Circadian rhythms – literally ‘around the clock’ – help living beings synchronise with the Earth’s rotation, becoming active during daylight hours and falling asleep at night (or vice versa, if the creature is nocturnal). Observant people have long noticed that these rhythms are determined not only by changes in light. In Jean-Jacques Dorthe de Meran’s famous experiment (1729), mimosa spread and contracted its leaves in a dark room where no daylight penetrated. Similar experiments were then repeated by many others. For example, at the beginning of the 20th century, German plant physiologist Erwin Bünning attached the leaves of a bean sprout to a kymograph and recorded their movements during normal day and night cycles and under constant lighting. Animals also demonstrated circadian rhythms, and people who lived for long periods in caves or enclosed spaces as part of a similar experiment fell asleep and woke up at a rhythm close to 24 hours (although some were ahead of the Earth’s day and others were slightly behind it). This means that both plants and animals have internal rhythm generators. After the DNA revolution in biology, it was natural to say, ‘Look for genes.’ The first fundamentally important studies were carried out shortly after the discovery of the double helix. In the 1960s and early 1970s, Seymour Benzer at the California Institute of Technology studied the genetics of Drosophila behaviour. Not everyone at the time accepted that animal behaviour could be rigidly determined by individual genes. Benser, however, believed that the simplest elements of behaviour could be as much a phenotypic trait dependent on genotype as the colour of a Drosophila’s eyes or body. (And if he had not died in 2007, he could well have been among the laureates.) He used chemical mutagenesis to obtain Drosophila lines with disrupted circadian rhythms. Three such lines were found: in one, the daily cycle was shortened to 19 hours, in another it was lengthened to 28 hours, and in the third line, daily activity varied randomly, and even the pupae of these flies did not hatch at a specific time of day, as is normal, but at random times. Ronald Konopka, one of Benzner’s students, studied these lines and discovered that the mutant gene in all three was located on the X chromosome (PNAS USA, 1971, 68, 2112–2116). The gene was named period, or per. Benzner and Konopka suggested (and, as it turned out later, they were right) that the gene of the arrhythmic flies had a nonsense mutation that interfered with the synthesis of the protein product, while mutations in the other two lines somehow altered its properties. Genes of circadian rhythms in drosophila and their products The period gene was cloned and sequenced in 1984 by Michael Rosebash and Jeffrey Hall at Brandeis University, as well as Michael Young at Rockefeller University (PNAS USA, 1984, 81, 2142-2146; Nature, 1984, 312, 752-754; Cell, 1984, 38, 701-710; Cell, 1984, 39, 369-376). The protein encoded by this gene was named PER. It remained to understand how it works. There were several more or less speculative hypotheses about this, until the following important observations were made in the laboratories of Hall and Rosbash (they became possible due to the appearance of antibodies to PER). It turned out that the concentration of this protein in drosophila nerve cells varies along a sinusoid throughout the day, with a peak at night. The concentration of messenger RNA (mRNA) of the per gene also changes in a similar way, and it reaches its peak several hours earlier than the concentration of the protein. In arrhythmic mutants, mRNA concentrations did not change, but the addition of a wild-type protein caused its expression. All this suggested some kind of feedback mechanism. And then it turned out that PER is a nuclear protein and moves from the cytoplasm to the nucleus. This suggested that it could be a regulator of transcription (transcription, that is, mRNA synthesis, occurs in the nucleus). In the 90s, Yang’s group discovered the timeless gene, or tim. The concentration of its mRNA also described a sinusoid with a period of 24 hours, and the product, TIM protein, bound to PER, thereby blocking its destruction and facilitating its release into the nucleus. Fruit flies with mutations in the tim gene were found to have a disrupted cycle of per expression, and the opposite was also true: the cycle of tim expression was disrupted in per mutants. Then other participants in this process were discovered – the clock and cycle genes (the Rozbash group; however, the clock gene in mice was first discovered by Joseph Takahashi from the Howard Hughes Medical Institute). The products of these CLK and CYC genes interact with each other, and then they land on the promoters of the tim and per genes and turn on their transcription. When there are a lot of PER and TIM proteins, the PER:TIM dimer turns off mRNA synthesis from the clock and cycle genes, thereby CLK and CYC proteins, and therefore, ultimately, its own mRNAs. The concentrations of PER and TIM, which had been increasing all this time up to the night peak, begin to fall, finally the “switch” – the PER:TIM dimer – disappears, and clock and cycle are activated again, so that their products turn on the tim and per genes again, and the daily cycle starts anew. This regulatory mechanism is called the “Transcription-Translation Feedback Loop” (TTFL). The drop in PER and TIM concentrations after the peak is provided by other proteins. The product of the doubletime gene (DBT), which Yang and colleagues discovered, is a kinase enzyme; it phosphorylates PER, that is, attaches a phosphate group to it and thereby accelerates its degradation. And the CRY protein, a product of the cryptochrome gene discovered by Rozbash’s group, is responsible for setting the biological clock on the sun. Cryptochromes are flavoproteins (that is, proteins containing riboflavin derivatives of nucleic acids) that are sensitive to blue light. The CRY protein is activated by light, interacts with TIM and triggers its degradation. And since TIM also stabilizes the “partner” protein PER, its decay also accelerates. A sleepy body illuminated by the sun, through CRY, feels that it’s time to get up, anyway. These are by no means all the genes of a biological clock, but their spring or pendulum is the main part that provides oscillations, that is, periodic fluctuations. The mechanism described above, as it turned out, is very conservative, similar genes and feedback loops are found in many higher organisms, including humans. The clock genes of mammals and drosophila are homologous, but in plants the genes are different, but they interact according to the same principle. Cyanobacteria are an exception to the general rule: their circadian oscillator does not depend on transcription, but on protein phosphorylation. Interestingly, human red blood cells (mature red blood cells are devoid of nuclei and DNA, they are sometimes impolitely called “hemoglobin bags”, respectively, there can be no transcription in them) have an oscillation system based on redox cycles of peroxyredoxins, antioxidant enzymes. And these cycles are even regulated by external signals, such as temperature. We should not forget about regulation at higher levels. The fruit fly is small, but the sun doesn’t really illuminate a person from the inside; “I think it’s black inside.” The main clock in mammals is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The retina of the eye transmits information about illumination to the brain, synchronizing the clocks of its neurons with the sun. And at the command of the CFS, the clock of the entire body is regulated through humoral factors (everyone has heard about the hormone melatonin) and the peripheral nervous system. According to the figurative expression of Carlos Ibanez, Professor of the Karolinska Institute, the author of a popular science story about the discoveries of Hall, Rozbash and According to J. Yang, posted on the Nobel website, “the circadian system of an animal is more like a clock shop than a single clock.” If you can imagine a store in which every morning the time on all the clocks is set according to one, the most correct one, but many clocks are also individually adjusted… Indeed, there is evidence that the peripheral clock can adjust its time based on external factors such as physical activity, air temperature, or nutrition. (And when a grandmother wakes her grandson up for school in winter and gives him a piece of apple “to wake up”, this is not pampering, but a deep understanding of human physiology.) The peripheral clock, in turn, regulates the metabolism of fats and glucose, the release of hormones, and these signals are perceived by the brain. The circadian clock influences our behavior, and by controlling our behavior, we help ourselves to wake up or fall asleep… In general, our sense of time is based on many feedback loops.   Daily changes in human physiology Can mutations in clock genes make us owls or larks? They can, but such cases seem to be much rarer than the habit of a particular regime created by upbringing or sloppiness. For example, the family syndrome of early falling asleep, advanced sleep phase syndrome (ASPS), is described. Such people go to bed before dark and wake up after dark. This syndrome can be hereditary, and it can be caused by mutations in the hReg2 gene (Science, 2001, 291, 1040-1043; the letter h in the name of the gene comes from human). And the family syndrome of late falling asleep, delayed sleep-phase syndrome (DSPS), when a born “owl” falls asleep at three in the morning and cannot wake up in the morning, is associated with the hPer3 gene, and now also with Cry1, which, again, was shown by Yang’s group (Cell, 2017, 169, 203-215.e213). Even the BHLHE41 gene, also known as DEC2, has been found, a point mutation in which correlates with the phenotype of short sleep. Humans and mice carrying this mutation need less time to get enough sleep. As for the connection between the biological clock and health, they are diverse. Circadian rhythm disorders cause not only sleep disorders (which is obvious), but also depression, bipolar disorders, and memory disorders. And chronic lifestyle inconsistencies with our internal clock readings can lead to serious illnesses, including cancer, metabolic disorders, and neurodegenerative diseases. It is assumed that research on circadian genes will help modern people find more delicate “screwdrivers” for our watches than melatonin, benzodiazepines and caffeine in all forms, and learn how to harmoniously combine work with rest. Have a good night’s sleep! Author: Elena Kleshchenko, “Chemistry and Life” No. 11, 2017. The article is published on the website “Elements”.