Mutations Mutations in bacteria Types of mutations Carcinogens Kind of mutagenic agents

1. Mutations
By
Dr. YALAVARTHI NAGARAJU

2. Introduction
• Term mutation was introduced by Hugo de Vries in 1900
• Systematic studies on mutation began in 1910 with the discovery of white eye
mutant of Drosophila by Morgan
• Mutagenic action of X rays in Drosophila was discovered by H.J Muller in 1927
• Mutagenic action of X rays in barley was discovered by by Stadler in 1929
• Mutagenic action of mustard gas was discovered by Auerbach and Robson in
1946

3. Important points
• Mutations are random
• Mutant alleles are recessive but some are dominant
• Most of the mutations are harmful
• Mutations are recurrent
• Mutations occur at very low frequencies in nature
• Some genes are show exceptionally high rates of spontaneous mutations and are known
as mutable genes
• Some genes increase the spontaneous mutation rates of some other genes of the
genome such genes are called mutator genes
• Certain other genes suppress the mutation rate of some other genes of the genome.
Such genes are termed as anti-mutator genes
• Highly mutable sites within a gene are commonly known as hot spots
• Many agents both physical and chemical increase the frequency of mutations by several
fold these agents are known as mutagens

4. Important points
• Most mutant alleles are pleiotropic
• Mutations occur in both forward and reverse directions
• Rates of forward mutations are much higher than the those of reverse
mutations
• Some mutant alleles do not mutate back

5. Mutations
1) Spontaneous mutation
2) Induced mutations

6. Spontaneous mutations
• Spontaneous mutations occur naturally without any apparent or known cause
• The possible source of origin of spontaneous mutations are as follows
a) Errors during replication
b) Mutagenic effects of the natural environments of organisms
c) Transposons and insertion sequences
d) Methylation, followed by spontaneous deamination of DNA bases, especially
cytosine

7. • Some solar radiations are likely contribute to spontaneous mutation
• UV rays in sunlight are highly mutagenic
• Both eukaryotic and prokaryotic genomes contain some transposable elements
e.g; insertion sequences (IS), transposons etc. these elements integrate at
specific sites in the genome and produce gene mutations
• Errors committed by DNA polymerases during DNA replication. It was estimated
that DNA polymerase incorporates bases with a frequency of 10-5 proof reading
reduce this to 10-10
• A major cause of spontaneous mutation in E. coli is the modification of DNA after
synthesis e.g methylation of cytosine at position 5 by a methylase enzyme
• The rate of spontaneous mutation is very low
• It is generally ranges between 10-4 to 10-6/ gene/ generation for eukaryotes
10-5 to 10-7 / gene/ generation for prokaryotes

8. MUTAGENS
• Agents capable of inducing mutations are called mutagens.
• Mutations produced either by treating with chemical or physical agents are called induced
mutations
• The capacity of an agent for inducing mutations is termed as mutagenic property
• The process of inducing mutations through treatment with a mutagen is called as mutagenesis
The different mutagenic agents may be classified into two broad group.
1.Physical mutagens
2.Chemical mutagens
Induced mutations are useful in two ways
a) Genetic and biochemical studies
b) Crop improvement

9. PHYSICAL MUTAGENS
• The different types of radiations having mutagenic properties are known as physical mutagens.
• These radiations are high energy radiations
• Radiations are grouped into the following two classes depending on the kind of effect they have on the atoms in their
path.
a) Ionizing b) Non-ionizing radiations.
a) Ionizing radiations
They may be either non-particulate.
E.g.:- X-Rays, Gamma Rays, alpha, beta, fast neutrons and thermal neutrons
Some are particulate
E.g.:- alpha-rays, Beta rays, etc….
b) Non ionizing radiations
Eg: UV radiation

10. NON-IONIZING RADIATIONS
• UV Rays are only Non-ionizing radiations with mutagenic properties.
• Their wavelength ranges from 10-400 nm.
• Produced from the mercury vapour lamps
• The maximum absorption of UV rays by DNA occurs at a wavelength of 254 nm
• This is relatively low energy radiation, with poor penetration capacity (penetrates only surface) but highly
mutagenic
• Direct effects : formation of pyrimidine dimers and pyrimidine hydrates
• Indirect effects : organic peroxidases and free radicles are produced
• UV Rays are sub-divided based on wavelength
 1.UV-C-100-280 nm (mutagenic)
 2.UV-B-280-315 nm
 3.UV-A-315-400 nm

11. Dimer Formation:-
• Two pyramidine molecules located next to each other in the same DNA strand.
• DNA polymerase is unable to catalyze the replication of a DNA molecule in the region containing a
pyramidine dimer.
Hydration of Pyramidines:-
Generally cytosine is hydrated, but hydration of thymine also occurs.
↓ UV rays
Cytosine +H2O…………………. Cytosine hydrate.
A major portion of the mutagenic action of UV rays is believed to be due to pyrimidine dimer formation.

12. • Exposure of organisms to visible light, particularly to blue light after UV irradiation greatly reduces the
yield of mutations because bacterial cells are readily killed (or) inactivated on exposure to UV rays.
• The frequency of inactivated cells drops sharply if UV-irradiated cells are exposed to visible light.
• Both the above phenomena are called photoreactivation (first mechanism)
• This mechanism breaks the bonds between thymine dimers.
• the second repair mechanism called excision-repair. It involves the removal of thymine dimers.
• The third mechanism of DNA repair in E.coli involves DNA replication & recombination between the
two sister molecules thus produced.
• A fourth mechanism is based on the enzyme Uracyl-DNA glycolase.

13. Ionizing Radiation
• Ionizing radiations cause ionization in the atoms present in their path.
There are two types of ionizing radiations:
 1. Particulate and
 2. non-particulate ionizing radiations.
Ionization:
• An ion is an atom carrying either a positive or negative charge.
• It is produced when an atom either gains or loses an electron.
• The process of ion production is known as Ionization.
Particulate radiation
• These consists of high energy atomic particles produced by radioactive decay.
• Beta-rays are high energy electrons produced by radioactive decay of 3H, 32P, 35S, etc…they are negatively charged.
• Alpha-rays are composed of alpha-particles having two protons and two neutrons each.
• Alpha-particles have double positive charge.
• Fast neutrons are produced in cyclotrons or atomic reactors due to radioactive decay of heavier elements
• The velocity of fast neutrons is reduced by graphite or heavy water to generate thermal and slow neutrons

14. Non-Particulate Ionizing Radiations
• These radiations are represented by X-rays and gamma-rays.
• They are high energy radiations composed of photons.
• The physical properties and biological effects of X-rays and gamma-rays are similar.
• These radiations only differ in the source of their origin.
• X-rays are produced by X-ray tubes, while gamma-rays are produced by decay of some radioactive
isotopes, e.g.,14C, 60Co, etc.
• Both X-rays and gamma –rays are highly penetrating in biological tissues and sparsely ionizing.

15. Genetic Effects
• The genetic effects of radiations result from their effect on DNA
• The genetic effects include – change in base (eg. Deaminution)
• Loss of base,
• Disruption of hydrogen bonds between complementary strands of DNA
• Single and double strand breaks in DNA
• Cross linkage of chromosomal proteins
• The genetic effects of radiations may be
1) direct – ionization of DNA molecules
2) Indirect – ionization of molecules other than DNA

16. Genetic Effects
• The efficiency of radiations in including mutations is affected by a number of factors,
e.g.,
1. The stage of cell division
2. O2 concentration,
3. Temperature,
4. The plant or animal species,
5. Moisture content of seeds, etc.
The radiation is generally measured in roentgen, “is the dose of radiation which produces
one electrostatic unit of charge in one cm3”

17. Chemical mutagens
• There are many chemical agents which produce mutations, and they have diverse effects too.
• Some of these compounds transfer chemical groups to the nitrogen bases, leading to alterations
in replication.
• Others interleave themselves among the pairs of DNA bases and deform the double helix
structure.
• And there are others which, given their analogy with a base, substitute the base and provoke
errors in the translation of the proteins.
1. Base Analogs
2. Alkylating agents
3. Intercalating agents
4. Metal ions
5. Other agents

18. Base analogues
• The base analogs are chemicals like the bases of DNA- purine, and pyrimidines
or structurally resemble the DNA bases.
• Bromouracil and aminopurine are two common base analogs incorporated into
DNA- rather than usual bases, during the method of replication.
• The 5-bromouracil are artificially synthesized molecules- a base analog
utilized within the genetic research which is incorporated in DNA in situ of the
thymine.
• Rather than the methyl of the thymine, the bromouracil contains Br group-
highly resembles the thymine.

19. 5-bromouracil
an analog of thymine
N
N
1
2
3
6
5
4
O
O
5BU
Br N
N
1
2
3
6
5
4
CH₃
T
O
O

20. N
N
1
2
3
6
5
4
Br
O
5BU
O
N
N
1
2
3
6
5
4
Br
O
5BU
OH
Keto
pairs with A
Enol
mispair with G

21. 2-amino purine (P)
 Base analog of adenine
 Normally pairs with thymine
 May mispair with cytosine
 Causes a transition mutation

22. Alkylating agents
• Ethyl nitrosourea, poison gas, diethyl sulphate (DES), dimethyl nitrosamine (DMN),
methylhydrazine, temozolomide, dacarbazine, busulfan, Ethyl ethane sulfate (EMS) and vinyl
chloride are common alkylating agents that add alkyl radical to the DNA and damages it.
• These agents result in ethylation or methylation of nitrogen bases and induce base-pairing
errors by increasing ionization and producing gaps within the DNA strand.
• It adds an ethyl group to guanine and produces 6-ethylguanine, which pairs with thymine and
leads to CG:TA transitions
• Also adds an ethyl group to thymine to produce 4-ethylthymine, which then pairs with
guanine, leading to a TA:CG transition
• Mutations produced by EMS can be reversed by additional treatment with EMS.
• The alkylated purine bases are removed by the phenomenon called depurination, although
depurination isn't mutagenic.
• It might be repaired by the DNA repair pathway.

23. Nitrous acid: causes deamination
Cytosine Uracil
N
N
CYTOSINE
1
2
3
6
5
4
NH2
O
H
N
N
1
2
3
6
5
4
O
o
HNo2
URACIL
H
Nitrous acid produces exclusively transition mutations
Both C.G T.A & T.A C.G transitions are produced
Thus mutations can be reversed with the nitrous acid

24. Hydroxl amine
 Specific base modifying mutagen which adds a hydroxyl group to cytosine
producing hydroxlamine cytosine which pairs with adenine instead of
guanine
 This Leads to C.G T.A tranisitions
 Acts only on cytosine thus can not revert the mutation produced

25. Intercalating agents
• The EtBr- ethidium bromide used during the agarose gel
electrophoresis is one in every one of the intercalating agents.
• Other intercalating agents like proflavine, acridine orange, or
daunorubicin, dioxin, operated by an identical mechanism like
the EtBr.
• The molecules intercalate between the bases of DNA and disrupt
its structure as they are about the same size as a nucleotide
• They distort the three-dimensional structure of the helix and
cause single-nucleotide insertions and deletions in replication
• If it's incorporated during the replication, it can cause frameshift
mutation. It's also going to block transcription.

26. Metal agents
• Metal ions are also dangerous to our DNA because it acts in styles of other
ways.
• Nickel, chromium, cobalt, cadmium, arsenic, chromium, and iron are a
number of the standard metal ions that cause mutations.
• The metal ions work by producing ROS (reactive oxygen species), hindering
the DNA repair pathway, causing DNA hypermethylation, or may directly
damage the DNA.

27. Biological mutagenic agents
Virus
• We all fathom HIV, right! A causative agent of AIDS. Infections are common mutagens that
are well-known to us and create lethal health issues. Viruses insert their DNA into our
genome and disrupt the traditional function of DNA or genes. Once it inserts DNA, the DNA
replicates, transcribes, and translates viral protein rather than our protein. Mature viral
particles form in an exceeding cell.
Bacteria
• Some bacteria also are dangerous for our DNA- cause inflammation. It provokes DNA
damage and DNA breakage.
Eg: Pseudomonas aeruginosa – cystic fibrosis
Transposons
• Lesser-known biological mutagens are transposons. The transposons are non-coding DNA
sequences, jump from one place to a different place in an exceedingly large genome, and
influence the function of genes.

28. Other chemical mutagens
• Reactive oxygen species,
• Benzene,
• Gum elastic,
• Rubber products,
• Sodium azide,
• Aromatic amines,
• Alkaloids,
• Deaminating agents, and
• PAH (Polycyclic aromatic hydrocarbons) are other mutagens that make different
mutations.

29. Mutation rates
 The frequency with which a gene changes from the wild type to a mutant is referred
to as the mutation rate.
 Expressed as the number of mutations per biological unit i.e. mutations per cell
division, per gamete per round of replication
e.g. mutation rate for achondroplasia (hereditary dwarfism) is about 4 mutations per
100,000 gametes
Mutation frequency:
 Incidence of a specific type of mutation with in a group of individual organism
e.g. for achondroplasia, the mutation frequency in united states is about 2x10⁻⁴

30. Somatic mutations
• Arise in the somatic cells
• Passed on to other cells through the process of mitosis
• Effect of these mutations depends on the type of the cell in which they occur & the
developmental stage of the organism
• If occurs early in development, larger the clone of the mutated cells
Germ line mutations
• They occur in the cells that produce gametes (meiosis)
• Passed on to future generations
• In multicellular organisms, the term mutation is generally used for germ line mutations

31. Forward mutation:
 a mutation that alters the wild type phenotype
Reverse mutation (reversion):
 a mutation that changes a mutant phenotype back in to the
wild type
Nonsense mutation: changes a sense codon into a nonsense codon.
Nonsense mutation early in the mRNA sequence produces a greatly
shortened & usually nonfunctional protein

32. Nonsense mutation: changes a sense codon into a
nonsense codon. Nonsense mutation early in the mRNA
sequence produces a greatly shortened & usually
nonfunctional protein
TCA
AGT
UCA
TGA
ACT
UGA
Ser
Stop codon

33. Silent mutation: alters a codon but due to degeneracy of
the codon, same amino acid is specified
TCA
AGT
UCA
TCG
AGC
UCG
Ser Ser

34. Neutral mutation: mutation that alters the amino acid
sequence of the protein but does not change its function as
replaced amino acid is chemically similar or the affected aa
has little influence on protein function.
CTT
GAA
CUU
ATT
TAA
AUU
Leu Ile

35. Loss of function mutations:
 Complete or partial loss of the normal function
 Structure of protein is so altered that it no longer works correctly
 Mutation can occur in regulatory region that affects transcription ,
translation or splicing of the protein
 Frequently recessive
Gain of function mutations:
 Produces an entirely new trait
 Causes a trait to appear in inappropriate tissues or at inappropriate times in
development
 Frequently dominant
Conditional mutations:
 Expressed only under certain conditions
Lethal mutations:
 Cause the death of the organism

36. Suppressor mutation:
 Suppresses the effect of other mutation
 Occurs at a site different from the site of original
mutation
 Organism with a suppressor mutation is a double mutant
but exhibits the phenotype of un mutated wild type
 Different from reverse mutation in which mutated site is
reverted back into the wild type sequence

37. Mutagenic Effect
• The mutagens are genotoxic- harmful to our DNA in some ways; some
directly affect the DNA some indirectly. And thus, the precise effect of every
mutagen remains unknown to us.
• At the chromosomal level, the mutagens can alter the structure or number of
chromosomes.
• As deletion, insertion, duplication, translocation, monosomy, and
nondisjunction are a number of the chromosomal abnormalities produced by
mutagens.
• The mutagens also affect or dysregulate the molecular central dogma
process- replication, transcription, and translation.
• At the molecular level, the mutagens create different gene mutations that
end up in the loss of function, altered function, or non-functional protein.

38. Mutagens vs. Carcinogens
• Carcinogens are compounds that induce serious mutations
to DNA that can lead to cancer.

39. Mutagen Screening:
Ames Test : Salmonella typhimurium
• Reverse mutation test.
• Mutant bacteria that requires histidine to grow. Mutation is from a single
nucleotide.
• Expose bacteria to potential mutagen and grow on a plate without media.
• Mutagens will randomly reverse the original mutation, allowing bacteria to
grow without histidine.

40. • If bacteria grow = compound is mutagenic
• If bacteria does not grow = non-mutagenic

41. Common Exposures to Mutagens and Carcinogens
• Polycyclic aromatic
hydrocarbons (PAH)
- burning fossil fuels
- creosote-soaked
wood
(wood pilings)
• Nitrosamines
- Meat preservatives
- Saliva
Why are my hot dogs always pink?

42. • Alleles-Different versions (sequences) of a gene.
• Mutant-Newly created allele made by mutagenesis.
• Genotype-The complete set of alleles for all genes carried by an individual.
• Wild type-Standard reference genotype. Most common allele for a certain trait.
• Phenotype-Observable trait specified by the genotype.
• Point mutation-A change in a single base pair (e.g., a G.C to A.T transition).
• Silent mutation-A point mutation in a codon that does not change the specified
amino acid.
• Missense mutation-A point mutation that changes the encoded amino acid.
• Nonsense mutation-A point mutation that introduces a premature stop codon
into the coding sequence of a gene.
• Recessive & dominant mutant alleles-(next slide)