[MUSIC] So now we're going to read a paper. You notice that slowly we progress in time. So this paper was published in 1966, so we may reach the mid-70s by the end of the course. This paper was communicated by Max Delbrück, done at Caltech by Edgar and Wood, and deals with the morphogenesis or the development of bacteriophage T4 in extract of mutant infected cells. This is biochemistry linked with genetics. And the power of molecular biology was actually combining these two approaches. So they know that there are many genes involved in making a virus particle. But the assembly pathway is not understood. And so their paper will be extremely simple, basic. They will infect cells with mutants. Lysis concentrated cells, lysis the cells and have a cell extract. And then they would have another cell infected with another mutant. Lysis cell, make an extract, and then they have two extracts. So one factory can make the engine, but because of some damage, the factory can no longer make the body. But there is another factory that can make the body and receive a different damage. It can no longer make the engine. None of these factories can make cars, because you need the body and the engine. But what would happen if you were to merge the two companies that own these two factories? Then you could ship the engine or ship the body. Or you could fuse the two factories and you would acquire the capacity to make cars using the engine made by one and the body made by the other. This is complementation. But this is not complementation by the book, by the gene. This is complementation by the product of the gene, and it's called in vitro complementation. So that's what they set out to do. And the first experiment, actually it's not a very large paper, but it's an amazing paper for the sum of experiments that were done and the amount of information that can be gathered. So the first thing they show is the map, the 1966 map, which is slightly more than the 1963 map. For instance you have still gene 33, and you have here gene 55, maturation defective. And now they know that they're fibers. And they know that all these genes are necessary to make fibers, tail fibers. So, they're going to make a mutant. And in fact, because they want to be completely clean, they will make a multiple mutant with the mutation that inactivate 34, 35, 36, 37. So they have four mutants. This guy can definitely did not make any fiber. But this guy can make heads and tails. What they show in the little cartoon here is what is seen in the electron microscope. That is, a head and a tail but no fiber. Now, they know that a mutant in gene 23 cannot make the head. No head, the defect is in the head, but it make the tail. You can see the tail in the electron microscope, and it's presumably and if you can see the fibers, it makes the fibers. So the idea of the experiment is very simple. Can I in vitro, not outside the cell, in broken cells, can I put together, mix together the two extract and see whether the fiber will attach to the particle? So that's their first experiment. This experiment is drawn on the cartoon on the left. This is a cartoon that was published in Scientific American by Edgar and Wood, a year later. And this is the fiber-less particles in red. The fiberless particle are made by the infection with a multiple mutant in gene 34, 35, 36, 37. No fibers, only this. Of course there are other things in the extract. But they purify the particles by sedimentation, and they have more or less clean particles. Not completely clean, because there are ribosomes and a few other things, but they are more or less clean particle. On the other hand they infect E. coli B that can not suppress amber mutant was a gene 23 mutant. No head. The fibers and the tail are shown in black. And now you ask, if I mix these two guys together, incubate them, will I get particles? And the answer is yes. On the right is a figure that shows the speed at which this process is occurring. This process is occurring extremely fast. It's practically done in 20 minutes. I mean you can wait until 60 minutes, but it's practically done in 20 minutes. And I draw your attention to one aspect that is often ignored. This is a logarithmic scale. You start with ten to the eighth particles, and you arrive at five times ten to the 11th, 5,000-fold. This is not an enzymatic reaction. This is not the worker that screws the fiber one by one with the screw driver. The fiber will attach continuously and will form an infectious particle. In the drawing, you'll notice that the infectious particle is red for the head and the tail and black for the fibers. The efficiency of this process is also made easy. The 100% efficiency would be, one, two, three, four, five, would be roughly here. So it's almost 90, 95% efficient. It's amazingly good. What we learned later was that a lot of these components are present in excess. And so, because it's better to produce Screws and bolts and knots in large quantities, because when you lose them they may roll under the car or whatever. So you produce a lot of things in excess. This is not a, the economy is not a cheap economy where you only make 20 if you need 20. You make enough to be quick. Be quick is more important. Okay, so they have this exponential acquisition of infectivity. Now, how do you distinguish whether the fibers are indeed black or whether they are red? This table we will not go into detail. But this is the crude assay that they had for answering this particular question. Because the ambers and the amber suppressor strain were all in K12. They were non in vie, so it was quite difficult to do every single possible experiment because of that particular detail. So what they did is they used two related phages, T2 and T4, that do not share the same receptor recognized by the fibers. The T2 fibers were recognized by one receptor, the T4 fibers are recognized by another receptor on the bacteria. So a T4 phage will absorb on a normal E. coli B, will absorb on an E. coli resistant to T2, but will not absorb on a bacteria resistant to T4. T2 will do the reverse image. It will absorb in a normal strain, not in absorb at T2 resistant, the bar two stands for T2 resistant, and absorb very well, very efficiently, on a B bar 4, S bar 4, in this case, T4 resistant host, T2 sensitive. So, with this, they can show that the particle that they make with the lysate from a T2 infected cell that makes T2 fibers is a T2 fiber. And these T2 fibers will attach to the T4 genome. And they will allow attachment on the T2 sensitive cell but not on the T4 sensitive cell. So they actually, this is a big experiment which took quite a lot of time. And just to show that what they have on the little drawing is correct, black fiber with red tail and head. Now, they wanted to see how is this process of phage attachment, how is it done. So what they did was using the electron microscope, and they looked at the, on the grid, in the electron microscope at different times and counted particles and counted how many fibers they had. Now, this is slightly dangerous, because the fibers are very delicate. And you can lose fibers when you put the phage on the microscope grid. So it's a minimum number of fibers that you should see. And they did this at three different times, zero time, 12 minutes, and two hours. At zero time, all but one or two of the particle had no fibers. Zero time means that you mix the two, and you put them on ice. It still takes some time. It's not absolute zero. But twelve minutes, you can see that you have at 12 minutes about 1.3 fiber average per particle. Some have zero, some have one, some have two, of course none has 1.3. 1.3 is an average. You either have one fiber or you don't have one fiber. And they never saw six fibers, because some of the fibers detached. But they have this kind of bell shape curve. And at 120 minutes, they have another bell shape curve with a mean roughly around three, 2.9. These curves, these bell shaped curves are very informative. They tell us that the reaction is independent. One fiber attaches, the other fiber attaches, the third fiber attaches. The fiber does not know and the phage does not know whether it has already attach fibers or not. That's called independent attachment. And this will give you rise to such a distribution. If the distribution had been cooperative, that is, once the first fiber attaches, the other will attach very quickly. This will be the same way as hemoglobin takes oxygen in a cooperative way. Then the curve should like instead of a bell shape, would look like, at time zero you have this, at 12 minutes you have this, and this. And at 12 minutes you have maybe this and this. That would be a cooperative binding. So the binding is not cooperative. So this is fine. This by itself would have been a paper. An in vitro reaction involved in the assembly of a virus, one assembly step of a virus, that would have been a paper. But they didn't stop there.
