LECTURER: DR.

OSUNDINAKIN MICHAEL

BIO 302/402: GENETICS AND EVOLUTION

Mutations

Mutation is a process which produces altered DNA sequence. It may be induced by radiations and

chemical mutagens in the environment or insult from endogenous reactive oxygen species (ROS)

or occur spontaneously due to endogenous factors (errors in chromosomal segregation,

recombination, DNA replication and repair, and also spontaneous chemical damage to DNA).

Mutations are inexorable. They may have adverse effects on organism’s health and survival.

Sometimes, they may have no obvious effects, or rarely, they may be beneficial for the bearer.

TYPES OF MUTATIONS

1. Macromutation: The large mutations that are recognized with certainty in a single plant, are

analogous to discontinuous or qualitative characters. All such mutations in which the change,

though inherited as a single unit of recombination, leads to several phenotypic consequences. They

may be the result of tight linkage, pleiotropy or the action of the gene commencing at a very early

stage in the ontogeny and thereby having an effect on the constellation of characters and hence on

the individual. Macromutations may be trans-specific (i.e. inter-specific, where induced mutation

in one species may be found in other species or genera also) or intra-specific. They include:

(a) Systemic mutations: They are mutations which either simulate an already existing taxon or

necessitate the creation of a new systematic unit by virtue of the character affected. In other words,

these mutations tend to affect a constellation of characters well-differentiated from other

taxonomic groups. For instance, in hexaploid wheat (Triticum aestivum), mutants simulating the

established species, like T. compactum, T. spherococcum, T. spelta, as T. vavilovi, etc. could be

obtained.
(b) Non-systemic mutations: These are the mutations associated, for example, with dwarf, erect

leaf types, early, late, disease resistant, better quality, high protein and gluten content, etc.

However, appropriate screening scales have to be employed to identify them. These may be

agriculturally useful and can be utilized by breeders.

2. Micromulations: These are small mutations affecting the polygenic or quantitative traits which

can only be detected in a group. They can be isolated and fixed only through the adoption of a

suitable biometrical scale.

Micro-mutants are either manifest or cryptic in their behaviour. Because of their nature,

micromutants are generally important from the plant breeding standpoint.

The above noted mutations are those that are visible, i.e. they are expressed in the phenotype and

can be measured. They are essentially the results of many categories of mutations occurring in the

cells, namely genome mutations (polyploidy), chromosome mutations-gene mutations and

structural rearrangements (deletion, duplication, inversion and translocation), and extranuclear

mutations.

Since mutations are recurrent and reproducible and many of these, particularly micromutations

and non-systemic mutations, are economically viable, they can be produced or artificially induced

with the help of appropriate mutagens.

Mutagenesis is defined as the process by which an organism's deoxyribonucleic acids (DNA)

change, leading to a mutation. Many agents (physical and chemical) have the mutagenic properties

to cause mutations. They are known as mutagens.

MUTAGENS AND THEIR PROPERTIES

For artificial induction of mutations, mutagens or mutagenic agents are employed. Muller (1927)

demonstrated for the first time that mutations can be induced in Drosophila by X-rays. Use of

certain chemicals for the same consequences came to light with the discoveries of Auerbach (1943)

in England, Oehlkers (1943) in Germany, and Rapoport (1946) in Russia.

Thus, two types of mutagens could be recognized: physical and chemical. Currently, an array of

both physical and chemical mutagens are under use for specific purposes and for different

organisms.

1. PHYSICAL MUTAGENS

Physical mutagens (also called radiation) essentially involve radioactive isotopes (or

radioisotopes), i.e. atoms of the same element with different molecular weights. Such atoms are

unstable because their neutron numbers are altered. To achieve stability, the atoms tend to split or

give off particles, thereby releasing a tremendous amount of bound energy.

For instance, carbon (C) atoms have six neutrons, six protons and six electrons, hence it is

symbolized as C12. On addition of two more neutrons, the weight of C becomes 14 rather than 12,

and then it is C 14. These additional neutrons render the C atom unstable and the heavy C 14 atoms

tends to emit radiation.

1. Particulate/corpuscular ionizing radiation

These are high potential fast moving charged atomic particles capable of transferring their kinetic

energy to any matter or organism they collide with and pass through. The primary collision

generally results in the ejection of an electron out of the orbit of the atom. Ionization is the

consequence. The atom left behind is a positively charged ion which pairs with a negatively

charged ion of another atom. The ejected secondary electrons further form ion pairs along their

track. Thus a chain process continues till the ejected secondary electrons are sufficiently slowed

down to be captured by an atom or molecule.

2. Electromagnetic (non-ionizing) radiation

These are high energy short-waves that can cause electric and magnetic disturbances in the

genotype of the organism. Electromagnetic radiation can cause secondary ionization. In fact, the

energy absorbed from waves creates a state of instability and thus the excess energy absorbed is

dissipated by forcing out one of the orbital electrons. This electron produces additional ionization

since it is a charged particle moving through the matter or organism.

Examples of physical mutagens are: X-rays, frays, B-rays, fast neutrons, thermal neutrons,

ultraviolet rays, infrared rays, etc.

EFFECTS OF IRRADIATION

Any type of irradiation produces the following effects on the cells of an organism

1. Inhibition of mitosis that impairs the auxin production, and hence stunted seedling height.

2. Disturbed mitosis as spindle formation becomes irregular due to chromosome stickiness. This

results in aneuploidy.
 3. Disturbed crossing over in both mitosis (somatic) and meiotic (gametic) cell divisions due to

increase or decrease in chiasma frequency, depending upon the dosages, and to repatternization of

chiasma sites promoting release of latent variability by recombination between distant genes.

4. Chromosomal aberrations like deletion, duplication, inversion and translocation.

CHEMICAL MUTAGENS

The history of chemical mutagens is perhaps as old as that of genetics itself. The discovery of

effective chemical mutagens was made during the period from the beginning of the Second World

War to early fifties. The first chemical mutagen, i.e. mustard gas was discovered in Germany

during World War II.

This followed the discovery of an array of chemical mutagens.

The number of chemical mutagens is very great and is continuously increasing. However, for

practical purposes of mutation induction in crop plants, so far only a few are really useful. Most

of these belong to the special class of alkylating agents and may be listed as: EMS (Ethyl Methane

Sulphonate), dES (DiEthyl Sulphate), El (Ethyleneimine), ENU (Ethyl Nitroso Urathane), ENH

(Ethyl Nitroso urea), and MNH (Methyle Nitroso urea).

Other alkylating mutagens are: sulphur mustards, nitrogen mustards, ethyl oxide, TEM

(Triethylene Melamine), dMS (dimethyl Sulphate), MMS (Methyl Methane Sulphonate), MNU

(N-methyle-N-nitroso urethane), etc. In addition to alkylating agents, there are azides (e.g. sodium

azide, etc.), acridines, nitrous acid, hydroxylamine, antibiotics (e.g. actinomycin-D, streptomycin,

mitomycin-C etc.), and base analogues e.g. 2-AP (e-aminopurine), 5-BUdR (5-bromo-

deoxyuridine), 5 BU (5-bromo-uracil), TMU (tetramethyluric acid), EOC (8-ethoxy caffeine), etc.]

Which are useful for specific purposes, particularly in genetical studies.

Mutagenic Pathway

The mutagens, whether radiation or chemical, interact with the DNA and cause mutagen

specificity. Such a specificity may be due to treatment effects on the fixation or expression of

potential mutations. This holds true because many cases of mutagen specificity arise at later levels

in the mutagenic pathway in the following sequence

1. Penetration of mutagen to DNA of nucleur gene

2. Production of a premutational lesion

3. Death from unrepaired damage

4. Repair of lesion, restoring the normal gene

5. Fixation of premutational lesion as mutated gene

6. Expression/formation of mutant cell

7. Death of mutant cell

8. Formation of clone of mutant cells.

Thus, the mutagens first contact DNA and then produce lesions which may then undergo any of

the three processes: (1) The damage due an lesions may be repaired, in it may not be repaired, (i)

the lesions may be fixed. The consequences are, the restoration of the normal gene, death or loss

of cell, and mutation of normal gene, respectively. The last one is followed by the formation of a

mutant clone.

The changes that reach the final stage are almost random and depend on the effectiveness and

efficiency of the mutagen under operation.

Based on the site of their occurrence, mutations can be divided into the following main categories

1. Genome mutations (e.g. polyploidy, haploidy, aneuploidy)

2. Chromosome mutations

(a) Structural rearrangements (e.g. translocations, inversions, duplications, deficiencies)

(b) Gene or point mutations (e.g. submicroscopic changes within the fine structure of a gene locus,

"cistron" such as changes in single-base radicals or "triplets" of nucleotide chain involved in a

DNA double helix- non-sense reading of triplet genetic code). However, several base pairs may

have to be altered before an observable phenotypic change (mutation) is created.

3. Extranuclear mutations, i.e. cytoplasmic

Thus, as a consequence of directed mutagenesis, genetic changes may occur at both nuclear

(chromosomal) and extranuclear (cytoplasmic) levels. The impact of such a mutagenesis is

reflected in the disturbance of normal biological processes, including physiological damages of

the organism subjected to mutagen. Physiological damage is perhaps of both chromosomal

(nuclear) and extra-chromosomal (cytoplasmic) origin.

PROPERTIES OF BIOLOGICAL SYSTEMS RESPONDING TO MUTAGENS

On the whole, mutagens may affect anywhere in the chain of genetic organization, starting from

DNA through gene, chromosome, cell, tissues, organ and the whole organism. The ultimate

consequences are abnormal expressions for growth, development (morphological/ developmental

differentiation), character and the individual.

1. The Physiological State: The mutagenic reaction is greatly influenced by the physiological state

of the test material, including diverse stages of growth, development and metabolism. In fact, only

active tissues, and not the dead or matured tissues respond to mutagenic treatment.
2. The Tissue- Structure: Nilan (1964) reported that different organs may have different

proportion of cells from which mutations may be recovered. But mutagens must come in contact

with those cells or tissues. Sometimes, seed-coats or hulls on seed present a mechanical resistance

to the diffusion of active mutagen towards mutationally responsive tissues. For instance, vapours

of mutagens, like DES, fail to affect dry seeds of barley.

3. The Genetic Make-up: All mutagens cannot be equally and universally effective and efficient

with all organisms. Profound variations exist both between and within species, though some are

sensitive to many mutagens and some to very restricted ones.

4. Capacity for Reproduction: The induction of mutation occurs necessarily when DNA synthesis

and chromosomal reproduction are going on. Matured or differentiated cells generally do not

undergo reproduction. Therefore, they are incapable of responding to mutagenic treatments. Thus,

the innate capacity of mutation differs greatly for various types of tissues, and probably differs

markedly with stages of cell-division and developments.

5. Differential Synthetic Capacities of Cells and Tissues: The synthetic capacity of cells to repair

or replace damaged components differs with their states of cell division or metabolic activity.

Obviously then, the yield of mutant clones, or mutated cell colonies, would depend upon the stage

of cell division when the mutagen hits that cell. Also, the fixation of mutations depends on the

time of synthesis, synthetic ability and the nutrition of mutagenically treated cells.

Furthermore, the responsiveness of biological systems to mutagens is the function of not only the

above noted properties of biological systems but also of several other factors associated with

mutagens and the treatment procedures together with external conditions.

SYSTEMS

Many factors influence the mutagenic reaction with biological materials.

1. Temperature: It influences the rate of most biochemical reactions. The temperature of plant

cells before, during and after irradiation may affect the total amount of genetic damage induced by

X- or gamma-rays. Similarly, the temperature of the mutagenic solution has considerable influence

on the mutagenic chemicals.

2. The pH and buffers: It is recognized that biological systems are sensitive to low pH and also

that at a certain H-ion concentration, buffer materials may play an active role as complexing

agents causing secondary effects. In fact, mutagens, particularly alkylating agents, react with

polar solvents (e.g. water) to produce acidic products.

3. Concentration of mutagen: The rate of diffusion of mutagen across the cell-membrane is

the function of the relative concentration of both mutagen and the solvent on either side of the

cell-membrane. It is a well-known fact that mutagen strength and duration of its treatment are of

great consequence in mutation experiments. For instance, high concentration of mutagens, like

EMS and DES are more damaging than low concentrations applied for a relatively longer time.

4. Chemical structure of mutagen: The structure of the mutagen, particularly those belonging

to alkylating group, may significantly influence its physical and chemical properties, and its

reaction with biological systems.

6. Presence of catalyst: The catalytic agents present in the solution tend to increase or decrease

the frequency of mutations through a synergistic process. For instance, copper and zink ions cause

a remarkable increase in chromosomal aberrations induced by EMS.

efficiency. But it depends on two factors: (i) concentration/strength, and (ii) rate of hydrolysis

reaction of mutagen. To obtain larger yields of mutations, a short treatment with mutagen at high

concentration and alternatively, a long treatment with mutagen at low concentration are

recommended.

8. Pre-treatment soaking: Diffusion of mutagens into seeds and other tissues Is facilitated

when they are fully hydrated. It is for this reason that soaking the plant organs, especially seeds,

before mutagenic treatment, creates a favourable condition for better yield of mutations since it

changes the physiological properties of the cells. First the cell membrane becomes permeable to

mutagen, thus reducing the experimental variability, and second, soaking prior to treatment with

higher concentration of mutagen tends to minimize injury, lethality and mutation yield.

9. Post-treatment drying: The primary purpose of post-treatment drying of seeds is to gain

convenience in handling treated seeds with regard to storage or mailing, or to easy machine

planting with uniform placement in fields. The consequences of drying underlie the delayed

effects of the active mutagen that is retained in the tissues after treatment. Drying increases the

in vivo mutagen concentration, and the rate of hydrolysis of mutagen determines the period over

which delayed effects can be expected degradation products. However, this can be offset by post-

treatment washing of seeds so as to extract active mutagen from the treated seeds.

10. Post-treatment washing: To mitigate delayed effects of mutagens treatment, post-treatment

washing of the treated material is in practice. Washing in flowing water (running water method)

is far more effective in reducing the delayed effects than soaking in still water (change method).
 11. Storage of treated seeds: Storing mutagenically treated seeds causes after-effects where

mutation-rate and biological damage increase with increasing storage-time, depending upon the

moisture content of the seeds, the mutagen-dose applied, storage-time and temperature. The

storage of EMS-treated seeds is a much greater problem. If the seeds are stored at room

temperature and at the 10-14 per cent moisture content, then the after effects may lead to lethality.

CARCINOGENS

The term "carcinogen" denotes a chemical substance or a mixture of chemical substances which

induce cancer or increase its incidence. Substances which have induced benign and malignant

tumours in well performed experimental studies on animals are considered also to be presumed or

suspected human carcinogens unless there is strong evidence that the mechanism of tumour

formation is not relevant for humans. Carcinogen promotes carcinogenesis which is due to the

ability to damage the genome or to the disruption of cellular metabolic processes.