In this last section of the last lecture, we're going to look at how the notion of the circadian clock and fitness has been investigated by chronobiology researchers. If clock mutant mice become ill then there must be some way to explore this experimentally, as a circadian principle. All clock mutants should have some deficiency, if we could just find it. I want to describe two experiments that have been done in the green world, in organisms that depend on photosynthesis. The first experiment involves cyanobacteria. This is the primary clock model organism from the prokaryotes, the bacteria. Cyanobacteria are terrifically diverse and abundant and were thought to be responsible for oxygenating the Earth's atmosphere billions of years ago. The species that chronobiologists use is Synechococcus elongatus which conveniently grows in fresh water. In a remarkable collaboration in the 90s, hundreds upon hundreds of Synechococcus mutants were generated. Like with Drosophila and Neurospora mutants and also mouse mutants for that matter, some mutants have a short free running period, some have a long one, and some are arrhythmic. This collection of mutants was used to map the three major clock genes in proteins in this bacterium -Kai A, Kai B, and Kai C. You may remember these from the description of the clock in the tube -whereby addition of these three proteins an ATP shows circadian rhythms and phosphorylation in vitro. These microbes grow as a sort of a slurry in water if given high intensity light for photosynthesis. They are pretty uncomplicated, dividing asexually, to form a dense solution of single cells. The kinds of things that can speed them up or slow them down in their growth are light intensity and temperature. But the circadian clock is -of course- temperature compensated. You can follow how quickly they divide either by counting the cells or using the spectrophotometer to determine their optical density. Under typical lab conditions, they have a pretty short generation time. They increase by about 100 fold over six days, meaning that they go through approximately one cell division a day. That's some basic background on these microbes. And more than you need to know to understand the experiment. The idea is to combine mutants and wild type Synechococcus in culture together and challenge them with various zeitgeber conditions. Then the question is which strain outgrows the other? Which has a fitness advantage? For THIS competition experiment, a wild type strain was co-cultured with a mutant that has a 28-hour free running period in constant light. Then, after days or weeks of co-culture, the cultures were analyzed for how many of the wild type and how many of the mutant cells were present. An important control for this experiment -before setting up the co-culture- was to show that the mutation doesn't have a pleiotropic effect, namely that the mutation doesn't slow the growth rate in addition to changing the free running period. Once this was confirmed, equal numbers of cells were mixed and either grown in constant light, or in light/dark cycles. In constant light, the cell ratios changed little. The relative abundance of the two strains stayed the same. In light cycles, however, if the cells were exposed to 12 hours of light and 12 hours of darkness -so a zeitgeber period of 24 hours- then the wild type strain overtook the culture. In some experiments, this occurred in less than 10 generations! You're probably thinking that by incubating in this particular condition, a weakness in the mutant was uncovered. But if the period of the light cycle is changed so that it is close to the period of the mutant, say 15 hours of light and 15 hours of darkness, then the mutant Synechococcus takes over. And just as fast as the wild type strain did. And the same thing occurs if you use other mutants, some with short periods incubated with the wild type and short light/dark cycles. The coincidence of the circadian period with the length of the entraining cycle -in THESE cases- leads to a fitness advantage. The big question is still why or how this occurs. One clue undoubtedly lies in the necessity to impose a pseudo-natural light/dark cycle on the cells in order to get the result. As we said before in this course, the circadian clock evolved under zeitgeber cycling conditions, not constant conditions. We think that what was observed in the test tube is an example of evolution. Not the part where a mutation is made, but the part where it is selected because it gives the organism characteristics that are better suited than those of its neighbor. When scientists looked at how the circadian clock of these cells behaves under entraining conditions, they found that the zeitgeber structure separated out gene expression to different times of day. When this gene was expressed at the end of the photo period the cells were more successful, more competitive. This is a graphic example of how zeitgeber structure interacts with the circadian clock leading to drastically different entrained phase -depending on the endogenous characteristics of the clock. Just emphasise that this observation might be a clock principle, not a one-off that is specific for cyanobacteria, an independent lab has performed similar experiments on the plant Arabidopsis, the plant that serves as a model genetic organism for circadian clock and plant biology in general. Similar to the cyanobacteria experiments, mutants with short or long periods were incubated in light dark cycles of different lengths -in so called T-cycles. In short light dark cycles, the short period mutant made more chlorophyll, fixed more carbon, and produced more leaf material. It outgrew or out competed the long period mutant in competition experiments. In the long cycles, the long period mutant fared better. This suggests that the fitness effect observed in cyanobacteria is found more broadly in circadian clocks in higher organisms. At least one implication of these two sets of experiments taken together with the associations of the circadian system with pathology, is that the clock IS a fitness factor. Further, the cyanobacteria experiments suggest that spontaneous mutations that help coping with the temporal features of the environment will make living things more successful than their relatives. This gives us an idea of how fast a clock might evolve in simple systems. Predictions from these works are that if the individuals that entrain early or late could live in a short or a long day, then they would be healthier or fitter. That is of course not realistic but it might give us some ideas on how to go forward to use zeitgeber structure to deliver a certain entrained phase that may be optimal. [SOUND] [SOUND]
