FIRST MAMMAL CLOCK GENE
By Sean Henahan, Access Excellence
EVANSTON, Ill., (May 16, 1997)- The first gene providing the
circadian rhythm of mammalian life has now been identified.
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
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.
Related information on the Internet
Center for Biological
AE: Jetlag and Bioclocks