Joseph S. Takahashi
Molecular Neurobiology and Genetics of Circadian ClocksA central question in the field of circadian rhythms concerns the molecular mechanism of circadian clocks. How are circadian oscillations generated and at what level of cellular organization do they originate? Two major approaches have been used to study the cellular and molecular mechanisms regulating circadian rhythms: 1) physiological analysis using perturbations with pharmacological and biochemical methods; and 2) genetic and molecular genetic analysis using circadian "clock mutants." While substantial progress have been achieved in our understanding of the neurobiology and physiology of circadian rhythms over the last two decades, much less is known about the molecular mechanisms that generate circadian rhythms in mammals (Takahashi 1995). Continued application of reductionist approaches has shown that the circadian clock is cell autonomous and requires periodic gene expression. However, it is not clear at this point in time whether such approaches alone will ultimately reveal the mechanism of the circadian clock in mammals. In the study of other cellular functions of comparable complexity, such as the cell cycle, pattern formation and signal transduction, the genetic approach has been essential in identifying the set of genes involved in regulating these processes. Given the likelihood that the set of genes regulating circadian rhythms in mammals is largely undescribed and therefore unknown, a forward genetic approach to mammalian circadian rhythms is clearly warranted.
it is generally assumed that classical mutagenesis and screening procedures are not feasible in the mouse. However' the development of high efficiency Hemline mutagenesis procedures with the alkylating agent, N ethyl N nitrosourea (ENU) by Russell and colleagues have made it feasible to undertake large scale mutant screens in the mouse (Rinchik 1991). Average forward mutation frequencies of 0.0015 per locus per gamete can be achieved in the mouse with ENU. This means that one has a 50% chance of finding a mutation, on average, in any single locus by screening about 655 gametes. With these considerations in mind, we initiated a behavioral screen for ENU induced mutations of the circadian system in the mouse. This effort is one of three major genetic screens in Arabidopsis, Drosophila and mice, which together form the "Clock Genome Project" within the NSF Center for Biological Timing. Because the majority of clock mutants isolated in other organisms have been semidominant, we screened G1 heterozygous offspring of ENU treated mice directly in a search for dominant mutations. In screening 300 Gil mice, we identified a clock mutation in the mouse (Vitaterna et al 1994). This mutation defines a gene, called Clock, that is essential for expression of normal circadian behavior. Under constant conditions, heterozygous Clock mutants express circadian periods that are about one hour longer than those seen in wild type mice. In contrast, homozygous Clock mutants initially express periods that are about four hours longer than normal and then completely lose circadian rhythmicity. Genetic linkage analysis shows that Clock segregates as a single gene that maps to the midportion of mouse chromosome 5 in a region with conserved synteny with human chromosome 4. The Clock mutation causes no obvious developmental or anatomical lesions, focusing its domain of action on behavior per se.
A number of questions are raised by the existence of the Clock mutation: What is the molecular nature of the gene product underlying the Clock mutation? What other genes interact with Clock? Is Clock a component of the molecular mechanism of the mammalian circadian clock? How many genes compose the core mechanism of the circadian pacemaking system? How do these genes interact to generate circadian oscillations? How do these genes ultimately regulate output pathways controlling physiology and behavior? An obvious goal for the near future is to clone Clock. This task should be reasonable in the mouse: Clock can by cloned by the method of positional cloning using the rapidly evolving genetic and physical mapping resources available with the "new mouse genomics" (Takahashi et al 1994). We have mapped Clock at high resolution (2000 meioses), have completed a 2 cM (4 Mb) YAC contig of the Clock region and are developing YAC expression methods in transgenic mice in order to rescue the mutation. Using a combination of physical mapping and functional rescue we should be able to identify the Clock gene.