Biological rhythms that occur daily are observed in most animals and plants and even some bacteria.
In 1971 Ronald Konopka, working in the laboratory of Seymour Benzer, was the first to identify
a gene that is involved in producing these complex behaviors.
As with all research, Konopka and Benzer began with a question.
Are daily biological rhythms determined by external cues or by an internal clock?
If such a biological clock exists, it must be genetically encoded.
Konopka hypothesized that if genes control circadian rhythms, then they could be mutated,
which would alter rhythmic behavior.
Konopka chose to study the fruit fly Drosophila melanogaster, which exhibits striking circadian
behavior that is easy to quantify.
Fruit flies are ideal for genetic research because they can be reared in small spaces
on inexpensive food and have very short generation times - making it easy to produce large, genetically
diverse populations and to track inheritance patterns over many generations.
In the 1970s, before DNA sequencing technology was developed, there were already sophisticated
genetic tools available to create, describe, and identify mutations in fruit files.
Drosophila has 4 pairs of chromosomes.
Like humans, male flies have an X and a Y chromosome that determine sex whereas females
have a pair of Xs.
Because there is only one copy of the X chromosome in males, mutations in genes on this chromosome
are fully expressed, even if recessive, and are therefore easier to identify than autosomal
mutations.
Konopka used this fact to his advantage through a clever genetic strategy: by using specific
genetic backgrounds, he could chemically mutate a male fly and search its male progeny for
phenotypes arising from that mutated X chromosome First, he added a mutagen, ethyl methane sulfonate,
to flies in their food, to introduce mutations randomly throughout the entire genome.
Then, he bred each mutagenized male to an “Attached-X” female.
In attached X females, the two X-chromosomes are joined at the centromere and unable to
separate during meiosis, so they are inherited as a unit.
The presence of two X’s, such as the attached X, is sufficient to make a fruit fly an egg-laying
female even though she also carries a Y.
Two possible combinations of this pairing, attached X plus X and Y plus Y, are not viable.
The viable offspring of a cross with an attached-X female will either be females with Mom’s
attached X and Dad’s Y, or males with Dad’s potentially mutated X and Mom’s Y.
If Dad’s X carries a mutant allele, all the males in the first generation will carry
the mutant gene, and none of the females will.
Using this approach, only male offspring will show a phenotype related to a mutation on
the X chromosome, and mutations on any other chromosome will be heterozygous.
In order for Konopka’s approach to work, heterozygous mutations on autosomal alleles
would not show a circadian phenotype, and one of the genes that controls rhythmic behavior
would have to be on the X chromosome.
Konopka generated more than 2000 mutant fly lines in the hope of finding X-linked mutations
that alter rhythmic behaviors, so he needed an efficient assay to see which of the families
of flies had changes in daily rhythms.
Drosophila is Greek for ‘dew lover,’ which nicely describes how flies, which evolved
in the tropics, emerge from their pupal cases early in the morning when humidity is at its
highest levels, allowing them to pump out their wings before they dessicate with the
midday heat.
Knopka observed each bottle that held the family from one cross and took note of the
general pattern of emergence of adult flies from their pupal cases, called eclosion.
. The best mutant candidates were lines of flies in which the attached X females emerged
in the early hours but males showed either no preference or emerged earlier or later
than the females.
The pupae from these lines, which carry the potentially mutated X, were transferred to
constant darkness to study eclosion rhythms without the influence of light, to make sure
that observed rhythms were driven by the internal clock rather than the environment.
To perform the eclosion screening, Konopka used automated “bang boxes”.
A bang box is an apparatus in which pupae are attached to a metal plate that is automatically
“banged” every hour, prompting the flies that have eclosed to fall through a funnel
into a chamber where they can be counted.
Normally, a population of flies will have an eclosion period of about 24 hours.
But Konopka observed three unusual patterns in the males of some lines: A short period,
with peaks in eclosion every 19 hours; a long period, with peaks every 28 hours, and males
with no observable rhythmic emergence.
We’ll refer to these fly lines as the long, short, and arrhythmic mutants.
Eclosion happens once in a fly’s lifetime, so patterns can be observed only in a population
of flies.
Therefore, Konopka turned to an assay of circadian locomotor rest-activity cycles that can be
measured in individual flies held in a chamber crossed by two infrared beams.
Every time the fly crossed the beam, an event was recorded.
Locomotor activity in constant darkness was measured in wild-type flies, where it followed
a pattern of activity that repeated about every 24 hours.
The flies that had the short eclosion pattern also had a short activity period of around
19 hrs.
Similarly, the long-period eclosion mutant had a long activity period of around 28 hrs,
and the arrhythmic eclosion mutant had no observable rhythmic pattern to its movements.
The observation of phenotypic changes from both assays is important because it suggests
that one gene controls the timing of both the one-time eclosion event and locomotor
activity throughout the fly’s lifespan.
Furthermore, the isolation of short and long period mutants is important, because there
are many possible ways in which a fly could be arrhythmic that are unrelated to timing.
Once he confirmed the three rhythm phenotypes, Konopka mapped the mutations to the X chromosome
by using their inheritance relative to observable visual markers
Drosophila geneticists take advantage of genes with known locations that produce visible
phenotypes to infer the locations of new mutations of interest.
This is because chromosomes undergo crossing over prior to the formation of haploid egg
and sperm cells, in which DNA is exchanged between paired chromosomes.
The closer two genes are, the less likely that crossing over will occur between them.
The more likely they are to cross over together
Konopka used visual markers with known locations on the X chromosome to see whether crossing
over occurred between the rhythm mutations and visual markers that were carried at different
locations on the female’s X chromosome.
Using this technique, Konopka discovered two important things.
The first was that the second-generation males that had a rhythm mutation never picked up
the two markers closest to the ends of the chromosome by crossing over, meaning that
the mutant gene and those visual markers must reside very close together on the DNA.
The second was that all three rhythm mutants mapped to the same area of the chromosome.
Was it possible that mutations in the same gene led to the short, long, and arrhythmic
mutants?
To test the hypothesis that three alleles lie in the same gene, Konopka performed genetic
crosses designed to see if they code for the same function.
First, he created females that represent all possible heterozygous combinations for the
4 X-chromosome allele - and then looked at the resulting rhythms.
Remember that female flies express genes from both of their X chromosomes.
When either the arrhythmic or the long-period allele was paired with a wild-type allele,
the phenotype was normal.
In other words, the arrhythmic and long-period alleles are recessive to the wild-type allele.
The short-period allele was determined to be partially recessive, because the heterozygote
had a slightly shorter period than wild type, but not as short as when the flies carried
two copies of the short-period allele.
However, when the short- and long-period alleles were both present in the heterozygote, the
rhythm was almost normal.
This result could either mean that their alleles are on the same gene and cancel each other
out in the phenotype, or that they are on different genes, and each mutant chromosome
has a wild-type allele of the other gene in question, rendering both recessive alleles
silent in the presence of a wild-type homolog.
Evidence for the same-gene hypothesis came from crosses of either the short or the long
period allele with the arrhythmic allele.
In each case, the resulting behavior was short or long, respectively.
This led the researchers to infer that the arrhythmic mutation represents a non-functional
copy of the same gene that was mutated in the long and short-period mutants.
Additional crosses supported the hypothesis that all three of these alleles lie in the
same small region of the X chromosome.
Through these experiments, the researchers showed that one single gene can affect the
fly’s timing of eclosion and locomotor activity.
The gene that this paper discovered came to be called period.
Today, it may seem obvious that genes control behavior; however, it was the pioneering work
of Konopka and Benzer that finally convinced the skeptical biological community that single
genes could determine complex behavior.
Before that work the only available evidence had indicated that behavior is under the control
of hundreds if not thousands of genes acting together.
Cloning of the period gene in 1984 in the laboratories of Michael Rosbash and Jeffrey
Hall at Brandeis University and Michael Young at Rockefeller University not only earned
them the Nobel Prize for Physiology or Medicine in 2017, but also initiated the ‘Golden
age’ of the molecular analysis of the circadian clock.
As we continue to make groundbreaking progress in the field of circadian biology, it is fascinating
to step back and see where it all started, 50 years ago.