M. and Hege, D.M. (1997) Circadian clock in Malpighian tubules. Nature 386, 664 34 Yamazaki, S. et al. (2000) Resetting of central and peripheral circadian oscillators in transgenic rats. Science 288, 682–685 35 Balsalobre, A. et al. (1998) A serum shock induces circadian gene expression in mammalian culture cells. Cell 93, 929–937 36 Pittendrigh, C.S. (1993) Temporal organization: reflections of a Darwinian clock-watcher. Annu. Rev. Physiol. 55, 16–54 37 Aschoff, J. (1960) Exogenous and endogenous components in circadian rhythms. Cold Spring Harbor Symp. Quant. Biol. 25, 11–28 38 Bargiello, T.A. et al. (1984) Restoration of circadian behavioural rhythms by gene transfer in Drosophila. Nature 312, 752–754 39 Reddy, P. et al. (1984) Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms. Cell 38, 701–710 40 Ikeda, M. and Nomura, M. (1997) cDNA cloning and tissuespecific expression of a novel basic helix–loop–helix/PAS protein (BMAL1) and identification of alteratively spliced variants with alternative translation initiation site usage. Biochem. Biophys. Res. Commun. 233, 258–264 41 Hogenesch, J.B. et al. (1998) The basic helix–loop–helix–PAS orphan MOP3 forms transcriptionally active complexes with circadian hypoxia factors. Proc. Natl. Acad. Sci. U. S. A. 95, 5474–5479 42 Vitaterna, M.H. et al. (1994) Mutagenesis and mapping of a mouse gene, clock, essential for circadian behaviour. Science 264, 719–725 43 King, D. et al. (1997) Positional cloning of the mouse circadian CLOCK gene. Cell 89, 641–653 44 Todo, T. et al. (1996) Similarity among the Drosophila (6–4) photolyase, a human photolyase homolog, and the DNA photolyase–blue light photoreceptor family. Science 272, 109–112 45 Hsu, D.S. et al. (1996) Putative human blue-light photoreceptors hCRY1 and hCRY2 are flavoproteins. Biochemistry 35, 13871–13877 46 Tei, H. et al. (1997) Circadian oscillation of a mammalian homologue of the Drosophila period gene. Nature 389, 512–516 47 Sun, Z.S. et al. (1997) RIGUI, a putative mammalian ortholog of the Drosophila period gene. Cell 90, 1003–1011 48 Shearman, L. et al. (1997) Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei. Neuron 19, 1261–1269 49 Takumi, T. et al. (1998) A light-independent oscillatory gene mPer3 in mouse SCN and OVLT. EMBO J. 17, 4753–4759

50 Zylka, M. et al. (1998) Molecular analysis of mammalian Timeless. Neuron 21, 1115–1122 51 Rutila, J.E. et al. (1998) CYCLE is a second bHLH–PAS clock protein essential for circadian rhythmicity and transcription of Drosophila period and timeless. Cell 93, 805–813 52 Allada, R. et al. (1998) A mutant Drosophila homolog of mammalian Clock disrupts circadian rhythms and transcription of period and timeless. Cell 93, 791–804 53 Stanewsky, R. et al. (1998) The cry b mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell 95, 681–692 54 Price, J.L. et al. (1998) double-time is a new Drosophila clock gene that regulates PERIOD protein accumulation. Cell 94, 83–95 55 Sehgal, A. et al. (1994) Loss of circadian behavioural rhythms and period RNA oscillations in the Drosophila mutant timeless. Science 263, 1603–1606 56 Myers, M.P. et al. (1995) Positional cloning and sequence analysis of the Drosophila clock gene, timeless. Science 270, 805–808 57 George, H. and Terracol, R. (1997) The vrille gene of Drosophila is a maternal enhancer of decapentaplegic and encodes a new member of the bZIP family of transcription factors. Genetics 146, 1345–1363 58 Blau, J. and Young, M.W. (1999) Cycling vrille expression is required for a functional Drosophila clock. Cell 99, 661–671 59 Schaffer, R. et al. (1998) The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering. Cell 93, 1219–1229 60 Wang, Z-Y. et al. (1997) A myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene. Plant Cell 9, 491–499 61 van Nocker, S. and Viestra, R.D. (1993) Two cDNAs from Arabidopsis thaliana encode putative RNA-binding proteins containing glycine-rich domains. Plant Mol. Biol. 21, 695–699 62 Heintzen, C. et al. (1997) AtGrp7, a nuclear RNA-binding protein as a component of a circadian-regulated negative feedback loop in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 94, 8515–8520 63 Carpenter, C.D. et al. (1994) Genes encoding glycine-rich Arabidopsis thaliana proteins with RNA-binding motifs are influenced by cold treatment and an endogenous circadian rhythm. Plant Physiol. 104, 1015–1025 64 Millar, A.J. et al. (1995) Circadian clock mutants in Arabidopsis identified by luciferase imaging. Science 267, 1161–1163 65 Feldman, J.F. and Hoyle, M. (1973) Isolation of circadian clock mutants of Neurospora crassa. Genetics 75, 605–613 66 McClung, C.R. et al. (1989) The Neurospora clock gene frequency shares a sequence element with the Drosophila clock gene period. Nature 339, 558–562 67 Perkins, D.D. et al. (1962) New data on markers and rearrangements in Neurospora. Can. J. Genet. Cytol. 4, 187–205 68 Ballario, P. et al. (1996) White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein. EMBO J. 15, 1650–1653 69 Linden, H. and Macino, G. (1997) White collar-2, a partner in blue-light signal transduction, controlling expression of light-regulated genes in Neurospora crassa. EMBO J. 16, 98–109 70 Kondo, T. et al. (1994) Circadian clock mutants of cyanobacteria. Science 266, 1233–1236 71 Ishiura, M. et al. (1998) Expression of a clock gene cluster kiaABC as a circadian feedback process in cyanobacteria. Science 281, 1519–1523 72 Fish, K.J. et al. (1995) Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family. J. Biol. Chem. 270, 14875–14883

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