The following points highlight the top four experimental evidences for DNA as genetic material. The experimental evidences are: 1. Transformation 2. Bacteriophage Infection 3. Transduction 4. Biochemical Analysis.
Experimental Evidence # 1. Transformation:
The genetic recombination in which naked DNA from one cell can enter and integrate in another cell is referred to as genetic transformation. Experiments on genetic transformation were conducted by Griffith which support that DNA is the genetic material.
These experiments are discussed as follows:
Griffith’s Experiment :
Mice and pneumonia causing bacteria Diplococcus pneumoniae.
The virulent strain causes pneumonia and death of mice, whereas avirulent strain does not cause pneumonia. The experiment is based on the principle of genetic transformation.
There are two types of strains in above bacteria, viz. virulent with smooth surface [having coating of polysaccharide] designated as SIII; and avirulent with rough surface [without coating] designated as RII.
The virulent strain causes pneumonia and death of mice, whereas avirulent strain does not cause pneumonia. Griffith injected strains of Diplococcus pneumoniae bacteria into mice in following four combinations and recorded the results.
i. Virulent strain SIII which causes pneumonia.
ii. Avirulent strain RII which does not cause pneumonia.
iii. Heat killed virulent strain SIII.
iv. Mixture of heat killed virulent strain SIII + avirulent strain RII.
The results suggest that the chemical from the heat killed bacteria transformed the avirulent bacteria into virulent which caused death of the mice. However, Griffith could not conclude that the transforming chemical was DNA.
Avery, Macleod and McCarty Experiment :
They used pneumonia causing bacteria Diplococcus pneumonia. These experiments provided the first evidence that DNA was the genetic material.
The extract from virulent strain will lead to transformation.
Avery, Macleod and McCarty isolated DNA from virulent strain (SIII) and applied the same in culture medium of avirulent strain [RII] in following four combinations and recorded the results.
(i) DNA extract from heat killed virulent strain SIII which contains all the components i.e. DNA, RNA and proteins.
(ii) DNA extract from heat killed virulent strain SIII treated with DNAase enzyme which digests DNA, so there are RNA and proteins but no DNA.
(iii) DNA extract from heat killed virulent strain SIII treated with RNAase enzyme which digests RNA, so there are DNA and Proteins but no RNA.
(iv) DNA extract from heat killed virulent strain SIII treated with protease enzyme which digests proteins, so there are DNA and RNA but no protein.
They concluded that DNA was the genetic material and not the proteins. Because the transformation occurred when DNA was present in the extract and there was no transformation when DNA was digested with DNAse enzyme.
Experimental Evidence # 2. Bacteriophage Infection:
Another evidence in support of DNA as the genetic material comes from the study of bacteriophage infection. Bacteriophages or phages are viruses which attack bacteria and kill them. The phage has two parts, viz. tail and head. The tail is composed of protein only. The head has outer coat of protein and inner core as DNA.
The phage infects bacteria by attaching its tail to the body of the bacteria. The DNA enters the bacterial cell and multiplies inside and protein portion remains outside the bacterial cell. After replication, phages come out of the bacterial cell and make the outer coating their own.
Hershey and Chase Experiment :
Hershey and Chase conducted their experiment with T2 phage of E. coli to find out whether DNA or protein is the genetic material of bacteriophages. They grew one population of phages in a medium containing radioactive sulfur, which radioactively labels the phage proteins. Another population of phages was grown in radioactive phosphorus, which labels the phage DNA.
The labelled component which will transfer the radioactivity to the next progeny is expected to involve in the transformation process.
They developed radioactive strains of T2 phage. In one sample radioactive phosphorus [P32] was used to label phosphorus which is component of DNA [not found in protein]. In another sample radioactive Sulphur [S35] was used to label sulphur which is component of protein [not found in DNA].
These DNA labelled and protein labelled T2 phages were used to infect two different samples of normal bacteria in culture medium. The newly multiplied viruses in the normal bacteria were examined for radioactivity.
It was observed that only the phages which were labelled with radioactive phosphorus [P32] transmitted radioactivity to their daughter pages. This provided a strong evidence that DNA is the genetic material and not the protein. They concluded that the genetic-material must be DNA, carrying the radioactivity inside the bacteria and to offspring phages.
Experimental Evidence # 3. Transduction:
The genetic recombination in bacteria in which DNA is transferred from one bacterial cell to another via the bacteriophage is known as transduction. Bacteriophages attack the bacteria, lyse them and multiply inside the bacteria. In this process, sometimes a small DNA segment of bacteria is contained by the bacteriophage.
When this bacteriophage attacks other bacteria, the DNA of previous bacteria from bacteriophage is integrated with the DNA of new bacteria and changes some of the features of new bacteria.
Transduction is of two types, viz. general and specialized. When any part of donor DNA is transferred to the recipient bacteria, it is known as general or unrestricted transduction. When a segment of donor DNA replaces a corresponding segment of host DNA, it is called complete transduction.
Sometimes donor DNA fails to recombine with host DNA, it is known as abortive transduction. When bacteriophage is able to transduce only a restricted portion of host DNA, it is referred to as restricted or specialized transduction. Transduction has been reported in several bacteria such as E. coli, Pseudomonas, Salmonella, etc.
Experimental Evidence # 4. Biochemical Evidences:
There are several biochemical evidences which support that DNA is the genetic material.
Some such evidences are briefly presented below:
1. The amount of DNA in the somatic tissues differs from species to species, but is constant for a species and cannot be altered by change either in the external or internal environment of the organism. The amount of DNA is also constant per diploid cell of an organism (Alfred Mirsky).
2. The amounts of each of the four nucleotide bases vary considerably from species to species, but the DNA of any given species contains equal amounts of adenine and thymine and equal amounts of cytosine and guanine (Chargaff).
3. The germ cells of higher plants and animals are haploid with only one set of chromosomes. Such cells contain half of the amount of DNA present in the somatic cells of the same species.
4. The amount of DNA in a species varies as per the ploidy level. The tetraploid will contain double the amount of DNA than diploid species.
5. The DNA is predominantly present in the chromosomes. This also suggests that DNA is the genetic material.
6. Some amount of DNA is also found in the cytoplasm especially in the chloroplasts and mitochondria. Such DNA controls the inheritance of cytoplasmic genes, because some characters exhibit cytoplasmic inheritance. Thus, biochemical estimation of DNA in various tissues and ploidy levels provides additional support for ‘DNA as the genetic material.
Denaturation and Renaturation:
The process of breaking of hydrogen bonds between the strands and separation of two strands on heating of DNA molecule is called denaturation. The DNA in disrupted state is known as denatured. Denaturation occurs at 100 Degree Celsius.
The process by which denatured DNA is changed into original DNA is called renaturation and reformed DNA is known as re-natured DNA. The cooling of denatured DNA gradually leads to renaturation. The optimum temperature for renaturation is 20-25 degree Celsius. The denaturation and renaturation ability of DNA is useful for developing hetero-duplexes.