The identification of the Clock gene was proven by restoring a functioning biological clock in a line of mutant mice which had lost normal circadian rhythms. The researchers accomplished this by inserting DNA for the gene into developing embryos, which not only grew to have normal biological clocks, but which incorporated them into their own genetic material, passing them on to their descendants.
"The identification of the Clock gene is definitive," said Joseph Takahashi, professor of neurobiology and physiology at Northwestern and senior author of two articles appearing in the Friday, May 16, issue of the molecular biology journal Cell. This is the first time that the discovery of a mammalian gene regulating behavior has been accompanied by a simultaneous proof that the gene has been located, by "rescuing" the lost function of the gene.
All life forms including humans possess internal 24-hour clocks, known as "circadian"(from the Latin "circa," about, and "dian," a day) clocks, which regulate our daily activities such as sleep and wakefulness. Difficulties in readjusting our clocks cause jet lag and shift work problems, as well as some types of sleep disorders. The circadian clock impacts almost every level of our bodily functions, from cognitive to molecular. The new understanding of the nature of the Clock gene may make it possible to discover new drugs that can regulate circadian rhythms and sleep.
"This landmark discovery holds great promise for better understanding what may be the genetic basis for individual differences in human sleep-wake behavior," said Dr. Charles Czeisler, associate professor of medicine at Harvard Medical School and chief of the Circadian Neuroendocrine and Sleep Disorders Section at Brigham and Women's Hospital in Boston. "It may further help to clarify the pathophysiology of a number of circadian sleep disorders," he said.
The biological clock gene is a distinctly new gene located in a segment of some 100,000 DNA base pairs. It has 24 separate "exons," or regions that code for the protein. The sequence of the protein indicates it is a "transcription factor," meaning that it regulates other genes.
The Clock gene, as it is called, is significant because it includes a DNA binding motif, an activation region, and sites designed to interact with other proteins, called "dimerization domains." These features give important clues to how the circadian clock might function in mice and humans.
"The fact that the Clock gene is a transcription factor provides direct evidence that clocks in mammals may be built using a 24-hour program in which genes are turned on and off once each day," Takahashi said. Such a molecular clock has been described in fruit flies and fungi, which were until now the only organisms in which clock genes had been cloned and identified at the molecular level.
The Takahashi lab used two separate but complementary research strategies to locate the gene. The first approach used "positional cloning." This involved using the mutant Clock mouse to locate the gene, first to a chromosome (published in Science April 29, 1994) then to progressively smaller and smaller regions of the genome. Eventually a set of new genes was identified in a 200,000 base pair region, and one of the genes proved to be the Clock gene.
The researchers also located the single base pair change from A to T that caused the mutation in mice whose biological clocks didn't work. That single nucleotide change caused the cell to skip over one of the crucial exons containing 51 amino acids, they determined. This research group included Fred Turek, professor and chair of neurobiology and physiology at Northwestern.
The second approach used a novel functional strategy called "rescue" to help locate the Clock gene. In this approach, the behavior of the mice is used to track down the responsible gene.
The team inserted a number of different bacterial artificial chromosomes (BAC) carrying normal DNA into the embryos of mutant mice, to see which might have an impact on the mouse's behavior. One of the BAC's proved to be able to restore the biological clock function in the mutated mice. This finding helped the first team to zero in on the precise location of the gene. This group included Lawrence Pinto, professor of neurobiology and physiology at Northwestern.
"The rescue experiments were very exciting because they showed us the gene was in one particular stretch of DNA and gave us the first breakthrough in finding the gene," Takahashi said.
"The way they put back the normal gene is incredibly impressive," said Jeffrey Hall, professor of biology at Brandeis University. "When they found that extra copies of the gene caused the clock to run a little faster, that's an added bonus for their conservative and thorough approach," he added.
The expression of the Clock gene was found to be very high in two tissues known to be able to generate circadian signals, the eye and the suprachiasmatic nucleus (SCN) of the hypothalamus. Surprisingly, Clock was also found to be expressed in other areas of the brain as well as in other tissues including the testis, ovary, liver, heart, lung and kidney. The widespread expression of Clock leads to the speculation that Clock may regulate the temporal organization at many different levels in cells and tissues in the body.
Examination of the DNA from other vertebrate species indicates that the Clock gene is highly conserved among vertebrates, including humans. The cloning and molecular characterization of the first clock gene in mammals provides an entree for elucidating the genetic and molecular mechanisms underlying the entrainment, generation and expression of circadian rhythms in higher organisms.
Taken together, the results bring to a successful conclusion the identification of the first mammalian circadian clock gene. This work is the result of the "Clock Genome Project" within the National Science Foundation Center for Biological Timing. The Project uses "forward genetics" to discover the genes regulating circadian clocks in mice, fruit flies and plants.
The research was supported by the NSF Center, an unrestricted grant from Bristol-Myers Squibb Foundation, the National Institutes of Mental Health and Northwestern University. -30- (Professor Takahashi can be reached at (847) 491-4598)