MUTATION

* any change in the nucleotide sequence of the

genetic material. [DNA or RNA]]
*Rearrangement of DNA molecule is called mutation (in
broad terms)

A change may be a simple substitution of 1

nucleotide, or the insertion or deletion of 1 or
more nucleotides within a normal DNA sequence
Can be classified in 3 main groups:
Genome Mutations: Affecting chromosome numbers (10 -2
/ division)

Chromosome Mutations: Affecting chromosome structures (6.10 -4

)

Gene mutations: Affecting gene organısation (10 -10

/bp/div., 10-5/locus/ gene.)
 standar
d
base-pa
iring
arrange
ment
Base Substitutions or Point Mutations

• the exchange of a nucleotide for another

• caused by chemicals and malfunctions of the DNA
replication

Two classifications:
1. Transitions
• if same types of nitrogenous bases exchange
• E.g. a purine replaces a purine (A ↔ G)
a pyrimidine replaces a pyrimidine (C ↔ T)

• Causes
 nitrous acid
 base mispairing
 mutagenic base analogs
2. Transversion
• if different types of nitrogenous bases
exchange
• E.g. a purine exchanges with a pyrimidine or
vice versa (C/T ↔ A/G)
Gene mutations occur in two ways:

They can be inherited from a parent or acquired during a person’s

lifetime. Mutations that are passed from parent to child are called
hereditary mutations or germline mutations
(because they are present in the egg and sperm cells,
which are also called germ cells).
This type of mutation is present throughout a person’s life in
virtually every cell in the body.

Acquired (or somatic) mutations occur in the DNA of individual

cells
at some time during a person’s life. These changes can be caused
by
environmental factors such as ultraviolet radiation from the sun,
or can occur if a mistake is made as DNA copies itself during cell
division. Acquired mutations in somatic cells
(cells other than sperm and egg cells) cannot be passed on to the
next generation.
There may be a problem during cell division
(proofreading)

Some different modifications can be made such as depurination,

demethylation or deamination.
These modifications can be induced by a chemical or can be
formed spontaneously.
Frame-shift mutation
In a frame shift mutation, one or more bases are inserted or
deleted.

Adding (insertion) or removing (deletion) one letter changes

each subsequent word (alter the reading frame : shifts the
reading frame, causes extensive missenses, causes immediate
nonsense) because our cells read DNA in three letter “words”

Original The fat cat ate the wee rat.

Frame Shift The fat caa tet hew eer at.

 Deletion : Mutations that result in missing DNA
Deletions can also cause frameshift mutations.

Original The fat cat ate the wee rat

Deletion The fat ate the wee rat.

Insertion Mutations that result in the addition of extra DNA are called
insertions. Insertions can also cause frameshift mutations,
and general result in a nonfunctional protein. Insertion of 3 bases
gets amplified and the protein will contain extra copies of amino
acid …… Huntington disease [ neurodegenerative disorder]

Original Ala Ala Ala Ala Ala Ala Ala Ala

5’-GCU GCU GCU GCU GCU GCU GCU GCU-3’
Frame shift Ala Ala Ser Cys Cys Cys Cys Cys
5’-GCU GCU AGC UGC UGC UGC UGC UGC-3’
Inversion

In an inversion mutation, an entire section of DNA is reversed.

A small inversion may involve only a few bases within a gene,
while longer inversions involve large regions of a chromosome
containing several genes.

Original The fat cat ate the wee rat.

Inversion The fat tar eew eht eta tac.

 WHAT
CAUSES
MUTATION?
Causes of Mutation:

1. Spontaneous mutations (molecular decay)

A. Tautomerism --- A base is changed by the repositioning of a

hydrogen atom, altering the hydrogen bonding pattern of that
base resulting in incorrect base pairing during replication.

B. Depurination --- Loss of a purine base (A or G) to form an apurinic

site (AP site)

C. Deamination --- Hydrolysis changes a normal base to an atypical

base containing a keto group in place of the original amine group.

D. Slipped strand mispairing --- Denaturation of the

new strand from the template during replication,
followed by renaturation in a different spot
("slipping"). This can lead to insertions or deletions.
2. Induced mutations caused by mutagens

A.Chemicals

B.Radiation
a. ultraviolet radiation (non ionizing radiation)
b. cytosine and thymine – are most vulnerable
to radiation that can change their properties

C. Viral infections
Molecular Basis of Mutation

• consider a gene as a linear sequence of nucleotide pairs

representing stored chemical information

• triplet nature of the genetic code --- each sequence of

three nucleotides specifies a single amino acid in the
corresponding polypeptide
ex. ATG codes for methionine

• any change that disrupts the coded information ----

sufficient basis for a mutation

• least complex change is the substitution of a single

nucleotide
ULTRAVIOLET RADIATION
Short Wave Ultraviolet radiation
( 200 – 300 nm) especially at  254 nm (DNA
maximum absorption) can cause genetic mutation:
A. Dimer production between pyrimidine bases which
is close to each other in the same DNA strand, or
between pyrimidine pairs on the opposite DNA
strand.
Dimer that could be produced : T-T, T-C, & C-C
B. Transition, transversion dan frameshift mutation
CHEMISTRY MUTAGENS
A.Nitric acid (deaminating agents)
 transition mutation

B. Hydroxylamine (Hydroxylating agents)

 transition mutation

C. Alkylating agent
transition, transversion dan frameshift
mutation
Hydroxylamine (NH2OH) reacts with
pyrimidine (T,C, & U)
Alkylating agents : EMS (ethyl methane sulphonate), MMS (methyl methane sulphonate),
NTG (Nitro-N-nitrosoguanidine)

4
Base Analogs
Interchalating agents
 Intercalating agents
• Flat molecules containing several polycyclic rings
that bind to the equally flat purine/pyrimidine
bases of DNA
 cause deletion /addition of a base pair even a
few base pairs
 insertion : by slipping between the bases in
the template strand, these mutagens cause the
DNA polymerase to insert an extra nucleotide
opposite the intercalated molecule
Informational Slide
 Chromosome
classification
• Metacentric : chromosome number:
• 1
• 3
• 16
• 19
• 20
 Chromosome
classification (2)
• SubMetacentric : chromosome number:
• 4
• 5
• 6-12
• X
• 17-18
 Chromosome
classification (3)
• acrocentric : chromosome number:
• 13-15
• 21-22
• Y
 Giemsa staining band
• Prior to 1960, when Moorehead and Nowell described the
use of Giemsa in their chromosome preparations,
conventional cytologic stains such as acetoorcein,
acetocarmine, gentian violet, hematoxylin, Leishman's,
Wright's, and Feulgen stains were used to stain
chromosomes. The Romanovsky dyes (which include Giemsa,
Leishman's, and Wright's stain) are now recommended for
conventional staining, because the slides can be easily
destained and banded by most banding procedures. Orcein-
stained chromosomes cannot be destained and banded;
therefore, orcein is generally not used in routine
chromosome staining. Giemsa stain is now the most popular
stain for chromosome analysis (Gustashaw, 1991).
 Giemsa Solutions
• Giemsa stain (Gurr's, Biomedical
Specialties cat. # 35086)
pH 6.8 phosphate buffer (Gurr's
tablets, Biomedical Specialties cat. #
33199)
Working stain: 4 mL Giemsa; 96 mL
pH 6.8 buffer
Giemsa staining band
 Giemsa staining band
Procedure
• Place slides in a Coplin jar or staining dish.
• Prepare the working stain and pour it over the
slides.
• Stain for 7 minutes.
• Rinse slides in two changes of distilled water.
• Air dry slides; mount them with a cover slip if
desired. (If sequential banding procedures are to
follow, coverslipping is not recommended.)
Quinacrine banding stain
 Q banding stain
a fluorescent stain for chromosomes that produces
specific banding patterns for each pair of homologous
chromosomes; centromeric regions of human
chromosomes 3, 4, and 13 are specifically stained, as
are satellites of some acrocentric chromosomes and
the end of the long axon of the Y chromosome;
banding patterns are similar to those obtained with G-
banding stain; similar fluorescent stain results are
seen with the antibiotics adriamycin and daunomycin,
as well as tertiary dyes (butyl proflavine and DAPI),
and the bisbenzimidazole dye Hoechst 33258.
 Reverse banding stain
• Reverse banding (R-banding) requires
heat treatment and reverses the
usual white and black pattern that is
seen in G-bands and Q-bands. This
method is particularly helpful for
staining the distal ends of
chromosomes
 C banding stain
• C-banding stains the constitutive heterochromatin,
which usually lies near the centromere

• Other staining technique :

• Nucleolar organizing region stains (NOR stains)
highlights the satellites and stalks of
acrocentric chromosomes
 summary
• C-banding stains centromeres.
• R-banding is the reverse of C-banding and stains
non-centromeric regions in preference to
centromeres
• G-banding is obtained with Giemsa stain (left
lower). It yields a series of lightly and darkly
stained bands.
• Q-banding is a fluorescent pattern obtained
using quinacrine for staining. The pattern of
bands is very similar to that seen in G-banding.
 Mutagenic agents
• physical : ionizing radiation (α, β, and γ
radiation, X-rays). In cells, it produces free
radicals (molecules with unpaired electrons),
 extremely reactive and can damage DNA.
• chemical effects,
• accidental errors in DNA replication and
recombination.
 Mutagenic agents

• Short-wavelength ultraviolet light (UV light) also

has mutagenic effects, mainly in skin cells
(sunburn). The most common chemical change due
to UV exposure is the formation of thymine
dimers, in which two neighboring thymine bases
become covalently linked to one another
• This results in errors when the DNA is read during
replication and transcription.
 Mutagenic agents
• Only a few examples of the group of chemical mutagens are shown
here. Nitrous acid (HNO2; salt: nitrite) and hydroxylamine (NH2OH)
both deaminate bases; they convert cytosine to uracil and adenine to
inosine.
• Alkylating compounds carry reactive groups that can form covalent
bonds with DNA bases. Methylnitrosamines release the reactive
methyl cation (CH3+), which methylates OH and NH2 groups in DNA.
The dangerous carcinogen benzo [a]pyrene is an aromatic hydrocarbon
that is only converted into the active form in the organism.
• Multiple hydroxylation of one of the rings produces a reactive
epoxide that can react with NH2 groups in guanine residues, for
example. Free radicals of benzo[a ]pyrene also contribute to its
toxicity.
 Effects
• Nitrous acid causes point mutations
• e.g. : C is converted to U, which in the next
replication pairs with A instead of G  The alteration
thus becomes permanent.
• Mutations in which a number of nucleotides not
divisible by three are inserted or removed lead to
reading errors in whole segments of DNA, as they
move the reading frame (frameshift mutations)
Fig. 2 using a simple example.
From the inserted C onwards, the resulting mRNA is
interpreted differently during translation, producing a
completely new protein sequence.
• A point mutation can be reversed by another point mutation.

Ways:
1.true reversion --- nucleotide is changed back to its original
state

2.second-site reversion --- a complementary mutation

elsewhere that results in regained gene functionality

• Point mutations that occur within the protein coding region

of a gene may be classified into three kinds:

1. Silent mutations --- which code for the same amino acid

2. Missense mutations --- which code for a different

amino acid  sickle cell anemia (glutamate 6 in the β-
globin  valine)

3. Nonsense mutations --- which code for a stop and can

truncate the protein
Always remember that there are 3 stop
codons:

UAA
UAG
UGA
Missense mutations

The new nucleotide alters the codon so as to produce an altered

amino acid in the protein product

Nonsense mutations

The new nucleotide changes a codon that specified an amino acid

to one of the stop codons (TAA, TAG, or TGA).

Silent mutations

Most amino acids are encoded by several different codons.

For example, if the third base in the TCT codon for serine is changed
to any one of the other three bases, serine will still be encoded.
Such mutations are said to be silent because they cause no change in
their product and cannot be detected without sequencing the gene
(or its mRNA).
Frameshift mutation : Insertions/deletions of 1/a small number of base
pairs that alter the reading frame

THE CAT SAW THE DOG

change of one
letter

THE BAT SAW THE DOG

THE CAT SAW THE HOG

THE CAT SAT THE DOG