DNA is a chemical substance that makes genes of every living organism from microorganism (bacteria) to higher organisms (humans). However, non-living organism such as virus contains DNA as genetic material. Universally, in around 1953, people accepted DNA as genetic material.
Nowadays, our modern science using DNA as fingerprint evidence for various practical claims, including paternity testing, forensic analysis, and genetic screening. Due to this characteristic feature of DNA, today many people are following the genetical analysis to clear their misconceptions.
After few decades, scientists were found that some viruses contain Ribonucleic acid (RNA) as the genetic material. The main concept of this article is that providing the information about DNA or RNA is the genetic material of most organisms and that was supported by the following scientific evidences.
- The nucleic acids DNA and RNA are long linear polymers carry genetic information in a form of chemical sequence from one generation to the another.
- Genetic information is stored in the sequence of nucleic acid bases along the
- A Nucleic acid is composite of four types of bases linked to a sugar-phosphate Backbone.
- RNA is linear and single helix, while DNA contains complementary strands and forms double helix.
- In case of DNA both the strands associated with chemical energy called hydrogen bond.
- The nucleic acid especially DNA contains two strands, which are complementary to each other and forms a double-helical structure.
- DNA is synthesized by DNA polymerases; this process is called DNA replication. The DNA replication provides the DNA copy to transfer the information to other cells or other generations.
- All types of RNA present in cells are synthesized by RNA polymerases that receives information from DNA templates, this process is called transcription and followed by translation, in which 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 EVIDENCES TO PROVE DNA AS GENETIC MATERIAL
The most convincing and conclusive evidences to support the DNA as the genetic material given by different scientist using the 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, today that we called as genetic transformation. As a part of experiment, Griffith used two related strains of bacteria, known as R and S.
S strain: This is a virulent strain. Colonies of this virulent bacterium, Diplococcus pneumoniae (pathogenic), that causes pneumonia was grown on nutrient agar, that grows as smooth (S) glistering appearance due to the presence of specific polysaccharide (a polymer of glucose and glucuronic acid) capsule.
S strain: This is a avirulent strain. This avirulent (non-pathogenic) bacterial strain lacks this polysaccharide capsule, hence they grow as dull and rough (R) colonies when grown on culture media.
The trait such as presence or absence of polysaccharide capsule genetically determines the characteristic of the bacterium either strain is Smooth (S) or rough (R). Both S and R forms found in numerous types and are termed as 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 separated from each other. The type of antigen produced is related to their genetical determination.
Interestingly, smooth (S) strains occasionally mutate to rough (R) strains, but this alteration has not been reversible. Considering this fact, in the course of his experiment, Griffith selected healthy laboratory mice and injected with live R-II pneumococci. After some time, he noticed that mice are still health, this is because of R-II pneumococci was avirulent. While, when he injected the 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 develop any pneumonia disease symptoms.
But, when he injected the mice with combination of living avirulent R-II and heat killed S-III virulent strain, then surprisingly the symptoms of pneumonia were developed and mortality results were high.
The autopsy (sectioning) of the dead mice was found that their heart blood 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 combination must be involved in the transformation of virulence substrate to living R-II bacteria to become a pneumonia causative strain. Besides proving the DNA as genetic material, the transformation process between two different strains really occurred or not has been clearly demonstrated by this experiment.
The obtained results proved as DNA that contains genes responsible for capsule production have been transferred during transformation, which occurred between heat-killed S-III bacteria to living R-II bacteria (activity of its own capsule producing gene was completely inhibited by mutations). From this experiment we can get some doubts how can DNA survive in heat killed S-III bacteria? the best answer for this question is DNA generally non-living chemical molecule that can survive even at more than 900 C but bacteria cannot survive at this temperature. The process of gene transfer was called “Griffith effect” or more popularly “bacterial trans-formation”. However, Griffith could not find the “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 large culture of the S-III bacteria cell extract (i.e., cell free extract) and confirmed that it was DNA. These scientists modified the known protocols for isolating DNA from S-III bacteria using different enzymes such as protease, lipase, RNase and DNase. Then the purified DNA was added to a live R bacterial culture. After a period of time, substance of S-III-containing R-II bacterial culture was spreader on an agar surface and incubated it few hours to form colonies. Finally, they observed the growth and surprisingly found the grown colonies contained few are related to S-III type (1 in 100). To conformed that transformation was a permanent genetic change, again they separated many of the newly developed S-III colonies and spreader them on a new agar surface and incubated them to grow. They found that the developed colonies were again related to S-III type. These experiments finally provided the evidence that purified trans-forming principle (substance) contain DNA. The following observations during experiment also provided the conclusive evidence:
- In their experiment, the chemical analysis showed that the major component involved in the disease development was deoxyribose sugar-containing nucleic acid (DNA).
- Physical measurement showed that the sample contained a highly viscous substance (polysaccharide) is due to presence of particular gene that codes for polysaccharide synthesis.
- Experiments demonstrated that transformation activity was not inhibited by reaction with either (a) purified proteolytic (protein degrading) enzymes trypsin, chymo-trypsin, or a mixture of both or (b) ribonuclease (an enzyme that depolymerizes RNA). The lipase also cannot inhibit the activity of transformation.
- However, the transforming activity was disturbed during treatment with material known as DNA-depolymerizing (DNAase) enzyme, that inactivated the transforming principle. Later on, transforming substance such as the DNA was found in a all type of prokaryotes including Hemo-philus influenzae, Shigella para-dysenteriae, Bacillus subtilus, etc. and in eukaryotic organisms.
b. Blender Experiment or Hershey-Chase experiment
The experimental results demonstrated by Avery and his colleagues were conclusive, however, many scientists were very reluctant 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 experiment, the well-evidenced and confirmed information was obtained in order to prove DNA as genetic material. In this experiment, they have used 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 progeny of phage particles. In the phage particle T2, the DNA was enclosed by protein shell. Since DNA is the only phosphorus containing substance in the bacteriophage particle T2 (virus), while, since protein shell is the only sulphur 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 sulphur (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 in two groups and inoculated one group in radio-active phosphate (32P) containing medium and another group in radio-active sulphur (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 were 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 radioactive32P 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 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, indicating that the phage protein never entered the bacterial cell. The conclusion is inescapable: 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 Blender Experiment is good validation. In which, the empty protein coat (ghost) left outside. Thus, Hershey and Chase experiment proved that DNA entering the host cell carries all the genetic information of phage particles, hence, is the sole genetic material in DNA bacteriophages.
c. Bacterial Conjugation
Another convincing evidence for DNA as the genetic material performed by Laderberg and Tatum in 1946. Their experimental phenomenon now days called as 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, latter, F— or ‘female’ cell was converted into a 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 EVIDENCES FOR DNA AS THE GENETIC MATERIAL
If we think in simple way, the indirect evidences 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 virus. To prove DNA only is the genetic material in higher organisms, some of the supportive evidences have been given bellow:
- The Feulgen techniques have stated that DNA completely remains limited to the chromosomes and it is major factor to form chromosomes.
- Various types of quantitative experiments on amount of DNA in different cells have revealed that amount of DNA determines the number of chromosomes in the cell, in other hand, 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 of haploid cells of the same species.
- The diploid amount of DNA is constant within the cells of same species but differs from one species to another.
- In 1950 Swift (Department of zoology, the university of Chicago) found the amount of DNA per latent nucleus is diploid (2n) and in interphase nucleus (mitotically active tissues) is tetraploid (4n) but in case of gametes it is haploid (1n), which means the normal human cell contains diploid chromosomes and when cell enters in cell cycle, in 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. EVIDENCES FOR RNA AS THE GENETIC MATERIAL OF SOME VIRUSES
Various experiments have shown that some of the viruses contains ribonucleic acid (RNA) as their genetic material. One of the best experiments on 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 significantly infected by inoculation with the RNA alone. This experiment revealed that RNA is the genetic material in some viruses.
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