The clock proteins, aging, and tumorigenesis.

Many aspects of mammalian physiology and behavior are driven by an intrinsic timekeeping system that has an important role in synchronizing various biological processes within an organism and coordinating them with the environment. It is believed that deregulation of this coordination may cause the development of various pathologies. However, recent studies using mice deficient in individual components of the circadian system clearly demonstrated more complex interaction of the circadian system with various biological processes. The growing amount of evidence suggests that in addition to their roles in the core clock mechanism, some of the components of the molecular oscillator are involved in modulation of such diverse physiological processes as response to genotoxic stress, regulation of the cell cycle, aging, and carcinogenesis. These new data provide a mechanistic link between deregulation of the circadian system and/or some of its core components and the development of various pathologies, suggesting novel strategies for the disease treatment and prevention.

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.

The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.

Mice express S and M opsins that form visual pigments for the detection of light and visual signaling in cones. Here, we show that S opsin transcription is higher than that of M opsin, which supports ultraviolet (UV) sensitivity greater than midwavelength sensitivity. Surprisingly, most cones coexpress both S and M opsins in a common cone cell type throughout the retina. All cones express M opsin, but the levels are graded from dorsal to ventral. The levels of S opsin are relatively constant. However, in the far dorsal retina, S opsin is repressed stochastically, such that some cones express M opsin only. These observations indicate that two different mechanisms control M and S opsin expression. We suggest that a common cone type is patterned across the retinal surface to produce phenotypic cone subtypes.

Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau.

The tau mutation is a semidominant autosomal allele that dramatically shortens period length of circadian rhythms in Syrian hamsters. We report the molecular identification of the tau locus using genetically directed representational difference analysis to define a region of conserved synteny in hamsters with both the mouse and human genomes. The tau locus is encoded by casein kinase I epsilon (CKIepsilon), a homolog of the Drosophila circadian gene double-time. In vitro expression and functional studies of wild-type and tau mutant CKIepsilon enzyme reveal that the mutant enzyme has a markedly reduced maximal velocity and autophosphorylation state. In addition, in vitro CKIepsilon can interact with mammalian PERIOD proteins, and the mutant enzyme is deficient in its ability to phosphorylate PERIOD. We conclude that tau is an allele of hamster CKIepsilon and propose a mechanism by which the mutation leads to the observed aberrant circadian phenotype in mutant animals.

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.

Transducin GTPase provides for rapid quenching of the cGMP cascade in rod outer segments.

The role of transducin GTPase in rapid cGMP phosphodiesterase quenching was studied by simultaneous registration of GTP hydrolysis and phosphodiesterase activity in the same rod outer segments (ROS) preparation. The results thus obtained allow the conclusion that: (i) phosphodiesterase quenching coincides with transducin-bound GTP hydrolysis independently of ROS concentration; (ii) an increase in the ROS concentration results in the acceleration of cascade quenching due to the existence of a GTPase accelerating mechanism in ROS; (iii) approximation to physiological conditions (protein concentration, temperature) provides a transducin GTPase rate equal to 1-2 turnovers per second i.e., sufficiently high for satisfying the real rate of photoresponse reversion in dark-adapted rods.

On the role of transducin GTPase in the quenching of a phosphodiesterase cascade of vision.

The rate of GTP hydrolysis in the active site of transducin and that of the release of the phosphate thus formed have been measured. The former step has been found to be a rate-limiting one. The rate constant for GTP hydrolysis is equal to 0.027 s-1 at 23 degrees C, and 0.07 s-1 at 37 degrees C. Besides, it has been shown that the rate of GTPase reaction on the transducin alpha-subunit does not depend on the concentration of a complex of transducin beta- and gamma-subunits or on the presence of cGMP phosphodiesterase and a 48 kDa protein from rod outer segments. According to the results, GTP hydrolysis on transducin proceeds too slowly to account for the rapid quenching of a phosphodiesterase cascade in rod outer segments.