In this article we will discuss about the history and induction of mutations.
History of Mutation:
The sudden heritable changes in genes, other than those due to Mendelian segregation and recombination constitute mutations. The idea of mutation first originated from observations of a Dutch botanist Hugo de Vries (in the 1880’s) on variations in plants of Oenothera lamarckiana (evening primrose) growing in Holland. This plant had been introduced from America and had grown wild in Europe.
De Vries collected seeds from Oenothera plants, raised plants from them, and analysed the progeny for transmission of traits showing variation. He found that heritable variations were distinct from environmental variations. He gave the name mutation (latin mutare meaning change) to heritable changes and in 1901 published a book entitled “The Mutation Theory”.
Although De Vries is credited with the discovery of the idea of mutations, it was realised almost half a century later that perhaps none of the plants studied by him actually showed a gene mutation. Instead, the heritable phenotypic variations observed by him were found to be due to rare crossovers between translocated chromosomes.
The early concepts of mutation therefore arose out of genetic studies of visible phenotypes. Later, by the middle of the twentieth century, the molecular basis of heredity began to be investigated. It became established that the transmission of hereditary traits takes place due to an accurate process of self-replication of the genetic material which is DNA.
Today gross structural changes in the genetic material at the level of chromosomes are classified under chromosomal aberrations. They are treated separately. Only alterations in a very localised region of the chromosome at the molecular level are called mutations. They may involve one or more genes or nucleotides in DNA.
Mutations therefore cause the substitution, deletion, addition or alteration of the sugar, base or phosphate of a nucleotide or of one more whole nucleotides. When mutations bring about a change in a single nucleotide they are called point mutations. When several nucleotides are altered, it results in gross mutations. The product of a mutation is called a mutant, and could be a genotype, cell, a polypeptide chain or an individual.
Mutations have been broadly categorized as somatic and germinal mutations. When a mutation occurs in a somatic cell, it does not change the whole organism, but produces a phenotypic change in the organ to which the mutant cell belongs. The resulting individual is a mosaic for mutant and normal tissues.
Navel oranges (so named because when, first discovered in South America, the orange had a shriveled, indented portion resembling the human navel), golden delicious apples, emperor seedless grapes, some horticultural varieties of flowering plants, and white sectors in the red eyes of Drosophila males are examples of somatic mutations. In plants somatic mutations are transmitted to the progeny by methods of vegetative propagation such as budding and grafting.
Germinal mutations take place in cells of the germ line. A classic example of germinal mutations and perhaps also the one first recorded is that of short-legged sheep. In 1791 a farmer named Seth Wright in New England, U.S.A. noticed a short-legged lamb which could not jump over the fence and run away like all the other sheep in his small flock. Considering this to be an advantage, he started breeding work on the short-legged lamb.
He had 15 ewes (females) and one ram (male) out of which he established a line of short-legged sheep. The name ancon was given to this breed (Greek meaning elbow), because the crooked looking forelegs showed resemblance to the human elbow.
The first short-legged lamb arose as the result of a recessive mutation in one of its recent ancestors. From the breeding work done in Wright’s farm the crosses indicated that this lamb was homozygous for the mutant gene and several of the ewes were heterozygous carriers. The same mutation is said to have occurred in a flock of sheep in Norway in 1925 and another breed of ancon sheep has been produced out of it.
Induction of Mutations:
In 1927 H. J Muller showed for the first time that mutations could be induced in Drosophila by use of external agents or mutagens. He was awarded Nobel Prize in 1946. When flies were irradiated with X-rays, he found that the offsprings showed new phenotypes which were similar to those produced by spontaneously occurring mutations.
He also found that increasing the dose of X-rays results in a linear increase in the frequency of mutations. By 1930s, it became established that physical agents such as X-rays, gamma rays, UV radiation, and some chemical agents are all effective as mutagens. At the same time L. J. Stadler demonstrated that X-rays could produce gene mutations in plants of barley. Mutagenesis by radiation and chemicals are discussed separately below.
Radiation:
Theoretical Background of Radiation:
An atom is composed of a positively charged nucleus and negatively charged electrons orbiting around the nucleus. The nucleus contains uncharged neutrons and positively charged protons. The electrons can move from one orbit to another.
An electron absorbs energy when it jumps to a high energy level, and releases energy when it moves to a lower energy level. The released energy is in the form of electromagnetic radiation. Visible and UV light, X-rays and gamma rays are all electromagnetic waves.
Radiation breaks chromosomes by a chemical reaction which requires energy. The mutagenic effect of radiation depends upon the amount and type of energy left in the tissue. Visible light is a less energetic radiation as it leaves energy in the form of heat. But the energetic radiations such as ultraviolet (UV) leave energy in the form of heat and activation which leads to chemical change.
Activation is the type of energy which makes an electron move from an inner to an outer orbit of the atom. X-rays and gamma rays are high energy radiations that not only heat and activate, but leave energy in the cell in the form of ionisation.
The Atomic Bomb Explosions at Hiroshima and Nagasaki:
On August 6, 1945 the atomic bomb exploded over Hiroshima killing over 78000 people and leaving many affected survivors who were studied for the effects of radiation on human beings. On August 9, a second atomic bomb was exploded over the city of Nagasaki. The words hibakusha (explosion affected person) and higaisha victim or injured person became understood to people in the world.
A number of individuals who had received extensive radiation had no children for several years. Many of the survivors have shown visible chromosome abnormalities such as breaks and translocations.
Persons over 30 years of age who had received more than 200 rads of radiation were found to be more sensitive to radiation than persons of the younger age group. About 2 per cent of survivors developed leukemia within the following decade. The children of survivors have not shown a detectable increase in genetic abnormalities.
UV Radiation:
E. Altenberg was first to show that ultraviolet radiation can induce mutations. UV rays have longer wavelengths than X-rays and gamma rays, hence they cannot penetrate tissues as deeply. Their mutagenic action is limited to bacteria, fungal spores or other free cells whose genetic material lies very near the irradiated surface. L.J. Stadler induced mutations in pollen grains of maize plants.
He found that UV rays with a wavelength of about 260 nm (Fig. 20.3) were more effective in mutagenesis. In fact the wavelength most readily absorbed by DNA also happens to be 260 nm, thus proving a direct correlation between UV induced mutations and DNA. The sun is a powerful source of UV.
As such the sun’s rays would be expected to cause widespread damage through mutations in all living organisms. But fortunately a layer of ozone in the upper atmosphere absorbs most radiation below 290 nm. The UV rays falling on DNA are absorbed by the pyrimidine bases especially thymine. Cross linking between adjacent pyrimidines takes place to form thymine dimers.
The two thymines join at their 4 and 5 positions to form a dimer. Dimer formation can take place between two thymine residues (TT), cytosine (CC) or uridine (UU) or two different pyrimidines (CT). When cytosine is exposed to UV a molecule of water is added across the double bond between the fourth and fifth carbon atoms (Fig. 20.4).
When heated or exposed to acidic conditions, the hydrated photoproduct can revert to the original form. If allowed to remain as such long enough, the hydrogen bonds between the pyrimidine and purine on the complementary strand break, leading to strand separation in that region. Both dimerisation and hydration of double stranded DNA affect DNA replication.
Chemicals as Mutagens:
Molecular Basis of Point Mutations:
Mutations which alter nucleotide sequences within a gene are of two types: base pair substitutions and frame shift mutations. In base pair substitution one base pair, for example AT may be replaced by another such as CG or GC. These are of two further types, namely transitions and transversions.
Transitions are base changes in which a purine is substituted by another purine as when A = T pair is replaced by a G = C pair or vice versa; or when a pyrimidine is replaced by a pyrimidine such as when T = A is replaced by C = G, or vice versa. Transversions are alterations in bases in which a purine is substituted by a pyrimidine, that is, when an A = T pair is replaced either by T = A or C = G, and vice versa.
In frame shift mutations, (so called because they shift the normal reading frame of base triplets in mRNA) single base pairs are deleted from or added to DNA in interstitial position. The genetic code requires reading of consecutive base triplets from a fixed starting point. If a single nucleotide is inserted or deleted, it shifts the reading frame, and all the subsequent triplets are read off differently.
The entire portion of the polypeptide chain after insertion or deletion is translated wrongly, resulting in a “non-leaky” phenotype. Frameshift mutations therefore differ from base pair substitutions which produce “leaky” phenotypes by altering only one nucleotide in a single triplet, so that only one amino acid is wrongly placed.
Nonsense and Missense Mutations:
When a mutation in a triplet changes the codon so that it is recognised by another amino acid, it is called missense mutation. But if the mutation changes the codon for a specific amino acid into one which signals chain termination (nonsense codon), it is called a nonsense mutation.
Missense mutations occur more frequently than nonsense mutations, and usually result in single amino acid replacements in the polypeptide chain. Such a chain may still have biological activity. Nonsense mutations result in premature termination of polypeptide chains so that only fragments of chains are formed. The lengths of fragments depend upon the distance of the nonsense mutation from the starting codon.
Mutations by Chemicals:
Alkylating Agents:
This is the most powerful group of mutagens. Chemicals of this group bind in vitro to the N-7 position of guanine. In vivo the 0-6 position of guanine and the 0-4 of thymine are preferred. They transfer alkyl groups to the nitrogen atoms of the bases in DNA. Alkylation of guanine causes ionisation of the molecule and changes its base pairing specificities. Alkylated guanine pairs with thymine instead of cytosine.
Thus on replication there is a transition from a G = C to an A = T base pair. Alkylation of purines leads to hydrolysis of the sugar base linkage so that the purine base is lost from the backbone of the DNA molecule producing apurinic gaps.
When DNA replication takes place, almost any base may be inserted in the gap. Insertion of a wrong base in a gap produces transitions as well as transversions. There are some bi-functional alkylating agents which form cross links between guanines on the same or opposite strands of the double helix. This causes more frequent production of apurinic gaps.
Examples of alkylating agents are nitrosoguanidine, mustard gas and mustard compounds, ethyl methane sulphonate (EMS) and ethyl ethane sulphonate (EES), alkyl halides, sulphuric and phosphoric esters, ethylene imines and amides, and others.
They are said to be electrophilic (electron deficient) reactants because they combine with nucleophiles which have electron rich centers. These compounds are also described as radiomimetic because their effects resemble those of ionising radiation.
EMS is one of the most powerful mutagenic agents known. It can add an ethyl group (- CH2 CH3) to a guanine, and much less frequently to adenine. Due to ethylation guanine pairs with adenine leading to A – T⇋ G – C transitions. EMS also produces apurinic gaps into which any of the four bases may be inserted giving rise also to transversions.
Base Substitution by Tautomerism:
The purine-pyrimidine base pairs in double helical DNA are determined mainly by the positions of hydrogen atoms which cross link the bases. Normally A pairs with T and G with C. The bases however, can exist in alternative forms due to rearrangements (tautomeric shifts) in the hydrogen atoms. Tautomers are rare and unstable and can revert to the common form.
Watson and Crick suggested that if a base was present in its tautomeric form at the time of DNA replication, then a wrong base would be synthesised in the new strand. At the next replication cycle, the tautomer would revert back to its normal form, the two strands would separate, and this time the normal correct base would be synthesised on the new strand. This would result in substitution of one base pair for another, i.e., A ⇋ G and T⇋ C transitions would occur.
Base Analogues:
These are chemicals with structure similar to bases in normal DNA and become substituted for normal bases during DNA replication. The first base analogue studied was 5-bromouracil (5-BU) which has chemical structure similar to that of thymine (5-methyluracil) except that the methyl group of thymine is replaced by bromine.
If 5-BU is supplied to the medium containing growing bacterial cells 5-BU gets incorporated into DNA instead of thymine. The cells remain alive and grow, and since 5-BU has the same pairing properties as thymine, it does not lead to mutation at all. However, there are two tautomeric forms of 5-BU, the normal keto form and the rare enol form (Fig. 20.5).
When the rare enol form becomes incorporated into DNA, it pairs with guanine instead of adenine due to its hydrogen- bonding properties. The keto form however pairs with adenine. The enol form is short-lived and will eventually return to the keto form.
When DNA undergoes replication in the presence of thymine, the strand containing 5-BU (now in keto form) will synthesise an adenine in the complementary strand. In the next round of replication, the A strand will synthesise a T strand opposite it. In this way a GC pair would be replaced by an AT pair resulting in mutation of the transition type.
A similar case is that of 2-amino-purine (2-AP) which is a chemical analogue of adenine inducing AT ⇌ GC transitions. In the common form 2-AP pairs with thymine and there is no mutation. But when it exists in its tautomeric form (imino form) it forms two hydrogen bonds with cytosine. 2-AP therefore acts by first replacing adenine by shifting to its imino form, and then pairs with cytosine during replication. It also induces reversion in 5-BU induced mutants.
Nitrous acid (HNO2) acts on non-replicating DNA by removing amino groups of nitrogenous bases, converting adenine to hypoxanthine, guanine to xanthine and cytosine to uracil (Fig. 20.6). The conversions lead to AT → GC and GC → AT transitions as explained in the Figure. In a similar way hydroxylamine (NH2OH) changes hydroxy-methyl-cytosine into uracil leading to AT → GC transition. These mutations are able to reverse their own effects (back mutation) as well as those of other base analogues.
The acridine dyes act by intercalating themselves in DNA. Intercalating agents are planar polycyclic molecules which act by inserting themselves between the stacked base pairs of the double-stranded DNA molecule. This results in doubling of the distance between adjacent base pairs. Acridine mutations show a high rate of spontaneous reversion.
Usually reversion is due to a second suppressor mutation within the same gene that carries the primary mutation. Acridine induced mutations are ‘non-leaky’ as they result in total loss of function of the gene product. Acridines include important fluorochromes and antiseptics, phenanthridines (like ethidium bromide) used as trypanocides, and polycyclic hydrocarbons which are important carcinogens.
Certain chemicals like ethoxy-caffeine, urethane and formaldehyde produce organic peroxides and free radicals leading to mutations. They probably cause destruction of nucleic acid bases resulting in breaks in single strands.
Sister Chromatid Exchanges:
Sister chromatid exchanges (SCEs) represent the interchange of DNA replication products at apparently homologous chromosomal loci. In the recent years SCE analysis has gained importance as a sensitive method for study of DNA damage; it seems that agents which induce SCEs are also active as mutagens and carcinogens (cancer causing agents). Some human genetic diseases deficient in DNA repair mechanisms show abnormalities in SCE formation and predisposition for cancer.
SCEs are induced in cells by incorporation of BrdU into DNA. The cells are treated with BrdU for one or two cycles. They are harvested at metaphase after the second cycle, stained and analysed. Besides BrdU, alkylating agents and proflavine also induce SCEs.
The exact mechanism resulting in SCE formation is not known. SCE analysis is useful for estimating the cytogenetic impact of some drugs given to patients in chemotherapy. A higher frequency of SCEs has been found in human beings exposed to environmental pollutants, or have cigarette smoking habits.