Novel Clockwork Controls Found

May 02, 1997

Dartmouth Medical School geneticists decoding the biological clocks that pace the daily activities of plants and animals have discovered new clues to what makes cells tick. Their discoveries, reported May 2 in the magazines Science and Cell, are expected to advance understanding of how both light and temperature regulate the circadian rhythm, the 24-hour cycle most organisms -- from plants to people -- share.

"Biological clocks are the cellular basis of the commonly known circadian rhythms that determine many of our body's functions, including when we go to sleep and wake up. Slow resetting by the clock is the underlying cause of jet lag, and clock malfunction has been linked to seasonal affective disorder and various sleep and mental disorders," explain researchers Jay Dunlap, PhD, professor of biochemistry, and Jennifer Loros, PhD, associate professor of biochemistry.

Their findings published in the May 2 issue of Science suggest a link in the evolutionary spectrum from light perception to time keeping that paves the way for detailing the gears of the modern clock in many organisms.

Another set of results, published in back-to-back papers in the May 2 issue of Cell, will help researchers unravel how biological clocks are assembled and how they keep time over the wide range of environmental conditions living things encounter.

Their work is supported by the National Institutes of Health (National Institute of General Medical Sciences and the National Institute of Mental Health), the National Science Foundation, and the U.S. Air Force.

Though they dictate diverse functions, all circadian clocks share common characteristics, including their 24-hour light-dark cycle. Visible light, along with temperature, is a major cue for the internal circadian rhythms that time behavior and metabolism in most plants and animals. Given the ubiquity, similarity and cellular basis of these biological clocks, findings in lower forms are likely to apply to humans.

Research by Loros and Dunlap, who for years have pieced together clocks in one of the best-known model systems, has helped explain how light resets all biological clocks and how the clockwork is built. Exploring cellular timekeepers that tell the bread mold, Neurospora, when to send out spores, the investigators were the first to demonstrate how light resets the circadian rhythm.

The Science findings, with Susan Crosthwaite, postdoctoral fellow, detail the actions of two clock proteins, White Collar-1 and White Collar-2, which regulate light responses. The researchers found that the two white collar proteins are also essential to the circadian clock, or oscillator, and that they work in the dark without light stimulation.

?"Previous activities ascribed to the proteins were associated only with light signals, so their the involvement time keeping came as a complete surprise," said Dunlap.

The dual function of the proteins opens an evolutionary window on the origin of biological timers from primitive proteins that rely on light.

"This provides a clear connection between molecules involved in light perception and in circadian rhythmicity in an organism, and strongly suggests an evolutionary link connecting ancient proteins involved in photoreception with modern proteins required for the assembly of circadian clocks in organisms ranging from fungi through mammals," Loros said.

Results reported in Cell explain some fundamental properties common to all biological clocks: their response to ambient temperature changes.

"Ambient temperature directly determines the presence or absence of rhythmicity in plants and animals," notes Dunlap, "and abrupt changes in temperature reset the clock very efficiently".

The white collar proteins discussed in Science are similar to others involved either in light responses in plants or separately in time keeping in insects. The sequence of amino acids comprising the proteins indicates ties to photoreception proteins in bacteria and plants and also to time keeping proteins in the fruit fly, Drosophila, evidence that all biological clocks may share common components, say the researchers.

The clockwork cycle is an intricate loop where products feed back to shut off activity, based on light relays. If clocks operated solely on a negative feedback delay, they would wind down quickly. The white collar proteins, particularly White Collar-2, are proteins with a known biochemical function that are involved in the time-keeping loop; they provide the positive feedback in the loop that was anticipated, but not previously found.

White Collar-1 is a clock-associated protein, required for sustained rhythmicity in the dark, but outside the feedback loop. White Collar-2, however, appears to be a clock component required to complete the loop. Unlike other clock components only associated with keeping time, White Collar-2 plays a distinct role in light response and is also necessary to operate the clock in constant darkness.

All organisms have exhibited a correlation between the ability to respond to light and the ability to keep time, and vertebrate tissues that can respond to light also have internal biological clocks.

In Cell, Dunlap and Loros, with postdoctoral fellows Norman Garceau and Yi Liu, report studies of the Frequency (FRQ) protein, a central cog in the Neurospora biological clock. The researchers found that ambient temperature determines both how much FRQ protein is made, and also the form; two forms of the protein arise from the single frequency (frq) gene. The organism has adapted to make the two different forms of the FRQ protein to control rhythm over a wide range of temperatures.

Ambient temperature regulates the ratio of the two forms in a way that expands the temperature range over which the clock will keep time. This explains in large part why organisms lose their ability to keep time at low temperatures.

"It's an aspect of biological timing that is common to a wide variety of living things," notes Loros.

The first paper describes how a single gene, frq, gives rise to the two forms of FRQ that are subsequently modified within cells in a time-of-day specific manner as a part of the operation of the clock. Eventually the products feed back to shut off the activity of their own gene. The feedback cycle thus generated is the molecular basis of the circadian clock in Neurospora, and similar clocks are believed to function in most, if not all, higher organisms, including people.

The second paper shows how ambient temperature regulates the ratio of the two forms of FRQ in a specific way to expand the temperature range over which the clock will function.

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The Geisel School of Medicine at Dartmouth

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