[MUSIC] So now that we have seen the explanation in outline, and please remember that for convenience they propose that the addition deletion are single base. But it doesn't have to be. One and the second, totally arbitrarily, they decided that was plus one base. It could have been minus one. They decided plus one. So then the question is, what is the phenotype of any double mutant we can make with this set of 80. So the theory predicts that any combination of a plus with a plus or a minus with a minus, will be R2 mutant. And this is what they tested. They have tested all these double. These are minus minus. These are plus plus. Minus minus and plus plus. They all mutant. Now, what you have to realize that this is the worst part of the work, is to make these double mutant. It looks strange but it is really hard. Why? Because in order to make say FC1 plus 21 what do you do? You cross an NFC1 mutant with an NFC21 mutant. When you do a cross, what is a progeny of the cross? It's the two parents and the two recombinant. So, this is a recombinant and this is a double one you have. Now, this is our plus, it will grow. But all the other ones are R. This R plus is usually 0.5%, around 0.5%. That means that 99.5% of the plaques cannot be distinguished morphologically. The only thing you can do is test by crossing with the parent, which is a little bit more cumbersome, because you want to isolate somebody who doesn't recombine with one and doesn't recombine with 21. That would be the double. So all of these are minus as predicted. So so far, this is totally compatible with the model. But then you may ask, why don't we have suppressors throughout the gene? Why does it stop somewhere? And there, they say, there are two possible explanation. One is nonsense codons, and the other one is unacceptable. Unacceptable means that the protein that is made can no longer perform the B-function for whatever reason, because it doesn't fold, because it's destroyed, for whatever reason. So these are the two unacceptable. These are the two, the wrong protein and the nonsense. Two reason why there's a limit to what region can be used. Okay. So and this will come immediately useful because the prediction is that if you take a plus and a minus, or a minus and a plus, they should be equal type or plus. That's the prediction from their analysis. Now, they know about nonsense. They know that nonsense exists. There's a notion about nonsense already coming around. It's not yet completely clear, but it will become clear next week. But they know that, they are nonsense. So they suspect that some of the plus minus or minus plus could be mutant. And so, they test a bunch of, they make a bunch doubles. Some are predicted to be Y type, some are known to be Y type, because for instance, FC0, FC1 they were isolated. FC1 was isolated as a suppressor of FC0. FC9 was isolated as a suppressor for FC0. And so was 21. These guys were isolated like that, so they know that they're suppressors. The other ones they didn't know. But they get this prediction that from this kind of data, there must be regions that are not favorable. And these are the regions that are not favorable, or actually, these are the possible regions. You can go from minus to plus from the beginning and to the end of the mutant, the region. But if you go from plus, minus to plus, on the ither direction, you're limited. And you're limited because they are barriers. For instance, there is no mutant that is a minus here and compensate for FC0. So somehow, here there must be a barrier. A barrier is a nonsense codon that was not present in the original frame but is revealed For instance, if you take a sequence that is GGG GUA ACC, this is a little piece, okay? That code's for three amino acids. Imagine that you delete this g, now you have g, g, g, u, a, a, c, c, etc. And this is a nonsense code. So, if you change the reading frame in such a way that you reveal a nonsense codon, you cannot have a suppressor. So the barriers are really predicted, and there's also one barrier which is predicted here. Because if you take FC 7, this is the limit, 4 +, you cannot have a + here. So Crick was extremely proud that they considered this to be a striking confirmation of their theory. And they point out that it may be of interest that the theory was constructed before these particular experimental results were obtained. Just on the basis of the original mutation they had. To think that the, they knew that they never got suppressor of FC21 further up. Never got a suppressor FC7 further up. Never got a suppressor of FC0 further down. So that was what their prediction before they experience. Now, now they may the triple mutant. The triple mutant sound terrible and difficult to make. In fact they're easy. The triple mutant you make by having the double mutant, having two double mutants. And this is how you make a triple. Because if you do this cross, you can get only two individuals out of this cross, two recombinant. You can get this, and you can get this. No other recombinant. The blue guy has the mutation dissenter. The green guy is the triple. So it's a simple cross. [COUGH] Leslie Barnett was helping Crick at the time, and so the final cross was duly carried out and the pile of petri dishes put in the incubator. We came back after dinner to inspect them. One glance at the crucial plate was sufficient. They check all the controls, everything was in order. I look at Ross, at Leslie. Do you realize, I said, that you and I are the only people in the world who know it is a triplet code? [LAUGH] It's totally delicious. So, these all based on this. It's probably a triplet code. Most likely a triplet code.