History Module: How Biological Clock
Genes Were First Discovered in Fruit Flies The study of
endogenous rhythms in human beings owes a great deal to the fruit fly, Drosophila,
because it was in this insect that genes homologous to those involved in the human
biological clock were first identified. Fruit flies offer many advantages
for genetic research. They are small, they reproduce rapidly, and thousands of
them can therefore be bred in the laboratory until interesting mutations occur.
That is exactly what the U.S. geneticists Ron Konopka and Seymour Benzer
began to do in the early 1970s. After administering a mutagenic substance to their
fruit flies, Konopka and Benzer examined the activity of 2000 of their descendants.
Most of them had normal, 24-hour circadian cycles, with about 12 hours of activity
and 12 hours of rest. But three mutant flies had very different circadian rhythms:
one had a 19-hour cycle, one had a 28-hour cycle, and one seemed to have no cycle
at all, with periods of activity and rest alternating in apparently random fashion.
It was only about 10 years later, in the early 1980s, that studies
in other laboratories showed that all three of these mutations occurred on the
same gene on the X chromosome. This gene, which the researchers named period
(abbreviation: per), manufactured a nuclear protein found in many
of the cells involved in expressing the circadian rhythm in the fruit fly. Thus
the per gene seemed not only to play a role in the circadian rhythm,
but also somehow to govern its duration. In 1990, U.S. biologist Michael
Rosbash and his team demonstrated that normal fruit flies displayed a circadian
(24-hour) cycle in their production of the messenger RNA for the per gene
and the resulting PER protein, whereas the mutant flies that had no circadian
rhythm showed no such cycle in their expression of the per gene.
At the same time, nearly 20 years after the per gene was discovered,
U.S. geneticist Michael Young identified a second gene with similar properties,
this time on chromosome 2. He named this gene timeless (abbreviation:
tim), because the flies that had this mutated gene had no circadian cycles.
In the mid-1990s, researchers determined that the proteins PER and TIM
produced by these two genes bind to each other. Rosbash and Young then demonstrated
the existence of a highly sophisticated feedback loop involving these two genes,
a loop that took 24 hours to complete one cycle. In simple terms, this loop works
as follows. The per and tim genes remain active, causing their
proteins, PER and TIM, to be produced in the cell’s cytoplasm, until the
concentration of these proteins in the cytoplasm becomes high enough for them
to bind to each other. This binding enables these proteins to enter the cell nucleus,
where they halt their own production by deactivating the per and tim
genes. After a few hours, enzymes break down the PER and TIM molecules that
have entered the nucleus. The per and tim genes then resume
their activity, and the cycle begins again. But what activates the per
and tim genes to begin with? In 1997, U.S. neurobiologist Joseph Takahashi
and his team provided part of the answer when they discovered the clock gene
in mice. Mice with a homozygotic mutation in this gene lose all circadian rhythms
when kept in constant light for a number of weeks. This gene causes the manufacture
of a transcription factor—the CLOCK protein—that binds to a strand
of DNA on another gene to transcribe copies of it in the form of messenger RNA.
In mice, the CLOCK protein binds to and thus activates the per gene.
In fruit flies, it binds to and activates not only the per gene, but
the tim gene as well. Rosbash and his colleagues also found a
gene that they named cycle, whose protein binds to the CLOCK protein
to activate the per and tim genes. Then, in
1998, yet another protein involved in the circadian rhythms of fruit flies was
discovered. This protein, DOUBLE-TIME, is a kinase that, through a phosphorylation
reaction, can add a phosphate group to other proteins. Specifically, DOUBLE-TIME
phosphorylates the PER protein. This structural change makes the PER protein unstable,
so that it cannot enter the nucleus and inhibit its own production. Meanwhile,
it was discovered that the level of the TIM protein is directly affected by the
intensity of the ambient light, via a protein called CRY. CRY is a member of a
class of proteins called cryptochromes—molecules that were first described
as light receptors. In the fruit fly, CRY, in response to light, interacts with
TIM, causing it to degrade. CRY thus prevents TIM from forming a complex with
PER that would otherwise enter the nucleus and inhibit the per and tim
genes. Once again, the overall effect is to activate the per and
tim genes by reducing the quantity of proteins inhibiting them. Thus
both DOUBLE-TIME and CRY alter the availability of two of the main proteins in
the fruit fly’s biological clock. As just stated, in fruit flies,
the CRY protein interacts with the TIM protein. But in mice, CRY binds to PER
to form a PER/CRY complex that is functionally analogous to the PER/TIM complex
in fruit flies. In other words, in fruit flies and in mice, CRY performs two nearly
opposite functions: activating the transcription of the per gene indirectly
in fruit flies but inhibiting this same gene in mice. Such problems are inherent
in comparing the results of studies of two different systems.
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