6. The Periodic Shift Worker
1.Note that the example of the lonely bunker worker is not the whole truth. The principal investigator, who worked together with Aschoff on the bunker experiments in Andechs, was Rüdger Wever. He performed experiments in which subjects were woken by a loud gong signal every day at the same time and found that he was able to entrain the human clock to twenty-four hours. He concluded that humans, unlike plants and animals, could be synchronized by social cues. It turned out much later that they were actually synchronized by a light–dark cycle, which they created themselves in synchrony with the gong: when they went to sleep they switched off the lights, and when the gong woke them up the next morning they switched them on again. The imaginative bunker subject would only behave the way I described here if he was forced to work all day and sleep all night under extremely dim continuous light, so that even closing his eyelids would hardly change the light level that reached his retinas.
2.Chiasm is Greek for “to mark with an X.”
3.The cortex is the outer layer of the brain that looks much like a bunch of tightly packed sausages.
4.Rodents are night-active—nocturnal.
5.The activity plots shown in this book are not the products of real experiments but are almost identical to what the results of real experiments look like.
6.The expression of a biological rhythm can be obscured by some conditions, such as light in the mouse’s case or darkness in the case of many birds, which stop hopping from perch to perch once placed in darkness in spite of their body clock, which still “tells” them to be active. Clock researchers call this phenomenon “masking” because the expression of the daily rhythm is masked by light or darkness.
7.M. S. Freedman, R. J. Lucas, B. Soni, M. von Schantz, M. Muñoz, Z. David-Gray, and R. G. Foster (1999). Regulation of mammalian circadian behavior by non-rod, non-cone, ocular photoreceptors. Science 284:502–504.
8.Melanopsin means “the black opsin.” Melas is the Greek word for ink, and melanos means “black.”
9.Practically all animals are built symmetrically, so that their right and left halves are the same except for some organs like the heart or the liver. Animal brains are also built in two symmetrical halves, so that most brain areas are present both on the left and the right with the exception of a few specialized regions that lie on the brain’s midline. The pineal is one of these. The pineal’s uniqueness, being present only once—unlike the rest of the brain—has historically inspired people to assign it special qualities. René Descartes, for example, thought that our soul resides in the pineal gland.
10.Melatonin is also derived from the Greek word for black.
11.Nucleus is a Latin word for “little nut” or core.
12.The human brain is estimated to consist of 100 billion neurons, which is five million times the SCN.
13.The rhythmicity of the animal that received the SCN would even reflect the individual period of the SCN of the donor animal.
7. The Fast Hamster
1.The activity of these hamsters was similar to Harriet’s in the previous chapter, except that these hamsters didn’t wake up later every day but considerably earlier. Whereas Harriet’s internal days had about twenty-five hours, the clock of these hamsters raced through short twenty-hour days.
2.Benzer used the fruit fly Drosophila melanogaster (Greek for “black-bellied dew lover”) as a genetic model organism.
3.To help you better understand the notion of mutation, here is a quick tutorial in molecular genetics: Chromosomes are extremely long DNA molecules forming double-stranded strings that are wound in spirals, super-spirals, and super-super-spirals, compacting into the dense structure of a chromosome that can be easily seen under the microscope. DNA consists of four different building blocks (nucleotides), abbreviated by the famous letters ACGT, which form an endless sequence in any possible combination of the four letters. Each gene is a defined stretch of nucleotides serving as a blueprint for making a protein (the tools and building blocks that a cell needs to function). Proteins also consist of strings of molecules, in this case, amino acids. While there are only four different “pearls” on the strings of DNA, in other words, ACGT, most organisms use twenty different amino acids, again in all different combinations, to make up the protein strings. Some proteins promote and control chemical reactions (enzymes); others are part of receptors (remember the opsins in the previous chapter), help to build the cell’s structure, switch genes on or off (transcription factors), or serve as “taxis” transporting other molecules around the cell and across the membranes of different cellular compartments. (These are just a few examples of the many functions of proteins.) The amino acid strings of a protein are coiled, clustered, knotted, and bent into a specific shape to serve its function as a cellular tool. DNA can be used as a blueprint because it encodes each amino acid with the help of three nucleotides. These three nucleotides are known as a triplet or codon: ATT, CTG, GGC, and so on. The encoding of amino acids is redundant: with all the possible permutations of the four letters and their sequence within a triplet, the DNA could make distinct codons for sixty-four amino acids, whereas cells generally only use twenty. Amino acids are, therefore, often encoded by several different triplets. Other triplets are used as stop and start signals for the molecular machinery that “reads” the DNA sequence to transcribe and translate it into a sequence of amino acids. Mutations occur when a nucleotide is changed. This can happen for several reasons, which I won’t go into here. Many mutations have no effect; they can either happen in a region of the DNA that does not encode a gene, or they can change a triplet in such a way that it still encodes the same amino acid (that’s where the redundancy I just mentioned comes into play), or they can change one amino acid to another without affecting the shape and function of the resulting protein. Some few mutations, however, do affect the function of a protein, and the effect may even be lethal.
4.The clock gene discovered in the bread mold was named frequency. This fungus, Neurospora crassa, is, like the fruit fly, a classic genetic model organism. Circadian clocks have been discovered in organisms of all phylogenetic kingdoms, from the simplest prokaryotes (cells that even lack a nucleus) to humans. This shows that the ability to predict the time of day must have huge advantages for survival, so that internal clocks probably arose very early in evolutionary history.
5.Christopher’s real name is Martin Ralph, who was at the time a graduate student in the laboratory of Michael Menaker.
6.Tau is the word for the Greek letter “T.” To create a relationship to “time,” clock researchers call the period of the environment synchronizing the body clock “T” (pronounced like the letter in English) and the period of the free-running body clock “t” (pronounced tau). Thus T refers to the length of the external day and t to the length of the internal day.
7.Classical genetics helps us understand the propagation of genes from one generation to the next, and how mutations in specific genes affect the apparent quality (trait or phenotype) they control. The genetic code resides in each cell’s nucleus (the core compartment of each nonbacterial cell). This code is packed into chromosomes. Humans have forty-six of them—twenty-three inherited from the mother and the other half from the father. Each of these chromosomes contains the information of thousands of genes. Since an offspring inherits one set of chromosomes from its mother and the other from its father, each gene is represented twice. An exception is the pair of sex chromosomes, X and Y, which are quite different in their gene composition. A child that inherits an X from the mother and an X from the father is going to be a girl. But if instead the father contributes a Y-chromosome, the child will be a boy.
Let’s suppose there is a mutation in a gene on a chromosome from, for example, the father. If it has an effect (very few mutations actually do), this effect may be counteracted (“rescued”) by the other, nonmutated copy of the gene inherited from the mother. In some cases, the rescue is perfect, and there is no visible difference between it
and an animal carrying a mutation on only one of its genes. In this case, we call the mutated gene nondominant or recessive. The nonmutated version of the gene is called dominant.
In other cases, the mutated gene is so powerful (dominant) in its effect that it cannot be rescued by the “normal” (recessive) copy of the gene. In this case, it wouldn’t be visibly obvious which animals were carrying two copies of the mutation and which were carrying only one copy of the mutation—both would be strongly affected. Occasionally, however, functions of the body rely on two intact copies of a gene to operate perfectly normally, so that if one gene is mutated, we would see an effect, but this effect would be smaller than if both the paternal and the maternal gene were to carry the same mutation. In this case the mutation is called semi-dominant.
8.A fertilized egg that carries the same version of a gene is called a homozygote (MM or NN in our example in the text); one carrying a mixture is called a heterozygote (MN or NM).
8. Dawn at the Gym
1.For those who (want to) know about these things: it is a casein kinase, phosphorylating, among other substrates, clock genes like period.
2.The mutation affects one of the amino acids that the kinase modifies by phosphorylation. Phosphorylation is the process whereby phosphate molecules are attached to amino acids in the chain that makes up the protein.
3.“The Biological Clock Has Been Cloned” was the headline of an article in the science section of an Austrian newspaper in March 1998.
4.To understand this simple hypothesis we should revisit the general dogma of modern biology. As you probably know, the information for the cell’s tools, the proteins, are stored in the cell’s nucleus. This DNA-encoded information about the exact sequence of amino acids (the recipe) that make a specific protein never leaves the nucleus; however, proteins are manufactured outside of the nucleus. A machinery of cell tools, therefore, makes a copy of the DNA sequence, a process called transcription. This copy is called messenger RNA (mRNA), another long molecule, which is very similar to DNA but consists, unlike its blueprint, of only a single thread (strand). This message is transported outside of the nucleus and can now be translated to a sequence of amino acids making up the protein. The transcriptional machinery contains the tools that help to copy the DNA sequence into a new mRNA. Several proteins, so-called transcription factors, control this process, so that transcription can be turned on and off as needed.
5.This modification is phosphorylation, defined in an earlier note.
6.To be able to control biochemical processes, it is important to keep the system dynamic. Most of the molecules, which are involved in cellular control, therefore have a relatively short life and are degraded (both the mRNA and the protein) once they have done their job. This process cleans the slate for future control tasks.
7.These scientists are: K. L. Toh, C. R. Jones, Y. He, E. J. Eide, W. A. Hinz, D. Virshup, L. J. Ptáček, and Y-H. Fu. Their article, published in 2001, is: An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome. Science 291:1040–1043.
9. The Elusive Transcript
1.Metabolism refers to all the biochemical reactions necessary for a cell, an organ, or an entire organism to function. These include reactions involved in the synthesis of necessary compounds as well as those that break them down (degrade them).
2.If you don’t know what these activities are, it doesn’t matter—they are part of the daily routine of someone who is working in a laboratory using techniques of molecular biology.
3.Western blots are a method of detecting proteins. A hot, gelatinlike substance is poured between two glass plates so that a thin layer solidifies after cooling with a row of pockets at the top, plastic stoppers at the sides, and a blunt open end at the bottom. These pockets are filled with the protein extracts of a tissue, and then the glass-gel-glass sandwich is fixed into an apparatus that allows a current to flow through the gel. In this current, proteins of different size and different charge (most chemical substances carry an electrical charge when in solution) travel at different speeds from one end of the gel (the pocket end) to the other (blunt) end. After being loaded in their pockets, the proteins of each liver extract travel in their own lane. The separated proteins are then transferred with the help of another electrical setup onto a membrane, which is then coated with an antibody to the protein of interest. This antibody is coupled to an agent that is either radioactive or produces light. A photographic film is finally exposed to this membrane, which turns black at the specific location of the protein of interest.
4.When we are sick with a fever, it also reaches its peak in the evening.
5.Our food preferences also have a strong cultural basis.
6.This statistic holds true even if we take into account the reduced number of cars on the road so early in the morning.
7.Oliver’s real name is Jérome Wuarin; the Canadian postdoc is Chris Mueller; they both worked in the laboratory of Ueli Schibler in Geneva. See J. Wuarin and U. Schibler (1990). Expression of the liver-enriched transcriptional activator protein DBP follows a stringent circadian rhythm. Cell 63:1257–1266.
8.This gene was a transcription factor called DBP (D-box binding protein), which regulates the activation of other genes.
9.Metabolites are those molecules that are involved in the chemical reactions of metabolisms.
10.Life is all about regulation—“being alive” means that something must be constantly regulated.
11.At present the human genome is estimated to hold the information for approximately 25,000 proteins. That seems not a lot, considering the vast amount of different tools cells need in the very different tissues of our body. Earlier estimates were as high as 150,000 protein-encoding genes or even higher.
10. Temporal Ecology
1.These invisible layers or barriers are called thermoclines.
2.During some nights one can see this bioluminescence in breaking waves or around the bow of a boat.
3.This process is called photosynthesis. The energy is gained directly from captured photons; the sugar molecules are synthesized by chemical reactions joining carbon dioxide (CO2) and water (H2O), thereby releasing oxygen.
4.Orientation toward a light source is called positive phototaxis. When an organism orients actively away from a light source we speak of negative phototaxis (from the Greek words phôs and taxis, meaning “light” and “orderly arrangement”).
5.The journey of these algae during their vertical migration is quite extraordinary, considering their size. A change of depth by ten meters is equivalent to us walking eighteen kilometers. We know that algae and other plankton can travel considerably more than fifty meters during their vertical migration down and back up, which would mean that we would have to travel 180 kilometers every day. The small creatures do not swim these large distances actively. Instead, they make themselves lighter or heavier than water by filling their cell with gas bubbles or getting rid of them.
6.These nutrients include nitrogen, phosphorus, sulfur, and others.
7.It also means that organisms that feed on these vertical travelers tend to follow their food. As a result, a huge biomass migrates toward the surface during the day and sinks back to considerable depths during the night.
8.These so-called cyanobacteria are among the oldest creatures on an evolutionary scale. Unlike the algae we heard so much about in this chapter, they don’t have a nucleus in their cells. They belong to the bacteria, as do the creatures that live in our gut and help us digest our food. Carl Johnson and I were both postdocs in Woody Hastings’s lab at Harvard, overlapping for several years. He now works at Vanderbilt University in Nashville, Tennessee. Johnson and his colleagues’ study on cyanobacteria is: O. Y. Yan, C. R. Andersson, T. Kondo, S. S. Golden, C. H. Johnson, and M. Ishiura (1998). Resonating circadian clocks enhance fitness in cyanobacteria. Proceedings of the National Academy of Sciences 95:8660–8664.
9.Mary E. Harrington (2001). Feedback. Journal of Biological Rhythms 16(3):277; reprin
ted with permission of SAGE Publications. Harrington currently teaches at Smith College in Northampton, Massachusetts.
11. Wait until Dark
1.He was interested in obtaining a variation of the Munich ChronoType Questionnaire, which is accessible via www.theWeP.org.
2.The famous Swedish botanist, physician, and zoologist Carl von Linné (1707–1778) constructed a round flowerbed in his garden in Uppsala, Sweden, that served as a clock, using the specific times of day when different plants opened and closed their flowers. A recent reconstruction of Linné’s flower clock has been created in the botanical gardens of the German castle Mainau, which is situated on an island in Lake Konstanz.
3.Ethology is the science that investigates behavior. Pioneers were Konrad Lorenz, Erich von Holst, and Niko Tinbergen.
4.A recent hypothesis for why we yawn is that yawning cools our brain.
12. The End of Adolescence
1.Biological rhythms that are shorter than a day, like the sleep–wake rhythms of babies and old people, are called ultradian, and those longer than a day are called infradian.
2.Activity is usually recorded with the help of actimeters, devices resembling wristwatches and worn similarly.
3.Besides the influence of genes, adult body height is influenced by several nongenetic factors, including nutrition.
4.The beginning and end of puberty was defined in an endnote to the first chapter. Once a girl or a boy has reached the end of puberty, they are—at least in our cultural setting—not adults yet. Adolescence is defined as starting with the onset of puberty but lasts beyond the end of puberty.
5.We published our results as: T. Roenneberg, T. Kuehnle, P. P. Pramstaller, J. Ricken, M. Havel, A. Guth, and M. Merrow (2004). A marker for the end of adolescence. Current Biology 14(24):R1038–R1039.
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