[MUSIC] Today, for this last session, we're going to discuss positive control of gene regulation. Positive control was a concept that took quite a while to be accepted. When the Pasteur group, Jacob, Monod, Pardee, Lwoff, proposed negative regulation, actually under the strong urge of Leo Szilard, the proposed model of the operon was based on two examples. Lambda, lysogenic phage, which we discussed last week, and the lactose system. Very quickly similar results were obtained with other systems, including arginine, methionine, tryptophan operon. And so it became obvious that everything could be explained by negative regulation. And the principle of parsimony pushed the scientist to discard anything that didn't fit with the negative regulation model. Now the work of which was started by Englesberg, and which led to the discovery of positive regulation, was not meant as a trial to disprove negative regulation. The work of Englesberg was concentrated on one of the catabolic system, the arabinose degradation system. If you recall, Monod had studied a number of adaptative systems, lactose, galactose, maltose, arabinose. All of these sugars were studied in the original experiments on dioxin. At the beginning there was no need to invoke any kind of special regulations for the arabinose system. What led to the positive control was a series of very careful experiments, which were basically forced the scientist to propose positive control. If you recall, the lac system had three phenotypes. There was the wild type phenotype, which was inducible, Lac+. Then there were mutants that were constitutive, those were called i-. And very soon after were the mutants that were defective, or superrepressing, they're called is allele, they were essentially Lac-. And those were superrepresseur. Superrepresseur, by superrepresseur we mean, Prodding that will stay on the DNA and prevent transcription even in the presence of the inducer. This were observed in the lac system, they were also observed in the lambda system, it was a lambda, a c1 mutant called ind-, not inducible by UV ray. It stayed lysogenic even after UV radiation. There was a constitutive, those were the c1. And the wild type was, of course, lysogenic. That's what we had. Now at that time, you had phenotypes of mutants, and very, very little information. So you had to interpret your results. Now, in the context of a positive regulation, A system that has lost the regulator would not be constitutive, it would be non inducible, or superrepressed. So, non inducible. And those would be the ara C-. Non-inducible. No positive activator. Of course there should be also constitutive mutants. And these constitutive mutants who are actually searched for, and identified, and characterized by Englesberg. So he had the non inducible, and he had the constitutive. At the time, the only kind of experiment you could do was to do dominance/recessive test. You could ask, is the mutant dominant, or is the mutant recessive to the wild type? And if you do this with the lac system, the constitutive, or at least the basic constitutive, i minus mutants, are recessive. Whereas the superrepresseur will bind to DNA no matter what, even when there is an inducer around, and so it's dominant over the i+. That's a prediction of the, Lac and lambda system, recessive and dominant. Now, a non inducible in the lac system is dominant and non inducible in the ara C system, if it's a loss of a function, should be recessive. In a constitutive, you don't know. Dominant or recessive depending on the model you built. So this is the context of the experiments that were performed by Englesberg. What you have to realize is that everything had to be done with his very, very simple means of making partial diploid, and looking at the phenotype, looking at the expression of the arabinose enzymes. The results of Englesberg were not questioned by the Pasteur people who were the leading theorists and experimentalists in the field. They were not questioned, nobody said that Englesberg didn't know how to do experiments, or that he was faking the results. What people said was that there were other interpretation, and that these other interpretation, in fact, were compatible with the negative regulation. What were the other interpretation? Okay so, the classical system has a repressor that will block expression of the enzymes, the operon. An activator will induce the expression of the enzymes. That's very simple. Now, how can you make a positive system, or a positive looking system, with negative regulation? Well it's very easy, it's very simple math. Plus is equal to minus minus. So, you can have a gene, let's call this gene C. C is a repressor that will repress gene R, and R is the repressor of the operon. Okay, this is a double negative control. This, this is a single negative control. Single positive and double negative. This will look like a positive control. So the C gene, which turn out to be the positive regulator, would repress a repressor. If the C gene is inactivated by a loss of function mutation, by a deletion, Then the repressor is always on and always preventing expression. The phenotype will be a C-, would have a phenotype of being Ara- in our system, which is exactly what was found. Now, the double negative implies that there must be a gene, R, in which one should be able to get mutants. Remember, these genes are purely, they're not real in the sense that you can touch them. They're postulated because of the phenotype of mutants. So you should get a mutant that is in the gene that is R, and those would be constitutive. The constitutive, the Ara constitutive should be R mutants, mostly. That's the prediction of the double control, double negative. There's another model that can be proposed to explain the result of Englesberg. And this model involves a step that was never discovered, by a chemical step that was never discovered, and that this hypothesis is that arabinose, which is a sugar you give to bacteria, is in fact not the true user. It has to be converted into an X product, and the X is the true user. And the enzyme Arac is doing this conversion, Arac is in fact an enzyme and it's doing the conversion of arabinose to X. And X is inducing because it inactivates a repressor. So this second model, this is model A, this is model B. This model supposed that arabinose is not the true user. C is an enzyme, And there still is an R gene, the true repressor. Negative regulation. Negative regulation's slightly more complicated, but still everything stays in this frame of thinking. So this is basically the challenge that Englesberg had to face.