Bacteriologists suspected the transforming factor was some kind of protein. The transforming principle could be precipitated with alcohol, which showed that it was not a carbohydrate like the polysaccharide coat itself. But Avery and McCarty observed that proteases - enzymes that degrade proteins - did not destroy the transforming principle.
Neither did lipases - enzymes that digest lipids. They found that the transforming substance was rich in nucleic acids, but ribonuclease, which digests RNA, did not inactivate the substance.
They also found that the transforming principle had a high molecular weight. According to the authors, the experimental ratios of those four elements supported the idea that the transforming principle was DNA, and not in protein. Enzymes are molecules that facilitate chemical reactions in cells by interacting with the substances that participate in the chemical reaction.
Enzymes are specific, meaning that a certain enzyme can only interact with certain substances. The authors state that enzymes that degrade ribonucleic acid, or RNA, a molecular similar to DNA, and enzymes that degrade protein had no effect on the transforming principle.
However, Avery, MacLeod, and McCarty write that when they exposed the transforming principle to an enzyme that degrades DNA, the enzyme broke down the transforming principle. The authors summarize their results of the enzyme tests in a table. The authors conclude that the results of their enzyme tests are further proof that the transforming principle is DNA. They state that the transforming principle sample did not contain any pneumococcal protein, the disease-causing substance of pneumococcus, and therefore the transforming principle must be structurally different from the pneumococcal protein.
The researchers also state that they used an analytical ultracentrifuge, a centrifuge that spins substances rapidly and can separate different components in a mixture, to see whether the transforming principle was a mixture of different types of substances. After centrifugation, Avery, MacLeod, and McCarty found that no parts of the transforming principle separated. Instead, it was consistent throughout, which, according to the authors, meant that the transforming principle likely only contained one kind of substance.
The authors identify that substance as DNA. Avery, MacLeod, and McCarty also write that the transforming principle absorbed the same type of light as DNA, an indication that the substances are likely the same. In that section, the authors interpret their experimental findings and conclude that the transforming principle was likely to be DNA. Avery, MacLeod, and McCarty state that, in their study, they successfully induced transformation in R form pneumococcus and that they extracted and purified DNA from the bacteria that caused R form bacteria to become S form bacteria.
They explicitly state that the DNA sample, containing no protein, could induce transformation in bacteria. The researchers also note when the bacteria transformed, they generated a capsule, or a protective outer layer made of sugar molecules. The authors discuss that capsule generation seemed to be related to the transforming principle even though the capsule substance was made of different chemicals than the transforming principle.
The authors state that for a substance to induce transformation in bacteria, it must have a high degree of biological specificity. Biological specificity is the idea that certain characteristics of organisms, like behaviors or biochemicals, vary depending on the species.
According to Avery, MacLeod, and McCarty, scientists had not explained how the DNA molecule, which had little structural variability when compared with proteins, could allow for biological specificity. For that reason, the authors do not claim that DNA is indisputably the cause of bacterial transformation. However, the researchers state that if other experiments support their findings, DNA must facilitate transformation in bacteria. In their paper, Avery, MacLeod, and McCarty did not completely rule out the possibility of some substance other than DNA causing the genetic changes required for bacterial transformation.
Historians have noted that, privately, Avery was more confident that DNA facilitated bacterial transformation. When people look at these two different molecules, they said hey, it's probably the proteins that are actually responsible for encoding the information of inheritance.
Proteins, people knew, were these complex molecules that in some ways you could say encoded information. Well, at the time, they thought that DNA were these kind of boring molecules that surely this couldn't encode information. And so the first evidence, strong evidence, that DNA is actually where the genetic information is encoded doesn't happen for several more decades.
And we start along that path with Griffith right over here, famous for Griffith's experiment, where he does something really interesting. And he by himself, his experiments in or that he publishes in or he actually he conducts and publishes in , they aren't responsible in and of themselves for establishing DNA to be the molecule that's actually the basis of inheritance, but they start an interesting path of inquiry where these gentlemen in are finally able to establish that DNA is where these heritable factors are actually encoded.
So what was Griffith's experiment? Well, he was studying strains of bacteria, and he saw that the same, the two variants on a certain strain or two variants of bacteria, you had the rough strain and the smooth strain, if he injected the rough strain into a mouse, the mouse lived. If he injected the smooth strain into a mouse, the mouse died. And it was because the smooth strain had this protective coating on it that made it harder to attack by the mouses immune system.
So that by itself, well, that's interesting, this is the virulent strain, this is the one that's actually going to kill the mice. Now if he took this smooth strain, the virulent strain and he heated up so those bacteria were killed and then he injected those, so this is the heat-killed smooth strain, if he injected those into the mouse, the mouse still lived because those bacteria were dead. But then he did something very, very, very interesting.
He took this, the heat-killed smooth strain, he took some of that and he took some of the live rough strain put together. Now common sense would tell you is like okay, this blue stuff, that's not going to kill the mouse, and this killed smooth strain, that's not going to kill the mouse either.
So if mix it up, that shouldn't kill the mouse, but it did kill the mouse, which was fascinating. And so he came up with this theory of a transformation principle. Even though he killed the smooth strain here, there must've been some type of materials, some type of molecule that still got transferred from the dead bacteria to the live bacteria and essentially transformed the live bacteria into the smooth strain, allowing them to kill the mouse.
And so he came up with this idea of some kind of transformation principle. And so you can imagine, and look, it took some time, over 10 years, now almost two decades, Avery, McCarty and McLeod said hey, what is this transformation principle?
Why don't we use Griffith's experiment and let's keep, instead of just taking you know the whole heat-killed smooth strain, let's try to break it up into its components and let's try to isolate the different components and keep doing the experiment until we have an isolated molecule or an isolated component that seems to do the trick.
So they were trying to isolate the transformation principle. And they did just as what I described. They took the heat-killed smooth strain, they would try to separate the different constituents out.
You can separate them out physically, you could use certain washes that would wash away certain components. You could use enzymes that would destroy certain components.
And eventually, and this is very meticulous work, so you can imagine they take the stuff, the whole dead heat-killed smooth strain and they start to separated it out into its various components. So that might be one component right over there, this is another, let me do it in these different colors, this is another component right over there, this is another component right over there.
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