Mutation

Any change in the DNA sequence

Mutation is a process that produces a gene or chromosome that differs from the wild type
(arbitrary standard for what “normal” is for an organism).
It is most commonly defined as a spontaneous permanent change in a gene or chromosome
which usually produces a detectable effect in the organism concerned and is transmitted to
the offsprings.
Mutated gene or chromosome results from a mutational process while the organism or cell
whose changed phenotype is attributed to a mutation is said to be a mutant.
The mutation may result due to changes either on the gene or the chromosome itself. Thus,
broadly mutation maybe:
Gene mutation where the allele of a gene changes.
Chromosome mutation where segments of chromosomes, whole chromosomes, or entire
sets of chromosomes change.
Mutagenesis is the process of inducing mutation by a number of physical, chemical or
biological agents.
The agents that causes mutation are called as mutagens. Mutation induced by mutagens is
called induced mutation. Sometime mutation occur spontaneously due to error during DNA
replication. However, mutagens increases the chances of mutation.
The development and function of an organism is in large part controlled by genes.
Mutations can lead to changes in the structure of an encoded protein or to a decrease or
complete loss in its expression. Because a change in the DNA sequence affects all copies of
the encoded protein, mutations can be particularly damaging to a cell or organism. In
contrast, any alterations in the sequences of RNA or protein molecules that occur during
their synthesis are less serious because many copies of each RNA and protein are
synthesized.

Geneticists often distinguish between the genotype and phenotype of an organism. Strictly
speaking, the entire set of genes carried by an individual is its genotype, whereas the
function and physical appearance of an individual is referred to as its phenotype. However,
the two terms commonly are used in a more restricted sense: genotype usually denotes
whether an individual carries mutations in a single gene (or a small number of genes), and
phenotype denotes the physical and functional consequences of that genotype.
 Mutations
Mutations Involve Large or Small DNA Alterations
A mutation involving a change in a single base pair, often
called a point mutation, or a deletion of a few base pairs
generally affects the function of a single gene (Figure 8-4a).
Changes in a single base pair may produce one of three types
of mutation:
Missense mutation, which results in a protein in which one
amino acid is substituted for another
Nonsense mutation, in which a stop codon replaces an amino
acid codon, leading to premature termination of translation
Frameshift mutation, which causes a change in the reading
frame, leading to introduction of unrelated amino acids into
the protein, generally followed by a stop codon
Small deletions have effects similar to those of frameshift
mutations, although one third of these will be in-frame and
result in removal of a small number of contiguous amino acids.
The second major type of mutation involves large-scale changes in chromosome structure
and can affect the functioning of numerous genes, resulting in major phenotypic
consequences. Such chromosomal mutations (or abnormalities) can involve deletion or
insertion of several contiguous genes, inversion of genes on a chromosome, or the exchange
of large segments of DNA between nonhomologous chromosomes.
https://www.ncbi.nlm.nih.gov/books/NBK21578/
Mutations Occur Spontaneously and Can Be Induced
Mutations arise spontaneously at low frequency owing to the chemical instability of purine
and pyrimidine bases and to errors during DNA replication. Natural exposure of an organism
to certain environmental factors, such as ultraviolet light and chemical carcinogens (e.g.,
aflatoxin B1), also can cause mutations.

A common cause of spontaneous point mutations is the deamination of cytosine to uracil in

the DNA double helix. Subsequent replication leads to a mutant daughter cell in which a T·A
base pair replaces the wild-type C·G base pair. Another cause of spontaneous mutations is
copying errors during DNA replication. Although replication generally is carried out with high
fidelity, errors occasionally occur.
 Induced Mutants
Spontaneous mutation rates are low; in most bacterial genes for example, the rate is
approximately 10–10 per generation per gene. The mutation rate can be greatly increased
by using mutagens, which are of two types. Physical mutagens include ultraviolet, γ and X
radiation; and chemical mutagens are compounds such as ethane methane sulphonate
(EMS), nitroso methyl guanidine (NTG), nitrous acid and acridine mustards.
Mutants are formed when the mutagens induce modifications of the base sequences of
DNA that result in basepair substitutions, frame-shift mutations or large deletions that go
unrepaired
Physical agents
(i) Ionizing radiations: X-rays, gamma rays, alpha-particles, and fast neutrons are ionizing
radiations and have all been successfully used to induce mutation. X-rays are produced by
commercially available machines. Gamma rays are emitted by the decay of radioactive
materials such as Cobalt. Fast neutrons are produced by a cyclotron or an atomic pile.
Ionizing radiations are so called because they knock off the outer electrons in the atoms of
biological materials (including DNA) thereby causing ionization in the molecules of DNA. As
a result, highly reactive radicals are produced and these cause changes in the DNA. Some
authors do not advise the use of ionizing radiations unless all other methods fail. This is
partly because the equipment is expensive and therefore not always readily available, but
also because ionizing radiations are apt to cause breakage in chromosomes.
(ii) Ultraviolet light: The mutagenic range of ultraviolet light
lies between a wavelength of 200 and 300 nm. ‘Low
pressure’ UV lamps used for mutagenesis emit most of
their rays in the 254 nm region. The suspension of cells or
spores to be mutagenized is placed in a Petri dish 2–3 cm
below a 15 watt lamp and stirred either by a rocking
mechanism or by a magnetic stirrer. The organisms are
exposed for varying periods lasting from about 300 seconds
to about 20 minutes depending on the sensitivity of the
organisms. The main effect of ultraviolet light on DNA is the
formation of covalent bonds between adjacent pyrimidine
(thymine and cytosine) bases. Thymine is mainly affected,
and hence the major effect of UV light is thymine
dimerization, although it can also cause thymine cytosine
and cytosine-cytosine dimers. Dimerization causes a
distortion of the DNA double strand. The ultimate effect is
to inhibit transcription, until finally the organism dies.
(ii) Ultraviolet light: What is UV Light?
Ultraviolet (UV) light is the part of the
electromagnetic spectrum between 200
and 400 nm, with shorter wavelengths than
violet of the visible spectrum (hence the
name, ultraviolet). This range is further
divided into short wave (200-280 nm, UV-
C), middle wave (280-315 nm, UV-B), and
long wave (315-400 nm, UV-A) light. The
shorter wavelength UV-C light carries with
it a lot more energy than its long wave UV-
A counterpart, and is much more damaging
to DNA.
UV-B and UV-C rays are two types of high-energy radiation that are capable of ionizing
(removing electrons from) molecules in a process called a photochemical reaction that
leads to new molecular products. The mutagenic range of ultraviolet light lies between a
wavelength of 200 and 300 nm. ‘Low pressure’ UV lamps used for mutagenesis emit most
of their rays in the 254 nm region.
UV damage occurs via two distinct types of mutations:

• Dimerizing mutations
• Oxidative mutations

The main effect of ultraviolet light on DNA is the

formation of covalent bonds between adjacent
pyrimidine (thymine and cytosine) bases. Thymine is
mainly affected, and hence the major effect of UV light
is thymine dimerization, although it can also cause
thymine cytosine and cytosine-cytosine dimers.
Dimerization causes a distortion of the DNA double
strand. The ultimate effect is to inhibit transcription,
until finally the organism dies.
Oxidative Mutations
UV exposure doesn’t always lead directly to mutations
in the DNA. In fact, UV-A radiation commonly causes
the creation of a free radical that then interacts with
and oxidizes DNA bases. These oxidized bases don’t
pair correctly during replication, causing mutations.

One example of this is a G to T transversion mediated

by reactive oxygen species. The oxidation of guanine
into 8-oxoguanine prevents the hydrogen bonding
required to base pair with cytosine. Instead, during
replication, 8-oxoguanine can base pair with adenine
via two hydrogen bonds. When the second strand is
synthesized, the base position originally occupied by a
guanine is then replaced with a thymine, leading to a G
to T transversion.
Chemical mutagens
(i) Chemicals acting on resting DNA
Some chemical mutagens, such as nitrous acid and nitrosoguanidine, work by causing
chemical modifications of purine and pyrimidine bases that alter their hydrogen-bonding
properties. For example, nitrous acid converts cytosine to uracil which then forms hydrogen
bonds with adenine rather than guanine. These chemicals act on the non-dividing cell and
include nitrous acid, alkylating agents, and nitrosoguanidine (NTG, also known as MNNG).
(a) Nitrous acid:
(b) Alkylating agents: These are compounds with one or more alkyl groups which can be
transferred to DNA or other molecules.
Induce transitions, transversions, frameshifts, and chromosome aberrations (break
chromosomes), Alkylation of bases can change base-pairing properties, Can also activate
error-prone DNA repair processes
(a) NTG—nitrosoguanidine: also known as 1-methyl-3-nitro-1-nitrosoguanidine— MNNG: It
is one of the most potent mutagens known. It is reported to induce mutation in closely
linked genes. It is widely used in industrial microbiology.
(d) Nitrogen mustards: The most commonly used of this group of compounds is methylbis
(Beta-chlorethyl) amine.
 Deaminating agents

AT to GC

CG to TA

Nitrous acid deaminates bases

(a) • Adenine to hypoxanthine, which pairs
with cytosine
(b) • Cytosine to uracil, which pairs with
adenine
(c) • Guanine to xanthine, which pairs with
cytosine
https://quizlet.com/ca/370512921/mutation-unit-10-part-2-flash-cards/
Hydroxylating agents

Alkylating agents
Chemical mutagens
(ii) Base analogues
These are compounds similar to base nucleotides in composition and can be incorporated
into a dividing DNA in place of the natural base. However, this incorporation takes place
only in special conditions. The best examples include 2-amino purine, a compound that
resembles adenine, and 5-bromouracil (5BU), a compound that resembles thymine. The
base analogues however do not have the hydrogen-bonding properties of the natural base.
Base analogues are not useful as routine mutagens because suitable conditions for their use
may be difficult to achieve. For example, with BU, incorporation occurs only when the
organisms is starved of thymine.
Chemical mutagens
(iii) Frameshift mutagens (also known as intercalating agents)
Frameshift or intercalating agents are planar three-ringed molecules about the same size as
a nucleotide base pair. During DNA replication, these compounds can insert or intercalate
between adjacent base pairs thus pushing the nucleotides far enough apart that an extra
nucleotide is often added to the growing chain during DNA replication.
A mutation of this sort changes all the amino acids downstream and is very likely to create a
nonfunctional product since it may differ greatly from the normal protein. Furthermore,
reading frames (i.e. the DNA base sequences) other than the correct one often contain stop
codons which will truncate the mutant protein prematurely.
Acridines are among the best known of these mutagens, which cause a displacement or
shift in the sequence of the bases. Acridine, C13H9N, is an organic compound consisting of
three fused benzene rings. Acridine is colorless and was first isolated from crude coal tar. It
is a raw material for the production of dyes. Acridines and their derivatives are DNA and
RNA binding compounds due to their intercalation abilities.
Biological mutagens:
transposons and insertion sequence (IS) elements are biological mutagens. Examples; mutator gene,
bacteriophage MU etc.
Transposons and IS elements are small sequence of DNA that moves from one site to another along DNA
strand and causes mutation. Transposons and insertion sequences are also known as jumping gene. These
sequence contains gene which codes the enzyme transposase which helps in transposition of these
sequence from one site to other.
Reversion and Second-Site Suppression
Suppressor is a second mutation that restores a function lost by the primary mutation. A
suppressor mutation that occurs within the same gene is called an "intragenic suppressor",
and a suppressor mutation that occurs in a different gene is called an "intergenic
suppressor". Suppressors can arise in many ways.

Reversion of a mutation refers to a second mutational event that changes the phenotype to
its original state. Thus, a mutant strain with a mutation in the lacZ gene that causes a Lac−
phenotype can be reverted to wild type, the Lac+ phenotype, by a second round of
mutagenesis. One type of reversion event changes the mutated base pair back to the
original wild-type base pair, which is called a true revertant.

However, mutations at other positions (second-site revertants) can also reverse the
phenotype. A second mutation in lacZ might change the amino acid sequence of the gene
product, β-galactosidase, so that the combination of the two altered amino acids is now a
functional protein. Thus, the second amino acid change suppresses the defect caused by the
first change. http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/rev-sup/Suppressor-types.html
Reversion
Point mutations are divided into two
classes based on their effect on
phenotype:
a. Forward mutations change the
genotype from wild type to mutant.
b. Reverse mutations (reversions or back
mutations) change the genotype from
mutant to wild-type or partially wild-
type.
i. A reversion to the wild-type amino acid
in the affected protein is a true reversion.
ii. A reversion to some other amino acid
that fully or partly restores protein
function is a partial reversion.

https://slideplayer.com/slide/5733502/
Suppressor mutations occur at sites different from the original mutation, and mask or
compensate for the initial mutation without actually reversing it. Suppressor mutations
have different mechanisms depending on the site at which they occur.
a. Intragenic suppressors occur within the same gene as the original mutation, but at a
different site. Two different types occur:
i. A different nucleotide is altered in the same codon as the original mutation.
ii. A nucleotide in a different codon is altered (e.g., an insertion frameshift is suppressed by
a nearby deletion event).
b. Intergenic suppressors occur in a different gene (the suppressor gene) from the original
mutation. Many work by changing mRNA translation.
i. Each suppressor gene works on only one type of nonsense, missense or frameshift
mutation.
ii. A given suppressor gene suppresses all mutations for which it is specific.
iii. Suppressor genes often encode tRNAs that
recognize stop codons and insert an amino acid,
preventing premature termination of translation.
polypeptide gene undergoes a mutation from TTC -
--> ATC The mRNA codon changes from AAG (lysine)
to UAG (stop).
A tRNA gene undergoes an anticodon mutation
from UAC (tyrosine) ---> UAG (stop), but still carries
tyrosine.
The mutant tRNA binds to the mutant mRNA stop
codon, and adds tyrosine instead of lysine. A
polypeptide is made with one amino acid
substitution.
(1) Full or partial function of the polypeptide may
be restored.
(2) The effect depends on how compatible the
substituted amino acid is with protein function.
iv. Nonsense suppressors fall into three classes, one for each stop codon (UAG, UAA and UGA)
v. Typical tRNA suppressor mutations are in redundant tRNA genes, so the wild- type tRNA
activity is not lost.
vi. Nonsense suppression occurs by competition between release factors and suppressor
tRNAs.
(1) UAG and UGA suppressor tRNAs do well in competition with release factors.
(2) UAA suppressor tRNAs are only 1–5% efficient.
vii. Suppression by a tRNA occurs at all of its specific stop codons (e.g., UGA or UAG), not just
the mutant one. This may produce read-through proteins, but they are not as common as
expected, possibly due to tandem stop codons (e.g., UAGUGA).

https://slideplayer.com/slide/5733502/
Ames Test
Ames test it is a biological assay to assess the mutagenic potential of chemical compounds.
It utilizes bacteria to test whether a given chemical can cause mutations in the DNA of the
test organism. The test was developed by Bruce N. Ames in 1970s to determine if a chemical
at hand is a mutagen.
Ames test uses several strains of bacteria (Salmonella, E.coli) that carry a particular
mutation.
Point mutations are made in the histidine (Salmonella typhimurium) or the tryptophan
(Escherichia coli) operon, rendering the bacteria incapable of producing the corresponding
amino acid.
These mutations result in his- or trp- organisms that cannot grow unless histidine or
tryptophan is supplied.
But culturing His- Salmonella is in a media containing certain chemicals, causes mutation in
histidine encoding gene, such that they regain the ability to synthesize histidine (His+). This
is to say that when a mutagenic event occurs, base substitutions or frameshifts within the
gene can cause a reversion to amino acid prototrophy. This is the reverse mutation.
These reverted bacteria will then grow in histidine- or tryptophan-deficient media,
respectively.
Procedure
I ) Isolate an auxotrophic strain of Salmonella Typhimurium for histidine. (ie. His-ve)
II) Prepare a test suspension of his-ve Salmonella Typhimurium in a plain buffer with test
chemical. Also add a small amount of histidine.

Note: small amount of histidine is required so bacteria starts growing. Once histidine is
depleted only those bacteria mutated to gain the ability to synthesize histidine form
colonies.

III) Also prepare a control suspension of His-ve Salmonella Typhimurium but without test
chemicals.
IV) Incubate the suspensions at 37°C for 20 minutes.
V) Prepare the two agar plate and spread the suspension on agar plate.
VI) Incubate the plates at 37°C for 48 hours.
VII) After 48 hours count the number of colonies in each plate.
A sample’s mutagenic potential is assessed by exposing amino acid-requiring organisms to
varying concentrations of chemical and selecting for the reversion event. Media lacking the
specific amino acid are used for this selection which allow only those cells that have
undergone the reversion to histidine / tryptophan prototrophy to survive and grow. If the
test sample causes this reversion, it is a mutagen.