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Cell Biology and Genetics
Genetic recombination and mutations
Sandip Das
Senior Lecturer
Department of Biotechnology
Faculty of Science
Jamia Hamdard (Hamdard University)
New Delhi 110062
Phone: 91-11-26059688
e-mail: [email protected]
[email protected]
Date of submission: April 24, 2006
Contents:
1. Genetic Variation
2. Mutation
a. Spontaneous
b. Induced
3. Transposable Genetic elements
4. DNA damage and repair
5. Recombination and crossing over
Significant Keywords: Genetic variation, Mutation, Evolution, DNA damage and repair,
Molecular markers, RFLP, PCR
Summary:
The immense morphological diversity or variation on this planet is a reflection of genetic variation. In other
words, morphological variation is an outcome of genetic variation. The study of cause and effect of genetic
variation in living beings has therefore become the central theme in the field of genetics. Genetic variants
arise as a result of either spontaneous or induced mutation. This genetic variation upon interaction with the
environment, leads to the generation of morphological or phenotypic diversity. Howsoever simple and
straightforward it may sound, it is enormously complex and far from being completely understood. The
means of generating diversity are many; they range from physical agents to chemical agents, and can be
broadly classified under the head of mutagens. The effect of the mutagenic agents can be subtle, i.e. a simple
base alteration or point mutation, to medium scale i.e. genomic changes to the order of few to several
hundred bases, or the effect could be drastic that are in the order of few to tens or hundreds of kilobases.
Biological agents such as transposable elements as an element of change are also responsible for large-scale
disruption and rearrangement of genetic material through their ability to move around in the genome.
One must examine the concepts in the light of the functional capability of the genome. As a safeguard against
the possible deleterious effects of mutation, nature has evolved an elaborate mechanism of damage control
through repair or rectification of mistakes that take place during the replication process. These DNA repair
mechanisms are an integral part of cellular machinery.
The present chapter attempts to cover these aspects of genetics with a special emphasis on the novelty that
has been generated either through induced mutation or that arose naturally; and how genetic variations can be
detected through the use of molecular tools.
Section 1:
GENETIC VARIATION:
Much before nucleic acid was recognized as the genetic material, Gregor J. Mendel through his seminal work
demonstrated that morphological features or traits are inheritable (i.e. capable of being passed on from
parents to offspring) and exist as (Mendelian) “factors”. Later work by researchers such as Griffith
(Transformation of Streptococcus), Avery, McLeod and McCarty (DNA as genetic material) and several
others established beyond doubt that such units are defined segments of nucleic acid, DNA and RNA, and
were termed as genes.
Have you ever observed a flowering plant over several generations? Observe a marigold plant in your
garden. Once the plant has borne orange flowers (in the case of marigold, each flower is actually an
inflorescence i.e. a collection of many flowers; and each petal is in fact an individual flower!) you could
collect the dry petals (flowers) and sow them. In the next generation, the plants will again bear orange
marigold flowers! This demonstrates the principle of inheritance (see figure 1a). However, a visit of the
neighbourhood park or the gardens will tell you that marigold flowers come in all shades of orange ranging
between light yellow to dark orange to red. If you are able to transfer the pollen (male gamete) from one of
the yellow flowers onto the stigma (receptor for the female gamete i.e. egg) of a red flower, then the plants of
the next generation are likely to bear orange flowers (caused due to mixing of alleles for yellow and red
flower, or heterozygous effect). Selfing (i.e. pollination of stigma with pollen derived from same flower) of
such an individual will produce plants in the subsequent generation that will bear red, orange or yellow
flowers (figure 1b). Numerous breeds of dogs (again a round of your neighbourhood) or the different types
of monkeys and butterflies (a visit to the zoo!) are examples of some other cases of variations (figure 1c).
Some of the other everyday examples of variations around you include difference in eye colour, skin colour,
height etc among humans. If genetic material is responsible for the morphological traits and characters, and
there are variations in terms of appearance and other characteristic features, this implies that these
phenotypic variants are due to differences in the genetic material. The differences in the sequences of the
nucleotide (in DNA or RNA) among a set of related organism can be termed as genetic variation. In other
words, variation at the genetic level upon interaction with environmental factors are responsible for the
differences in morphology, physiology, or behaviour among individuals of a species.
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Genetic variation manifested as the morphological differences or the “difference visible to the naked eye”
among the members of any species (humans, for example) are ultimately caused and derived from the
existing differences at the nucleotide level and also from differences that arises as an outcome of differential
gene
regulation.
Figure 1: Genetics and Variation:
A. The morphological features or characteristics of any organism are inherited from one generation to the next, provided the parents
with similar features (both homozygous dominant) contribute the gamete, demonstrating the Law of Inheritance.
B. In the instance where alleles are mixed, the first generation is likely to be an intermediate. However, upon selfing, parental traits are
recovered back along with the intermediates in the next generation.
C. A look around in the garden or in the zoo will exemplify the variation that exists in any organism, butterflies, for instance.
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As we have just learnt that genetic variation refers to the existence of differences in the genetic material in
related organism, it is therefore important for us to understand how does this variation arise i.e. the cause?
How does this variation spread? What does this variation do i.e. the effect? What mechanisms are available
to the organism “to tackle this variation”? We would also like to explore how to detect this variation?
Genetic variation arises primarily through the route of mutations and spreads among the members of the
population during reproductive process either through sexual reproduction or asexual reproduction, also
termed as gene flow.
Section 2
MUTATION
The answer to the question as to “How does the genetic variation arise?” lies in understanding the
phenomena of mutation. Mutation can be defined as the occurrence of any change in the sequence of nucleic
acid or any change in the chromosomal structure. Mutations can also be defined as heritable changes in the
genetic material. This point becomes important in multicellular organisms where we must distinguish
between changes in gametes (germline mutations) and changes in body cells (somatic mutations). The former
are passed on to one's offspring; the latter are not. The term mutation would also include the processes that
are involved or are responsible for inducing any change. A point to be kept in mind is that when we talk of
any changes, the alterations are relative to “wild type (most widely occurring)” nucleotide sequence or
chromosomal structure.
Mutations usually arise due to the errors that occur during the replication process and are retained or carried
forward if not rectified. The chance that a mutation occurs is rare in nature and may vary from organism to
organism. The rate at which mutation occurs also varies between different genomic segments, i.e. occurs at
different frequencies in various segments of the same genome. The rate of mutation can vary between 10-4 to
10-8 per gene per generation. For example, a gene that is much larger than other has a higher chance of
undergoing mutation, and a gene whose function is not so important can accumulate mutation to a higher
degree as compared to other genes. This is termed as the natural or spontaneous mutation rate. However, in
the presence of certain external and internal agents, the rate of mutation goes up tremendously and is usually
several folds higher than its natural rate. The agents that cause mutation in the nucleic acid sequences and
chromosomal structure are termed as mutagens and the organism affected by mutation are termed as mutant.
Mutation can occur at the nucleotide level (fine changes) or at the chromosomal level (gross changes). The
alterations at the nucleotide level are exemplified by point mutations such as insertions, deletions, transitions
and transversions. At the chromosomal level, alterations could be numerical (aneuploidy, disomy, trisomy
etc) or structural in nature (duplication, deletion, translocation, inversion etc).
Mutagens can be divided into chemical agents and physical agents. In addition, transposable elements that
occur in genomes are potent mutagens i.e. they can cause major alterations in the gene structure and
sequence.
Chemical mutagens: These are chemical moieties that interact with nucleic acids and bring about
changes in their sequences.
The chemical mutagens act through a variety of means. Some of the chemical mutagens mimics the structure
of the nucleotide and are thus termed as base analogs. Due to their similar structure, some of the base analogs
can be incorporated during replication and cause mutation by altered base pairing. For example, 5-BromoUridine (5-BU) is a base analog of thymine and normally pairs with Adenine as per the Watson-Crick basepairing rule. However, at times, 5-BU undergoes further small structural alterations due to tautomeric shift
and then pairs with guanine. In such a situation, the T:A pair is eventually replaced by a G:C pair upon
replication (figure 2a).
Apart from mimicry, some chemical moieties, taking advantage of their small size, insert themselves
between bases and disrupt the replication process. Upon intercalation, such mutagenic agents cause insertion
of a single base in the middle of two existing nucleotides resulting in a frameshift mutation. Ethidium
bromide is one such chemical which causes mutation through insertion / intercalation into double-stranded
DNA.
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Figure 2: Mutagens and mode of action
Mechanism of action of some chemical mutagens and UV light (physical mutagen).
A. Incorporation of base-analogs, such as 5-bromo uracil, an structural analog of thymine leads to replacement of a T:A pair with a G:C
pair.B. Agents such as nitrous acid causes de-amination and alters base pairing properties.
C. Physical agents such as UV-light, can catalyze formation of dimers between neighbouring Thymine residues which if not corrected
can cause mutation due to improper processing of genetic information.
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A third category of chemical moieties induces mutation through modification of bases. For example, methyl
methane sulfonate adds methyl group to bases at different positions and can convert guanine into 7-methyl
guanine and O6-methyl guanine, and adenine to 3-methyl adenine. The modified bases then pairs with
thymine leading to replacement of a G:C pair with an A:T nucleotide pair. Similarly, nitrous acid causes
deamination of cytosine to produce uracil that eventually leads to conversion of a C:G base pair into a A:T
base pair post-replications (Figure 2b). Such deamination can also occur spontaneously even in the absence
of mutagens like nitrous acid and causes conversion of a natural methylated cytosine (5-methyl cytosine) to
produce thymine. Therefore, the process of deamination on cytosine or methyl-cytosine produces uracil and
thymine resp. and leads to replacement of a G:C base-pair to A:T base pair (Figure 2b).
Alkylation is also one among several spontaneous base modification processes. For example, EMS and
Nitrosoguanidine modify guanine through addition of ethyl and methyl group respectively.
Another type of spontaneous DNA damage that occurs frequently is damage to the bases by free radicals of
oxygen. These arise in cells as a result of oxidative metabolism and also are formed by physical agents such
as radiation. An important oxidation product is 8-hydroxyguanine, which mispairs with adenine, resulting in
G:C to T:A transversions.
Some of the common chemical mutagens are listed in table 1.
Table 1: Common chemical mutagens and their mode of inducing mutation
Mode of inducing mutation
1.
Chemical (Common
name/Abbreviation)
5-Bromo uridine (5-BU)
2.
2-Amino Purine (2-AP)
3.
Ethyl Methanesulfonate (EMS)
4.
Nitrosoguanidine (NG)
An analog of adenine; upon protonation can
mispair with cytosine and therefore can lead to
conversion of A:T base-pair to G:C base-pair
Alkylating agent; adds ethyl group (alkyl group)
and oxygen at the 6th position of guanine to create
O6-alkyl guanine leading to mis-pairing with
thymine followed by replacement of G:C base-pair
with A:T
Alkylating agent; adds methyl group (alkyl group)
to Guanine
5.
Proflavin
6.
Acridine orange
7.
Ethidium bromide
8.
Nitrous Acid
An analog of thymine; the keto form of 5-BU pairs
with adenine and enol form with guanine eventually
causing conversion of A:T base-pair to G:C pair
Intercalating agent; Flat planar molecule that
mimics bases and is able to intercalate between
stacked nitrogen bases and cause single nucleotide
insertion or deletion
Intercalating agent; Flat planar molecule that
mimics bases and is able to intercalate between
stacked nitrogen bases and cause single nucleotide
insertion or deletion
Intercalating agent; Flat planar molecule that
mimics bases and is able to intercalate between
stacked nitrogen bases and cause single nucleotide
insertion or deletion
Deamination; causes conversion of cytosine to uracil
and 5-methyl cytosine to thymine
Physical mutagens: These are physical agents such as X-rays, gamma rays (γ-rays), UV-rays, beta rays
(β-rays), fast neutron etc that are known to induce changes in nucleic acid sequence either through base
changes or through modification in gross chromosome structure. UV rays, for example are known to
catalyze the formation of thymine dimmer, which if not repaired, impairs the proper flow of genetic
information either during replication or during transcription (Figure 2c).
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The ultraviolet portion of the light spectrum is in the range of wavelengths 100 nm to 400 nm. Prolonged
exposure of nucleic acid molecules in the 260 nm - 270 nm range is the most harmful and can cause DNA
damage. UV light catalyzes the formation of thymine dimers between adjacent thymine bases on the same
DNA strand through covalent linkages (see figure 2c). If not corrected, the thymine dimer prevents
complimentary base-pairing during replication and therefore leads to premature termination of the replication
of that DNA strand. X-rays and gamma rays, which are ionizing radiations, are potentially much more
damaging than UV as these have much more energy and penetrating power. Due to their high energy, X-rays
and gamma-rays ionize water and other molecules to form radicals (molecular fragments with unpaired
electrons) that can break DNA strands and alter purine and pyrimidine bases.
Physical mutagens also lead to alterations or gross changes in the chromosome structure (discussed in
subsequent section).
Occurrence of mutation:
Mutation can occur in any individual, organ, tissue or cell type and can either be artificially introduced
(Induced mutation) or can occur naturally (Spontaneous mutation), and are perpetuated through cell
division. Genetic changes in the vegetative cells and the germ cell are referred to as somatic and germinal
mutation respectively. As the products of vegetative cells are not inherited, the mutation is also lost in the
subsequent generation; however, the progenies of the somatic cell do carry the mutation. In contrast, any
mutation in the germ lines or cells will be inherited and passed onto next generation. Mutations in the germ
cells have different fate depending upon the stage of occurrence. For instance, a mutation in the germ cell
primordia is passed on to all the progenies i.e. all the germ cells; in contrast a mutation in the germ cell itself
is limited to that particular cell only and has lesser chances of being inherited unless it takes part in the
process of reproduction. The other factor that determines the transmission and expression of the mutation is
the dominant or recessive nature. A dominant mutation will be expressed in the next generation whereas a
recessive mutation is likely to be simply inherited and can express when in a homozygous recessive state, in
a diploid organism. The effects of mutation can be felt immediately or after several generations depending
upon the ploidy level; a haploid organism (such as a microbe) will express the mutant phenotype irrespective
of whether the mutation is dominant or recessive whereas in a diploid organism, a dominant mutation will
express itself immediately while a homozygous recessive state is required to exhibit the recessive mutation.
It is practically impossible to distinguish between either spontaneous or induced as the origin of mutation.
One of the ways to differentiate between these two routes is to estimate the frequency of occurrence as
spontaneous mutation are far less likely to happen as compared to induced mutation in a population by a
magnitude of several thousand in a given gene or genetic locus. Spontaneous mutations are the result of
inherent errors in the DNA replication process where not all the mis-incorporations or errors are corrected by
the proofreading enzymes. In contrast, induced mutations are caused by the deliberate actions of physical or
chemical mutagens.
The process of mutation is a completely random process; however, the rate at which some regions of the
genome undergo mutation may be several times more than other sections of the genome. Such regions of the
genome are termed as “Hotspots of Mutation”. Similarly, any mutation event that occurs in the coding
region or regulatory regions of the genome are far likely to have a major impact than a mutation in the noncoding heterochromatin region or in the repeated DNA sequences.
NUCLEOTIDE LEVEL MUTATION:
Two different sub-classes of point mutations are recognized, i) Base substitutions and ii) Base addition or
deletions. Base substitutions can further be of two types- transition and transversion. A diagrammatic
representation of the concepts is provided in figure 3 and figure 4.
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Figure 3: Point mutation
Any change that replaces a purine with another purine is termed as transition and purine to pyrimidne is termed as transversion.
Transitons are indicated with solid and transversions are indicated with dahsed lines.
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Figure 4: Mutation and consequences
A schematic representation of the various mutational events.
A: Shows transversion and transition and how a second mutation can restore the wild type sequence.
B: Silent mutation where a nucleotide alteration at a position corresponding to the third position of the codon nullifies the mutation due
to degeneracy.
C: A point mutation can introduce a stop codon and thus a trancated protein.
D and E: Insertion and deletion of a single or two nulceotide causes frameshift and the entire polypeptide sequence is altered
downstream of the site where mutation has taken place.
F and G: A point mutation (base replacement) can be synonymous (replacement of an amino acid with another of similar nature) or nonsynonymous (replacement of an amino acid by one with an entirely different chemical or physical nature).
Types of mutation:
Forward mutation: Any change in the “wild type” DNA to a mutant form is termed as forward
mutation.
Reverse mutation: A second mutational event in the mutant to restore the wild type form is termed as
reverse mutation. This might take place via two different routes, namely Back mutation and Suppressor
mutation, as defined below.
•
•
Back mutation: A second mutation that takes place at the same site as the first mutation in the
mutant to restore the wild type.
Suppressor mutation: A second mutation in the mutant, in another location in the genome, that as
dominant compensates the effects of the first mutation and thus suppresses the effect of the first
mutant.
A back mutant restores the sequence of the gene whereas a suppressor mutation does not.
Figure 4 explains the above-mentioned mutagenic processes with the help of examples and illustrates the
outcome at the amino acid level.
Mutations can be classified based on several criteria. Prominent among these are the effect on the function of
the sequence.
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Mutations that lead to complete or substantial loss-of-function of genes creates a null allele, and the
mutation is termed as amorphic mutation.
In contrast, some mutations may confer new or additional function to the gene product. Such mutations are
termed as gain-of-function mutation that usually are dominant in nature.
Mutations may also lead to products that have an antagonistic effect to the wild type allele. Such mutations
are termed as Dominant negative mutations (also called antimorphic mutations).
Some of the mutations do not allow the organism to survive and are thus lethal mutations.
Molecular Basis of mutation:
•
•
•
•
Point mutation: These involve changes at a specific single base level, and may be further
sub-divided as base-substitution (transition or transversion) and base-addition or deletion.
Transition: A transition event is when a base is replaced by another base of similar nature
i.e. a purine being replaced by another purine or a pyrimidine being replaced by another
pyrimidine (A → G or C →T);
Transversion: In transversion, a purine is replaced by a pyrimidine and vice versa (A or
G→ C or T, and C or T → A or G) (figure 3).
Insertions/Deletions (Indels): As the term explains, additon or deletion of a single base in
the sequence is creates an Indel.
The most common form of genetic variation is a single nucleotide polymorphism, or SNP. A SNP is a
variation in a single position in a DNA sequence and are a result of point mutations including base changes
or Indels. It is estimated that the human genome contains between three and six million SNPs.
Consequences of Mutation:
Most of the consequences or effects of mutation are felt at the level of amino acid sequence or at the protein
level. The following are the primary consequences of differences at the amino acid level (figure 4).
• Silent substitution: A substitution mutation that replaces a nucleotide with another one
but does not alters or change the amino acid sequence (at the third position of the codon
leading to the degeneracy of code) belongs to this category. As the name suggest, such
mutation are ‘silent’ i.e. do not have any effect on the protein structure or function.
•
•
Missense mutation: When a mutation in the DNA – addition, deletion or substitution
(transition/transversion) alters the amino acid sequence. Missense mutation which is of
substitution type may be further divided into
i)
Synonymous type - replacement of one amino acid with a amino acid of
similar chemical nature (basic type with basic type, acidic type with
acidic type) such that there is minimal or least likely structural
disturbance of protein structure and function;
ii)
ii) Non-synonymous type -replacement of one amino acid with another
type belonging to different chemical nature (acidic type with basic type,
for example), and such non-synonymous changes are likely to have
drastic effect on the structure and function of the resultant protein.
Nonsense mutation: Any alteration in the nucleotide sequence that introduces a stop
codon leading to synthesis of mRNA with a premature translation termination for the
resultant protein. Such a change is deemed to be hazardous for the organism unless the
truncated polypeptide proves beneficial for the organism.
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•
Frameshift mutation: Frameshift mutation is the consequence of indels when insertion or
deletion of a single or two bases alters the reading frame downstream of the mutation. This
consequence can be minimized when nucleotides are inserted or deleted in multiples of
three that leads to insertion or deletion of amino acids in the resultant protein.
A point mutation, either substitution, or addition or deletion causes single nucleotide polymorphism (SNP).
CHROMOSOMAL LEVEL MUTATION:
Chromosome as a genetic heritable unit is also subjected to various types of mutational events that might
affect the structural integrity of the chromosome or alter the numerical balance (figure 5).
Figure 5: Chromosomal Mutation
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Mutations can be classified into several categories depending upon the level at which it occurs. Chromosomal level mutations can bring
about change in chromosomal numbers or structure, while nucleotide level changes involve point mutations and substitutions.
The various forms of structural changes occurring at the chromosomal level are depicted with the help of line diagrams.
Structural level changes:
The structural changes at the chromosomal level are diverse and include addition, deletion, translocation,
inversion and duplication events. The causal agents of such gross changes are chemical or physical
mutagens, and transposable elements. The ionizing radiations induce changes in chromosome structure
through chromosomal breakage (complete or partial loss of chromosomal arm). The broken segment or arm
of a chromosome can undergo translocation (movement of complete or partial chromosome segment from
one chromosome to another) or lost during subsequent cell division. Translocation could be sub-divided into
reciprocal- or non-reciprocal translocation. Other chromosome level changes include inversion (reorientation of partial or complete chromosome arms relative to its original orientation or direction), deletion
(complete or partial loss of genetic material) and addition (partial or complete gain of chromosomal
material). See figure 5 for an overview of the chromosomal level changes.
Deletion leads to loss of substantial part of the chromosome that might be lost during cell division taking
place subsequently. A deletion event may create a hemizygous condition where, in a diploid organism, only
one allele is present as the corresponding allele might be lost. A part of the chromosome that has been
physically detached from one chromosome may also get attached to another chromosome (homologous or
non-homologous), and as a consequence, the recipient chromosome gains additional tracts of genome. Such
additional regions may remain un-paired during crossing-over events.
Chromosomal translocation refers to exchange of genomic segments between chromosomes, usually
mediated through homologous recombination between homologous chromosomes or via illegal
recombination between non-homologous chromosomes. A translocation event could be reciprocal where the
genomic material is exchanged, whereas in a non-reciprocal translocation, the flow of genomic segment is
unidirectional. Another form of chromosomal level changes is inversion, where the order of genetic units
located on the chromosome is inverted. Inversion may involve only one arm (paracentric) or centromere and
the flanking regions from both the arms (pericentric) of the chromosome. The final form of structural
alteration is duplication where a defined stretch of the chromosome is present more than once either in
tandem or in a dispersed manner. A diagrammatic representation of the structural changes is given in figure
5.
All the different types of structural mutation at the chromosome level are harmful to the organism. In a few
cases, such alterations have been found responsible for serious genetic disorder in humans. (Table 2).
Numerical level changes:
Non-disjunction or the failure of chromosome to separate during cell division causes changes in the
numerical composition of daughter cells that are different from the mother cell. In the present context we are
only concerned about aneuploidy as a chromosomal alteration, which does not involve complete
chromosome complements (euploidy). In aneuploids, a single or pair of homologous chromosomes is lost or
gained. In the event of failure of separation of chromosomes during cell division, one of the daughter cells
becomes hypoploid (lost one partner, or a pair of chromosome) and the other daughter cell is a hyperploid
(gained one partner or a pair of chromosome; Figure 5).
Both hypoploidy and hyperploidy have been implicated to be responsible to several genetic disorders in
humans (Table 2).
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Table 2: Human genetic disorders caused due to structural and numerical alterations. A few
representative examples are given.
Structural alteration
(Karyotype)
46, XX, 5p46, XX, 9q+, 22q-
Cri-du-chat
Philadelphia chromosome
46, 17p33
Miller-Dieker syndrome
Numerical alteration
(Type)
Hyper-ploidy
Chromosome nomenclature and
formula
47, +21 (2n+1)
47, +13 (2n+1)
47, +18 (2n+1)
47, +XXY (2n+1)
48, +XXXY (2n+2)
48, +XXYY (2n+2)
49, +XXXXY (2n+3)
45, X (2n-1)
Hypo-ploidy
Disorder
Cause
Short arm of chromosome 5 is deficient
Long arm of chromosome 22 translocated
on long arm of chromosome 9
Micro-deletion in short arm of
chromosome 17, band 33
Disorder
Down’s syndrome
Patau’s syndrome
Edward’s syndrome
Klinefelter’s syndrome
Klinefelter’s syndrome
Klinefelter’s syndrome
Klinefelter’s syndrome
Turner’s syndrome
SECTION 3
TRANSPOSON OR TRANSPOSABLE ELEMENTS:
The existence of mobile genetic elements that have a significant effect on genomic stability was for the first
time inferred from the work of Barbara McClintock during the 1940-50s as controlling elements in maize
for which she received the Nobel Prize in 1983. These mobile genetic elements were later shown to be
segments of DNA that can move around in the genome. Due to their ability of moving around or jumping
from one location to another in the genome, these controlling elements were also termed as “jumping
genes”. Such mobile genetic elements or Transposable elements have been found to occur in almost all
organisms, both prokaryotic as well as eukaryotic, such as bacteria, fungi, vertebrates (fish, birds, animals
etc) and plants studied thus far. Transposable elements may be present in any part of the genome, and in
multiple copies. For example, the human genome contains almost 300,000 copies of the Alu SINE element,
and 20,000 copies of the L1 LINE element (for details on SINE and LINE, see following text).
Transposable elements have been divided into various main classes depending upon their organization,
structure and content. The mobility of transposable elements is an enzyme catalyzed process, and involves
the enzyme transposase. The ability to synthesize transposase is used as a criterion for classification.
Based on this system of classification, transposons are categorized as autonomous i.e. they code for their
own transposase enzyme, and non-autonomous system where the transposable elements depend on another
transposon in the same genome for transposase enzyme and therefore transposition. Another system
classifies these as either transposon or retro-transposon (figure 6). Retrotransposons move via an RNA
intermediate whereas transposons move via a “cut-paste” mechanism and do not involve any RNA
intermediate, and are also grouped in Class I (Retrotransposon) and Class II (transposon) respectively. A
diagrammatic representation of the classification along with the organization is provided in Figure 6.
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Figure 6: Retrotransposon organisation:
Structural organization of typical transposon and retro-transposon.
A and B. Retro-transposons are classified based on the organization of their gene modules. At a broader level, retroelements are
distinguished based on the presence or absence of terminal repeats (LTR and non-LTR). Within the LTR, the order of genes may vary
giving rise to Ty-1 or Ty-3 type elements. The non-LTR types are differentiated based on their size (LINE and SINE) and gene content.
C. Transposon: The bacterial transposon system consists of several coding regions / genes flanked by insertion sequences (including
inverted repeats). Within the IS / IR flanks are present genes coding for transposase (the enzyme required for transposition), a repressor
and drug (antibiotic) resistance.
PBS: Primer binding site; PPT: Polypurine tract
Retrotransposon:
Retrotransposon are the major type of mobile elements and may constitute upto 80% of the genome as in
maize. Depending upon the structural type, retro-elements are further sub-divided into LTR (Long-terminal
repeat, Figure 6a) or non-LTR types (figure 6b). The LTR-type retro-element contains Gag and Pol genes;
the former coding for Capsid-like protein and the later for protease, Reverse transcriptase, Integrase and
RNaseH proteins (Figure 6). The non-LTR type of retroelements comprises of LINEs (Long Interspersed
Repetitive Elements) and SINEs (Short Interspersed Repetitive Elements).
DNA sequences that move or are copied from one genomic location to another are termed as transposable
elements. Depending upon the mechanism of transposition and the intermediate molecule, these are classified
as retrotrasposon (RNA, class I) or transposon (DNA, class II); another system of classification uses the basis
of whether transposons code for their own products (transposase) that catalyze transposition (autonomous
TEs) or the transposition catalyst is provided by another Transposable Element present in the same host
(nonautonomous TEs). As the two classes can transpose either in an autonomous or non-autonomous fashion,
the requirement for such an activity is different. Retrotransposable elements are designated so as they are
first transcribed to mRNA and then reverse-transcribed into a new locus as a DNA copy. Being autonomous,
the retroelements code for capsid protein (gag gene), and protease, reverse transcriptase and integrase protein
(from pol gene). The presence of these genes with coding capabilities indicates their close relationship with
retro-viruses, exception being the absence of env gene for envelope protein in retrotransposons. Such
retrotransposable elements contain long terminal repeats (LTRs) at their 5’ and 3’ end. The other type of
retrotransposons are devoid of LTRs at their ends and are thus termed as non-LTR types. Instead, such retroelements contain long and short interspersed repeat elements (LINEs and SINEs). LINEs contain genes for
nucleocapsid protein, endonuclease and reverse transcriptase (GAG, EN and RT resp.). SINEs are usually
short (around 100-300 bp) and contain sequences similar to RNA pol III promoter (figure 6). Although SINE
14
rely on reverse transcriptase for transposition but themselves do not code for reverse transcriptase and thus
may not be formally categorized under retro-elements. RNA polymerase III promoters are known to initiate
transcription of tRNA, small nuclear RNA and 5sRNA, and the presence of pol III promoter and tRNA like
sequences is indicative of the origin of SINE from reverse transcription of tRNA or other class of small RNA
in the past.
Transposons:
Transposable elements differ from retro-transposable elements in not requiring any RNA intermediate and
are thus able to move as DNA elements. These TE require genes coding for the transposase that recognize
the flanking repeat sequences (Terminal Inverted Repeat: TIR) and facilitate the transposition via a cut-andpaste (conservative) or copy-and-paste (replicative) mechanism. In the replicative mechanism, the parental
copy is retained at the original location and a new copy of the transposable element is generated which
moves to the new location. In conservative (non-replicative) mode, the parental element is excised from its
original location and transposed elsewhere.
Although transposable elements were discovered in plants, but a thorough understanding about transposons
was revealed upon analysis from bacterial systems. In bacterial systems, these are termed as Insertion
Sequences (IS). Several different types of insertion sequences are now characterized from the bacterial
genomes, notably E.coli. For instance, the E.coli genome contains eight copies of IS1 (800 bp long IS), five
copies of IS2 (1350 bp long IS) and five copies of IS3. Transposons have been shown to be present on both
chromosomal as well as plasmid DNA in the bacteria. The transposons coded by the plasmids in the
pathogenic bacteria carry genes coding for antibiotic resistance, for example to penicillin, tetracycline,
streptomycin, chloramphenicol etc. Such plasmids are termed as R (resistance) plasmids. The R plasmids
carrying antibiotic resistance can be passed through conjugation among different bacteria consequently
leading to rapid spread of resistance gene.
At a molecular level, the bacterial transposon consists of the antibiotic resistance gene flanked on either side
by Insertion Sequences (figure 6c). The genes for drug resistance are located between the inverted repeats
and are called as transposon elements or Tn elements. A number of Tn elements have been isolated and
shown to confer tolerance to various types of drugs. Tn1-Tn3 confers resistance to Ampicillin, Tn4 to
Ampicillin and Streptomycin, Tn5 and Tn6 to Kanamycin, Tn9 to Chloramphenicol and Tn10 to
Tetracycline.
A characteristic feature of the transposon is the presence of repeat sequences of usually 10-200 bp long at
their end. These repeats are a part of the insertion sequences in many cases and can be organized as direct
repeats or as inverted repeats. In the case of inverted repeats, the ends are mirror image of each other in terms
of sequence composition. Such direct or inverted repeat sequences can be used as sequence footprint as
identifying landmark for transposon location in the genome.
The movement of transposable and retro-transposable elements has two major implications:
i)
Inducing mutation through disruption of genes or regulatory sequences,
ii)
Create novel combination of genes and regulatory sequence, and
iii)
Contribute to genetic diversity
Insertion of the transposon alone can either disrupt a functional gene or the transposon can bring additional
stretches of new sequence that hitchhike along with it, and such new sequences may have regulatory
function. Most of such insertional events are deleterious, however, in a small proportion of cases, such
changes may prove beneficial for the organism. The transposable elements have been shown to have a
preference for sites for insertion in the genome. Chromosomal rearrangements such as inversion or
translocation of large segments of DNA are also another major outcome of transposon activity. Such
rearrangements can lead to mis-pairing between homologous chromosomal pairs and incompatibility may
ultimately give rise to a new species.
Transposable elements have contributed significantly towards expansion and contraction of genomes. For
example, it is estimated that retro-elements have been largely responsible towards doubling of genome size
15
in maize from 1.2 to 2.4 billion basepairs, and that too in an evolutionarily short duration of only two-tothree million years!
SECTION 4
MUTATION DETECTION TOOLS:
The great bulk of mutation or the genetic variation at the nucleotide level may not be visible at the
phenotypic level. To overcome such a handicap, molecular tools for the detection of variation or mutation
have been developed. These tools can be based either on biochemical assays, which measure the outcome of
these changes i.e. at the protein or metabolite level, or on DNA-based assays such as those based on
hybridization and PCR. Molecular mutation detection tools are the basis of disease diagnostic kits for various
genetic disorders, and provide the advantage of early screening even before the onset of the disease.
Molecular or DNA based markers offer many advantages over conventional phenotypic and biochemical
markers. They are heritable, easy to score, free from developmental and environmental influence, detectable
in all tissues and insensitive to genetic interactions (epistatic or pleiotropic). Tools that directly screen the
mutation at the level of DNA include hybridization based Restriction Fragment Length Polymorphism
(RFLP) and PCR based tools such as Allele Specific Oligonucleotides (ASO). Besides these, highthroughput technique of micro-array be used to simultaneously analyse a large number of loci (wild type and
mutant) through the re-sequencing chip.
The basic principles of DNA based assays are discussed.
Molecular tools:
•
RFLP: Restriction fragment length polymorphism is the occurrence of DNA fragments of varying
lengths, caused due to mutational event. Mutation can create, destroy or alter the frequency or
distribution of restriction endonuclease cleavage sites. Therefore when such a region is compared
between wild type and mutant individual, the changes can be visualized as RFLP. The mutational
events could be in the form of point mutation (addition, deletion or substitution of bases), insertion
or deletion of large genomic tracts (for example caused due to movement of transposable/retrotransposable elements), inversion or duplication events.
•
RFLP essentially involves isolation of genomic DNA, digestion with a restriction enzyme followed
by electrophoretic separation via agarose gels. In the event of the genome being small enough to be
resolved in a standard agarose gel such as a bacterial or viral or organellar genome, any alteration in
the nucleotide sequence resulting in fragment length variation between organisms can be visualized.
In the case of the genome being large or too complex, we need the assistance of a probe that is
complimentary to the region likely to harbour the change. Such a probe, tagged to a radio-label or a
fluorescent moiety, can therefore bind to the region following Watson-Crick base pairing rule and
highlight the polymorphism. The polymorphism can be visualized either with the help of
autoradiography or through detection of fluorescent signals.
For example, the point mutation in the β-globin gene creates/eliminates a site for the restriction
enzyme Mst II. Such a change can be detected via the use of RFLP tool (see figure 7).
•
•
PCR: Polymerase chain reaction has now become the tool of choice for mutation detection for the
ease and speed of assay. Polymerase Chain Reaction is an in-vitro DNA amplification reaction
where a small amount of template DNA can be amplified million fold through the use of
thermostable DNA polymerase, dNTPs, buffer and primers. PCR based assays for detection of
genetic variation includes tools such as Random Amplification of Polymorphic DNA (RAPD) and
Amplified Fragment Length Polymorphism (AFLP).
RAPD detects nucleotide sequence polymorphisms between two or more DNA samples by using a
primer of arbitrary or random nucleotide sequence. AFLP is a combination of RFLP and PCR and
involves both restriction and PCR. Polymorphisms between the samples are created due to the
presence or absence or redistribution of primer binding site which are actually an outcome of
16
•
•
mutational process. Both RAPD and AFLP are non-targetted assays (i.e. are not specific to any
particular segment of the genome).
Most of the mutation detection depends on designing allele specific oligonucleotide or ASO.
Polymerase chain reaction (PCR) relies on the in-vitro amplification of target DNA using sequence
specific primers. Correct base-pairing at the 3’ end of the primers is critical for the initiation of
primer-dependent chain elongation. Therefore primers can be designed carrying nucleotide
complimentary to either the normally occurring or the mutated nucleotide. These are termed as
Allele specific Oligonucleotides (ASO). ASO are able to amplify either the wild type or the
mutated allele for screening purposes (Figure 7).
DNA microarray: The latest weapon in the armoury of molecular biologist is the high density
DNA chips specially designed for re-sequencing purposes. Using a single array, hundreds of
mutational events could be discovered in a single experiment. This is termed as mutation detection
through re-sequencing array.
17
Figure 7: Mutation Detection
A.
B.
An A -->T point mutation in the first exon of the ß-globin gene deletes a restriction site for Mst II apart from altering the
amino acid sequence. The altered restriction site gives rise to a fragment length polymorphism between the normal and the
mutated alleles and can be detected as a RFLP.
Use of Allele specific Oligonucleotide (ASO) in Polymerase Chain Reaction. The primers are designed in such a way so as
to be specific to the wild type allele and thus amplify only the wild type fragment. Primers can also be designed for the
mutated allele which will only amplify the mutated allele and not the wild type.
18
SECTION 5
DNA REPAIR MECHANISM:
Genetic variation serves as the raw material on which evolution works. In other words, the process of
mutation creates variants in sequence of nucleic acid or genetic variation which is then selected or rejected
by the evolutionary forces.
If all the changes that occur in the genome were to accumulate, this might prove disastrous for the organism.
Eventually, then over a few generations, the composition of the genetic material in terms of sequence would
entirely change and the organism would no longer remain the same organism as it was before! The genome
must therefore have enough safeguards built in that would rectify most of the errors, while few may be left
out. This achieves some kind of balance between the identity of the genome (in terms of retaining the
sequence originality and fidelity) and allowing a few changes or variability to arise on which evolutionary
selective forces can act.
In order to rectify any changes that are inadvertently introduced, the cell possesses an elaborate system of
DNA repair mechanism. The DNA repair mechanism operates at two levels, during replication and postreplication. Any mis-match or mutation incorporated during the replication process is usually corrected with
the help of the proof-reading function of the polymerases.
Several distinct molecular pathways/mechanism for DNA damage repair system have been established
primarily based on studies on the bacterium E.coli, and all of these mechanisms are also found in mammals,
except the Photo-activation system. In comparison, mammals may possess additional DNA-repair systems
that are not found in lower organisms. See figure 8 for a diagrammatic representation of DNA-repair
systems.
1.
Light-Dependent repair or photoreactivation system
The process is named light-dependent or photoreactivation as it depends on light as a source of
energy to catalyze the repair reaction using the enzyme DNA photolyase. The enzyme binds to
nucleotide dimers such as thymine–thymine, cytosine-cytosine and cytosine-thymine dimers
and cleaves the covalent cross-linkages between the nucleotides utilizing light as a source of
energy (figure 8A).
2.
Excision Repair:
The excision repair system can be further divided into the Base-excision repair (BER) and the
nucleotide-excision repair (NER). BER process repairs mutation / alterations caused by
alkylation, oxidation or deamination of bases, whereas NER repairs larger pieces of damaged
DNA such as nucleotide-dimers formed as a result of exposure to ultraviolet light, or even
oligomers.
The excision repair system relies on correct excision of the mutagenized bases with the help of
endonuclease containing enzyme complex followed by filling in of the gap thus created with
the proper nucleotide catalyzed by DNA polymerase and finally sealing or re-joining of the
phosphor-di-ester backbone with the help of DNA ligase. The entire process can be further
elaborated. One of the mutagenic processes involves deamination which leads to base-pair
mismatch. For example, deamination of cytosine residue converts it into uracil and introduces a
G:U mis-pairing. If not corrected, a G:C pair will be converted into a T:A pair upon DNA
replication. To prevent such a mutational process, the correctional procedure involves the
recognition of the uracil by Uracil DNA glcosylase enzyme which cleaves the glycosidic bind
between the de-aminated converted nucleotide (uracil in the present example). This catalytic
activity leads to the formation of a Apurinic or Apyrimidinic (AP) sites, where the base is
missing. Such AP sites are further acted upon by two different enzymes, AP-endonuclease and
phospho-di-esterases to remove the sugar-phosphate group and create a ‘blank’ position. Upon
successful removal of the mutated base along with the backbone, DNA polymerase replaces the
correct base following Watson-Crick base pairing rule restoring the original sequence. The final
19
step in this pathway takes place with the help of the DNA ligase which re-joins the
phosphodiester bond. This particular mechanism is termed as base excision repair system.
Another form of excision repair system is termed as nucleotide excision system (NER; figure
8B) that deals with larger pieces of damaged DNA such as nucleotide-dimers formed as a result
of exposure to ultraviolet light, or even oligomers. Although the basic mechanism remains
similar, this process recruits a different set of enzymes and enzyme complex to achieve the
repair. In the first step, an enzyme complex consisting of two different enzymes, namely UvrA
and UvrB recognizes and binds to the dimeric nucleotides. Once the dimer has been recognized
correctly, one of the enzyme from the complex, UvrA dissociates and instead another enyme,
UvrC binds to the already present UvrB-dimer complex. The nucleotide dimer is then cleaved
on either side by the UvrB and UvrC in the 3’ and 5’ end of the dimer; and the cleaved
oligomer (as both UvrB and UvrC cleave 5th and 8th phophodiester bond from the site of dimer
formation) is released by the action of DNA helicase II, a product of UvrD gene. Therefore,
even for a dimer, the cleaved product is a 12-base oligomer. Post-release, the next two steps i.e.
filling-up and re-joining of the phosphodiester bonds in the DNA strand, are catalyzed with the
help of DNA polymerase and DNA ligase as in the previous mechanism.
Whereas the first mechanism i.e. photoreactivation is light-dependent, the excision repair is light independent
and can operate in dark.
1.
Mismatch Repair: This mechanism of DNA repair identifies and replaces normal deoxy-nucleotide
monohosphates that have been incorporated at an incorrect position (such as incorporation of dA oppostite
dC or dG) leading to a mis-match. This is indicative of the post-replicative nature of the mechanism.
Additionally, MMR removes small insertion / Deletion Loops (IDLs). Such errors have not been detected
and corrected by the proof-reading function of DNA polymerases during the replication process, and relies
on the differential methylation status i.e. hemimethylated status, of the template and the newly synthesized
daughter strand to identify the correct sequence (since both the nucleotides are ‘normal’ and the wild type
and the mutant form need to be differentiated. The template strand will contain methylated DNA whereas the
newly synthesized strand will contain unmethylated DNA, a difference that is utilized by the cellular
machinery to initiate the mismatch repair process.
A multi-enzyme complex is required to carry out the mismatch repair process of DNA damage repair. In
E.coli, the various enzymes involved in MMR include MutS, MutH and MutL products (figure 8C). Mut H is
responsible for recognizing the non-methylated strands of DNA that are generated upon replication. The
hemimethylated status is then used as a discrimination marker and to initiate the MMR mechanism. MMR is
a bi-directional process indicating that the nicking and degradation can begin in either 5’- 3’ or 3’ - 5’
direction of the mismatch.
20
Figure 8: DNA damage repair mechanisms
A.
B.
C.
Photo-reactivation repair: The enzyme DNA photolyase can catalyze the repair of thymidine and other nucleotide dimers
formed as a result of prolonged exposure to UV radiation. As the term indicates, the enzyme uses light as a source of energy
to cleave the covalent bond between the nucleotides.
Nucleotide-excision repair: In the NER system, UvrA and UvrB enzyme complex to bind to the damaged site, and guides
addition of another enzyme UvrC. Before the actual repair, UvrA disassociates, and instead UvrD enzyme creates nicks on
either side of the damaged region. Eventually, the damaged fragment and UvrC are released, and DNA polymerase fills in
the gap to restore the DNA in its original state.
Mismatch repair: A multi-enzyme system repairs mis-incorporations in the DNA. For example, in this figure the A::C
base-pairing is a result of failure of proof reading. In order to restore the actual sequence, the hemi-methylated base (where
the original strand is methylated while the newly synthesized daughter strand is not) is recognized by MutS. Later, MutH and
MutL also bind to the mutated region and introduce a nick. An exonuclease then uses the nick to remove the mutated strand
followed by synthesis of the correct sequence by DNA polymerase.
In eukaryotes, MutH homologues have not yet been identified so far. This raises the question as to how the
new and old strands are distinguished. It is presumed that the discrimination between the old and the new
strand may be mediated by the replication accessory PCNA (proliferating cell nuclear antigen) or through
recognition of nicks and gaps or free 3’ strands during replication. Once the faulty strand is identified, the
MMR takes over by degrading the strand through the Exo1 enzyme followed by synthesis of correct strand
by the DNA synthesis / replication machinery (DNA polymerase δ and DNA polymerase ε) and finally
ligation to rejoin the nick. The stretch that may undergo MMR may be in the range of 100-1000 nucleotides.
Table 3: DNA-repair systems
DNA-REPAIR SYSTEM
DAMAGE REPAIRED
Replication error
KEY ENZYMES INVOLVED
MutS, MutL, MutH
Photo-reactivation
Pyrimidine dimers
DNA phtolyase
Base-excision
Damaged bases
DNA glycolase
Nucleotide excision
Pyrimidine dimers
UvrA, UvrB, UvrC and UvrD
Mismatch repair
21
SECTION 6
RECOMBINATION
The exchange and reshuffling of genes and genetic variation across generations is a result of genetic
recombination. Broadly, recombination involves pairing of homologous chromosomes followed by physical
exchange of genetic material through crossing over. Exchange of genetic material between similar or
homologous chromosomes is termed as homologous recombination. Cytologically, recombination occurs
during first division in meiosis.
There are several variants to the basic model of recombination first proposed by Robin Holliday in 1964.
which is known as the Holliday Model of crossing over. The other important model is known as DoubleStrand-Break-Repair (DSB) model of crossing over. The establishment of the cross-over structure known
as Holliday junction is common to all the models, and the difference lies in the way these strands are cleaved
or nicked and interact.
Homologous recombination proceeds via several well established steps (figure 9): The first step involves
pairing of homologous chromosomes or chromosomal segments with similar or identical sequences. Nonsister chromatids of sister chromosomes are involved in the process of pairing. This implies that genetic
alleles, which have slightly different sequence, can also pair and participate in genetic exchange. Therefore,
homologous recombination can lead to re-shuffling of genetic alleles, generating novel combination of genes.
Following successful pairing, the next step creates breaks in the DNA. As the pairing involves similar
sequences, the breakpoint serves as a region where pairing between DNA strands originating from sister
chromosomes can begin. This pairing between complimentary strands of the duplex molecules leads to a
cross (“X”) shaped structure termed as Holliday junction, and the process involved is designated as strand
invasion. The Holliday junction can, through repeated denaturation (melting) and renaturation (base-pairing),
move along the length of the chromatids through branch migration. The final step in recombination is
cleavage when the Holliday junction is cleaves to separate and release the two chromatids after the genetic
exchange has taken place, a process known as resolution (figure 9a). Depending upon where the nick
cleaves the Holliday junction, various configurations of cross-over products can be obtained (figure 9a, b).
The Double-strand break (DSB) repair model differs from the earlier discussed model in that the
recombination process is initiated through break in both the strands of the chromatids of one of the DNA
molecules, while the other molecule remains intact. The Spo11 protein introduces double-stranded break in
eukaryotic system and no specific enzyme that introduces DSB in the prokaryotic (E.coli) counterpart has not
yet been identified. Instead, DSB are the outcome of DNA damage and failure of replication fork. The
double stranded break, the DNA strand is processed by RecBCD (helicase / nuclease) in E.coli and by MRX
protein in eukaryotes to generate partial single stranded DNA (ssDNA) molecules. These ssDNA then forms
a D-loop through pairing with the intact double stranded DNA. The ssDNA then acts as a primer to initiate
DNA synthesis complimentary to the intact DNA molecule at the region of pairing. Rec A protein in E.coli
and Rad51 in eukaryotes facilitates and catalyzes the process of pairing of homologous chromosomes and
strand invasion. Further, RecBCD and RecFOR in E.coli, and Rad52 and Rad59 in eukaryotes catalyze
strand assembly. The Continued progress of the newly synthesized DNA strands eventually leads to
formation of two Holliday junctions. The two Holliday junctions move apart through branch migration,
catalyzed by RuvAB complex and eventually are resolved to complete the process of recombination with the
help of RuvC enzyme. The Holliday junctions can be resolved via different permutation and combination of
nicking, i.e. both junctions cleaved via North-South or East-West cut, or North-South + East-West or EastWest + North-South. Depending upon the combination of nicking process, assortments of rearranged DNA
segments are generated (Figure 9b).
22
Figure 9: Recombination and crossing over
Holliday Model: Recombination starts with the pairing of homologous regions of DNA. The sequence need not be identical but
sufficiently similar to initiate pairing. Single strand breaks on both chromatids act as a site where pairing between
complimentary regions of DNA. Such complimentarity leads to formation of Holliday junction (an ‘X’ shaped structure) to
initiate crossing-over. The formation of Holliday junction creates partial heteroduplex and partial homoduplex molecules.
The junction can slide along the paired region by a process termed as branch migration. The recombination process is
completed by a process of resolution that nicks at the junction either in the North-South or East-West fashion.
Double-Strand Break model: This model differs from the earlier described basic Holliday model in that the nick is
introduced in one of the chromosome, but on both the strands. DNA from the nicked regions are removed to generate
single stranded DNA (ssDNA). The ssDNA then invades the intact DNA molecule to form a D-loop, and a Holliday
junction. Base pairing leads to extension of the heteroduplex and formation of a second cross-over point. The two
Holliday junction can then move away through branch migration and eventually is completed through different
combinations of North-South and East-West nicks.
Recombination and crossing-over leads to exchange of genetic material between homologous chromosomes
thereby leading to generation of new allele combinations. For example, when homologous chromosomes
carrying genes ‘ABCD’ and ‘abcd’ (where the alleles are A and a, B and b, and so on) in linear arrangements
undergo recombination, various allele permutations can be obtained. A crossing over event between A-B and
a-b (not including CD/cd) can generate ‘AbCD’ and ‘aBcd’; recombination between ‘BC’ and ‘bc’ similarly
generates ABcD and abCd. More than one cross-over event in this region can lead to even more possible
permutations of alleles. The chance of occurrence of crossing over between two genetic loci is a function of
the physical distance between the loci; the larger the distance, higher is the chance of a crossing over.
Another factor guiding recombination is the physical location in the genome; some regions in the genome are
more prone to recombination or are highly reombinogenic (hot-spots of recombination), while parts of
genome are deficient in recombination (cold-spots) for example, regions in and around centomeres and
telomeres are poorly recombining. For instance, RecBCD (composed of recB, recC and recD products) that
peforms helicase and nuclease activity and therefore catalyzes and facilitates unwinding and creation of
ssDNA of the DSB region is guided by specific DNA sequence termed as chi sites (cross-over hotspot
instigator). Chi sites are known to increase the recombination frequency. The over-representation of chi
sequences in a certain genomic segment can make that segment highly prone to recombination or hot spots of
recombination. Similarly, fewer chi sites than normal can make a genomic segment cold-spot for
recombination. The frequency of two or more genes being inherited together or separated during crossing
over event depends upon the physical distance between them, and their chromosomal location. This feature
forms the basis of recombination-based genetic maps, where the genetic distance between loci can be
estimated by their co-inheritance and separation among a set of individuals. Such a genetic linkage analysis
23
can be used to generate linkage maps displaying the order and genetic distance (in centiMorgan units)
between the loci. This aspect of recombination / crossing over being used in applied genetics will be
discussed elsewhere.
CONCLUSION
Mutations are changes in the DNA. A single mutation can have a large effect, but in majority of the cases,
evolutionary change is based on the accumulation of many mutations. Mutations provide a source of natural
genetic variation that is acted upon by the evolutionary forces through selection procedure. Any variation
that is beneficial to the organism is carried forward to future generation, whereas mutations having
deleterious effects are lost or are maintained in heterozygous condition in nature. The cellular system has
devised several DNA damage and repair mechanism to rectify the errors. However a small fraction of the
mistakes manage to “slip through” the tight corrective procedure and are inherited. Such mutations are the
source of variation on which natural selection acts. Characterization of the mutation provides for the basis on
which molecular diagnosis employing RFLP, PCR or direct sequencing is based. Humans have learnt to
exploit the naturally occurring or induced mutations to their advantage by utilizing these alterations in
breeding high yielding crop varieties and in animal / livestock improvement programmes.
The beneficial effects of mutations are several. Mutation breeding program have been used to develop
improved crop varieties, and were the cornerstone of Indian green revolution. For example, red-grained
Mexican wheat varieties were subjected to mutation to develop light or amber grained wheat varieties
“Sharbati Sonora” and “Pusa Lerma” from “Sonora 64” and “Lerma Rojo 64A” respectively. Presently,
atleast three hundred crop (cereals, fruits, vegetables, ornamentals) varieties are grown in India that have
been bred or improved through induced mutation! These include 46 different types of Chrysanthemum, 41
varieties of rice, 15 types of roses, 12 varieties of mung bean plus varieties of wheat, groundnut, Brassica,
sugarcane, brinjal etc.
More than animal systems, the effect of numerical alterations in chromosome numbers is visible in plants.
Most of the cultivated plant/crop species are either autopolyploid or allopolyploids. Common bread wheat
(Triticum aestivum) contains three different genomes from T.monococcum, T.searsii and T.tauschii, denoted
by A, B and D. Similarly, mustard or Brassica juncea (AABB) contains two different genomes, from
Brassica rapa (AA) and Brassica nigra (BB).
The harmful effects of mutations are manifested in the form of large number of genetic disorders among the
humans. Some of these are haemophilia, sickle cell anaemia, ß-Thalessemia, familial form of cancer, Down’s
syndrome, Phenyl ketonuria, Cystic fibrosis etc.
Eventually, genetic alleles that first arise as a result of spontaneous or induced mutation can spread in the
population through the reproductive cycle. Recombination helps the alleles enter into novel permutation and
combination through shuffling of loci through crossing –over. Therefore, recombination plays a very
significant role in generating novel allelic combinations, finally leading to creation and spread of variation in
the population.
24
Suggested Reading:
1.
Modern Genetic Analysis. Griffiths, Gelbart, Miller and Lewontin. W. H. Freeman and company
(1999)
2.
Introduction to Genetic Analysis. (7th ed). Griffiths, Miller, Suzuki, Lewontin, Gelbart. W. H.
Freeman and Company (1999)
3.
Genomes. 2nd ed. Brown, T. A. Oxford, UK: BIOS Scientific Publishers, Ltd; (2002)
4.
Molecular Biology of the Gene. Watson, Baker, Bell, Gann, Levine and Losick (5th ed). Pearson
Education publishers, Singapore (2004)
25