[MUSIC] So now we arrive at the final experiment. The final experiment is the same cross as before. z+i+ HfrA, z-i- female and in this case, they decide to use a trick that Hayes pioneered, which is to use phage T6. So this strain is streptomycin resistant, streptomycin sensitive, T6 sensitive, and T6, resistant. Cannot be killed by the phage. So, they killed a male twice. They killed the male by having T6 infecting the male and they killed the male by having Streptomycin. Okay, so they're only left with the female. And then they have four cultures. Two are with inducer, and two are without inducer. And this is the magnificent result. The female now after several hours becomes phenotypically i+. Early on, the phenotype is i-. Later, the phenotype is i+. At early time, it's as if i- was dominant over i+. And we know that at later times, i+ is dominant over i-. And this takes time. This is zygotic induction with lactose. And so in 58, the dumped the experiment, they still did not understand exactly how to interpret it. And during the summer, Jacob was alone in Paris. He went to a movie with his wife, and the movie was bad. He kindly doesn't remember the title of the movie. And so he thought about his experiments and he was the only one who could make the connection. Because he had identified zygotic induction of the phage, of landa. When landa is transferred from a male to a female, it's induced. When lactose is transferred, the lacz gene are transferred from a male to a female. It is induced first and then it is repressible. It's the same phenomenon. You cannot observe this with landa, because landa would have killed the cell by that time. So basically with a very simple experiment and this is the experiment published in [INAUDIBLE] this is the experiment that is in many books All the controls, all the rest is not there. But it's only understandable if you really see how the idea develops. Now, this was 1959 and we move, okay, the paper was sent on March 59 and now this paper was read in February 1960. It's submitted by Trefouel, member of the Academy of Science and the director of the Pasteur. Jacob is there, Perrin and Sanchez, who were collaborators also on the paper, and Monod is the last author. This is again, a very short note. Two pages, one table. It's not what we would call today a paper. Right, it's maybe a letter to a journal. What did they do here? Here, they identify the names that will remain. Operon is a new name that resembles cistron, neutron, etc. It's a group of genes whose expression is coordinated. And they need to use a notion which is an operator. So with this note, the model takes its real shape. The regulator and the structorology. So they talk about a double genetic determinism because there are two genes that are involved in the control. The structural gene and the gene that encodes the regulator And a third entity, a third piece of the DNA as we know it today, which will be the operator. So, structural genes, regulatory genes, and regulatory sites. The prediction is that this operator is responsible for expression. So in that paper they define the operator as a region necessary for expression and control. We'll see later that in fact these two regions are separated. There is something now we call it the promoter and the operator itself. This is a piece of DNA where the repressor will sit and prevent transcription, that's the operator. This they claim is identified by mutation that they call O0 because they have a beautiful name for them. They call them physiological deletions because they prevent expression of two genes even though they are point mutations. And the operator mutant, the re-operated mutant are the Oc, operator constitutive. Now in this paper, They have, they take advantage of progress that was made in Monod's group, which is to find certain Lac Z mutants. There are Lac Z+, Lac Z+ allele, there are Lac Z- allele, and there are some Lac Z- allele. Which are called CZ, or CRM for Cross Reacting Material. They make a protein that can be recognized by antibodies. But the protein is inactive. The interest of having these allele is that you can look now and to deploy it at the contribution of each chromosome. And that's of fundamental importance. And in the previous paper they were using HFR. In this paper, which are transient diploids, in this paper they use stable diploids which are called now F primel ac. Its the same F factor but which contain a piece of the lac region. Or piece of the chromosome that has the lac region. So three notions here, first notion is there is an operator region a site that will be defined in fact by the, this is wrong, but the operator constitutive is true. They have two technical tricks that they will use. So, let's look at the table. First line, very easy, i+o+z+y+, wild-type. No induction, no bacteries galactosidase, no permit. Induction, and permease are present, at what they call 100% value. Second line, so you look at all the genes, o+o+y+y+i-i-z-z+. What do you look here? You look at dominance and recessive character of the i+ and the z+. No enzyme, no induction. No enzyme, that means i+ is dominant. z+ in the u induites and you have z and permease so z+ is dominant. But in fact, in this condition you have the CZ protein is made. So, both alleles are expressed. Okay, let me try to get a good example of the two allele that are expressed. Okay, you have a white allele that make white petals, and you have wild-type allele that make red petals. And then you make a hybrid between these two plants and the F1 has pink petals. So both are dominant, they're both co-dominant. Neither win, it's not all white or all red, it's in between. That's called codominance. Both alleles expressed, this is what you see with CZ. Now, the next one is this one and this one is important i-i+. We have seen that i+ is dominant over i-. So now we can ask, what is Oc versus O+? And so you ask in absence of inducer, who is constitutive? In absence of inducer, Lac Z is constitutive and permease is constitutive. If not 100% constitutive, this constitutive, it's coordinated and going to get another color, z1- here, this allele is not expressed not detected. You have the two chromosomes, daddy's chromosome, mommy's chromosome and you can see which one is expressed or whether they're both expressed. And in this case both, they're not both expressed. Only the Oc chromosome is expressed. So, Oc would be called here cis-dominant, cis because it's on the same chromosome. The next line is another Oc allele, which is slightly more constitutive and it gives the same result. Basically, with this system they have identified two type of mutations that are constitutive. So, you have inductible i+ o+ or heterozygote i+ o+/ i- o+. Those are inductible. Constitutive, either you have i- constitutive or you have Oc. These are recessive and these are cis-dominant. Now, what was the situation with landa? With landa, you have mutants that are constitutive, that cannot make lysogens. Or for landa, you can change color. For lambda, constitutive means no lysogen. And it's called a clear plaque because it doesn't make turbid plaques like the lysogens. And in lambda, those no lysogen clear plants can be distinguished. And how do you distinguish them? You do a recessive dominance test. You take a lambda that doesn't make repressor is called a c1-. It doesn't make no lysogen or you take what we call a lambda vir, which is essentially a lambda o mutant, also makes no lysogen. But with these viruses, you can infect a non-lysogenic strain and he will not make a lysogen or you can infect a lambda lysogenic strain. If this guy enters, this chromosome enters a lambda lysogenic stain that makes repressor. When you infect a lambda lysogen, no plaque. With this guy that doesn't bind the repressor, doesn't care whether the repressor is there or not. Because it doesn't bind it. This will grow in a lambda lysogen. Hence it's name, virulent. So these mutants existed, the same mutation with the same phenotype were detected both in the lac system and in lambda. And that made the proposal so attractive. That it was actually the same system, the same mechanism of regulation, the operon model.