Circadian rhythms of activity and physiology are evident across the animal kingdom as well as in plants and some bacteria. The scientific study of biological clocks goes back almost 300 years to a French astronomer called Jean-Jacques d’Ortous De Mairan, who discovered that the diurnal closing of Mimosa leaves persisted under conditions of constant darkness. Whether this was due to mysterious “magnetic rays”, or to the presence of an equally mysterious internal clock in the plant was controversial, but it later became clear that light-independent twenty-four hour clocks could also be found in animals. The mechanisms underlying such clocks were a long-standing puzzle until the Shaw Prize Laureates of 2013, Jeffrey C Hall, Michael Rosbash and Michael W Young discovered two key components of the endogenous clock mechanism of the fruit fly, Drosophila melanogaster. Over the course of the last twenty-five years, thanks to the work of these pioneers, details of the clock mechanism in animals have steadily emerged. It is a much more complicated molecular machine than any theorist had imagined.

The crucial first step for the molecular understanding of biological clocks came in 1971, when Ronald Konopka and Seymour Benzer identified three mutant strains of fruit flies that showed heritably altered circadian rhythms. Mapping the mutations revealed a single gene, Period, or Per, that could be mutated to give either shorter or longer cycles of activity, or no rhythmic activity cycles at all. Clearly, Per was intimately connected with the clock. But how the clock worked could only be a matter for speculation until the Per gene was cloned, a challenging feat that was achieved in 1984 by Michael Young at Rockefeller University and, independently, by a collaboration between Jeffrey Hall and Michael Rosbash at Brandeis University. But the deduced protein sequence of Per did not at first reveal its nature or function.

The truth began to dawn in 1988 and 1990, when the Hall and Rosbash labs measured Per protein and mRNA levels in the flies’ heads during the day and night. High levels of Per were found at night, and low levels by day. Crucially, these circadian oscillations continued even when the animals were kept in constant darkness, suggesting that Per was truly a central component of the clock mechanism. The most revealing finding was that the level of Per messenger RNA was maintained at a constant high value in arrhythmic Permutants, implying that Per shut off its own synthesis. This turned out to be quite right: Per is a transcriptional repressor.

The next clue came from Young’s lab in 1994, where a fresh genetic screen for flies with abnormal circadian rhythms revealed a second key component of the clock, called Timeless, or Tim. Remarkably, their studies revealed that levels of Tim protein and mRNA oscillate in parallel with Per. Even more telling, in Timmutant flies with disrupted circadian rhythm, Per protein failed to enter the nucleus where it normally accumulates, indicating a critical interaction between Tim and Per.

Subsequent studies provided additional information on the core clock mechanism discovered by Hall, Rosbash, and Young. Further genetic screens by the two groups uncovered a number of proteins needed for proper functioning of the core mechanism, including Doubletime, a kinase discovered by Young’s lab that regulates the half-life of the Per protein, Cryptochrome, a light sensor uncovered by the Hall and Rosbash labs that allows entrainment, or resetting, of the clock, and other proteins involved in the transcription of Perand Tim genes, or the stability or nuclear localization of Per and Tim proteins. Other groups also began to contribute to the growing understanding of the Drosophila clock and, beginning with the cloning of a mouse clock gene by Joseph Takahasi’s group, information began to appear about a related clock mechanism in mammals.

With time, a detailed picture has emerged in which the protein products of the Per and Tim genes associate and are transported to the nucleus. There, the Per-Tim complex inhibits Cycle and Clock, two factors needed for Per and Tim gene transcription, resulting in a drop in the production of Per and Tim mRNAs and proteins. A further decline in nuclear Per-Tim complexes occurs owing to specific kinases and phosphatases that alter protein stability and/or transport to the nucleus. As a result, the repression of Per and Tim gene transcription is relieved, transcription restarts, and the whole process is then repeated. The correct twenty-four hour timing of the cycle is ensured by built-in delays between transcription and translation, as well as by the enzymes that regulate the stability of Per-Tim complexes and their nuclear transport.

The pioneering studies of Hall, Rosbash and Young on circadian rhythms constitute a major contribution to our understanding of a fundamental biological process. They discovered the core components and mechanisms of the Drosophila circadian clock and then went on to make numerous additional contributions to our present knowledge of how these mechanisms are regulated to ensure proper twenty-four hour cycles of physiology and behavior. We now know that many of the genes involved in the Drosophila circadian clock are present not only in insects, but also in mammals, where they appear to play similar roles. The human circadian clock is associated with sleep as well as other processes, including daily fluctuations in hormones and metabolism. Two human counterparts of the Drosophila Period gene have now been associated with hereditary syndromes that affect circadian patterns of sleep in humans, suggesting that these and other clock genes first discovered in fruit flies could ultimately shed light on mechanisms that control sleep as well as other important processes under circadian control in humans.

Life Science and Medicine Selection Committee
The Shaw Prize

23 September 2013   Hong Kong