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.

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.

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