[MUSIC] Good morning. Now you have read Avery's paper and we're going to go through it. This first slide is taken from a paper also published in the Journal of Experimental Medicine 50 years after Avery's paper. And this is typical of Rockefeller, Discovering the Genetic Role of DNA, which is correct, but The Experiment That Transformed Biology. It looks flamboyant, and it's also a joke, because the paper is about transformation. And you have pictures of the three authors. Colin MacLeod as slightly before the paper, Avery, who was 65 years old when the paper was published, and McCarty, the last surviving member of the team, the editor-in-chief of the Journal of Experimental Medicine for many, many years. He succeeded actually Peyton Rous, who received the manuscript from Avery. So this is the beginning of the paper. The chemical nature of the substance inducing transformation. It's a biochemistry paper, but at the time biochemistry was not really a real subject per se, and so the chemical nature. And as a matter of fact, Beadle and Tatum, which we've seen in the other class, got the Nobel Prize for the chemical nature of enzymatic reactions. So that's a general title, it's a pretty dry title. I mean, you would send this to Nature or Science Today, the paper would be rejected before it reaches any editor. DNA is still called desoxyribonucleic acid, not yet DNA, isolated from Pneumococcus Type III, and this is very correct, this is the material that they have used, purified and transformed with. The three authors, the hospital and the institute, because at that time, again, it was not yet an university. And the date of when the manuscript was received, Nov 43, the paper was published in 44. What I show you on some of this slide is the type of colonies that would be discussed throughout the paper, the R and the S. On the left you have an R colony or a series of R colony, this is an R colony. This is an R colony, this is an R colony, this is an R colony. All of these dots are R colonies. Now, a colony is between 1 and 10 million bacteria. It's also called a clone, because each of the bacteria in this population is derived from a single common ancestor. This is the R strain that it used, R36A, derived from a Type II. And these are the smooth colony of a Type S, smooth, virulent, and this is a Type III. And this is the kind of colony they see when they analyze their transformed cells, this is the criteria. Okay, so this picture is taken from a notebook, and is the way people represented in their mind the transformation. This is the donor cell, extract, plus minus verification. This is the recipient cell and this is the result of the transformation, the result of the transformation event. This is one bacterium or one diplococcus that received the DNA. Of course, the capsule is sightly exaggerated because that's what they're interested in. And we always exaggerate what you're interested in, whenever we present things on a real physical scape. Okay, so in the paper they start by stating their aim and putting their work in perspective. We want to induce predictable and specific changes that could be transmitted as hereditary characters. That's what they want. Now, after sort of a general introduction about the system, they described Griffith's experiment very carefully. Actually, they don't, except for the number of bacteria injected into the mice, they give almost all the details that are available and if people are interested, there are three other people who repeated basically with his results. Then the in vitro and immediately you will find the notion that you will use serum. The sera can be, they're called anti-R or anti-S I, II, III, etc. For example, SII or anti-SIII. Now, in order to classify a serum, you have to assay, this serum was different bacteria, was cultures of different bacteria. And see whether you get a precipitation of the cells with the antibody or not. You can use rabbit sera, you can use mice sera, in not very large volumes, human sera, horse sera, whatever. Pneumococcus, which was also called diplococcus, which is now called streptococcus pneumonia. Pneumococcus is very prevalent. That means that most animals and people have antibodies against one or the other form of pneumococcus. So it's not always easy to find, if you want a serum that's completely defective for antibody's against pneumococcus, you have to work pretty hard. But you can, if you assay a number of sera you finally get some. So that's important, because you can use these sera for the experiment. They call this material the transforming principle or the active principle, depending on where and when. And certainly in the lab jargon it was called TP. Transforming Principle 1, TP1, TP44 means that 44th experiment with TP. That's the jargon. And at the beginning of the experimental part, they described in an incredibly precise detail what they need to get consistent and reproducible results. Now, what they get were convincing results, but they were certainly not very reproducible and they were not that consistent. Because the system was a very crude system and it's not like you put a balance, your kitchen balance, and you put a glass of water on top, full of water on top of the balance. You do it once, twice, three times, you get more or less the same weight. This is a much more complicated system. But they recognize in this system three elements, the fourth being purification. And for them each of these element is as important as the others, because they all required to do what they want to do. They have to grow the R strain, so the nutrient growth is critical and not all conditions work. They have to use a serum or serous fluid, usually serum. And they go through a lot of experiments to really try to understand what protein, or what fraction in the serum is responsible for the effect, and they don't know, and still today people don't know. Serum is a very complicated mixture. And finally, they describe the R strain. The R strain, R36A, which means that you've taken a Type II Pneumococcus in a tube, you've added the Type IIS, plus an antiserum against Type IIS. And you let that stay at 37 degree for a couple of days. At the end, what you will have is normally a tube with a solid phase at the bottom and the solid phase is the immunoglobulin, the antibodies plus the S cells. And then you get the top phase. Now the top phase can be clear or turbid. It's clear if there are no bacteria growing, it's turbid if bacteria are growing. If it's turbid, then you take the supernatant, you go on a Petri dish and you look for an R colony. You pick that colony, and you do it all over again. And you do this cycle 36 times. And at the 36th time you have a plate, and on this plate they have a number of colonies and they call them R36A, B, C, D. And one of them is A. A does not revert strain, so they're prudent, they say relatively fixed, but it has never spontaneously reverted, okay. So, we're pretty happy with this. Now, here there is the only thing that is a true scientific mistake in the wording used by Avery, the change, the variant, is not induced, the variant is selected. Induced means you have added a mutagen, or you've done something. There, what you've done is you've precipitated all the SLs, and you let the R grow on the top. So this is just, and you pick those, it's a selection. It's not an induction. This is the only word that would be in-acceptable today if you hand out a written exam somewhere. And some of these are mutants, are deletions, we'll see that later. In the case of R36A we have a deletion and some are point mutant that can revert. So then they claim that have been able to observe transformation using extract of different strains, 1, 3, 6, 14, but they will not purify these because they have not been able to do so. So far the only one they were able to purify is the Type III, and we'll see why the Type III was so easy or apparently easy compared to the others.
