It took some time to get a handle on how PERIOD was working in the clock. The first clues that gave a hint as to the function of period protein were descriptions of gene expression. When was the RNA expressed? When was the protein expressed? The RNA was expressed at high levels in the subjective afternoon and evening. The first half of the subjective night. The word subjective is a special circadian term to indicate that we're working or looking in constant conditions so there's no actual morning or evening as indicated by light or dark. But if you would count back to in this case when the fly was last exposed to light, then it should be afternoon or evening. So it's subjective afternoon or evening as predicted by the circadian clock of the fly. If the fly were alive it would be finishing its big activity bout of the day - you remember that from the first lecture. To measure RNA expression then the fly is sacrificed. And in this case the head is collected and RNA is purified. Back to gene expression. The period RNA was found at high levels in fly heads in the subjective afternoon and evening. And at low levels in the subjective late night and morning. The relative amounts over the course of 24 hours looks something like this. Time is on the x axis and the amount of RNA is on the y axis. You see one peak and one trough over 24 hours. When you look at protein expression from the period gene you see a similar pattern except for one thing. The timing of protein expression is offset by four to six hours. What kinds of mechanisms could explain such an expression pattern? A relatively simple principle was invoked to explain the findings, namely negative feedback. Feedbacks are everywhere in biology. They're part of a mechanism keeping signalling pathways from spiralling out of control. Something can be stimulated or induced, and then a feedback is activated, leading to a return to the previous state. Feedback is used to keep biological states within certain limits that are permissive for basic functions. By way of example, on the surface of any given cell, there are receptors that recognize the environment. The largest family of receptors by far is the so called G protein coupled receptors. These receptors are activated by binding something from their environment such as an odorant or a neurotransmitter like dopamine or a hormone like melatonin. Their activation leads to the influx of calcium into the cell or the transcription of certain genes. Within very defined limits these are important limits to the cell but too much calcium or uncontrolled gene expression can be damaging. In order to stop these effects and prevent the cell from being flooded with calcium or with certain tightly controlled proteins the G protein coupled receptor is modified on the inside of the cell, a molecule feeds back onto the receptor itself, and the receptor is internalized into the cell. From there it might be degraded or it might be recycled, but it's protected, sequestered away from the molecule that stimulated it in the first place. The circadian feedback hypothesis was proposed as follows. When the PERIOD protein levels reach a certain level, the RNA levels decrease. It was suggested that the protein was actively feeding back to repress the amount of transcription. Transcription of the period gene itself. Here's a schematic of how a feedback loop might work. The RNA is expressed, necessarily driven by a transcriptional activator. The RNA is translated into protein. Protein feeds back negatively on its own transcriptional activator, decreasing RNA production, and leading to decay of protein levels and decay of the negative feedback. Thus, allowing a restart of the process with a new round of RNA production. This would lead to the oscillations in RNA and protein that were observed and as shown here. This is a 24 hour time course of a clock protein in constant conditions where you see large amounts which first decrease and then increase again. How is the feedback hypothesis tested? The key event to demonstrate, is the process of negative feedback itself, and the way to do this, would be to over-express the period gene ectopically. That means to put a period gene into the genome, just about anywhere, but so that you can manipulate its expression. For that, you would need to replaces it's promoter. Lets step back for a minute and talk about how genes are organized and how they're expressed and made into proteins. Most of you scientists would know this but for the students who don't have a biology background it might help to understand some basic concepts to be able to grasp the experiments that I'm about to describe. The genome is the entire collection of genes in a given organism. Humans have about 21 thousand genes. Drosophila the fruit fly has about 13,000 Neurospora crassa, the fungus, has 10,000 and yeast, another fungus some of us know as bakers yeast has about 6,000 genes. Bacteria generally have fewer genes, with E.coli having over 4,000, and the photosynthetic bacteria, cyanobacteria, that we mentioned in the first lecture, having about 2,500 genes. Genes are organized within strands of DNA, sort of like beads on a string, one after another. Once the DNA sequence of a genome is determined, then it can be analyzed for which parts encode a protein, and which parts are in between, where the beads are, and where the string is. The genes themselves are made up of several elements. There's the DNA sequence that encodes protein, this part is called the open reading frame, or ORF. This bit of DNA along with some surrounding sequences upstream and downstream is used as a template for the production of RNA, another string like molecule made up of nucleic acids. The RNA has a slightly different composition and is much smaller than the genomic DNA, since it most often is coating for a single protein. There are many specific signal sequences contained within the RNA sequence that determine where this molecule goes and how much protein is made. The same piece of RNA has the potential, in some cases, to make one of several forms of a protein. Outside of the open reading frame, are sequences that control how much of the gene's DNA is made into RNA. And importantly for us when transcription occurs. There's a molecular machinery that controls transcription and it includes the molecules or enzymes that actually fuse the molecules together that make the RNA but these molecules called polymerase are rather generic. They each work on many genes. The specificity of transcription comes mainly from so called transcription factors. Transcription factors direct polymerase and the transcription machinery to a specific gene, and they can either stimulate or activate transcription or decrease or repress transcription. In one case a transcriptional activator and in the other a transcriptional repressor. I've just given you a highly simplified version of gene regulation. There are many, many exceptions to the general concepts that I just described. But for now this is the basic information that should allow you to appreciate how the experiments that are coming up were done. We were discussing how one could test the negative feedback hypothesis. And the concept that I described was to put a copy of the gene into the genome so that you could manipulate its expression. If you could turn it on and off, then you could ask questions such as, what happens to the normal circadian rhythm. At the time that the feedback hypothesis was introduced, actually at a Gordon conference here in Germany in 1991, there were very poor tools for manipulating genes in <i>Drosophila</i>. - for reverse genetics, the situation where you have identified a gene and you want to go back into the organism, and change expression of the gene to learn more about its function. This is where <i>Neurospora crassa</i> comes in. I showed you the circadian rhythm in <i>Neurospora</i> in the first lecture. The ideal tools were available to test the feedback hypothesis using <i>Neurospora</i>. It had a circadian phenotype, a cloned clock gene, a strain with a mutation in that clock gene that showed no circadian rhythm and an unrelated promoter that was inducible in an exquisitely quantitative manner and inducible in a way that did not impact the wild type circadian phenotype. That's a lot of information. You've seen the circadian phenotype. I'll try to explain the rest step by step. The cloned clock gene. The first clock gene identified in <i>Neurospora</i> was called <i>frequency</i>. It was just as enigmatic as period, in that its sequence didn't reveal much about its function. Similar to the story with the period gene a number of mutants were identified all in the same gene. Mutations in the frequency gene also called <i>frq</i> show either a short period, a long period, or no obvious circadian rhythm. As for the period gene the <i>frq</i> RNA and FRQ protein oscillate with a circadian rhythm that's about four to six hours out of phase with one another. With the protein following the RNA like with the period gene products in the fly. That it showed these similarities to the period gene meant that it was an appropriate experimental model to use to test the feedback loop hypothesis. What about the inducible promoter? The last tool that was required was the inducible promoter and for this the promoter from the gene called <i>qa-2</i>, was chosen, because it was induced with a metabolite found in tree bark called quinic acid. <i>Neurospora</i> is often found in nature, growing from burnt trees, prolifically exploding out of the blistered bark. It uses quinic acid as the carbon source. The <i>qa-2</i> gene in <i>Neurospora</i> is expressed at high levels in the presence of quinic acid. A dose response curve is highly reproducible at concentrations between ten to the minus six and ten to the minus four molar as shown in this graph. At lower levels the gene is not detectable. A dose response curve refers to a graphical representation of an experiment that titrates a compound into a system. The amount of compound is plotted on the x axis, and the response, it could be gene expression, physiology, behavior, whatever you're measuring is plotted on the y axis. The shape of a dose response curve is usually some variation of an S. Going from no response to maximum response. There is usually a plateau at the upper end of the S. In developing pharmaceuticals dose response characteristics are clearly very important. Here we're discussing using this to characterize an experimental system. We can then take the amounts of compound that are required to stimulate weak expression, moderate expression, and high expression and use them for experiments. The results of a dose response curve could also help to understand the ecology of the organism. In this case one could correlate the expression of metabolic pathways with the actual amounts of quinic acid encountered in the environment. By taking a promoter of the <i>qa-2</i> gene and attaching it to the open reading frame of another gene, the quinic acid induced expression characteristics can be transferred. I guess you can see where we're going with this. So what experiments were performed with this toolkit of clock gene and inducible promoter? First, an artificial gene was constructed. The <i>qa-2</i> promoter was fused to the open reading frame of the <i>frq</i> gene. This was then inserted into the genome of a normal wild type <i>Neurospora crassa</i> strain. Then a simple question was asked what happens to conidiation with no induction. This would be the control condition where a normal circadian rhythm is expected and then what happens with strong induction? That would be the experimental condition where a disruptive circadian rhythm is expected, due to nonrhythmic, over-expression, of a clock protein. The results are shown here: At high concentrations of quinic acid, ten to the minus fifth molar, conidiation in the wild type strain, with no artificial transgene, is normal. This is the important control. In the strain with the inducible transgene, the fusion of the inducible promoter and the <i>frq</i> open reading frame, all you see is arrhythmic conidiation, no organization of conidiation whatsoever. As the the amount of quinic acid is titrated down to levels that should only weakly activate the promoter, conidial bands gradually reappear. This result could be meaningful, it could reflect disruption of the circadian clock mechanism or it could also indicate that an output regulating conidiation has been disrupted without affecting the timing mechanism. Based on the series of <i>frq</i> mutants that were recovered there was a strong indication that it was the former explanation. So this first result set up the follow-up experiment which tested what happens to expression levels of the <i>frq</i> gene when the FRQ protein is over-expressed. To look at this, one of the most convincing experiments was to over-express FRQ protein in the arrhythmic mutant strain. In this strain, the <i>frq9>/i> strain, the <i>frq</i> RNA expression level was extremely high, much higher than ever seen in normal wildtype strains. This in itself is more evidence supporting the feedback loop hypothesis. Without a functional feedback protein, the RNA levels should not be suppressed. So what happens to the elevated <i>frq9</i> RNA levels when the transgene the <i>qa-2</i> promoter fused to the <i>frq</i> open reading frame is inserted into the <i>frq9</i> genome. On induction of quinic acid the levels of <i>frq</i> RNA disappeared although levels without the transgene remained high. This experiment showed a molecular basis for the negative feedback principle. This molecular mechanistic model served as a framework for many experiments to follow. Questions like if period and frequency are negative feedback elements what are the transcriptional activators of these clock genes. Perhaps related to this question, what are they molecular clock components in mammals? How can a circa 24 hour free running period be organized around the feedback loop mechanism? And how can this loop be entrained, with zeitgebers? [SOUND]
