In this article we will discuss about Modern Concept of Gene:- 1. Introduction to the Modern Concept of Gene 2. Definition of Genes 3. The Gene as a Unit of Function (Cistron) 4. Gene as the Unit of Recombination (Recon) 5. The Gene as a Unit of Mutation (Muton) 6. Identification of Genetic Material.
- Introduction to the Modern Concept of Gene
- Definition of Genes
- The Gene as a Unit of Function (Cistron)
- Gene as the Unit of Recombination (Recon)
- The Gene as a Unit of Mutation (Muton)
- Identification of Genetic Material
1. Introduction to the Modern Concept of Gene:
Mendel while explaining the result of his breeding experiment pointed out that the hereditary characters were governed by some particulate genetic determiners present in the germ cells. The genetic determiners of heredity characters are now called ‘genes’. The term gene was coined by Johannsen (1909). De Vries used the term ‘pangen’ and perhaps, from this word the term gene was derived.
In the old books of genetics the term ‘gene’ is treated as merely something which affects the phenotype and behaves as a particle. It should be noted here that nothing was said about the size, shape and chemical constitution of gene in old definitions.
After the rediscovery of Mendelian laws of heredity, the cytology advanced rapidly and reached to the stage where Mendel’s hypothetical hereditary factors could soon be correlated with the chromosomes on following grounds:
1. Like the chromosomes, Mendelian factors are carried singly in mature germ cells.
2. Like the chromosomes, the hereditary factors are brought together in pairs by fertilization.
3. Like the chromosomes they separate or segregate at the time of germ cell formation in different generations.
The only objection in treating the chromosomes equivalent to Mendelian factors is that the number of Mendelian factors in the organisms far exceeds the number of chromosomes. In 1902, Sutton and Boveri independently suggested the way out for this objection and considered the chromosomes as containers of Mendelian hereditary units.
They did not treat chromosome as single heredity unit. In 1914, Thomas Morgan was working with the fruitfly (drosophila melanogaster). He noted that the fruitfly in question had 4 pairs of chromosomes and the hereditary characters were too many. This fact led Morgan to propose gene theory.
The theory states that:
(i) Chromosomes are bearers of hereditary units or genes and each chromosome carries hundreds or thousands of genes.
(ii) The genes are arranged on the chromosomes in the linear order and on the specific regions or loci.
If it is considered that gene is specific particle, then it follows that the chromosome would be a linear array of such particles bound together by non-genetic linkers of some type. Such a morphological differentiation can be observed in the chromosomes under some special circumstances. When a chromosome is observed in the early stages of first meiotic prophase, it appears as a ‘string of beads’.
The thickened parts or beads of chromosomes are called chromomeres and the string or linker joining the chromomeres is known as chromonema (Fig. 19.1). At one time, chromomeres were treated as genes. But, this is an oversimplified view and is no longer held.
The banding pattern of salivary gland chromosome of Drosophila (Fig. 19.2) and chromosomal loops in the lampbrush chromosome (Fig. 19.3) of amphibian eggs are also suggestive of functional discontinuity.
Actually, the genie material is not different from other non-genic material in the chromosome.
It is, perhaps, suggested by the fact that genetic recombination can and does take place within genie as well as non-genic areas.
2. Definition of Genes:
As both the operation and the physicochemical nature of gene are not known clearly, the gene can be defined best in terms of its effects. Efforts to formulate theoretical models or hypothesis of gene action have resulted in highly divergent opinions.
There are two schools of thought which define genes in different ways:
(i) According to one school of thought, the specific molecule of genie material controls each character.
The exponents of this school are Dobzhansky (1955) and Beadle (1955).
(ii) The other school under the leadership of Goldschmidt holds the view that the interactions between all the components of genome are responsible for the manifestation of characters.
Stadler (1955) points out that the differences of opinions arise because one school defines genes as “operational units” whose actions can be demonstrated experimentally and the other school defines the gene in a hypothetical physicochemical sense.
In hypothetical sense the gene of classical genetics is visualised as a discrete particle inherited in Mendelian fashion or as unit of function that occupies a definite locus in the chromosome and is responsible for the expression of a specific phenotypic character, e.g., genes for white colouration of flower in pea or those for length of pea plant etc.
According to Benzer’s classical concept, gene is the unit of function (cistron), a unit of recombination (recon) and a unit of mutation (muton).
A gene does not produce character by itself but it exercises the major control on the development of character. The genes produce gene products that, in turn, determine specific phenotype.
3. The Gene as a Unit of Function (Cistron):
The first link between a gene and gene product was established in 1909 when A.E. Garrod suggested that the gene product was a protein. This hypothesis ‘one gene-one protein’ was largely ignored until around 1940 when it was conclusively demonstrated by Beadle and Tatum (1941) that the genes directed the protein synthesis.
Encouraged by this link between gene and protein, investigators worked out the mechanisms by which information in DNA was translated into the structure of protein. Soon it became clear that DNA is first transcribed into RNA, which is subsequently translated into protein.
Garrod as well as some other investigators suggested that genes produce proteins that act as enzymes. One gene-one enzyme hypothesis has been interpreted in different ways.
Gene represents a particular sequence of nucleotides along DNA molecule (or occasionally RNA in certain viruses) that acts as a unit of inheritance. Since gene forms a messenger RNA molecule it can be said one gene-one messenger RNA. But in prokaryotes several genes form a single mRNA (polycistronic gene).
The concept expressed as one genes one protein has now been changed to one gene-one polypeptide, since an enzyme or a protein may contain one or more polypeptides. Stahl had defined gene as a polynucleotide sequence of DNA controlling the expression of a particular trait.
The gene as a unit of function thus represents a segment of DNA molecule and consists of a linear sequence of nucleotides which controls some cellular function.
The number of nucleotides in a gene may vary in different organisms. In Escherichia coli the cistron may contain 1500 base pairs but in some others it may contain as many as 30,000 nucleotides. Each cistron is responsible for coding one mRNA molecule which in tum codes for a polypeptide chain (enzyme or protein).
A cistron may have hundreds of units of mutation (mutons) and units of recombination (recons) within it. Therefore, cistrons occupy much greater area in chromosome as compared to mutons and recons.
4. Gene as the Unit of Recombination (Recon):
The classical studies on the genetics of Drosophila indicated that gene was the shortest segment of chromosome controlling phenotypic characters which could be separated from its adjacent segments during crossing over, i.e., the genes were those parts of chromosomes between which crossing over was possible and the crossing over was not supposed to take place within the gene.
But recent studies based on the tests for recombination in viruses have shown that in viral DNA strand crossing over could occur not only between the genes but also within the genes. One of the sub-units of gene has been called recon. It is the smallest unit capable of genetic recombination. Recombination studies on microbes indicate that structurally recon may have one or two pairs of nucleotides.
Benzer (1955) demonstrated crossing over within the gene in T4 bacteriophage. Phage T4 contains one linear chromosome. There are two strains of T4 phage wild strain producing smooth edged plaques and the r II mutant strain producing rough edged plaques. r-Il mutants are of several types.
Benzer found that two adjacent genes r ll A and r ll B were responsible for rough edged plaque character. Each gene forms a polypeptide and the two polypeptides form a protein. A change in the polypeptide chain would result in a change in the phenotypic expression.
R ll A and r ll B mutants may cross in E. coli. The progeny produced from the cross were analysed for the frequency of recombination and it was found that some phage particles were of normal type. This was possible only if crossing over occurred within the genes by which segments of A and B genes united to form normal wild genes.
5. The Gene as a Unit of Mutation (Muton):
Gene has also been defined as an unit of mutation. The smallest chromosomal unit capable of undergoing mutation has been called the muton. At molecular level a muton consists of one or many pairs of nucleotides within the DNA molecule. Mutation thus may be caused by a change in one or more nucleotides in the DNA molecule.
Muntzing (1961) defines a gene as small segment of chromosome having a unitary biochemical function and specific effect on the properties of individual. According to him the genes may also occur in the cytoplasmic bodies that are sometimes associated with chromosomes or even sometimes occurring free in the cytoplasm.
6. Identification of Genetic Material:
For understanding the nature of genes it is necessary to know the chemical and physical nature of genetic material. At the moment most of our knowledge about this comes from the studies on fungi, bacteria and viruses. Here everybody seems generally agreed on the fact that the genetic materials of lower organisms are similar to those of higher ones.
Before deciding the nature of hereditary material one should keep in mind the following three principal characteristics of the material responsible for inheritable traits:
1. It must contain all the information’s regarding cell structure, function, development and reproduction.
2. It must be able to replicate accurately so that the progeny cells inherit the same genetic features as the parent cells.
3. It must be capable of undergoing variation through recombination and mutations and exist in infinite forms, otherwise organisms would not be capable of change, and adaptations and evolution would not be possible.
Much of the chemistry of the genetic material was known before its significance in the genetics was recognised. In mid to late nineteenth century and early twentieth century scientists believed that the genetic instructions from the nucleus were carried to the cytoplasm by protein molecules that were folded into specific configuration.
But several spectacular discoveries during the past four decades have changed the old ideas and now Deoxyribonucleic acid (DNA) is considered as the genetic material. Following publication of Mendel’s work Johann Friedrich Miescher in 1869 isolated a novel phosphorus bearing compound and named that as nuclein.
Miescher and other scientists subsequently demonstrated that the nuclei of all cellular organisms contain nuclein and nuclein was an important constituent of chromosomes. Chromosomes consist of 60% protein and 40% DNA. The following seven direct and indirect evidences from different sources have established that DNA is the essential genetic material (Lima de Faria and Moses, 1966 and others).
1. In prokaryotic organisms (organisms lacking well organized nucleus and chromosomes) to which operationally viruses can also be included, the genetic material is DNA.
2. Deoxyribonucleic acid is confined almost to the chromosomes in the nuclei of eukaryotes. DNA is a stable macromolecule, the content of which is directly related to the chromosome number.
3. The amount of DNA per nucleus is species characteristic and the amount of DNA found in the gamete is only half of the amount of DNA present in the somatic cells of all the members of a species. The diploid condition is restored only after fertilization.
Careful measurements of the amount of DNA and proteins in somatic cells and sperm cells indicated that DNA only played hereditary role .and not protein. The measurements indicated that the amounts of protein in sperms and somatic cells bore no definite relationship with each other whereas the amount of DNA in sperms was exactly one-half that in somatic cells.
4. Genes are characterized as self-perpetuating units, DNA molecules too replicate themselves and in the process of replication the old DNA acts as template for new one.
5. The highest efficiency of mutagenesis by ultraviolet light is at the wavelength of2600 A which is also wavelength of peak absorption by DNA.
6. The genetic transformation in bacteria is mediated by DNA. The most convincing evidence in support of the fact that genie material is DNA could appear in 1944 when Avery, MacLeod, and McCarty reported their work on transformation in Pneumococcus.
7. Hershey and Chase (1952) confirmed that in bacterial viruses (bacteriophages) the genetic material is DNA. Certain RNA viruses are exceptions in which the transfer of genetic information has been taken over by ribonucleic acid. Fraenkel Conrat (1955) working on the reproduction of Tobacco mosaic virus (TMV) proved that the hereditary material in that virus was definitely RNA.
The major experiments which established that DNA is genetic material are discussed here in to brief:
1. Transformation Experiment in Bacteria:
Frederick Griffith (1928) for the first time demonstrated genetic transformation in bacterium diplococcus pneumoniae, now named Streptococcus pneumoniae and Avery, MacLeod and McCarty (1944) later showed that DNA and not the protein, was the carrier of hereditary characters in diplococcus pneumoniae or Pneumococcus. This bacterium causes pneumonia in mice and men. There are two strains of Pneumococcus.
In one strain, capsule layer (slime coat) is formed of a polysaccharide material and colonies are shining and “smooth” (‘S’ strain). In other strain, cells lack polysaccharide slime layer and colonies formed by such cells are irregular or “rough” (‘R’ strain). Smooth (S) cells are virulent and cause Pneumonia but rough (R) cells are non-virulent. ‘S’ Pneumococci are classified into types, such as, type I-S, type II-S, type III-S and so on.
The cell of ‘S’ colony may change occasionally into ‘R’ bacterium but the reverse change (i.e., from R to S) is almost never seen. R cells upon division always give rise to R cells. In the course of his experiment. Griffith injected mice with living II-R Pneumococci and found that the mice did not suffer.
But when the mice were injected with live III-S type they suffered by pneumonia and died and when heat killed III-S bacteria were injected the mice did not suffer.
However, when the mixture of living cells of non-virulent II-R and heat killed III- S cells were injected into the mice, the mice unexpectedly developed pneumonia and died.
Postmortem examination of dead mice showed the presence of both II-R and III-S type of pneumococci in the heart blood and this led Griffith to conclude that something released from the heat killed III-S cells was taken up by the avirulent R type cells which might have caused genetic transformation of living II-R bacteria into virulent cells (Fig. 19.4).
Avery and his associates at the Rock-feller Institute earnestly pursued to identify the transforming principle. Between 1930 and 1933 they demonstrated transformation in vitro rather than in the mice. They disintegrated encapsulated cells of type III-S and separated various chemical components (carbohydrates, proteins, fats, DNA, RNA etc.).
Then they took R-H cells derived from type II-S and mixed them separately with different chemical components of III-S cells. Avery and his associates observed that the DNA fraction was able to change some of the R cells (non-capsulated) to encapsulated S cells. The transformed R-II cells were like III-S and were similar in all respects to the cells from which the DNA fraction was taken (Fig. 19.5).
In 1944, Avery, MacLeod and McCarty published a remarkable paper in which they reported that they had purified the transforming principle. Analysis of the molecular composition and weight indicated that transforming factor was DNA.
They took S-type bacterium separated by centrifugation, killed them by heating, ruptured the cells in water and filtered. Filtrate salvaged was tested for its transforming ability, that was found to be positive. Filtrate was then treated with protease to degrade all proteins in that and tested for transforming ability which was found to be positive.
Next, the filtrate was treated with ribonucleic (RNAase) enzyme that digested all ribonucleic acid (RNA).
The filtrate salvaged was tested for its transforming power that was found to be positive. Finally the filtrate was treated with deoxyribo-nuclease (DNAase) which totally destroyed the DNA. The DNAase treated filtrate was tested for transforming ability, that was no longer able to convert R-type cells into virulent cells like III-S type.
The result of the experiment made avery and his colleagues to conclude that the transforming principle must be DNA. The idea was supported not only by the results of enzymatic digestion experiments but also by the analysis of purified transforming principle.
These experiments clearly indicate that the whole transforming ability resides in DNA. For this reason DNA of the cells is called the “transforming principle “. A variety of other hereditary characters in different species of bacteria have also been demonstrated to be governed by DNA.
The bacterial strains which are sensitive to antibiotics like penicillin or streptomycin can acquire permanent resistance to these antibiotics by transformation of DNA.
2. Bacteriophage Multiplication:
Another evidence in support of the fact that DNA is genetic material comes from the study of bacteriophage multiplication.
The DNA is infective material in the virus. In the infection, the phage attaches itself to the bacterial cell wall and injects its DNA strand into the bacterial cytoplasm. Inside the bacterial cell the phage particle multiplies. During the process of phage multiplication many new strands of DNA and protein sheaths are synthesised and hundred or so new phage particles resembling the original one are formed.
Phage multiplication is immediately followed by death or lysis of the infected bacterial cell. The newly born phage particles attach with the other adjacent bacterial cells and produce a region of lysis in the bacterial colony.
If the two components (DNA and Protein) of bacteriophage are separated mechanically and they are separately injected in the uninfected bacterial cells, then the new phage particles with both DNA and protein sheath will continue to be produced inside the cell in which virus DNA was injected, whereas no viral particle will be produced from the cell in which viral protein was injected.
The “Waring Blender Experiment” by Hershey and Chase in which they utilized the radioactive isotope P32 to label DNA and s35 to label protein of the bacterial virus T2 and showed that during infection of bacterial cell by virus only DNA entered the cell while the protein remained outside (Fig. 19.6). It is evident from the experiment that DNA is hereditary material and protein is non- hereditary.
If two genetically different phage particles differing in such characters as the sizes of the plaques of dead bacteria they form simultaneously infect the same bacterial cell, recombinant phage particles may be formed. Since in the infection only the DNA strands of virus particles entered the bacterial cell, it can be inferred that only DNA is genetic material.
3. Bacterial Conjugation:
A convincing evidence for DNA as genetic material has come from the process of bacterial conjugation. Laderberg and Tatum (1946) found that when F+ (male) strains of Escherichia coli conjugate with F– (female) cells there is an unidirectional flow of F+ factor from male cells to F– female cells so that the latter is converted into F– from male strain.
The F+ factor is found to be a fragment of DNA molecule which occurs in the cytoplasm of bacterium.
4. RNA as the Genetic Material in Some Viruses:
Tobacco mosaic virus (TMV) which causes mosaic disease in tobacco consists of an outer protein coat and an inner core of nucleic acid RNA (Fig. 19.7). In 1955, Fraenkel Conrat fractionated RNA and protein of TMV and injected them separately into two healthy tobacco plants.
In this experiment they observed that viral RNA, completely free from its protein, could produce new virus particles which also caused mosaic disease in tobacco plant. The inoculation of viral protein did not produce the disease symptoms at all. This experiment showed that viral RNA alone can direct the formation of normal virus (Fig. 19.7).
Different strains of TMV are now known which produce different inherited lesions on tobacco leaves. The common virus (TMV-common) produces green, mosaic disease and a mutant TMV strain, TMV-HR (holmes ribgrass) produces ring-spot lesions. The amino-acid compositions of the protein sheaths of two strains are found to be different.
Heinz Fraenkel Conrat and Bea Singer isolated the RNA and proteins of two different strains of TMV and then they developed techniques to reassemble or reconstitute functional viruses with the RNA from TMV-common enclosed in protein sheath of TMV-HR and vice-versa (Fig. 19.7).
When the reconstituted viruses were allowed to infect tobacco leaves, the progeny viruses obtained from the infected leaves were always found to be the phenotypically and genotypically identical to parent strain from which RNA had been obtained.
The reconstituted viruses with RNA of TMV-common and protein sheath of TMV-HR produced green mosaic disease symptoms of TMV-common and protein characteristic of recovered progeny resembled that of TMV-common.
Similarly the reconstituted viruses with RNA of TMV-HR and Protein sheath of TMV-common produced ring spot lesions and the virus progeny had protein characteristic of TMV-HR. Thus it becomes clear that the source of RNA determined the nature of mosaic infection as well as the nature of proteins of progeny viruses. Hence, it is RNA in the plant viruses that carry genetic information and not the proteins.
The above experiments provide evidence in support of the fact that usually DNA, (excepting a few RNA viruses) is the genetic substance.