Contents
Biology Topics related to disease and health provide critical insights into human physiology and medicine.
The Genetic Material – Discovery of DNA & RNA as the Hereditary Material
The study of genes, genetic variations, and heredity in living organisms, is considered as genetics. It is generally considered a field of biology that is strongly linked with the study of information systems. The molecular inheritance mechanism of genes is still the primary principle of genetics. The field of modern genetics has expanded beyond inheritance in studying the function and behaviour of genes. Within the context of the cell of an organism, its gene structures, functions, variations, and distributions are studied. The molecular basis for genes is DNA which is composed of chains with 4 types of nucleotides – Adenine (A), Cytosine (C), Guanine (G), and Thymine (T). The genetic information exists in the sequence of these nucleotides and genes exist as stretches of sequences along the DNA chain.
Search for Genetic Material
Long before the time of Mendel, people had observed the physical resemblances between parents and their offspring in many cases and realized that something might have been transmitted from parents to their progenies. Mendel perceptively described the transmission of “Characters”, but he did not know their chemical composition. Today we can visualize the genetic material with the help of powerful microscopes.
The search for the chemical identity of genetic material began about a century ago. Swiss biochemist Friedrich Miescher in 1869 for the first time, isolated the nucleus of white blood cells obtained from pus and chemically analyzed the contents of a cell’s nucleus by his own method. Miescher called it ‘nuclein’ in his 1871 paper. In the nucleus, he discovered an unusual acidic substance containing nitrogen and phosphorus. In later years nuclein was called nucleic acid.
In 1909, Levene deciphered the structure of nucleic acids. He identified the 5-carbon sugar ribose as part of some nucleic acids and in 1929, he discovered a new, similar sugar, deoxyribose in other nucleic acids. Levene then discovered that the three parts of a nucleic acid – a sugar, an N2-containing group, and a phosphorus-containing component occur in equal proportions. Levene revealed a major chemical distinction between RNA (ribonucleic acid) and DNA (deoxyribonucleic acid), which are the two types of nucleic acids.
In later years, nucleic acid was found to be associated with various proteins, mostly histone, and protamine, in combinations called nucleoproteins. When the nucleic acid was separated from the protein, Levene (1925) showed that nucleic acid could be broken down further into smaller units called nucleotides. Around the year 1935 Alexander Todd confirmed Levene’s work and synthesized DNA building blocks. In 1950, Erwin Chargaff, by chemical analysis showed that DNA contains equal amounts of purine to that of pyrimidine. Next in early 1953, Maurice Wilkins and Rosalind Franklin by using the X-ray diffraction technique showed the structural pattern of DNA molecules that revealed the repeating structure of building blocks. Finally in 1953, James Watson and Francis Crick first unrevealed the unique structure of DNA to build a replica of the DNA molecule using ball-and-stick models that showed that the genetic material consists of long strands of DNA that entwine to form a double helix, which resembles a twisted ladder.
Crick pointed out that ‘‘a genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way”. Now, the questions arise where exactly is DNA located and what is the relationship between DNA and chromosomes? Chromosomal theory of heredity strongly revealed that the chromosomes are transmitted from parental generation to filial generation through sexual reproduction. An individual has both paternal chromosomes and maternal chromosomes in equal numbers.
The events of cell division and reproductive cycles indicate that chromosomes are the carriers of genetic material for four significant reasons:
- Chromosomes duplicate and divide equally in mitosis furnishing each daughter cell with a full complement of chromosomes.
- Chromosomal behaviour in meiosis reflects the principle of heredity that it is due to contributions from both parents.
- Crossing over between homologues during meiosis provides a source of variation in individuals.
- Chromosomes and their aberrations are associated with the inheritance of specific characteristics.
It becomes, therefore, a logical corollary to know the composition of chromosomes and also to identify and isolate the genetic material from the chromosomes, if they carry the genetic material. Mirsky and Ris in the 1950s showed the chemical composition of chromosomes by isolating chromosome material from the calf thymus gland using biochemical methods. They showed that the chromosomes consist of two fractions: major – 90% and minor – 10%. The major fraction consists of 45% DNA and the remainder are histone proteins. The minor or “residual” chromosome fraction has 1.5 – 2.6% DNA, 7.5 – 14% RNA, and about 80% heavy protein.
Various quantitative studies on DNA were made by isolating DNA from the nucleus and one of the important results was “constancy” of DNA amount in all diploid tissues of a species. Another result showed that the amount of DNA involved in the gametes is as expected of the genetic material of meiotic products. Results also showed that the number of chromosomes and amount of DNA remains unchanged even against adverse conditions. These facts strongly support the genetic role of DNA.
Of all the characteristics that distinguish the living from the non-living, the one that is most important for the continuance of life is the ability to reproduce. At the cellular level, reproduction occurs by duplication of a cell be it a single-celled organism or a cell lining of the intestine. At the molecular level, reproduction depends upon a biomacromolecule that has dual abilities: to direct the specific activities of that cell and to produce an exact replica of itself. Today it has been well established that deoxyribonucleic acid or DNA is a multifunctional molecule that can replicate and orchestrate cellular activities by encoding and controlling protein synthesis.
DNA as Genetic Material
The cellular material capable of self-replication with the power of storage of information for life and also the power of transferring the information from parent to progeny is the genetic material. In living organisms, DNA carries such a property. However, in some viruses, RNA also acts as genetic material (e.g., TMV, HIV, and other retroviruses). The fact that DNA acts as genetic material is supported by several direct and indirect evidence. So far there are two experimental pieces of evidence available as direct evidence in support of this. One is concerned with bacterial transformation and the other is related to viral reproduction in bacterial cells.
Properties of Genetic Material
The properties of the genetic material show-
1. Universality:
The universality of genetic material is manifested by its presence in almost all types of organisms, starting from viruses to higher animals and plants.
2. Variability:
Lives on earth are characterized by variations. Therefore, genetic material should have the potency to show variation in its organization. Four different nitrogenous bases, i.e., A, T, G, and C appear to be the constituting variable bases in DNA. These bases in DNA may exhibit innumerable variations in their sequence of arrangement. Thus DNA has enormous potential to develop variation.
3. Autocatalytic Property:
The power of replication of a chemical substance is known as autocatalysis. DNA possesses this autocatalytic property and prior to cell division, the amount of DNA present in a cell is doubled by replication. A genetic material should contain this autocatalytic property.
4. Heterocatalytic Property:
DNA can not only show self-replication, but it can also help in the synthesis of other materials needed for the expression of phenotypes. Such a property is known as hetero-catalysis. With the help of hetero-catalytic properties, DNA promotes the production of RNA and proteins. Such a property of DNA is supportive of its becoming the genetic material.
5. Mutation:
Various features of character are characterized by change and such changes are relative to the structural changes of DNA. Various environmental agents as X-rays, UV-rays, chemical mutagens, etc., can alter the constitutional variation of DNA causing mutation. Hence, the property of DNA to be mutated is also supportive of DNA as genetic material.
DNA as Molecular Basis of Inheritance
Quantity of DNA:
Every body cell of the living organism is the same. The body cells are descendants of a single cell and they carry the same amount of DNA as present in the originating single cell. Further, the gametes of the organism carry half the amount of DNA present in the original cell. This unique distribution of DNA in all types of living species makes genetic material.
Artificial Gene:
Artificially a gene may be constructed by combining the nucleotides. Not only that, the artificially synthesized gene also functions inside a living body. Artificially synthesized genes represent a sequence of DNA. Therefore, the probability of DNA being genetic material is more.
Bacterial Transformation and Evidence in Support of DNA as the Genetic Material
Transformation
The mechanism by which one bacterial strain, by taking up free DNA from the external medium may be transformed into another strain, is known as transformation.
1. Experiment of Griffith:
In 1928, Frederick Griffith carried out experiments on mice and two strains of Diplococcus pneumoniae, which proved that one bacterial strain may be converted into another strain. The two strains of D. pneumoniae Griffith took, were D. pneumoniae strain Sill and D. pneumoniae strain RII. The Sill bacteria are virulent and may cause pneumonia in mice, whereas the RII strain of bacteria is non-virulent and cannot cause pneumonia. In an experiment, he injected some mice with heat-killed Sill bacteria and found that the rats remained alive. In the same way, inoculation of RII bacteria in mice failed to develop pneumonia. Next, he mixed the heat-killed Sill bacteria and live RII bacteria together and inoculated the mixture in the mice. As a result, he found that the mice developed pneumonia and died.
Inference: Having seen this he came to the conclusion that in the presence of heat-killed Sill D. pneumoniae, the RII bacteria were transformed into Sill type. Griffith called this conversion of a strain a transformation.
Differences between S III and R II Strain of Diplococcus Pneumoniae:
| SIII Type D. Pneumoniae | RII Type D. Pneumoniae |
| 1. Presence of a cell coat or capsule outside the cell. | 1. No cell coat or capsule is present. |
| 2. Colony appears smooth. | 2. Colony appears rough. |
| 3. Capable of causing pneumonia, so virulent. | 3. Incapable of developing pneumonia, so nonvirulent in nature. |
2. Experiment of Avery, MacLeod, and McCarty:
Following the experiments of Griffith, in 1944 three scientists Avery, MacLeod, and McCarty designed a protocol for experiment and their experiments as per protocol could prove that DNA is the genetic material.
Experiment:
- The Sill type bacteria taken from a liquid culture were killed with the application of heat and DNA was isolated from the bacteria.
- The DNA obtained was inoculated in a solid culture of RII bacteria. This resulted in transformation and developed Sill bacteria in the culture.
- Then they treated the DNA of Sill bacteria with RNase to remove any contamination of RNA in the DNA obtained. This treated DNA was inoculated in the RII culture on the Petri dish. They
- found that Sill bacteria were also developed this time.
- They also treated the DNA of Sill bacteria with protease to remove any protein contamination and inoculated the treated material in the culture of RII bacteria as before. The development of Sill bacteria was observed this time too.
- The DNA was then treated with DNase that disintegrated the DNA material and now the treated material was inoculated into the RII culture. This time no Sill bacteria was developed on the culture plate, indicating that in the absence of DNA of Sill, no transformation became possible.
Inference: Avery, MacLeod, and McCarty came to the conclusion that DNA acted as a transforming principle, i.e., DNA is the genetic material.
3. Experiment on Viral Reproduction:
A.D. Hershey and M. Chase in 1952 carried out one experiment with T2 bacteriophage and E. coli and their experimental observation conclusively proved that DNA is the genetic material. For this experiment, they were awarded Nobel Prize in 1969. The experiment of Hershey and Chase was based on the principle that DNA contains phosphorus but lacks sul¬phur, and protein contains sulphur but does not contain phosphorus.
Experiment:
- Two groups of T2 bacteriophages were labeled with radioactive 32P and 35S separately. In the case of labelling with 32P, only the DNA of the phages was labelled, whereas in the case of labelling with 35S only protein capsids were labeled.
- In one experimental set up the 32P labelled phages were allowed to infect the E. coli cells. After a few minutes, the inoculated culture of bacteria was agitated to separate the virus and E. coli cells. Following autoradiography it was found that most of the radioactivity entered in the bacterial cells. The usual bacterial lysis occurred with the production of progeny phages.
- In a second experiment, the 35S labelled phages were allowed to infect some E. coli cells and after a few minutes, the culture was agitated with a blender to separate the bacteria and the virus particles. Following autoradiography it was found that the radioactivity was retained outside the bacterial cells. In this case, also lysis of the bacterial cells occurred with the production of progeny phages.
Inference: From the observations of their experiments they came to the conclusion that during infection the virus injects its DNA into the bacterial cell and the protein capsid is left outside. DNA only can promote the production of complete progeny phages containing DNA and protein capsid. Therefore, DNA acts as the genetic material in the T2 phages.
4. Evidence from Genetic Engineering:
At the present state of advancement of science with the use of techniques of genetic engineering specific genes in the form of DNA fragments may be introduced in an organism to produce transgenic organisms. The transgenic organism may produce a desired product of our interest. For example, transgenic bacteria now produce human insulin, GH, and interferon. The transgenic mice would be produced by introducing the human growth hormone (GH) gene in the fertilized egg of mice.
Differences between Transformation and Transduction:
| Transformation | Transduction |
| 1. Introduction of free DNA into a bacterium from the medium promoting change of character. | 1. Introduction of DNA into a bacterium mediated by a virus with the change of character. |
| 2. The DNA that enters a bacterium remains in the culture medium of the bacteria. | 2. The DNA that enters a bacterium remains in the viral genome in integrated condition. |
| 3. The bacterium can take up DNA in competent condition. | 3. The bacterium gets the DNA when infected by a virus containing a specific gene. |
Differences between Transformation and Transgenesis:
| Transformation | Transgenesis |
| 1. Natural process. | 1. Artificial process. |
| 2. Usually occurs in prokaryotes as bacteria. | 2. It may be achieved both by the use of prokaryotes and eukaryotes. |
| 3. No special direction in occurrence. | 3. Happens as per our desire. |
| 4. Usually more than one gene is introduced. | 4. Normally one gene is introduced. |
RNA as the Genetic Material
Some viruses do not contain DNA. But internally they contain one RNA and their body is formed of RNA and protein capsid. Such RNA-containing viruses are called reovirus. Tobacco mosaic virus (TMV) and Human Immuno Deficiency virus (HIV) are the two RNA-containing viruses. In these viruses, RNA acts as genetic material. H. Frankel Conrad and B. Singer carried out an experiment on TMV and proved that TMV RNA serves as the genetic material. They collected two types of TMV and observed that the virus particles differ by their protein capsids.
Experimentally, they could have produced two types of reconstituted virus, when RNA from one type of virus was made to be enclosed with the protein capsid of another type of virus. They found that when proteins from one type of virus were mixed with RNA from another type of virus, then the RNA might get enclosed into proteins obtained from different viruses. Therefore, new virus particles produced in the laboratory carried RNA and proteins of dissimilar categories. Now with the use of a reconstituted virus, they developed an infection on tobacco plants and observed that progeny virus particles produced following such infection carried RNA and protein capsid of compatible category. From this experiment, Frankel Conrad and B. Singer could be able to prove that RNA was the genetic material in TMV.