This article provides an experiment on DNA.
As early as 1848, Wilhelm Hofmeister, a German botanist, has observed that cell nuclei resolve themselves into small, rod-like bodies during mitosis. Later, these structures were found to absorb certain dyes and so came to be called chromosomes (colored bodies). In 1869, Friedrich Miescher, a Swiss physician, isolated a substance from cell nuclei that he called nuclein – now known as DNA.
The chromosomes of eukaryotes contain a variety of proteins in addition to DNA. Until the early 1950s most biologists were inclined to believe that the proteins were the chief carriers if heredity. Nucleic acids contain only four different unitary building blocks, but proteins are made up of 20 different amino acids. Proteins therefore appeared to have a greater diversity structure, and the diversity of the genes seemed first likely to rest on the diversity of the proteins.
In 1928, Frederick Griffith, an English army doctor, wanted to make a vaccine against Streptococcus pneumoniae, which caused pneumonia. Since the time of Pasteur, about 50 years before, vaccines had been made using killed microorganisms which could be injected into patients to elicit the immune response of live cells without risk of disease. Though, he failed in making the vaccine he stumbled on a demonstration of the transmission of genetic instructions by a process we now call the “transformation principle”.
In his experiments, Griffith used two strains that are distinguishable by the appearance of their colonies when grown in laboratory cultures. In one strain, a normal virulent type, the cells are enclosed in a polysaccharide capsule, giving colonies a smooth appearance; hence, this strain is labeled ‘S’. In Griffith’s other strain, a mutant non-virulent type that grows in mice but is not lethal, the polysaccharide coat is absent, giving colonies a rough appearance; this strain is called ‘R’. The R bacteria were harmless, but the S bacteria were lethal when injected into mice. Heat-killed S cells were also harmless – the same effect seen by Pasteur.
However, surprisingly when live R cells were mixed with killed S cells and injected into mice, the mice died, and the bacteria rescued from the mice had been “transformed” into the S type (Fig. 7.1). Griffith announced that it was because of a phenomenon other than mutation, which he called transformation. This experiment strongly implied that genetic material had been transferred from the dead to the live cell. It was hard to be certain of this, or to know what exactly genetic material was transferred and was responsible for the transformation process.
In 1944, the team of Avery, MacLeod and McCarty revised the above experiment. They extracted and purified DNA, proteins and other materials from Streptococcus pneumoniae S bacteria, they mixed R bacteria with these different materials. Only those mixed with DNA were transformed into S bacteria (Fig.7.2). This led to the conclusion that the chromosome of S-bacteria causes the transformation and not the capsule. So they announced that bacterial transformation involves transfer of a part of DNA from the dead bacterium (donor) to the active living bacterium (recipient), which expresses the character of the donor cell, and so is called a recombinant.
This experiment strongly implied that DNA is the “transforming factor” and not proteins or other materials and this demonstrated what is known to us as the transforming principle – that genes are made of DNA. Amazingly, not everyone was convinced by the experiments of Avery’s, MacLeod’s and McCarty’s. It was the experiments of Hershey and Chase (1952) that finally proved that DNA was the genetic material and not protein.
The Hershey-Chase experiment, which demonstrated that the genetic material of phage is DNA and not protein (Fig. 7.3). They reasoned that phage infection must entail the introduction (injection) into the bacterium of the specific information that dictates viral reproduction. The phage is relatively simple in molecular constitution. Most of its structure is protein, with DNA contained inside the protein sheath of its “head.” The experiment uses two sets of T2 bacteriophages. In one set, the protein coat is labeled with radioactive sulfur (35S), not found in DNA.
In the other set, the DNA is labeled with radioactive phosphorus (32P), not found in protein. When the 32P-labeled phages were used, most of the radioactivity ended up inside the bacterial cells, indicating that the phage DNA entered the cells. 32P can also be recovered from phage progeny. When the 35S-labeled phages were used, most of the radioactive material ended up in the phage ghosts, indicating that the phage protein never entered the bacterial cell. Only the 32P is injected into the E. coli, indicating that DNA is the agent necessary for the production of new phages.
