By Sean Henahan, Access Excellence

Researchers at Northwestern University used a kind of reverse-knockout approach to identify the Clock gene. Working with a line of mutant mice lacking normal circadian rhythms, they restored a functioning biological clock in a line of by inserting DNA for the gene into developing embryos. The mice not only grew to have normal biological clocks, but incorporated the genetic information into their own DNA.

"The identification of the Clock gene is definitive," said Joseph Takahashi, professor of neurobiology and physiology at Northwestern. "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."

The newly identified biological clock gene is 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. 

The Clock gene 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 team utilized two complementary research strategies to locate the gene. Using "positional cloning" they used 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.

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.

"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.

Two Clock gene research articles appearing in the May 16, 1997 issue of journal Cell.