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The Molecular Genetics of Circadian Clocks
(Released August 1999)

  by Wilma Ek, M.S.  
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The daily rhythm of life is maintained by a circadian clock in organisms ranging from bacteria to humans. The time kept by a circadian clock enables the organism to respond physiologically and influences its behavior to daily environmental fluctuations. Perhaps the best known rhythms are those that display an approximate 24 hour period length, entrainment of the clock in response to light-dark transitions and a temperature compensation of the period.

Circadian rhythms are thought to contain at least three elements: a) input pathways(s) relay environmental information to a circadian pacemaker (clock) b) the circadian pacemaker generates the oscillation and c) output pathway(s) through which the pacemaker regulates various output rhythms. Rapid strides have been made in the field of circadian biology at the physiological level and more recently, at the molecular level. A recognizable pattern that is emerging is based on autoregulatory transcription/translation feedback loops. In this hot topic, we will summarize our understanding of the molecular mechanisms underlying circadian clocks.

Cyanobacteria: The circadian clock in Synechococcus controls processes such as photosynthesis, nitrogen fixation, cell division, respiration, amino acid uptake and carbohydrate synthesis. A gene cluster comprising three clock genes was cloned viz., kaiA, kaiB and kaiC. Gene regulation and phenotypic analysis of mutant alleles indicate that the kaiA gene product is a transcription activator. The kaiC gene product functions as a negative element of the oscillator. To date, the role of the kaiB gene product is unknown. Gene expression of the kai gene cluster is not greatly affected by light, providing scarce information on entrainment of this clock.

Fungi: In Neurospora crassa, the frequency (frq) gene satisfies both input and oscillator criteria. Conidiation enables the monitoring of circadian rhythms. Both, light and temperature contribute to entrainment of the oscillator. We will focus on light as an environmental signal. At midnight, frq transcript and FRQ isoform levels are low. Transcript levels rise and peak around mid-morning. Two transcription factors, WC-1 and WC-2 are responsible for the increase in frq transcription. FRQ proteins appear around dawn and levels peak in the early afternoon. Either of the FRQ isoforms is partially phosphorylated. FRQ enters the nucleus and interacts with WC-2. Around midday, the amount of nuclear FRQ falls but rises in the cell. In the afternoon, frq levels decline and increasingly phosphorylated FRQ levels fall through the early night, starting the cycle over.

Drosophila: The period (per), timeless (tim) and doubletime (dbt) genes have been identified as clock components. Additional clock-related genes that have been cloned include the Clock (Clk), cycle (cyc) and cryptochrome (cry) genes. In flies, the gene product of the cryptochrome gene, CRY, sets the clock to a 24-hour light-dark cycle. The circadian cycle begins around noon when per and tim mRNA levels rise. Transcription activation is largely due to the PAS protein heterodimer of CLK and CYC. The protein encoded by the dbt gene, DBT, regulates accumulation of per and tim RNA. After sunset, the accumulated RNA is translated, PER and TIM proteins associate to form dimers and enter the nucleus. PER and TIM become increasingly phosphorylated through the night. Approximately four hours before dawn, the level of PER/TIM protein complexes peaks. This event signals the cells to terminate per and tim transcription. Around dawn, TIM is rapidly degraded and PER is released from the complex. Per and tim transcription commences once again, by midday. Light is able to reset the clock by the interaction of CRY with TIM. Recent studies suggest both, CRY and visual input regulate the main clock responsible for behavior in Drosophila.

Plants: In plants, the circadian clock controls processes like photosynthesis, flowering and the passage of seasons. Photosynthesis progresses optimally during the day. A gene encoding components of the light harvesting chlorophyll a/b complex (LHC) is regulated by the circadian clock. Both Lhc transcription and LHC protein synthesis occur during the day peaking around noon. Another gene, the toc-1 gene has been identified in Arabidopsis as a component of the clock. Mutation in this gene affects several circadian outputs. Using luciferase as a reporter gene, sequences responsible for light/dark regulation were identified in the promoter of the Arabidopsis lhcb1*1 gene. A number of DNA-binding proteins bound to these sequences. One of them, a myb-like transcription factor CCA1 oscillates in a circadian fashion. The LHY (late elongated hypocotyl) gene also encodes a myb-like transcription factor. Overexpression studies of both genes indicate that LHYand CCA1 are likely candidates of negative feedback loops. In Arabidopsis, the red light-sensitive phytochromes and the blue light-sensitive cryptochromes are involved in setting the clock.

Mammals (mice): The suprachiasmatic nucleus (SCN) is the site of the central pacemaker in mice. Cloning of the Clock gene identified the protein as containing the protein-protein-binding (PAS) domain in addition to a DNA-binding domain. Molecular studies further identified CLOCK's partner, a transcription activator named BMAL1/MOP3. Three per gene homologs and one tim homolog, mPer1, mPer2, mPer3and mTim1 have also been characterized. mPer1 transcript levels increase toward late night. The interaction between CLOCK and BMAL1/MOP3 is partially responsible for this increase. Soon after, mPer2 and mPer3 levels increase. The levels of the three mPers peak at different times of the day. Unlike Drosophila, the mPER-mPER interaction is stronger than the mPER-mTIM interaction thereby defining a different role for mTIM in the circadian machinery. Presumably, the PERs enter the nucleus, interact via their PAS domains, repress transcription by disruption of the BMAL1/CLOCK activation of their promoters, get phosphorylated and turn over. The ubiquitous BMAL1/CLOCK heterodimers activate mPer transcription again and the cycle starts over. In mice, the Cry1 and Cry2 proteins have been implicated in light entrainment of the clock.

Several inroads have been made in the enigmatic phenomenon of circadian rhythms. Evolutionarily, nature has cooperated using fairly common molecular mechanisms. Regulatory loops and PAS domain proteins are unifying features in eukaryotes as are regulatory loops and PER and TIM proteins in animals. Several genes and their role in the input and output pathways beg elucidation. Future research will enable a lucid dissection of individual components in the clock in relation to entrainment and the final effects on biological rhythms.



Wilma Ek, M.S.

  • CSA Senior Life Science Editor

  • B.Sc., University of Bombay

  • M.Sc. (Microbiology and Biochemistry), University of Bombay

  • M.S. (Genetics), Pennsylvania State University

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