Experimental Evidence for dna's Role as Genetic Material

Two experiments are cited to prove I that DNA is the genetic material.

1. Griffith's and Avery's Transfor­mation Experiments. The first direct evidence that the DNA transmits genetic informa­tion came from the experiments of Griffith and Avery.

Griffith's Experiment. In 1928, S.F. Griffith, an English bacteriologist, experimented with Strep­tococcus pneumoniae (Diplococcus), a bacterium that causes pneumonia in mammals, including humans, and is commonly called pneumococcus. There are two different strains of this bacterium : a smooth, virulent strain (S-strain), which is protected by a polysaccharide capsule, forms smooth glistening colonies when grown on agar medium, and causes pneumonia; and a rough, harmless strain (R-strain) without a capsule and forming rough colonies.

He injected live, rough bac­teria into mice, the mice did not die. The mice injected with live, smooth bacteria died, but the mice injected with heat-killed, smooth bacteria sur­vived. Griffith then injected mice with a mixture of live, rough bacteria and heat-killed, smooth bac­teria. Although neither of these alone was harmful, their mixture caused pneumonia and killed the mice. He found live, smooth bacteria in the blood of dead mice. From this, he concluded that some trans­forming principle (a chemical substance) from the dead, smooth bacteria had entered the living, rough bacteria and transformed the latter into smooth bacteria. These transformed smooth bacteria recovered from the dead mice, when grown in a culture, produced smooth bacteria. Thus, transfor­mation was a permanent genetic change

Griffith's Problems. Griffith could not know a) the role of mice in the change of bacterial strain, and (b) what component of S-strain bacteria polysaccharide, protein or DNA) caused virulence.

No Role of Mice. A few years later, some other scientists found that when smooth, heat-killed bacteria and live, rough bacteria were kept together in a culture medium, the former transmitted virulence to the latter which got transformed into smooth, virulent bacteria. This proved that mice cells had no role to play in the change of one strain of bacteria into another.

Role of Bacterial Extract. Still later, it was found that an extract of the smooth, heat-killed bacteria also transformed the live, rough bacteria into smooth, virulent bacteria in culture media. This finding proved that some component of the bac­terial cell, rather than the whole cell, was the trans­forming agent.

Experiment of Avery and Others. In 1944, O.T. Avery, C. Macleod and M. Mc Carty separated the extract of smooth, virulent bacteria into protein, DNA and carbohydrate fractions. Each fraction was separately added to a cul­ture medium containing live rough bacteria. Only the culture that received the DNA fraction of the extract from virulent bacteria produced smooth bac­teria. This proved that DNA was the transforming agent. When DNA fraction was treated with deoxyribonuclease (an enzyme that digests DNA), it became inactive and incapable of transforming the rough strain into the smooth strain. This confirmed that DNA is the genetic material. It is not destroyed if the cells are killed with heat.

Transformation. It is a permanent, in­heritable change produced in one strain of bacteria by a substance isolated from another strain of the same kind of bacteria. A substance which brings about transformation is called a transforming agent, for example, DNA in the above experiment.

Conclusive evidence that DNA is the only genetic material in organisms has since been ob­tained in several direct and indirect ways. Perhaps the best evidence is provided by a bacteriophage experiment.

2. Bacteriophage Experiment. Bac­teriophages are viruses that infect bacteria and kill them. In 1952, Alfred.D. Hershey and Martha Chase experimented with the bacterium Es­cherichia coli and bacteriophage T₂, both inhabiting

the human intestine. Bacteriophage T₂ has a DNA core and a protein coat. When it comes in contact with a living bacterium, its DNA core enters the cell but its protein coat remains outside. Hershey and Chase wanted to confirm it. They used different radioactive isotopes to label DNA and protein. First they grew T₂ with£. coli in the presence of radioac­tive sulphur ³⁵S. Because protein, but not DNA, contains sulphur, radioactive atoms were incor­porated into the protein coat of T₂ only. These bacteriophages were used to infect normal bacteria. The protein coats were separated from the bacterial cell walls by shaking and centrifugation. The protein coats, being lighter, stay in the supernatant and the infected bacteria settle down in the centrifuge tube. It was found that almost the entire radioactivity was associated with protein coats in the supernatant and almost none with the bacterial cell. This showed that the bacteriophage protein coat did not: enter the bacterial cell during infection.

Hershey and | Chase then repeated ; the experiment by making the DNA core of the bacteriophages labelled with atoms of radioactive phosphorus ³²P. Because DNA, but not protein, contains phosphorus, the T₂ protein coat remained unlabelled. This experi­ment showed radioac­tivity in the bacterial cell fraction instead of in the protein coat su­pernatant. This proves that only the bac­teriophage DNA enters the bacterial cell.

DNA is the Genetic Material of T₂ Phage. There was another rather more important finding of the experiment. The bac­teria, which had the bacteriophage DNA in them but were cleared of the bacteriophage protein coats from their sur­face, produced a new generation of normal bac­teriophages in them. Since the DNA of the infecting bacteriophages has given rise to a fresh crop of bacteriophages, it is clear that the DNA functions as the hereditary material in the bacteriophage T₂.

Additional Evidence for DNA being Genetic Material. In eukaryotes, a diploid set of chromosomes has twice as much DNA as the haploid set present in the gametes of the same organism. Also, prior to mitosis, a eukaryotic cell exactly doubles its DNA content, and during mitosis, the DNA is distributed exactly half and half to the two daughter cells.