[MUSIC] Good morning. Today we are going to discuss a paper that was very important and somewhat controversial, certainly not accepted at face value at the time he was published. Namely the paper by Avery et al that convincingly showed that DNA is a genetic material. Avery was not a molecular biologist. He was not a molecular biologist at the time. Avery was a doctor, a medical doctor. And he started to work on a disease which is not so much of a worry today in developed country. But which was a very very dreadful disease called bacterial pneumonitis. And up to 50-60% of the affected people, particularly that were in not such good health to start with, died from this disease. At the time there was no antibiotics. But some people have noticed that if you take serum from a patient that recovered from that pneumonitis, some of the survivors, this serum could sometimes help a patient and allow its better and quicker recovery. There was the beginning of something called Zero Therapy. Zero Therapy is less used today than it was used at the time but it's still used, for instance in some cases of tetanus, it's can be used. It's still used for instance if you get snake bites, the only treatment today is zero therapy. So Avery was a young doctor, well not so young, but he was a doctor. And he was working at the Rockefeller Hospital and Institute. It was not yet a University. And he was in 1913 trying to sort serum. And that basically led to the nomenclature that would be used later, type one, type two, type three, you have approximately 60 types of pneumococcus. It's basically the same bacteria, but each of the type carries a different set of genes that confer pathogenicity. So Avery was typing patients, and since you cannot draw that much blood from a patient they went to bigger animals, like horses, in the hospital of the institute, and the hospital had a horse stable. Not for riding but a horse stabled, and each horse was labeled, Type One, Type Two, Type Three, and would provided serum if needed for the patients. Avery was a very curious man, and he was interested in what makes Type One different from Type Two. It's the same, basically it's the same disease. On the microscope the bacteria looks the same, so what is the difference? And during the time between the first and the second World War, Avery in his lab isolated something that is called the capsule. The capsule is a kind of gluey wax around the pneumococcus. And the capsule is a very strong pathogenic factor, or virulence factor. Basically what the capsule does is it prevents polymorphonuclear or neutrophils to eat, by phagocytosis, the bug. And destroy them. So the capsule is like a coat, a protective coat for the bacteria. And obviously there are different types of capsules. And so Avery decided to identify and to purify this capsule to identify the factor. In the twenties he managed to purify the type three capsule because the type three capsule is the simplest capsule, was the simplest capsule and is still the simple known capsule. It consists of only two sugars that are polymerized sugar a, sugar b, sugar a, sugar b, sugar a, sugar b. So it's a very simple capsule. And these sugars, we'll come back to the sugars later, are simple sugars. And sugars are also called carbon hydrates. it's an old fashioned word for sugars because you can write their formula as c h20 times one, times two, times three, times six. Glucose is C6, H12, O6. And that was a big, big, big surprise because at that time people believed that the only antigen that existed were proteins. Proteins. Major ingredient of life, enzymes. So people didn't believe Avery and Heildelberger and since 1930 Avery was nominated, among many, many scientists, was nominated for a Nobel prize. And the verdict was there must be some protein contaminating his sugar, his capsule. And the same criticism, as you will see, was raised against his evidence for DNA as being the gene. So, they were trying to figure out different structure of different capsules and they were working. Relatively slowly but they were working. And then in 1928 came. A real bombshell. 1928, a doctor named Griffith was working in England, published a very long paper. And you will not have to read that very long paper because it's, yeah, it's almost 60 pages long and so, you will not have to read that long paper. Griffith studied a lot of cases of pneumonitis, bacterial pneumonitis. And he noticed a few things, that were going to be important later on. The first thing he noticed is that it's not always one type that infects one patient. And because he took sputum from patients and isolated the bacteria directly arising with antiserum against the different types. And so he found very often you had two types. And the main thinking of time is one bacterium, one disease, so if there's one bacterium infecting, you should have one disease, if you have one disease, you should have only one bacteria. And Griffith was smart enough to realize that he also analyzed the nose and the throat of healthy people. And in particular of healthy family members of patients. And in the throat and nose he recovered streptococcus pneumonia. That was pretty harmless. But was on the same type as one of the two types he recovered from the patients. So his notion and the thing he proposed was that there is a primary code of infection which is one time of pneumococcus and when the sputum goes up the airways and will come out, it carries along the other bug. So it's really one disease, one agent. Then Griffith noticed that if you plate, if you put on a petri dish. And you try to make a culture of streptococcus you will get colonies, you will see colonies later. And he noticed that these colonies very often gave rise to morphological variance. The colonies were called Raf, and the Y tag was called smooth, S, R. And it was easy to get R variance. They were not yet called mutants at the time. And Griffith noted that these R variants are not virulent. You can give them to mice and the mice will not die. Of course if you give the appropriate dose. Now that, it was called an avirulent strain. What Griffith noted [COUGH] that there are two type, two general kind of R, there are mutants that are stable. You can give ten million bacteria to a mouse. You can give ten million bacteria to one mouse each and then the same dose to ten mice, to 20 mice. You'll never get disease. And there were other part, when you injected a million, which was a normal dose, you would not get disease. If you inject 10 million, the mice would die. Maybe a couple of days later. So the notion was that some Rs are unstable, and some Rs are stable. The stable R today are called delicion and the unstable R today are called point mutant, and then Griffith did one experiment which was he developed one tool, sorry. Before that he developed one tool which was quite useful. He developed the tool where you would grow a bacterium for instance an S strain with anti S serum. If it's an S2 with anti S2 serum. And what you observe is the anti S2 serum would attach to the S2 bacteria, and bring them down at the bottom of the tube. It would be a pellet and the culture medium on top would be clear. But sometimes you got bacterial growth in the culture, in the medium. And those mutants that grew in the mediums where R mutants, when you plated them, they have the R. So he had these tools, and then he did one experiment which is shown on this slide. Which is actually a very small part of his paper, but this is a very critical experiment. This experiment has been Included in many, many, many, many, many textbooks. So, the experiment is the following, you start with a virulent strain, virulent A-strain, you inject a million, hundred thousand bacteria into a mouse and the mouse is dead after a few days. And if you take the heart of this mouse, just before it's dying, you take the blood from the heart and from that blood you can isolate S bacteria of the same type as the original type. If this was a type two, this is also a type two. Experiment One. Second experiment, the non-virulent R strain derived from the S, when it's injected, the mice survived. And if you take the blood from the surviving mice, the blood from the heart, there's no bacteria. It's sterile. If you take the blood from your heart, your heart, my heart, I hope it's sterile, it should be sterile. Now, the next experiment is also very simple. Griffith heated the bacteria, the suspension of bacteria, S bacteria. He heated them for long enough time to kill all the bacteria. And so injected the kill bacteria. And he asked what happens with the kill bacteria? And the kill bacteria survived. The kill bacteria did not give rise to an infection in the blood and the heart because the kill bacteria was dead. Now if you do this with a culture of tetanus or phlosphatium the toxin will be there and it will kill the mouse. So this is very specific for this particular kind of bacteria and the bacteria itself, by itself alive, has to be alive to be virulent and to kill the mouse. And now that's the Griffith experiment. Remember an experiment is useless unless they're all the control. Sometimes you have 23 lanes of controls and one experimental lane. And the one experimental lane is meaningful because all the other controls are what you expected them to be. So, in the D mouse where received two things, a mixture of killed S bacteria and R bacteria. They should not die because the R are a virulent. They should not die because the S are killed. But if you mix them together in the animal, something happens. The mice died. That's surprising, because the S virulent bacteria are dead, so the mouse should survive. The R bacteria are avirulent so the mice should survive. But when you put the two together, you end up with the disease with a dead mouse and with S virulent bacteria in the blood of that dead mouse. Now you may say okay, you just told us that some of the Rs can go back to virulent. But when an R goes back to virulent, it's always of the same type. If the R is derived from, if you have a IIs and you get an R, if this R can come back to where we went, we would say revert, it would be a IIs, never any other type. So what he did was actually to mix killed IIIs that are dead plus R derived from two. And now I'm asking you, what do you think? What will be the genotype of these virulent bacteria that recover here? Two or three, or if you're completely poetic, any number. But this is science. This is very basic biology. The bug, the virulent bacteria, the smooth colony forming bacteria are all of type III. You've taken a dead III, and the dead III has given something that the R picked to become an S. That's the result of Griffith. The result of Griffith is extremely important. Now the way you interpret the result is difficult because there are lots of interpretation. One of the interpretation, which was favored by Griffiths, is that the dead cells release a little piece of material, a pabulum. And that this little piece of dust, type three dust, will serve, will be incorporated by the R bacterium. And serve as a starting point for the growth of a capsule. So it's like you start with one little crystal and then around this little crystal, all the other atoms arrange themselves and you get a big crystal. So this is this notion of crystallization. Crystal growth is, for instance from the physics or geology point of view, perfectly understandable. And from a biological point of view, it's also perfectly understandable. But Griffith stopped. And he stopped his research and that was it. But of course, Avery was extremely interested by that result. And he had two colleagues, the first colleagues was Dusen, and Dusen decided that the mouse is a complicate case test tube. We should get a simpler test tube. So what Dusen did is he mixed the killed S and the R, but not in a mouse, but in a tube. And he cooked it for awhile and looked for the presence of virulent type three bacteria. And he found him.