MUTAGENS

MUTAGENESIS

• Sudden uncorrected change (de Vries,1903)

MUTATION

• Sudden change of genetic information able to be

inherited (Knippers)

• Change in nucleotide sequence of a genome by

substitution, deletion or insertion

(terminological genetic commission)

• Sudden change of genetic information, out of range

of polymorphism

MUTATION

MUTANTorganism keeping mutation

MUTAGEN

physical, chemical or biological

factor causing mutation

- gained or inherited mutation?

- selection?

GENOTOXICOLOGYScientific discipline of genetics. It covers mutagenic

effects and mutagen classification, and based on gene

expression forming, identifies changes in gene

expression that serve as a tool in foretelling sensitivity

to medicinal drugs and chemicals.

ANTIMUTAGEN

Substance reducing the frequency of mutations =

ANTIOXIDANT (vitamins C and E, carotenoids, selenium and

others.)

MUTATION RATE (SPEED)

Probability of mutation during the whole generation.

EPIMUTATIONThis is not a change in genetic material. It is a change

in gene expression, which can change a phenotype,

exactly as the „real" mutation.

They arise as a result of functioning of epigenetic cell

mechanisms.

Mutations

• hereditary changes showing as permanent

• they are unique changes in traits and characteristics of an organism that are determined by changes in their DNA

• they are sudden, undirected, permanent and unique

• they are always connected with a genotype change, however, they might not show in phenotype

• they are an important biological process accompanying evolution

• they are the basis for the origin of new species in the nature

Individuals within a certain species differ in the

sequence of nitrogen bases in their DNA

(genetic code degeneration).

CAGTCGATCCGAGG

CAGTCGGTCCGAGG

CAGTCGTTCCGAGG

Polymorphism of DNA coding region

Variability of human genome

Average difference in the genome of two people is 0,1 %

• vast majority are neutral polymorphisms, without a phenotype effect

• However, certain part of changes causes patology of various severity (from minor cosmetic defects to conditions incompatible with life) > mutations

Mutations in regard to origin:

1. SPONTANEOUS

2. INDUCED

- unknown reason

- known mutagen

- mutations cause many diseases

SPONTANEOUS MUTATIONS

- mutation is a random event (10-5-10-9gen/generation)

- mutations are essential for evolution

Formation of mutations that carry over to other

generations has low frequency in the nature.

• they form in organisms, in the nature or in the in vitro system without an action of man (but not without a reason)

• they form without deliberate use of mutagens

• mainly point mutations of the substitution type

• there are inter-population differences in the incidence, mutation scale and proportion of individual mutations

• they spread due to different population and genetic factors: gene drift, the founder effect, genetic isolation, inbreeding, selection, etc.

• low proportion of de novo mutations

Spontaneous mutations

replication system – if the DNA polymerase includes a wrong

nucleotide during DNA replication (bases complementarity)

recombination system – if the recombination running in the

prophase of the first meiotic division during sex cell formation

is incorrect

reparation system – if the enzymes repairing defects in DNA do

not work, their activity is reduced or they make a mistake

Spontaneous mutations are connected with the defect:

• DNA is most vulnerable during replication.

• DNA polymerase includes approximately 1 incorrect: 10 000 nt.

• Real frequency of mutations 1 incorrect: 107 - 1011 nt (proofreading).

• Other reparation mechanisms focus on the detection of mispairing and

substitution of the one nucleotide from the double-strand DNA, which

has already been replicated or modified by a chemical mutagen.

• In case of an extensive DNA damage the cell prefers apoptosis to

wasting energy and means on the correction of numerous mutations or

risking that the cell would transform to a tumorous one.

Substitution of one base in replication

Mispaired bases

gap

break

The same mutation has different consequences

HGMD: Human Gene Mutation Databasewww.hgmd.cf.ac.uk/ac/index.php

Gene function

Heredity type Enzymes Protein function

modulators

Receptors Transcription

factors

AR 78 % 45 % 48 % 25 %

AD 12 % 45 % 48 % 65 %

X linked 10 % 10 % 4 % 10 %

Age of the start of

disease

in utero 10 % 10 % 14 % 45 %

up to 1 year of life 40 % 12 % 27 % 20 %

1 year - puberty 29 % 30 % 33 % 15 %

puberty – 50 years 19 % 43 % 24 % 17 %

over 50 years 2 % 5 % 2 % 3 %

Distribution of the most frequently mutated genes according to the gene product function

The original gene is labelled as wild–type in English.

Aminoacids are labelled with capital letters, e.g. K is lysine, L

is leucine, G is glycine, R is arginine and X is stop codon.

Sometimes three-letter symbols are used, e.g. Arg (arginine)

and His (histidine).

G52R or Gly52Arg – number 52 amino acid glycine has changed to arginine

K320X – number 320 amino acid has changed from the original lysine to stop codon

nt400(C→G) – number 400 cytosine has changed to guanine

nt5445(del4) – deletion of four nucleotides from the position 5445

∆F508 – deletion of codon for phenylalanine on the position 508

nt317(insA) – adenine has been included after the nucleotide on the position 317

Cystic fibrosis

About 1300 mutations are known in CFTR gene

Asthma

more than 55 different mutations in gene ADAM 33

Mutations causing diseases need not to be single !

Hemoglobin

400 different variants of normal hemoglobin are known

Mutation need not to cause disease !

S : CCT GTG GAG

Pro Val Glu

A : CCT GAG GAG

Pro Glu Glu

Sickle cell anemia

146 AA in β-globin strand

in hemoglobin

substitution – adenin is

replaced by tymin in 6. AA:

RFLP Method

Restriction Fragment Length Polymorphism

1. DNA isolation

2. DNA cleavage by restriction enzyme

3. DNA fragments division in electrophoresis

4. DNA fragments denaturation

5. Southern´s blot

6. Preparation of the probe

7. Hybridization

8. Autoradiography and development of the RTG film

– physical mutagens

– chemical mutagens

– biological mutagens

INDUCED MUTATIONS

- They form deliberately, after the mutagens have acted upon a

cell or organism

- They are responsible for the formation of mutations

- Substances or factors increasing the number of mutations

- Classification:

Mutagens

1. PHYSICAL – various types of ionization and non-ionization radiation, radiotherapy

(UV radiation, X-ray radiation)

2. CHEMICAL – medicaments (e.g. aminopterin, reserpin)

– substances acquired through food (conservation agents, mycotoxins)

– pesticides (plants protection)

– chemotherapy

– other chemicals (paints, diluents, solvents ... )

3. BIOLOGICAL – viruses (e.g. oncogenic viruses)

Mutation origin

• ability of mutagens to react with DNA either directly or through their metabolic products

Mutagen penetrates the nucleus and reacts with the DNA

A gene with pre-mutation lesion forms; it either returns to the initial state (reparation) or stabilizes (mutation)

A change in genetic information occurs in the cell

Biochemical properties of mutated cells change

Depending on the degree of cell´s damage – the cell dies (lethal mutation) or reproduces and a clone forms

• can stimulate mutation origin

Pyrimidine dimers

• major lesion done by UV-B and UV-C

• covalent bonds of adjacent pyrimidinesin one strand

• can block replication and transcription

Physical mutagens:

UV

it mainly impairs genetic material of skin cells

Chemical mutagens:

- substance decreasing activity of repair enzymes

- substances damaging DNA

- base analogues

Biological mutagens:

retroviruses and other mobile genetic

elements (insertions mutations)

INDUCED MUTATIONs

Mutations in regard to target cell

– in developing somatic tissue will result in

mutant cells population

• GAMETIC (germinal)– in germ line - cell mutations are heritable

• SOMATIC

• Gametic mutations are more frequent than somatic

• It is due to a very high frequency of the sex cells formation

• The more the genetic material is used (cell division – replication),

the greater the probability that mutations will occur

Somatic mutation

4. Death of cell

earlier later

Possible consequences:

1. None (silent mutation)

2. Developmental defects (miscarriage, still-birth)

3. Malignant transformation of cell (cancer)

In regard to genome organisation

• GENE – point mutation

• GENOME – numerical aberration

• CHROMOSOMAL – structural

aberration

Genome mutations

• Changes in the number of chromosomes

• Polyploidy: - occurs when the usual number of chromosome sets increases(they occur for instance by an error in reduction division)

- embryos with polyploidy die soon

- it is a natural condition in some plants

- it is an important source of variability in the evolution and breeding

• Haploidy: - typical for sex cells

- in some species it is a perfect, phylogenicly fixed feature

- in mammals and man it is incompatible with life

• Aneuploidy: - it occurs due to a change in the number of chromosomes, whichis not a multiple of the haploid number (monosomy, trisomy)

GENOME Trisomy 21- Down´s syndromeMUTATIONS

GENOME Trisomy 13 – Patau´s syndromeMUTATIONS

GENOME Trisomy 18 – Edwards´ syndromeMUTATIONS

CHROMOSOME Syndrome Cri du ChatMUTATIONS - aberrations

deletion, duplication, inversion,

translocation, isochromosome, insertion

• Inversions: GATC → GTAC

GENE MUTATIONs

• Transitions: Pu → Pu

Py → Py

• Transversions: Pu → Py

Py → Pu

• Substitutions: GATC → CATC

• Insertions: GATC → GGATC

• Deletions: GATC → GTC

• Duplications: GATC → GAGATC

Types:

GENE MUTATIONs

• Frameshift

Substitutions

duplication, deletion, insertion

• Instable repeats of trinucleotides CpG

• Samesense: No effect (silent mutation)UAU-UAC

Tyr-Tyr• Missense: Results –AA substitution

UAU-UCC

Tyr-Ser

• Nonsense: Results in a stop codon (TAG,TAA,TGA)UAU-UAA

Tyr-STOP

GENOME MUTATIONS ACCORDING TO FUNCTION

CHANGES

NONSENSE

By their influence the termination codon is created:

UAA = ochre mutations

UGA = opal mutations

UGA = amber mutations

Proteosynthesis stops, which puts the gene completely out of

operation

MISSENSE

By their influence a codon for other AA, than the originally coded

one, is created, by which a slightly altered protein forms that only

partially fulfills the function of the original protein

GENE MUTATIONS ACCORDING TO THE FUNCTION

CHANGES

SUPRESSOR

Mutation in one gene is eliminated by mutation in the other gene,

so called supressor gene (it eliminates the effect of the first

mutation)

AUXOTROPHIC

They result in inability to synthesize a low-molecular metabolite,

e.g. a vitamin, AA

THERMOSENSITIVE

Slightly altered protein is synthesizes by their influence, which is

non-functional in a certain thermal range.

Pathogenic potential CpG

- Mammals´ genome contains a great proportion of repetitive sequences

- Tandem repeats of short sections are also found in genes

- These areas are prone to slipped strand mispairing

- VNTR/STR in the non-coding areas

- deletions/additions in the coding areas

- expansions (if they exceed certain limit)

- so far only described in the human genome

- basic sequence is trinucleotide

- stable and without a pathological effect in a certain range

- unstable with a pathogenic effect after a certain limit has been exceeded

- expansion mechanism unknown

- anticipation – severity of disease escalates (or the age of start decreases)

in following generations (myotonic dystrophy)

Instable repeats of trinucleotides

FRAXA

Normal

GCAGCG...CGG...CTGGG

6-54x

Premutation

GCAGCG...CGG...CTGGG

55-200x

Mutation

GCAGCG...CGG...CTGGG

200-1300x

Number of repetitions:

Normal

Premutation

Mutation

FRAXA

FRAXA Huntington Myotonic

disease dystrophy

Incidence

1:2000 1:15000 1:8000

Heritability

X AD AD

Localisation

Xq27.3 4p16.3 19q13.3

CpG islets

CGG CAG CTG

Normal 5-58 9-35 5-35

Premut. 58-230 36-38 50-100

Mutat. 230-2000 39-100 50-2000

Diseases caused by trinucleotide expansion

disease localization CpG number

Huntington´s dis. gene CAG 9 - 35

Kennedy´s dis. gene CAG 17 - 24

Spinocerebral ataxy 1 (SCA1) gene CAG 19 - 36

Spinocerebral ataxy 3 (SCA3) gene CAG 12 - 36

FRAXA 5’ UTR CGG 6 - 54 (200 <)

Myotonic dystrophy 3’ UTR CTG 37 - 50 (50 <)

Mutations - according to localization

nucleus – they originate in the DNA located in the cell nucleus

off-nucleus – they originate in the v DNA present in

mitochondrions, chloroplasts

Mutations - according to compatibility with life

vital – they do not affect how an individual experiences life

lethal – mutated genotype does not allow survival to its carrier

(mutations of essential genes)

According to the mutation´s direction

direct – when a mutated allele is forming from a normal one

reverse – when a mutated genotype turns back to a wild genotype

Reverse mutations

• Pseudoreversionrecovering of polypeptide chain function

AGG -TGG -AAGArg Trp Lys

• Exact reversiontotal recovering of mutated sequence to original one

AGG -TGG -AGGArg Trp Arg

• Equivalent reversion (degeneration of genetic code)

AGG -TGG -CGGArg Trp Arg

According to the degree of phenotype expression

dominant – mutated allele is superior to normal allele

recessive – mutated allele exspresses itself in the homozygote

recessive form only (most of the mutations)

According to the place of localization

in coding sequences

in non-coding sequences (introns) – mutations resulting in splicing, directly in the cut places (splice sites of introns)

• GT – transcription of intron into mRNA

• AG – deletion of the respective exon

in regulation sequences

on the 5’ end

• in TATA-, CAAT-boxes, in other areas of promoter, in enhancers – they affect the transcription speed

reduced amount of product

on the 3’ end

• Mutations of poly(A) signal – instable mRNA

reduced amount of product

1. Product with decreased to zero function (loss of function)

- recessive

- the gene product is most frequently enzyme

- frequent types of the missense mutation are duplications,

extensive deletions, inversions, insertions, STOP completely

eliminate allele´s function

2. Product with abnormal function (gain of function)

- dominant

- altered polypeptide gains a new function

- the product is most frequently non-enzymatic protein

- frequent in case of tumours (somat. mutations), rare in case of

monogene diseases

- vast majority of mutations are expansions and gene fusion, gene

deletion does not apply (it does not lead to a new function)

Accroding to the effect on gene product

Review of tests for potential

mutagens will be the

content of practicals!