Unit 6: Mutation

 Chapter 1: Mutation
6.1.1 Mutation

An inheritable spontaneous deviation from the “wild type” is called “mutation” and the
organism in which it occurs is called a “mutant”. Genetic recombination, through
independent assortment and reciprocal crossing-over, reshuffles existing alleles into
new combinations and in sexually reproducing organisms is an effective method of
promoting genetic variation. However, genetic recombination cannot create new
alleles in the first instance: the ultimate source of all genetic variation is mutation.

Mutations are caused by alterations in the structure, arrangement or quantity of DNA.

Characteristically they are random(almost), rare, recessive, and harmful.

Types of Mutation

 Chromosome mutation may change the order of the genes within the
chromosome, e.g., by deficiency, deletion, inversion, duplication, or
translocation.
 Gene or point mutations may result from changes in the base sequence
in a gene.
6.1.2 Chromosome mutations

Normally chromatids (mitosis, meiosis) or chromosomes (meiosis), separate at

anaphase by the action of spindle fibres. Failure to separate (nondisjunction) results
in abnormal ploidy levels.

 Thus, the phenomenon in which two homologous chromosomes fail to

separate or disjoin with each other during anaphase of either mitotic or
meiotic cell division is called non-disjunction.
 In Drosophila and man various cases of sex-chromosomal non-
disjunctions have been reported.
 They basically cause trisomic and monosomic aneuploids which
ultimately affect the sexual phenotype and metabolism of the organisms.
 Sometimes this failure is caused by a malfunction of the cell.
 It can also be induced by chemicals such as colchicine, which cause
rapid depolymerization of the microtubules.
 Aneuploids, are usually deleterious.
 Similarly, autopolyploids and polyploids with odd numbers of sets of
chromosome are usually sterile, because pairing at meiosis may result
in unbalanced gametes with aneuploid numbers of chromosomes.
 Chromosome imbalance is also the reason why polyploidy is rare in
animals. XXXX or XXY individuals are usually sterile.
 6.1.2.1. Mutations caused by changes to ploidy level

Changes in the number of chromosomes can also cause mutation.

Aneuploidy: Gain/loss of individual chromosome, from a set. There are several
reports of aneuploids in salmonids. E.g., a male brooktrout that was trisomic for a
chromosome carrying a LDH-B enzyme locus was reported. This fish was fertile and
produced euploid and trisomic offspring in equal frequencies; the trisomics were
similar in size, appearance, and viability to the normal fish. “A few” trisomic and
monosomic rainbow trout were also reported. These aneuploids also had no obvious
differences from normal fish. Thus aneuploid salmonids seem to be common and
viable.

1.Nullisomics (2n-2) - both of a pair of homologous missing from a diploid set

2. Monosomics (2n-1) - one chromosome from a diploid set missing.

3.Trisomics (2n+1) - one extra chromosome.

6.1.2.2. Polyploidy

Gain/multiplication of whole sets of chromosomes. The genomic formula for

autopolyploids is BB BB; the genomic formula for allopolyploids is BB CC. These two
types have different origins.

 Autopolyploidy originates when in a dividing cell, spindle formation fails

to occur, daughter chromosomes fail to segregate and the nucleus is
reconstituted with double the normal chromosome complement.
 Allopolyploidy arises as a consequence of hybridization between
species which are sufficiently closely related to produce viable hybrids
but whose genomes have diverged as a result of chromosome structural
change and differentiation at the gene level. As a result of such changes
the F1 interspecific hybrid may be completely sterile.
1.
- duplicated sets from the same species (triploid tilapia)
Autopolyploidy

2. individual containing sets of chromosomes originating from

-
Allopolyploidy different species (Mrigal- common carp hybrid)

6.1.2.3.Polyploidy in fishes

Haploid individuals among fishes are non-viable.

 When the egg development is stimulated by spermatozoa with

destroyed nuclei, almost all the developing embryos become haploid,
the development proceeding with malformations (haploid syndrome)
and resulting in embryonic death at later stages of embryogenesis.
Triploids in fishes appear quite frequently.
  Apparently, in most fish species the reductional division may be
omitted during meiosis (predominantly in females), and the resulting
gametes are diploid.
 The fertilization of such an egg by a normal spermatozoan (as well as
the fertilization of the normal egg by a diploid spermatozoan) leads to
the appearance of triploid organisms.
 They can be quite viable : for example, certain varieties of Carassius
auratus gibelio and of the viviparous
fishes Poeciliopsis and Poecilia are triploid.
 Triploid organisms have recently been found in the rainbow trout.
 The decreased fertility of the triploids in bisexual fishes (associated
with the irregular segregation of chromosomes in meiosis) apparently
explains the absence of such forms in the natural populations of most
species.
 Triploids appear in fishes from time to time as a consequence of distant
hybridization.
 Probably tetraploids also appear from time to time as a result of fusion
of the diploid gametes, but there are no data on the frequency of these
chromosomal mutations in nature.
 6.1.2.4. Chromosomal aberration

Four main types of chromosome aberrations are known.

1. Deletion – Fragment of chromosome is missing
2. Duplication – Fragment of chromosome becomes duplicated.
3. Inversion – Fragment detaches and reinserts in the reverse order
4. Translocation – A segment from one chromosome becomes connected to a non-
homologous chromosome.
Deletions occur among fishes more frequently than duplications, but lost of the
deletions lead to a drastic loss of viability,and the organisms carrying these
deletions are rapidly eliminated from the populations.
Duplication of chromosomal regions is known to occur in fishes, although these
events are probably infrequent.
 The presence of duplicated genes has been established by purely
genetic techniques, since duplications cannot be detected under the
microscope ;
 the most probable duplication mechanism involves unequal crossing
over, i.e., the exchange by portions of imperfectly
conjugated chromosomes.
 According to many authors, duplications play a particularly important
role in the evolution of fishes.
Inversions can be classified into two main types.
 Paracentric inversions which do not involve the
centromeric regions are difficult to detect. They do apparently occur
in fishes quite frequently, but their presence can only be established
by analyzing the inheritance of the linked genes.
 Pericentric inversions involving the centromere are quite
frequent. If the two breakage events take place at equal distances
from the centromere one cannot detect the inversion without the
analysis of marker genes. When the sites of breakage are located
asymmetrically, the relative length or even the absolute number of
chromosomal arms will be changed.
Robertsonian translocations or centric fusions are very important and are
apparently fairly frequent.
 The breakage of one acrocentric chromosome occurs near the
centromere and another acrocentric chromosome is joined to the site of
the breakage.
 One or two small regions adjacent to the centromere (with one of the
centromeres) are lost and two acrocentric chromosomes fuse into a
single metacentric one.
 The number of chromosomal arms remains unchanged.
 The reverse process of centric fissions is rarer because an additional
centromere is required.
 According to recent data, however, the direct division of one
centromere into two may be possible.
6.1.3 Gene Mutations

Most information is available about point or gene mutations. Through base

substitution, a base pair in the wild type allele may be replaced by another base in the
mutant allele. Several kinds of changes are recognized.

 A transition is an exchange of a purine with another purine or a

pyrimidine with another pyrimidine .
 A transversion refers to the substitution of a pyrimidine with a
purine or vice versa .
 One characteristic of point mutation is that they can revert.
 Another category includes those called frameshift mutations,which
result when one nucleotide or more is inserted or deleted, thus altering
the reading frame in the following transcription and translation process,
and leading to changed amino acid sequence in the resulting protein.
 Gene mutations result from alterations to the number, type, and
arrangement of bases within a gene.
 Watson and Crick suggested that the base sequence in DNA acted as a
code which determined the amino acid sequence of polypeptides.
 Triplet of bases determine a single amino acid. GAA specifies Leucine,
but alteration of the first base to A converts the triplet to AAA, which
specifies phenylalanine.
During DNA replication, old strands act as templates for the new ones, and base
pairing is highly specific. Provided the bases never alter in any way, accurate
replication is thus ensured. However, they do alter, and at an alarmingly high
frequency. There are three kinds of alteration.

1. Depurination. A and G may break off, leaving gaps in the double helix.
TCAGAAA TC -- GAAA TCGAAA
 AGTCTTT AG T CTTT AGCTTT 1Bp shorter

2. Deamination. C may lose its amino group, and so become U.The latter
pairs with A, not G, so a switch from C:G to U:A may occur.
TCAGAAA TUAGAAA TUAGAAA

AGTCTTT AGTCTTT AATCTTT (A instead of G)

3. Tautomerism. Like many organic molecules, the bases may change

their shape (isomerism).
 Thus, C may become CR (-R= rarestate), and since CR pairs with A, this
can also result in a C:G to T:A switch. All these alterations are due to
random chemical reaction occurring between DNA and the many
substance found in the nucleus.
The following structural changes occur in DNA
1. Pyrimidine dimers, in which two adjacent pyrimidines on a DNA strand
are coupled by additional covalent bonds and thus lose their ability to
pair.
2. Chemical changes of single bases, such as alkylation or deamination,
thus causing changes in the pairing properties of the DNA.
3. Crosslinks between the complementary DNA strands, which prevent
their separation in replication.
4. Intercalation of mutagenic agents into the DNA, causing frameshift
mutations.
5. Single-strand breaks.
6. Double-strand breaks.
6.1.4 Induced Mutation

The frequency of mutations in fishes can be markedly increased by X-rays and

chemical treatment.

 X-irradiation of fish gametes results in the appearance of various genic

and chromosomal mutations.
 X-rays induce mutations in the platyfish, and these mutations impair
the precision of regulation of action of the gene coding for the
development of black pigment cells - melanophores. As a result, these
fishes develop a tumour, a premelanoma, resembling that which
emerges after the hybridization of the platyfish with the swordtail.

 Chemical mutagens, particularly nitrosoethylurea (NEU) are highly

effective in inducing mutations in fishes. If spermatozoa or eggs are
treated with NEU,embryos with many chromosomal defects are usually
found : these defects may be easily observed when mitotic figures are
inspected at the blastula stage. These observations lead to the
conclusion that the frequency of chromosomal mutations induced by
such treatment is very high.
  The frequency of genic mutation was measured in the carp using
the S and n scale genes as a model: it was equal to 0.02% - 0.04%
after dimethyl sulphate treatment.
 In one experiment with NEU 40 mutations of the n gene were observed
among the 11500 fishes examined, the frequency being equal to
0.36%.
 It is quite probable,however, that the mutation induced in this gene ( n
) is a chromosomal aberration(deletion); then the surprisingly
high mutation frequency in the latter case is due to the induction of
deletions in the region of the gene n and is not a consequence of point
(genic) mutations.
 The rate of mutation can be increased dozens or even hundreds of
times by chemical mutagen.
 X-irradiation of fish spermatozoa in large doses and their treatment by
high doses of chemical mutagen result in gynogenesis, that is the
development in the absence of the male parent’s chromosomes.
 In such conditions embryos are haploid, but up to 1% of the embryos
turn out to be spontaneous diploids.
 The use of a temperature shock (incubation at lowered temperatures)
has made it possible to markedly increase the yield of diploid larvae
due to gynogenesis in the loach and in the common carp.
 Cooling of eggs also resulted in an increase in the number of triploids
after normal fertilization be mentioned that cytochalazine increases the
number of polyploid embryos in salmonids.
6.1.4.1 Mutagens

A mutagen, by definition, is an agent which increases the frequency

of mutation. Mutation which occur at a frequency above the base (spontaneous rate)
are described as induced.

Physical mutations

 short-wavelength ultraviolet irradiation (254 nm) affects DNA in a

number of ways, but a well-established action is the formation of
thymine dimers, means that adjacent Ts will bond together, instead of
with As in the complementary chain. This weakens the double helix
and leads to short breakage.
 Chromosome aberrations are therefore typically associated with UV
mutagenesis. UV (265mm), g , and X rays are known as physical
mutagens.
 Ionizing radiation includes X-rays, g , ß-rays, which act by causing
ionization of the medium through which they pass.
 These rays are usually used for mutagenesis only if other mutagens
cannot be used (e.g. for cell material impermeable to UV rays).
 Single- and double-strand breaks occur with a significantly higher
probability than with all other mutagens.
 Double-strand breaks result in major structural changes, such as
translocation, inversion or similar chromosome mutations.
  Muller found that the frequency of induced mutation is directly
proportional to the intensity of the mutagen treatment.
 The effect of dosage is cumulative, so exposure to 1rad/year for ten
year is equivalent to 10 rads/year.
6.1.4.2 Chemical mutations

A variety of chemicals are known which are mutagenic, and these may be classified
into three groups according to their modes of action.

1. Mutagens which affect nonreplicating DNA.

2. Base analogs, which are incorporated into replicating DNA due to their
structural similarity with one of the naturally occurring bases.
3. Frame shift mutagens,which enter into DNA during replication or repair
and through this intercalation cause insertion or deletion of one or a few
nucleotide pairs.
Nitrous acid- Promotes deamination and mispairing.

Base analogues- resemble normal bases, but cause mispairing.

 No data are as yet available on the frequency of gene mutations in

fishes, but this frequency appears not to be high.
 For example, after the examination of 260 thousand individuals of the
common carp,no single mutation affecting
the S and N genes responsible for the pattern of scales had been found.
 The populations of many fish species appear to be saturated by mutant
forms of genes coding for the synthesis of many different proteins.
6.1.5 Mutation hotspots

Mutation frequencies vary significantly along nucleotide sequences such that

mutations often concentrate at certain positions called hotspots.

 Mutation hotspots in DNA reflect intrinsic properties of

the mutation process, such as sequence specificity, that manifests itself
at the level of interaction between mutagens, DNA, and the action of the
repair and replication machineries.
 The hotspots might also reflect structural and functional features of the
respective DNA sequences.
 When mutations in a gene are identified using a particular experimental
system, resulting hotspots could reflect the properties of the gene
product and the mutant selection scheme.
 Analysis of the nucleotide sequence context of hotspots can provide
information on the molecular mechanisms of mutagenesis.
 However, the determinants of mutation frequency and specificity are
complex, and there are many analytical methods for their study.
 There are computational approaches for analyzing mutation spectra
(distribution of mutations along the target genes) that include many
mutable (detectable) positions.