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.
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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