▪ Mutation is any permanent change in the sequence of nucleotides in DNA resulting in
changes either on the gene or the chromosome. Mutation is induced due to either errors during replication, or by radiation, toxins, and other environmental factors.
▪ Cells use different DNA-repair mechanisms to undo the changes that occurred in the DNA strand like excision repair, photoreactivation repair, mismatch repair and many more. But, the accumulation of mutation can cause cancer.
Thus, broadly mutation maybe:
1- Gene mutation where the allele of a gene changes.
2- Chromosome mutation where segments of chromosomes, whole chromosomes, or
entire sets of chromosomes change Mutations due to the Type of Cell Involved 1- Somatic mutations
▪ Mutations that are in the somatic tissues of the body.
▪ Mutations are not transmitted to progeny.
▪ The extent of the phenotypic effect depends upon whether the mutation is dominant or recessive
(dominant mutations generally have a greater effect).
2- Germinal mutations
▪ Mutations that are in the germ tissues of the body.
▪ Mutations may be transmitted to progeny
▪ Dominant mutations are seen in first generation after the mutation occurs
▪ If a female gamete containing an X-linked mutation is fertilized, the males will show mutant phenotype Mutations due to Mode of Origin
1- Spontaneous mutations ▪ The spontaneous mutations occur suddenly in the nature and their origin is unknown.
▪ They are also called “background mutation” and have been reported in many organisms such as, Oenothera, maize, bread molds, microorganisms (bacteria and viruses), Drosophila, mice, man, etc.
2- Induced mutations
Besides naturally occurring spontaneous mutations, the mutations can be induced artificially in the living organisms by exposing them to abnormal environment such as radiation, certain physical conditions i.e., temperature, radiations and chemicals. Mutations due to Direction
1- Forward mutations In an organism when mutations create a change from wild type to abnormal phenotype, then that type of mutations are known as forward mutations. Most mutations are forward type.
2- Reverse or back mutations
The forward mutations are often corrected by error correcting mechanism, so that an abnormal phenotype changes into wild type phenotype. Mutations due to Magnitude of Phenotypic Effect 1- Dominant mutations ▪ The mutations which have dominant phenotypic expression are called dominant mutations.
▪ For example, in man the aniridia (absence of iris of eyes) occurs due to dominant mutant gene.
2- Recessive mutations ▪ Most mutations are recessive in nature and so they are not expressed phenotypically immediately.
▪ The phenotypic effects of mutations of recessive gene is seen only after one or more generations.
Mutations due to Type of Chromosome Involved
According to the types of chromosomes, the mutations may be of following two kinds:
1- Autosomal mutations. This type of mutation occurs in autosomal chromosomes.
2- Sex chromosomal mutations. This type of mutation occurs in sex chromosomes. Structural changes in chromosomes:
Changes in number of genes-
1. Loss: Deletion which involves loss of a broken part of a chromosome.
2. Addition: Duplication which involves addition of a part of chromosome.
Changes in gene arrangement:
1. Rotation of a group of genes 1800 within one chromosome: Inversion in which broken segment reattached to original chromosome in reverse order.
2. Exchange of parts between chromosomes of different pairs: Translocation in which the broken segment becomes attached to a non- homologous chromosome resulting in new linkage. Numerical Changes in chromosomes
Euploidy ▪ It involves the loss, or gain, of whole chromosome set. ▪ Euploidy designates genomes containing chromosomes that are multiples of basic number (x). ▪ The number of chromosomes in a basic set is called the monoploid number, x. ▪ Those euploid types whose number of sets is greater than two are called polyploid. ▪ Thus, 1x is monoploid, 2x is diploid; and the polyploid types are 3x (triploid), 4x (tetraploid), 5x (pentaploid), 6x (hexaploid) and so on. ▪ Mutation due to Euploidy refers to the state of having a chromosome number that is an exact multiple of a basic chromosome set. This means the number of chromosome sets is increased in euploidy. Polyploidy Addition of one or more sets of chromosomes. They may be further: 1- Autopolyploidy- The autopolyploidy involves polyploidy, in which the same basic set of chromosomes are multiplied.
2- Allopolyploidy- The polyploidy results due the doubling of chromosome number in a F1 hybrid which is derived from two distinctly different species. Aneuploidy ▪ It involves the loss, or gain, of a part of the chromosome set. ▪ Hence, the number of chromosomes can be greater or less than that of wild type. ▪ Various types of aneuploidy can be identified as: nullisomy, monosomy, and trisomy.
1. Nullisomy (2n-2) is the loss of both chromosomes of the homologous pair.
2. Monosomy (2n-1) is the loss of a single chromosome of the homologous pair. 3. Trisomy is the gain of an extra chromosome (2n+1). Mutations according to Change in Amino Acid Sequence
1. Point mutations occur when a single nucleotide or nucleotide pair in the DNA is changed. There are three main types of point mutations: silent, missense, and nonsense.
I. Silent mutations occur when a base change results in the same amino acid being added to the protein. II. Missense mutations are a result of a base change that causes a different amino acid to be used to build the protein. III. Nonsense mutations occur when a base change results in the addition of a stop codon, which results in truncation of the protein. 2- Frameshift mutation- ▪ The addition or deletion of a base or bases such that the coding sequence of reading frame is changed. ▪ This leads to the changes in the amino acid sequences of the proteins. ▪ The altered codon may code for a different amino acid and change property of protein. ▪ The effect may be seen in altered phenotype which in turn changes the property of the protein. Base Pair Substitution Mutations
A. Transition: Mutation causing substitution of a purine by another purine or pyrimidine by another
pyrimidine.
Transition may be caused by (i) Tautomerization (ii) Deamination (iii) Base analogs
B. Transversion: Mutation causing substitution of a purine by pyrimidine or vice-versa.
Transversion is caused by (i) Depurination (ii) Alkylating agent
Causes of mutation Causes of mutation- Mutation is caused by the mutagenic agents, which are the factors responsible for the mutation. Mutagenic agents induce mutations in either of the following ways.
1. They may replace a base in the DNA.
2. They may alter the base in such a way which its specifically mis-pairs with another base.
3. They may damage the base so much that it can no longer pair with any base.
4. They may intercalate themselves in the DNA paving way for addition or deletion of base. Types of mutagens-
1. Physical mutagens.
2. Chemical mutagen Physical Mutagenesis- ▪ It refers to the phenomena that cause mutation in the DNA structure through physical energy sources like ionizing radiations, non-ionizing radiations and heat.
Physical mutagens-
▪ Ionizing radiations (like X-rays) primarily cause chain breaks in DNA.
▪ non-ionizing radiation (like UV-rays) causes covalent linkage between the adjacent bases. ▪ Heat causing DNA denaturation.
▪ Physical mutagens mainly include sources of electromagnetic radiations like X-rays,
α-rays, β-rays, UV-rays etc. and thermal heat, which can cause either direct DNA- damage or indirect DNA damage via highly reactive free radicals. Causes of physical mutagenesis-
1- Single stranded breakage in DNA:
▪ It results due to the mismatched 5′ and/or 3′ termini of DNA. Double-stranded breakage becomes lethal and results in the case of two single-strand breaks in the opposite DNA strands.
2- Base damage: ▪ It is the indirect DNA damage induced by the ionizing radiations through the formation of reactive free radicals. ▪ It involves mismatched base pairing due to addition, deletion and incorporation of the nitrogenous bases.
3- Dimerization of bases: ▪ Pyrimidine dimers commonly form under exposure to UV-light. ▪ Thymine-thymine dimer is the most evident pyrimidine dimer among the thymine-cytosine, cytosine-cytosine, thymine-uracil and uracil-uracil.
4- Crosslinking: ▪ Ionizing radiations cause interstrand crosslinking in the non-hybridized sequence of DNA. Ionizing Radiations-
▪ Ionizing radiations includes- X-rays, α-rays, β-rays and γ-rays, which can induce DNA damage due to their high ionizing property.
▪ Ionizing radiations have a low wavelength and high energy or frequency.
▪ They produce reactive ions, atoms or molecules on ionization, damaging the sugar- phosphate backbone or the phosphate ester linkages.
▪ High-energy radiations directly damage the DNA by causing chromosomal breakage
(either one or both the strands), chromosomal rearrangement and DNA crosslinking.
▪ However, ionizing radiations indirectly damage the DNA through the formation of free radicals. Non-ionizing Radiation ▪ UV-radiation is a non-ionizing radiation, which induce DNA damage because of the skin cells’ high absorption capacity.
▪ Nucleic acid (DNA) readily absorbs UV-light of wavelength 280 nm. Thus, DNA acts as a target molecule of the UV-rays.
▪ UV-radiation naturally originates from the sunlight. It has a high wavelength and low energy or penetration.
▪ Non-ionizing radiations result in DNA excitation due to photon (UVA and UVB) absorption by the cells, resulting in crosslinking, base damage, dimer formation.
▪ Dimerization in the pyrimidine bases is most evident than the purine bases. Photoreactivation- Photoreactivation is a unique mechanism that utilizes blue light to inverse the UV-induced photo adducts into its normal state by well-conserved DNA repair enzymes, Photolyases (PLs). Heat-
• Heat causes cause lethal effects on the DNA integrity and causes DNA denaturation.
• Under the temperature (>95 degrees Celsius), the double-stranded DNA denatures into single stranded DNA.
• The DNA denaturation results due to breakage in the N-glycosidic bond.
• Heat stress causes single-strand breaks that result in the cessation of the DNA replication.
• Besides this, heat stress also causes double-strand breaks and inhibits the DNA – repair system. Chemical mutagens. ▪ Many chemical substances have been responsible to increase the mutability of genes.
▪ Chemical mutagens are classified into major groups on the basis of their specific reaction with DNA.
1. Base analogs
2. Alkylating agents
3. Intercalating agents
4. Deamination
5. Hydroxylamine BASE ANALOGS- ▪ Base analogs are the chemical mutagens whose structures is similar to those of any of the four standard bases of DNA.
▪ DNA polymerases cannot distinguish these analogs from the standard bases, so if base analogs are present during replication, they may be incorporated into newly synthesized DNA molecules.
▪ 5-bromouracil (5BU) is an analog of thymine; it has the same structure as thymine except that it has a bromine (Br) atom on the 5-carbon atom instead of a methyl group.
▪ Normally, 5-bromouracil pairs with adenine just as thymine does, but it occasionally mispairs with guanine, leading to a transition (T • A → 5BU • A → 5BU • G → C • G).
▪ Through mispairing, 5-bromouracil can also be incorporated into a newly synthesized DNA strand opposite guanine. 2- ALKYLATING AGENTS-
▪ Alkylating agents are chemicals that donate alkyl groups, such as methyl (CH3) and ethyl (CH3–CH2) groups, to nucleotide bases.
▪ For example, ethylmethylsulfonate (EMS) adds an ethyl group to guanine,
producing O6-ethylguanine, which pairs with thymine.
▪ Thus, EMS produces C • G → T • A transitions.
▪ EMS also add an ethyl group to thymine, producing 4-ethylthymine, which then pairs with guanine, leading to a T • A → C • G transition.
▪ Because EMS produces both C • G → T • A and T • A → C • G transitions,
mutations produced by EMS can be reversed by additional treatment with EMS. 3- DEAMINATION- ▪ Deamination is the release of amine group by some chemicals.
1. Nitrous acid deaminates cytosine-
creating uracil, which in the next round of replication pairs with adenine, producing a C • G → T • A transition mutation.
2. Nitrous acid also changes adenine into hypoxanthine-
which pairs with cytosine, leading to a T • A → C • G transition.
which pairs with cytosine just as guanine does; however, xanthine pair with thymine, leading to a C • G → T • A transition. 4- HYDROXYLAMINE-
▪ Hydroxylamine is a very specific base-modifying mutagen that adds a hydroxyl
group to cytosine, converting it into hydroxylaminocytosine.
▪ This conversion increases the frequency of a rare tautomer that pairs with adenine instead of guanine and leads to C • G → T • A transitions. 5- INTERCALATING AGENTS- ▪ Proflavin, acridine orange, ethidium bromide, and dioxin are intercalating agents, which produce mutations by sandwiching themselves (intercalating) between adjacent bases in DNA,
▪ Intercalating agents distort the three-dimensional structure of the helix and causing single-nucleotide insertions and deletions in replication.
▪ These insertions and deletions frequently produce frameshift mutations, so the
mutagenic effects of intercalating agents are often severe.
▪ Because intercalating agents generate both additions and deletions, they can reverse the mutations they produce. Ames test The Ames Test or Bacterial Reverse Mutation Test
▪ The Ames test is commonly used to test whether a particular chemical can cause mutations in the DNA of the test organism.
▪ This test is based on the principle of reverse mutation or back mutation. So, the test is also known as bacterial reverse mutation assay.
▪ A positive result indicates that chemical is mutagenic and therefore act as a carcinogen.
▪ This test serves as quick and convenient assay to estimate the carcinogenic potential of a compound. PRINCIPLE OF TEST:
▪ This test is based on the Salmonella typhimurium histidine (His) reversion system, involves mutation
of histidine locus in the genome.
▪ Auxotrophic mutant of Salmonella Typhimurium carry mutation in gene that encodes histidine.
▪ This mutant loss the ability to synthesize histidine are known as His- and require histidine in growth
media.
▪ Auxotrophic mutant of Salmonella Typhimurium salmonella is grown in media containing certain
chemicals, causes mutation in histidine encoding gene.
▪ In presence of mutagen His (-) regain the ability to synthesize histidine by reverse mutation.
▪ Such chemicals responsible to revert the mutation is actually a mutagen.
Screening procedures for isolation of mutants Replica plating technique
▪ Replica plating technique was invented by J. Lederberg (1952).
▪ Microspore are grown in complete media lacking streptomycin, both wild type and mutant were grown.
▪ These colonies are imprinted on velvet plate by inverting the petri plate so that all colonies are
imprinted and leave spores at corresponding positions on.
▪ Another plate having streptomycin is now pressed on velvet plate to get an impression.
▪ In streptomycin plate only mutant for streptomycin resistant will grow.
▪ Knowing the position of mutant colony, corresponding colon in original plate without streptomycin can
be marked and used for isolation and multiplication of mutant.
Filtration enrichment technique-
▪ Filtration enrichment technique was invented by Woodward (1954).
▪ Concentration of auxotrops (nutritional mutants) can be increased by
filtration of a culture grown in suspension culture with minimal media
on which only prototrophs will grow.
▪ During filtration due to filamentous growth of fungi, the prototrophs
will be filtered out and only non-growing auxotrops mutant spores
