DNA as Genetic Material-Experimental Proof-Part I-Dr C R Meera

 

DNA as Genetic Material-Experimental Proof

Part I- The Transformation Experiments- Dr. C R Meera

Three simple experiments, done with great care, identified DNA as genetic material. They included:

1.    The transformation experiments (Griffith/Avery, McLeod & McCarty)

2.    The chemical experiments (Chargaff)

3.    The Blender experiments (Hershey & Chase)

I.     The Transformation Experiments

The first experimental proof for DNA as genetic material was by Fred Griffith in 1928. He was studying the human bacteria causing pneumonia, Streptococcus pneumoniae or Pneumococcus. Its virulence is attributed to its polysaccharide capsule that protects it from body defense. On Nutrient agar media, the organisms produced colonies with smooth edges due to the presence of capsules and are called “S colonies”. When these organisms were injected into mice, it caused the death of the animal. This means, S colonies are lethal.

Griffith could isolate mutants of the organisms which were producing rough-edged colonies and named them “R bacteria”. They were non-encapsulated and non-lethal when injected into the mice.

He further experimented with “heat-killed S” colonies which were again non-lethal as “R colonies”. A significant observation made by Griffith was that when he injected the mixture of  “R live” and “heat-killed S”, it resulted in the death of the mice. He also isolated the bacteria from the dead mice and surprisingly it was found to be “S live”. He thought that the “R live” was replaced or transformed into “S live” forms within the mice.  

After several years, it was found that mice are not necessary for this transformation. The same experiments were carried out in In vitro models.  “R live” and “heat-killed S” cells were grown together in culture media and “S live” cells could be isolated from the same. And it was concluded that “R cells” restored the viability of “S cells”. But this idea was eliminated later due to another experiment in which “R cells” and “cell extract of heat-killed S cells”(extract was prepared from broken S cells and freed from both intact cells and capsular polysaccharides) produced “S live” strain. This experiment concluded the “cell extract” as the “transforming principle” nature of which was unknown at that time. 

 

Fig.2. Experiment with live “R cells” and “cell-free extract of lysed S cells”

The next development occurred some 15 years later in 1944, when Oswald Avery, Colin McLeod, and Maclyn McCarty partially purified the transforming principle from cell extract and demonstrated that it was DNA.

However biochemical investigation of DNA had begun in 1868 with Freidrich Miescher.

Ø  Miescher isolated a phosphorous-containing substance from nuclei of pus cells or leukocytes in the discarded surgical bandages. He named it “nuclein”. Nuclein consisted of acidic and basic portions. The acidic portion is now known as DNA and the basic portion is protein.

Ø  Later, Miescher discovered the same “acidic” substance in the heads of the sperm cells of salmon. However, he could partially purify nuclein and studied its properties. However, its primary covalent structure was not known till 1940s.

The first direct evidence for DNA as the bearer of genetic information came in 1944 by the experiments of Oswald Avery, Colin McLeod, and Maclyn McCarty. They extracted DNA from heat killed S strain (virulent), protein was removed as much as possible. It was then mixed with non-virulent R strain. DNA gained entrance into the R cells by the process of transformation (transformation is defined as the genetic alteration of a cell which is caused by the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane). In this experiment “non-virulent R strain” was permanently transformed into the “virulent S strain”.   The procedure for purifying DNA at that time was not perfect and contained many impurities. Hence, necessary evidence for transformation by DNA was given by Avery, McLeod, and McCarty using the following procedures.

1.    Chemical analysis of transforming principle- Chemical analysis revealed the major component as deoxyribose containing nucleic acids

2.    Physical measurement – The sample contained highly viscous substance with physical properties of DNA

3.    Enzyme reactions- Transforming principle was treated first with proteolytic enzymes like trypsin, chymotrypsin, or both.

Also treated with ribonuclease (RNA depolymerizing enzyme). Transforming activity was not lost when the treated transforming principle was mixed with the R strain, indicating that neither proteins nor RNA is the active principle.

Treatment with DNase (catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone and degrade DNA) inactivated the transforming principle which was a shred of clear evidence for DNA as genetic material.

Through these procedures, Avery, McLeod, and McCarty concluded that the transforming principle they isolated is DNA.

But the scientific community of that time was not ready to accept their conclusions. Because, whatever the genetic material was, it was expected to be a substance capable of the enormous variation in order to contain information carried by the huge number of genes. At that time, DNA was known as a tetranucleotide only (tetranucleotide hypothesis), so could not be considered the sole material of genetic information.  

Early experiments suggested that 4 bases (adenine (A), cytosine (C), guanine (G), and thymine (T)) occur in equal ratios in nucleotides. After discovering the location of  nucleic acids on chromosomes, the tetranucleotide hypothesis was put forward by Phoebus Levene in 1929. Ribose sugar and Deoxy ribose sugar were discovered by him in 1909 and 1929, respectively.    In the tetranucleotide hypothesis, Levene suggested that nucleic acids are repeating tetramers.  In other words, DNA is composed of repeating sequences of four nucleotides. He called the Sugar-Base-Phosphate unit a nucleotide. Also said that the simplicity of this structure of DNA was too uniform to contribute to a complex genetic variation. Hence, DNA could not be the genetic material.  Thereafter, attention focused on proteins as the probable hereditary substance.

Fig 3. Tetranucleotide hypothesis: Repeating tetranucleotide unit

(Image courtesy: en.wikipedia.org)