Discovery of DNA
The Search for the Genetic Material
Long before DNA and RNA were known to carry genetic information, scientists realized that living organisms contain some substance—a genetic material—that is responsible for the characteristics that are passed on from parent to child. Geneticists knew that the material responsible for hereditary information must have three key characteristics:
1. It must contain, in a stable form, the information about an organism’s cell structure, function, development, and reproduction.
2. It must replicate accurately, so that progeny cells have the same genetic information as the parental cell.
3. It must be capable of change. Without change, organisms would be incapable of variation and adaptation,and evolution could not occur.
The Swiss biochemist Friedrich Miescher is credited with the discovery, in 1869, of nucleic acid. He isolated a substance from white blood cells of pus in used bandages during the Crimean War. At first he believed the substance to be protein; but chemical tests indicated that it contained carbon, hydrogen, oxygen, nitrogen, and phosphorus, the last of which was not known to be a component of proteins. Searching for the same substance in other sources, Miescher found it in the nucleus of all the samples he studied—and, therefore, he called it nuclein. At the time, its function was unknown, and its exact location in the cell was unknown.
In the early 1900s, experiments showed that chromosomes—the threadlike structures found in nuclei—are carriers of hereditary information.
Chemical analysis over the next 40 years revealed that chromosomes are composed of protein and nucleic acids, which by this time were known to include DNA and RNA. At first, many scientists believed that the protein in the chromosomes must be the genetic material. They reasoned that proteins have a great capacity for storing information because they were composed of 20 different amino acids. (Note: Twenty amino acids were known at the time. A twenty-first amino acid was identified in the 1970s, and a twenty-second was identified in 2002.) By contrast, DNA, with its four nucleotides, was thought to be too simple a molecule to account for the variation found in living organisms. However, beginning in the late 1920s, a series of experiments led to the definitive identification of DNA as genetic material.
Griffith’s Transformation Experiment
In 1928, Frederick Griffith, a British medical officer, was working with Streptococcus pneumoniae (also called pneumococcus), a bacterium that causes pneumonia. Griffith used two strains of the bacterium: the S strain, which produces smooth, shiny colonies and is virulent (highly infectious); and the R strain, which produces rough colonies and is nonvirulent (harmless). Although this distinction was not known at the time, the virulence of the S strain is due to the presence of a polysaccharide coat—a capsule— surrounding each cell. The coat is also the reason for the smooth, shiny appearance of S colonies. The R strain is genetically identical except that it carries a mutation that prevents it from making the polysaccharide coat.
A mutation is a heritable change in the genetic material. In this case, a mutation in a gene affects the ability of the bacterium to make the coat and, hence, alters the virulence state of the bacterium. There are several types of S strains, each with a distinct chemical composition of the polysaccharide coat. Griffith worked with IIS and IIIS strains, which have type II and type III coats, respectively. Occasionally, S-type cells mutate into R-type cells, and R-type cells mutate into Stype cells. The mutations are type-specific—meaning that, if a IIS cell mutates into an R cell, then that R cell can mutate back only into a IIS cell, not a IIIS cell. Griffith injected mice with different strains of the bacterium and observed their effects on the mice. When mice were injected with IIR bacteria (R bacteria derived by mutation from IIS bacteria), the mice lived. When mice were injected with living IIIS bacteria, the mice died, and living IIIS bacteria could be isolated from their blood. However, if the IIIS bacteria were killed by heat before injection, the mice lived. These experiments showed that the bacteria had both to be alive and to have the polysaccharide coat to be virulent and kill the mice. In his key experiment, Griffith injected mice with a mixture of living IIR bacteria and heat-killed IIIS bacteria.
The mice died, and living IIIS bacteria were present in the blood. These bacteria could not have arisen by mutation of the R bacteria, because mutation would have produced IIS bacteria. Griffith concluded that some IIR bacteria had somehow been transformed into smooth, virulent IIIS bacteria by interaction with the dead IIIS bacteria. Geneticmaterial from the dead IIIS bacteria had been added to the genetic material in the living IIR bacteria. Griffith believed that the unknown agent responsible for the change in the genetic material was a protein; but this was a hunch, and he turned out to be wrong. He had no experimental evidence one way or the other as to the material acting as the agent bringing about the genetic change. Griffith called this agent the transforming principle.
Avery’s Transformation Experiment
In the 1930s and 1940s, American biologist Oswald T. Avery, along with his colleagues Colin M. MacLeod and Maclyn McCarty, tried to identify Griffith’s transforming principle by studying the transformation of R-type bacteria to S-type bacteria in the test tube. They lysed (broke open) IIIS cells with a detergent and used a centrifuge to separate the cellular components—the cell extract— from the cellular debris. They incubated the extract with a culture of living IIR bacteria and then plated cells on a culture medium in a Petri dish. Colonies of IIIS bacteria grew on the plate, showing that the extract contained the trans-forming principle, the genetic material from IIIS bacteria capable of transforming IIR bacteria into IIIS bacteria. Avery and his colleagues knew that one of the macromolecular components in the extract—polysaccharides, proteins, RNA, or DNA—must be the transforming principle. To determine which, they treated samples of the cell extract with enzymes that could degrade one or more of the macromolecules. After an enzyme treatment, the researchers tested to see if transformation still occurred. They found that the extract failed to bring about transformation only when DNA had been degraded, despite the presence of all other remaining macromolecules in the extract. By contrast, any enzyme treatment that did not lead to digestion of the DNA did not eliminate the transforming principle. These results showed that DNA, and DNA alone, must have been the transforming principle (the genetic material). That is, removing DNA from the cell extract was the only change that could eliminate the ability of the extract to provide the IIR bacterium with genetic material. The starting point is a mixture of DNA and RNA purified from a cell extract of IIIS cells. Samples of the mixture are treated separately with two different kinds of nucleases, enzymes that degrade nucleic acids. The samples are then tested to see if they can transform IIR bacteria to IIIS. For the mixture treated with ribonuclease(RNase), which degrades RNA and not DNA, DNA is unaffected and IIIS transformants resulted. For the mixture treated with deoxyribonuclease (DNase), which degrades DNA and not RNA, RNA is unaffected but DNA is digested, and no transformants resulted. The results show that DNA is the transforming principle. Although Avery and his colleagues’ work was important, it was criticized at the time by scientists who were supporters of the hypothesis that protein was the genetic
material. These scientists argued that the preparations of the various enzymes the researchers had used were only crudely purified. If proteins were the genetic material, they might have escaped digestion when protein-digesting enzymes were tested, but they might have been digested accidentally when DNases were tested.
Hershey and Chase’s Bacteriophage Experiment
In 1953, Alfred D. Hershey and Martha Chase published a paper that provided more evidence that DNA was the genetic material. They were studying a bacteriophage called T2.
Bacteriophages (also called phages) are viruses that attack bacteria. Like all viruses, the T2 phage must reproduce within a living cell. T2 reproduces by invading an Escherichia coli (E. coli) cell and using the bacterium’s molecular machinery to make more viruses. Initially the progeny viruses are assembled inside the bacterium; but eventually the host cell ruptures, releasing 100–200 progeny phages.
The suspension of released progeny phages is called a phage lysate. The in which a phage infects a bacterial cell and produces progeny phages that are released from the broken-open bacterium is known as the lytic cycle.
Hershey and Chase knew that T2 consisted of only DNA and protein, and their working hypothesis was that the DNA was the genetic material. T2 phages are very simply put together. They have an outer shell that surrounds their genetic material. When they infect a bacterium, they inject their genetic material inside the host cell but leave their outer shell on the surface of the bacterium. Once the genetic material has been injected into the host cell, the empty outer shell that is left is sometimes referred to as a phage ghost. To prove that the phage genetic material was made up of DNA and not protein, Hershey and Chase grew cells of E. coli in media containing either a radioactive isotope of phosphorus or a radioactive isotope of sulfur. They used these isotopes because DNA contains phosphorus but no sulfur, and protein contains sulfur but no phosphorus. The E. coli tookup whichever isotope was provided and incorporated the into all the nucleic acids made inside the cell or incorporated the into all the proteins made inside the cell. Any phage inside the bacteria would use its host bacterium’s nucleic acids and proteins to construct progeny phages. Hershey and Chase then infected the bacteria with T2 and collected the progeny phages. At this point, the researchers had two batches of T2, one with DNA labeled radioactively with and the other with protein labeled with . Next, they infected two cultures of E. coli with one or other of the two types of radioactively labeled T2. When the infecting phage was -labeled, most of the radioactivity was found within the bacteria soon after infection. Very little was found in the phage ghosts released from the cell surface after the cells were agitated in a kitchen blender. After completion of the lytic cycle, some of the was found in the progeny phages. In contrast, after E. coli were infected with -labeled T2, almost none of the radioactivity appeared within the cell or in the progeny phage particles, while most of the radioactivity was in the phage ghosts. Hershey and Chase reasoned that, because it was DNA and not protein that entered the cell—as evidenced by the presence of and the absence of 35S inside the bacterial cells immediately after the phage had begun the infection process by injecting their genetic material inside their host.
Source:
iGENETICS-A Molecular Approach, third edition, Peter J. Russel.