In the first two lectures, you learned about basic circadian concepts, including free-running rhythms, temperature compensation and circadian entrainment. We talked about circadian organization, and how the clock is ticking in each of our cells, although it's doing a specific job, depending on which cell we're talking about. In this lecture we're going to review what's known about molecular mechanisms of the circadian clock. What molecules are going up and down each 24 hours, specifically to keep the circadian clock ticking. In the next lecture, the fourth, we'll discuss more about clock controlled processes in cells and organisms, but here we want to focus on how the internal clock keeps ticking. We're going to start out by using an historical approach. First you'll hear about identification of the first clock gene. Then how it was put into a functional context. How it might encode time and then how the molecular clock network was expanded to the complex picture that it is today. Like all other victims of genetics research, it looks like just so much spaghetti, but we'll try to put it into order and make some sense of it. Specifically, we'll cover the negative feedback hypothesis, adding more loops to the model, feedbacks from non-transcriptional levels, exceptions to the feedback loop model and how zeitgebers figure into the molecular mechanism. At the end we'll touch briefly on the role of protein degradation in the clock mechanism. A lot of methods are going to be coming at you in this lecture, and also of course we'll discuss the protocols that are typically used for circadian experiments even ones that focus on genetics questions. We know that the background of the students taking this course is pretty broad. So there might be some terms that are not familiar to you. If I say something that you don't recognize, it would be a good idea to just stop the video and look it up. The clock gene story started with Seymour Benzer, a physicist who recognized the power of genetics and as a result he switched his research field. He devoted most of his career to applying genetics to understand behaviour. At the time there was massive skepticism from the scientific establishment. The genetics that had been used to tease apart enzymatic pathways could be applied to behavior. Indeed, I'd heard stories that the pioneers of circadian biology thought that the problem of circadian timing was too complicated, to be solved by genetics. I agree with them in so far as solved is too big a word, but genetic methods undoubtedly delivered the first big clues as to the identity of clock molecules in cells. The general idea of genetics as a method of research, is that you manipulate a single gene, you make more or less of it or none of it. And then asked what's changed in the behavior that I'm studying. Specific functions can then be associated with a given gene. The first genetic approach that was used for the circadian clock problem is called a mutant screen. This is a method that's still widely used to find genes involved in many processes. Benzer used mutant screens to find genes that regulate complex behaviors, specifically, he looked for genes that regulate mating, learning and memory, and circadian timing. He was successful on all three fronts, finding genes called fruitless, dunce, and period. I think you can figure out which gene corresponds to which behavior. If you want to read more about these first experiments in behavioral genetics, there's a very good book out there called Time, Love and Memory by Jonathan Weiner. I can recommend it as very readable for anyone. I mentioned the Benzer's key tool was a mutant screen. In a mutant screen, a mutagenic agent is applied to reproductive tissue. In practical terms, this could mean treating mouse sperm with a chemical called ethyl nitrosourea (ENU) or treating fungal spores or cells with high doses of mutagenic ultraviolet light. Or by introducing a piece of DNA called a transposon that inserts itself more or less randomly throughout the genome. So that eventually, if you looked at enough mutants, almost all genes would be disrupted. By now, mutant screens have been used thousands of times on many model genetic organisms to identify genes that are responsible for many functions. So what did Seymour Benzer do in his mutant screen? He chose a small animal to work on, one of the best experimental systems for doing genetics research, namely <i>Drosophila melanogaster</i>, the fruit fly. Why was <i>Drosophila</i> so good for this experiment? It has a very clear robust circadian rhythm. In the first lecture we saw the rhythm in eclosion from pupae. We saw that it entrained to light dark cycles and that it ran free with a circa 24 hour period in constant darkness. We also saw that it was temperature compensated. So it fulfills important characteristics of a circadian clock that can be expressed in numbers, a good feature for genetics. Just as important as the circadian clock phenotype is the model organism itself. <i>Drosophila</i> is a wonderful choice for mutant screens for many reasons. It has a relatively small genome, 123 million base pairs, and around 13,000 genes. And many of their genes are homologous to those of humans. It's an animal, it has a small compact brain with only 100,000 neurons. This may sound like a lot, but it's Lilliputian in comparison with mice, obviously one can do much more definitive work in a system with fewer neurons. Another advantage of <i>Drosophila</i> is that it's relatively cheap to maintain. That turns out to be an important consideration. There are a couple of disadvantages of this system, also. One is that scientists still haven't figured out how to preserve strains, for example, by freezing, like we can do with cells or mouse embryos. Once you have a mutant fly, or any strain of interest, you have to maintain it by continually passing progeny to fresh media. This means that many <i>Drosophila</i> researchers spend time passing flies. So, back to Seymour Benzer and his post-doc Ron Konopka. They had the phenotype, and they had the genetic model organism. They performed mutagenesis, isolated mutant flies, and grew them up and looked at the timing of their eclosion. Interestingly they used a two step procedure to find clock mutants. The first was to look simply at approximate numbers of flies that are closed in a light- dark cycle. When the distribution was skewed, they took these populations, grew them up again, and put the pupae into constant conditions. A constant darkness screen, where they could access the period of the circadian rhythm, not just if eclosion occurred in the night or day. They rather quickly isolated three mutants all in the same gene. This was a real stroke of luck, statistically it shouldn't have happened, but it did. They found a short-period mutant, a long-period mutant, and an arrhythmic one, they called the gene period. It was another decade until the sequence of the period gene was revealed. What did the DNA sequence, or the predictive protein sequence, tell us? This was in the 80s, the 1980s, and it was difficult to put the sequence into a good context. The many protein motifs that are so well-known now were mostly unheard of comparisons of the predicted protein of the period gene with other predicted proteins revealed a structural motif called a PAS domain, with P coming from the period gene, A coming from Arnt and S coming from single-minded. Arnt and Sim are genes that regulate development in flies, and prompt the other processes as well. The significance of this structural motif didn't become clear until much later, which we'll discuss in a few minutes. [SOUND]
