[MUSIC] We've discussed the deletions that were very useful to identify the operator site in the arabinose system. Now, they also use deletion in this paper, but these deletions were constructed in a slightly different way. Remember that if you have the ara region, the C gene, the B, the A and the D. In the D gene, they had two mutants. 53 and 139. Now, our D mutants that are C+, B+, A+, D- have a useful phenotype. They cannot grow on a rich medium plus a ribinose. They will not grow because one of the products of the enzyme cascade is toxic to the cell. So, this is extremely useful, because this started with an araD 53 strain, and they isolated mutants that grow on this rich plus arabinose median. So, they select arabinose resistant mutants and they get about 200. And these mutants, some are B-, some are A-, some are C- but they also get deletions. And the deletions are identified, because they do not recombine with mutants in B, and A, and C. So the deletions. And these deletions. Are shown on this slide. Two of these deletion, deletion 201, and 204. These deletion are bad, of course, for ARABAD because they cover most of our D and B and A and C. But, they are defective for permeate expression. That means if you remove this gene by a deletion, you do not get permeate. That's the best loss of function you can think of. A deletion, you don't add something, you remove. And if you remove it and you still get the function, then this is compatible with the repressor model, negative control. But if you remove C and you don't get our E, the permease express, our E at the other end of the chromosome. If you don't get our expression, this is a very, very strong evidence for positive control of gene expression. These mutants are also RE minus. They don't express the permeate. So, you need RSC to express the permeate. And that's what will lead to the final model. And on this slide, I put for you the model proposed by in 65 and the model, the current model as written in 2005, so 40 years later side by side. They had, of course, in 2005 the drawing quality was better. So, what is the model? The model states that there are three genes, B A D. Now, they propose that the protein produced by RSC exist in two conformations, P1 and P2, an activator and a repressor. This is, this notion is still true. RSC exists as a repressor. And so, the repressor of this would be P1 and as an activator P2. That has not changed. And the transition between the two is plus and minus arabinose. What has changed is the position of the site, the operator and the initiator. Now, for some reason, probably because he was influenced by the weight of the school, the operator is drawn in large and the initiator is drawn small. In fact, they're equivalent sizes. They're drawn into the B gene for reasons that are not worth discussing. That I was completely wrong they in fact in between a, b, and c. So, that part is wrong. What's right is that, control of transcription of a messenger RNA, that's right. Then they put the premiase which is activated by the activator. And because they feel that all genes have to be expressed under negative control, they introduced an adog, a repressor, for the ORG, for the EG. Which has absolutely no existence. It's completely useless. And it just, They have no evidence for it. They discuss it in a way to make it more palatable to the other member of the community. Because it took so long to accept positive control gene expression. And if you think of it in terms of the logic, when people isolated the so-called promoter mutants of the lack. The promoter mutants isolated by Beckwith and by the people, in fact were mutants in an initiate or lac region in the lac, near the lac promoter. The positive control of lambda repressor synthesis was under the eyes of the Pasteur people, Jacques O and Eisen and all these people. They had that under their eyes and they didn't see it. Epstein and his colleagues had under their eyes, a mutant for which they knew there was a loss of function. Because there was an amber. And they knew that that was a loss of function. That could not be a gain of function. And this loss of function muted in gene 33 was incapable of making tail, head, fibers, any particle, any late gene expression. So they had the positive control, it was there. But just people could probably not see it because it, the intellectual weight of the repressor model was so strong that people just didn't see it. So, this story of these 30 years of classical molecular genetics are full of things that were accepted, things that were not accepted, things that were extremely useful even they are not exactly right. And how much insights people got from very, very simple experiments. And of course, when you do genetic experiments, there usually more than one explanation, but you tend to favor the explanation that this is simplest And so, this is a system were intellectual strength, experimental design and psychological frame played all together a big role. So, that you can see it as a major cathedral being built from scratch to the top. But in fact there were trials and errors. And it's a little bit more, I think it's nice to end with this positive and negative controlling coming together when they have been. Like enemies for some time. And now, and there are many other examples of that, so in the development of science. And, actors of the war usually tends to, well, sometime they recognize years after that they were not entirely right, but usually they just stick to what they've said and written. It is normal. It's very hard for people who have been so deeply involved in the science to really accept. It's very funny because the Malto's regulation which came out a few years later was developed and studied and developed in the Pasteur lab with people who were under the weight of and they had positive control. They also spent an enormous amount of time to convince people that they had positive control. So, now the paper totally accepted and everything, is fine. But now, you have the opposite, because people who work on eukaryotic gene expression, tend to believe that everything has to be positive. And if you show them evidence for negative control they say, whoa. [INAUDIBLE] So that's life. That's it.
