The mouse Clock locus: sequence and comparative analysis of 204 kb from mouse chromosome 5.

The Clock gene encodes a basic helix-loop-helix (bHLH)-PAS transcription factor that regulates circadian rhythms in mice. We previously cloned Clock in mouse and human using a battery of behavioral and molecular techniques, including shotgun sequencing of two bacterial artificial chromosome (BAC) clones. Here we report the finished sequence of a 204-kb region from mouse chromosome 5. This region contains the complete loci for the Clock and Tpardl (pFT27) genes, as well as the 3' partial locus of the Neuromedin U gene; sequence analysis also suggests the presence of two previously unidentified genes. In addition, we provide a comparative genomic sequence analysis with the syntenic region from human chromosome 4. Finally, a new BAC transgenic line indicates that the genomic region that is sufficient for rescue of the Clock mutant phenotype is no greater than 120 kb and tightly flanks the 3' end of the Clock gene.

Molecular cloning and characterization of the human CLOCK gene: expression in the suprachiasmatic nuclei.

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.

Mammalian circadian autoregulatory loop: a timeless ortholog and mPer1 interact and negatively regulate CLOCK-BMAL1-induced transcription.

We report the cloning and mapping of mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals.

Functional identification of the mouse circadian Clock gene by transgenic BAC rescue.

As a complementary approach to positional cloning, we used in vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice to identify the circadian Clock gene. A 140 kb BAC transgene completely rescued both the long period and the loss-of-rhythm phenotypes in Clock mutant mice. Analysis with overlapping BAC transgenes demonstrates that a large transcription unit spanning approximately 100,000 base pairs is the Clock gene and encodes a novel basic-helix-loop-helix-PAS domain protein. Overexpression of the Clock transgene can shorten period length beyond the wild-type range, which provides additional evidence that Clock is an integral component of the circadian pacemaking system. Taken together, these results provide a proof of principle that "cloning by rescue" is an efficient and definitive method in mice.

Positional cloning of the mouse circadian clock gene.

We used positional cloning to identify the circadian Clock gene in mice. Clock is a large transcription unit with 24 exons spanning approximately 100,000 bp of DNA from which transcript classes of 7.5 and approximately 10 kb arise. Clock encodes a novel member of the bHLH-PAS family of transcription factors. In the Clock mutant allele, an A-->T nucleotide transversion in a splice donor site causes exon skipping and deletion of 51 amino acids in the CLOCK protein. Clock is a unique gene with known circadian function and with features predicting DNA binding, protein dimerization, and activation domains. CLOCK represents the second example of a PAS domain-containing clock protein (besides Drosophila PERIOD), which suggests that this motif may define an evolutionarily conserved feature of the circadian clock mechanism.

Molecular cloning of chick pineal tryptophan hydroxylase and circadian oscillation of its mRNA levels.

We have previously shown that the level of [35S]methionine incorporation into tryptophan hydroxylase (TPH) shows a circadian rhythm in cultured chick pineal cells. The TPH protein oscillation persists in constant darkness, peaks in the early night and can be phase-shifted by light, in parallel to the effect of these treatments on melatonin synthesis. We have cloned and sequenced a full-length cDNA for chick pineal TPH. Levels of TPH mRNA show a robust diurnal oscillation both in vivo and in vitro. The rhythm in TPH mRNA also persists in constant darkness, suggesting that TPH mRNA synthesis and/or turnover is regulated by an endogenous circadian clock in cultured chick pineal cells. The circadian oscillation of TPH constitutes the first described circadian rhythm of a chick pineal gene at the mRNA level.