July 14, 1998 A newly discovered gene called double-time regulates the molecular cycles underlying circadian rhythms, scientists from The Rockefeller University report in two papers featured on the cover of the July 10 issue of Cell. The researchers also identified the molecular mechanism that allows this gene to work.
"We've identified a gene in the fruit fly Drosophila that times the pairing of two proteins essential for circadian rhythms," says senior author Michael W. Young, Ph.D., professor and head of the Laboratory of Genetics at The Rockefeller University. Young also directs the National Science Foundation (NSF) Science and Technology Center for Biological Timing at Rockefeller.
Earlier studies have indicated that the genes and proteins governing circadian rhythms in Drosophila play a similar role in humans. In humans, daily circadian rhythms underlie many functions, including the sleep/wake cycle, body temperature, mental alertness, pain sensitivity and hormone production. In natural conditions, many rhythms have a 24-hour period related to sunlight, but though light can affect the rhythm, it does not cause the cycle. In fact, in the absence of light or other environmental clues, rhythms continue and most adapt to periods slightly longer or shorter than 24 hours.
In the fly, the circadian rhythm requires the pairing of two proteins, PER and TIM, made by the period (per) and timeless (tim) genes, respectively. All cells of the fly have per and tim genes, but the brain cells set the body clock. PER and TIM proteins accumulate in the nuclei of light-sensitive eye cells, called photoreceptors, as well as pacemaker cells of the central brain. Scientists at the California Institute of Technology discovered per in 1971, while Young's group identified tim in 1994.
In 1995, Young's laboratory showed how PER and TIM partner to control the fly's body cycle. The fly circadian cycle begins around noon when the per and tim genes become active, making RNA--molecules essential to create the PER and TIM proteins--but only after sunset does the accumulated RNA prompt the cell to stockpile the PER and TIM proteins. At night, the proteins pair in the cytoplasm and then migrate into the nucleus, home to cells' genetic material. It is this movement to the nucleus that signals the per and tim genes to stop making RNA and, hence, new PER and TIM proteins. Near dawn, the old PER/TIM protein pairs disintegrate. With the proteins depleted, the per and tim genes begin to make RNA again by midday.
The pace of the clock is set by a time lag: while the per and tim genes are freed to make RNA early in the day, PER and TIM protein pairs form at night.
Previous work by Young's laboratory hinted that this delay was caused by the late arrival of one of the two protein partners, PER. Accumulation of PER is postponed because PER proteins are rapidly broken down in the cytoplasm when they are not paired with TIM. The new gene, double-time, regulates this process.
Young and co-workers named double-time after the way in which it affects the length of the fly's daily cycle. Both fruit flies and humans have activity rhythms that adapt perfectly to a 24-hour cycle of night and day, but the researchers identified three mutants of double-time that alter this cycle. The first produces an 18 hour clock, among the fastest of all other fruit fly clock mutants. The second mutation slows down the fly's clock, to about 28 hours. The third mutant blocks the circadian cycle altogether. The last mutant, which lacks the protein product of the double-time gene, provided the clues to deciphering the mechanism behind the circadian oscillations.
The researchers found that, in the mutants lacking double-time protein, very high levels of PER protein accumulate in the cytoplasm and these PER proteins no longer disintegrate when not paired with TIM, meaning that both PER and TIM proteins are produced at the same time. Without the usual time lag, PER and TIM can pair and move into the nucleus prematurely.
"In other words, the double-time gene regulates the buildup of PER in the cell. This determines the time it takes to complete the cycle, or whether there is any cycle at all," says Young.
Young and his co-workers cloned the double-time gene and found that it produced an enzyme called a kinase, a protein that phosphorylates, or places phosphate molecules on, other proteins. Scientists have known for some time that PER proteins are phosphorylated with a rhythm. In the double-time mutants, however, PER proteins are overproduced and are not phosphorylated.
"We think that the way double-time regulates this cycle is to hold down the rate of accumulation of PER protein by phosphorylating it as soon as it's made," says Young. "Phosphorylation is often a mark put on a protein that labels it for degradation. Because the double-time protein is made constantly, unlike TIM and PER, the only way for PER to survive is for TIM to come in and rescue it by attaching to it."
Young thinks that the partnership between PER and TIM either prevents a phosphorylation event that causes the instability of PER, or prevents destruction of PER even if its phosphorylated. Either way, says Young, TIM becomes a protector of PER under these conditions.
Young's co-authors on the first paper, "double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation," are Jeffrey L. Price, Ph.D., formerly of Rockefeller and now at West Virginia University, and Justin Blau, Ph.D., Adrian Rothenfluh, Dipl., Marla Obodeely, B.A., and Brian Kloss, Ph.D., of Rockefeller.
Young's co-authors on the second paper, "The Drosophila clock gene double-time is a protein closely related to human casein kinase Ie," are Kloss, Lino Saez, Ph.D., Blau, Rothenfluh, Cedric S. Wesley, Ph.D., of Rockefeller and Price.
The work was supported by NSF and by the National Institute of General Medical Sciences and the National Institute of Mental Health, both part of the federal government's National Institutes of Health. Blau was supported by the Human Frontier Science Program and Rothenfluh was supported by a Beckman fellowship.
Rockefeller began in 1901 as The Rockefeller Institute for Medical Research, the first U.S. biomedical research center. Rockefeller faculty members have made significant achievements, including the discovery that DNA is the carrier of genetic information and the launching of the scientific field of modern cell biology. The university has ties to 19 Nobel laureates, including the president, Torsten N. Wiesel, M.D., who received the prize in 1981. The university recently created six centers to foster collaborations among scientists to pursue investigations of Alzheimer's disease, of biochemistry and structural biology, of human genetics, of immunology and immune diseases, of sensory neurosciences and of the links between physics and biology.
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