DNA

Let’s talk about DNA. The genetic code is the blueprint used to build our bodies and that of every living being. At the very beginning of the 20th century, it was already known to scientist that the code was in genes, which in turn resided in chromosomes. In this series of articles I want to get through the incredible history and see how this most interesting of molecules works.

• Discovering the Genetic Code: We today know that chromosomes are made of DNA, but how that became a known fact? We must begin by going back to the earlier part of the 20th century, to the work of an English physician named Frederick Griffith. This experiment that I am about to describe really provided the first insight into the chemical nature of genetic information.
  
  
• Proteins Vs. DNA: In the early part of the 20th century, when Griffith published his work, there was generally an assumption that the genetic material must be a protein. Why did they think that? They thought it because pretty much everything that happens in the cell is done by a protein. It makes sense that if you got something complex and important that is being done in the cell, like providing information, it is probably going to be a protein.
  
  
• The Code is in DNA: In the early 1950’s, Hershey and Chase took a novel approach in trying to found out what the genetic material might be made of, by looking at how a particular kind of virus worked.

How it Works

• The Building Blocks: The building blocks of nucleic acids are called nucleotides. There are only four types of nucleotides. This is one of the reasons why nucleic acids seem relatively simple compared to proteins. Each nucleotide has a sugar that forms a ring.
  
  
• The DNA Structure: After the work of Hershey and Chase, biologists in the early 1950’s became convinced that DNA was what they needed to look at to understand the genetic code. They actually had no idea how DNA could possibly act as a mechanism for genetic inheritance.
  
  
• Watson and Crick’s Double Helix: James Watson was a young American, who had just completed his PhD. He was interested in protein structure. He moved to Cambridge, England, and began working with Francis Crick, who was a physicist familiar with x-ray crystallography and how to interpret it. The story goes that Watson happened to visit London for a seminar, and saw the x-ray diffraction patterns that Rosalind Franklin had obtained from Maurice Wilkins’ purified DNA. Watson made some notes, rushed back to Cambridge and told Crick what he had seen.

Understanding Replication

• Theories of Replication: The first alternative suggested that the DNA double helix must remain completely intact when it is replicated. That is, the two strands do not separate. The entire molecule is somehow used as a template for making more DNA. A second alternative suggested that the original DNA molecule becomes completely broken down during replication, with the newly copied DNA assembled by some unknown mechanism. In other words, the DNA double helix would actually be irrelevant. The mechanism that Watson and Crick proposed became known as the semi-conservative model of DNA replication. This was called semi-conservative because it predicts that during replication, the double helix unzips and the new daughter helixes would both have one strand of the old helix.
  
  
• Watson and Crick had it right: Watson and Crick’s semi-conservative model contrasted with a couple of other possibilities for how DNA could possibly replicate. There is the conservative model, which suggests that both strands in the original DNA double helix stay together during replication. Then there is the dispersive model, which suggests that both strands are not only separated, but even broken up into smaller pieces during replication. Deciding which of these models was the correct one seemed to be pretty easy, because they make very different predictions. It was not obvious how to prove it in the laboratory, however.
  
  
• The Process of Replication: In 1957, Arthur Kornberg made a really interesting discovery. He showed that DNA can be replicated outside of a cell, in a laboratory test tube. Kornberg wasn’t much interested in which model of replication was right. Instead, he was interested in specifically how replication occurred. Watson and Crick had suggested that the replication of DNA may not actually require an enzyme. If you could somehow unzip DNA, they thought that new DNA might just self-assemble, because the complimentary base-pairing would bring in all the appropriate nucleotides. Kornberg thought, though, that there must be some enzyme involved. He set out to figure out what that enzyme was.

A History Of DNA, Part III: The Code is in DNA

In my last post we saw how Avery and his colleagues demonstrated (but not conclusively to the scientific community) that the molecule which holds the genetic material in living things is DNA. Now I want to look at a very interesting experiment that really changed the minds of biologist in the matter. In the early 1950’s, Hershey and Chase took a novel approach in trying to found out what the genetic material might be made of, by looking at how a particular kind of virus worked.

Let me give you some background. Viruses are not true cells. They are made of an outer coat of protein with an inner core of nucleic acid. Viruses are made of just two things. The way a virus makes its living is by attaching to a cell, say a bacteria cell, injecting something into that cell and taking over the machinery of the cell. Here is a great introduction to viruses by Salman Khan(Sal), I really recomend you to watch it to understand viruses better.



Hershey and Chase were working with a particular kind of virus, called the T2 phage. This is a bacteria-eating virus, which makes its living by taking over a bacteria and using the protein-synthesizing machinery of the bacteria to make more viruses. Viruses can’t replicate themselves, they have to take over another cell. Clearly, then, what a virus must be doing, is injecting some information. It’s the information that would cause the cell to be taken over. What Hershey and Chase set out to do was to ask, what is it that these T2 viruses are actually putting inside the bacteria? There were only two candidates, proteins and nucleic acids.

The trick was to figure out how to determine which part was being injected. It is a very simple experiment to propose conceptually, but like many experiments in science, the devil is in the details. Hershey and Chase developed a very clever way to figure that out. They did this by radioactively labeling the proteins and the DNA that the virus was made of. In proteins, sulfur is a fairly common element. There is a radioactive form of sulfur (S-35). So, they could grow some T2 viruses in a medium that had a lot of this radioactive sulfur in it. What would happen is that as the viruses reproduce, they would incorporate sulfur into their protein codes. That meant that you could ask not where did the protein go, but where did the radioactivity go.

Alternatively, they could label the DNA. They could grow the same kind of virus in a medium that had radioactive phosphorus (P-32). Phosphorus is not found in proteins, but it is a major chemical constituent of DNA.

So, they grew viruses in a medium that either had radioactive sulfur or radioactive phosphorus. This resulted in some viruses having their proteins radioactively labeled, and others their DNA radioactively labeled.

In separate experiments, they added either the radioactively labeled sulfur viruses (with the radioactive protein), or the radioactively labeled phosphorus viruses (with the radioactively labeled DNA). In both cases they would give these viruses just a couple of minutes. Enough time for them to attach to bacteria and inject whatever they are injecting. Then they would stop the whole process. They were given enough time to inject but not enough time to take over the cell and cause it to build more viruses.

They gave the viruses just 20 minutes, and then they would put the solution in a blender. Then they put this solution in a centrifuge, which spins it around. Because of the action of the centrifuge, the heavier stuff would go down to the bottom of the tube. This would be the relatively large bacterial cell bodies. The lighter stuff, which would be the outer coats of the tiny viruses, would remain up in the solution. If you centrifuge them just right, you’ll get a little lump of stuff at the bottom of the tube, that’s going to be all the bacteria. Then you’ll have the rest of the fluid in the tube, which would include the viral coats.

They then would ask, where is the radioactivity? Is the radioactivity at the bottom, or at the rest of the fluid? What they found was that if they radioactively labeled the sulfur, marking the proteins, the radioactivity was found in the fluid, where the viral coats were. If you radioactively labeled the DNA with phosphorus, the radioactivity was found at the bottom, where the bacteria were. This was a very simple result but took the world by storm, because it showed incontrovertibly that what these viruses were injecting in the bacteria (and happened to be the genetic material), was DNA.

Hershey and Chase published these results in 1952, and it really caused a lot of interest. Biologists began to take a closer look at nucleic acids. That is what I want to do in my next post, look at the structure of DNA.

A History Of DNA, Part I: Before the Discovery

Proteins are the biological molecules that make things work in living systems. We could say that they are involved in every process in the cell. They are controllers of biochemical reactions, structural elements that hold parts of the cell together, motors that make things move, signals, and so forth. The function of a protein depends almost entirely on its shape. Its three dimensional shape determines its physical and chemical properties, which in turn allow the protein to serve its unique function. The three dimensional shape of a protein, in turn, depends almost entirely on the linear sequence of the building blocks of life, the amino-acids.

There are 20 kinds of amino-acids. An average protein might have a few hundred amino-acids. So, proteins are the work horses, and their function is determined by its chemical sequence of amino-acids. Now, how do we get a protein of a particular sequence built? To answer this question we need to address two things. First, what is the blueprint that is used to build proteins? Second, how does that molecule actually work?

It is the first question that I want to talk about today. Before we can understand how the code works, we need to understand what the code is made of. I think that we all know the answer today: DNA. Interestingly, though, that was one of the questions that defined molecular biology in the 20th century.

At the very beginning of the 20th century, it was already known to scientist that the code was in genes, which in turn resided in chromosomes. This was known from the work of early cell biologists, Walter Sutton and Theodor Boveri being the most important, who discovered that the particular movement of chromosomes that occurs when cells divide corresponded to patterns of transmission of traits between parents and their offspring. These patters of trait transmission had actually been discovered earlier by the Austrian monk Gregor Mendel.

Cell biologists knew about Mendel’s work, how chromosomes moved, and they developed what now is called the chromosomal theory of inheritance. This theory basically says that the way chromosomes move is somehow related to the way that inheritance occurs, therefore, chromosomes are related to information in cells.

We today know that chromosomes are made of DNA, but how that became a known fact? We must begin by going back to the earlier part of the 20th century, to the work of an English physician named Frederick Griffith. This experiment that I am about to describe really provided the first insight into the chemical nature of genetic information.

Griffith’s Experiment

Griffith was a physician and he wasn’t interested in the molecular basis of inheritance. Instead, he was working on a much more applied problem. He was studying Streptococcus pneumoniae, which is a bacteria that causes pneumonia in humans. What Griffith wanted to do was to develop a vaccine against this particular organism, because the pneumonia caused by it often proved fatal. This was before the advent of antibiotics, of course.

As often it is the case for disease-causing bacteria, there were different strains that varied in their virulence. They varied in how likely they were to induce the disease and cause death. Griffith was working with two strains of Streptococcus pneumoniae. He was working with what we call the S strain on the one hand. This was a very virulent strain. It is called S because if you grow it in a colony, it actually looks kind of shiny and smooth. He had another strain, which he called the R strain, which was non-virulent. If you got the R strain, you might be a little sick but you wouldn’t die. It is called the R strain because the bacteria look kind of rough on the surface.

The important thing to note is that these strains did breed true. In other words, as they reproduced, their offspring had the same properties. S strain bacteria gave rise to more S strain bacteria and so on. It is inferred from that, that the difference between the S strain and the R strain bacteria somehow must be genetically encoded.

What did Griffith do? Griffith was using the approach pioneered by Louis Pasteur, which was to take the organism that you want to develop a vaccine for and kill it. This organism could no longer harm you, but nonetheless, would perhaps induce some sort of immune response if injected in a subject. The idea was to take S strain bacteria, kill them by heating them up, and then take these dead S strain bacteria and inject them into a laboratory mouse, and see if that mouse develops immunity. The mouse wouldn’t die if you injected dead S strain bacteria, but the parts of the bacteria that are injected might nonetheless induce an immune response.

This is a great idea, but it didn’t work. It often doesn’t work. There wasn’t enough left of these dead S strain bacteria to induce an immune response. If you injected these in a mouse, and then injected live S strain bacteria, the mouse would die.

Another common technique that was used then and now to develop vaccines was to eject not only the dead offending organism, but some related organism that was less virulent. In this case, we are talking about the R strain bacteria. The idea is that the live R strain bacteria, because they are alive, would induce the organism to develop a full immune response. That development of a full immune response would somehow pickup some immunity to the dead S strain bacteria. This is a common technique and it often works.

What Griffith did then? He killed some S strain bacteria and injected them in with living R strain bacteria. What he hoped would happen is that the mouse would develop immunity to the S strain, but what happened instead was unexpected, and unfortunate for the mouse, the mouse died. This is a surprising result. What’s being injected into the mouse are dead S strain bacteria, that wouldn’t kill the mouse; and live R strain bacteria, that wouldn’t kill the mouse neither. Nothing was injected in the mouse that should kill it, and yet the mouse died.

What Griffith found out when he dissected the poor dead mouse was that inside it were living S strain bacteria. He injected dead S strain, and when he took the mouse apart, he found living S strain. What Griffith concluded from this work, and correctly, was that somehow the living R strain bacteria had taken up something from the dead S strain bacteria, and incorporated it into them. That somehow transformed the R strain into the S strain.

Griffith later showed that he didn’t need the mouse. You can do this in a beaker. If you put dead S strain and live R strain bacteria in a beaker, some of the live R strain would become transformed into live S strain.

What’s really interesting from this experiment, in my opinion, is that the material that they incorporated must somehow be genetic material. It must somehow have information in it. How else could the R strain bacteria now become virulent like an S strain bacteria? The difference between S and R had to have something to do with genetically transmitted information. What Griffith did was he left it there. It was only 1929 when he was doing this work. He said that he had discovered what he called the transforming principle.

Our interest in this, and of scientist interested in genetics, is that somehow this transforming stuff must involve genetic material. In my next post we will see how this experiment helped in discovering that DNA is the molecule that holds genetic material in living things.