2 Introduction to Nucleic acids -Historical Perspective

Prof. Sunil Kumar Khare

1. Objectives 

• History of genetic material
• Historical experiments-DNA as a hereditary material
• Understanding the timeline of DNA discovery

2. Concept Map

3. History of Genetic Material

3. 1. Search of DNA

The history of deoxyribonucleic acid (DNA) or genetic material originated with some basic and fundamental discoveries. Depiction of DNA dates back to 1869, with its initial finding by Friedrich Miescher. On February 26, 1869, he shared the isolation of this mysterious substance (now known as DNA), with his uncle Wilhelm His (who was a renowned physician and professor of anatomy and physiology at University of Basel; he had discovered neuroblasts and coined the term “dendrite) in a letter. In this letter he wrote, “In my experiments with low alkaline liquids, precipitates formed in the solution after neutralization that could not be dissolved in water, acetic acid, highly diluted hydrochloric acid or in a salt medium and therefore do not belong to any known type of protein”.

He reported the isolation of a material in the nuclei of human white blood cells, which was weakly acidic in nature and whose function was not yet known. He named this material “nuclein”. In another few years, with more research Miescher was able to separate nuclein into protein and nucleic acid components. Further research in the last half of the 20th century, the implications of nuclein as genetic material and function as the bearer of hereditary characteristics was discovered.

3.2. Evidences towards the DNA as hereditary material-historical experiments

Till many years after Miescher’s death, nuclein had received very little attention around the world. Majority of researchers were convinced that protein is the genetic material because enormous hereditary information can only be stored in complex substances. While DNA was made up of only four different molecules, proteins were composed of 20 different amino acids. Scientist thus remained confident about proteins having more complex structures and storing genetic information.

3.2.1 Griffith’s Experiment

In the year 1928, Frederick Griffith, who was a Medical Officer in British ministry of Health, performed several experiments with different strains of Diplococcus pneumoniae (now named as Streptococcus pneumoniae). These strains were of two types i.e. Smooth (S) and Rough (R).

• Smooth (S): Smooth cells were found virulent (infectious) as it caused pneumonia in vertebrates (especially human and mice). There virulence was mainly due to the polysaccharide capsules, which also led to formation of smooth colonies on agar plate.

• Rough (R): In this type, cells lacked polysaccharide capsules and were non-virulent (non-infectious). Due to absence of polysaccharide capsule, they showed dull rough colonies on agar plates.

This specific feature allows the microbiologist to easily differentiate the virulent and non-virulent strains using simple microbiological culture techniques. Each strains of Diplococcus having several serotypes like S-I, S-II, S-III, R-I, R-II, R-III etc. The specificity of the serotype again depends upon the detailed chemical structure of the polysaccharide capsule, which can be identified through different immunological techniques.

Griffith selected serotypes S-III and R-II for his work, which led to the generation of the new concept of genetic material. Griffith already knew the fact that pneumonia is only caused by living smooth cell. If the heat killed virulent bacteria will be injected in mice there should ideally be no sign of infection as similar as the non-virulent live cells. Strategically Griffith designed his experiment, which is provided in Table 1 below.

Griffith performed his experiments by using above sets of bacteria and injecting them into mice and found the below results (Fig 4):

a) When he injected with live cells of virulent S-III strain into the mice; the mice developed pneumonia and finally died.

b) When live cells of non-virulent R-II bacteria were injected into the mice, the mice did not develop any sign of illness and survived, thus confirming the nature of non-virulent strain.

c) Heat killed cells of the virulent S-III bacteria were also not able to develop pneumonia into mice and thus mice survived. He concluded that after heat treatment bacteria must be dead due to which no infection occurs.

d) When Griffith injected a mixture of both heat killed cells of virulent S-III and live cells of non-virulent R-II into the mice; the mice suffered from pneumonia and died. After dissection of mice, it was observed that mice blood cells were having both R-II and S-III strains of bacteria. He concluded that some factor must have been passed from heat killed virulent S-III strain to the live non-virulent R-II strains, which empowered them with the ability to produce polysaccharide capsule and make them virulent type. Griffith hypothesized that the transforming factor was an S-III protein and called the phenomena as Transforming Principle.

Several other physician and bacteriologists like Henry Dawson in 1931, Lionel J. Alloway in 1933 further carried on work in the support of Griffith’s work.

After 16 years of Griffith’s work, in 1944 Ostwald T. Avery, Colin MacLeod and Maclyn McCarty reported that DNA of the virulent S-III strain served as genetic material and was responsible for the Griffith’s result.

They started their work with large culture volume of virulent S-III strain. The cells were pelleted down by centrifugation and killed by heating at 65 oC. Further pellets were homogenized with the supernatant and extracted with the detergent deoxycholate, after which they obtained a soluble filtrate having transformation capability. To remove the protein from the soluble active filtrate, several rounds of chloroform extraction was done and subsequently to remove the polysaccharides , enzymatic digestion was also carried out. At last by ethanol precipitation, a fibrous mass was collected which had the ability to induce the transformation in non-virulent R-II strain.

To solidify their finding, they planned to eliminate all the contaminants like protein, RNA etc which is present in the soluble active fraction.

In their first experiments, soluble extract were directly tested for transformation with live non-virulent R-II type strain and injected in mice. It was observed that mice developed the infection and died. Both S-III and R-II type strains were found in the blood of mice.

In the next experiment, to remove the proteins, soluble extract were treated with proteases and further transformation assay was carried out along with non-virulent R-II type strain. As a result it was observed that mice died due to pneumonia and both strains were present. Further to remove the RNA, Avery, MacLeod and McCarty treated the soluble extract with RNase. The mixture of RNase treated extract and non-virulent R-II strain was injected in mice and it was found that mice developed the infection.

The final inference came with the experiment where DNA digesting enzyme DNase was used to treat the soluble extract. The mixture of DNase treated extract and R-II strain was inject and it was found that no transformation occurs in non-virulent R-II strain. Further, they emphasized that once transformation occurs, the polysaccharide capsules will be formed in the successive generation. Therefore, transformation is heritable. This confirmed the finding that DNA is the transforming factor, which acts as a hereditary genetic material.

In support of DNA as the genetic material, another good piece of evidence was provided during the study of bacteriophage T2 and its target bacterium Escherichia coli by Alfred Hershey and Martha Chase in 1952. The bacteriophages T2 are simply referred as Phage, a kind of virus which contain DNA as core and surrounded by the protein coat. The electron micrograph of T2 phage is shown in Fig.8.

The life cycle of phage T2 is briefly described in Fig.9. T2 Phages were first adsorbed on the bacterial cell surface and subsequently injected its chromosome inside the bacterial cell. Inside bacterial cell, degradation of the chromosome was initiated by phage specific enzymes. Following the infection step, the viral information “commandeers” the cellular machinery of the host and directs viral reproduction. Within a very short time, many new phages emerged from a single bacterial cell and the bacterial cell is lysed. It seemed that some molecular components of the phage (whether DNA, or protein or both) enter inside the bacterial cell and directs the viral reproduction.

As it is well known fact that DNA contains phosphorus (P) and not sulphur(S), whereas in proteins only sulphur (S) is present, Hershey and Chase strategically designed their experiments where they had used radioisotope 32P and 35S to label DNA and protein respectively. This was a key determining point in their experiments. They grew E. Coli in two separate radioactive medium containing 32P and 35S and phages were allowed to attack the radiolabeled E. Coli bacteria.

As a result, the progeny of Phages were also become radiolabeled. In some of the phages, progeny DNA gets labelled and some phages protein coat gets labelled with their respective radioisotope. When these radiolabeled phases were allowed to attack on general without radiolabeled E. coli bacteria, the progeny showed a remarkable result in that phases whose DNA was radiolabeled got a radiolabeled progeny but the phages whose protein was labelled got a non-radiolabeled progeny. This ultimately confirms that none other than the DNA serves as hereditary genetic material.

3.3 Timeline of DNA discovery

Apart from the above mentioned three classical experiments, several researchers have also contributed significantly in the search of DNA and nucleic acid. Some of them are listed in Table-2.

Summary:

In this lecture we learnt about

• The initial discovery of nuclein
• How different scientist proved that protein is not containing the genetic information
• How DNA was discovered as genetic material

The timeline of DNA related discoveries