Being a lark or being an owl definitely has a genetic basis, but the genetics are complex—which is no surprise considering the number of genes that are involved in building body clocks at the molecular level. You have read here about clock genes and their products, the clock proteins. Beyond these clock genes, there are many other genes that make people early or late; for example, those that convey light information to the clock, such as melanopsin.
Sarah’s family tree, genetically expressed. Circles represent women, squares represent men; filled circles and squares represent affected individuals, empty circles and squares represent unaffected individuals. Generations are represented by the roman numerals on the left. Redrawn from K. L. Toh et al. (2001). An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome. Science 291(5506):1040–3, with permission from Science.
The circadian system is not merely a neuronal center that ticks away in our brain. It is an entire pathway with inputs, clocks, and outputs. This pathway starts by detecting light or darkness in the eyes and sending this information to the clock neurons in the SCN. These in turn inform other parts of the brain (for example, sleep centers) or organs like the liver about what internal time of day it is. All the components of this pathway are important in determining our chronotype.
9
The Elusive Transcript
To his great frustration, Oliver’s experiments had utterly failed for several weeks now. He was working on a project that concerned the biochemical and molecular regulation of liver metabolism, continuing the experiments of a Canadian postdoctoral fellow who had recently left the lab for his first job as a professor and scientist.1 The experiments focused on a gene that was switched on in liver cells under certain conditions. Although Oliver had always meticulously followed the experimental protocols, he was never able to find the products that should have been present if the gene had been activated. Oliver had always been an excellent student with considerable lab experience. He had done a superb job in the experimental work that eventually had led to his final thesis. For weeks he had been trouble-shooting his procedures, but he couldn’t find the reason why his predecessor had always found huge amounts of the expected gene products while he drew a blank every time.
It was approaching noon. Oliver had been in the lab since six in the morning, constantly working at the bench. As the son of farmers, he was used to getting up very early and loved being in the big lab all on his own before the others arrived. That way he could fully concentrate on his experiments while listening to his favorite music (some of his co-workers who populated the lab later in the day didn’t share his taste). Now the lab was full of his team members, but in spite of his exhaustion, he decided to do another extraction of liver tissue. After several hours of grinding, extracting, measuring optical densities, pipetting, amplifying, loading gels, and running them in the electrophoresis set-up,2 he was back where he had started. The expected gene products were undetectable.
It was getting late. He was almost alone again in the lab, looking through the animal records and checking once more whether there was a difference in the rats that had been used by his predecessor compared with those he sacrificed to harvest liver tissue. But as he expected, he did not find any indication that this could be the source of his problems. He began to hate the ghost of the guy who had done the initial successful experiments and who was quite a legend in the lab, one of the few who had received a summa cum laude for his thesis. The experiments Oliver was trying to replicate were only a side project that was left unfinished when the Canadian had moved. His daily habits had apparently been the opposite of Oliver’s. He hardly ever arrived in the lab before noon, and he experimented throughout the night—until almost the time Oliver usually arrived for his day’s work.
Oliver went out to get some fast food with a lot of coffee and decided to try his luck one last time. He would start from scratch, sacrificing a new rat and going through all the experimental procedures again before he confronted his professor with his long line of negative results. He was certainly not looking forward to this meeting, especially since his negative results contradicted those of a star experimenter. He was just a Ph.D. student, and the other fellow had been a longtime postdoc. Either he was a complete loser, or his highly praised predecessor had made a big mistake—both possibilities would be difficult to communicate to his boss. But, on the other hand, he was sure that he had made no mistakes in his experiments, so the other guy must have blundered. He was still frantically working through the laborious protocols when his coworkers started to arrive. They were not astonished to see him—they were used to Oliver already being there when they came to the lab. But since they had left him the evening before, still working hard, they all knew that he must have pulled an all-nighter. When Oliver had finished loading his gels and had started the electrophoresis apparatus, which would take several hours to run, he finally went home to sleep, making sure his blinds were shut as tight as possible so as not to let in any daylight.
He woke up at noon, showered, got dressed, and made himself the first real meal he’d had for almost thirty hours. He desperately wanted to get to the lab. He almost got run over by a car on his way because he could only think of the outcome of the experiment he had started earlier that day. When he finally arrived at the institute, he threw his knapsack onto the desk in his small cubbyhole and went to check his gel. To his great surprise and relief, it showed a large amount of the expected gene product. He went out for a long walk, trying to make sense of why he could sometimes see the wretched protein and sometimes not. What had he done differently? How could all this make sense? Suddenly he stopped, turned around, and jogged back to the institute. He had come up with a hypothesis and had thought of how to verify or falsify it—the best kind of experiment a scientist can think of. He was glad that he had slept because the experiment he was now planning would take more than twenty-four hours—and then at least another day would be needed to do all the preps and analyses. Two-and-a-half days later he sat at his desk completing his lab book. Finally, he pasted the photograph of his last western blot on the last page of his notes, looked at it for a long time, and smiled.3 Suddenly, he was very much looking forward to the meeting with his professor.
Oliver’s western blot, showing the protein of interest. This western blot is the work of the real “Oliver.” Reprinted from J. Wuarin and U. Schibler (1990). Expression of the liver-enriched transcriptional activator protein DBP follows a stringent circadian rhythm. Cell 63: 1257–1266, with permission from Elsevier.
The discovery of the SCN as the clock-center in mammals was a huge step toward understanding internal timing. This small group of SCN neurons appears to contain all the necessary features required to control the daily events of our body. It receives light information via the optic nerves from the eyes and can thereby synchronize to day and night. If it is lesioned, the regularity of the daily activity rhythm is disrupted. What else do we need in order to create an internal timing system?
So far, you have read about the daily changes in our behavior, about when we sleep or are active, about the fact that our body’s temperature swings from a low in the late night to a high in the evening.4 Together with the curious astronomer de Mairan, you discovered the daily movements of plant leaves. Surely, the clock has not evolved only to control these few qualities in the life of an organism. We know from experience that there are many more functions that change over the course of the day: at certain times, we prefer to exercise; at others, we are hungry; and our preferences for food change drastically from morning to evening.5 The times when we can best concentrate, solve puzzles, and speak foreign languages also change over the course of the day. At lunchtime, we react to alcohol differently than in the evening. Statistics even show that people prefer certain times of day to have sex. Other studies reveal that the pain we suffer when going to the dentist is not the same throughout the day. Road accidents are highest at around 4 A.M.6 Many of us experience a postlunch dip and woul
d like to have a little power nap (or if we lived in Mediterranean regions, we would have an extended siesta). Many kinds of medication should be taken at a specific time of day (recall Sarah’s thoughts in the last chapter about her blood pressure pills). You probably have encountered the medical routine of asking patients to fast before coming to the clinic at eight in the morning for a blood draw. Many components in the blood go up and down over the course of a day. The hormone cortisol is a good example of such a component. Its concentrations in the blood are highest in the morning, decline with some wiggles over the course of the day, reach their minimum during the first half of the night, and then start to rise again toward their morning peak. Although cortisol concentrations depend on many factors, their daily rhythm is controlled by the body clock. So if we measure its concentration at 8 A.M., the results will very much depend on the patient’s chronotype. The science that investigates when to take what medication (chronopharmacology) is a very important branch of clock research. Its aim is to find the right time of day for a drug—when a minimal concentration exerts the maximal effects—thereby also reducing any undesired side effects of the drug. Again, the optimal time of a drug will vary greatly with the patient’s chronotype.
The aim of this chapter is to show how profound the influence of circadian timing is. The simple reason for Oliver’s initial failure to replicate his predecessor’s results had to do with his daily routine.7 While he, the doctoral student, arrived in the lab well before the rest of the team, sacrificing rats for the extraction of liver tissue in the early morning, the Canadian postdoc came to the lab late and worked well into the night, sacrificing the rats for his experiments in the evening. The gene they investigated was only activated toward the end of the rat’s sleep phase, which, in a nocturnal animal, corresponds to the late afternoon of our day.8 Oliver came to this conclusion on his long walk and planned an extensive experiment in which he would sacrifice rats every four hours over the course of twenty-four hours and prepare the respective extracts for the detection of the binding protein, DPB. The results clearly verified his hypothesis. The first time point at which he could detect the protein was at 4 P.M. At eight the protein was even more abundant, but it was already declining around midnight, becoming undetectable again at 4 o’clock in the morning. No wonder Oliver was unable to detect the protein whereas the Canadian postdoc found bucketloads whenever he made an experiment.
The consequences of this discovery were substantial but retrospectively not really surprising. This is true for so many important findings—once we have been made aware of them, they make so much sense in the context of what we already know. Oliver’s findings indicated that the circadian clock apparently controls the activation and deactivation of individual genes. Many of the metabolites collected in the Andechs bunker experiments went up and down in synchrony with the internal days that subjects experienced in their time-free existence.9 Clearly, metabolism is under the control of the circadian clock. The regulation of practically all functions involves, directly or indirectly, the regulation of genes—they are the templates for the cell’s tools that organize metabolism.10 So it is not surprising that the circadian clock also “uses” the regulation of gene expression to organize metabolism appropriately within the twenty-four-hour day. But still, this had to be shown, and Oliver’s results showed how profoundly cells use gene regulation as a circadian mode of regulation. Depending on the tissue, 15–40 percent of the genes in our genome are switched on and off at different times of the day (these estimates are still quite rough).11 This is not surprising, because different tissues have different specialized functions for which they need different tools; thus, not all genes in our genome are used in all cells of our body. Why should a liver cell, for example, produce opsins, like the retinas of our eyes? There are, however, some genes that appear to go up and down in most cells of our body. Among them are those that encode the clock proteins, which can produce a circadian rhythm at the cellular level. This suggests that a circadian clock is potentially ticking away in every cell of our body. Clock researchers have known for decades that single cells can produce daily rhythms because they could observe them in single-cell organisms.
10
Temporal Ecology
The tiny creature danced, together with millions of others, below the surface of the ocean. They swam toward the sun, many of them to the same location, forming dense clouds. When the clouds reached a certain density, they suddenly seemed to become heavier than water and sank away from the water’s surface, glittering in the morning sun. The clouds dispersed into single creatures again, then immediately reformed and swam back up toward the sun. This repeating ritual created a tiny current, similar in shape to a magnetic field, drawing in other creatures at the surface. This accumulation made the clouds even bigger and denser, sending them down even farther. Currents of dancing creatures formed in many places, and neighboring currents started to move toward each other, the bigger ones drawing in the smaller ones. When the clouds were close enough, they fused into one, gradually forming long sheets, like streaming curtains or polar lights hanging from the ocean’s surface.
This marine ballet continued throughout the day until, late in the afternoon, the clouds became thinner and the strength of the currents lessened. The little creatures seemed to have lost their urge to swim upward, and the swarming curtains began to dissolve and disappear. More and more creatures were now beginning to sink—endlessly, it must have seemed to them—until they reached the coastal bottom or a layer where waters of different temperatures met, forming an invisible blanket that was impenetrable to the tiny particles.1 When the sun had set and darkness surrounded the large mats of creatures meeting at the bottom or at the invisible blanket, a spectacular display began. The swarm started to give off an ever-so-faint glow. When they bumped into each other, they emitted flashes of blue-green light that were a thousand times brighter than the faint background glow.
The tiny creatures were often hunted by others that were many thousand times larger than themselves. When these hunters swam into them they collided with thousands of their population mates, triggering a fireworks of flashes. Some hunters were startled, stopped, and forgot to eat, others turned around and swam away because they feared the fireworks would give them away, so that their own enemies would find and eat them.
The tiny creatures loved to swim toward light. At night, they made their own light in the otherwise pitch-dark depth of the ocean, and it seemed as if this self-made light at night helped them to stay together. If each of them swam toward that light, none would get lost. During the day, staying together was easy thanks to their gregarious dance, which herded them by their self-produced currents.
When winter came and the ocean’s temperatures fell, the little creatures sank to the bottom, surrounded themselves with a hard coat, and settled in the mud, where they hibernated until the water temperatures rose again in spring. Once they detected the advent of warmer days, they got rid of their hard winter coats and got ready to mate. Throughout the year, their population grew by mere duplication of themselves. Having sex was their way of celebrating spring.
So far, we have been looking at the biological clock mainly in the context of our own existence, even if we have occasionally used mice and hamsters, or even bread mold and fruit flies, as models for our mammalian body clock. I hope to have convinced you in the last few chapters that the body clock is a profound biological function. It involves clock genes and their protein products giving it individual heritable qualities (for example, chronotype); and it controls the timing of our body on practically all levels, from switching genes on or off to changing our behavior. But how can we, who think of clocks predominantly as mechanical or electronic devices that help us keep appointments, understand the importance of this internal clock for survival?
To fully appreciate the importance of biological clocks and how they allow organisms to occupy niches they otherwise couldn’t cope with, we do best to turn away from our own species. That
is why I chose a quaint and anthropocentric way of telling a story about little ocean creatures. The protagonist is a marine alga, no more than a single cell armored by hard plates covered with little holes, as if it had been hit by a shotgun. If we would line up thirty of its kind, they would only measure one millimeter.
The alga’s current scientific name is Lingulodinium polyedrum. I worked with this alga for more than fifteen years, first in Woody Hastings’s lab at Harvard University and then in my own lab in Munich. At that time, it was called Gonyaulax polyedra, and had become a model organism for clock research because the single cells can produce light by a biochemical reaction called bioluminescence, which they do exclusively at night.2 This bioluminescence could readily be used to record the biological clock ticking away in a single cell. The genus Gonyaulax belongs to the phylum of dinoflagellates, microscopic unicellular plants that can actively swim with the help of flagella, which they use like propellers.
The bioluminescent marine alga Lingulodinium polyedrum (formerly known as Gonyaulax polyedra).
Clock researchers have to look after their experiments at all times of the twenty-four-hour day. When I worked at Harvard, I entered the air-conditioned rooms where we cultured our Gonies—as we fondly called them—around the clock. I noticed that the cells swam near the surface when the lights were on but that they formed an inactive carpet on the bottom of their flasks in darkness, when they also displayed their self-made light. While Gonyaulax’s bioluminescence was an established tool for studying the biological clock, no one seemed to be interested in the behavior of these creatures. I was fascinated by their drastic changes in behavior day after night after day. I was even more fascinated by the fact that these living dust particles had any behavior at all and that their behavior appeared to control the entire population. I began to experiment with time-lapse recordings. What followed was a long line of research that offered many insights into the ecological significance of biological clocks and finally led to the amazing discovery that a single cell could contain more than one circadian clock.
Internal Time: Chronotypes, Social Jet Lag, and Why You’re So Tired Page 8