Fall 1995 Volume 4, Number 4
An inescapable fact of human life is its organization into a cycle of activity and rest that corresponds to the environmental cycle of day and night. Obvious to the physician and physiologist, if not the casual observer, is the parallel underlying organization of numerous neurological, endocrine, and physiological processes into precise daily rhythms. Most, if not all of these are directed by a central timekeeping system in the brain.
This basic pattern of daily rhythms in physiology and behavior is a widespread property of life, found in the most diverse organisms -- birds, fish, and reptiles; insects, crustaceans, and molluscs; flowering plants, weeds, and trees; and even in single-celled forms, such as algae and fungi.
What makes these daily biological rhythms truly remarkable is that they persist, often indefinitely, in conditions of constant lighting and temperature. The first to report this phenomenon was the French astronomer de Mairan, who, in 1729, demonstrated that the daily leaf movements of a plant continued on schedule after the plant was moved to a basement and kept in total darkness.
In recent decades, intensive experimental work in species from fungi to humans has demonstrated that daily biological rhythms are generated internally. In artificial constant conditions, these rhythms adopt a cycle of slightly more or less than 24 hours, the exact deviation being characteristic of a given species. For example, intrinsic human rhythms are typically 24.2 hours, whereas a laboratory strain of the fruitfly Drosophila melanogaster, widely used in genetic studies, has a cycle of 23.7 hours.
These endogenous rhythms are referred to as "circadian," meaning "approximately one day." This is not the same as imprecision: The intrinsic period of circadian rhythms in humans and other mammals, for example, varies only several minutes per cycle, a precision much greater than 99 percent. Such precision allows the circadian system to give the organism a highly accurate measure of elapsed time.
The Earth's daily light-dark cycle is not required for circadian rhythms, but it strongly influences the circadian system. It causes the circadian system to be re-set each day so that an organism's intrinsic period becomes precisely synchronized, or "entrained," to the 24-hour day. This entraining puts the internal cycle in a fixed and proper relationship to the external light-dark cycle, allowing the circadian system to serve as a true biological clock. Thus, particular internal signals produced by the circadian system will always occur at a particular time relative to the external day. For example, peak levels of the hormonal signal, melatonin, will be produced in the middle of night, and the minimum core body temperature will occur in early morning, just prior to waking.
The circadian system is evidently responsible for the essential functions of coordinating various tissues with one another and controlling the daily timing of complex behavioral activities, such as sleeping, waking, and feeding. And whether a species is active during the day (as humans are) or active during the night (as mice are) almost certainly reflects a long history of predator-prey relationships, food availability, and other environmental factors that have shaped the circadian system by natural evolutionary selection.
How are intrinsic biological rhythms generated? What genes and proteins form the components of the clock mechanism in the cell? How can the circadian clock be so precise, and how is it entrained to the daily light-dark cycle? Answers to these questions have begun to emerge from circadian clock studies by scientists using organisms amenable to genetic analysis, particularly fruitflies and fungi
More than 20 years ago, in a screening experiment for mutant fruitflies exhibiting fast, slow, or absent circadian rhythms, at least one of each type of clock mutant was isolated. This led to the cloning of the first circadian clock gene, named period, some ten years ago. The fruitfly's period gene and the protein it makes (called period protein) revealed a striking pattern: In clock cells in the brain, period gene activity and the abundance of period protein are rhythmic, with the same intrinsic period as that of other circadian rhythms in the fruitfly.
Here's how studies indicate this clock gene works: Period protein gains admission, at a particular time, to the cell nucleus, which contains the organism's genes, and it then directs the temporary inactivation of its own gene (and perhaps others). After some ten hours in the cell nucleus, the period protein is destroyed, and the period gene again becomes active, making new period protein to accumulate outside the cell nucleus. After a delay, the newly-synthesized period protein enters the nucleus, and the cycle repeats. Thus the period gene and its protein product form a central element in a self-regulating feedback loop -- in a manner of speaking, a self-sustaining molecular rhythm generator within the circadian clock cells of the brain.
Recently, we and our collaborators isolated the second fruitfly circadian clock gene, called timeless, and we analyzed the function of its protein product. Like the period gene, timeless exhibits a circadian rhythm in its activity. Strikingly, the timeless protein binds directly to the period protein and this binding of the two proteins seems to govern the time the period protein enters the cell nucleus. Thus, timeless protein determines, in part, the length of time required for one turn of the molecular cycle.
Do circadian clocks in humans and other organisms work in this way, with one or more clock genes rhythmically inactivated by their own protein products? Maybe: The only other clock gene cloned and analyzed so far, the frequency gene from the fungus Neurospora crassa, also has a circadian rhythm of activity, and its protein acts to inactivate its own gene. Indirect experiments suggest that a similar mechanism could operate in mammalian circadian clocks.
Deepening understanding of clock genes from fruitflies and fungi -- and the
recent identification of hamster and mouse clock mutants -- opens the door to
isolation of
the first mammalian clock genes. Isolation of human circadian
clock genes is surely not far off -- the opening of a long awaited chapter in
biology.*
Dr. Weitz is assistant professor of Neurobiology at Harvard Medical School.