Mutation and Repair of Damaged DNA in Bacteria

The following points highlight the twelve main aspects of mutation and repair of damaged DNA in bacteria. Some of the aspects are: 1. Nature of Bacterial Variations 2. Spontaneous and Induced Mutations 3. Molecular Mechanism of Mutagenesis 4. Physical Mutagenic Agents 5. Mutation Rate and Mutant Frequency 6. Phenotypic Changes Due to Mutation and Others.

Aspect # 1. Nature of Bacterial Variations:

Till early 1940s, it was generally believed that variations occurring in bacteria were due to adaptation to the environmental conditions, rather than due to true genetic changes, i.e. mutations. Although it is true that bacteria can quickly adapt to changes in the environment, it is also true that mutations do occur in bacteria.

This was proved by a classical experiment conducted by Luria and Delbrtick in 1943. The experiment, known as fluctuation test, proved that the changes occurring in bacteria are true mutations, just as they occur in higher organisms. Because of its historical importance, the experiment and the conclusions drawn from it are briefly discussed next.

Fluctuation Test:

The objective of the experiment was to assess whether a variation in the character of bacteria, e.g. development of resistance to bacteriophage, was an adaptive phenomenon due to the presence of bacteriophage, or the phage resistance developed spontaneously, independent of the external factor i.e. bacteriophage.

E. coli B was selected as the experimental organism and the character examined was resistance to coliphage T₁. The organism is normally sensitive to the phage and on infection by the phage, the cells undergo lysis with release of large number of phage particles.

When a bacterial lawn of E. coli B in a Petri dish is exposed to a suspension containing large number of Ti phage particles, it is observed that most of the bacteria are killed and only a small number of bacteria survive to form isolated colonies on the plates. These colonies are derived from the phage resistant cells. When the bacteria from these resistant colonies are cultured and again exposed to the same phage, they are no longer killed.

This means that some bacteria have become permanently resistant to the phage. Therefore, the phage resistance property is a heritable character acquired by the bacteria. The question is how the bacteria, acquire this property.

According to the adaptation hypothesis, the property is acquired because the bacteria come in contact with the phage and the resistance is de sloped in some of them due to contact with the phage which acts as the causal agent for the change.

On the other hand, evidence to support the mutation hypothesis requires that the change from phage-sensitivity to resistance occurs spontaneously in bacteria irrespective of their contact with the phage. The main point of contention is whether the change occurs before or after the contact with the phage.

In a typical Luria and Delbruck experiment, an outline of which is shown in Fig. 9.48, a suspension of a fresh culture of E. coli B was prepared in nutrient broth containing 10³ cells/ml. The suspension was divided in two parts. From one part, aliquots of 0.5 ml each were distributed in 40 tubes to constitute a set of independent cultures and from the other part 20 ml was taken in a single tube which constituted a bulk culture.

The tubes of both type were incubated for 36 hr at 37°C to allow the bacteria to multiply. As all the cultures initially contained the same number of bacteria/ml, and they were grown under identical conditions, it could be expected that after incubation they would have more or less similar population density.

Next, small aliquots (0.1 ml) from each tube of the independent cultures, as well the bulk culture were spread on large number of replicate plates coated with the phage T₁. After incubation for 24 hr at 37°C, the number of colonies growing on each plate was counted.

Colonies on phage-coated plates were evidently produced by the phage-resistant bacteria originally present in the aliquotes spread on each plate. It was observed that the number of phage-resistant colonies in the 40 independent cultures showed a much greater fluctuation (i.e. a wider variation) than the number of resistant colonies growing on the plates prepared from the aliquots taken from the bulk culture.

The observation was interpreted as follows:

The greater fluctuation in the independent cultures was due to the origin of spontaneous mutations (from phage sensitivity to phage resistance) arising independently in the 40 tubes at different times. Each mutant multiplied to form progeny of resistant bacteria during the incubation period, so that the final number of resistant bacteria in different tubes was widely variable at the time of plating.

In contrast, the bulk culture contained a uniform population containing both sensitive and resistant bacteria. Any fluctuation that is observed in the plates prepared from the bulk cultures was due to chance variation. Thus, the wider fluctuation observed in the independent cultures could only he explained by assuming that phage-resistance arose due to spontaneous mutation at different times during incubation in different cultures before the bacteria came in contact with the phage.

If the adaptation hypothesis had been correct, the differential fluctuation in the independent cultures and the bulk culture would not have occurred, because the adaptation hypothesis holds that resistance develops only after the bacteria come in contact with the phage.

A more straight-forward evidence to prove that phage-resistance develops in a bacterial population which has never come in contact with the phage was provided by the replica-plating technique developed by Lederberg and Lederberg (1952).

When a dilution plate containing discrete colonies of E. coli is replicated once on a plate without phage and then on a phage-coated agar plate, it is observed after incubation that the phage-coated plate contains only few colonies of phage resistant bacteria.

The other plate contains colonies of both phage sensitive and phage resistant E. coli. By comparing the two plates, the phage-resistant colonies from this latter plate can be identified. When such a resistant colony is picked up and cultured, the bacteria exhibit phage-resistance property, even though they have never come in contact with the phage (Fig. 9.49).

Aspect # 2. Spontaneous and Induced Mutations:

A mutation involves a change in the sequence of bases of DNA. Such a change is reflected in the base sequence of m-RNA, provided the segment of DNA containing the changed sequence codes for a polypeptide. A change in the sequence of m-RNA may result in incorporation of an incorrect amino acid in the polypeptide chain and may lead to formation of an abnormal protein. An individual with such an abnormal protein is a mutant.

A change in the base sequence in DNA resulting in a mutation may occur spontaneously, or it may be induced artificially by treatment with physical or chemical agents. The factors which cause spontaneous mutations are not well known. One of the possible factors has been thought to be the abilities of some nucleic acid bases, like adenine and thymine to exist in two tautomeric forms as a result of which their normal base-pairing relations change. As a consequence, base sequence in the daughter strands during DNA replication is altered.

Mutations involving a single base-pair of DNA are called point mutations. Both spontaneous and induced mutations may change only a single base-pair. Point mutations may be of four different types— transition, trans-version, insertion and deletion. In a transition mutation, a purine base is replaced by another purine and a pyrimidine by another pyrimidine e.g. an A-T base-pair is replaced by a G-C base-pair and vice versa.

Transitions may be induced in bacteria using base-analogues, like 5-bromouracil or 2-amino purine as mutagen. The second type of point mutation is trans-version which involve replacement of a purine by a pyrimidine, or vice versa.

The alkylating agents, like ethyl methane sulfonate (EMS) which is extensively used as a mutagen in eukaryotic systems and nitrosoguanidine in bacteria are known to cause trans versions, though these agents generally cause transition type of mutation.

The third type of point mutation is insertion involving addition of an extra base into one strand of DNA which causes a complete change in the reading frame from the point of insertion downstream. Insertion causes a frame-shift mutation.

Insertions are caused by acridines. The fourth type of point mutation involves deletion of a base from one DNA strand. During replication the daughter strand may incorporate any one of the four nucleotides against the blank space in the template strand. As a result, the sequence in the daughter strand becomes different from the original. Deletion of bases is caused by alkylating agents when they act as depurinating agents and also by acridines.

The four types of point mutations are schematically shown in Fig. 9.50. It should be understood that all point mutations are not invariably expressed, because a single base substitution as it occurs in transition or trans-version, may not necessarily result in a change of an amino acid in the polypeptide chain, because of degeneracy of the genetic code.

In case the changed base sequence codes for the same amino acid as the wild-type sequence, there would be no change in the amino acid sequence of the polypeptide. Thus, even if a mutation occurs in DNA, it is not expressed (same sense mutation)

Even when an amino acid is changed (substituted) due to a mutation, the polypeptide and the protein containing it may still be functional, provided that the amino acid substitution does not affect its conformation, i.e. if the vital parts of the molecule are not affected. Such mutations which do not result in a change of the phenotypic character are known as silent mutations.

An example is provided by the protein haemoglobin. In course of evolution, haemoglobin molecule of mammals has suffered many mutations resulting in amino acid substitutions without affecting its overall function. But a single substitution of glutamic acid coded by (GAA/GAG) through valine (GUA/GUC/GUG/GUU) results in a defective haemoglobin — known as sickle-cell haemoglobin — which is the cause of sickle-cell anaemia.

A point mutation involving trans-version of adenine to uracil is enough to change the normal haemoglobin molecule to a defective one, because the change affects a vital portion of the complex molecule. Sickle-cell haemoglobin is defective in oxygen transport and the normal disc-shaped reticulocytes assume a sickle-shape under low oxygen tension.

Aspect # 3. Molecular Mechanism of Mutagenesis:

Various physical and chemical agents, known as mutagens, are used to induce mutation in different prokaryotic and eukaryotic organisms. Such agents act in different ways on DNA resulting in change of its base sequence.

The molecular mechanisms involved in mutagenesis by some of these agents are discussed:

(i) Nucleic Acid Base Analogues:

Analogues are compounds having structural similarity. Two well-known analogues of nucleic acid bases are 5-bromouracil, an analogue of thymine and 2-amino purine, an analogue of adenine (Fig. 9.51).

Due to structural similarity, 5-BU can be incorporated into replicating DNA as a substitute of thymine, so that the normal A-T base pair is replaced by A-BU. Due to the presence of a stronger electro-negative Br atom in place of a methyl group in thymine, 5-BU can assume an enol form which has a greater tendency to pair with guanine during replication.

As a consequence, during replication, guanine pairs with BU rather than adenine. In the next replication cycle, guanine pairs with its normal complementary base cytosine giving rise to a transition from an A-T base pair to a G-C pair (Fig. 9.52).

In a similar way, 2-amino purine which in its normal form pairs with thymine, but in a rare imino form, it pairs with cytosine. After replication, cytosine pairs in a normal way with guanine. Thus, A-T pair changes to G-C pair through transition (Fig. 9.53).

Base pairing of 5-bromouracil and 2-amino purine in their enol and imino-forms with guanine and cytosine, respectively, is shown in Fig. 9.54:

(ii) Chemical Modification of Bases:

Some compounds act as mutagens by modification of nucleic acid bases, so that their base-pairing relation is changed. Two important agents of this class of compounds are nitrous acid (or nitrite) and hydroxylamine. They can alter the bases in non-replicating DNA and can act even in isolated DNA.

Nitrous acid (HNO₂) acts on bases containing amino (-NH₂) groups, such as adenine, guanine and cytosine causing oxidative deamination. Thus, adenine is deaminated to hypoxanthine, guanine to xanthine and cytosine to uracil. As a result, the original base pairing relations are changed, because hypoxanthine pairs with cytosine and uracil pairs with adenine, whereas xanthine continues to pair with cytosine.

The outcome is A-T pair undergoes transition to G-C pair and G-C pair to A-T. Deamination of guanine to xanthine does not change the base pairing relation and hence is not mutagenic.

The base pairing relations and their consequences are shown in Fig. 9.55 and Fig. 9.56:

Another mutagenic agent of this class is hydroxylamine (NH₂OH) which has a limited mutagenic effect, because it can chemically modify only pyrimidines and has no effect on purines. Between the two pyrimidine bases, cytosine is specially affected and thymine is only negligibly.

Cytosine molecule is modified by addition of a hydroxyl group to the amino group. The modified cytosine molecule can no longer pair with its normal partner, guanine and instead pairs with adenine as shown in Fig. 9.57.

This results in a transition of a C-G base pair to T-A pair:

(iii) Alkylating Agents:

There are a number of chemical compounds which act as alkylating agents and can cause mutation. These agents add an alkyl group, like methyl, ethyl etc. to DNA bases.

Some of these compounds with their structures are given:

These compounds add an alkyl-group to the hydrogen-bonding oxygen atom which causes the bases to mispair with other bases. For example, alkylation of thymine causes it to pair with guanine and alkylation of guanine leads to pairing with thymine, rather than cytosine.

The sites of alkylation are shown in Fig. 9.58:

Mis-pairing of alkylated bases leads to transitional mutation A-T G-C. Besides transition, alkylating agents also cause removal of bases from DNA by depurination. These agents particularly attack guanine at N-7 (shown by an asterisk in above figure), causing labialization of the N-glycosidic bond (N-9) between the base and deoxyribose, so that alkylaguanine is removed from DNA creating a gap. During replication, any one of the four bases may be incorporated in the gap.

But usually an adenine is inserted into the daughter strand opposite the gap and a thymine pairs with it resulting in a trans-version. Thus, depurination may result in a trans-version, because guanine (purine) is replaced by thymine (pyrimidine). In case, the gap is filled by a purine, a transition may also occur, or if the gap is filled by guanine, it results in the restoration of the original sequence.

This is diagrammatically shown in Fig.9.59:

(iv) Acridines:

Mutagens produce mutation by base-substitutions, causing mostly transition and less commonly transversion. The acridines, like acridine orange or proflavin (structures shown below) act as mutagen by a quite different mechanism.

The three-ringed heterocyclic molecules of acridine dyes have a planar structure i.e. the three rings lie flat in a single plane. Acridines interact with the double helix of DNA by a process of intercalation. X-ray diffraction studies suggest that by virtue of their hydrophobic nature and planar structure, acridine molecules become interacalated between two adjacent bases with consequent extension and unwinding of the phosphodiester bond of the DNA double helix.

Thereby, the normal gap of 3.4A between two adjacent bases is extended to 6.8 a, because the thickness of the acridine molecules is approximately 3.4A. The intercalation of acridine at a low concentration may result either in addition of an extra base in one strand during replication, or may cause deletion of one base depending on whether acridine is bound to the template strand or the new strand that is being synthesized. The first event is known as insertion which is more common and the second event, deletion, is less common.

In case, an acridine molecule becomes intercalated between two adjacent bases of the template strand during DNA replication, a gap is created on the new strand while it is being synthesized. The gap can be exactly filled by insertion of an extra base thereby the daughter strand acquires an insertion of a base. This inserted extra base is retained during the next replication and a complimentary base occupies the position opposite it (Fig. 9.60A).

On the other hand, if an acridine molecule is intercalated in the new strand while it is being synthesized during DNA replication, the acridine molecule will lie opposite to a base of the template strand preventing entry of the complimentary base in the daughter strand, thereby one base will be missing from the daughter strand.

After shedding of the acridine molecule, the daughter strand will have one base deleted from it. When the daughter strand will replicate normally, it will form a complimentary strand with one base short. Thus, acridine may also result, though less frequently in deletion of one base from DNA (Fig. 9.60B).

As a result of insertion or deletion, the reading frame from the point of insertion or deletion is changed as in Fig 9.38. This type of mutation, known as frame-shift mutation, is quite different from the base-substitution mutations caused by base-analogues and nitrous acid. In the latter, substitution of a single base-pair affects only a single triplet and results in only one amino acid change in the polypeptide chain.

Such a change may or may not affect the function of the protein. In contrast, in frame-shift mutation, a whole range of triplets are changed leading to a totally non-functional protein. It may also lead to premature chain termination in case of shifting — a termination codon appears in the reading frame.

Another important difference between the base-analogue-induced mutation and acridine-induced mutation is that the former class of mutants can be easily reverted to their wild-type by treatment with base-analogues. But frame-shift mutants are not reverted by treatment with base-analogues, though they can be reverted by acridines.

Aspect # 4. Physical Mutagenic Agents:

Different types of radiations are known to affect biological functions of living cells including genetic changes.

Radiations are generally distinguished into two main types:

i. Ionizing radiations, like X-rays and y-rays, and

ii. Non-ionizing radiations like ultraviolet rays.

Various components of cells can absorb radiations in the form of photon quanta.

The energy content of photons depends on the wave­length of radiations and the two are inversely proportional i.e. shorter the wave length greater is the energy content of photons. For example, X-rays have 10⁴ times more energy than UV-photon.

From the point of view of mutagenesis, absorption of radiations by the DNA is of special significance. Several evidences support that DNA acts as the primary target of radiations in causing mutation. For example, it has been demonstrated that sensitivity to X-rays depends on the base composition of DNA. The resistance to X-rays is inversely related to the G-C content of DNA. On the other hand, resistance to UV increases with G-C content. The mechanism by which radiations induce mutation is best understood in case of ultraviolet light.

(i) Ionising Radiations:

X-rays and gamma rays (emitted by decay of radioactive cobalt Co⁶⁰) have been extensively used as mutagenic agents, particularly in eukaryotic systems, like plants and drosophila. X-rays cause extensive damage to a large number of cellular components including nucleic acids, but it is difficult to analyse the effects on individual components. However, the number of induced mutants among the survivors has been found to be directly proportional to the dosage of X-irradiation (Fig. 9.61).

X-rays and y-rays are known to affect DNA causing breaks in purine and pyrimidine rings. They also produce single-strand and double-strand breaks in the phosphodiester backbone of DNA. Strong ionizing radiations, while passing through water, lead to formation of free radicals which can cause multiple alterations in the nucleic acids. Many of these damages prove lethal to the cells. But biological organisms also possess mechanisms to repair some of these damages. As a result, some of the survivors may exhibit permanent genetic changes or mutations.

(ii) Ultraviolet Light:

Ultraviolet light having wavelength ranging between 200 to 300 nm has much less penetrability than X-rays. It is highly bactericidal and also mutagenic. Nucleic acids can absorb ultraviolet light and maximum absorption takes place at 260 nm. The bactericidal activity is maximal at 254 nm.

The absorption spectrum of nucleic acids and. action spectrum of bactericidal activity are nearly overlapping (Fig. 9.62) which suggests that absorption of ultraviolet light by nucleic acids is largely responsible for its lethal action.

Although the overlapping curves shown in Fig. 9.62 suggest that nucleic acids are the target of ultraviolet light in bacterial cells, they do not explain the mechanisms of either lethality or mutagenicity. One of the earliest observations showed that DNA synthesis (replication) was inhibited by ultraviolet light. However, the best known effect of UV-light on DNA is the formation of thymine- dimers.

The dimers are formed by coalescence of two adjacent thymine molecules of the same strand of DNA. The intrastrand dimerization results from the activation of the 5: 6 double bonds as shown in Fig. 9.63 to form a cyclobutane ring.

The fusion of two thymine’s of the same strand draws the two molecules closer to each other, thereby causing a local distortion in the normal double-helix structure of DNA. Also, the H-bonding’s between thymine of the affected strand and adenine of the other strand are broken. These factors seem to influence the normal DNA replication, more or less in a comparable way as a zipper gets jammed.

Distortion of the DNA double strand is schematically represented in Fig. 9.64:

Evidence has been produced to show that thymine dimers are formed both in vitro and in vivo. When a thymine solution is frozen and exposed to UV, thymine dimer formation takes place as shown by change in absorption properties. Thymine dimers have also been recorded when isolated DNA is exposed to UV.

Again, thymine dimers have been extracted from UV-irradiated bacteria. Therefore, so far as biological effects of ultraviolet light are concerned, thymine-dimer formation is thought to be the primary effect of photochemical reaction.

As UV-light has much longer wave-length than X-rays, it has restricted penetrability. UV-light has its biological effect mainly on microorganisms and viruses. In case of multicellular organisms, its activity is limited only to the surface layers.

An interesting phenomenon observed in connection with the bactericidal effect of UV-light is that the lethal effect of UV-irradiation can be significantly nullified when the irradiated bacteria are exposed to visible light immediately afterwards. This phenomenon has been called photo reactivation, because the bacteria which are inactivated by UV light are reactivated and made viable by exposure to visible light.

UV-light in the range from 218 nm to 313 nm has bactericidal property, whereas light in the range from 313 nm to 549 nm has photo reactivation property. The maximum effect is found at 380 nm. It appears, therefore, that exposure of UV-treated cells to visible light results in repair of the damage inflicted by UV-light.

Another way of reactivation of UV-treated cells has also been observed. The probability of survival of such cells increases significantly when they are kept in a liquid, such as a buffer solution, without any nutrients for some time. This is commonly known as liquid-holding recovery. It does not involve light of any sort.

Aspect # 5. Mutation Rate and Mutant Frequency:

In a growing population, mutations occur spontaneously and at random. At any given time, such a population contains a variable number of mutants of a particular genetic character. The number depends on the rate of mutation of that character. Mutation rate is defined as the probability of an individual cell to undergo mutation per generation.

On the other hand, mutant frequency is the total number of mutants present at any given time in the total population i.e. number of mutants/total number of cells in the population. The two parameters, viz. mutation rate and mutant frequency, are different, though the latter depends on mutation rate.

The data that are required to calculate mutation rate are the number of mutants arising in a given time period, the number of generations in that time period, and the mean number of organisms present at the beginning and at the end of that time period.

From these data, mutation rate can be determined using the following formula:

m = log_e2 (M₂-M₁)/(N₂-N₁)

where m=mutation rate, M₁, and M₂ are the number of mutants at the beginning and the end of the time period, and N₁, and N₂ are the mean number of organisms at the beginning and the end of the time period.

Mutation rates vary widely in the same organism depending on the character concerned. For example, in E. coli, resistance to bacteriophage has a mutation rate of 10^-7 to 10^-9; resistance to streptomycin and penicillin occurs at a rate of 1CT¹⁰ and 10^-7, respectively. On the other hand, auxotrophy for histidine occurs at a much greater rate of 10^-6.

Similarly, mutation rates of the same character in different organisms may be quite different. From the mutation rates it should be clear that mutations in general are rare events and, on the average, only one organism in a million or ten million turns out to be a mutant.

Aspect # 6. Phenotypic Changes Due to Mutation:

All phenotypic characters are subject to alteration by mutation, because such characters are controlled by genes and mutations result from changes in the genes. With special reference to bacteria, the phenotypic changes may affect morphological characters including both cell morphology and colony morphology as well as physiological, biochemical, serological characteristics, as also pathogenic properties.

The morphological mutations may affect formation of glycocalyx of bacterial cells giving rise to capsulated and non-capsulated variants. A well-known example is provided by the R and S strains of Streptococcus pneumoniae which were used for the classical experiment on transformation. Mutations may change many other morphological characters, such as loss of motility due to mutation in the gene(s) controlling synthesis of flagellar protein, production of pigments, sporogenesis etc.

Bacterial mutants that are of practical interest arc those affecting resistance to drugs and affecting production of useful metabolites, like antibiotics, amino acids, organic acids, vitamins etc. Another class of bacterial mutants has proved invaluable in scientific research in biochemistry and genetics of microorganisms. These are auxotrophic mutants which are deficient in synthesis of a particular enzyme required for production of an essential metabolite.

As a result, these mutants are unable to grow in a medium (minimal medium) which sustains growth of the parent organism (wild-type). These mutants obligately require supplementation of the minimal medium with the metabolite which the mutant is unable to synthesise.

Such a medium which supports growth of auxotrophic mutants is called a complete medium and the specific metabolite which is added to the minimal medium to make it complete is called a growth factor. Auxotrophy has been induced by various mutagens in numerous bacteria and fungi for understanding the metabolic pathways and the problems relating to gene expression.

Just as a forward mutation can change a phenotypic character, a mutation in the same gene can revert the change in the opposite direction restoring the lost character to its parental type. Such mutations are known as back mutation or reversion. An important application of revertants is utilized in the Ames test for detecting carcinogenic activities of various chemical compounds.

In the Ames test, the histidine auxotroph’s (his^–) of Salmonella are employed for testing the abilities of various chemical compounds to revert the histidine auxotroph’s back to their parental forms i.e. from his^– to his⁺, because carcinogenic chemicals are also mutagenic. The revertants of auxotroph’s are generally known as prototroph. Reversion of auxotroph’s to prototroph also occurs spontaneously.

Therefore, Salmonella his auxotroph’s are incubated under appropriate condition both in presence of the chemical to be tested for its carcinogenic activity, as well as in its absence. From the number of revertants growing on plates containing a minimal medium from both sets, an indication is obtained regarding the mutagenicity and therefore of carcinogenicity of the chemical tested.

Many potential carcinogens are converted to active carcinogens in the body by enzymatic reactions. For this purpose, the Salmonella mutants are incubated in a medium containing such enzymes, e.g. in presence of rat liver extracts which is a rich source of such enzymes.

Reversion of a mutant phenotype may also be due to mutations other than back-mutation of the same gene involving the same site. For example, a second mutation at a different site may also restore the wild-type phenotype. In other words, a second mutation may suppress the expression of the mutant phenotype. This type of mutation is called a suppressor mutation.

A suppressor mutation may be intragenic when it occurs in the same gene (but not at the same site), or intergenic when it occurs in a different cistron or gene. For example, treatment with acridine causes insertion or deletion of a single base in one of the DNA strands resulting in frame-shift mutation. When in such a mutant caused by insertion, another mutation occurs by deletion in a nearby site, then the reading frame is restored to a large extent and the protein coded by the gene retains more or less the properties of the wild type. This constitutes a reversion caused by intragenic suppressor mutation (Fig. 9.65).

An intergenic suppressor mutation may occur in two different cistrons coding for two polypeptides of the same protein molecule. A mutation may occur in one cistron to make a protein defective and non-functional. A second mutation may occur in the second cistron to produce a polypeptide which restores the activity of the first polypeptide. In that case, that second mutation is an intergenic suppressor mutation.

A quite different type of suppressor mutation occurs due to a suppressor t-RNA. Normally, polypeptide synthesis stops due to one of the three termination codons, UAG, UAA and UGA when they occupy the A-site of the m-RNA-ribosome complex. Sometimes, chain termination fails to occur due to the presence of a mutant t-RNA having an anticodon which can pair with one of the termination codons.

Normally, there is no t-RNA which can pair with the termination codons. Such mutant t-RNAs are known as suppressor t-RNAs. For example, a t-RNA having an anticodon CUU which is specific for lysine may be altered due to a transcriptional error to CUA by trans-version of the third base U to A.

The t-RNA with the altered anticodon sequence can pair with the termination codon UAG and it will incorporate lysine in the polypeptide chain where chain termination should have occurred. Thus, chain termination is suppressed by incorporation of an amino acid.

Aspect # 7. Conditional Mutations:

In some mutations, the mutant phenotype is expressed under certain environmental conditions and in other conditions the mutant behaves like the wild-type. Such mutations are known as conditional mutations. A well-known example of this type is temperature-sensitive mutants of many bacteria (the Ts-mutants).

A Ts-mutant behaves like the wild-type at 30°C to 34°C, but at 39°-40°C the mutant phenotype is expressed. Such a behaviour of a Ts-mutant is ascribed to the inactivation of the gene product (some enzyme protein), rather than to a change in the gene itself. The conditional mutants behave as normal organisms under permissive conditions and as mutants under non-permissive environmental conditions.

Aspect # 8. Mutational Hot-spots:

A cistron consists of a long DNA sequence. Theoretically, a change of base sequence may occur at any spot constituting a mutation. But the classical genetical study of the rII gene of bacteriophage T₄ conducted by Benzer (1961) revealed for the first time that point mutations were not distributed evenly, but restricted to certain sites. These sites were designated as hot spots.

Whereas in other sites only a few mutations could be induced, two hot spots produced about 300 in one and 500 or more mutations in the other. Why some sites are more susceptible to mutation than others is not exactly known. One of the possible explanations might be that all point mutations do not affect the amino acid sequence of a protein equally and such mutations may go undetected.

The same-sense mutations do not change the amino acid sequence due to degeneracy of the genetic code. Even all missense mutations are not phenotypically expressed (silent mutations). Only such missense mutations which lead to an amino acid substitution affecting the vital portions of a polypeptide chain are phenotypically expressed and are detectable. The hot spots might code such vital amino acids.

Another probable factor contributing to higher mutability of certain portions of DNA is the presence of methylcytosine. About 1 to 5% of cytosine in normal DNA in both prokaryotes and eukaryotes is methylated. Methylcytosine spontaneously deaminates to produce thymine. During DNA replication methylcytosine-guanine base-pair changes to thymine-adenine base-pair constituting a transitional mutation. So, the sites of DNA rich in methylcytosine tend to mutate at a higher frequency compared to other sites.

Aspect # 9. Site-directed Mutation:

Both spontaneous and induced mutations occur at random and it is generally not possible to get mutants of any desired genes at the will of the experimenter. To obtain a mutant of a specific gene, it becomes necessary to select a desired mutant from a large population of the wild-type and mutants involving many characters. The development of modern recombinant DNA technology has now made it possible to induce site-specific mutations. A variety of procedures for inducing mutation at specific sites of DNA has now been developed.

The general principle in inducing a site-directed mutation in a desired gene is to isolate the gene and manipulate it to introduce an altered base sequence. The segment of DNA so manipulated is next introduced into a plasmid by recombinant DNA technology. The recombinant plasmid is then introduced into the host bacterium for replication and gene expression.

Aspect # 10. Phenotypic Lag:

A mutational event generally causes a change in base pair of a segment of DNA sequence present in a cistron. In diploid eukaryotic organisms, a gene of only one homologous chromosome becomes mutated, while the corresponding gene of the other homologous chromosome remains unaltered. The organism becomes heterozygous in relation to the particular gene.

Unless the mutation is of dominant type, the mutant phenotype is not expressed immediately after the incidence of mutation. The mutant phenotype can be expressed only after inbreeding for several generations which lead to segregation of the mutant gene to produce a homozygous mutant individual. This time-lag between incidence of mutation of a gene and its ultimate expression as a mutant phenotype is known as phenotypic lag.

In case of prokaryotic organism having only one chromosome, the phenotypic lag should have been absent. But in practice, phenotypic lag occurs also in prokaryotes. A peculiar feature of prokaryotic organisms, like bacteria is that DNA replication takes place at a faster rate than cell division.

As a result, most bacteria growing exponentially possess more than one copy of DNA, usually four per cell. Of these DNA molecules, only one contains a mutant gene. For expression of the mutant gene, unless it is a dominant one, it requires several cycles of cell divisions to reach a homozygous condition (Fig. 9.66).

Aspect # 11. Selection of Mutants:

As mutation is a rare phenomenon, the number of mutants of a particular character is so small in comparison to the wild-type that the detection of a mutant is often a difficult task. It is comparatively easier when the mutation imparts a property which is not possessed by the wild-type e.g. resistance to bacteriophage or antibiotics, ability to utilize a substrate which is not utilized by the wild-type, ability to produce metabolites, like amino acids, vitamins etc., and auxotrophy to prototrophy. But in case of mutations which develop a negative trait, it is much more difficult to select a mutant. Special techniques have been developed to detect such mutants.

Aspect # 12. Mechanisms of Repair of Damaged DNA:

All the changes caused either spontaneously or induced by artificial agents in the base sequence of DNA do not result in mutations, because organisms possess in-built mechanisms for repairing the damages in DNA.