In this article we will discuss about Genetic Material:- 1. Properties of Genetic Material 2. Evidence of Genetic Material.
Properties of Genetic Material:
A living cell is composed of several inorganic and organic components. Among them, one will obviously act as genetic material responsible for controlling hereditary characters. Identification of this genetic material remained controversial for a long time.
Now if any component is to be genetic material, it must fulfil a number of basic properties:
i. Genotypic function or replication or auto-synthesis.
ii. Phenotype function or expression or hetero catalysis.
iii. Mutation.
The first property states that the genetic material must be capable of storing hereditary information and replicate with high efficiency in successive cell generations forming the basis for transmission of hereditary characteristics it controls.
The second property is a fundamental one involved in gene action which through a series of chemical reactions results in the ultimate expression of the characteristics within the organism. The third property states that the genetic material does undergo occasional heritable changes called mutation.
It creates variations among the organisms besides recombination. Variations, on the other hand, are the important source of raw materials for evolution.
Besides the above-mentioned important properties of genetic material, the gene substance also shows the following additional properties:
a. To control the innumerable diversities in the characteristics of organism available in nature, the genetic material must show a very wide diversity in form.
b. Since phenotype character is the final expression of a chain of reactions initiated at the gene level, obviously the genetic material must be a chemically unique entity.
Before 1900 several biologists proposed that hereditary material must be in the chromosome of the cell nucleus. In 1903, Sutton and Bovery postulated that genes were located in chromosome. In eukaryotic system, chromosomes are made of mainly protein and nucleic acid (DNA and RNA) and one of them obviously constitute the genetic material.
But which one would be the most suitable candidate for the position of genetic material remained controversial for a long time.
Early molecular biologists have assigned the properties of genetic material to the chromosomal proteins because they found nucleic acid too simple to carry genetic information. Besides this, nucleic acid is made of monotonous chemical components like sugar, phosphate and base.
On the other hand, protein showed a highly complex structure composed of a variety of amino acid. So a wide range of diversities is possible in protein structure to fulfill the diversity required in the genetic material for controlling the countless diversities in the characteristic of organism.
The controversy about the assignment of gene substance either to chromosomal protein or to nucleic acid, existed up to 1950 when finally it was unanimously accepted that the genetic information resides in the nucleic acids rather than in proteins.
More specifically, several elegant experiments showed that DNA is the genetic material of most microorganisms and higher organisms. Later on, RNA was found to be the genetic material of some viruses where DNA is absent.
Evidence of Genetic Material:
The concept that DNA or RNA is the genetic material of most organisms has been developed and supported by following evidence:
i. Direct evidence;
ii. Indirect evidence.
i. Direct Evidence:
(a) Transformation in Pneumococcus:
The first direct evidence showing that the genetic material is DNA rather than protein or RNA was published by O. T. Avery, C. M. Macleod and M. McCarthy in 1944. They discovered that the substance of the cell responsible for the phenomenon of transformation in the bacterium Diplococcus pneumoniae is DNA.
Transformation is the mode of exchange or transfer of genetic information (recombination) from one strain of bacterium to another strain of bacterium without involving any direct contact between them. The process of transformation was first discovered by Frederick Griffith in 1928.
This was called as Griffith’s effect. The experiment of Griffith demonstrated transformation but he could not recognise the transforming principle.
Different strains of Pneumococci shows the genetic variability that can be recognised by existence of different phenotypes. Griffith initially conducted his experiment on two strains of pneumococci which were phenotypically distinct.
When they are grown artificially on nutrient agar medium, they form two types of colonies:
(1) Smooth and
(2) Rough.
The cells of strains forming smooth (S) colonies have a smooth glittering appearance due to presence of strain-specific polysaccharides (a polymer of glucose and glucuronic acid) capsule. Such strains are able to produce pneumonia and are called virulent.
The polysaccharide capsule is required for virulence since it protects the bacterial cell against phagocytosis by leucocytes. But the cells of stain lack this capsule and they produce dull rough (R) colonies. Such stains are termed as avirulent since they cannot produce pneumonia.
Therefore smooth (S) and rough (R) phenotypic characteristic are directly related to the presence or absence of the capsule and this trait is known to be genetically determined.
Both S and R forms occur in several subtypes and are designated as S I, S II, S III, etc. and R I, R II, R III, etc., respectively, on the basis of antigen properties of the polysaccharides present in their capsule. This property ultimately depends on the genotype of the cell.
The experiments of Griffith (Fig. 12.1) are briefly described below stepwise on the basis of his observation:
Step I:
Griffith injected live cells of the virulent type III S into mice, all the mice died due to pneumonia and live type III S cells were recovered from the serum of blood of the dead bodies of mice.
Step II:
When live cells of the avirulent type II R were injected into a separate group of mice, none of the mice died and live type II R were isolated from the serum of blood of all mice.
Step III:
When mice were injected with heat-killed virulent type III S pneumococci alone, again none of the mice died, showing that virulence is lost after heat-killing.
Step IV:
When mice were injected heat-killed type III S pneumococci (virulent when alive) plus live type II R pneumococci (non-virulent), some of the mice died due to pneumonia; pneumococci cells isolated from the dead mice were of the type III S.
Since it is known that non-capsulate type R cells can mutate back to virulent encapsulated type S cells, the resulting cell will be type II S, not type III S. Thus the transformation of non- virulent type II R cells to virulent type III S cells cannot be explained by mutation, rather, some component of the dead type III S cells (the “transforming principle”) must convert living type II R cells to type III S.
This leads to a change in the trait of cells and helps to bring some new characters in the transformed cell. Hence the transforming principle must contain some genetic material.
(b) Transforming Principle is DNA:
Avery, Macleod and McCarthy experimentally proved that the transforming principle was DNA. They showed that if DNA extract from type IIS pneumococci was mixed with type IIR pneumococci in vitro, some of the pneumococci were transformed to type III S.
But DNA extract from type III S may be contaminated with a few molecules of proteins, RNA and this contaminating protein and RNA may be responsible for transformation from type II R to type III S. So Avery, Macleod and McCarthy demonstrated the most definitive experiment using bacterial culture system and specific enzymes that degrade DNA, RNA and protein.
In separate experiments (Fig. 12.2) DNA extract from type III S cells was treated with:
i. DNAse which degrades DNA.
ii. RNAse which degrades RNA.
iii. trypsin, a protease which degrades protein; and then tested the treated DNA extract for its ability to transform type II R pneumococci to type III S.
iv. They observed that the treatment with RNAse or trypsin had no effect on the ability of the DNA extract to transform type II R to type III S. But DNAse treatment destroyed the transforming activity of the DNA preparation and II R cells were not transformed into III S cells. This established beyond any doubt that DNA is the transforming principle.
But these findings of Avery and co-workers was not able to explain the molecular mechanism of transformation. So some biologists were unable to appreciate the significance of these findings and they were hesitant in accepting them as an incontrovertible evidence for DNA being the genetic material.
(c) The Experiment of Hershey-Chase:
Another direct evidence indicating that DNA is the genetic material was demonstrated by A. D. Hershey and M. Chase in 1952. They first studied the life cycle of T2 bacteriophages of Escherichia coli. T2 bacteriophages are composed of hexagonal box-like head coat and tail made of protein. The DNA is packed inside the proteinaceous head coat.
Bacteriophages are acellular and do not contain cytoplasm, organelles and nucleus. The DNA is present in high pure form and is not associated with RNA and protein. Bacteriophage are obligate parasite since they can reproduce only within bacterium using as host cell.
Hershey and Chase showed that, during the reproduction of bacteriophages, the DNA of the phage entered the host cell whereas most of the protein head and tail portion remained absorbed on the outside of the cell. Hence it is strongly implied that the genetic information necessary for viral reproduction was present in DNA.
DNA contains phosphorus (P) but no sulphur (S), while proteins of head and tail contains sulphur (S) but no phosphorus.
Hershey and Chase were able to specifically label the phage DNA by its growth in a medium containing the radioactive isotope of phosphorus, i.e., 32P in place of normal phosphorus. Similarly, in another group of phage, the protein coats were labelled by growth in a medium containing radioactive sulphur 35S in place of normal sulphur.
E. coli cells were then infected with 32 P labelled T2 bacteriophage and, after being allowed 10 minutes for infection, they were agitated in a blender which sheared off the phase coats. The phase coats and the infected cells were then separated by centrifugation (Fig. 12.3).
Radioactivity was then measured of the sediment and in phage coat suspension. Most of the radioactivity was found in the cells. When the same experiment was done using phage with 35S-labelled protein coat, most of the radioactivity was found in the suspension of phage coats; very little entered the host cells.
Since phage reproduction (both DNA synthesis) occurs inside the infected cells, and, since only the phage DNA enters the host cell, the DNA—not the protein—must carry the genetic information. As a result of the findings of Hershey and Chase led to the universal acceptance of DNA as the genetic material.
(d) Bacterial Conjugation:
Another direct evidence for DNA as the genetic material comes from the phenomenon of conjugation of bacteria. Conjugation was discovered by J. Lederberg and E. I.
Tatum in 1946. During conjugation DNA is transferred from a donor bacterial cell to a recipient bacterial cell through conjugation tube that forms between them. The donor cell—also called male—contains a F factor or fertility factor whereas recipient cells—or female cells, lack F factor, i.e., F– cell (Fig. 12.4).
In male, the F factor can exist in two different states:
(1) Autonomous state and
(2) Integrated state (Fig. 12.4) where the F factor is inserted with main DNA and thus the male become Hfr male (Fig. 12.5).
The F factor is a mini-circular DNA molecule. Beadle and Tatum observed that when a F+ male E. coli cell conjugated with a F– female E. coli cell, an unidirectional transfer of F+ factor of male cell to F– or female cell took place, so that the latter was covered into a F+ or male strain.
The F factor is actually a fragment of DNA molecule that replicates during transfer. Thus mixing a population of F+ or Hfr cells with a population of F+ cells results in virtually all the cells in the new population becoming F+ or Hfr (Fig. 12.6).
ii. Indirect Evidence:
The fact that DNA is the genetic material of higher organisms has also been supported by some indirect evidences:
(a) Localisation:
The genetic substance should have a fixed location within the cell. If it has no fixed location, then the genes are not able to function properly. It is known that the DNA, as a gene substance, is always located primarily within the chromosome in the nucleus of the eukaryotic cell.
The specific location of DNA can be studied in situ by the Feulgen reaction—which is regarded as the most specific one for DNA. Feulgen staining stains chromosome magenta colour against the clear cytoplasmic background. This technique has shown that DNA entirely remains restricted to the chromosome and it forms the major component of chromosomes.
(b) Stability:
Various macromolecules present within the cell are continuously being anabolised and catabolized. But this is not desirable for a genetic substance containing valuable hereditary information. If it happens, the genetic function will be lost. Of all the macro- molecules in the cell, DNA is the metabolically stable.
(c) Sensitivity to Mutagens:
Mutation is an important characteristic feature of the genetic material. The agents capable of inducing mutation are called mutagens. Different types of radiation (UV-ray, X-ray, y-ray) and a variety of chemical compounds acts as mutagens. When the cells of an organism are treated with mutagens, they cause a change in the structure of gene.
Since genes are DNA segments, the gene mutation include changes in the number and arrangement of nucleotide. Sometimes mutation causes the breaks in the DNA molecule. The changes in the DNA structure ultimately reflect the changes of the organism’s hereditary character. Therefore sensitivity of DNA to mutagens is an indirect evidence for DNA being the genetical materials.
(d) DNA Content:
One of the striking features of the genetic material is the correlation between DNA content and the number of chromosome sets. Various quantitative assay methods have shown that diploid cells contain twice as much DNA as do haploid cells of the same species (Table 12.1).
Similarly, tetraploid and octaploid cells contains four times and eight times DNA as compared with DNA content of the haploid cells. Even the DNA content of sperm cells shows a correlation with the same or different tissues of different organisms (Table 12.2).
Thus the parallelism of behaviour in DNA and chromosome indirectly indicates that DNA is the genetic material.
(e) RNA as Genetic Material:
The genome of viruses may be DNA or RNA. Most of the plant viruses have RNA as their hereditary material. Fraenkel-Conrat (1957) conducted experiments on tobacco mosaic virus (TMV) to demonstrate that in some viruses RNA acts as genetic material.
TMV is a small virus composed of a single molecule of spring-like RNA encapsulated in a cylindrical protein coat. Different strains of TMV can be identified on the basis of differences in the chemical composition of their protein coats. By using the appropriate chemical treatments, proteins and RNA of RNV can be separated.
Moreover, these processes are reversible by missing the protein and RNA under appropriate conditions—reconstitution will occur yielding complete infective TMV particles. Fraenkel-Conrat and Singer took two different strains of TMV and separated the RNAs from protein coats, reconstituted hybrid viruses by mixing the proteins of one strain with the RNA of the second strain, and vice versa.
When the hybrid or reconstituted viruses were rubbed into live tobacco leaves, the progeny viruses produced were always found to be phenotypically and genotypically identical to the parental type from where the RNA had been isolated (Fig. 12.7). Thus the genetic information of TMV is stored in the RNA and not in the protein.