[MUSIC] So now we've gone through the easy part, attaching the fibers on a particle. The next step was quite bold. The next step was to start with extracts that do not make tail and extract that do not make head, And see whether they can get a complete particle. The extract that do not make tail make head. The extract that do not make head make tail. Are those functional? Can those join when you mix the two cells? Of the cell extract. Okay, so of course all of these numbers of genes are not very easy but we go slowly. Let's take, for instance, a mutant in gene 23. And you mix this extract with itself. You have 3.8 particles. That's the background. But if you mix this extract with an extract from 5, 6, 10, 27 and 29, you get over 100 particles. The extract 5 by itself makes nothing, the 6 makes nothing, the 10 makes nothing. The 23 which is no head can use the heads provided by the 5 and the 6 and the 10 and the 27 and the 29. Of course if you mix two extracts that make no tail, either one makes no tail, you will never make a phage particle. All of these extracts make no tail. No tail, okay? And of course the 31, 23, and 2 are head genes. All those three are head genes. When you mix them with any of the, and they haven't done all the mixes, with any of the other extract they get particle, when they mix them together, the 2, the 23 and the 21, they get nothing. So these are mixing extracts that make no head, and you have no head and you have no phage. This is non-complementing. These are non-complementing, and the green are complementing. And the green are these guys, all these guys are complementing. This is illustrated in this cartoon. Again, from the article, to extract one that had heads, and it's easy because the heads are these big red things. Those are the heads, and tails are in black. Both extracts make fibers. They mix, and they get active particle that have heads red, tail black, and the fibers can be either black or red. They didn't test this in this particular experiment. They didn't verify this, but they've done it before. So now, first they had the heads, tail, plus the fibers. Now they have the head, the tail, plus the fibers, and they get phage. So they have two reactions that occur in the test tube without the cell, When you do this mixing by complementation. So this is quite impressive. Of course it's not perfect because there's a little bit of background and the complementation doesn't give you as good result as with the tail fiber attachment. The efficiency is about 100 fold versus about 1,000 fold or more than a 1,000 fold for the tail fiber. It's not as efficient, but it works. So this is a step further. A step further because they are going to see that in fact, sometimes what they see in the electronic microscope is not the exactly what happened in, what is active in the extract. So here they take two extracts. For instance, they take the first one is an easy one. This one has heads and no tail. This one has tail and no head. They mix them, they get particles, and all these particle have the genotype of A. Because this makes the head. So if you take this first line, the heads are giving the genes, the geno. This extract is only giving the tail. That's an easy one. You can take another easy one is gene 6, which you mix with gene 2. Gene 6 is a gene involved in the tail. Gene 2 is a gene involved in the head. So in the extract you have heads and you have tails, and you get everybody has the aging of that. The head is giving the genome. You don't have a mixing of the genome. You only have one genome recovered in the particles. And that is the genome in the head, already preformed in the cell. Some of the problems come with genes like gene 14, because gene 14, in the microscope, you see both head and tails. In this gene you see both the heads and you see tails. In the microscope you don't know if they're functional, you see them. When you mix this with various extract, like a 23 minus extract, all the heads should come from gene 14 and they do. But when you mix it with six, they extract from the six which doesn't make tail but makes heads. The heads here can give rise to a particle, 9 and 11. About half the phage are of genotype A and half a stage of genotype B. Which means that the heads provided by gene 14 is a heads that is capable of being activated. But the tail given in gene 14 cannot. So this is in a nutshell, some of the result that they observed, and I'll go into, they tested all the extract that they had, and I spare you the big table because it's a very large table with all the genotypes. I just made some extract. I just took some bits and pieces from the table. So, let's start with gene 5, 6, and 7. This is the name of the amber mutant, you don't care. This is a number of particles of active viruses produced in the cell, it is the background. This is the background, and then they extract this. They incubate this extract with a gene 23 extract, which gives the tail because it has no head. And it works, but it doesn't work with a head donor which doesn't need a tail. Of course, the 6 and the 6 don't work together, that's obvious. But, it doesn't work with the 5 and the 7. So this is 0. This means that this phage have fibers and heads but no tail. They have heads component in the microscope, in the electron microscope, and those heads are functional. That's The fibers, or the tails, sorry. Now we're going to take a series of genes that are involved in what is called the head formation. All these genes are involved in the head formation. In the microscope, what you see are tails and fibers. Now what is active in this? This mutants cannot complimented 23 extract obviously because the 23 extract makes no add. So it's logical that you don't have complementation here. But the compliment that was a gene 6 extract, and mutant 6 extract, which can provide a head, what is active is what is present in the microscope. Now, those are the easy ones, but they also had genes that behaved in a bit of a funny way. These genes are called heads completion. 2, 64, 50, 65, and 4. This is head formation, and this is head finishing completion. So that the head, after these genes, can go and attach to the tail. So what happens with this? Self, not much. You can see that sometimes some of these mutants have a high background, like this mutant in gene 50, this mutant in gene 4, they have the higher background. You also had that which is gene 21. But they are not. I didn't cut them to show only the ones that are good or simple. But with all these guys, when you add a heads of 23 extract, which gives tail, that has no head, you don't get any phage. These heads are not functional. These heads are not finished. They look like heads, but chemically they're not finished heads. Of course you can incubate this with a head donor and now the head accumulate. So the tail are functional. So with these mutants the tail are functional and the head that you see in the microscope are non functional. These are non functional. We should have, but now we can put a little star or surround them or do something, but that's what they saw in the microscope. So, here, I showed you only three of the five or six kind of phenotypes that they had. But in a few years they heads, they reached this pathway of virus assembly. Now, of course, this virus assembly is more complex for a phage like T4 than for tobacco mosaic virus. Tobacco mosaic virus is a single RNA with a few hundred of code protein. And basically, you can take the RNA individual, the protein individual, and you reassemble the virus. That's a very simple assembly. In this case, the assembly's quite complex. The genome size is also quite complex. So these viruses have involved something which is a little bit like what Henry Ford did with his car manufacturing, the assembly line. You have a line for the body, you have a line for the engine, etc, etc. It's a little bit like that. You have an assembly line for the fibers. This is the line for the fibers. And at the end, once you've reached the phage, this reaction is happened by itself. Doesn’t need anything. If you have an extra that has the fibers and then all they exited in the fiberless particle, it's a spontaneous event. There are couple of the events that are necessary to go from the fiberless particle to the. to a fiberless particle that in accept the fibrous. This involve one of this gene. But, let's ignore this. There's one assembly line for the head and one assembly line for the tail. That's a tail, and this is the head. Once you have both, this is spontaneous. These two things are spontaneous. Well actually, they can occur in the extract. They have not yet been done with highly purified component. So in terms of biochemistry, it's still pretty crude. You've seen things with a head. You've seen that their genes that are necessary to make the head formation. This is 23 and the others, it's the start. Once you've passed this step, all these genes are active. All these proteins are functional. You have something that you can see in the end. This is an EM-Head, not yet a functional head. For the functional head, you need to have this step with this six or seven genes to go from the EM-Head to the functional head. Functional being capable of attaching to a tail, in this particular asset. And all of this is measured at the end by having a particle that will infect a bacterium and will form a plaque. And you count the plaques. The plaques that we're seeing by the rail in the early 1900 and we'll use to count phage particles. So this is quite a brilliant series of experiments because Epstein and Amber and Edgar and Vitiesse gave you the tools to do these experiments. And these tools were used in a fantastically clever way to ask the question, how do you build this Lego construction? How do you, which one goes first? Which one goes after? Which step do you need to do together otherwise it doesn't work. And it's like an assembly, this is not a random process of things floating around and finding each other. This is the very order regulated, this is the first developmental pathway that was understood that the genetic and biochemical level.
