DNA I: _{The genetic material}

The chemical basis of heredity

In the mid-19th century, Gregor Mendel completed his now classic experiments on genetics (see our Mendel and Inheritance module). Mendel proposed that the "characters" that controlled inheritance exhibited certain patterns of behavior. Specifically, they seemed to operate in pairs and separated independently during reproduction. The work that Mendel did established some trustworthy rules and properties about genetics and heredity, but no one had any idea what Mendel's "characters" were and how features were passed from generation to generation. Scientists were convinced that the basis of genetics and heredity could be found somewhere in the chemistry of our cells.

In the early 1900s, scientists began to focus on a recently discovered structure in cells called chromosomes (named by Walther Flemming from the Greek words for "colored bodies" because they selectively absorbed a red dye that Flemming used to color cells). Curiously, chromosomes seemed to behave in a manner similar to Mendel's "characters." Specifically, they were seen to line up randomly, separate, and then segregate from each other just prior to cell division, reminiscent of Mendel's laws of independent assortment and segregation (Figure 1). Gradually, scientists began to suspect a connection between chromosomes and heredity.

DNA or protein?

While biologists were becoming convinced that chromosomes were the physical seat of genetics and inheritance, chemists were claiming that these structures were made of both protein and DNA. So, which was the genetic molecule housing all the hereditary information? Many scientists of the day actually thought it was protein because there are 20 different amino acids for building a protein polymer, while DNA polymers are made of only four nucleotide bases.

Consider it this way: The genetic molecule works like a language for storing information consisting of words that are made of individual "letters." The "language" of the DNA polymer would only have four different "letters" to work with (the four nucleotide bases), while "protein language" would have twenty possible letters – the twenty different amino acids. Imagine making a language using only four letters! Thus, because it offers far more complexity, most scientists in the early 20^th century believed that protein was the component of chromosomes that housed the genetic information. Regarding the DNA, they thought that perhaps it acted as structural support for the chromosomes, like the frame of a house.

Griffith discovers "transformation"

Clarification came during the First World War. During the war, hundreds of thousands of servicemen died from pneumonia, a lung infection caused by the bacterium Streptococcus pneumoniae. In the early 1920s, a young British army medical officer named Frederick Griffith began studying Streptococcus pneumoniae in his laboratory in the hopes of developing a vaccine against it. As so often happens in scientific research, Griffith never found what he was looking for (there is still no vaccine for pneumonia), but instead, he made one of the most important discoveries in the field of biology: a phenomenon he called "transformation."

Dr. Griffith had isolated two strains of S. pneumoniae, one of which was pathogenic (meaning it causes sickness or death, in this case, pneumonia), and one which was innocuous or harmless. The pathogenic strain looked smooth under a microscope due to a protective coat surrounding the bacteria and so he named this strain S, for smooth. The harmless strain of S. pneumoniae lacked the protective coat and appeared rough under a microscope, so he named it R, for rough (Figure 2).

Dr. Griffith observed that if he injected some of the S strain of S. pneumoniae into mice, they would get sick with the symptoms of pneumonia and die, while mice injected with the R strain did not become sick. Next, Griffith noticed that if he applied heat to the S strain of bacteria, then injected them into mice, the mice would no longer get sick and die. He thus hypothesized that excessive heat kills the bacteria, something that other scientists, including Louis Pasteur, had already shown with other types of bacteria.

However, Dr. Griffith didn't stop there – he decided to try something: He mixed living R bacteria (which are not pathogenic) with heat-killed S bacteria, and then he injected the mixture into mice. Surprisingly, the mice got pneumonia infections and eventually died (Figure 3).

Dr. Griffith examined samples from these sick mice and saw living S bacteria. This meant that either the S bacteria came back to life, an unlikely scenario, or the live R strain was somehow "transformed" into the S strain. Thus, after repeating this experiment many times, Dr. Griffith named this phenomenon "transformation." This discovery was significant because it showed that organisms can somehow be genetically "re-programmed" into a slightly different version of themselves. One strain of bacteria, in this case the R strain of S. pneumoniae, can be changed into something else, presumably because of the transfer of genetic material from a donor, in this case the heat-killed S strain.

Scientists around the world began repeating this experiment, but in slightly different ways, trying to discover exactly what was happening. It became clear that, when the S bacteria are killed by heat, they break open and many substances are released. Something in this mixture can be absorbed by living bacteria, leading to a genetic transformation. But because the mixture contains protein, RNA, DNA, lipids, and carbohydrates, the question remained – which molecule is the "transforming agent?"

Avery, MacLeod, and McCarty discover the transforming agent

This question was examined in several ways, most famously by three scientists working at The Rockefeller Institute (now Rockefeller University) in New York: Oswald Avery, Colin MacLeod, and Maclyn McCarty. These scientists did almost exactly what Griffith did in his experiments but with the following changes. First, after heat-killing the S strain of bacteria, the mixture was separated into six test tubes. Thus, each of the test tubes would contain the unknown "transforming agent." A different enzyme was then added to each tube except one – the control – which received nothing. To the other five tubes, one of the following enzymes was added: RNase, an enzyme that destroys RNA; protease, an enzyme that destroys protein; DNase, an enzyme that destroys DNA; lipase, an enzyme that destroys lipids; or a combination of enzymes that breaks down carbohydrates.

The theory behind this experiment was that if the "transforming agent" was, for example, protein – the transforming agent would be destroyed in the test tube containing protease, but not the others. Thus, whatever the transforming agent was, the liquid in one of the tubes would no longer be able to transform the S. pneumonia strains. When they did this, the result was both dramatic and clear. The liquid from the tubes that received RNase, protease, lipase, and the carbohydrate-digesting enzymes was still able to transform the R strain of pneumonia into the S strain. However, the liquid that was treated with DNase completely lost the ability to transform the bacteria (Figure 4).

Thus, it was apparent that the "transforming agent" in the liquid was DNA. To further demonstrate this, the scientists took liquid extracted from heat-killed S. pneumoniae (S strain) and subjected it to extensive preparation and purification, isolating only the pure DNA from the mixture. This pure DNA was also able to transform the R strain into the S strain and generate pathogenic S. pneumoniae. These results provided powerful evidence that DNA, and not protein, was actually the genetic material inside of living cells.

Hershey and Chase further investigate DNA vs. protein

Despite this very clear result, some scientists remained skeptical and continued to think that proteins were likely the genetic molecule. Eight years after the famous Avery, MacLeod, and McCarty experiment was published, two scientists named Alfred Hershey and Martha Chase performed an entirely different type of genetic experiment. For their experimental system, they selected an extremely small virus called a bacteriophage (or just phage), which only infects bacterial cells. At that time, scientists knew that when these phage infect a bacterial cell, they somehow "reprogram" the bacterium to transform itself into a factory for producing more phage. They also knew that the phage itself does not enter the bacterium during an infection. Rather, a small amount of material is injected into the bacteria and this material must contain all of the information necessary to build more phages. Thus, this injected substance is the genetic material of the phage.

Hershey and Chase designed a very simple experiment to determine which molecule, DNA or protein, acted as the genetic material in phages. To do this, they made use of a technique called radioactive labeling. In radioactive labeling, a radioactive isotope of a certain atom is used and can be followed by tracking the radioactivity (radioactivity is very easily detected by laboratory instruments, even back in the 1940s, and remains a very common tool in scientific research). So, what Hershey and Chase did was to grow two batches of phage in their laboratory. One batch was grown in the presence of radioactive phosphorous. The element phosphorous is present in large amounts in DNA, but is not present in the proteins of bacteria and phage. Thus, this batch of phage would have radio-labeled DNA. The second batch of phage was grown in the presence of radioactive sulfur. Sulfur is an element that is often found in proteins, but never in DNA. Thus, the second batch of phage would have radio-labeled proteins.

Then, Hershey and Chase used these two batches of phage separately to infect bacteria and then measured where the radioactivity ended up. What they observed was that only those bacteria infected by phage with radio-labeled DNA became radioactive, bacteria infected by phage with radio-labeled protein did not. Thus Hershey and Chase concluded that it is DNA, and not protein, that is injected into the bacteria during phage infection and this DNA must be the genetic material that reprograms the bacteria.

The blueprint of life

Taken together, these experiments represented strong evidence that DNA is the genetic material. Other scientists later confirmed these result in many different kinds of experiments, including showing that eukaryotic, and even human cells can be "transformed" by the injection of DNA. The result of these findings was to convince the scientific and lay communities that the molecule of heredity is indeed DNA. It turns out that the initial instincts of many scientists were exactly backward: They assumed that protein was the genetic material of chromosomes and DNA merely provided structure. The opposite turned out to be true. The DNA molecule houses genetic information, and proteins act as the structural framework of chromosomes.

The discovery that DNA was the "transforming agent" and the genetic component of human chromosomes was one of the greatest discoveries of science in the 20th century. However, the mechanism of how DNA codes for genetic information was initially a complete mystery and became the focus of intense scientific study (see our DNA II module). Still today, the study of how DNA functions comprises an entire discipline of science called molecular biology. Originally an offshoot of biochemistry, the field of molecular biology joins biologists, chemists, anthropologists, forensic scientists, geneticists, botanists, and many others who are working to shed light onto the immense complexity of DNA, the so-called blueprint of life.