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Genetic heterogeneity underlies many phenotypic variations observed in circadian rhythmicity. Continuous distributions in measures of circadian behavior observed among multiple inbred strains of mice suggest that the inherent contributions to variability are polygenic in nature. To identify genetic loci that underlie this complex behavior, we have carried out a genome-wide complex trait analysis in 196 (C57BL/6J X BALB/cJ)F2 hybrid mice. We have characterized variation in this panel of F2 mice among five circadian phenotypes: free-running circadian period, phase angle of entrainment, amplitude of the circadian rhythm, circadian activity level, and dissociation of rhythmicity. Our genetic analyses of these phenotypes have led to the identification of 14 loci having significant effects on this behavior, including significant main effect loci that contribute to three of these phenotypic measures: period, phase, and amplitude. We describe an additional locus detection method, genome-wide genetic interaction analysis, developed to identify locus pairs that may interact epistatically to significantly affect phenotype. Using this analysis, we identified two additional pairs of loci that have significant effects on dissociation and activity level; we also detected interaction effects in loci contributing to differences of period, phase, and amplitude. Although single gene mutations can affect circadian rhythms, the analysis of interstrain variants demonstrates that significant genetic complexity underlies this behavior. Importantly, most of the loci that we have detected by these methods map to locations that differ from the nine known clock genes, indicating the presence of additional clock-relevant genes in the mammalian circadian system. These data demonstrate the analytical value of both genome-wide complex trait and epistatic interaction analyses in further understanding complex phenotypes, and point to promising approaches for genetic analysis of such phenotypes in other mammals, including humans.

Many aspects of normal retinal physiology are controlled by a retinal circadian clock. In Xenopus laevis, the photoreceptor cells within the retina contain a circadian clock that controls melatonin release. In this report we present the cloning and characterization of the Xenopus homolog of the Clock gene, known to be critical for normal circadian behavioral rhythms in the mouse. The Xenopus Clock gene is expressed primarily in photoreceptors within the eye and is expressed at constant levels throughout the day. Analysis of other tissues revealed that, as in other species, the Xenopus Clock gene is widely expressed. This characterization of the Clock gene provides a useful tool for further exploration of the role of the circadian clock in normal retinal function.

The Clock gene is an essential regulator of circadian rhythms. It encodes a member of the basic helix-loop-helix/PER-ARNT-SIM family of transcription factors known to play a central role in the control of diverse cellular events. Previously we described the functional identification and molecular isolation of the Clock gene in the mouse, its interaction with the BMAL1 protein, and the role of this complex as a transcriptional activator in the circadian pacemaker. Here, we report the cloning, exon organization, chromosomal location, and mRNA expression of the human CLOCK gene. The coding sequence of human CLOCK extends for 2538 bp and is 89% identical to its mouse ortholog; its deduced amino acid sequence is 846 residues long and is 96% identical to mouse CLOCK. Radiation hybrid mapping localized human CLOCK to the long arm of human chromosome 4 (4q12). Direct sequencing of a genomic CLOCK clone indicated that the coding sequence of human CLOCK extends over 20 exons and that its intron/exon organization is identical to that of the mouse ortholog. Northern blot analysis indicated widespread expression of two major transcripts of 8 and 10 kb, and in situ hybridization of human brain tissue revealed elevated expression of CLOCK mRNA in the suprachiasmatic nuclei, the locus of circadian control in mammals, and in the cerebellum. Comparison of cDNA clones revealed two single nucleotide polymorphisms in noncoding sequence flanking the CLOCK open reading frame. The central role of Clock in the organization of circadian rhythms suggests that it will be a useful candidate gene for genetic analyses of disorders associated with dysfunction of the circadian system.