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: sdas@jamiahamdard.ac.insd66@rediffmail.com

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-Bromo-Uridine (5-BU) is a base analog of thymine and normally pairs with Adenine as per the Watson-Crick base-pairing 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 Chemical (Common

name/Abbreviation) Mode of inducing mutation

1. 5-Bromo uridine (5-BU) 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

2. 2-Amino Purine (2-AP) 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

3. Ethyl Methanesulfonate (EMS) 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

4. Nitrosoguanidine (NG) Alkylating agent; adds methyl group (alkyl group) to Guanine

5. Proflavin Intercalating agent; Flat planar molecule that mimics bases and is able to intercalate between stacked nitrogen bases and cause single nucleotide insertion or deletion

6. Acridine orange Intercalating agent; Flat planar molecule that mimics bases and is able to intercalate between stacked nitrogen bases and cause single nucleotide insertion or deletion

7. Ethidium bromide Intercalating agent; Flat planar molecule that mimics bases and is able to intercalate between stacked nitrogen bases and cause single nucleotide insertion or deletion

8. Nitrous Acid 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 non-coding 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 non-synonymous (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 (re-orientation 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)

Disorder Cause

46, XX, 5p- Cri-du-chat Short arm of chromosome 5 is deficient 46, XX, 9q+, 22q- Philadelphia chromosome Long arm of chromosome 22 translocated

on long arm of chromosome 9 46, 17p33 Miller-Dieker syndrome Micro-deletion in short arm of

chromosome 17, band 33 Numerical alteration

(Type) Chromosome nomenclature and

formula Disorder

Hyper-ploidy 47, +21 (2n+1) Down’s syndrome 47, +13 (2n+1) Patau’s syndrome 47, +18 (2n+1) Edward’s syndrome 47, +XXY (2n+1) Klinefelter’s syndrome 48, +XXXY (2n+2) Klinefelter’s syndrome 48, +XXYY (2n+2) Klinefelter’s syndrome 49, +XXXXY (2n+3) Klinefelter’s syndrome Hypo-ploidy 45, X (2n-1) 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 retro-elements 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

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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-and-paste (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

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in maize from 1.2 to 2.4 billion basepairs, and that too in an evolutionarily short duration of only two-to-three 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, high- throughput 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/retro-transposable 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

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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.

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Figure 7: Mutation Detection A. 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.

B. 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.

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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 post-replication. 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

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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.

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Figure 8: DNA damage repair mechanisms A. 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.

B. 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.

C. 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 KEY ENZYMES INVOLVED

Mismatch repair Replication error MutS, MutL, MutH

Photo-reactivation Pyrimidine dimers DNA phtolyase

Base-excision Damaged bases DNA glycolase

Nucleotide excision Pyrimidine dimers UvrA, UvrB, UvrC and UvrD

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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 Double-Strand-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. Non-sister 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 East-West + North-South. Depending upon the combination of nicking process, assortments of rearranged DNA segments are generated (Figure 9b).

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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

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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.

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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)

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