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DNA as Genetic Material – Evidence

DNA is a chemical substance that makes genes of all organisms ranging from prokaryotes (bacteria) to higher organisms (humans). Interestingly, the non-living organism such as virus also contains DNA as genetic material. Universally, in 1953, people accepted DNA as genetic material after a plethora of experimental evidence.

DNA is a chemical substance that makes genes of all organisms ranging from prokaryotes (bacteria) to higher organisms (humans). Interestingly, the non-living organism such as virus also contains DNA as genetic material. Universally, in 1953, people accepted DNA as genetic material after a plethora of experimental evidence.

Currently, our modern technology using DNA as fingerprint evidence for several practical claims, including paternity testing, forensic analysis, and genetic screening. Due to this characteristic feature of DNA, today many people are following a genetic analysis to clear their misconceptions.

Later, scientists were found that some viruses also contain ribonucleic acid (RNA) as the genetic material. The main goal of this article is to provide a piece of scientific information which was previously analyzed by various scientist that DNA or RNA is the genetic material of all organisms and that was supported by the following scientific evidence.

Key points:

  1. The nucleic acids DNA and RNA are long linear polymers that carry genetic information in a form of chemical sequence from one generation to another.
  2. Genetic information is stored in the sequence of nucleic acid bases along the chromosomes.
  3. Nucleic acid is a composite of four types of bases linked to sugar and phosphate as a backbone.
  4. RNA is a linear and single helix, while DNA contains complementary strands and forms a double helix.
  5. In the case of DNA both the strands are associated with chemical energy called a hydrogen bond.
  6. The nucleic acid especially DNA contains two strands, which are complementary to each other and forms a double-helical structure.
  7. DNA is synthesized by DNA polymerases; this process is called DNA replication. DNA replication provides a DNA copy to transfer it as genetic information to other cells or other generations.
  8. All types of RNA are found in all living cells, which are synthesized by RNA polymerases and that receive information from DNA templates, this process is called transcription and followed by translation. In translation, the synthesis of proteins occurred according to instructions provided by mRNA.  Thus, the flow of genetic information or gene expression takes place in normal cells.

1. Direct Evidence for DNA as Genetic Material

The most convincing and conclusive evidence to support the DNA as the genetic material given by different scientists using different experiments such as bacterial transformation, bacterial conjugation, and mode of infection by bacteriophages.

a. Transformation Experiments

The British bacteriologist, Frederick Griffith in 1928 discovered a phenomenon, now that we called as genetic transformation. As a part of the experiment, Griffith used two related strains of bacteria, known as R and S.

R strain: This is an avirulent strain (non-pathogenic) and that lacks the polysaccharide capsule. Hence, the bacteria appear like dull and rough (R) colonies when they grew on culture media.

S strain: This is a virulent strain (Diplococcus pneumoniae). This virulent bacterium that causes pneumonia was grown on nutrient agar. It grows like a smooth (S) glistering appearance due to the presence of a specific polysaccharide (a polymer of glucose and glucuronic acid) capsule.

DNA as Genetic Material Griffith Transformation Experiments
Figure: Griffith Transformation Experiments

The phenotypic trait such as presence or absence of polysaccharide capsule genetically determines the characteristic feature of the bacterium, either the strain is smooth (S) or rough (R). Both S and R forms are found in numerous types and are termed S-I, S-II, S-III, etc., and R-I, R-II, R-III, etc., respectively. Based on the antigen production, all these subtypes of S and R bacteria were classified. The type of antigen production is related to their genetic determination.

In general, smooth strains occasionally mutate to rough strains; however, this alteration has not been reversible. Considering this feature of the bacterium, in the course of his experiments, Griffith selected healthy laboratory mice and injected them with live R-II pneumococci. Later, he noticed that mice are still healthy, this is because R-II pneumococci were avirulent.

But, when he injected the live virulent S-III pneumococci into the mice, then the mice got sick due to pneumonia disease, and eventually, mice were dead. However, when mice were injected with heat-killed S-III bacterial strain, they did not exhibit any symptoms of pneumonia.

But, when he injected the mice with a combination of living avirulent R-II and heat-killed S-III virulent strain, then surprisingly, the symptoms of pneumonia appeared and mortality results became high.

The autopsy (sectioning) of the dead mice was revealed that the heart blood of the mice contained both R-II and S-III pneumococci. Considering these comprehensive results, Griffith finally concluded that the presence of heat-killed S-III bacteria in the combination must be involved in the transformation of virulence substrate (DNA) to living R-II bacteria to become a causative strain of pneumonia. Besides proving the DNA as genetic material, the possibilities of transformation between these two different strains have been clearly demonstrated by his experiment.

The obtained results were stated that the DNA which is responsible for capsule production has been transferred during the transformation and changed heat-killed S-III bacteria to living S-III bacteria from the R-II strain.

However, from this experiment, we can get some doubts that “how can DNA survive in heat-killed S-III bacteria?”. The best answer for this question is that “generally the DNA is a non-living chemical molecule that can be stable even at more than 900 C, but bacteria cannot survive at this temperature.

The process of gene transformation is called the “Griffith effect” and more popularly “bacterial gene transformation”. However, Griffith could not find the exact “transforming principle or substance” to understand the cause of bacterial transformation.

The same principle was initially observed by Oswald Avery, Colin MacLeod, and Maclyn McCarty in 1944. In their experiment, they partially purified the transforming substance (principle) from the large culture of the S-III bacterial extract (i.e., cell-free extract) and confirmed that it was DNA.

In their experiment, the scientists slightly modified the known protocols and isolated the DNA from S-III bacteria using different enzymes such as protease, lipase, and RNase (except DNase). Then the purified DNA was added to the live R-II bacterial culture.

After a period of time, the bacterial culture containing a substance of S-III was spread on the agar surface containing R-II strain, and the plates were incubated for few hours at room temperature.

Finally, there was bacterial growth and surprisingly, they found that the grown colonies are mixed of R-II and S-III strains (1 in 100).

To confirm if the transformation is a permanent genetic change, they again isolated few newly developed S-III colonies and grown them on a new agar surface. Finally, they found that the developed colonies were again related to the S-III type.

These experiments finally provided evidence that the purified trans-forming principle (substance) was DNA.  Moreover, during the experiment, the following observations also provided conclusive evidence:

  1. In their experiment, the chemical analysis showed that the major component involved in the disease development was deoxyribonucleic acid (DNA) not RNA.
  2. Physical measurements showed that the viscous nature of the bacteria culture (polysaccharide) is due to the presence of specific genes involved in polysaccharide synthesis.
  3. Experiments demonstrated that the transformation process was not inhibited by purified proteolytic enzymes (trypsin, chymotrypsin, or a mixture of both), ribonuclease (an enzyme that depolymerizes RNA), or lipase.
  4. However, the transforming process was completely disturbed with the treatment of the DNA-depolymerizing enzyme (DNAase).
  5. After this experiment, scientists were tried to identify this transforming substance (DNA) in all species of prokaryotes, (e.g., Hemophilus influenza, Shigella para-dysenteriae, Bacillus subtilus) and in eukaryotes.

b. Blender Experiment or Hershey-Chase experiment

The experimental results demonstrated by Avery and his colleagues were conclusive, however, many scientists were very opposite to accept DNA (rather than proteins) as the genetic material. Hence, the further experiment was performed by Alfred Hershey and Martha Chase in 1952. Through blender experiments, the well-evidenced and confirmed information was obtained in order to prove DNA as genetic material.

In this experiment, they have used a kitchen waring blender as a major piece of apparatus, hence the name, blender experiment. They found that the DNA injected by a phage particle into a bacterium contains all the information needed to synthesize the progeny of phage particles. In the phage particle T2, the DNA was enclosed by a protein shell. Since DNA is the only phosphorus-containing substance in the bacteriophage particle T2 (virus), while the protein shell is the only sulfur atoms containing substances (especially amino acids methionine and cysteine) in bacteriophage particle T2; in this experiment, they have used radio-active phosphate (32P) medium and radio-active sulfur (35S) medium to distinguish that which substance acts as genetic material (either protein or DNA). As a part of their experiment, initially, they have separated the phages into two groups and inoculated one group in radio-active phosphate (32P) containing medium and another group in radio-active sulfur (35S) containing medium. After sufficient time, they have isolated both newly formed phage particles (one group contains 32P in their DNA and group contains 35S in their protein) and used these particles to infect newly growing E. coli cells.

After some time, the mixer of bacteria and phage particles was agitated by using waring blender for injection to take place. Finally, to separate the bacterial cells from the phage ghosts, they used centrifugation and then measured the radioactivity in the two fractions. After separation, only radioactive 32P was found linked with bacterial cells, while, 35S was found only in supernatant but not in bacterial cells. It was concluded that, when the 32P-labeled phages were used, most of the radioactivity was ended up inside the bacterial cells but less in phage progeny, which is indicating that the phage DNA entered the cells. When the 35S-labeled phages were used, most of the radioactive material ended up in the phage ghosts but not in bacteria, suggesting that the phage protein never entered the bacterial cell. The conclusion is very clear: DNA is the hereditary material; the phage proteins are mere structural packaging that is discarded after delivering the viral DNA to the bacterial cell.

Hence, the Waring Blender Experiment is good validation. In which, the empty protein coat (ghost) was left outside. Thus, Hershey and Chase’s experiment proved that the DNA entering the host cell carries all the genetic information of phage particles, hence, DNA is the sole genetic material in DNA bacteriophages.

c. Bacterial Conjugation

Another convincing evidence for DNA is the genetic material was produced by Lederberg and Tatum in 1946. Their experimental phenomenon is currently called bacterial conjugation. In their experiment, they have found that when an F—(‘female’) E. coli cell conjugated with an F+ (‘male’) E. coli cells, the unidirectional transfer of F+ factor of ‘male’ cell to F— or ‘female’ cell took place. Thus, the latter, F— or ‘female’ cell was converted into an F+ or ‘male’ strain. The F+ factor was found to be an extracellular fragment of DNA molecule which laid in the bacterial cell cytoplasm.

2. Indirect Evidence for DNA as Genetic Material

If we think in a simple way, the indirect evidence for DNA as the genetic material is generally the higher organisms also contain DNA as their genetic material in the form of chromosomes. However, it was derived from smaller organisms. In higher organisms, manipulation of genetic material is not as easy as bacteria and viruses. To prove DNA only is the genetic material in higher organisms, a few of the supportive pieces of evidence have been given below:

  1. The Feulgen techniques have stated that DNA completely remains limited to the chromosomes and it is a major factor to form chromosomes.
  2. Various types of quantitative experiments on the amount of DNA in different cells have revealed that the amount of DNA determines the number of chromosomes in the cell, in the other hands, there is a correlation between the amount of DNA and the number of chromosome sets (ploidy) in the cell. Diploid cells have DNA, which is twice the haploid cells of the same species.
  3. The diploid amount of DNA is constant within the cells of the same species but differs from one species to another.
  4. In 1950 Swift (Department of Zoology, the University of Chicago) found the amount of DNA per latent nucleus is diploid (2n) and in the interphase nucleus (mitotically active tissues) is tetraploid (4n) but in the case of gametes it is haploid (1n), which means the normal human cell contains diploid chromosomes and when the cell enters in cell cycle, in the interface it contains tetraploid to give four haploid gamete cells. From this, he concluded that DNA duplication occurred during the interphase. This parallelism of behavior in DNA and chromosomes clearly demonstrated that DNA only is the genetic material even in higher organisms.

3. Evidence for RNA as Genetic Material of some Viruses

Various experiments have shown that some of the viruses contain ribonucleic acid (RNA) as their genetic material. One of the best experiments on the Tobacco Mosaic Virus (TMV) shown in the 1930s to be composed of protein and RNA. However, no DNA or its fragments were found in this virus particle. In 1956, Gierer and Schramm purified the RNA particle from this virus by using mild alkali treatment and analyzed their effect on tobacco plants. Interestingly, they have found that tobacco plants are significantly infected by inoculation with the RNA alone. This experiment revealed that RNA is the genetic material in some viruses.

References:

  1. Nucleic Acids as Genetic Material. Wiley Online Library. 19 April 2001
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  3. Avery, O. T., MacLeod, C. M., and McCarty, M. (1944). Studies on the chemical nature of the substance inducing transformation of Pneumococcal types.  Exp. Med.79(2), 137-158. Retrieved from.
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