David P. King

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.

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.

Forward Genetic Approaches to Circadian Clocks in Mice

The study of mammalian circadian rhythms has a rich history of physiological understanding, but the molecular elements of the mammalian clock remain to be discovered. Availability of effective mutagenesis procedures and mouse molecular genetics resources sufficient for the task of positional cloning give cause for hope that the Clock gene, as well as other components of the mammalian circadian system, will be identified soon. The isolation of Clock in a deliberate mutagenesis screen provides an important proof-of-principle that forward genetics can be successfully applied toward the discovery of novel genes underlying complex behaviors in mice.