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YTISREVINUALAGAPPA
APPAGALAUNIVERSITY
M.Sc. [Microbiology]
elcyC drihT eht ni )46.3:APGC( CA[Accredited
AN yb edarGwith
’+A’’A+’
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cA[ (CGPA:3.64) in the Third Cycle
]CGU-DRHM yb ytisrevinU I–yrogeand
taC Graded
sa dedarasG Category–I
dna University by MHRD-UGC]
300 036 – IDUKIARA
KARAIKUDI
K – 630 003
364 21 NOITACUDE ECNATSIDDIRECTORATE
FO ETAROTCEOF
RIDDISTANCE EDUCATION

MICROBIAL GENETICS
II - Semester

MICROBIAL GENETICS
M.Sc. [Microbiology]
364 21

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TISREVINUALAGAPPA
APPAGALAUNIVERSITY
M.Sc. [Microbiology]
lcyC drihT eht ni )46.3:APGC( CA[Accredited
AN yb edarGwith
’+A’’A+’
htiwGrade
detidby
ercNAAC
cA[ (CGPA:3.64) in the Third Cycle
]CGU-DRHM yb ytisrevinU I–yrogeand
taC Graded
sa dedarasG Category–I
dna University by MHRD-UGC]
300 036 – IDUKIARA
NOITACUDE ECNATSIDDIRECTORATE
KARAIKUDI
K
FO ETAROTCEOF
– 630 003
RIDDISTANCE EDUCATION
MICROBIAL GENETICS
II - Semester
 ALAGAPPA UNIVERSITY
[Accredited with ‘A+’ Grade by NAAC (CGPA:3.64) in the Third Cycle
and Graded as Category–I University by MHRD-UGC]
(A State University Established by the Government of Tamil Nadu)
KARAIKUDI – 630 003

Directorate of Distance Education

M.Sc. [Microbiology]
II - Semester
364 21

MICROBIAL GENETICS
Authors
Dr Geetika Singh, Assistant Professor, Department of Botany, Govt. College for Girls, Panjab University, Chandigarh, India
Units (1.2, 3, 4, 11)
Dr Richa Sharma , Assistant Professor , Department of Microbiology, Shaheed Rajguru College of Applied Science for Women,
University of Delhi
Dr Rekha Mehrotra, Associate Professor , Department of Biology , Shaheed Rajguru College of Applied Science for Women,
University of Delhi
Units ( 7 & 8)
Dr. Sushil Kumar Upadhyay, Assistant Professor, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be
University), Mullana, Ambala- (Haryana), India
Units (2 & 5)
Meena Kumar, M.Sc Botany,B.ED , TGT , Senior Science Teacher ,Delhi Government School
Units (6, 12, 13, 14)
Dr. Helianthous Verma, Assistant Professor, Department of Zoology, Ramjas College, University of Delhi, Delhi
Units (9.2-9.3)
Dr. Pradeep Kumar, Research Associate-III, Department of Pediatrics, Army Hospital Research & Referral, New Delhi
Units (9.4-9.5, 10.2)
Vikas® Publishing House: Units (1.0 - 1.1, 1.3 to 1.9, 9.0-9.1, 9.6-9.10, 10.0-10.1, 10.3 to 10.8)

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 SYLLABI-BOOK MAPPING TABLE
Microbial Genetics
Syllabi Mapping in Book

BLOCK I: MUTATION, DNADAMAGE AND REPAIR

Unit 1: Mutation and its Types, Mutation Rate and its Determination Unit 1: Mutation
and Mutagenesis. (Pages 1-23);
Unit 2: Mutagens-Types, Physical Mutagens, DNA Reactive Chemicals, Unit 2: Mutagens and its Types
Base Analogs, Intercalating Agents, Metals and Biological Agents. (Pages 24-52);
Unit 3: DNA Damages- Deamination of Bases, Alkylation, Damage Due Unit 3: DNA Damages
to Reactive Oxygen, UV Induced Damage. (Pages 53-70);
Unit 4: Repair Pathways (Methyl-Directed Mismatch Repair, Nucleotide Unit 4: Repair Pathways
Excision Repair, Base Excision Repair, Recombinational Repair, SOS (Pages 71-91)
Inducible Repair, Specific Repair for Oxidative DNA Damage, Pyrimidine
Dimers and Alkylation Induced Damage and Adaptive Response).

BLOCK II: BACTERIALRECOMBINATION

Unit 5: Recombination- Types of Recombinations, Models for Unit 5: Recombination
Homologous Recombination, Molecular Mechanism of Homologous and its Types
Recombination, Homologous Recombination in Eukaryotes, Mating Type (Pages 92-123);
Switching. Molecular Mechanism for Site-Specific Recombination. Unit 6: Conjugation
Biological Roles of Site Specific Recombination. and F-Factor
Unit 6: Conjugation- Conjugation by E. coli F Factor, Structure of (Pages 124-141);
F-Factor, Regulation of F-Factor Fertility, Establishment of Cell Contact, Unit 7: Transformation
DNA Mobilization, Transfer and Separation of Mating Pair, Hfr (Pages 142-157);
Conjugation and Chromosomal Transfer, Interrupted Mating and Unit 8: Transduction
Conjugational Mapping. and its Types
Unit 7: Transformation- Mechanism of Natural Competence and (Pages 158-171)
Transformation in Bacillus Subtilis and Streptococcus Pneumoniae.
Transformation by Inducing Artificial Competence, Gene Linkage and
Mapping by Transformation.
Unit 8: Transduction- Generalized and Specialized Transduction.

BLOCK III: OPERON CONCEPT AND EXTRA CHROMOSOMAL

MATERIALS
Unit 9: Gene Concept- Regulation of Bacterial Gene Expression. Lactose Unit 9: Gene Concept and
System - Coordinate Regulation, Lac Components, Positive, Negative Lactose System
Regulation and Catabolite Repression. (Pages 172-205);
Unit 10: Tryptophan Operon - Attenuation. Arabinose Operon and its Unit 10: Tryptophan Operon
Regulation. and Arabinose Operon
Unit 11: Plasmids- Types of Plasmids - F, R & Col Plasmids. Properties (Pages 206-219);
of Plasmids - Sex Factors, Drug Resistant, Colicinogenic. Agrobacterium Unit 11: Plasmids: Types
Ti and Broad Host Range Plasmid. and its Properties
(Pages 220-242)
BLOCK IV: TRANSPOSABLE ELEMENTS AND EPIGENETICS
Unit 12: Detection and Purification of Plasmid DNA. Transfer of Plasmid Unit 12: Detection, Purification
DNA. Replication of Plasmid. Control of Copy Number, Plasmid and Transfer of Plasmid DNA,
Amplification, Curing and Incompatibility. and Plasmid Replication
Unit 13: Transposable Elements- Types of Transposable Elements, (Pages 243-265);
Structure, Genetic Organization and Mechanism of Transposition of Tn5, Unit 13: Transposable Elements
Tn3 and Related Transposons, Bacteriophage Mu, Tn7 and IS911, (Pages 266-290);
Integrons, Retrotransposons. Unit 14: Epigenetics
Unit 14: Epigenetics- Definition, Molecular basis, Mechanisms, (Pages 291-310)
Functions and Epigenetics in Bacteria.
 CONTENTS
INTRODUCTION

BLOCK I: MUTATION, DNA DAMAGE AND REPAIR

UNIT 1 MUTATION 1-23
1.0 Introduction
1.1 Objectives
1.2 Mutation and its Types
1.3 Mutation Rate and its Determination
1.4 Mutagenesis
1.5 Answers to Check Your Progress Questions
1.6 Summary
1.7 Key Words
1.8 Self Assessment Questions and Exercises
1.9 Further Readings
UNIT 2 MUTAGENS AND ITS TYPES 24-52
2.0 Introduction
2.1 Objectives
2.2 Mutagens: History and Causes
2.2.1 History of Mutagens
2.2.2 Causes of Mutagens
2.3 Types of Mutagens
2.3.1 Physical Mutagens
2.3.2 Chemical Mutagens
2.3.3 Biological Mutagens
2.4 Tests System for Mutagens
2.5 Consequences of Mutagens Exposures
2.6 Answers to Check Your Progress Questions
2.7 Summary
2.8 Key Words
2.9 Self Assessment Questions and Exercises
2.10 Further Readings
UNIT 3 DNA DAMAGES 53-70
3.0 Introduction
3.1 Objectives
3.2 DNA Damages
3.2.1 Deamination of Bases
3.2.2 Alkylation
3.2.3 Damage Due to Reactive Oxygen
3.2.4 UV Induced DNA Damage
3.3 Answers to Check Your Progress Questions
3.4 Summary
3.5 Key Words
3.6 Self Assessment Questions and Exercises
3.7 Further Readings
UNIT 4 REPAIR PATHWAYS 71-91
4.0 Introduction
4.1 Objectives
4.2 DNA Damage and Repair Pathways
4.2.1 An SOS Repair System or Recombinational Repair
4.2.2 Methyl Directed Mismatch Repair
4.2.3 Nucleotide Excision Repair
4.2.4 Recombinational Repair
4.2.5 SOS Inducible Repair
4.2.6 Specific Repair for Oxidative DNA Damage
4.2.7 Pyrimidine Dimers
4.2.8 Alkylation Induced Damage and Adaptive Response
4.3 Answers to Check Your Progress Questions
4.4 Summary
4.5 Key Words
4.6 Self Assessment Questions and Exercises
4.7 Further Readings

BLOCK II: BACTERIAL RECOMBINATION

UNIT 5 RECOMBINATION AND ITS TYPES 92-123
5.0 Introduction
5.1 Objectives
5.2 Recombination and its Types
5.2.1 Types of Recombination
5.3 Homologous Recombination
5.3.1 Models for Homologous Recombination
5.3.2 Molecular Mechanism of Homologous Recombination
5.3.3 Enzymes of Homologous Recombination
5.3.4 Role of Reca Protein in Homologous Genetic Recombination
5.3.5 Homologous Recombination in Prokaryotes and Eukaryotes
5.3.6 In Vitro Homologous Recombination
5.4 Site-Specific Recombination
5.4.1 Types of Site-Specific Recombination
5.4.2 Molecular Mechanism of Site-Specific Recombination
5.4.3 Biological Roles of Site-Specific Recombination
5.5 Answers to Check Your Progress Questions
5.6 Summary
5.7 Key Words
5.8 Self Assessment Questions and Exercises
5.9 Further Readings
UNIT 6 CONJUGATION AND F-FACTOR 124-141
6.0 Introduction
6.1 Objectives
6.2 Conjugation
6.2.1 Conjugation by Escherichia coli F Factor
6.2.2 Fertility Factor or F Factor
6.2.3 Hfr Conjugation and Chromosomal Transfer
6.2.4 The F′ (F Prime) Factor
6.2.5 Interrupted Mating and Conjugational Mapping
 6.3 Answers to Check Your Progress Questions
6.4 Summary
6.5 Key Words
6.6 Self Assessment Questions and Exercises
6.7 Further Readings
UNIT 7 TRANSFORMATION 142-157
7.0 Introduction
7.1 Objectives
7.2 Discovery of Transformation
7.3 Competence
7.4 Natural and Artificial Transformation
7.4.1 Natural Transformation
7.4.2 Artificial Transformation
7.5 Gene Linkage and Mapping by Transformation
7.6 Answers to Check Your Progress Questions
7.7 Summary
7.8 Key Words
7.9 Self Assessment Questions and Exercises
7.10 Further Readings
UNIT 8 TRANSDUCTION AND ITS TYPES 158-171
8.0 Introduction
8.1 Objectives
8.2 Bacteriophage and its Types
8.3 Transduction: Discovery and Types
8.3.1 Types of Transduction
8.3.2 Generalized Transduction
8.3.3 Specialized Transduction
8.3.4 Transduction Mapping
8.4 Answers to Check Your Progress Questions
8.5 Summary
8.6 Key Words
8.7 Self Assessment Questions and Exercises
8.8 Further Readings

BLOCK III: OPERON CONCEPT AND EXTRA CHROMOSOMAL MATERIALS

UNIT 9 GENE CONCEPT AND LACTOSE SYSTEM 172-205
9.0 Introduction
9.1 Objectives
9.2 Gene Concept
9.2.1 Gene Expression in Eukaryotes
9.3 Regulation of Bacterial Gene Expression
9.4 Lactose System - Coordinate Regulation
9.5 Lac Components
9.6 Answers to Check Your Progress Questions
9.7 Summary
9.8 Key Words
9.9 Self Assessment Questions and Exercises
9.10 Further Readings
UNIT 10 TRYPTOPHAN OPERON AND ARABINOSE OPERON 206-219
10.0 Introduction
10.1 Objectives
10.2 Tryptophan Operon
10.3 Arabinose Operon and its Regulation
10.4 Answers to Check Your Progress Questions
10.5 Summary
10.6 Key Words
10.7 Self Assessment Questions and Exercises
10.8 Further Readings
UNIT 11 PLASMIDS: TYPES AND ITS PROPERTIES 220-242
11.0 Introduction
11.1 Objectives
11.2 Plasmids: Types and Properties
11.2.1 Types of Plasmids
11.2.2 Properties and Characteristics
11.3 Answers to Check Your Progress Questions
11.4 Summary
11.5 Key Words
11.6 Self Assessment Questions and Exercises
11.7 Further Readings

BLOCK IV: TRANSPOSABLE ELEMENTS AND EPIGENETICS

UNIT 12 DETECTION, PURIFICATION AND TRANSFER OF
PLASMID DNA, AND PLASMID REPLICATION 243-265
12.0 Introduction
12.1 Objectives
12.2 Plasmid DNA
12.3 Detection and Purification of Plasmid DNA
12.3.1 Isolation and Purification of Plasmid DNA
12.4 Transfer of Plasmid DNA
12.5 Replication of Plasmid
12.6 Plasmid Copy Number
12.7 Answers to Check Your Progress Questions
12.8 Summary
12.9 Key Words
12.10 Self Assessment Questions and Exercises
12.11 Further Readings
UNIT 13 TRANSPOSABLE ELEMENTS 266-290
13.0 Introduction
13.1 Objectives
13.2 Transposable Elements
13.2.1 Types of Transposable Elements
13.3 Genetic Organization and Mechanism of Transposition
13.3.1 Tn3 Transposon
13.3.2 Tn5 Transposon
 13.3.3 Bacteriophage Mu
13.3.4 Insertion Sequence–Tn7 and IS911
13.3.5 Integrons and Retrotransposons
13.4 Answers to Check Your Progress Questions
13.5 Summary
13.6 Key Words
13.7 Self Assessment Questions and Exercises
13.8 Further Readings
UNIT 14 EPIGENETICS 291-310
14.0 Introduction
14.1 Objectives
14.2 Epigenetics
14.2.1 Definitions of Epigenetics
14.2.2 Molecular Basis of Epigenetic
14.2.3 Epigenetics Mechanisms
14.2.4 Functions and Consequences
14.2.5 Epigenetics in Bacteria
14.3 Answers to Check Your Progress Questions
14.4 Summary
14.5 Key Words
14.6 Self Assessment Questions and Exercises
14.7 Further Readings
Introduction
INTRODUCTION
Microbial genetics is a subject area within microbiology and genetic engineering.
NOTES Microbial genetics studies microorganisms for different purposes. The
microorganisms that are observed are bacteria, and archaea. Some fungi and
protozoa are also subjects used to study in this field. The studies of microorganisms
involve studies of genotype and expression system. Genotypes are the inherited
compositions of an organism. Genetic Engineering is a field of work and study
within microbial genetics. The usage of recombinant DNA technology is a process
of this work. The process involves creating recombinant DNA molecules through
manipulating a DNA sequence. That DNA created is then in contact with a host
organism. Cloning is also an example of genetic engineering.
After the discovery of DNA transfer in bacteria, bacteria became objects
of great interest to geneticists because their rate of reproduction and mutation is
higher than in larger organisms, i.e., a mutation occurs in a gene about one time in
10,000,000 gene duplications, and one bacterium may produce 10,000,000,000
offspring in 48 hours. Conjugation, transformation, and transduction have been
important methods for mapping the genes on the chromosomes of bacteria.
Microbial genetics is principally a branch of genetics concerned with the
transmission of hereditary characters in microorganisms. Within the usual definition,
microorganisms include prokaryotes like bacteria, unicellular or mycelial
Eukaryotes, for example yeasts and other fungi, and viruses, notably bacterial
viruses (bacteriophages). Because of their relative simplicity, microbes are ideally
suited for combined biochemical and genetic studies, and have been successful in
providing information on the genetic code and the regulation of gene activity.
Microbial genetics has played a unique role in developing the fields of molecular
and cell biology and also has found applications in medicine, agriculture, and the
food and pharmaceutical industries. Microbial genetics also has applications in
being able to study processes and pathways that are similar to those found in
humans, such as drug metabolism.
This book, Microbial Genetics, is divided into four blocks that are further
divided into fourteen units which will help to understand the basic concepts of
mutation, DNA damage and repair, mutagenesis, mutagens, DNA reactive
chemicals, DNA damages, repair pathways, bacterial recombination, models for
homologous recombination, conjugation, F factor, Hfr conjugation and
chromosomal transfer, transformation, transformation by inducing artificial
competence, gene linkage and mapping by transformation, transduction -
generalized and specialized transduction, gene concept, lactose system, tryptophan
operon, arabinose operon, plasmids, transposable elements and epigenetics.
The book follows the self-instruction mode or the SIM format wherein
each unit begins with an ‘Introduction’ to the topic followed by an outline of the
‘Objectives’. The content is presented in a simple and structured form interspersed
with ‘Check Your Progress’ questions and answers for better understanding. A list
of ‘Key Words’ along with a ‘Summary’ and a set of ‘Self Assessment Questions
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and Exercises’ is provided at the end of the each unit for effective recapitulation.
 Mutation
BLOCK - I
MUTATION, DNA DAMAGE AND REPAIR

NOTES
UNIT 1 MUTATION
Structure
1.0 Introduction
1.1 Objectives
1.2 Mutation and its Types
1.3 Mutation Rate and its Determination
1.4 Mutagenesis
1.5 Answers to Check Your Progress Questions
1.6 Summary
1.7 Key Words
1.8 Self Assessment Questions and Exercises
1.9 Further Readings

1.0 INTRODUCTION

At the simplest level, a mutation is a change or transformation. In biology, mutations

refer to changes in chromosomes and genes, which typically manifest physically.
The effect of a mutation can depend on the region in which the sequence of genetic
material has been changed. The simplest and the most harmless are substitutions
of a single base pair with another, with no effect on protein sequence. At the other
end are insertion or deletion mutations that lead to non-functional gene products.
Mutations can also occur on a large scale, with long stretches of DNA (or RNA
when it is the genetic material) being inverted, inserted, duplicated, deleted,
transposed or translocated.
The result of a mutation could be harmful, beneficial, neutral or even silent.
Mutation can lead to the loss or gain of a specific function, to changes to the
expression levels, or in extreme cases, even embryonic lethality. Mutations can be
classified in various ways depending on the cause of the mutation, its effect on the
function of the gene product or the kind of changes to the structure of the gene
itself.
In genetics, the mutation rate is the frequency of new mutations in a single
gene or organism over time. Mutation rates are not constant and are not limited to
a single type of mutation, therefore there are many different types of mutations.
Basically, the mutation rates are given for specific classes of mutations. Point
mutations are a class of mutations which are small or large scale insertions or
deletions. The rate of these types of substitutions can be further subdivided into a
mutation spectrum which describes the influence of the genetic context on the
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Mutation mutation rate. Mutagenesis is a process by which the genetic information of an
organism is changed, resulting in a mutation. It may occur spontaneously in nature,
or as a result of exposure to mutagens. It can also be achieved experimentally
using laboratory procedures. In nature mutagenesis can lead to cancer and various
NOTES heritable diseases, but it is also a driving force of evolution.
In this unit, you will study about the mutation and its types, mutation rate
and its determination, and mutagenesis.

1.1 OBJECTIVES

After going through this unit, you will be able to:

Discuss what mutation is
Explain radiation induced mutations
Analyze practical applications of mutations
Describe gene mutations and its types
Explain about the mutation rate
Determine the mutation rate
Understand mutagenesis

1.2 MUTATION AND ITS TYPES

The term mutation refers both to the change in the genetic material and to the
process by which the change occurs. An organism exhibiting a novel phenotype as
a result of the presence of mutation is referred to as mutant. Mutation refers to any
sudden, heritable change in the genotype of an organism not explainable by
recombination of preexisting genetic variability. Such genotypic changes includes
change in chromosome number (euploidy and aneuploidy), gross changes in the
structure of chromosomes (chromosomes aberration) and changes in individual
genes. The term mutation refers to changes in individual genes.
Many mutations involve changes in single base pairs, the substitution of one
basepair for another, or duplication or deletion of single base pairs, such mutation
is referred as point mutation.
Mutation is the ultimate source of all genetic variation, it provides the raw
material for evolution,(Refer Figure 1.1). Recombination (independent assortment
plus recombination of genetic variability present in individual chromosomes)
rearranges this genetic variability into new combinations and natural selection
preserves the combinations adapted to existing environmental conditions. Without
mutation, all genes would exist on only one form. Alleles would not exist and
genetic analysis would not be possible. Organisms would not be able to evolve
and adapt to environmental changes.
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 Mutation

NOTES

Fig. 1.1 Diagrammatic Representation of Mutations

Mutations are inherited alterations in the DNA sequence. DNA is a highly

stable molecule that is replicated with amazing accuracy but changes in DNA
structure and errors of replication do occur, (Refer Figure 1.2). A mutation is
defined as an inherited change in genetic information, the descendants may be
cells or organisms.

Fig. 1.2 DNA Incoming UV Structure Before and After Incoming UV Photon

The appearance of a new mutation is a rare event. Most mutations that

were originally studied occurred spontaneously. This class of mutation is
termed spontaneous mutations. Historically, geneticists recognized these in nature.
The mutations were collected, and the inheritance of these mutations were analyzed.
But these mutations clearly represent only a small number of all possible mutations.
To genetically dissect a biological system further, new mutations were created by
scientists by treating an organism with a mutagenizing agent. These mutations are
called induced mutations.
Mutagens are chemical compounds or forms of radiation (such as, UltraViolet
(UV) light or X-rays) that cause irreversible and heritable changes (mutations) in
the cellular genetic material, DeoxyriboNucleic Acid (DNA). In this way,
mutagenesis becomes a cumulative process, stretching over the lifetime of an
organism. Self-Instructional
Material 3
Mutation The biological consequences of a mutation depend upon many critical factors,
such as the target loci, size of the mutation, timing during the cell cycle, and
compounding effects of preexisting mutations. Mutagenic lesions persist when they
escape detection by protective cellular DNA repair mechanisms, when mistakes
NOTES occur in the repair process, or when repair mechanisms are overwhelmed by
extensive damage. Upon subsequent cellular replication, these mutations become
fixed in the genome and are inherited by all daughter cells. Thus, a mutagenic
event occurring in a nonfunctional area of DNA will have no effect (silent mutation),
whereas a similar change in an actively transcribed region may profoundly affect
gene expression and phenotype or even lead to cell death (lethal mutation).
There are many chemical mutagens which can react with nucleus and the
DNA, for example, base analogues which are derivatives of normal bases. This
can cause direct mutagenesis. Similarly alkylating agents like
methylmethanesulphonate, ethylmethanesulphonate and ethylnitrosourea are
capable of producing DNA lesions which are repaired to prevent any disruption
to process of transcription and replication. Nitrous acid deaminates cytosine to
produce Uracil (U) which can base pair with adenine creating transitions. Similarly,
5 bromouracil can lead to mutations. Transition is where a pyrimidine changes to
another Pyrimidine or Purine to another Purine while in transversion change involves
Purine to Pyrimidine or Pyrimidine to Purine.
The spontaneous mutation rate varies. Large gene provide a large target
and tend to mutate more frequently. A study of the five coat color loci in mice
showed that the rate of mutation ranged from 2 x 10-6 to 40 x 10-6 mutations per
gamete per gene. Data from several studies on Eukaryotic organisms shows that
in general the spontaneous mutation rate is 2-12 x 10-6 mutations per gamete per
gene. Given that the human genome contains 100,000 genes, we can conclude
that we would predict that 1-5 human gametes would contain a mutation in some
gene. Spontaneous mutations occur infrequently although the observed frequencies
vary from gene to gene and from organism to organism.
Phenotypic Effects of Mutation
Mutations cause some detectable phenotypic change for their presence to be
recognized. The effects of mutations on phenotype range from alterations so minor
that they can be detected only by special genetic or biochemical techniques to
gross modifications of morphology. A gene is a specific sequence of nucleotide
pairs coding for a particular polypeptide. Any mutation occurring within a given
gene will produce a new form. Because of the degeneracy of the genetic code,
some base pair changes do not change the protein products encoded by the genes
in any way. Genes containing mutations with small effects can be recognized by
special techniques called isoalleles. Mutations may be either recessive or dominant.
In haploid organisms like viruses or bacteria, both recessive and dominant mutations
can be recognized by their effects on the phenotype of the organism in which they
originated. In diploid organisms, recessive mutation will be recognized only when
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present in the homozygous condition. Most recessive mutations in diploids will not Mutation

be recognized at the time of their occurrence, since they are present in heterozygous
state. Sex linked recessive mutations are an exception, since they will be expressed
in hemizygous state in the heterogametic sex. The most useful mutations for genetic
analyses of many biological processes are conditional lethal mutations. NOTES

Somatic and Germinal Mutations

Mutation occur in any cell and at any state in the cell cycle. The immediate effect
of mutation and its ability to produce phenotypic change are determined by its
dominance, the type of cell in which it occurs and when it happens relative to life
cycle of the organism.
If the mutation occurs in a somatic cell, which can produce cell like itself but
not the whole organism, the mutant change will be perpetuated only in somatic
cells that descend from original cell in which mutation occurred. The Delicious
apple and the navel orange were mosaics in somatic tissues. Changes that give
these two fruits their desirable qualities followed spontaneous mutation in single
cells. If dominant mutation occur in germ cells, their effects may be expressed
immediately in progeny. If mutations are recessive, their effects are obscured in
diploids. Germinal mutations may occur at any stage in the reproductive cycle of
the organism, but they are common during some stages than others.
Back Mutation and Suppressor Mutation
The mutation of a wild type gene to form that results in a mutant phenotype is
referred as forward mutation. In a population composed entirely of brown eyed
individual, the allele for blue eyes is thought as mutant allele. Mutation events are
reversible. Mutation may occur that restores the original wild type phenotype.
This is referred to as back mutation, reverse mutation or reversion. Reversion may
occur by true back mutation at the same site in the gene as the original mutation,
restoring the wild type nucleotide sequence or by the occurrence of a second
mutation at a different location in the genome, which compensates for first mutation,
these mutations are called suppressor mutations. It may occur at distinct sites in
the same gene as the original mutation or in different genes, even in different
chromosomes.
Radiation Induced Mutations
That portion of the electromagnetic spectrum containing wavelengths that are
shorter and of higher energy than the visible light can be divided into ionizing
radiation(X rays, gamma rays , cosmic rays) and non-ionizing radiations (UV light).
Ionizing radiations are of high energy and are useful for medical diagnosis because
they penetrate the living tissue. UV rays having lower energy penetrate only the
surface layer of cells in higher plants and animals and do not induce ionizations.
UV rays dissipate their energy to atoms that they encounter raising the electrons in
the outer orbitals to higher energy levels referred as excitation. Molecules containing
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Mutation atoms in their ionic or excited forms are chemically more reactive than those
containing atoms in their normal stable state. The increasing reactivity of atoms
present in DNA molecules is the basis of the mutagenic effects of UV light and
ionizing radiations. UV radiations are readily absorbed by substances like purines
NOTES and pyrimidines which enter a more reactive or excited state. The two major
products of UV absorption by pyrimidines appear to be pyrimidine hydrates and
pyrimidine dimmers.
Chemically Induced Mutations
The first chemical mutagen discovered was mustard gas. When C. Auerbach and
her associates discovered the mutagenic effects of mustard gas and related
compounds during world war II. These compounds are examples of large class of
chemical mutagens that transfer alkyl group to the bases in DNA, called as alkylating
agents.
Chemical mutagens are divided into two classes: those that are mutagenic
to both replicating and non-replicating DNA such as alkylating agent and nitrous
acid., those that are mutagenic only to replicating DNA, which mainly includes
acridine dyes, which bind to DNA and increase the probability of mistakes during
replication and base analogs, purines and pyrimidines with structure similar to
normal bases of DNA. The base analogs must be incorporated into DNA chains
in place of normal bases during replication to exert their mutagenic effects.
Base Analogs: The base analogs that are mutagenic have structures similar to the
normal bases so that they are incorporated into DNA during replication. The two
most commonly used base analogs are 5 Bromo Uracil (U) and 2 Amino Purine.
Nitrous Acid: It is a very potent mutagen that acts directly on either replicating or
non-replicating DNA by oxidative deamination of the bases that contain amino
groups- Adenine (A), Guanine (G) and Cytosine (C). Conversion of the amino
groups to keto groups changes the hydrogen bonding potential of the bases.
Acridines: The positively charged acridine intercalate between the stacked base
pairs in DNA. They increase the rigidity and alter the conformation of the double
helix, causing kinks in the molecule. When DNA molecules containing intercalated
acridines replicate, addition and deletions of one or few base pairs occur. These
small additions and deletions of single base pair result in reading frameshifts for the
portion of the gene distal to the mutation.
Alkylating and Hydroxylating Agents: Alkylating agents, such as Nitrogen and
Sulphur mustards, Methyl and Ethyl methanesulfonate have several effects on DNA.
Nitrosoguanidine, one of the most potent chemical mutagens has been found to
induce clusters of closely linked mutations in the segment of the chromosome that
is replicating during mutagenic treatment. The hydroxylamine has a very specific
mutagenic effects. It induces only GC- AT transitions.

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 Mutation

NOTES

Fig. 1.3 Chromosome Segments

Practical Applications of Mutations

Mutations are invalueable to the process of evolution since they provide the raw
material required for its occurrence. Mutation provide the alleles required for various
types of genetic analysis, from Mendel’s two factor crosses to chromosome mapping
to studies of genetic structures of populations. Even though most mutations make
the organism less efficient and disadvantageous, the possibility of developing new
desirable traits through induced mutations has intrigued many plant breeders. Plant
breeders have reported induced mutants in barley, wheat, oat, soyabeans, tomatoes,
fruit trees that improve cultivated strains. Barley mutants have been obtained that
provide increased protein content and hull less seeds. One application of induced
mutations came from efforts to improve the yield of penicillin by the mold penicillium.
When penicillin was first discovered, yield was low and production was limited.
Then millions of spores were irradiated and few of the surviving colonies produced
more penicillin than the average. Such mutant overproducers of penicillin have
proven invaluable in the commercial production of this important antibiotic.
Mutations and Humans
Purposeful artificial selection is not practiced in humans and therefore the possible
advantages cited for domestic animals and plants do not apply to humankind.
Variations do exist in populations, however and presumably they originated through
past mutations. Since mutations are detrimental, it would seem advantageous from
the stand point of short term effects for humans to avoid excessive exposure to
mutagenic agents. In case of acute irradiation, two types of dangers are considered.
i.e. the immediate damage to the exposed person, more damage to the DNA in his
or her reproductive cells, which would affect future generations. The immediate
damage is indicated by burns and other direct or secondary effects on body tissues.
When doses are on the order of 50 mr or lower, no immediate damage can be
detected, although some harmful effects such as induction of leukemia and shortening
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Mutation of life span may occur. Effects of the second type of damage will be observed in
only future generations. This is a reason to believe that exposure to high energy
radiations at any dosage level is potentially harmful. In the few results to date,
mutations have been proportional to the dosage and the effects have been
NOTES cumulative.
The relation between dosage and effects cannot be accurately measured in
humans at present because of the complexity of the subject and difficulties of dealing
with the genetics of humans. Preliminary reports including data on children born to
parents who survived the Nagasaki bombing have revealed a significant increase in
incidence of leukemia. The normal sex ratio has been altered, and changes have
been interpreted as resulting from induced sex linked lethals. Most of the data available
on mutation rates and the nature of mutations have come from other organisms and
only by inference are they applied to humans. However the genetic material in humans
is DNA, just as it is in most other organisms. It should not be surprising that the
effects of irradiation are comparable to most organisms.
Mutations can be induced by several methods. The three general approaches
used to generate mutations are radiation, chemical and transposon insertion. The
first induced mutations were created by treating Drosophila with X-rays. Using
this approach Mueller to induce lethal mutations. In addition to X-rays, other
types of radiation treatments that have proven useful include gamma rays and fast
neutron bombardment. These treatments can induce point mutations (changes in a
single nucleotide) or deletions (loss of a chromosomal segment).
Chemical mutagens work mostly by inducing point mutations. Point mutations
occur when a single base pair of a gene is changed. These changes are classified
as transitions or transversions. Transitions occur when a purine is converted to a
purine (A to G or G to A) or a pyrimide is converted to a pyrimidine (T to C or C
to T). A transversion results when a purine is converted to a pyrimidine or a
pyrimidine is converted to a purine. A transversion example is adenine being converte
d to a cytosine.
Two major classes of chemical mutagens are routinely used. These
are alkylating agents and base analogs. Each has a specific effect on DNA.
Alkylating agents (such as, ethyl Methane Sulphonate (EMS), Ethyl Ethane
Sulphonate (EES) and Mustard Gas) can mutate both replicating and non-replicating
DNA. By contrast, a base analog (5-bromouracil and 2-aminopurine) only mutate
DNA when the analog is incorporated into replicating DNA. Each class of chemical
mutagen has specific effects that can lead to transitions, transversions or deletions.
Scientists are now using the power of transposable elements to create new
mutations. Transposable elements are mobile pieces of DNA that can move from
one location in a geneome to another. Often when they move to a new location,
the result is a new mutan t. The mutant arises because the presence of a piece of
DNA in a wild type gene disrupts the normal function of that gene. As more and
more is being learned about genes and genomes, it is becoming apparent that
Self-Instructional transposable elements are a power source of or creating insertional mutants.
8 Material
Gene Mutations and its Types Mutation

Germ line mutations arise in cells that ultimately produce gametes. A germ line
mutation can be passed to future generations producing individual organisms that
carry the mutation in all their somatic and germ line cells. Mutations have been NOTES
partitioned into those that affect a single gene, called gene mutations and those that
affect the number or structure of chromosomes called chromosome mutations.
Base Substitutions: The simplest type of gene mutation is a base substitution, the
alteration of a single nucleotide in the DNA. Base substitution are of two types. In
transition, a purine is replaced by different purine or pyrimidine is replaced by a
different pyrimidine. In transversion, a purine is replaced by a pyrimidine or a
pyrimidine is replaced by a purine.
Insertions and Deletions: It mainly includes the addition or the removal of one
or more nucleotide pairs. Insertions and deletions within sequences that encode
proteins may lead to frameshift mutations, changes in the reading frame of the
gene. It alter all amino acids encoded by nucleotides following mutation and have
drastic effects on the phenotype.
Expanding Nucleotide Repeats: Mutations in which the number of copies of a
set of nucleotides increases in number are called expanding nucleotide repeats.
This type of mutation was observed in 1991 in a gene called FMR-1 which causes
fragile X syndrome, the most common cause heredity cause of mental retardation.
Expanding nucleotide repeats are found in almost 30 human diseases. The number
of copies of the nucleotide repeat correlates with the severity or age of onset of
disease.
The detailed knowledge of the structure and funciton of transposable elements
is now being applied in the pursuit of new mutations. Stocks are created in which
a specific type of element is present. This stock is then crossed to a genetic stock
that doe s not contain the element. Once that element enters the virgin stock, it can
begin to move around that genome. By carefully observing the offspring, new
mutants can be discovered. This approach to developing mutants is termed
insertional mutagenesis.

1.3 MUTATION RATE AND ITS DETERMINATION

In genetics, the mutation rate is the frequency of new mutations in a single gene or
organism over time. Mutation rates are not constant and are not limited to a single
type of mutation, therefore there are many different types of mutations. Mutation
rates are given for specific classes of mutations. Point mutations are a class of
mutations which are small or large scale insertions or deletions. There are also
Missense and Nonsense mutations, which are variations of point mutations. The
rate of these types of substitutions can be further subdivided into a mutation
spectrum which describes the influence of the genetic context on the mutation rate.
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Mutation There are several natural units of time for each of these rates, with rates
being characterized either as mutations per base pair per cell division, per gene
per generation, or per genome per generation. The mutation rate of an organism is
an evolved characteristic and is strongly influenced by the genetics of each organism,
NOTES in addition to strong influence from the environment. The upper and lower limits to
which mutation rates can evolve is the subject of ongoing investigation. However,
the mutation rate does vary over the genome. Over DNA, RNA or a single gene,
mutation rates are changing.
When the mutation rate in humans increases certain health risks can occur,
for example, cancer and other hereditary diseases. Having knowledge of mutation
rates is vital to understanding the future of cancers and many hereditary diseases.
Measurement
An organism’s mutation rates can be measured by a number of techniques.
1. One way to measure the mutation rate is by the fluctuation test, also known
as the Luria–Delbrück experiment. This experiment exhibits in bacteria
mutations that occur in the absence of selection instead of the presence of
selection.
2. This is very important to mutation rates because it proves experimentally
mutations can occur without selection being a component. Therefore,
mutations occur at random in bacteria and other organisms.
Substitution Rates
Many sites in an organism’s genome may admit mutations with small fitness effects.
These sites are typically called neutral sites. Theoretically mutations under no
selection become fixed between organisms at precisely the mutation rate. Fixed
synonymous mutations, i.e., synonymous substitutions, are changes to the sequence
of a gene that do not change the protein produced by that gene. They are often
used as estimates of that mutation rate, despite the fact that some synonymous
mutations have fitness effects. As an example, mutation rates have been directly
inferred from the whole genome sequences of experimentally evolved replicate
lines of Escherichia coli B.
Mutation Accumulation Lines
A particularly labor-intensive way of characterizing the mutation rate is the mutation
accumulation line.
Mutation accumulation lines have been used to characterize mutation rates
with the Bateman-Mukai Method and direct sequencing of, for example intestinal
bacteria, round worms, yeast, fruit flies, small annual plants.

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Variation in Mutation Rates Mutation

Mutation rates differ between species and even between different regions of the
genome of a single species. These different rates of nucleotide substitution are
measured in substitutions (fixed mutations) per base pair per generation. For NOTES
example, mutations in intergenic, or non-coding, DNA tend to accumulate at a
faster rate than mutations in DNA that is actively in use in the organism (gene
expression). That is not necessarily due to a higher mutation rate, but to lower
levels of purifying selection. A region which mutates at predictable rate is a candidate
for use as a molecular clock.
If the rate of neutral mutations in a sequence is assumed to be constant
(clock-like), and if most differences between species are neutral rather than
adaptive, then the number of differences between two different species can be
used to estimate how long ago two species diverged. In fact, the mutation rate of
an organism may change in response to environmental stress. For example, UV
light damages DNA, which may result in error prone attempts by the cell to perform
DNA repair.
The human mutation rate is higher in the male germ line (sperm) than the
female (egg cells), but estimates of the exact rate have varied by an order of
magnitude or more. This means that a human genome accumulates around 64 new
mutations per generation because each full generation involves a number of cell
divisions to generate gametes. Human mitochondrial DNA has been estimated to
have mutation rates of ~3× or ~2.7×10–5 per base per 20 year generation
(depending on the method of estimation); these rates are considered to be
significantly higher than rates of human genomic mutation at ~2.5×10–8 per base
per generation. Using data available from whole genome sequencing, the human
genome mutation rate is similarly estimated to be ~1.1×10–8 per site per generation.
The rate for other forms of mutation also differs greatly from point mutations.
An individual microsatellite locus often has a mutation rate on the order of 10–4,
though this can differ greatly with length.
Some sequences of DNA may be more susceptible to mutation. For example,
stretches of DNA in human sperm which lack methylation are more prone to
mutation.
Figure 1.4 illustrates the reported estimates of the human genome-wide
mutation rate. The human germ line mutation rate is approximately 0.5 × 10–9 per
base pair per year.

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Mutation

NOTES

Fig. 1.4 Estimation of the Human Genome-Wide Mutation Rate

In general, the mutation rate in unicellular Eukaryotes (and Bacteria) is roughly

0.003 mutations per genome per cell generation. However, some species, especially
the ciliate of the genus Paramecium have an unusually low mutation rate. For
instance, Paramecium tetraurelia has a base-substitution mutation rate of ~2 ×
10–11 per site per cell division. This is the lowest mutation rate observed in nature
so far, being about 75 × lower than in other eukaryotes with a similar genome size,
and even 10 × lower than in most prokaryotes. The low mutation rate in
Paramecium has been explained by its transcriptionally silent germ line nucleus,
consistent with the hypothesis that replication fidelity is higher at lower gene
expression levels.
The highest per base pair per generation mutation rates are found in viruses,
which can have either RNA or DNA genomes. DNA viruses have mutation rates
between 10–6 to 10–8 mutations per base per generation, and RNA viruses have
mutation rates between 10–3 to 10–5 per base per generation.
Mutational Spectrum
The mutation spectrum of an organism is the rate at which different mutations
occur at different sites. Typically two sites are considered, each of which may
have three mutations, resulting in six total rates for most mutation spectra. The two
sites are the two correct pairs possible in DNA, the A:T pairs and the C:G pairs.
Figure 1.5 illustrates the Transitions (Alpha) and Transversions (Beta).

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 Mutation

NOTES

Fig. 1.5 Transitions (Alpha) and Transversions (Beta)

There is a systematic difference in rates for transitions (Alpha) and

transversions (Beta).
Spontaneous Mutations
Spontaneous mutations are mutations that occur in the absence of exogenous agents.
They may be due to errors made by DNA polymerases during replication or repair,
errors made during recombination, the movement of genetic elements, or
spontaneously occurring DNA damage. The rate at which spontaneous mutations
occur can yield useful information about cellular processes.
The mutation rate is the expected number of mutations that a cell will sustain
during its lifetime. The mutant fraction or frequency is the proportion of cells in a
population that are mutant. Even though mutant frequencies can be adequate
indicators of the rate at which mutations are induced by DNA damaging agents,
they are inadequate indicators of spontaneous mutation rates. This is because the
population of mutants is composed of clones, each of which arose from a cell that
sustained a mutation.
Determination of Mutation Rate
Broadly, there are two methods for determination of the mutation rate: Mutation
Accumulation and Fluctuation Analysis. Mutant accumulation methods have the
advantage that they are very accurate, but they are complicated and time-consuming
to perform because the culture is sampled at multiple time points. The methodology
depends on growing bacteria exponentially until probability dictates that a mutant
will be present. If the assumption is made that the growth rates of wild-type and
mutant bacteria are the same, then the proportion of mutants will increase linearly
with time. Furthermore, if the number of mutants and the total number of bacterial
cells are known at each time point, then the mutation rate (m) can be calculated
from the slope of the line describing the relationship between the number of mutants
against the generation number. The mutation rate can be determined by using the
equation,
= [(r2/N2) – (r1/N1)] × ln (N2/N1) = (f1 “ f2) × ln (N2/N1)
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Mutation Where r1 is the observed number of mutants at time point 1, r2 is the observed
number of mutants at the next time point, and N1 and N2 are the numbers of cells
at time points 1 and 2, respectively, while f1 and f2 are the mutant frequencies at
points 1 and 2.
NOTES
For this method to be accurate, a very large difference in the total cell
number is required between N1 (the number of cells at the first time point) and N2
(the number of cells at the second time point). Serial dilutions can help in estimating
this, but this includes sampling errors. Alternatively, the continuous culture can be
used for the selection of waves of bacteria, each suited than the generation before
to take over the culture.
Following is the description of the terms used in the determination of mutation
rate:
m Number of Mutations Per Culture
Mutation Rate; Probability of Mutation Per Cell Per Division or Generation
N Number of Cells
N0 Initial Number of Cells in a Culture
Nt Final Number of Cells in a Culture
r Observed number of Mutants in a Culture
r˜ Median Number of Mutants in a Culture
f Mutant Fraction or Frequency = r/N
V Volume of a Culture
C Number of Cultures in Experiment
p0 Proportion of Cultures without Mutants
z Dilution Factor or Fraction of a Culture Plated
pr Proportion of Cultures with r Mutants
Cr Number of Cultures with r Mutants
Pr Proportion of Cultures with r or More Mutants
Q1 Value of r at 25% of the Ranked Series of r
Q2 Value of r at 50% of the Ranked Series of r, the Median
Q3 Value at of r at 75% of the Ranked Series of r
Standard Deviation
CL Confidence Limit = (1 ) × 100
Level of Statistical Significance, usually 0.05 or 0.01

It is significant to distinguish between m which is the mean number of

mutations that occur during the growth of a culture, and which is the mutation
rate, specifically the mean number of mutations that occur during the lifetime of a
cell. Almost all methods to calculate mutation rates start by determining m and
then obtaining by dividing m by some measure of the number of cell-lifetimes at
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risk for mutation, usually Nt. It is also significant to distinguish between the number Mutation

of mutations per culture, m, and the number of mutants per culture, r. r divided by
Nt is the mutant fraction or mutant frequency, f.
Calculating the Mutation Rate NOTES
The mutation rate, , is the mean number of mutations, m, normalized to some
measure of the number of cells at risk for mutation. Three such measures are used,
each of which is based on different assumptions about the underlying mutational
process. If the probability of mutation is constant over the cell cycle, then m should
be divided by the number of cell divisions that have taken place. Since the final
number of cells in a culture, Nt, arose from Nt –1 divisions, the mutation rate is
(Luria and Delbrück, 1943):
= m / (Nt -1) H” m / Nt

1.4 MUTAGENESIS

Mutagenesis is a process by which the genetic information of an organism is

changed, resulting in a mutation. It may occur spontaneously in nature, or as a
result of exposure to mutagens. It can also be achieved experimentally using
laboratory procedures. In nature mutagenesis can lead to cancer and various
heritable diseases, but it is also a driving force of evolution. Mutagenesis as a
science was developed based on work done by Hermann Muller, Charlotte
Auerbach and J. M. Robson in the first half of the 20th century.
Mutagenesis may occur endogenously, for example, through spontaneous
hydrolysis, or through normal cellular processes that can generate reactive oxygen
species and DNA adducts, or through error in replication and repair. Mutagenesis
may also arise as a result of the presence of environmental mutagens that induce
changes to the DNA. The mechanism by which mutation arises varies according
to the causative agent, the mutagen, involved. Most mutagens act either directly,
or indirectly via mutagenic metabolites, on the DNA producing lesions. Some,
however, may affect the replication or chromosomal partition mechanism, and
other cellular processes.
Mutagenesis may also be self-induced by unicellular organisms when
environmental conditions are very restrictive, for instance, in presence of toxic
substances like antibiotics or, in yeasts, in presence of an antifungal agent or in
absence of a nutrient.
Many chemical mutagens require biological activation to become mutagenic.
An important group of enzymes involved in the generation of mutagenic metabolites
is cytochrome P450. Other enzymes that may also produce mutagenic metabolites
include glutathione S-transferase and microsomal epoxide hydrolase. Mutagens
that are not mutagenic by themselves but require biological activation are called
promutagens.
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Mutation Many mutations arise as a result of problems caused by DNA lesions during
replication, resulting in errors in replication. In bacteria, extensive damage to DNA
due to mutagens results in single-stranded DNA gaps during replication. This induces
the SOS response, an emergency repair process that is also error-prone, thereby
NOTES generating mutations. In mammalian cells, stalling of replication at damaged sites
induces a number of rescue mechanisms that help bypass DNA lesions, but which
also may result in errors. The Y family of DNA polymerases specializes in DNA
lesion bypass in a process termed TransLesion Synthesis (TLS) whereby these lesion-
bypass polymerases replace the stalled high-fidelity replicative DNA polymerase,
transit the lesion and extend the DNA until the lesion has been passed so that normal
replication can resume. These processes may be error-prone or error-free.
Transposons and viruses may insert DNA sequences into coding regions or
functional elements of a gene and result in inactivation of the gene.
Mutagenesis in the laboratory is the significant technique whereby DNA
mutations are deliberately engineered to produce Mutant Genes, Proteins, or Strains
of Organism. Various constituents of a gene, such as its control elements and its
gene product, may be mutated so that the functioning of a gene or protein can be
examined in detail. The mutation may also produce mutant proteins with interesting
properties, or enhanced or novel functions that may be of commercial use.
Early methods of mutagenesis produced entirely random mutations; however,
later methods of mutagenesis may produce site-specific mutation.
Adaptive Mutagenesis Mechanisms
Adaptive mutagenesis has been defined as mutagenesis mechanisms that enable
an organism to adapt to an environmental stress. For instance, in bacteria, while
modulation of the SOS response and endogenous prophage DNA synthesis has
been shown to increase Acinetobacter baumannii resistance to Ciprofloxacin.
Resistance mechanisms are presumed to be linked to chromosomal mutation
untransferable via horizontal gene transfer in some members of Enterobacteriaceae
family, such as Escherichia coli, Salmonella spp., Klebsiella spp., and
Enterobacter spp. Chromosomal events are also relevant to this adaptive
mutagenesis in bacteria.

Check Your Progress

1. Explain the term mutation.
2. What is silent mutation and lethal mutation?
3. What is a gene?
4. What is phenotypic effects of mutation?
5. Define the term germ line mutation. Differentiate between gene mutations
and chromosome mutations.
6. Explain mutation rate with reference to genetics.
7. Define mutation spectrum of an organism.
8. What is mutagenesis? How it occurs?
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 Mutation
1.5 ANSWERS TO CHECK YOUR PROGRESS
QUESTIONS

1. The term mutation refers both to the change in the genetic material and to NOTES
the process by which the change occurs. An organism exhibiting a novel
phenotype as a result of the presence of mutation is referred to as mutant.
Mutation refers to any sudden, heritable change in the genotype of an
organism not explainable by recombination of pre-existing genetic variability.
Such genotypic changes includes change in chromosome number (euploidy
and aneuploidy), gross changes in the structure of chromosomes
(chromosomes aberration) and changes in individual genes.
2. A mutagenic event occurring in a nonfunctional area of DNA will have no
effect (silent mutation), whereas a similar change in an actively transcribed
region may profoundly affect gene expression and phenotype or even lead
to cell death (lethal mutation).
3. A gene is a specific sequence of nucleotide pairs coding for a particular
polypeptide.
4. Mutations cause some detectable phenotypic change for their presence to
be recognized. The effects of mutations on phenotype range from alterations
so minor that they can be detected only by special genetic or biochemical
techniques to gross modifications of morphology. A gene is a specific sequence
of nucleotide pairs coding for a particular polypeptide. Any mutation
occurring within a given gene will produce a new form.
5. Germ line mutations arise in cells that ultimately produce gametes. A germ
line mutation can be passed to future generations producing individual
organisms that carry the mutation in all their somatic and germ line cells.
Mutations have been partitioned into those that affect a single gene, called
gene mutations and those that affect the number or structure of chromosomes
called chromosome mutations.
6. In genetics, the mutation rate is the frequency of new mutations in a single
gene or organism over time. Mutation rates are not constant and are not
limited to a single type of mutation, therefore there are many different types
of mutations. Point mutations are a class of mutations which are small or
large scale insertions or deletions. The rate of these types of substitutions
can be further subdivided into a mutation spectrum which describes the
influence of the genetic context on the mutation rate.
There are several natural units of time for each of these rates, with rates
being characterized either as mutations per base pair per cell division, per
gene per generation, or per genome per generation. However, the mutation
rate does vary over the genome. Over DNA, RNA or a single gene, mutation
rates are changing.
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Mutation 7. The mutation spectrum of an organism is the rate at which different mutations
occur at different sites. Typically two sites are considered, each of which
may have three mutations, resulting in six total rates for most mutation spectra.
The two sites are the two correct pairs possible in DNA, the A:T pairs and
NOTES the C:G pairs.
8. Mutagenesis is a process by which the genetic information of an organism is
changed, resulting in a mutation. It may occur spontaneously in nature, or as
a result of exposure to mutagens. It can also be achieved experimentally
using laboratory procedures. In nature mutagenesis can lead to cancer and
various heritable diseases, but it is also a driving force of evolution.
Mutagenesis may occur endogenously, for example, through spontaneous
hydrolysis, or through normal cellular processes that can generate reactive
oxygen species and DNA adducts, or through error in replication and repair.
Mutagenesis may also arise as a result of the presence of environmental
mutagens that induce changes to the DNA. The mechanism by which mutation
arises varies according to the causative agent, the mutagen, involved.

1.6 SUMMARY

The term mutation refers both to the change in the genetic material and to
the process by which the change occurs.
An organism exhibiting a novel phenotype as a result of the presence of
mutation is referred to as mutant.
Mutation refers to any sudden, heritable change in the genotype of an
organism not explainable by recombination of pre-existing genetic variability.
Such genotypic changes includes change in chromosome number (euploidy
and aneuploidy), gross changes in the structure of chromosomes
(chromosomes aberration) and changes in individual genes.
Sudden, heritable changes in the genetic material are called mutations.
It refers to the process by which such changes are produced.
It may occur spontaneously or may be induced by agents that interact with
DNA and RNA.
At the simplest level, a mutation is a change or transformation. In biology,
mutations refer to changes in chromosomes and genes, which typically
manifest physically.
The result of a mutation could be harmful, beneficial, neutral or even silent.
Mutation can lead to the loss or gain of a specific function, to changes to the
expression levels, or in extreme cases, even embryonic lethality.
Mutations can be classified in various ways depending on the cause of the
mutation, its effect on the function of the gene product or the kind of changes
Self-Instructional to the structure of the gene itself.
18 Material
A mutation is defined as an inherited change in genetic information, the Mutation

descendants may be cells or organisms.

The appearance of a new mutation is a rare event. Most mutations that
were originally studied occurred spontaneously.
NOTES
Mutagens are chemical compounds or forms of radiation (such as ultraviolet
(UV) light or X-rays) that cause irreversible and heritable changes
(mutations) in the cellular genetic material, deoxyribonucleic acid (DNA).
The biological consequences of a mutation depend upon many critical factors
such as the target loci, size of the mutation, timing during the cell cycle, and
compounding effects of pre-existing mutations.
Mutations cause some detectable phenotypic change for their presence to
be recognized.
Mutation occur in any cell and at any state in the cell cycle. The immediate
effect of mutation and its ability to produce phenotypic change are determined
by its dominance, the type of cell in which it occurs and when it happens
relative to life cycle of the organism.
The portion of the electromagnetic spectrum containing wavelengths that
are shorter and of higher energy than the visible light can be divided into
ionizing radiation(X rays, gamma rays , cosmic rays) and non-ionizing
radiations (UV light).
The first chemical mutagen discovered was mustard gas. When C. Auerbach
and her associates discovered the mutagenic effects of mustard gas and
related compounds during world war II.
The positively charged acridine intercalate between the stacked base pairs
in DNA. They increase the rigidity and alter the conformation of the double
helix, causing kinks in the molecule.
Mutations are invaluable to the process of evolution since they provide the
raw material required for its occurrence.
Mutation provide the alleles required for various types of genetic analysis,
from Mendel’s two factor crosses to chromosome mapping to studies of
genetic structures of populations.
The relation between dosage and effects cannot be accurately measured in
humans at present because of the complexity of the subject and difficulties
of dealing with the genetics of humans.
Germ line mutations arise in cells that ultimately produce gametes. A germ
line mutation can be passed to future generations producing individual
organisms that carry the mutation in all their somatic and germ line cells.
In genetics, the mutation rate is the frequency of new mutations in a single
gene or organism over time.

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Mutation Mutation rates are not constant and are not limited to a single type of mutation,
therefore there are many different types of mutations.
Mutation rates are given for specific classes of mutations. Point mutations
are a class of mutations which are small or large scale insertions or deletions.
NOTES
There are several natural units of time for each of these rates, with rates
being characterized either as mutations per base pair per cell division, per
gene per generation, or per genome per generation.
The mutation rate of an organism is an evolved characteristic and is strongly
influenced by the genetics of each organism, in addition to strong influence
from the environment.
The upper and lower limits to which mutation rates can evolve is the subject
of ongoing investigation. However, the mutation rate does vary over the
genome. Over DNA, RNA or a single gene, mutation rates are changing.
Mutation rates differ between species and even between different regions
of the genome of a single species. These different rates of nucleotide
substitution are measured in substitutions (fixed mutations) per base pair
per generation. For example, mutations in intergenic, or non-coding, DNA
tend to accumulate at a faster rate than mutations in DNA that is actively in
use in the organism (gene expression).
The mutation spectrum of an organism is the rate at which different mutations
occur at different sites. Typically two sites are considered, each of which
may have three mutations, resulting in six total rates for most mutation spectra.
The two sites are the two correct pairs possible in DNA, the A:T pairs and
the C:G pairs.
The mutation rate is the expected number of mutations that a cell will sustain
during its lifetime. The mutant fraction or frequency is the proportion of
cells in a population that are mutant.
Mutagenesis is a process by which the genetic information of an organism is
changed, resulting in a mutation. It may occur spontaneously in nature, or
as a result of exposure to mutagens. It can also be achieved experimentally
using laboratory procedures.
In nature mutagenesis can lead to cancer and various heritable diseases,
but it is also a driving force of evolution.
Mutagenesis as a science was developed based on work done by Hermann
Muller, Charlotte Auerbach and J. M. Robson in the first half of the 20th
century.
Mutagenesis may occur endogenously, for example, through spontaneous
hydrolysis, or through normal cellular processes that can generate reactive
oxygen species and DNA adducts, or through error in replication and repair.

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 Mutagenesis may also arise as a result of the presence of environmental Mutation

mutagens that induce changes to the DNA. The mechanism by which mutation
arises varies according to the causative agent, the mutagen, involved.
Most mutagens act either directly, or indirectly via mutagenic metabolites,
NOTES
on the DNA producing lesions. Some, however, may affect the replication
or chromosomal partition mechanism, and other cellular processes.
Adaptive mutagenesis has been defined as mutagenesis mechanisms that
enable an organism to adapt to an environmental stress.

1.7 KEY WORDS

Mutation: It is a permanent alteration of the nucleotide sequence of a

genome.
Ionizing radiation: These are radiations that consist of X-rays, gamma
rays with sufficient energy to cause ionization in a medium.
Mutagens: These are either physical or chemical agents that causes genetic
mutation.
Somatic mutations: It is a change that appears in somatic cells and it
shows mutant change.
Germinal mutations: If mutations originate in a gamete, if dominant mutations
occur in germ cells, effect is expressed immediately in the progeny.
Spontaneous mutations: It is a kind of mutations that occurs without any
cause.
Induced mutations: It arise due to exposure of mutagenic agents, such as
ionizing radiations or various chemicals.
Mutation rate: In genetics, the mutation rate is the frequency of new
mutations in a single gene or organism over time.
Mutation spectrum: The mutation spectrum of an organism is the rate at
which different mutations occur at different sites.
Mutagenesis: It is a process by which the genetic information of an
organism is changed, resulting in a mutation, it may occur spontaneously in
nature, or as a result of exposure to mutagens.

1.8 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What are the phenotypic effects of mutation?
2. Write a note on somatic and germinal mutations. Self-Instructional
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Mutation 3. Discuss back mutation and suppressor mutation.
4. What are chemically induced mutations?
5. Write a note on mutations and humans.
NOTES 6. What is radiation induced mutations?
7. List a few practical applications of mutations.
8. Discuss gene mutations and its types.
9. Explain the significance of mutation rate.
10. What is mutation spectrum?
11. How mutagenesis occurs?
Long Answer Questions
1. What do you mean by somatic and germinal mutations? Also discuss the
effects of mutation.
2. From your learning of the text, differentiate between back mutation and
suppressor mutation.
3. How are radiation induced mutations different from chemical induced
mutation? Discuss.
4. “Purposeful artificial selection is not practiced in humans and therefore the
possible advantages cited for domestic animals and plants do not apply to
humankind.” Discuss.
5. Briefly discuss the concept of mutation rate.
6. Explain the concept of mutation spectrum giving example.
7. “The mutation rate is the expected number of mutations that a cell will sustain
during its lifetime. The mutant fraction or frequency is the proportion of
cells in a population that are mutant.” Discuss.
8. Explain the method of determining the mutation rate with the help of an
example.
9. Discuss the concept of mutagenesis giving examples.
10. Why mutagenesis is the significant technique in the laboratory? Explain.

1.9 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
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Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H. Mutation

Freeman & Company.

Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
NOTES
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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Mutagens and its Types

UNIT 2 MUTAGENS AND ITS

TYPES
NOTES
Structure
2.0 Introduction
2.1 Objectives
2.2 Mutagens: History and Causes
2.2.1 History of Mutagens
2.2.2 Causes of Mutagens
2.3 Types of Mutagens
2.3.1 Physical Mutagens
2.3.2 Chemical Mutagens
2.3.3 Biological Mutagens
2.4 Tests System for Mutagens
2.5 Consequences of Mutagens Exposures
2.6 Answers to Check Your Progress Questions
2.7 Summary
2.8 Key Words
2.9 Self Assessment Questions and Exercises
2.10 Further Readings

2.0 INTRODUCTION

A mutagen is defined as any chemical that can cause changes in the DNA sequence
of an organism. These changes are called mutations. There are various types of
well-known mutagens, for example UV radiation, intercalating agents, metals, base
analogs, benzene, X-rays, 2,4 – diaminoanisole, radioactivity, most aromatic
amines, alkylating agents, etc. Thus mutagens may be of physical, chemical or
biological origin. They may act directly on the DNA, causing direct damage to the
DNA, and most often result in replication error, or mutations. In genetics,
a mutagen is a physical or chemical agent that changes the genetic material, usually
DNA, of an organism and thus increases the frequency of mutations above the
natural background level. As many mutations can cause cancer, mutagens are
therefore also likely to be carcinogens, although not always necessarily so.
All mutagens have characteristic mutational signatures with some chemicals
becoming mutagenic through cellular processes. Not all mutations are caused by
mutagens, the so-called ‘spontaneous mutations’ occur due to spontaneous
hydrolysis, errors in DNA replication, repair and recombination.
In this unit, you will study about history and introduction of mutagens, types
of mutagens and effects of exposure of mutagens on the survival of organisms in
detail.

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 Mutagens and its Types
2.1 OBJECTIVES

After going through this unit, you will be able to:

Discuss the history and introduction of mutagens NOTES
Understand the types of mutagens
Analyse the effects of exposure of mutagens on the survival of organisms

2.2 MUTAGENS: HISTORY AND CAUSES

Mutagens are chemical compounds or forms of radiation, such as UltraViolet

(UV) light or X-rays that cause irreversible and heritable changes (mutations) in
the cellular genetic material, DeoxyriboNucleic Acid (DNA). Mutagens are not
necessarily carcinogens, and vice versa. Sodium azide for example may be
mutagenic (and highly toxic), but it has not been shown to be carcinogenic.
Mutagenic lesions persist when they escape detection by protective cellular DNA
repair mechanisms, when mistakes occur in the repair process, or when repair
mechanisms are overwhelmed by extensive damage. Upon subsequent cellular
replication, these mutations become fixed in the genome and are inherited by all
daughter cells. In this way, mutagenesis becomes a cumulative process, stretching
over the lifetime of an organism. In addition, the accumulation of mutations over
time, leading to gradually less efficient cellular repair capabilities, has been linked
to the aging process and associated degenerative diseases. While the health of a
particular individual can be affected by mutations in somatic cells, mutagenic events
in germ cells (germline mutations) lead to the transmission of genetic diseases to
subsequent generations. Genetic predispositions to certain breast and colon cancers,
cystic fibrosis, and Huntington’s disease are included among many examples of
such inheritable diseases. Although many dietary and environmental agents have
been classified as mutagens, cells are constantly subjected to a barrage of
spontaneous DNA damage.
UV has enough energy to destroy and break molecular bonds. As a result,
if a material is exposed to UV radiation, there always exists the possibility that the
chemical structure of that material is changed. This fact, couples with the fact that
DNA strongly absorbs UV radiation of some frequencies (especially the 260–
280 nm and below 100 nm bands, means that in any living organism exposed to
UV, there will be a relatively chance of DNA damage occurring during exposure.
There are multiple types of UV-induced DNA damage, such as base loss where
the base is severed from the deoxyribose, resulting in an abasic site, thymidine
dimers where two adjacent or near-adjacent thymidine molecules are bonded
together, single-strand and double-strand breaks, etc. Figure 2.1 shows different
types of mutagens and plant materials used for mutagenic treatment.

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Mutagens and its Types

NOTES

Fig. 2.1 Different types of Physical and Chemical Mutagens and

Plant Materials used for Mutagenic Treatment

We are regularly exposed to another example of a DNA-damaging agent.

It is called Ethidium Bromide (EtBr). It is used as a DNA stain due to its enormous
affinity towards DNA. EtBr works efficiently because it is capable of inserting
itself in between two adjacent DNA bases, a so-called ‘intercalating agent’. When
it inserts itself like that, it cans fluorescence intensely under UV or green light,
making it a near-ideal agent for visualizing DNA. However, this intercalation means
that whenever DNA Polymerase tries to replicate DNA, it malfunctions at the site
of EtBr intercalation and can insert an inappropriate base, or skip that base
altogether or ‘jam’ in place, not only failing to synthesize any more DNA, but also
halting DNA replication in the middle of the strand until it is removed by other
repair proteins or the protein p53 (the shepherd of the genome) commits the cell
to apoptosis.

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 One mutagen that is either essential or deadly is the ‘Oxygen’. It is one of Mutagens and its Types

the most potent mutagens known, in the form of Reactive Oxygen Species or
ROSs. These ROSs arise from multiple sources but the primary sources are all
related to energy metabolism. One particularly dangerous ROS is superoxide, O2
(-) where the ‘-’ in parentheses is the charge. It can attack almost all organic NOTES
substances in the cell and has a very high potency to cause damage to DNA by
producing hydroxyl radicals, which can then attack DNA bases and damage them.
Luckily the cells have a line of defense against superoxide in the form of SuperOxide
Dismutase (SOD). SOD can take two superoxide molecules and oxidize one to
molecular oxygen and reduce the other to peroxide, O2 (-2). While peroxide itself
is dangerously toxic, it is far less toxic than superoxide. Peroxide is then destroyed
by catalase, which converts it to water and molecular oxygen.
The biological consequences of a mutation depend upon many critical factors,
such as the target loci, size of the mutation, and timing during the cell cycle, and
compounding effects of preexisting mutations. Thus, a mutagenic event occurring
in a nonfunctional area of DNA will have no effect (silent mutation), whereas a
similar change in an actively transcribed region may profoundly affect gene
expression and phenotype or even lead to cell death (lethal mutation). The influence
of mutations in human health is underscored by several human disease states caused
by mutations that disrupt regulatory regions or gene coding sequences, resulting in
altered gene expression and protein function. For example, mutations in genes that
promote or inhibit growth and cellular replication (proto-oncogenes and tumor
suppressor genes, respectively) or code for components of DNA repair pathways
are important contributors to the multistage development of cancer.
Many natural constituents of food are mutagenic and are produced by plants
as defense agents. However, additional food borne mutagens can be present as
residues of compounds used during food production or leached from packaging
materials. Mutagenic compounds can also be produced during food cooking and
preparation. Foods also contain many compounds that can modulate the activity
of mutagens. In order to minimize exposure to mutagens, the majority of developed
countries have instituted regulatory protocols governing the introduction of new
foods and food-associated chemicals before they are accepted into the
marketplace. With advances in the understanding of mutagenic processes, existing
foods and food contact items may also be reassessed for acceptability.
2.2.1 History of Mutagens
The first mutagens to be identified were carcinogens, substances that were shown
to be linked to cancer. Tumors were described more than 2,000 years before the
discovery of chromosomes and DNA in 500 B.C., the Greek Physician
Hippocrates named tumors resembling a crab karkinos (from which the word
‘Cancer’ is derived via Latin), meaning crab. In 1761, J. Hill made the first direct
link of cancer to chemical substances by noting that excessive use of snuff may

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Mutagens and its Types cause nasal cancer. In 1775, Sir P. Pott wrote a paper on the high incidence of
scrotal cancer, and suggested chimney soot as the cause of scrotal cancer. In 1915,
Yamagawa and Ichikawa showed that repeated application of coal tar to rabbit’s
ears produced malignant cancer. Subsequently, in the 1930s the carcinogen
NOTES component in coal tar was identified as a PolyAromatic Hydrocarbon (PAH),
benzo[a]pyrene.
The association of exposure to radiation and cancer had been observed as
early as 1902, six years after the discovery of X-ray radioactivity. The mutagenic
property of mutagens was first demonstrated in 1927, when H. Muller discovered
that X-rays can cause genetic mutations in fruit flies, Drosophila, producing
phenotypic mutants as well as observable changes to the chromosomes, visible
due to presence of enlarged ‘polytene chromosomes’ in fruit fly salivary glands.
Similar work related to UV radiation showed the mutational effect on maize in
1936. The effect of sunlight had previously been noted in the nineteenth century
where rural outdoor workers and sailors were found to be more prone to skin
cancer. Chemical mutagens were not demonstrated to cause mutation until the
1940s, when C. Auerbach and J.M. Robson found that mustard gas can cause
mutations in fruit flies. A large number of chemical mutagens have since been
identified, especially after the development of the Ames test in the 1970s by B.
Ames that screens for mutagens and allows for preliminary identification of
carcinogens.
2.2.2 Causes of Mutagens
Mutagens can cause changes to the DNA and are therefore genotoxic. They can
affect the transcription and replication of the DNA, which in severe cases can lead
to cell death. The mutagen produces mutations in the DNA, and deleterious mutation
can result in aberrant, impaired or loss of function for a particular gene, and
accumulation of mutations may lead to cancer. Mutagens may therefore be also
carcinogens. However, some mutagens exert their mutagenic effect through their
metabolites, and therefore whether such mutagens actually become carcinogenic
may be dependent on the metabolic processes of an organism, and a compound
shown to be mutagenic in one organism may not necessarily be carcinogenic in
another.
Different mutagens act on the DNA differently. Powerful mutagens may
result in chromosomal instability, causing chromosomal breakages and
rearrangement of the chromosomes, such as translocation, deletion and inversion.
Such mutagens are called cladogens. Mutagens may also modify the DNA
sequence; the changes in nucleic acid sequences by mutations include substitution
of base pairs, insertions and deletions of one or more nucleotides in DNA
sequences. Although some of these mutations are lethal or cause serious disease,
many have minor effects as they do not result in residue changes that have significant
effect on the structure and function of the proteins.

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 Chemical mutagens, such as Ethyl-MethaneSulfonate (EMS) and X-rays Mutagens and its Types

can both be used to induce pseudorandom mutations into the genome of the fly.
According to Greenspan, EMS typically causes single base-pair changes while
X-rays often result in deletions and gross chromosomal rearrangements. EMS is
highly toxic and mutagenic for humans and all manipulations should be performed NOTES
in a fume hood with the appropriate protection. Offspring of the EMS- or X-ray-
mutagenized males are tested for the presence of relevant mutations using the
screening protocol and found to significant.
When chemical mutagens or radiation damage DNA during G1 phase of cell
cycle, DNA replication is postponed until the damage is repaired. The key molecular
species that operates this checkpoint is a protein called p53. The p53 is a
transcription factor that induces the expression of DNA-repair genes. It also acts
indirectly to inhibit the activity of the cyclin–CDK complex that normally drives
the cell from G1 to S. Mutations in p53 interfere with its checkpoint function,
leading to chromosomal rearrangements and gene amplification. These
rearrangements predispose to cancer. Furthermore, in the presence of p53,
irreparably damaged cells commit suicide via apoptosis. In the absence of p53,
damaged cells may proliferate, thereby being another predisposing factor toward
malignancy.
Some mutagens can produce modified DNA bases that appear
‘uninformative’ to the polymerase. These may be bypassed in an error-prone
manner with a random selection of a base opposite the modified base. The ultimate
example of this class is an abasic site in which the template base has been completely
removed. Polymerase can synthesize over such lesions, albeit inefficiently. In
Escherichia coli, there appears to be a preference for the insertion of an Adenine
opposite the uninformative lesion, but this is not absolute, while in mammalian
cells such a preference is not evident.
According to Bishop and Shelby (1990), Andler et al., (2007) many
mutagens (DNA damaging agents) have been tested for effects in germ cells,
although the database for effects on male germ cells is much larger than for oocytes.
The dominant lethal test for germ cell mutagenecity can be applied to males or
females. The resulting pregnant females are then examined late in gestation for
numbers of living or dead fetuses, or implantation sites of embryos that died shortly
after implantation, as well as numbers of Corpora lutea to be used as a measure
of the number of potential embryos (oocytes ovulated). Pre- and post-implantation
loss can then be calculated. Although this test cannot distinguish between embryo
mortality due to genetic defect in the oocyte and pregnancy loss due to maternal
toxicity, non-genetic oocyte toxicity, or even failed fertilization, it can be used in
conjunction with other measures of oocyte function (such as chromosome analysis,
zygote morphology and cleavage, or blastocyst formation) to characterize acute
effects of chemicals on the oocyte and its developmental potential.

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Mutagens and its Types However, many mutagens are highly toxic to proliferating cells, and they are
often used to destroy cancer cells. Alkylating agents, such as cyclophosphamide and
cisplatin, as well as intercalating agent, such as daunorubicin and doxorubicin may
be used in chemotherapy. However, due to their effect on other cells which are
NOTES also rapidly dividing, they may have side effects, such as hair loss and nausea.
Research on better targeted therapies may reduce such side-effects. Ionizing
radiations are used in radiation therapy.

Check Your Progress

1. What is mutagen?
2. Expand EtBr. How EtBr works efficiently?
3. From where ROSs arises from?
4. What are biological consequences of a mutation?

2.3 TYPES OF MUTAGENS

Mutagenesis is the process of inducing mutation by a number of physical, chemical

or biological agents. The agents that cause mutations are called as mutagens. So
that mutagens may be of physical, chemical or biological in their origin. They may
act directly on the DNA, causing direct damage to the DNA, and most often
result in replication error. Some however may act on the replication mechanism
and chromosomal partition. Many mutagens are not mutagenic by themselves, but
can form mutagenic metabolites through cellular processes, for example through
the activity of the cytP450 system and other oxygenases, such as cyclooxygenases.
Such mutagens are called promutagens. Mutation induced by mutagens is called
induced mutation. Sometime mutation occurs spontaneously due to error during
DNA replication. However, mutagens increase the chances of mutation.
There are three types of mutagens (Refer Figure 2.2).
Physical Mutagens
Chemical Mutagens
Biological Mutagens

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 Mutagens and its Types

NOTES

Fig. 2.2 Cladistic Representation of Types of Mutagens

2.3.1 Physical Mutagens

Physical mutagen is a mutation agent which is in the form of physical substances,
such as short waves, particles and radioactive elements. Physical mutagens are
either ionizing which includes particulates, such as -rays, -rays, fast neutrons,
ionic elements/metals (Cobalt-60 and Cesium-137) and non-particulate matters
( -rays, cosmic rays and X-rays) or non-ionizing UV-rays/UV light. The ionizing
radiations have capacity to ionize water of the cell to release hydroxyl free radical
(OH). The hydroxyl radical is a powerful oxidizing agent and oxidizes the
phosphodiester bond of DNA. Higher dose of X-rays can even causes death of
an organism. However, the non-ionizing physical mutagens lead the formation of
Thymine dimer (Pyrimidine dimer). The occurrence of two Thymines together in
one strand of DNA, UV light causes fusion to form Thymine dimer. Nitrogenous
bases absorb UV lights and the absorption is maximum at 260 nm. At the site of
Thymine dimer confirmation of DNA is changed, so rate of error during DNA
replication is high. The physical mutagens may forward as radioactive decay, such
as 14C in DNA which decays into nitrogen and affect the normal biological activities.
Radiations as Mutagen
Radiation is the most important among the physical mutagens and damaging the
DNA molecules fall in the wavelength range below 340 nm and photon energy
above 1 electro-Volt (eV). The destructive radiation consists of UltraViolet (UV)
rays, X-rays, -rays, alpha ( ) rays, beta ( ) rays, cosmic rays, neutrons, etc.
(Refer Figure 2.3).

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Mutagens and its Types

NOTES

Fig. 2.3 Outline of Wavelengths and Photon Energy of Various Radiations

Radiation induced damage can be categorized into the three broad types:
lethal damage (killing the organisms), potentially lethal damage (can be lethal under
certain ordinary conditions) and sub-lethal damage (cells do not die unless radiation
reaches to a certain threshold value). The effect of damage is at molecular level
(proteins, lipoproteins, DNA, carbohydrates, etc.) in a live cell is caused directly
by ionization/excitation, or indirectly through highly reactive free radicals produced
by radiolysis of cellular water. DNA stores genetic information’s so any damage to
it assumes great dimension. It can perpetuate genetic effects and, therefore, the
cellular repair system is largely devoted to its welfare.
When the Bacteria are exposed to radiation they gradually lose the ability to
develop colonies. This gradual loss of viability can be expressed graphically by
plotting the surviving colonies against the gradually increasing exposure time. This
dose-response graph is called survival curve. The survival curve is analyzed by a
simple mathematical theory called hit theory. According to this theory each organism
possesses at least one sensitive site which is known as target site. Radiation photons
(particles of light) damage or hit the target site and inactivate the organisms. One
can derive the equation based on this theory. The equations help to calculate the
survival curve for many kinds of populations of N identical organisms exposed to
dose D of radiation causing damage. The number dN damaged by a dose dD is
proportional to the initial population that has not received radiation; hence
dN = KN; where, K is the constant which measures the effectiveness of dose
(Refer Figure 2.4).

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 Mutagens and its Types

NOTES

Fig. 2.4 Standard UV Rays Survival Curve for a Bacterium

UltraViolet (UV) Radiations as Mutagen

Ionization usually occurs because the radiation source has very large energy, for
example, radiation from radioactive substances and cosmic ray, if hit the DNA
molecules, the DNA chain will lose so that the DNA chain cannot function in
protein synthesis. Ultraviolet ray generally do not cause ionization, however, the
energy from UltraViolet ray will be absorbed by purine and pyrimidine so that the
atom becomes more reactive (the electron undergoes excitation). Consequently,
DNA double-helix becomes in disorder and inhibits replication, one of the effects
caused by ultraviolet ray is skin cancer.
UV radiation causes damage in the DNA duplex of the Bacteria and Phages.
The UV rays are absorbed and cause excitation of macromolecules. The absorption
maxima of nucleic acid (280 nm) and protein (260 nm) are more or less similar.
The DNA molecule is the target molecule for UV rays but not the proteins. However,
absorption spectrum of RNA is quite similar to that of DNA. The excited DNA
leads to cross-linking, single strand breaks and base damage as minor lesion and
generation of nucleotide dimer as a major one. Purines are generally more radio–
resistant than the pyrimidine of the latter, Thymine is more reactive than Cytosine.
Hence, the ratio of Thymine-Thymine (TT), Thymine-Cytosine (TC) and Cytosine-
Cytosine (CC) dimer is 10:3:3, respectively. A few dimers of TU and UU also
appear. The initial step in pyrimidine dimerization is known to be hydration of their
4: 5 bonds (Refer Figure 2.5).

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Mutagens and its Types

NOTES

Fig. 2.5 UV Radiation induced Formation of Pyrimidine Dimer

Formation of Thymine-Thymine (TT) dimer causes distortion of DNA helix

because the Thymines are pulled towards one another. The distortion results in
weakening of hydrogen-bonding to Adenines in the opposing strand. This structural
distortion inhibits the advance of replication fork.
X-Rays as Mutagen
The X-rays cause breaking of phosphate ester linkages in the DNA. This breakage
occurs at one or more points. Consequently, a large number of bases are deleted
or rearranged in the DNA molecule. The X-rays may break the DNA either in one
or both strands. If breaks occur in both strands, it becomes lethal. The DNA
segment between the two breaks is removed resulting in deletion. Since both the
X-rays and UV rays bring about damage in DNA molecule, they are used in
sterilization of Bacteria and Viruses.
2.3.2 Chemical Mutagens
Chemical mutagens are compounds that increase the frequency of some types of
mutation. Among them are alkylators, including Ethyl-MethaneSulfonate (EMS),
Methyl MethaneSulfonate (MMS), DiEthylSulfate (DES), and Nitrosoguanidine
which tend to de-acidify the acidic properties of DNA. They also have the property
of inducing error-prone DNA repair. This stimulation of error-prone repair allows
all sorts of mutation types to occur. Nitrous Oxide can also cause mutations in the
DNA affecting protein synthesis. Thus chemical mutagens are standard tools for
mutagenesis in a variety of organisms, and they are a primary means of creating
mutations in phenotype-based screens in most genetic systems. Although varied in
the experimental design, all whole animal screens involve the generation of lines
harboring mutated chromosomes followed by the examination of the resulting
phenotypes in the heterozygous or homozygous state. In contrast, gene-based
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screens rely on the identification of lines that carry mutations in specific genes, Mutagens and its Types

prior to any phenotypic examination. The ability to perform gene-based screens

has been made possible by recent technological advances in the high-throughput
detection of subtle mutations.
NOTES
Table 2.1 Various Classes of Chemicals as Mutagenic Agents for Living Beings

Chemical mutagens have also been used successfully in the mutagenesis of

mouse Embryonic Stem (ES) cells. This approach holds a variety of advantages
over mutagenesis in the whole animal. Cell culture–based mutagenesis is
distinguished from whole animal mutagenesis in its ability to use a variety of mutagens,
to monitor and modulate mutation load, and to screen a large number of lines. The
possibility of generating and characterizing mutations in cell culture enables the
rapid identification of many mutations in genes, which can be transmitted to a
mouse line. In this unit, we describe the merits and methods of gene-based screens
to identify chemically induced mutation in ES cells. Several chemical mutagens
have been successfully used to mutagenize mouse ES cells, and the potential exists
for the use of almost any mutagen.
Base Analogs as Chemical Mutagen
These chemical are morphologically similar to that of normal nitrogen bases. So
during replication these molecules are incorporated instead of normal nitrogen
bases and hence causes mutation, for example 2-aminopurine is analogue to
Adenine, and 5-bromourcail is analogue to thymine. So, base analog mutagens
are chemicals that mimic bases to such an extent that they can be incorporated
into DNA in place of one of the normal bases but in doing so lead to an increase
in the rate of mutation. To be mutagenic, a base analog must mispair more frequently
than the normal base it replaced. This mispairing can occur either during the initial
incorporation into DNA, or during subsequent rounds of replication when the
base analog is used as a template. Most of these mutagens typically induce only Self-Instructional
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Mutagens and its Types base pair substitutions (and not other types of mutation). They are usually not
highly toxic nor do they increase rates of recombination (Refer Figure 2.6).

NOTES

Fig. 2.6 Schematic Representation of Base Analogs Insertion during Replication

Thus a base analogue is a chemical compound similar to one of the four

bases of DNA. It can be incorporated into a growing polynucleotide chain when
normal process of replication occurs’. These compounds have base pairing
properties different from the bases. They replace the bases and cause stable
mutation. A very common and widely used base analogue is 5-BromoUracil
(5-BU) which is an analogue of thymine. The 5-BU functions like Thymine and
pairs with Adenine. The 5-BU undergoes tautomeric shift from keto form to Enol
form caused by bromine atom. The Enol form can exist for a long time for 5-BU
than for Thymine. If 5-BU replaces a Thymine, it generates a Guanine during
replication which in turn specifies Cytosine causing G: C pair (Refer Figure 2.7).

Fig. 2.7 Base Analog (5-BromoUracil) Induced Mutagenesis, Keto Form of 5-BU Pairs
Self-Instructional with Adenine, however Tautomerised Enol with Guanine
36 Material
 During the replication, keto form of 5-BU substitutes for T and the replication Mutagens and its Types

of an initial AT pair becomes an A: BU pair. The rare Enol form of 5-BU that pairs
with G is the first mutagenic step of replication. In the next round of replication G
pairs with C. Thus, the transition is completed from AT GC pair. The 5-BU can
also induce the conversion of GC to AT. The Enol form infrequently acts as an NOTES
analogue of Cytosine rather than thymine. Due to error, GC pair is converted into
a G: BU pair which in turn becomes an AT pair. Due to such pairing properties
5-BU is used in chemotherapy of Viruses and cancer. Because of pairing
with guanine it disturbs the normal replication process in microorganisms. The
5-bromodeoxyuridine (5-BDU) can replace thymidine in DNA molecule.
The
2-amino-purine (2-AP) and 2, 6-di-amino-purine (2, 6-DAP) are the purine
analogues. The 2-AP normally pairs with Thymine but it is able to form a single
hydrogen bond with Cytosine resulting in transition of AT to GC. The 2-AP and 2,
6-DAP are not as effective as 5-BU and 5-BDU (Refer Figure 2.8).

Fig. 2.8 Replication during 5-BU Induced Mutagenesis; (A). AT-GC Replication and;
(B). GC-AT Replication

Chemical Altering the Hydrogen Specificity as Mutagen

There are many chemicals that after incorporation into DNA change the specificity
of hydrogen-bonding. Those which are used as mutagens are nitrous oxide (HNO2),
HydroxylAmine (HA) and Ethyl-MethaneSulphonate (EMS).
Nitrous Oxide (HNO2): Nitrous oxide converts the amino group of bases
into keto group through oxidative deamination. The order of frequency of
deamination (removal of amino group) is Adenine > Cytosine > Guanine.
Deamination of Adenine: Deamination of Adenine results in formation of
hypoxanthine, the pairing behaviour of which is like Guanine. Hence, it pairs
with Cytosine instead of Thymine replacing AT pairing by GC pairing (Refer
Figure 2.9).

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Mutagens and its Types

NOTES

Fig. 2.9 Nitrous Oxide induced Deamination of Adenine to Hypoxanthine

Deamination of Cytosine: Deamination of Cytosine results in formation

of Uracil by replacing – NH2 group with -OH group. The affinity for
hydrogen bonding of Uracil is like Thymine; therefore, C-G pairing is replaced
by U-A pairing (Refer Figure 2.10).

Fig. 2.10 Nitrous Oxide Induced Deamination of Cytosine to Uracil

Deamination of Guanine: Deamination of Guanine results in formation of

xanthine, the later is not mutagenic. Xanthine behaves like Guanine because
there is no change in pairing behaviour. Xanthine pairs with Cytosine.
Therefore, G-C pairing is replaced by X-C pairing (Refer Figure 2.11).

Fig. 2.11 Nitrous Oxide Induced Deamination of Guanine to Xanthine and 6-

Methylcytocine to Thymine
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 Hydroxylamine (NH2OH): It hydroxylates the C4 nitrogen of Cytosine Mutagens and its Types

and converts into a modified base via deamination which causes to base
pairs like Thymine. Therefore, GC pairs are changed into AT pairs.
Alkylating Agents as Chemical Mutagen NOTES
Addition of an alkyl group to the hydrogen bonding oxygen of Guanine (N7 position)
and Adenine (at N3 position) residues of DNA is done by alkylating agents. As a
result of alkylation, possibility of ionization is increased with the introduction of
pairing errors. Hydrolysis of linkage of base-sugar occurs resulting in gap in one
chain. This phenomenon of loss of alkylated base from the DNA molecule (by
breakage of bond joining the nitrogen of purine and deoxyribose) is called
depurination. Depurination is not always mutagenic. The gap created by loss of
a purine can effectively be repaired.
Following are some of the important widely used alkylating agents:
Dimethyl Sulphate (DMS)
Ethyl-MethaneSulphonate (EMS)
Ethyl-EthaneSulphonate (EES)
EMS has the specificity to remove Guanine and Cytosine from the chain
and results in gap formation. Any base (A, T, G, C) may be inserted in the gap.
During replication chain without gap will result in normal DNA. In the second
round of replication gap is filled by suitable base. If the correct base is inserted,
normal DNA sequence will be produced. Insertion of incorrect bases results in
transversion or transition mutation. Another example is methylnitrosoguanidine that
adds methyl group to Guanine causing it to mispair with Thiamine. After subsequent
replication, GC is converted into AT transition.
Intercalating Agents as Mutagen
The chemical intercalate or slip in between two base pair in double stranded DNA
helix and hence alter the morphology of DNA at that position. The chances of
error during replication are higher at this position causing mutation. There are
certain dyes, such as acridine orange, proflavine and acriflavine which are three
ringed molecules of similar dimensions as those of purine pyrimidine pairs. In
aqueous solution these dyes can insert themselves in DNA (i.e., intercalate the
DNA) between the bases in adjacent pairs by a process called intercalation (Refer
Figure 2.12).

Fig. 2.12 Chemical Structure of Two Mutagenic Acridine Derivatives

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Mutagens and its Types Therefore, the dyes are called intercalating agents. The acridines are planer (flat)
molecules which can be intercalated between the base pairs of DNA; distort the
DNA and results deletion or insertion after replication of DNA molecule. Due to
deletion or insertion of intercalating agents, there occur frameshift mutations (Refer
NOTES Figure 2.13).

Fig. 2.13 Intercalation of an Acridine Molecule during Replication

DNA Reactive Chemicals as Mutagen

These chemical mutagens react directly with the nitrogenous bases of DNA and
chemically modify the DNA causing mutation. A large number of chemicals may
interact directly with DNA. However, many PAHs, aromatic amines, benzene are
not necessarily mutagenic by themselves, but through metabolic processes in cells
they produce mutagenic compounds.

Reactive Oxygen Species (ROS): These may be superoxide, hydroxyl

radical and hydrogen peroxide, and large number of these highly reactive
species are generated by normal cellular processes, for example as a by-
products of mitochondrial electron transport, or lipid peroxidation. As an
example of the latter, 15-hydroperoxyicosatetraenocic acid, a natural
product of cellular cyclooxygenases and lipoxygenases, breaks down to
form 4-hydroxy-2(E)-nonenal, 4-hydroperoxy-2(E)-nonenal, 4-oxo-2(E)-
nonenal, and cis-4,5-epoxy-2(E)-decanal; these bifunctional electophils are
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 mutagenic in mammalian cells and may contribute to the development and/ Mutagens and its Types

or progression of human cancers. A number of mutagens may also generate

these ROS. These ROS may result in the production of many base adducts,
as well as DNA strand breaks and crosslinks.
NOTES
Ethylnitrourea: The ethylnitrosourea compounds transfer methyl or ethyl
group to bases or the backbone phosphate groups. Guanine when alkylated
may be mispaired with thymine. Some may cause DNA crosslinking and
breakages. Nitrosamines are an important group of mutagens found in
tobacco, and may also be formed in smoked meats and fish via the interaction
of amines in food with nitrites added as preservatives. Other alkylating
agents include mustard gas and vinyl chloride.
Aromatic Amines: The aromatic amines and amides have been associated
with carcinogenesis since 1895 when German physician L. Rehn observed
high incidence of bladder cancer among workers in German synthetic
aromatic amine dye industry. The 2-acetylaminofluorene originally used as
a pesticide but may also be found in cooked meat, may cause cancer of the
bladder, liver, ear, intestine, thyroid and breast.
Alkaloids: The alkaloids from plants, such as those from Vinca species,
may be converted by metabolic processes into the active mutagen or
carcinogen.
Others: Bromine and some compounds that contain bromine in their
chemical structure, sodium azide, psoralen with UV radiation and benzene
may also acts as potent mutagenic agents when exposed to living beings.
Metals as Mutagen
Many metals, and their compounds may be mutagenic, but they may act, however,
via a number of different mechanisms. Arsenic, chromium, iron, and nickel may be
associated with the production of ROS, and some of these may also alter the
fidelity of DNA replication. Nickel may also be linked to DNA hypermethylation
and histone deacetylation, while some metals, such as cobalt, arsenic, nickel and
cadmium may also affect DNA repair processes, such as DNA mismatch repair,
base and nucleotide excision repair. There are large numbers of metals for mutagenic
in Bacteria and phases for example, compounds of copper, manganese,
molybdenum, platinum and selenium. The compounds containing aluminum,
antimony, arsenic, cadmium, copper, lead, mercury, and tellurium have been shown
to induce chromosomal aberrations or abnormal cell divisions in animal and plant
cells. The genetic evidence suggests that arsenic, chromium, and molybdenum
compounds may influence the accuracy of DNA repair processes in microorganisms.
Several metals are known mutagens and carcinogens. These metals effectively
displace acridine orange from DNA when measured by fluorescence polarization.
The displacement of 50% of the acridine orange is obtained with less than 0.5mM
concentrations of lead, manganese, cobalt, zinc, cadmium, nickel, iron, copper,
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Mutagens and its Types and cis platinum. In contrast, greater than 80mM concentrations of lithium, sodium,
and potassium are required to displace an equivalent amount of acridine orange
from calf thymus DNA. Although cis platinum shows the best DNA reactivity in
this assay, the interaction between this metal and DNA does not occur immediately,
NOTES as it does for the other metals tested. These results indicate that the acridine orange
displacement assay provides a relative measure of the interaction of metals with
DNA, and this DNA reactivity shows a positive correlation with mutagenic/
carcinogenic potential.
2.3.3 Biological Mutagens
Biological mutagen is a mutation agent in the form of Virus or Bacteria which can
induce mutation in every living organism. When cell divides, Virus will change the
genetic material (DNA) Composition of the attacked cell I order to damage the
cell and tissues toxin produced by bacteria can also cause disorder or damage on
genetic Material or certain cell and tissues. Hepatitis, chickenpox, measles, yellow
Fever, or food poisoning (botulism) may begin from genetical material change
induced either by Virus or Bacteria. The horticulturists achieve a specific mutation
in plant cuttings with the application of colchicine, to the reproductive parts of the
flowers. The colchicine is an extract from plants of the Autumn crocus. The mutation
which occurs is to double the number of chromosomes, which results in plant with
parts twice as large as normal. In this way a plain wild flower can be turned into a
showy flower twice as large as the original. Another biological mutagen is simply
aging of the egg or sperm. Damage to DNA in both plants and animals causes
mutations, such as mongoloidism in humans when the egg has begun to deteriorate
from age. There are many biological organism and parts of organism mat acts as
mutagenic agents given below:
Virus: Virus DNA may be inserted into the genome and disrupts genetic
function. Infectious agents have been suggested to cause cancer as early as
1908 by Vilhelm Ellermann and Oluf Bang, and 1911 by Peyton Rous who
discovered the Rous sarcoma Virus.
Bacteria: Some Bacteria, such as Helicobacter pylori cause inflammation
during which oxidative species are produced, causing DNA damage and
reducing efficiency of DNA repair systems, thereby increasing mutation.
Transposon and Insertion Sequence (IS): Transposon is a section of
DNA that undergoes autonomous fragment relocation/multiplication. Its
insertion into chromosomal DNA disrupts functional elements of the genes.
These elements (Transposon and IS) are small sequence of DNA that moves
from one site to another along DNA strand and causes mutation, also known
as jumping gene. These sequences contain gene which codes the enzyme
transposase which helps in transposition of these sequence from one site to
other. IS element are simplest type of transposable element. They are short
DNA about 1000 nucleotide long and typically contain inverted repeats of
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 10-50 base pair. The only gene they posses is the gene for enzyme Mutagens and its Types

transposase. IS element are found in both chromosome and plasmid of

Bacteria and archaea as well as some bacteriophage. The transposons are
larger than IS element but similar as IS element because it also has two
essential component; contains inverted repeats and encodes transposases. NOTES
The transposons and Insertion Sequence (IS) elements are biological
mutagens, for example; mutator gene, bacteriophage MU, etc.

Check Your Progress

5. Define the term mutagenesis?
6. Name the types of mutagens.
7. What is physical mutation?

2.4 TESTS SYSTEM FOR MUTAGENS

Many different systems for detecting mutagen have been developed. Animal
systems may more accurately reflect the metabolism of human, however, they are
expensive and time-consuming (may take around three years to complete), they
are therefore not used as a first screen for mutagenicity or carcinogenicity.
I. Bacterial Tests
Ames Test: This is the most commonly used test, and Salmonella
typhimurium strains deficient in histidine biosynthesis are used in this test.
The test checks for mutants that can revert to wild-type. It is an easy,
inexpensive and convenient initial screen for mutagens.
Resistance to 8-Azaguanine in Salmonella typhimurium: Similar to
Ames test, but instead of reverse mutation, it checks for forward mutation
that confer resistance to 8-azaguanine in a histidine revertant strain.
Escherichia coli Systems: Both forward and reverse mutation detection
system have been modified for use in Escherichia coli. Tryptophan deficient
mutant is used for the reverse mutation, while galactose utility or resistance
to 5-methyltryptophan may be used for forward mutation.
DNA Repair: Escherichia coli and Bacillus subtilis strains deficient in
DNA repair may be used to detect mutagens by their effect on the growth
of these cells through DNA damage.
II. Yeast Tests
Systems similar to Ames test have been developed in yeast. Saccharomyces
cervisiae is generally used. These systems can check for forward and reverse
mutations, as well as recombinant events.

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Mutagens and its Types III. Drosophila for Sex-Linked Recessive Lethal Test
Males from a strain with yellow bodies are used in this test. The gene for the
yellow body lies on the X-chromosome. The fruit flies are fed on a diet of test
NOTES chemical, and progenies are separated by sex. The surviving males are crossed
with the females of the same generation, and if no males with yellow bodies are
detected in the second generation, it would indicate a lethal mutation on the X-
chromosome has occurred.
IV. Plant Assays
Plants such as Zea mays, Arabidopsis thaliana and Tradescantia have been used
in various test assays for mutagenecity of chemicals.
V. Cell Culture Assay
Mammalian cell lines such as Chinese hamster V79 cells, Chinese Hamster
Ovary (CHO) cells or mouse lymphoma cells may be used to test for mutagenesis.
Such systems include the HRT assay for resistance to 8-azaguanine or 6-
thioguanine, and OUAbain-resistance (OUA) assay. Rat primary hepatocytes may
also be used to measure DNA repair following DNA damage. Mutagens may
stimulate unscheduled DNA synthesis that results in more stained nuclear material
in cells following exposure to mutagens.
VI. Chromosome Check Systems
These systems check for large scale changes to the chromosomes and may be
used with cell culture or in animal test. The chromosomes are stained and observed
for any changes. Sister chromatids exchange is a symmetrical exchange of
chromosome material between sister chromatids and may be correlated to the
mutagenic or carcinogenic potential of a chemical. In micronucleus test, cells are
examined for micronuclei, which are fragments or chromosomes left behind at
anaphase, and is therefore a test for clastogenic agents that cause chromosome
breakages. Other tests may check for various chromosomal aberrations such as
chromatid and chromosomal gaps and deletions, translocations, and ploidy.
VII. Animal Test Systems
Rodents are usually used in animal test. The chemicals under test are usually
administered in the food and in the drinking water, but sometimes by dermal
application, by gavage, or by inhalation, and carried out over the major part of the
life span for rodents. In tests that check for carcinogens, maximum tolerated dosage
is first determined, then a range of doses are given to around 50 animals throughout
the notional lifespan of the animal of two years. After death the animals are examined
for sign of tumors. Differences in metabolism between rat and human however
means that human may not respond in exactly the same way to mutagen, and
dosages that produce tumors on the animal test may also be unreasonably high for
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a human, i.e., the equivalent amount required to produce tumors in human may far Mutagens and its Types

exceed what a person might encounter in real life.

Mice with recessive mutations for a visible phenotype may also be used to
check for mutagens. Females with recessive mutation crossed with wild-type males
NOTES
would yield the same phenotype as the wild-type, and any observable change to
the phenotype would indicate that a mutation induced by the mutagen has occurred.
Mice may also be used for dominant lethal assays where early embryonic deaths
are monitored. Male mice are treated with chemicals under test, mated with females,
and the females are then sacrificed before parturition and early fetal deaths are
counted in the uterine horns.

Check Your Progress

8. What is Ames test?
9. Write a short note on Escherichia coli systems.
10. How can DNA repair be defined?
11. What is cell culture assay?

2.5 CONSEQUENCES OF MUTAGENS EXPOSURES

Mutagenesis arises due to mutagens not only from the errors in the replication but
also from damage to the DNA. Some damage is caused by environmental factors,
such as radiation and chemicals like mutagens, which are chemical agents that
increase the rate of mutation. DNA also undergoes spontaneous damage from the
action of water. The mechanism which causes DNA damage and mutagenesis
include: hydrolysis (deamination and depurination), alkylation, oxidation, radiation
reactions, and base analogue and intercalating agents.
Consequences Due to Radiation Exposure
Radiation damages the spermatogonia and the damaged germ cells could
occur for a very long time, perhaps a lifetime.
Radiation also induces recessive and dominant point mutations.
Sometimes gross chromosomal damage may also occur.
Majority of the mutations after radiation exposure will be of recessive type
and, therefore, not affect the phenotype in the first generation.
Mature oocytes are more susceptible regarding the radiation induced
mutation than spermatogonia.
If conception has taken place shortly after radiation exposure it will be
more dangerous.

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Mutagens and its Types Consequences Due to Chemical Exposure
The effects of chemical mutagens are less easy to generalize about than those of
the radiation mutagen. One important thing is to be remembered that chemical
NOTES mutagen is very stage specific and, accordingly, chemical mutagens can be classified
into two classes:
Chemicals which are mutagenic to both replicating and non-replicating DNA.
Chemicals which are mutagenic only to replicating DNA. It has long been
recognized that most of the strongly chemical mutagenic agents are also
carcinogenic agents, because most of the geneticists agree that somatic
mutation can cause cancer. There are a number of chemicals which can act
as a potential mutagenic agent in humans and some of these chemicals are
also used as drugs for curing some diseases.
Most of cytostatic, antimetabolite, hallucinogenic drugs and some antibiotics also
act as potential mutagenic agents in normal therapeutic doses. Therefore, if a patient
is treated with a high dose of an unusual or potentially dangerous drug, the doctor
must take some careful measures like the recommendation of the use of
contraceptives during the period of therapy and at least 8-10 weeks after the
therapy, etc.

Check Your Progress

12. From where does mutagenesis arises?
13. Name the mechanism which causes DNA damage and mutagenesis.
14. What are the consequences that are caused due to radiation exposure?
15. In how many classes chemical mutagens can be classified?

2.6 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. Mutagens are chemical compounds or forms of radiation, such as UltraViolet

(UV) light or X-rays that cause irreversible and heritable changes (mutations)
in the cellular genetic material, DeoxyriboNucleic Acid (DNA).
2. Ethidium Bromide (EtBr) works efficiently because it is capable of inserting
itself in between two adjacent DNA bases, a so-called ‘intercalating agent’.
3. Reactive Oxygen Species (ROSs) arise from multiple sources but the primary
sources are all related to energy metabolism.
4. The biological consequences of a mutation depend upon many critical factors
such as the target loci, size of the mutation, and timing during the cell cycle,
and compounding effects of preexisting mutations.

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 5. Mutagenesis is the process of inducing mutation by a number of physical, Mutagens and its Types

chemical or biological agents.

6. There are three types of mutagens:
Physical Mutagens NOTES
Chemical Mutagens
Biological Mutagens
7. Physical mutagen is a mutation agent which is in the form of physical
substances, such as short waves, particles and radioactive elements.
8. Ames test is the most commonly used test, and Salmonella
typhimurium strains deficient in histidine biosynthesis are used in this test.
The test checks for mutants that can revert to wild-type. It is an easy,
inexpensive and convenient initial screen for mutagens.
9. Escherichia coli Systems, both forward and reverse mutation detection
system have been modified for use in Escherichia coli. Tryptophan deficient
mutant is used for the reverse mutation, while galactose utility or resistance
to 5-methyltryptophan may be used for forward mutation.
10. Escherichia coli and Bacillus subtilis strains deficient in DNA repair may
be used to detect mutagens by their effect on the growth of these cells
through DNA damage.
11. Cell Culture Assay: Mammalian cell lines such as Chinese hamster V79
cells, Chinese Hamster Ovary (CHO) cells or mouse lymphoma cells may
be used to test for mutagenesis. Such systems include the HRT assay for
resistance to 8-azaguanine or 6-thioguanine, and OUAbain-resistance
(OUA) assay. Rat primary hepatocytes may also be used to measure DNA
repair following DNA damage. Mutagens may stimulate unscheduled DNA
synthesis that results in more stained nuclear material in cells following
exposure to mutagens.
12. Mutagenesis arises due to mutagens not only from the errors in the replication
but also from damage to the DNA.
13. The mechanism which causes DNA damage and mutagenesis include:
hydrolysis (deamination and depurination), alkylation, oxidation, radiation
reactions, and base analogue and intercalating agents.
14. Consequences due to radiation exposure are as follows:
Radiation damages the spermatogonia and the damaged germ cells could
occur for a very long time, perhaps a lifetime.
Radiation also induces recessive and dominant point mutations.
Sometimes gross chromosomal damage may also occur.
Majority of the mutations after radiation exposure will be of recessive
type and, therefore, not affect the phenotype in the first generation.
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Mutagens and its Types Mature oocytes are more susceptible regarding the radiation induced
mutation than spermatogonia.
If conception has taken place shortly after radiation exposure it will be
more dangerous.
NOTES
15. Chemical mutagens can be classified into two classes:
Chemicals which are mutagenic to both replicating and non-replicating
DNA.
Chemicals which are mutagenic only to replicating DNA. It has long
been recognized that most of the strongly chemical mutagenic agents
are also carcinogenic agents, because most of the geneticists agree that
somatic mutation can cause cancer. There are a number of chemicals
which can act as a potential mutagenic agent in humans and some of
these chemicals are also used as drugs for curing some diseases.

2.7 SUMMARY

Mutagens are chemical compounds or forms of radiation, such as UltraViolet

(UV) light or X-rays that cause irreversible and heritable changes (mutations)
in the cellular genetic material, DeoxyriboNucleic Acid (DNA).
Mutagens are not necessarily carcinogens, and vice versa. Sodium azide for
example may be mutagenic (and highly toxic), but it has not been shown to
be carcinogenic.
Mutagenic lesions persist when they escape detection by protective cellular
DNA repair mechanisms, when mistakes occur in the repair process, or
when repair mechanisms are overwhelmed by extensive damage.
Mutagenesis becomes a cumulative process, stretching over the lifetime of
an organism.
In addition, the accumulation of mutations over time, leading to gradually
less efficient cellular repair capabilities, has been linked to the aging process
and associated degenerative diseases.
Genetic predispositions to certain breast and colon cancers, cystic fibrosis,
and Huntington’s disease are included among many examples of such
inheritable diseases.
Although many dietary and environmental agents have been classified as
mutagens, cells are constantly subjected to a barrage of spontaneous DNA
damage.
In genetics, a mutagen is a physical or chemical agent that changes the genetic
material, usually DNA, of an organism and thus increases the frequency of
mutations above the natural background level.

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Ethidium Bromide (EtBr) is used as a DNA stain due to its enormous affinity Mutagens and its Types

towards DNA.
EtBr works efficiently because it is capable of inserting itself in between
two adjacent DNA bases, a so-called ‘intercalating agent’.
NOTES
One mutagen that is either essential or deadly is the ‘oxygen’. It is one of
the most potent mutagens known, in the form of Reactive Oxygen Species
or ROSs.
ROSs arise from multiple sources but the primary sources are all related to
energy metabolism.
One particularly dangerous ROS is superoxide, O2 (-) where the ‘-’ in
parentheses is the charge.
O2 (-) can attack almost all organic substances in the cell and has a very
high potency to cause damage to DNA by producing hydroxyl radicals,
which can then attack DNA bases and damage them.
The first mutagens to be identified were carcinogens, substances that were
shown to be linked to cancer.
Tumors were described more than 2,000 years before the discovery of
chromosomes and DNA in 500 B.C., the Greek.
Physician Hippocrates named tumors resembling a crab karkinos (from
which the word ‘cancer’ is derived via Latin), meaning crab.
Many mutagens are highly toxic to proliferating cells, and they are often
used to destroy cancer cells.
Alkylating agents such as cyclophosphamide and cisplatin, as well as
intercalating agent such as daunorubicin and doxorubicin may be used in
chemotherapy.
Research on better targeted therapies may reduce such side effects. Ionizing
radiations are used in radiation therapy.
Many metals, and their compounds may be mutagenic, but they may act,
however, via a number of different mechanisms.
Arsenic, chromium, iron, and nickel may be associated with the production
of ROS, and some of these may also alter the fidelity of DNA replication.
Nickel may also be linked to DNA hypermethylation and
histone deacetylation, while some metals such as cobalt, arsenic, nickel and
cadmium may also affect DNA repair processes such as DNA mismatch
repair, base and nucleotide excision repair.
There are large numbers of metals for mutagenic in Bacteria and phases for
example, compounds of copper, manganese, molybdenum, platinum and
selenium.

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Mutagens and its Types The compounds containing aluminum, antimony, arsenic, cadmium, copper,
lead, mercury, and tellurium have been shown to induce chromosomal
aberrations or abnormal cell divisions in animal and plant cells.
The genetic evidence suggests that arsenic, chromium, and molybdenum
NOTES
compounds may influence the accuracy of DNA repair processes in
microorganisms.
Biological mutagen is a mutation agent in the form of Virus or Bacteria
which can induce mutation in every living organism.
When cell divides, Virus will change the genetic material (DNA) Composition
of the attacked cell I order to damage the cell and tissues toxin produced
by Bacteria can also cause disorder or damage on genetic material or certain
cell and tissues.
Transposon is a section of DNA that undergoes autonomous fragment
relocation/multiplication. Its insertion into chromosomal DNA disrupts
functional elements of the genes.
Transposon and IS elements are small sequence of DNA that moves from
one site to another along DNA strand and causes mutation, also known
as jumping gene.
Mutagenesis arises due to mutagens not only from the errors in the replication
but also from damage to the DNA.
Some damage is caused by environmental factors, such as radiation and
chemicals like mutagens, which are chemical agents that increase the rate of
mutation. DNA also undergoes spontaneous damage from the action of
water.
The mechanism which causes DNA damage and mutagenesis include:
hydrolysis (deamination and depurination), alkylation, oxidation, radiation
reactions, and base analogue and intercalating agents.

2.8 KEY WORDS

Mutagen: A mutagen is defined as any chemical that can cause changes in

the DNA sequence of an organism.
Biological mutagen: Biological mutagen is a mutation agent in the form of
Virus or Bacteria which can induce mutation in every living organism.
Transposon: Transposon is a section of DNA that undergoes autonomous
fragment relocation/multiplication.

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 Mutagens and its Types
2.9 SELF ASSESSMENT QUESTIONS AND
EXERCISES

Short Answer Questions NOTES

1. What are mutagens? How is it caused?

2. Explain what physical mutagens are.
3. Write a note on base analogs as chemical mutagen.
4. Explain DNA reactive chemicals as mutagen.
5. What are biological mutations?
6. Name the different tests systems for mutagens.
7. What are the consequences due to radiation exposure?
Long Answer Questions
1. What is mutagenesis? Discuss in details about the mechanism of mutagenesis
based on exposure of physical mutagens.
2. In what ways the base analogs act as mutagen. Discuss in detail with
necessary diagrams.
3. What are chemical mutagens? Give detail accounts of any two chemicals
with mutagenic efficiency studied by you.
4. What are the major consequences of physical and chemical mutagens?
5. Discuss in details about the different types of tests for the assessment of
mutagenic potential of various mutagens.

2.10 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.

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Mutagens and its Types Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
NOTES
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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 DNA Damages

UNIT 3 DNA DAMAGES

Structure NOTES
3.0 Introduction
3.1 Objectives
3.2 DNA Damages
3.2.1 Deamination of Bases
3.2.2 Alkylation
3.2.3 Damage Due to Reactive Oxygen
3.2.4 UV Induced DNA Damage
3.3 Answers to Check Your Progress Questions
3.4 Summary
3.5 Key Words
3.6 Self Assessment Questions and Exercises
3.7 Further Readings

3.0 INTRODUCTION

DNA damage is distinctly different from mutation, although both are types of error
in DNA. DNA damage is an abnormal chemical structure in DNA, while a mutation
is a change in the sequence of standard base pairs. DNA damages cause changes
in the structure of the genetic material and prevents the replication mechanism
from functioning and performing properly. DNA damage and mutation have
different biological consequences. While most DNA damages can undergo DNA
repair, such repair is not 100% efficient. Unrepaired DNA damages accumulate in
non-replicating cells, such as cells in the brains or muscles of adult mammals, and
can cause aging. In replicating cells, such as cells lining the colon, errors occur
upon replication past damages in the template strand of DNA or during repair of
DNA damages. These errors can give rise to mutations or epigenetic alterations.
Both of these types of alteration can be replicated and passed on to subsequent
cell generations. These alterations can change gene function or regulation of gene
expression and possibly contribute to progression to cancer.
Throughout the cell cycle there are various checkpoints to ensure the cell is
in good condition to progress to mitosis. The three main checkpoints are at G1/s,
G2/m, and at the spindle assembly checkpoint regulating progression through
anaphase. G1 and G2 checkpoints involve scanning for damaged DNA. During
S phase the cell is more vulnerable to DNA damage than any other part of the cell
cycle. G2 checkpoint checks for damaged DNA and DNA replication
completeness. DNA damage is an alteration in the chemical structure of DNA,
such as a break in a strand of DNA, a base missing from the backbone of DNA,
or a chemically changed base such as 8-OHdG. DNA damage can occur naturally
or via environmental factors. The DNA Damage Response (DDR) is a complex
signal transduction pathway which recognizes when DNA is damaged and initiates
the cellular response to the damage.
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DNA Damages In this unit, you will study about DNA damages, deamination of bases,
alkylation, damage due to reactive oxygen and UV induced damage in detail.

NOTES
3.1 OBJECTIVES

After going through this unit, you will be able to:

Explain various DNA damages
Discuss about deamination of bases and alkylation
Understand the damage caused due to reactive oxygen
Analyse UV induced damage

3.2 DNA DAMAGES

Charles’s Darwin theory (1859) was based on slow variations exhibited by

individuals of same race. Variations increase the adaptability of the individuals and
make them better fitted in the struggle for existence and play role in natural selection.
All variations were called as Darwin’s continuous variations. There are two sources
of variations in populations, i.e., recombination and mutation. The most significant
consequence of oxidative stress in the body is thought to be damage to DNA.
DNA may be modified in a variety of ways, which can ultimately lead to mutations
and genomic instability. This could result in the development of a variety of cancers
including colon, breast, and prostate. Here we discuss the various types of damage
to DNA, including oxidative damage, hydrolytic damage, DNA strand breaks,
and others. Oxidative DNA damage refers to the oxidation of specific bases. 8-
hydroxydeoxyGuanosine (8-OHdG) is the most common marker for oxidative
DNA damage and can be measured in virtually any species. It is formed and
enhanced most often by chemical carcinogens. A similar oxidative damage can
occur in RNA with the formation of 8-OHG (8-hydroxyGuanosine), which has
been implicated in various neurological disorders.
Hydrolytic DNA damage involves deamination or the total removal of
individual bases. Loss of DNA bases, known as AP (APurinic/APyrimidinic) sites,
can be particularly mutagenic and if left unrepaired they can inhibit transcription.
Hydrolytic damage may result from the biochemical reactions of various metabolites
as well as the overabundance of reactive Oxygen species. Ultraviolet and other
types of radiation can damage DNA in the form of DNA strand breaks. This
involves a cut in one or both DNA strands; double-strand breaks are especially
dangerous and can be mutagenic, since they can potentially affect the expression
of multiple genes. UV-induced damage can also result in the production of
Pyrimidine Dimers, where covalent cross-links occur in Cytosine and Thymine
residues. The most common Pyrimidine Dimers are Cyclobutane Pyrimidine
Dimers (CPD) and Pyrimidine - (6-4) Pyrimidine Photoproducts (6-
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4PP). CPD and 6-4PP are the most frequent DNA mutations found in the p53 DNA Damages

protein in skin cancers. Pyrimidine dimers can disrupt polymerases and prevent
proper replication of DNA.
DNA damage may also result from exposure to Polycyclic Aromatic
NOTES
Hydrocarbons (PAHs). PAHs are potent, ubiquitous atmospheric pollutants
commonly associated with oil, coal, cigarette smoke, and automobile exhaust fumes.
A common marker for DNA damage due to PAHs is Benzo(a)Pyrene Diol
Epoxide (BPDE). BPDE is found to be very reactive, and known to bind covalently
to Proteins, Lipids, and Guanine residues of DNA to produce BPDE adducts. If
left unrepaired, BPDE-DNA adducts may lead to permanent mutations resulting
in cell transformation and ultimately tumor development.
DNA is a highly stable and versatile molecule. Though sometimes the damage
is caused to it, it is able to maintain the integrity of information contained in it. The
perpetuation of genetic material from generation to generation depends upon
keeping the rates of mutation at low level. DNA has many elaborate mechanisms
to repair any damage or distortion. The most frequent sources of damage to DNA
are the inaccuracy in DNA replication and chemical changes in DNA. Malfunction
of the process of replication can lead to incorporation of wrong bases, which are
mismatched with the complementary strand.
The damage causing chemicals break the backbone of the strand and
chemically alter the bases. Alkylation, oxidation and methylation cause damage to
bases. X-rays and gamma radiations cause single or double stranded breaks in
DNA. A change in the sequence of bases if replicated and passed on to the next
generation becomes permanent and leads to mutation. At the same time mutations
are also necessary which provide raw material for evolution. Without evolution,
the new species, even human beings would not have arisen. Therefore a balance
between mutation and repair is essential.
Types of Damage
Damage to DNA includes any deviation from the usual double helix structure.
Simple Mutations: Simplest mutations are switching of one base for another
base. In transition one pyrimidine is substituted by another Pyrimidine and Purine
with another Purine. Transversion involves substitution of a Pyrimidine by a Purine
and Purine by a Pyrimidine, such as T by G or A and A by C or T. Other simple
mutations are detection, insertion of a single nucleotide or a small number of
nucleotides. Mutations which change a single nucleotide are called point mutations.
Deamination: The common alteration of form or damage includes deamination
of Cytosine (C) to form Uracil (U) which base pairs with Adenine (A) in next
replication instead of Guanine (G) with which the original Cytosine (C) would
have paired. As Uracil (U) is not present in DNA, Adenine (A) base pairs with
Thymine (T). Therefore C-G pair is replaced by T-A in next replication cycle.
Similarly, hypoxanthine results from Adenine (A) deamination.
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DNA Damages Missing Bases: Cleavage of N-Glycosidic bond between Purine and sugar causes
loss of Purine base from DNA. This is called Depurination. This Apurinic site
becomes non-coding lesion.
Chemical Modification of Bases: Chemical modification of any of the four
NOTES
bases of DNA leads to modified bases. Methyl groups are added to various bases.
Guanine (G) forms 7- Methylguanine, 3-Methylguanine. Adenine (A) forms 3-
Methyladenine. Cytosine (C) forms 5- Methylcytosine.
Replacement of Amino group by a Keto group converts 5-Methylcytosine
to Thymine (T). Following is Thymine (T) Dimer in one strand.

Formation of Pyrimidine Dimers (Thymine (T) Dimers): Formation of

Thymine (T) dimers is very common in which a Covalent Bond (Cyclobutyl Ring)
is formed between adjacent Thymine (T) bases. This leads to loss of base pairing
with opposite stand. A bacteria may have thousands of dimers immediately after
exposure to ultraviolet radiations.
Strand Breaks: Sometimes phosphodiester bonds break in one strand of DNA
helix. This is caused by various chemicals like peroxides, radiations and by enzymes
like DNases. This leads to breaks in DNA backbone. Single strand breaks are
more common than double strand breaks. Following is the double strand breaks.

Sometimes X-rays, electronic beams and other radiations may cause

phosphodiester bonds breaks in both strands which may not be directly opposite
to each other. This leads to double strand breaks. Some sites on DNA are more
susceptible to damage. These are called hot-stops.
Repair Mechanisms
Most kinds of damage create impediments to replication or transcription. Altered
bases mispairing can cause permanent alteration to DNA sequence after replication.
In order to maintain the integrity of information contained in it, the DNA has various
repair mechanisms.
Direct Repair: The damage is reversed by a repair enzyme which is called photo-
reactivation. This mechanism involves a light dependent enzyme called DNA
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Photolyase. The enzyme is present in almost all cells from bacteria to animals. It DNA Damages

uses energy from the absorbed light to cleave the C-C bond of Cyclobutyl Ring of
the Thymine (T) dimers. In this way Thymine (T) dimers are monomerized.
Excision Repair: It includes base excision repair and nucleotide excision repair.
NOTES
Base excision repair system involves an enzyme called N-Glycosylase which
recognizes the abnormal base and hydrolyses Glycosidic bond between it and
sugar.
Another enzyme, an endonuclease cleaves the DNA backbone on the 52 -
side of the abnormal base. Then the DNA polymerase by its exonucleases activity
removes the abnormal base. DNA polymerase then replaces it with normal base
and DNA ligase seals the region.
Nucleotide repair system includes three steps, incision, excision and synthesis.
Incision is done by endonuclease enzyme precisely on either side of the damaged
patch of the strand. In this way damaged portion of the strand is cleaved.
Endonuclease enzymes involved are UvrA, UvrB which recognizes the
damaged stretch of the strand. UvrC makes two cuts (incision) on either side.
Exonuclease removes the damaged strand. Enzyme involved is UvrD.
Later, DNA polymerase synthesizes the new strand by using complementary
strand as a template. DNA ligase forms phosphodiester bonds which seal the
ends on newly synthesized strand.
Mismatch Base Repair: Sometimes wrong bases are incorporated during
replication process, G-T or C-A pairs are formed. The wrong base is always
incorporated in the daughter strand only. Therefore in order to distinguish the two
strands for the purpose of repair, the Adenine (A) bases of the template strand are
labelled or tagged by Methyl groups. In this way the newly replication DNA helix
is hemimethylated. The excision of wrong bases occur in the non-methylated or
daughter strand.
Recombination Repair or Retrieval System: In Thymine (T) dimer or other
type of damage, DNA replication cannot proceed properly. A gap opposite to
Thymine (T) dimer is left in the newly synthesized daughter strand. The gap is
repaired by recombination mechanism or retrieval mechanism also called sister
strand exchange.
During replication of DNA two identical copies are produced. Replicating
DNA molecule has four Strands A, B, C and D. Strands A and C have same
DNA, sequence. Strands B and D also have same sequence as they are identical.
A Thymine (T) dimer is present in strand A. The replication fork passes the dimmer
as it cannot form hydrogen bonds with incoming Adenine (A) bases, thus creating
a gap in the newly synthesized Strand B.
In recombination repair system a short identical segment of DNA is retrieved
from Strand D and is inserted into the gap of Strand B. But this creates a gap in
Strand D which is easily filled up by DNA polymerase using normal Strand C as a
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DNA Damages template. This event is dependent on the activity of a special Protein RecA. The
RecA protein plays its role in retrieving a portion of the complementary strand
from other side of the replication fork to fill the gap. RecA is a strand exchange
protein.
NOTES
After filling both gaps, Thymine (T) is monomerised. So in this repair
mechanism a portion of DNA strand is retrieved from the normal homologous
DNA segment. This is also known as daughter strand gap repair mechanism.
SOS Repair Mechanism: Sometimes the replicating machinery is unable to repair
the damaged portion and bypasses the damaged site. This is known as translesion
synthesis also called bypass system and is emergency repair system. This
mechanism is catalyzed by a special class of DNA polymerases called Y-family of
DNA polymerases which synthesized DNA directly across the damaged portion.
There are different types of DNA damage and therefore different molecular
pathways of DNA repair to correct them, including non-homologous end joining,
homologous recombination, mismatch repair and nucleotide excision repair.
Numerous proteins and pathways have been involved in these processes.
The ATM and ATR kinases, as well as DNA-PK, are key for detection of the
DNA lesions. Chromatin remodelers participate in making sure that the chromatin
environment is accessible to the DNA repair apparatus, and key repair factors,
such as RPA, Rad51 and the Fanconi Anemia proteins directly act in repairing
the DNA lesions. Fanconi anemia is a rare disease passed down through families
(inherited) that mainly affects the bone marrow. It results in decreased production
of all types of blood cells. Importantly the p53 network, the RAS
GTPase superfamily, and the ubiquitin system are also involved in different aspects
of the DNA damage response (Refer Figure 3.1).

Fig. 3.1 DNA Damage

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 Direct DNA damage can occur when DNA directly absorbs a UVB photon, DNA Damages

or for numerous other reasons. UVB light causes Thymine (T) base pairs next to
each other in genetic sequences to bond together into Pyrimidine Dimers, a
disruption in the strand, which reproductive enzymes cannot copy. It
causes sunburn and it triggers the production of Melanin. NOTES
Other names for the ‘Direct DNA Damage’ are:
Thymine Dimers
Pyrimidine Dimers
Cyclobutane Pyrimidine Dimers (CPDs)
UV Endonuclease Sensitive-Sites (UPESS)
Due to the excellent photochemical properties of DNA, this nature made
molecule is damaged by only a tiny fraction of the absorbed photons. DNA
transforms more than 99.9% of the photons into harmless heat (but the damage
from the remaining < 0.1% is still enough to cause sunburn). The transformation of
excitation energy into harmless heat occurs via a photochemical process
called internal conversion. In DNA, this internal conversion is extremely fast, and
therefore efficient. This ultrafast (subpicosecond) internal conversion is a
powerful photo-protection provided by single nucleotides. However, the Ground-
State Recovery is much slower (picoseconds) in G–C–DNA duplexes and
hairpins. It is presumed to be even slower for double-stranded DNA in conditions
of the nucleus.
The absorption spectrum of DNA shows a strong absorption for UVB
radiation and a much lower absorption for UVA radiation. Since the action spectrum
of sunburn is indistinguishable from the absorption spectrum of DNA, it is generally
accepted that the direct DNA damages are the cause of sunburn.
While the human body reacts to direct DNA damages with a painful warning
signal, no such warning signal is generated from indirect DNA damage.
3.2.1 Deamination of Bases
Cytosine (C) susceptible to hydrolysis deaminated to Uracil (U). If left uncorrected,
the conversion of Cytosine (C) to Uracil (U) mutations migration occurs. It is a
base for foreign DNA, Uracil (U) will change back enzyme specific Cytosine (C)
to Uracil (U) DNA Glycosylase or UDG.
Deamidation is by removing the Amino groups of the molecule. Enzyme
that catalyzes this reaction, known as deaminase. The human body, deamination
takes place mainly in the liver, but glutamic acid, is deaminated in the kidney. If
there is an excess of proteins, is a process when the deamidation amino acid is
split. It is removed from an Amino acid, and an Amino group is converted to
Ammonia. Consists essentially of Carbon and Hydrogen, the rest of the Amino
Acid sequence is Oxidized or recycled as energy. Ammonia is toxic to the human
body, the enzyme converts the uric acid and urea by adding a molecule (which is
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DNA Damages not considered in the deamidation process) Carbon Dioxide in the Urea Cycle,
which takes place in the liver. Uric acid and Urea, can be safely after diffused in
the blood and excreted in the urine.
Release of Ammonia in the process, deamination spontaneous reaction is a
NOTES
hydrolysis of Cytosine (C) to Uracil (U). Instead of the bisulfite to convert Cytosine
(C), and can be prepared by in vitro of using the 5 –Methylcytosine. This property
has been allowed for researchers to distinguish between Methylated Cytosine (C)
(shown as Uracil (U)) Unmethylated Cytosine (C) to sequence methylated DNA.
The DNA, the deamination spontaneous is adjusted removal of Uracil (U). DNA
glycosylase generator of abasic (AP) site (rather than Cytosine (C) deamination,
the product of the portion of the DNA) by. By replacing Cytosine (C) another
basic sites obtained is recognized by the (AP endonuclease) enzymes that degrade
to allow repair of the lesion, the phosphodiester bonds of DNA. DNA polymerase
is able to fill the reaction of the polymerase activity by nick translation terminal
excision reaction due to the 3 5 exonuclease activity, to perform this substitution
followed. DNA ligase to form a phosphodiester bond sealing the two strand nicks
resulting product contains a new, correct Cytosine 5-methylcytosine is a methylated
form of the DNA base Cytosine (C), which can be incorporated in the regulation
of gene transcription.
5-Methylcytosine is a methylated form of the DNA base Cytosine (C) that
may be involved in the regulation of gene transcription. When Cytosine (C) is
methylated, the DNA maintains the same sequence, but the expression of methylated
genes can be altered. 5-Methylcytosine is incorporated in the nucleoside 5-
methylcytidine. In 5-methylcytosine, a methyl group is attached to the 5th atom in
the 6-atom ring, counting counterclockwise from the NH nitrogen at the six o’clock
position, not the 2 o’clock. This methyl group distinguishes 5-methylcytosine from
Cytosine (C).
When trying to separate the bacterial toxins responsible for tuberculosis,
separating the nucleic acids called Tuberculinic Acid in 1898 by a new
Mycobacterium tuberculosis. It was first identified by a German chemist W.G.
Ruppel in 1898. The nucleic acid, in addition to Methylated Nucleotides, Guanine
(G), Cytosine (C) and Thymine (T), can be included in it. Coghill and Johnson
(1925), successfully detected a minor amount of a Methylated Cytosine derivative
as a product of hydrolysis of Tuberculinic Acid with Sulfuric Acid. This report was
criticized for the identification of those based on the optical properties of crystalline
picric acid exclusively rapidly, other scientists did not have the ability to reproduce
the same results. Using a paper chromatography to establish a Methylated Cytosine
(C) unique completely different Uracil (U) and Cytosine (C) conventional, when it
is divided into the nucleic acid of calf thymus DNA, and finally, Stapler, facts
1948. After 70 years, the precise role is unclear, but also, it should be a common
feature of the RNA molecule in a variety was found.
Life, seems to be designed to minimize the error. The universal nature of
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60 Material
C / T will all agree on this point. However, in spite of the logic of this design, DNA Damages

Cytosine (C), is particularly sensitive to deamination, such as removal of the exocyclic

Amino group is interesting, but there, it will be a (base, which is normally found in
RNA) Uracil (U). Typically the Uracil (U) in DNA is not present, it can be removed
by detecting the repair enzymes it effectively. If it is detected, it is not deleted, base NOTES
pair with Adenine (A), which means that you specify the Adenine (A) during DNA
replication can be it. Thymine (T) in set in subsequent rounds of replication of
Adenine (A) in order. The conclusion is that the deamination spontaneous Cytosine
(C), there is a possibility that base substitutions called transition, is replaced by
(being substituted with the other strand of the DNA and G) T is C occurs. The
conversion to C half-life single-stranded DNA of Cytosine (C) for specific about
200 years certain Cytosine (C) deamination degree, since such mutations are a
common occurrence at 37 degrees C. In fact, the high rate of these deamidation,
led researchers from the pool. Al complain of ‘Confusion Cytosine’.
3.2.2 Alkylation
Alkylation lesions in DNA and RNA result from endogenous compounds,
environmental agents and alkylating drugs. Simple methylating agents, for example
methylnitrosourea, tobacco-specific nitrosamines and drugs like Temozolomide
or Streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions
are mainly repaired by direct base repair, base excision repair, and to some extent
by Nucleotide Excision Repair (NER). The identified carcinogenicity of O (6)-
methylGuanine (O (6)-meG) is largely caused by its miscoding properties. Mutations
from this lesion are prevented by O (6)-alkylG-DNA alkyltransferase (MGMT or
AGT) that repairs the base in one step. However, the genotoxicity and cytotoxicity
of O (6)-meG is mainly due to recognition of O(6)-meG/T (or C) mispairs by the
MisMatch Repair (MMR) system and induction of futile repair cycles, eventually
resulting in cytotoxic double-strand breaks. Therefore, inactivation of the MMR
system in an AGT-defective background causes resistance to the killing effects of
O (6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating
agents, such as ChloramBucil or Carmustine (BCNU), are commonly used anti-
cancer drugs. DNA lesions caused by these agents are complex and require
complex repair mechanisms. Thus, primary chloroethyl adducts at O (6)-G are
repaired by AGT, while the secondary highly cytotoxic Interstrand Cross-Links
(ICLs) require nucleotide excision repair factors for incision and homologous
recombination to complete repair. Recently, Escherichia coli protein AlkB and
human homologues were shown to be oxidative demethylases that repair cytotoxic
1-methylAdenine (1-meA) and 3-methylCytosine (3-meC) residues. Numerous
AlkB homologues are found in Viruses, Bacteria and Eukaryotes, including eight
human homologues (hABH1-8). These have distinct locations in subcellular
compartments and their functions are only starting to become understood.
Surprisingly, AlkB and hABH3 also repair RNA. An evaluation of the biological
effects of environmental mutagens, as well as understanding the mechanism of
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DNA Damages action and resistance to alkylating drugs require a detailed understanding of DNA
repair processes (Refer Figure 3.2).

NOTES

Fig. 3.2 Damage due to Alkylation of Bases

3.2.3 Damage Due to Reactive Oxygen

About 100 Oxidatively generated base lesions and 2-Deoxyribose modifications,
including initially formed Thymidine Hydroperoxides and Diastereomeric
nucleosides, have been isolated and identified in model studies. The number of
products detected in cellular DNA is much lower, owing to several limitations and
difficulties. These include, among others, the lack of sensitivity of available methods
for detecting lesions produced in low yields, instability of some modifications,
such as base hydroperoxides, optimization of assays that may require the synthesis
of internal standards labeled with stable isotopes, and finally, artefactual oxidation
of overwhelming normal nucleosides during DNA extraction and subsequent
workup.
Hydroxyl Radical
The hydroxyl radical (•OH) is a highly Reactive Oxygen Species (ROS) that
efficiently reacts with nearby biomolecules at diffusion-controlled rates of reaction.
The reaction volume of •OH is less than 2 nm in cells and tissues; thus, it reacts
essentially at the site of generation. The most likely source of •OH in cells is the
Fenton reaction which involves the reaction of reduced redox active metal ions,
such as Ferrous and Cuprous Ions, with metabolically produced H2O2. For this
reason, the main lines of defense against ROS by aerobic organisms include metal-
binding chelators and proteins (for example, Ferritin) to minimize the concentration
of labile metal ions, together with catalase and peroxidases to minimize the
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concentration of H2O2. The generation of •OH by Fenton-like reactions is believed DNA Damages

to take place in a site-specific manner, for example, involving metal ions in close
proximity or bound to DNA. •OH is also generated by the radiolysis of water
molecules according to the so-called indirect effect of ionizing radiation.
NOTES
Thymine
Two main reactions mediated by •OH have been shown to take place with Thymine
(T) nucleobases in cellular DNA: addition across the 5,6-pyrimidine bond and H-
atom abstraction from the methyl group. Model studies have shown that •OH
preferentially adds to C5 and to a lesser extent to C6, giving rise to reducing C6-
yl and oxidizing C5-yl radicals, respectively. In the case of nucleoside Thymidine
(T), O2 rapidly adds to the radical site, giving rise to the corresponding hydroperoxyl
radicals that subsequently convert into eight cis and trans diastereomers of 5-
hydroxy-6-hydroperoxy-5,6-dihydrothymidine and 6-hydroxy-5-hydroperoxy-
5,6-dihydrothymidine. The major radiation-induced base degradation products
so far detected in cellular DNA are the cis and trans diastereomers of 5,6-
dihydroxy-5,6-dihydrothymine. These products may be explained by stereospecific
reduction of intermediate Thymine Hydroperoxides. Typically, the Thymine
Hydroperoxides may also decomposed by Pyrimidine Ring cleavage to 5-hydroxy-
5-methylhydantoin derivatives (Hyd-Thy), which was recently detected in irradiated
cells .The second major pathway of •OH-mediated decomposition of Thymine
(T) and its derivatives, including DNA in solution, involves H-atom abstraction
from the methyl group. This leads to the 5-(Uracilyl) methyl radical, which is readily
converted into the corresponding peroxyl radical after O2 addition and
hydroperoxide after subsequent reduction and protonation. In turn, hydroperoxide
decomposes by reduction and competitive dehydration to 5-HydroxyHethylUracil
(5-HmUra) and 5-FormylUracil (5-FoUra) derivatives, respectively. The latter
products are major oxidation products detected in cellular DNA by HPLC coupled
to Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS). The 5-
(Uracilyl) methyl radical can also react with neighboring Guanine (G) and Adenine
(A) bases to produce intrastrand or possibly interstrand cross-links connected
between the methyl group of Thymine (T) and the C8 position of either Guanine
(G[8-5 m]T) or Adenine (A) DNA intrastrand cross-link.
3.2.4 UV Induced DNA Damage
Induction of DNA damage by solar UVR is a key event that drastically influences
the normal life processes of all organisms. A number of endogenous factors, such
as free radicals generated during metabolic processes as well as exogenous factors,
such as UV or ionizing radiations are known to interfere with genome integrity
(Refer Figure 3.3).
DNA damage results in:
Misincorporation of bases during replication process.

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DNA Damages Hydrolytic damage, which results in deamination of bases, depurination,
and depyrimidination.
Oxidative damage, caused by direct interaction of Ionizing Radiations
(IR) with the DNA molecules, as well as mediated by UV radiation-
NOTES
induced free radicals or reactive oxygen species.
Alkylating agents that may result in modified bases.
The hydrolytic deamination (loss of an Amino group) can directly convert
one base to another; for example, deamination of Cytosine (C) results in Uracil
(U) and at much lower frequency Adenine (A) to hypoxanthine. In depurination/
depyrimidination, there are complete removals of Purine/Pyrimidine bases, leaving
the deoxyribose sugar depurinated/depyrimidinated that may cause breakage in
the DNA backbone. The exposure of UVR, IR, and certain genotoxic chemicals
may result in single as well as double DNA strand breaks. Among different types
of damages, DNA Double Strand Breaks (DSBs) are the most deleterious, since
they affect both strands of DNA and can lead to the loss of genetic material. At
high concentrations oxygen-free radicals or, more frequently, Reactive Oxygen
Species (ROS) can induce damage to cell structure, lipids, proteins as well as
DNA and results in oxidative stress which has been implicated in a number of
human diseases. The hydroxyl radicals (OH°) can damage all components of DNA
molecules, such as Purine and Pyrimidine bases and also the deoxyribose backbone,
inhibiting the normal functions of the cell.

Fig. 3.3 UV Induced DNA Damage

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 DNA Damages

Check Your Progress

1. What does hydrolytic DNA damage involves?
2. Where can DNA damage result? NOTES
3. What is simple mutations?
4. Define deamination.
5. Write in short about direct repair.
6. Give the other names for the ‘direct DNA damage’.
7. What are DNA damage results?

3.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. Hydrolytic DNA damage involves deamination or the total removal of

individual bases. Loss of DNA bases, known as AP (APurinic/APyrimidinic)
sites, can be particularly mutagenic and if left unrepaired they can inhibit
transcription. Hydrolytic damage may result from the biochemical reactions
of various metabolites as well as the overabundance of reactive oxygen
species.
2. DNA damage may also result from exposure to Polycyclic Aromatic
Hydrocarbons (PAHs). PAHs are potent, ubiquitous atmospheric pollutants
commonly associated with oil, coal, cigarette smoke, and automobile exhaust
fumes.
3. Simple mutations are switching of one base for another base. In transition
one Pyrimidine is substituted by another Pyrimidine and Purine with another
Purine. Transversion involves substitution of a Pyrimidine by a Purine and
Purine by a Pyrimidine, such as T by G or A and A by C or T. Other simple
mutations are detection, insertion of a single nucleotide or a small number
of nucleotides. Mutations which change a single nucleotide are called point
mutations.
4. The common alteration of form or damage includes deamination of Cytosine
(C) to form Uracil (U) which base pairs with Adenine (A) in next replication
instead of Guanine (G) with which the original Cytosine (C) would have
paired.
5. The damage is reversed by a repair enzyme which is called photo-
reactivation. This mechanism involves a light dependent enzyme called DNA
photolyase. The enzyme is present in almost all cells from bacteria to animals.
It uses energy from the absorbed light to cleave the C-C bond of Cyclobutyl
Ring of the Thymine (T) dimers. In this way Thymine (T) dimers are
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DNA Damages 6. Other names for the ‘Direct DNA Damage’ are:
Thymine Dimers
Pyrimidine Dimers
NOTES Cyclobutane Pyrimidine Dimers (CPDs)
UV Endonuclease Sensitive-Sites (UVESS)
7. DNA damage results in:
Misincorporation of bases during replication process.
Hydrolytic damage, which results in deamination of bases, depurination,
and depyrimidination.
Oxidative damage, caused by direct interaction of Ionizing Radiations
(IR) with the DNA molecules, as well as mediated by UV radiation-
induced free radicals or Reactive Oxygen Species (ROS).
Alkylating agents that may result in modified bases.

3.4 SUMMARY

Charles’s Darwin theory (1859) was based on slow variations exhibited by

individuals of same race.
Variations increase the adaptability of the individuals and make them better
fitted in the struggle for existence and play role in natural selection. All
variations were called as Darwin’s continuous variations.
The most significant consequence of oxidative stress in the body is thought
to be damage to DNA.
DNA may be modified in a variety of ways, which can ultimately lead to
mutations and genomic instability.
Oxidative DNA damage refers to the oxidation of specific bases. 8-
hydroxydeoxyguanosine (8-OHdG) is the most common marker for
oxidative DNA damage and can be measured in virtually any species.
A similar oxidative damage can occur in RNA with the formation of 8-
OHG (8-hydroxyguanosine), which has been implicated in various
neurological disorders.
Hydrolytic DNA damage involves deamination or the total removal of
individual bases.
Loss of DNA bases, known as AP (APurinic/APyrimidinic) sites, can be
particularly mutagenic and if left unrepaired they can inhibit transcription.
Hydrolytic damage may result from the biochemical reactions of various
metabolites as well as the overabundance of Reactive Oxygen Species
(ROS).
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Ultraviolet and other types of radiation can damage DNA in the form of DNA Damages

DNA strand breaks. This involves a cut in one or both DNA strands; double-
strand breaks are especially dangerous and can be mutagenic, since they
can potentially affect the expression of multiple genes.
NOTES
UV-induced damage can also result in the production of Pyrimidine dimers,
where covalent cross-links occur in Cytosine (C) and Thymine (T) residues.
The most common pyrimidine dimers are Cyclobutane Pyrimidine
Dimers (CPD) and Pyrimidine - (6-4) Pyrimidine Photoproducts (6-4PP).
CPD and 6-4PP are the most frequent DNA mutations found in the p53
protein in skin cancers.
Pyrimidine dimers can disrupt polymerases and prevent proper replication
of DNA.
DNA damage may also result from exposure to Polycyclic Aromatic
Hydrocarbons (PAHs).
PAHs are potent, ubiquitous atmospheric pollutants commonly associated
with oil, coal, cigarette smoke, and automobile exhaust fumes.
A common marker for DNA damage due to PAHs is Benzo(a)Pyrene Diol
Epoxide (BPDE). BPDE is found to be very reactive, and known to bind
covalently to proteins, lipids, and guanine residues of DNA to produce
BPDE adducts.
The perpetuation of genetic material from generation to generation depends
upon keeping the rates of mutation at low level. DNA has many elaborate
mechanisms to repair any damage or distortion.
The most frequent sources of damage to DNA are the inaccuracy in DNA
replication and chemical changes in DNA.
Malfunction of the process of replication can lead to incorporation of wrong
bases, which are mismatched with the complementary strand.
The damage causing chemicals break the backbone of the strand and
chemically alter the bases.
Alkylation, Oxidation and Methylation cause damage to bases. X-rays and
gamma radiations cause single or double stranded breaks in DNA.
A change in the sequence of bases if replicated and passed on to the next
generation becomes permanent and leads to mutation.
Simplest mutations are switching of one base for another base. In transition
one Pyrimidine is substituted by another Pyrimidine and Purine with another
Purine.
Transversion involves substitution of a Pyrimidine by a Purine and Purine
by a Pyrimidine, such as T by G or A and A by C or T. Other simple

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DNA Damages mutations are detection, insertion of a single nucleotide or a small number
of nucleotides.
Mutations which change a single nucleotide are called point mutations.
NOTES The common alteration of form or damage includes deamination of Cytosine
(C) to form Uracil (U) which base pairs with Adenine (A) in next replication
instead of Guanine (G) with which the original Cytosine (C) would have
paired.
Cleavage of N-Glycosidic bond between purine and sugar causes loss of
purine base from DNA. This is called depurination. This apurinic site
becomes non-coding lesion.
Chemical modification of any of the four bases of DNA leads to modified
bases. Methyl groups are added to various bases. Guanine forms 7-
Methylguanine, 3-Methylguanine.
The damage is reversed by a repair enzyme which is called photo-
reactivation. This mechanism involves a light dependent enzyme called DNA
Photolyase.
Another enzyme, an endonuclease cleaves the DNA backbone on the 52 -
side of the abnormal base. Then the DNA polymerase by its exonuclease
activity removes the abnormal base.
DNA polymerase then replaces it with normal base and DNA ligase seals
the region.
Endonuclease enzymes involved are UvrA, UvrB which recognize the
damaged stretch of the strand. UvrC makes two cuts (incision) on either
side.
The absorption spectrum of DNA shows a strong absorption for UVB
radiation and a much lower absorption for UVA radiation.
Deamidation is by removing the amino groups of the molecule. Enzyme that
catalyzes this reaction, known as deaminase. The human body, deamination
takes place mainly in the liver, but glutamic acid, is deaminated in the kidney.
Two main reactions mediated by •OH have been shown to take place with
Thymine nucleobases in cellular DNA: addition across the 5,6 - pyrimidine
bond and H-atom abstraction from the methyl group.
Induction of DNA damage by solar UVR is a key event that drastically
influences the normal life processes of all organisms.
A number of endogenous factors, such as free radicals generated during
metabolic processes as well as exogenous factors, such as UV or ionizing
radiations are known to interfere with genome integrity.

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 DNA Damages
3.5 KEY WORDS

Malfunction: Malfunction of the process of replication that can lead to

incorporation of wrong bases, which are mismatched with the complementary NOTES
strand.
Point mutations: Mutations which change a single nucleotide are called
point mutations.

3.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is DNA damage?
2. What are the types of DNA damages?’
3. How can missing bases be defined?
4. Draw the structure of Thymine (T) dimer in one strand.
5. Distinguish between direct repair and excision repair.
6. What is SOS repair mechanism?
7. Explain UV induced DNA damage.
Long Answer Questions
1. Discuss about DNA damages and its types in detail.
2. Write a note on formation of pyrimidine dimers.
3. What is repair mechanism? Explain.
4. Discuss about mismatch base repair.
5. Explain deamination of bases.
6. Write in detail about alkylation.
7. Explain damage due to reactive Oxygen.

3.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
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DNA Damages Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
NOTES
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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 Repair Pathways

UNIT 4 REPAIR PATHWAYS

Structure NOTES
4.0 Introduction
4.1 Objectives
4.2 DNA Damage and Repair Pathways
4.2.1 An SOS Repair System or Recombinational Repair
4.2.2 Methyl Directed Mismatch Repair
4.2.3 Nucleotide Excision Repair
4.2.4 Recombinational Repair
4.2.5 SOS Inducible Repair
4.2.6 Specific Repair for Oxidative DNA Damage
4.2.7 Pyrimidine Dimers
4.2.8 Alkylation Induced Damage and Adaptive Response
4.3 Answers to Check Your Progress Questions
4.4 Summary
4.5 Key Words
4.6 Self Assessment Questions and Exercises
4.7 Further Readings

4.0 INTRODUCTION

DNA repair processes are of crucial importance for the maintenance of the genetic
information of all organisms. The stability of the genome is constantly endangered
by environmental agents, endogenous metabolic processes, for example reactive
species inside cells, and errors of cellular processes involving DNA. Modifications
of DNA can lead to mutations, which alter the coding sequence of DNA and can
lead to cancer in humans and other mammals. Other DNA lesions interfere with
normal cellular transactions, such as DNA replication or transcription, and are
deleterious to the cell. To counteract DNA damage, organisms have evolved various
damage prevention and repair systems. These systems ensure the stability of DNA
and accurate transmission of genetic information by protecting the genome against
a large number of different chemical and structural alterations. At the same time,
random changes in DNA are viewed as a main source of genetic variability, and
thus a driving force for evolution. In multicellular organisms changes in the DNA
sequence and structure are responsible, for example differential production of
antibodies by the immune system. Therefore, DNA repair mechanisms have to
balance the noxious against the beneficial effects of alterations in the genome
sequence and chemical structure.
DNA repair is a very complicated process, involving many factors. For
instance to date, 168 genes that encode proteins involved in DNA repair have
been identified in the human genome. They are involved in diverse processes,
starting from detection of a damage site in the DNA, through several steps of
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Repair Pathways enzymatic transformation of the damaged DNA, to recombination and signaling to
stop the cell cycle or initiate apoptosis. Another form of dealing with the DNA
damage is lesion bypass, which facilitates continuation of replication even when
irremovable modifications occur, but does not guarantee proper recreation of the
NOTES original sequence and frequently leads to mutation generated by TransLesion
Synthesis (TLS) polymerases.
In this unit, you will study about repair pathways, methyl directed mismatch
repair, nucleotide excision repair, base excision repair, recombinational repair,
SOS inducible repair, specific repair for oxidative DNA damage, pyrimidine dimers
and alkylation induced damage and adaptive response in detail.

4.1 OBJECTIVES

After going through this unit, you will be able to:

Discuss about repair pathways and its types
Explain methyl directed mismatch repair
Understand what nucleotide excision repair is
Explain base excision and recombinational repair
Describe SOS inducible repair and specific repair for oxidative DNA damage

4.2 DNA DAMAGE AND REPAIR PATHWAYS

DNA damage is a change in the basic structure of DNA that is not itself replicated
when the DNA is replicated. A DNA damage can be a chemical addition or
disruption to a base of DNA (creating an abnormal nucleotide or nucleotide
fragment) or a break in one or both chains of the DNA strands. When DNA
carrying a damaged base is replicated, an incorrect base can often be inserted
opposite the site of the damaged base in the complementary strand, and this can
become a mutation in the next round of replication. Also, DNA double-strand
breaks may be repaired by an inaccurate repair process leading to mutations. In
addition, a double strand break can cause rearrangements of the chromosome
structure (possibly disrupting a gene, or causing a gene to come under abnormal
regulatory control), and, if such a change can be passed to successive cell
generations, it is also a form of mutation. Mutations, however, can be avoided if
accurate DNA repair systems recognize DNA damages as abnormal structures,
and repair the damages prior to replication (Refer Figure 4.1).

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 Repair Pathways

NOTES

Fig. 4.1 DNA Damage

DNA Damage occurs due to:

Single Base Change: Such changes takes place due to conversion of one
base to other, for example deamination of 5-methylcytosine to Thymine
(T).
Structural Distortion: Such distortion take place due to single strand nick,
removal of a base or introduction of a covalent link between bases of same
or different strands, for example formation of Thymine (T) dimer due to
UV.
There are several types of damage to DNA due to endogenous cellular processes
(Refer Figure 4.2):
Oxidation of bases, for example 8-oxo-7, 8-dihydroGuanine (8-oxoG) and
generation of DNA strand interruptions from reactive oxygen species.
Alkylation of bases (usually methylation), such as formation of
7-methylguanosine, 1-methyladenine, 6-O-Methylguanine.
Hydrolysis of bases, such as deamination, depurination, and
depyrimidination.
Mismatch of bases, due to errors in DNA replication, in which the wrong
DNA base is stitched into place in a newly forming DNA strand, or a DNA
base is skipped over or mistakenly inserted.

Fig. 4.2 Types of DNA Damages

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Repair Pathways Damage caused by exogenous agents comes in many forms. Some examples are:
UV-B light causes crosslinking between adjacent Cytosine and Thymine
bases creating pyrimidine dimers. This is called direct DNA damage.
NOTES UV-A light creates mostly free radicals. The damage caused by free radicals
is called indirect DNA damage.
Ionizing radiation such as that created by radioactive decay or in cosmic
rays causes breaks in DNA strands. Intermediate-level ionizing radiation
may induce irreparable DNA damage (leading to replicational and
transcriptional errors needed for neoplasia or may trigger viral interactions)
leading to pre-mature aging and cancer.
Thermal disruption at elevated temperature increases the rate
of depurination (loss of purine bases from the DNA backbone) and single-
strand breaks. For example, hydrolytic depurination is seen in
the thermophilic bacteria, which grow in hot springs at 40–80°C. The rate
of depurination (300 purine residues per genome per generation) is too high
in these species to be repaired by normal repair machinery, hence a possibility
of an adaptive response cannot be ruled out.
Industrial chemicals such as vinyl chloride and hydrogen peroxide, and
environmental chemicals such as polycyclic aromatic hydrocarbons found
in smoke, soot and tar create a huge diversity of DNA adducts ethane
bases, oxidized bases, alkylated phosphotriesterase and crosslinking of
DNA, just to name a few.
UV damage, alkylation/methylation, X-ray damage and oxidative damage are
examples of induced damage. Spontaneous damage can include the loss of a
base, deamination, sugar ring puckering and tautomeric shift. Constitutive
(spontaneous) DNA damage caused by endogenous oxidants can be detected as
a low level of histone H2AX phosphorylation in untreated cells.
Nuclear Versus Mitochondrial
In human cells, and eukaryotic cells in general, DNA is found in two cellular
locations – inside the nucleus and inside the mitochondria. Nuclear DNA (nDNA)
exists as chromatin during non-replicative stages of the cell cycle and is condensed
into aggregate structures known as chromosomes during cell division. In either
state the DNA is highly compacted and wound up around bead-like proteins
called histones. Whenever a cell needs to express the genetic information encoded
in its nDNA the required chromosomal region is unravelled, genes located therein
are expressed, and then the region is condensed back to its resting conformation.
Mitochondrial DNA (mtDNA) is located inside mitochondria organelles, exists in
multiple copies, and is also tightly associated with a number of proteins to form a
complex known as the nucleoid. Inside mitochondria, Reactive Oxygen Species
(ROS) or free radicals, byproducts of the constant production of Adenosine
TriPhosphate (ATP) via oxidative phosphorylation, create a highly oxidative
74
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environment that is known to damage mtDNA. A critical enzyme in counteracting
the toxicity of these species is superoxide dismutase, which is present in both the Repair Pathways

mitochondria and cytoplasm of Eukaryotic cells.

Senescence and Apoptosis
Senescence, an irreversible process in which the cell no longer divides, is a NOTES
protective response to the shortening of the chromosome ends. The telomeres are
long regions of repetitive non-coding DNA that cap chromosomes and undergo
partial degradation each time a cell undergoes division. In contrast, quiescence is
a reversible state of cellular dormancy that is unrelated to genome damage.
Senescence in cells may serve as a functional alternative to apoptosis in cases
where the physical presence of a cell for spatial reasons is required by the
organism, which serves as a ‘last resort’ mechanism to prevent a cell with damaged
DNA from replicating inappropriately in the absence of pro-growth cellular signaling.
Unregulated cell division can lead to the formation of a tumor, which is potentially
lethal to an organism. Therefore, the induction of senescence and apoptosis is
considered to be part of a strategy of protection against cancer.
DNA Repair System
The mechanism of DNA repair in different species exhibit similarities as well as
differences. There are many methods for DNA repair. Every method repairs a
particular type of damage. Some of the repair systems are as follows (Refer Figure
4.3):

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Repair Pathways Photoreactive Repair or Direct Repair: In direct repair of DNA damage no
fresh synthesis of DNA is required and process of DNA damage is reversed.
Albert Kelner (1949) reported the direct repair of UV induced DNA damage in
Escherichia coli. He observed that DNA damage process can be reversed if
NOTES damaged cells were exposed to light in blue range of visible spectrum. The photo-
reactivation is due to enzyme photo-reactivation enzyme and temperature
controlled. The enzyme cleaves the bond between Thymine dimers and due to this
effect of UV radiations on DNA is reversed.
Excision Repair: It is light independent repair system by Paul Howard Flanders
in 1960’s. A group of genes called Uvr genes (UvrA, UvrB, and UvrC) code for
the components of repair enzyme endonuclease which is involved in the process
of excision repair. Another enzyme UvrD is also needed for helicase activity. The
mechanism involves the following steps:
Recognition and Cleavage: The distorted or affected of strand caused
by UV induced dimer is recognized and enzymatically clipped out by
endonuclease enzyme that cleaves phosphodiester bond. This excision may
include several nucleotides adjacent to dimer as well and leaves a gap in the
helix.
Gap Filling: DNA polymerase I fills this gap by inserting the ribonucleotides
complementary to those on the intact strands. The enzyme adds these bases
to the 3OH end of the clipped DNA.
The enzyme DNA ligase seals the final nick that remains at the 3OH end of
the last base inserted thus closing the gap. The process of excision repair
can be activated in response to any damage to DNA that distorts the helix
provided the distortion may be recognized, for example DNA glycosylases
recognizes the presence of Uracil when it is a part of DNA.
Excision repair system is absent in some human beings causing rare disease
Xeroderma Pigmentosum (XP). Such persons cannot tolerate sunlight.
Mismatch Repair: To cope up with the errors that remain after ‘Proof Reading’
another mechanism called mismatch repair was proposed by Robin Holliday like
other DNA lesions. It has following features:
Detection of Mismatch or Alteration.
Removal of Incorrect Nucleotide.
Replacement with Correct Base.
But a special problem exists in the recognition of correct template, which contains
the mismatch, for example there is mismatch in base pair as GC-GT then a repair
system may give rise to GC or AT, wild type or mutant type. The repair system has
to distinguish the new strand and to restore the wild type.
In Escherichia coli, this process has been elucidated and is based on the
process called DNA methylation. The bacteria contain an enzyme called Adenine
(A) Methylase that recognizes DNA sequence as substrate. Upon recognition, a
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methyl group is added to each of the Adenine (A) residues. Following replication, Repair Pathways

the newly synthesized strand remains unmethylated for a short period and at this
point, the repair enzyme recognizes the mismatch and starts the process of repair.
4.2.1 An SOS Repair System or Recombinational Repair NOTES
It was first discovered in an excision defected strain of Escherichia coli. This
system is thought to respond when damaged DNA escaped repair and process of
replication is interrupted. About 20 different gene products are involved in this
repair mechanism of which LexA product is of particular interest. The SOS repair
response is initiated by the interaction of RecA protein with LexA repressor coded
by gene RecA and LexA, respectively. It is also called recombinational repair
(Refer Figure 4.4).
Following steps are involved in SOS repair system or recombinational repair:
DNA damage activates RecA proteins (a protease).
RecA interacts with LexA and causes it to undertake autocatalytic
cleavage. This inactivates the repression function of LexA and all the
operon bound to LexA are induced.
Since LexA represses its own synthesis its own synthesis and that of
RecA, its proteolytic degradation leads to amplification of both RecA
and LexA proteins.
When damage signal is removed, RecA loses induction and free LexA
protein quickly accumulates in the uncleaved forms and turn off SOS
genes.

Fig. 4.4 SOS Repair System

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Repair Pathways 4.2.2 Methyl Directed Mismatch Repair
DNA mismatch repair is a system for recognizing and repairing erroneous insertion,
deletion, and misincorporation of bases that can arise during DNA replication and
NOTES recombination, as well as repairing some forms of DNA damage.
Mismatch repair is strand-specific. During DNA synthesis the newly
synthesised (daughter) strand will commonly include errors. In order to begin repair,
the mismatch repair machinery distinguishes the newly synthesised strand from the
template (parental). In Gram-Negative Bacteria, transient hemimethylation
distinguishes the strands (the parental is methylated and daughter is not). However,
in other prokaryotes and eukaryotes, the exact mechanism is not clear. It is suspected
that, in eukaryotes, newly synthesized lagging-strand DNA transiently contains
nicks (before being sealed by DNA ligase) and provides a signal that directs
mismatch proofreading systems to the appropriate strand. This implies that these
nicks must be present in the leading strand, and evidence for this has recently been
found. Recent work has shown that nicks are sites for RFC-dependent loading of
the replication sliding clamp PCNA, in an orientation-specific manner, such that
one face of the donut-shape protein is juxtaposed toward the 3'-OH end at the
nick. Oriented PCNA then directs the action of the MutL alpha endonuclease to
one strand in the presence of a mismatch and MutS alpha or MutS beta. Any
mutational event that disrupts the superhelical structure of DNA carries with it the
potential to compromise the genetic stability of a cell. The fact that the damage
detection and repair systems are as complex as the replication machinery itself
highlights the importance evolution has attached to DNA fidelity.
Examples of mismatched bases include a G/T or A/C pairing. Mismatches
are commonly due to tautomerization of bases during G2. The damage is repaired
by recognition of the deformity caused by the mismatch, determining the template
and non-template strand, and excising the wrongly incorporated base and replacing
it with the correct nucleotide. The removal process involves more than just the
mismatched nucleotide itself. A few or up to thousands of base pairs of the newly
synthesized DNA strand can be removed.
DNA MisMatch Repair (MMR) is an evolutionarily conserved process
that corrects mismatches generated during DNA replication and escape
proofreading. MMR proteins also participate in many other DNA transactions,
such that inactivation of MMR can have wide-ranging biological consequences,
which can be either beneficial or detrimental. We begin this review by briefly
considering the multiple functions of MMR proteins and the consequences of
impaired function. We then focus on the biochemical mechanism of MMR replication
errors. Emphasis is on structure-function studies of MMR proteins, on how
mismatches are recognized, on the process by which the newly replicated strand
is identified, and on excision of the replication error.

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4.2.3 Nucleotide Excision Repair Repair Pathways

In both prokaryotes and eukaryotes, a major cellular mechanism for the removal
of DNA damage is nucleotide excision repair (excision repair), an enzymatic
pathway that recognizes and corrects a wide spectrum of structural anomalies NOTES
(DNA lesions) ranging from bulky, helix-distorting adducts to non-helix-distorting
lesions. The modifications that transform normal bases into damaged bases
corrected by nucleotide excision repair are so diverse that it is unlikely that a specific
chemical structure is recognized. Rather, it appears that any abnormal DNA
structure that destabilizes (denatures) the double helix is recognized as damage
both in Escherichia coli and human cells.
The primary function of nucleotide excision repair is removal of bulky adducts
generated by chemicals or UV radiation, while base excision repair is the major
pathway for correction of non-helix-distorting lesions, such as those introduced
by ionizing radiation or cellular metabolic events. Additional pathways exist for
direct reversal of certain types of damage (for example, photolyase and
methyltransferase), correction of mismatched bases, removal of interstrand
crosslinks, and repair of DNA strand breaks. Excision repair involves removal of
a damaged nucleotide by dual incisions bracketing the lesion, this is accomplished
by a multisubunit enzyme referred to as the excision nuclease or excinuclease (Refer
Figure 4.5).

Fig. 4.5 Nucleotide Excision Repair

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Repair Pathways The basic mechanism of excision repair involves:
Damage Recognition.
Subunit Assembly.
NOTES Dual Incisions that result in Excision of the Damage-Containing Oligomer.
Resynthesis to fill in the Gap.
Ligation to Regenerate an Intact Molecule.
In Prokaryotes: Uvr Proteins
A schematic representation of models for the nucleotide excision repair pathway
controlled by Uvr proteins.
The process of nucleotide excision repair is controlled in Escherichia coli by
the UvrABC endonuclease enzyme complex, which consists of four Uvr proteins
– UvrA, UvrB, UvrC, and DNA Helicase II (sometimes also known as UvrD in
this complex). First, UvrA-UvrB complex scans the DNA, with the UvrA subunit
recognizing distortions in the helix, caused for example by pyrimidine dimers. When
the complex recognizes such a distortion, the UvrA subunit leaves and an UvrC
protein comes in and binds to the UvrB monomer and, hence, forms a new UvrBC
dimer. UvrB cleaves a phosphodiester bond 4 nucleotides downstream of the DNA
damage, and the UvrC cleaves a phosphodiester bond 8 nucleotides upstream of
the DNA damage and created 12 nucleotide excised segment. DNA Helicase II
(sometimes called UvrD) then comes in and removes the excised segment by
actively breaking the hydrogen bonds between the complementary bases. The
resultant gap is then filled in using DNA Polymerase I and DNA Ligase. The basic
excision process is very similar in higher cells, but these cells usually involve many
more proteins – Escherichia coli is a simple example.
TC-NER, i.e., Transcription-Coupled Nucleotide Excision Repair also exists
in bacteria, and is mediated by the TRCF, i.e., Transcription Repair Coupling
Factor (Mfd, i.e., Mutation frequency decline) protein. TRCF is an SF2 ATPase that
uses ATP hydrolysis to translocate on dsDNA upstream of the transcription bubble
and forward translocate RNA polymerase, thus initiating dissociation of the RNA
polymerase ternary elongation complex. TRCF also recruits the UvrABC nucleotide
excision repair machinery by direct physical interaction with the UvrA subunit.
4.2.4 Recombinational Repair
In cases where DNA is severely damaged, a cell will engage in a phenomenon
called the SOS response in an effort to salvage a functioning set of genetic
information. This response, also called error prone repair, represents a last ditch
response to salvage a chromosomal information system. In addition, recombinational
repair systems act to allow one copy of the replicating DNA at a replication fork
to supply information to the other daughter chromosome. Recombinational repair
is a way of using one copy of the cell’s information to ensure that the overall
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 The biochemical process of recombination occurs by breaking and rejoining Repair Pathways

DNA strands. The key reaction is strand displacement initiated at a nick in the
chromosome. Then a protein called RecA (which stands for recombination; rec
bacteria are unable to recombine their DNA information and therefore are
abnormally sensitive to UV radiation) binds to a single stranded DNA fragment NOTES
and catalyzer its exchange with the same sequence of the duplex. RecA protein is
a strand displacement protein.
RecA preferentially binds to single stranded DNA in a cooperative fashion;
this cooperativity means that RecA will cover an entire single stranded DNA
molecule rather than bind to several molecules partially. RecA then
aligns homologous segments (those with complementary information) to form base
pairs. The key reaction of RecA coated DNA is the movement of the single stranded
regions of the DNA to form a joint molecule—a process called strand displacement.
This reaction involves ATP hydrolysis (Refer Figure 4.6).

Fig. 4.6 Homologous Recombination DNA Repair Pathway

In homologous recombination, two double helices align and are nicked.

Then RecA catalyzes the invasion of each double helix by one strand of the other.
This forms a crossed structure called a Holliday junction. If the Holliday structure
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Repair Pathways were simply broken at the point where it was formed, no genetic recombination
could occur because the two original DNA molecules would simply reform. Instead,
the junction migrates by displacement of one strand of DNA. Finally, the displaced
Holliday junction is broken and rejoined, or resolved. The exact type of
NOTES recombination between the two strands depends on which of the strands is broken
and rejoined.
Note: Each recombination event involves two breaking and rejoining events - one to initiate
strand displacement and one to resolve the Holliday junction.

4.2.5 SOS Inducible Repair

Chromosomal DNA is exposed to continuous damage and repair. Cells contain a
number of proteins and specific DNA repair systems that help maintain its correct
structure. The SOS response was the first DNA repair system described
in Escherichia coli induced upon treatment of bacteria with DNA damaging agents
arrest DNA replication and cell division. Induction of the SOS response involves
more than forty independent SOS genes, most of which encode proteins engaged
in protection, repair, replication, mutagenesis and metabolism of DNA. Under
normal growth conditions the SOS genes are expressed at a basal level, which
increases distinctly upon induction of the SOS response. The SOS-response has
been found in many bacterial species (for example, Salmonella
typhimurium, Caulobacter crescentus, Mycobacterium tuberculosis), but not
in Eukaryotic cells.
However, species from all kingdoms contain some SOS-like proteins taking
part in DNA repair that exhibit amino acid homology and enzymatic activities
related to those found in Escherichia coli, but are not organized in an SOS system.
4.2.6 Specific Repair for Oxidative DNA Damage
The integrity of DNA is constantly challenged by damaging agents and chemical
modifications. Base oxidation is a frequent insult that can arise from endogenous
metabolic processes as well as from exogenous sources such as ionizing radiation.
At background levels, a human cell is estimated to undergo 100 to 500 such
modifications per day, most commonly resulting in 8-oxo-7, 8-dihydro guanine
(8-oxoG) and related products, which are then processed into repair intermediates.
At steady state, up to 2400 8-oxoG sites per cell are reported. However, estimates
differ widely due to differences in methodology.
Oxidative damage is processed in a two-step process through the Base
Excision Repair (BER) pathway. The damaged base is first recognized and excised
by 8-OxoGuanine DNA Glycosylase 1 (OGG1), leaving an Apurinic Site (AP-
site). Glycohydrolysis is highly efficient, with an 8-oxoG half-life of 11 minutes.
AP-sites are removed through backbone incision by AP lyase (APEX 1), and end
processing through Flap-Endonuclease 1 (FEN1), and the base is subsequently
replaced with an undamaged nucleotide. Alternatively, in short-patch base excision
repair, replacement is dependent on polymerase beta. Other sources of AP-sites
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include spontaneous depurination and excision of non-oxidative base modifications, Repair Pathways

such as Uracil. Cells are reported to typically present with a steady state of
~ 15,000 to ~30,000 AP-sites per cell, which includes the associated beta-
elimination product. Left unrepaired, 8-oxoG can compromise transcription, DNA
replication, and telomere maintenance. Also, AP-sites can lead to genomic instability NOTES
and compromise genomic processes. Moreover, damaged sites provide direct
and indirect routes to C-to-A mutagenesis.
Ionizing radiation is one of the most relevant exogenous sources of high-
level oxidative DNA damage and DNA strand breaks. Each gray (Gy) is estimated
to lead to ~ 106 ionization events in the nucleus, only ~ 2000 of which are supposed
to target DNA directly. Most DNA damage from ionizing radiation occurs indirectly
from radiolysed water and 60–70% can be prevented through radical scavenging.
While absolute numbers differ throughout the literature, Lehnert estimates 1000–
2000 base modifications per gray, 250 alkali labile sites, 1000 Single-Strand Breaks
(SSB), and 40 double-strand breaks. Others report base modifications to be
threefold more prevalent than SSBs or even several orders of magnitude increased.
Interestingly, direct formation of AP-sites however has been shown not to increase
more than 5% from background levels. Therefore, after ionizing radiation, most
AP-sites likely arise from excision of oxidized bases, which comprise mostly of 8-
oxoG and the related modification FaPy-Guanine (G). Though originally
controversial, there is now broad acceptance that mutation rates vary across different
genomic regions. Background mutation rates in Escherichia coli were shown to
vary non-randomly between genes by an order of magnitude, with highly expressed
genes displaying lower mutation rates. In cancer genomes, Single Nucleotide
Variants (SNVs) tend to accumulate preferentially in heterochromatin. More
recently, it was reported that SNV densities in cancers are lower in regions
surrounding transcription factor binding but are elevated at the binding sites
themselves and at sites with high nucleosome occupancy. These variabilities likely
arise through a combination of regional differences in damage sensitivity and the
accessibility to the DNA repair machinery.
However, since mutations represent the endpoint of mutagenesis, it is
impossible to tease apart the contributions from damage and repair through
resequencing alone. The role of oxidative damage in regional differences of
mutagenesis remains largely unclear. Repair intermediates remain unexplored, but
the genome-wide distribution of 8-oxoG has been studied through chemical
enrichment and immunoprecipitation. The specificity of 8-oxoG antibodies,
however, remains questionable and the studies using chemical enrichment also
arrive at disparate conclusions. Both Wu et al., and Ding et al., find 8-oxoG
enriched at telomeres in yeast and mouse embryonic fibroblasts, respectively.
However, Wu et al., find 8-oxoG largely depleted at promoters, while Ding
et al. report increased damage at these sites. Therefore, we reassessed the raw
data and did not find evidence for increased 8-oxoG at promoters. Using antibodies,
however, peaks of 8-oxoG accumulation under conditions of hypoxia have been
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Repair Pathways reported in active promoters linked to specific transcription factors on a larger
scale, studies found accumulation in GC and CpG island rich, early replicating
DNA, but also in gene deserts and the nuclear periphery. Some of these apparently
contradicting conclusions may be explained through different levels of resolution,
NOTES experimental systems, and methodology. So far, ionizing radiation-induced
oxidative damage has not been addressed genome wide. In addition, base
modifications, which have been processed into the more persistent AP-sites remain
hidden from the previously used techniques.
4.2.7 Pyrimidine Dimers
Pyrimidine dimers are molecular lesions formed from Thymine or Cytosine bases
in DNA via photochemical reactions. Ultraviolet (UV) light induces the formation
of covalent linkages between consecutive bases along the nucleotide chain in the
vicinity of their Carbon–Carbon double bonds.
The dimerization reaction can also occur among pyrimidine bases in dsRNA
(double-stranded RNA)—Uracil (U) or Cytosine (C). Two common UV products
are Cyclobutane Pyrimidine Dimers (CPDs) and 6–4 photoproducts. These
premutagenic lesions alter the structure and possibly the base-pairing. Up to 50–
100 such reactions per second might occur in a skin cell during exposure to sunlight,
but are usually corrected within seconds by photolyase reactivation or nucleotide
excision repair. Uncorrected lesions can inhibit polymerases, cause misreading
during transcription or replication, or lead to arrest of replication. Pyrimidine dimers
are the primary cause of melanomas in humans (Refer Figure 4.7).

Fig. 4.7 Pyrimidine Dimers

4.2.8 Alkylation Induced Damage and Adaptive Response

Alkylation damage to DNA occurs when cells encounter alkylating agents in the
environment or when cellular metabolism produces active alkylators. To cope
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with DNA alkylation, cells have evolved genes that encode proteins with alkylation- Repair Pathways

specific DNA repair activities. In Escherichia coli, the main response specific for
alkylation damage has been called the adaptive response. The adaptive response
genes are induced upon exposure to exogenous alkylators by Ada-dependent
induction, and also during stationary phase by rpoS-dependent gene expression, NOTES
possibly to prevent accumulation of DNA damage due to increased endogenous
production of alkylating agents. Recent studies of the regulatory mechanisms of
Ada protein and the various responses of the individual promoters regulated by
this protein has revealed a complexity of regulation not initially recognized.

Check Your Progress

1. What is DNA damage?
2. Give the causes of DNA damage.
3. What is mismatch repair? Give its features.
4. List the steps involved in SOS repair system or recombinational repair.
5. What are the basic mechanism of excision repair?
6. Define pyrimidine dimers.
7. How does alkylation damage to DNA occur?

4.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. DNA damage is a change in the basic structure of DNA that is not itself
replicated when the DNA is replicated. A DNA damage can be a chemical
addition or disruption to a base of DNA (creating an abnormal nucleotide
or nucleotide fragment) or a break in one or both chains of the DNA strands.
2. DNA damage occurs due to:
Single Base Change: Such changes takes place due to conversion of
one base to other, for example deamination of 5-methylcytosine to
Thymine (T).
Structural Distortion: Such distortion take place due to single strand
nick, removal of a base or introduction of a covalent link between bases
of same or different strands, for example formation of Thymine (T) dimer
due to UV.
3. To cope up with the errors that remain after ‘Proof Reading’ another
mechanism called mismatch repair was proposed by Robin Holliday like
other DNA lesions. It has following features:
Detection of Mismatch or Alteration.
Removal of Incorrect Nucleotide.
Replacement with Correct Base.
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Repair Pathways 4. Following steps are involved in SOS repair system or recombinational repair:
DNA damage activates RecA proteins (a protease).
RecA interacts with LexA and causes it to undertake autocatalytic
NOTES cleavage. This inactivates the repression function of LexA and all the
operon bound to LexA are induced.
Since LexA represses its own synthesis its own synthesis and that of
RecA, its proteolytic degradation leads to amplification of both RecA
and LexA proteins.
When damage signal is removed, RecA loses induction and free LexA
protein quickly accumulates in the uncleaved forms and turn off SOS
genes.
5. The basic mechanism of excision repair involves:
Damage Recognition.
Subunit Assembly.
Dual Incisions that result in Excision of the Damage-Containing Oligomer.
Resynthesis to fill in the Gap.
Ligation to Regenerate an Intact Molecule.
6. Pyrimidine dimers are molecular lesions formed from Thymine or Cytosine
bases in DNA via photochemical reactions. UltraViolet (UV) light induces
the formation of covalent linkages between consecutive bases along
the nucleotide chain in the vicinity of their Carbon–Carbon double bonds.
7. Alkylation damage to DNA occurs when cells encounter alkylating agents
in the environment or when cellular metabolism produces active alkylators.
To cope with DNA alkylation, cells have evolved genes that encode proteins
with alkylation-specific DNA repair activities.

4.4 SUMMARY

DNA damage is a change in the basic structure of DNA that is not itself
replicated when the DNA is replicated.
A DNA damage can be a chemical addition or disruption to a base of DNA
(creating an abnormal nucleotide or nucleotide fragment) or a break in one
or both chains of the DNA strands.
When DNA carrying a damaged base is replicated, an incorrect base can
often be inserted opposite the site of the damaged base in the complementary
strand, and this can become a mutation in the next round of replication.
A double strand break can cause rearrangements of the chromosome
structure (possibly disrupting a gene, or causing a gene to come under
abnormal regulatory control), and, if such a change can be passed to
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Mutations, however, can be avoided if accurate DNA repair systems Repair Pathways

recognize DNA damages as abnormal structures, and repair the damages

prior to replication.
Mismatch of bases, due to errors in DNA replication, in which the wrong
NOTES
DNA base is stitched into place in a newly forming DNA strand, or a DNA
base is skipped over or mistakenly inserted.
Ionizing radiation such as that created by radioactive decay or in cosmic
rays causes breaks in DNA strands.
UV damage, alkylation/methylation, X-ray damage and oxidative damage
are examples of induced damage.
In human cells, and eukaryotic cells in general, DNA is found in two cellular
locations – inside the nucleus and inside the mitochondria. Nuclear DNA
(nDNA) exists as chromatin during non-replicative stages of the cell
cycle and is condensed into aggregate structures known
as chromosomes during cell division.
Mitochondrial DNA (mtDNA) is located inside mitochondria organelles,
exists in multiple copies, and is also tightly associated with a number of
proteins to form a complex known as the nucleoid.
Inside mitochondria, Reactive Oxygen Species (ROS), or free radicals,
byproducts of the constant production of Adenosine TriPhosphate (ATP)
via oxidative phosphorylation, create a highly oxidative environment that is
known to damage mtDNA.
A critical enzyme in counteracting the toxicity of these species is superoxide
dismutase, which is present in both the mitochondria and cytoplasm of
Eukaryotic cells.
Senescence, an irreversible process in which the cell no longer divides, is a
protective response to the shortening of the chromosome ends.
The telomeres are long regions of repetitive non-coding DNA that cap
chromosomes and undergo partial degradation each time a cell undergoes
division.
Senescence in cells may serve as a functional alternative to apoptosis in
cases where the physical presence of a cell for spatial reasons is required
by the organism, which serves as a ‘last resort’ mechanism to prevent a cell
with damaged DNA from replicating inappropriately in the absence of pro-
growth cellular signaling.
Unregulated cell division can lead to the formation of a tumor, which is
potentially lethal to an organism.
The mechanism of DNA repair in different species exhibit similarities as
well as differences.

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Repair Pathways The distorted or affected of strand caused by UV induced dimer is recognized
and enzymatically clipped out by endonuclease enzyme that cleaves
phosphodiester bond.
DNA Polymerase I fills this gap by inserting the ribonucleotides
NOTES
complementary to those on the intact strands.
DNA mismatch repair is a system for recognizing and repairing erroneous
insertion, deletion, and misincorporation of bases that can arise during DNA
replication and recombination, as well as repairing some forms of DNA
damage.
During DNA synthesis the newly synthesised (daughter) strand will commonly
include errors.
In Gram-Negative Bacteria, transient hemimethylation distinguishes the
strands (the parental is methylated and daughter is not).
DNA MisMatch Repair (MMR) is an evolutionarily conserved process
that corrects mismatches generated during DNA replication and escape
proofreading.
MMR proteins also participate in many other DNA transactions, such that
inactivation of MMR can have wide-ranging biological consequences, which
can be either beneficial or detrimental.
Emphasis is on structure-function studies of MMR proteins, on how
mismatches are recognized, on the process by which the newly replicated
strand is identified, and on excision of the replication error.
In both prokaryotes and eukaryotes, a major cellular mechanism for the
removal of DNA damage is nucleotide excision repair (excision repair), an
enzymatic pathway that recognizes and corrects a wide spectrum of structural
anomalies (DNA lesions) ranging from bulky, helix-distorting adducts to
non-helix-distorting lesions.
The modifications that transform normal bases into damaged bases corrected
by nucleotide excision repair are so diverse that it is unlikely that a specific
chemical structure is recognized.
The primary function of nucleotide excision repair is removal of bulky adducts
generated by chemicals or UV radiation, while base excision repair is the
major pathway for correction of non-helix-distorting lesions such as those
introduced by ionizing radiation or cellular metabolic events.
The process of nucleotide excision repair is controlled in Escherichia coli by
the UvrABC endonuclease enzyme complex, which consists of four Uvr
proteins: UvrA, UvrB, UvrC, and DNA Helicase II (sometimes also known
as UvrD in this complex).

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TC-NER, i.e., Transcription-Coupled Nucleotide Excision Repair also exists Repair Pathways

in bacteria, and is mediated by the TRCF, i.e., Transcription Repair Coupling

Factor (Mfd, i.e., Mutation frequency decline) protein.
TRCF also recruits the UvrABC nucleotide excision repair machinery by
NOTES
direct physical interaction with the UvrA subunit.
In cases where DNA is severely damaged, a cell will engage in a phenomenon
called the SOS response in an effort to salvage a functioning set of genetic
information.
The biochemical process of recombination occurs by breaking and rejoining
DNA strands.
RecA preferentially binds to single stranded DNA in a cooperative fashion;
this cooperativity means that RecA will cover an entire single stranded DNA
molecule rather than bind to several molecules partially.
Chromosomal DNA is exposed to continuous damage and repair. Cells
contain a number of proteins and specific DNA repair systems that help
maintain its correct structure.
The integrity of DNA is constantly challenged by damaging agents and
chemical modifications. Base oxidation is a frequent insult that can arise
from endogenous metabolic processes as well as from exogenous sources
such as ionizing radiation.
Ionizing radiation is one of the most relevant exogenous sources of high-
level oxidative DNA damage and DNA strand breaks.
Pyrimidine dimers are molecular lesions formed from Thymine or Cytosine
bases in DNA via photochemical reactions. Ultraviolet (UV) light induces
the formation of covalent linkages between consecutive bases along
the nucleotide chain in the vicinity of their Carbon–Carbon double bonds.
The dimerization reaction can also occur among pyrimidine bases in dsRNA
(double-stranded RNA)—Uracil (U) or Cytosine (C).
Uncorrected lesions can inhibit polymerases, cause misreading
during transcription or replication, or lead to arrest of replication.
Alkylation damage to DNA occurs when cells encounter alkylating agents
in the environment or when cellular metabolism produces active alkylators.
In Escherichia coli, the main response specific for alkylation damage has
been called the adaptive response.
The adaptive response genes are induced upon exposure to exogenous
alkylators by Ada-dependent induction, and also during stationary phase
by rpoS-dependent gene expression, possibly to prevent accumulation of
DNA damage due to increased endogenous production of alkylating agents.

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Repair Pathways
4.5 KEY WORDS

DNA damage: DNA damage is a change in the basic structure of DNA

NOTES that is not itself replicated when the DNA is replicated.
DNA mismatch repair: DNA mismatch repair is a system for recognizing
and repairing erroneous insertion, deletion, and misincorporation of bases
that can arise during DNA replication and recombination, as well as repairing
some forms of DNA damage.
Senescence: Senescence, is an irreversible process in which the cell no
longer divides, is a protective response to the shortening of the chromosome
ends.

4.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is senescence and apoptosis?
2. Explain the types of DNA damages with the help of diagram.
3. What is DNA repair system?
4. Define what excision repair is.
5. Draw a well-labelled diagram to show nucleotide excision repair.
6. Briefly explain Prokaryotes: Uvr proteins.
7. What are SOS inducible repair?
8. What are pyrimidine dimers? Explain with the help of example.
Long Answer Questions
1. Write a detailed note on DNA damage and repair pathways.
2. Draw a well-labelled diagram to show DNA damage.
3. What is photoreactive repair or direct repair? Explain.
4. Elaborate a note on SOS repair system.
5. Write in detail about methyl directed mismatch repair.
6. Explain nucleotide excision repair and recombinational repair.
7. Discuss in detail about specific repair for oxidative DNA damage.
8. What are pyrimidine dimers? Explain briefly.
9. What are alkylation induced damage and adaptive response?

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 Repair Pathways
4.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing NOTES
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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Recombination
and its Types BLOCK - II
BACTERIAL RECOMBINATION

NOTES
UNIT 5 RECOMBINATION
AND ITS TYPES
Structure
5.0 Introduction
5.1 Objectives
5.2 Recombination and its Types
5.2.1 Types of Recombination
5.3 Homologous Recombination
5.3.1 Models for Homologous Recombination
5.3.2 Molecular Mechanism of Homologous Recombination
5.3.3 Enzymes of Homologous Recombination
5.3.4 Role of Reca Protein in Homologous Genetic Recombination
5.3.5 Homologous Recombination in Prokaryotes and Eukaryotes
5.3.6 In Vitro Homologous Recombination
5.4 Site-Specific Recombination
5.4.1 Types of Site-Specific Recombination
5.4.2 Molecular Mechanism of Site-Specific Recombination
5.4.3 Biological Roles of Site-Specific Recombination
5.5 Answers to Check Your Progress Questions
5.6 Summary
5.7 Key Words
5.8 Self Assessment Questions and Exercises
5.9 Further Readings

5.0 INTRODUCTION

Recombination, in genetics, regrouping of the maternal and paternal genes during

the formation of gametes, the sex cells. Recombination occurs randomly in nature
as a normal event of meiosis, the process by which gametes are produced.
Recombination is enhanced by the phenomenon of crossing over, in which
gene sequences called linkage groups are disrupted, resulting in an exchange of
segments between paired chromosomes that are undergoing separation. Thus,
although a normal daughter cell produced in meiosis always receives half of the
genetic material contained in the parent cell, recombination acts to ensure constant
variability: no two daughter cells are identical, nor are any identical in genetic
content to the parent cell.
In eukaryotic cells, which are cells with a nucleus and organelles,
recombination typically occurs during meiosis. Meiosis is a form of cell division
that produces gametes, or egg and sperm cells. During the first phase of meiosis,
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the homologous pairs of maternal and paternal chromosomes align. During the Recombination
and its Types
alignment, the arms of the chromosomes can overlap and temporarily fuse, causing
a crossover. Crossovers result in recombination and the exchange of genetic material
between the maternal and paternal chromosomes. As a result, offspring can have
different combinations of genes than their parents. Genes that are located farther NOTES
apart on the same chromosome have a greater likelihood of undergoing
recombination, which means they have a greater recombination frequency.
In this unit, you will study about history and introduction of recombination,
types of recombination, mechanism and effects of homologous recombination and
mechanism and biological role of site-specific recombination in detail.

5.1 OBJECTIVES

After going through this unit, you will be able to:

Understand the history and introduction of recombination
Explain the types of recombination
Discuss the mechanism and effects of homologous recombination
Explain the mechanism and biological role of site-specific recombination

5.2 RECOMBINATION AND ITS TYPES

The genetic recombination involves the exchange or sharing of genetic information

between multiple parental sequences to create new ‘Progeny’ genes. A key benefit
of using recombination to generate genetic diversity is the fact that the parental
sequence fragments being combined correspond to pieces of related and functional
proteins (the homologues provide ‘functional diversity’). Compared to random
mutagenesis, where increasing the number of mutations drastically reduces the
probability of obtaining a folded or functional protein, recombination allows for a
higher probability of a greater number of amino acid changes (relative to a given
parent sequence) to retain the protein’s ability to fold and carry traits from both
parents, as well as potentially new properties. The in vitro recombination for the
sake of creating gene libraries is classified as either homologous, when the parent
genes share a high level of sequence similarity, or non-homologous, when the
parents are not homologues or share low sequence similarity.
The Genetic recombination (also known as genetic reshuffling) is the
exchange of genetic material between different organisms which leads to production
of offspring with combinations of traits that differ from those found in either parent.
In eukaryotes, genetic recombination during meiosis can lead to a novel set of
genetic information that can be passed on from the parents to the offspring. Most
recombination is naturally occurring. During meiosis in eukaryotes, genetic
recombination involves the pairing of homologous chromosomes. This may be
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Recombination followed by information transfer between the chromosomes. The information
and its Types
transfer may occur without physical exchange (a section of genetic material is
copied from one chromosome to another, without the donating chromosome being
changed); or by the breaking and rejoining of DNA strands, which forms new
NOTES molecules of DNA. The recombination may also occur during mitosis in eukaryotes
where it ordinarily involves the two sister chromosomes formed after chromosomal
replication. In this case, new combinations of alleles are not produced since the
sister chromosomes are usually identical. In meiosis and mitosis, recombination
occurs between similar molecules of DNA.
In meiosis, non-sister homologous chromosomes pair with each other so
that recombination characteristically occurs between non-sister homologues. In
both meiotic and mitotic cells, recombination between homologous chromosomes
is a common mechanism used in DNA repair. The recombination can be artificially
induced in laboratory (in vitro) settings, producing rDNA (recombinant DNA) or
purposes including vaccine development. The V(D)J recombination in organisms
with an adaptive immune system is a type of site-specific genetic recombination
that helps immune cells rapidly diversify to recognize and adapt to new pathogens.
The V(D)J recombination occurs in the primary lymphoid organs and in a nearly
random fashion rearranges Variable (V), Joining (J), and in some cases, Diversity
(D) gene segments.
The genetic recombination is catalyzed by many different enzymes, for
example, recombinase (catalyze the strand transfer step during recombination found
in Escherichia coli), RAD51 and DMC1 required for mitotic and meiotic
recombination found in yeast. The non-homologous recombination can occur
between DNA sequences that contain no sequence homology. This can cause
chromosomal translocations, sometimes leading to cancer. In genetic engineering
recombination can also refer to artificial and deliberate recombination of disparate
pieces of DNA, often from different organisms, creating what is called rDNA. A
prime example of such a use of genetic recombination is gene targeting, which can
be used to add, delete or otherwise change an organism’s genes. This technique is
important to biomedical researchers as it allows them to study the effects of specific
genes. Techniques based on genetic recombination are also applied in protein
engineering to develop new proteins of biological interest. The achiasmy is the
phenomenon where autosomal recombination is completely absent in one sex of a
species. Achiasmatic chromosomal segregation is well documented in male fruit
fly. The heterochiasmy occurs when recombination rates differ between the sexes
of a species. This sexual dimorphic pattern in recombination rate has been observed
in many species. In mammals, females most often have higher rates of
recombination. The ‘Haldane-Huxley Rule’ states that achiasmy usually occurs in
the heterogametic sex. Thus the DNA recombination involves the exchange of
genetic material either between multiple chromosomes or between different regions
of the same chromosome. This process is generally mediated by homology; that
is, homologous regions of chromosomes line up in preparation for exchange, and
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some degree of sequence identity is required. Various cases of non-homologous Recombination
and its Types
recombination do exist, however (Refer Figure 5.1).

NOTES

Fig. 5.1 Model of Meiotic Recombination Started with dsDNA Breaks

followed by Pairing with Homologous Chromosomes

The role of recombination during the inheritance of chromosomes was first

demonstrated through experiments with maize. Specifically, in 1931, Barbara
McClintock and Harriet Creighton obtained evidence for recombination by
physically tracking an unusual knob structure within certain maize chromosomes
through multiple genetic crosses. Using a strain of maize in which one member of
a chromosome pair exhibited the knob but its homologue did not, the scientists
were able to show that some alleles were physically linked to the knobbed
chromosome, while other alleles were tied to the normal chromosome. McClintock
and Creighton then followed these alleles through meiosis, showing that alleles for
specific phenotypic traits were physically exchanged between chromosomes (Refer
Figure 5.2). Evidence for this finding came from the fact that alleles first introduced
into the cross on a knobbed chromosome later appeared in offspring without the
knob; similarly, alleles initially introduced on a knob less chromosome subsequently
appeared in progeny with the knob. The recombination also occurs in Prokaryotic
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Recombination Cells, and it has been especially well characterized in Escherichia coli. Although
and its Types
bacteria do not undergo meiosis, they do engage in a type of sexual reproduction
called conjugation, during which genetic material is transferred from one bacterium
to another and may be recombined in the recipient cell. As in Eukaryotes,
NOTES recombination also plays important roles in DNA repair and replication in
Prokaryotic organisms.

Fig. 5.2 McClintock and Creighton’s Model Showing Physical

Evidences of Recombination

Recombination
Genetic recombination occurs when genetic material is exchanged between two
different chromosomes or between different regions within the same chromosome.
We can observe it in both Eukaryotes (like animals and plants) and Prokaryotes
(like archaea and bacteria). Keep in mind that in most cases, in order for an
exchange to occur, the sequences containing the swapped regions have to be
homologous, or similar, to some degree. The process occurs naturally and can
also be carried out in the lab. Recombination increases the genetic diversity in
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sexually reproducing organisms and can allow an organism to function in new ways. Recombination
and its Types
The genetic recombination occurs naturally in meiosis. Meiosis is the process of
cell division that occurs in eukaryotes, such as humans and other mammals, to
produce offspring. In this case, it involves crossing over. What happens is that two
chromosomes, one from each parent, pair up with each other. Next, a segment NOTES
from one crosses over, or overlaps, a segment of the other. This allows for the
swapping of some of their material, as you can see in the illustration below. What
we end up with is a new combination of genes that did not exist before and is not
identical to either parent’s genetic information. Note that recombination is also
observed in mitosis, but it does not occur as often in mitosis as it does in meiosis
(Refer Figure 5.3).

Fig. 5.3 Generalized Recombination between Non-Sister

Chromatids of Homologous Chromosomes

The cell can undergo recombinational repair also, for example, if it notices
that there is a harmful break in the DNA which is a kind of break that occurs in
both strands. What we observe is an exchange between the broken DNA and a
homologous region of DNA that will fill the gaps. There are also other ways that
recombination is used to repair DNA. Thus genetic recombination refers to the
rearrangement of DNA sequences by some combination of the breakage, rejoining,
and copying of chromosomes or chromosome segments. It also describes the
consequences of such rearrangements, i.e., the inheritance of novel combinations
of alleles in the offspring that carry recombinant chromosomes. Genetic
recombination is a programmed feature of meiosis in most sexual organisms, where
it ensures the proper segregation of chromosomes. Because the frequency of
recombination is approximately proportional to the physical distance between
markers, it provides the basis for genetic mapping. Recombination also serves as
a mechanism to repair some types of potentially lethal damage to chromosomes.
Genetic recombination is often used as a general term that includes many
types of DNA rearrangements and underlying molecular processes. Recombination
in meiosis is reciprocal, because each participating chromosome receives
information comparable to what it donates to the other partner, since all the
information on both sides of the effective break has been exchanged.
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Recombination 5.2.1 Types of Recombination
and its Types
Scientists have observed the following types of recombination in nature:
Homologous (General) Recombination: As the name implies, this type
NOTES occurs between DNA molecules of similar sequences. Our cells carry out
general recombination during meiosis.
Non-Homologous (Illegitimate) Recombination: Again, the name is self-
explanatory. This type occurs between DNA molecules that are not
necessarily similar. Often, there will be a degree of similarity between the
sequences, but it is not as obvious as it would be in homologous
recombination.
Techniques have been devised for the artificial transfer of DNA fragments
from any source into cells of many different species, thus conferring new
properties upon them. In bacteria and the yeast (Saccharomyces cerevisiae
or S. cerevisiae), integration of such DNA into the genome (on which the
stability of transformation generally depends) requires substantial sequence
similarity between incoming DNA and the recipient site. However, cells of
other fungi, higher plants, and animals are able to integrate foreign DNA
into their chromosomes with little or no sequence similarity. These organisms
appear to have some unidentified system that recombines the free ends of
DNA fragments into chromosomes regardless of their sequences. It may
have something in common with the mechanism, equally obscure, whereby
broken ends of chromosomes can heal by nonspecific mutual joining.
Site-Specific Recombination: This is observed between particular, very
short, sequences, usually containing similarities. Bacteriophages, plasmids,
bacteria, and unicellular eukaryotes provide many examples of differentiation
through controlled and site-specific recombination of DNA segments.
Invertebrates, a controlled series of deletions leads to the generation of the
great diversity of gene sequences encoding the antibodies and T-cell receptors
necessary for immune defense against pathogens. All these processes depend
upon interaction and recombination between specific DNA sequences,
generally but not always with some sequence similarity, catalyzed by site-
specific recombinase enzymes. The molecular mechanisms may have some
similarities with those responsible for general meiotic recombination, except
that the latter does not depend on any specific sequence, only on similarity
(homology) of the sequence recombined.
Mitotic Recombination: This does not actually happen during mitosis,
but occurs during inter-phase. The process is similar to that in meiotic
recombination, and has its possible advantages. But it is usually harmful and
can result in tumors. This type of recombination is increased when cells are
exposed to radiation. Crossing-over between homologous chromosome
pairs can also occur during the prophase of mitotic nuclear division. The
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 frequency is very much lower than in meiosis, presumably because the mitotic Recombination
and its Types
cell does not form the synaptic apparatus for efficient pairing of homologs.
Mitotic crossing-over has been studied in the fruit fly Drosophila
melanogaster, in the filamentous fungus Aspergillus nidulans, and
in Saccharomyces yeast. In these species it is detected through the formation NOTES
of homozygous clones of cells in an initially heterozygous diploid. There is a
50% chance of homozygosity in daughter cells whenever a cross-over occurs
between chromatids in the interval between the marker and the centromere,
in the chromosomal site attachment to the mitotic spindle. The frequency of
mitotic crossing-over is greatly increased by radiation.
Conservative Recombination: In conservative recombination events, the
number of copies of the interacting chromosomes or DNA sequences is
maintained throughout the process.
Non-Conservative Recombination: In non-conservative events, two
original copies are reduced to one in the product. This distinction of
conservative and non-conservative events can be made for both homologous
and non-homologous recombination.
Ectopic Recombination: Essentially analogous events may take place
between homologous sequences that are present at different locations on
non-homologous chromosomes is often called ectopic recombination.
The prokaryotic cells can undergo recombination through one of these three
processes:
Conjugation: Conjugation is where genes are donated from one organism
to another after they have been in contact. At any point, the contact is lost
and the genes that were donated to the recipient replace their equivalents in
its chromosome. What the offspring ends up having is a mix of traits from
different strains of bacteria.
Transformation: This is where the organism acquires new genes by taking
up naked DNA from its surroundings. The source of the free DNA is another
bacterium that has died, and therefore, its DNA was released to the
environment.
Transduction: Transduction is gene transfer that is mediated by viruses.
Viruses called bacteriophages attack bacteria and carry the genes from
one bacterium to another.

Check Your Progress

1. Define the term genetic recombination.
2. What does genetic recombination involves?
3. When does genetic recombination occur?

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Recombination
and its Types 5.3 HOMOLOGOUS RECOMBINATION

Homologous recombination can occur between homologous chromosomes or sister

NOTES chromatids in meiotic cell and mitotic cells as well. The homologous recombination
is also called general recombination. Smith (1989) reviewed the homologous
recombination in prokaryotes. General recombination in Escherichia coli is guided
by base pairing interactions between the complementary strands of two homologous
DNA molecules. Double helix of two DNA molecules breaks and the two broken
ends join to their opposite partners to reunite to form double helix. The site of
exchange can occur anywhere in the homologous nucleotide sequence where a
strand of one DNA molecule becomes base paired to the second strand to yield
heteroduplex just between two double helices.
In the heteroduplex no nucleotide sequences are changed at the site of
exchange due to cleavage and rejoining events. However, heteroduplex joints can
have a small number of mismatched base pairs. In bacteria and viruses general
recombination is carried out by the products of Rec genes, such as RecA Protein.
The RecA Protein is very important for DNA repair, therefore, it is RecA dependent
recombination (Refer Figure 5.4).

Fig. 5.4 Formation of Heteroduplex Joint during Homologous Recombination

5.3.1 Models for Homologous Recombination

I. Holliday Model for Homologous Recombination

Holliday (1974) presented a model to show the homologous or general
recombination. According to this model recombination occurs in seven steps, such
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as strand breakage, strand pairing, strand invasion/assimilation, branch pairing, Recombination
and its Types
chiasma (crossing-over) formation, breakage and reunion, and mismatch repair
(Refer Figure 5.5).
Figure 5.5 (A) illustrates the Holliday model for homologous or general
NOTES
recombination while the Figure 5.5 (B) illustrates the Holliday model for reciprocal
general recombination.

Fig. 5.5 (A) Holliday Model for Homologous or General Recombination

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Recombination
and its Types

NOTES

Fig. 5.5 (B) Holliday Model for Reciprocal General Recombination

1. Strand Breakage: General recombination occurs through crossing over

by pairing between the complementary single strands of DNA duplex (A).
Two homologous regions of DNA double helix undergo an exchange
reaction. The homologous region contains a long sequence of complementary
base pairing between a strand from one or two original double helices and
a complementary strand from the other. However, it is unknown how the
homologous region of DNA recognizes each other. The RecBCD proteins
of RecBCD or RecJ genes are required for recombination in Escherichia
coli. This protein enters the DNA from one end of double helix, and travels
along the DNA at double helix, at the rate of about 300 nucleotides per
second. It creates a loop of ssDNA along travelling DNA (B). It uses energy
derived from hydrolysis of ATP molecules. A special recognition site (a)
sequence of eight nucleotides scattered throughout Escherichia coli
chromosome (b) is nicked in the travelling loop of DNA formed by RecBCD
protein.
2. Strand Pairing: The RecBCD proteins act as DNA helicase because these
hydrolyze ATP and travel along DNA helix. Thus, the RecBCD proteins
result in formation of single stranded whisker at the recognition site which is
displaced from the helix (c). This initiates a base pairing interaction between
the two complementary sequences of DNA double helix.
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3. Strand Invasion/Assimilation: A single strand (whisker) generated from Recombination
and its Types
one DNA double helix invades the other double helix (d). In Escherichia
coli, RecA gene produces RecA protein which is important for recombination
between the chromosomes like Single Strand Binding (SSB) Proteins, The
RecA protein binds firmly to single stranded DNA to form a nucleoprotein NOTES
filament. Roca and Cox (1990) have reviewed the structure and function of
RecA protein. RecA protein promotes rapid renaturation of complementary
ssDNA hydrolyzing ATP in the process. RecA protein has several binding
sites, therefore, it can bind to ssDNA and subsequently a dsDNA. Basically,
the RecA protein binds first to ssDNA, then search for homology between
the donor strand and the recipient molecule.
Due to the presence of these sites RecA protein catalyses a multistep reaction
(called synapsis) between the homologous region of ssDNA and a DNA
double helix. Escherichia coli SSB protein helps the Rec protein to carry
out these reactions. When a region of homology is identified by an initial
base pairing between the complementary sequences, the crucial step in
synapsis occurs.
The in vivo experiments have shown that several types of complexes are
formed between ssDNA covered with RecA protein and a dsDNA helix.
First a non-base paired complex is formed which is converted into a three
stranded structure (ssDNA, dsDNA and RecA Protein) when a homologous
region is found. This complex is unstable and spins out a DNA heteroduplex
plus a displaced ssDNA from the original helix. Once the homologous
regions are encountered and the ssDNA and dsDNA are complexed, a
stable D-Loop is formed (d).
4. Branch Migration: The next step is the assimilation of strand and nick
ligation (e). The donor strand gradually displaces the recipient strand which
is called branch migration. After formation of synapsis, the heteroduplex
region is enlarged through protein-directed branch migration catalysed by
RecA protein. RecA protein directed branch migration proceeds at a uniform
rate in one direction due to addition of more RecA protein to one end of
RecA protein filament on the ssDNA. Branch migration can take place at
any point where two single strands with the sequence make attempts to
pair with the same complementary strand. An unpaired region of other single
strand results in movement of branch point without changing total number
of DNA base pairs. Special DNA helicases that catalyze protein directed
branch migration are involved in recombination. In contrast, the spontaneous
branch migration proceeds in both the directions almost at the same rate.
Therefore, it makes a little progress over a long distance.
5. Chiasma or Crossing Over Formation: Exchange of a single strand
between two double helices is a different step in a general recombination
event. After the initial cross strand exchange, further strand exchanges
between the two closely opposed helices is thought to proceed rapidly. A Self-Instructional
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Recombination nuclease cleaves and partly degrades the D-Loop at some points. At this
and its Types
stage possibly different organisms follow different pathways.
However, in most of the cases an important structure called cross-strand
exchange (also called Holliday Juncture or chi form or chiasmas, is formed
NOTES
by the two participating DNA helices (g). A chi form of single stranded
connections in the cross over region has also been observed under the
electron microscope by Dressier and Potter (1982). The chi form of two
homologous helices that initially paired and held together by mutual exchange
of two of the four strands where one strand originates from each of the
helices (g). The chi form has two important properties, i.e., as follows:
The point of exchange can migrate rapidly back and forth along the
helices by a double branch migration.
It contains two pairs of strands, one pair of crossing strands and the
other pair of non-crossing strands.
6. Breakage and Reunion: The chi structure can isomerizes several rotations
(h). This results in alteration of two original non-crossing strands into the
crossing strands, and the crossing strands into the non-crossing strands. In
order to regenerate two separate DNA helices, breakage and reunion in
two crossing strands are required. If breakage and reunion occur before
isomerization the two crossing strands would not occur. Therefore,
isomerization is required for the breakage and reunion of two homologous
DNA double helices resulting from general genetic recombination.
Breakage and reunion occur either in the vertical or horizontal plane. If
breakage occurs horizontally the recombinants would contain genotype AB/
ab with a little change in base sequences at the inner region (i). However, if
breakage occurs vertically the recombinants would contain Ab/aB (J). The
RurC protein and RecG protein expressed from RuvC and RecG genes,
respectively, are thought to be alternative endonucleases specific for Holliday
structure.
7. Mismatch Repair (Mismatch Proof Read­ing System): It is such a
repair system which corrects mismatched base pairs of unpaired regions
after recombination. This system recognizes mis­matched function of DNA
polymerase. The mecha­nism involves the excision of one of the other
mismatched bases along with about 3,000 nucleotides. This RecFJO is
involved in the repair of short mismatch either in the initial stage or at the
end of recombination. The two proteins MutS and MutL are present in
Bacteria and Eukaryotes. The MutS protein binds to mismatched base pair,
whereas MutL scan the DNA for a nick.
When a nick is formed MutL triggers the degradation of the nicked strand all the
way back through the mismatch, because the nicks are largely confined to the
newly replicated strands in eukaryotes, replication errors are selectively removed.
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In bacteria the mechanism is the same except that an additional protein MutH Recombination
and its Types
nicks the un-methylated GATC sequences and begins the process (Refer
Figure 5.6).

NOTES

Fig. 5.6 Mechanism of Removal of Error in Newly Made Strand

by Mismatch Repair System

It has been demonstrated in yeast and bacteria that the same mismatch repair
system which removes replication errors also interrupts the genetic recombination
events between imperfectly matched DNA sequences. It is known that homologous
genes in two closely related bacteria (Escherichia coli and Salmonella
typhimurium or S. typhimurium) generally will not recombine, even after having
80% identical nucleotide sequences. However, when mismatch repair system is
inactivated by mutation, the frequency of such interspecies recombination increases
by 100-folds. This mechanism protects the bacterial genome from sequence changes
that would be caused by recombination with foreign DNA molecules entering in
the cell.
II. Two Holliday Junctions for Homologous Recombination
The double stranded breaks in both strands of one DNA molecules occur quite
frequently. During DNA repair of double stranded breaks, homologous
recombination’s occurs. This type of genetic recombination is called double stranded
break repair mechanism. These types of breaks may be caused by ionizing
radiations and various damaging agents. Degeneration of broken strands and
generation of single strand tails (ss tails) with 3 ends (Refer Figure 5.7).
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Recombination
and its Types

NOTES

Fig. 5.7 Double Strand Break Repair Model with two Holliday Junctions

Enzymes RecBCD further degrades the broken DNA strands. It generates single
stranded tails with 32 ends. One 32 end tail invades the other unbroken homologous
DNA molecule, displacing one of the strands. The invading 32 end serves as a
primer for new DNA synthesis. The displaced strand serves as a template to fill
the gap left in the first DNA. If different alleles are present at the site of break, they
are permanently lost as regeneration involves homologous DNA as template which
has different alleles. This process is called gene conversion because genes of the
broken strand are replaced by genes of the homologous DNA. In double strand
break repair, two Holliday junctions are created. These Holliday junctions move
laterally by branch migration and the cleavage resolves and separates the two
DNA molecules forming a crossover and a non-crossover structure. This is similar
to the resolution in the single Holliday junction.
5.3.2 Molecular Mechanism of Homologous Recombination
The most important features of organisms are to adapt in the environment and to
maintain their DNA sequence in the cells generation to generations with very little
alterations. In long term survival of organisms depends on genetic variations, a
key feature through which the organism can adapt to an environment which changes
with time. This variability among the organisms occurs through the ability of DNA
to undergo genetic rearrangements resulting in a little change in gene combination.
Rearrangement of DNA occurs through genetic recombination.
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 Thus, recombination is the process of formation of new recombinant Recombination
and its Types
chromosome by combining the genetic material from two organisms. The new
recombinants show changes in phenotypic characters. Most of the eukaryotes
show a complete sexual life cycle including meiosis, an important event that
generates new allelic combinations by recombination. It is made possible through NOTES
chromosomal exchange resulting from crossing over between the two homologous
chromosomes containing identical gene sequences. Much work was done on
eukaryotic genetics until 1945 that laid the foundation of classical genetics. The
work on bacterial genetics was done between 1945 and 1965 that advanced the
understanding of microbial genetics at molecular level. Basically, there are three
theories, viz., breakage and reunion, breakage and copying and complete copy
choice that explain the mechanism of recombination (Refer Figure 5.8).

Fig. 5.8 Schematic Presentation of the Three Possible Mechanisms of Recombination

Breakage and Reunion: Two homologous duplex of chromosome laying

in paired form breaks between the gene loci ‘a’ and ‘b’, and ‘a+’ and ‘b+’ (A).
The broken segments rejoin crosswise and yield recombinants containing
‘a’ and ‘b+’ segment, and ‘a+’ and ‘b’ segment. This type of recombination
does not require the synthesis of new DNA. This concept has been used to
explain genetic recombination.
Breakage and Copying: One helix of paired homologous chromosome,
‘ab’ and ‘a+ b+’ breaks between ‘a’ and ‘b’ (B). Segment ‘b’ is replaced by
a newly synthesized segment copied from ‘b+’ and attached to ‘a’ section.
Thus the recombinants contain and ‘ab+’ and ‘a+ b+’.
Complete Copy Choice: In, 1931, Belling proposed this theory for
recombination of chromosome in higher animals. However, it has been
questioned by several workers. Therefore, it has only historical importance.
According to this theory a portion of one parental strand of homologous
chromosome acts as template for the synthesis of a copy of its DNA molecule.
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Recombination The process of copying shifts to the other parental strand. Thus, the
and its Types
recombinants contain some genetic information of one parental strand and
some of the other strand.

NOTES 5.3.3 Enzymes of Homologous Recombination

There are various proteins that catalyze various steps in the process of homologous
recombination in Escherichia coli. Enzymes RecBCD load onto one end of DNA
of double stranded break and move along DNA (Rec-recombination). In the
process it unwinds DNA (helical activity) and degrades one or both DNA strands
(nuclease activity). RecBCD is an endonuclease enzyme. It is encoded by three
genes, RecB, RecC and RecD. RecBCD continues its degrading activity until it
reaches a chi site (%). At this point activities of RecBCD are stopped. The chi site
has eight nucleotides (52 GCTGGTGG32 ) which promote recombination.
The single strand DNA tails generated by BCD enzymes are coated by
RecA enzyme. RecA stimulates pairing or synopsis between two homologous DNA
molecules RecA also promotes strand invasion, displacing one strand of unbroken
DNA molecule and forming D-loop. The displaced strand invades the broken
DNA molecule. The missing portions of DNA strands are synthesized using
homologous strand as template and gaps are sealed by ligase enzyme.
RuvAB enzymes recognize and bind to Holliday junction and perform branch
migration. RuvC enzyme cuts DNA strands at Holliday junction and causes
separation and resolution of Holliday junction. Different biological processes like
replication, recombination and repair occur in a co­ordinated manner. In this way
new DNA can be synthesized, damaged DNA repaired and genetic recombination
takes place. Nucleotide sequences can be replaced through heteroduplexes and
gene conversion.
5.3.4 Role of RecA Protein in Homologous Genetic Recombination
In the various homologous genetic recombination models, the central features are
similar in all recombination models. These include:
Breaks or nicks in DNA molecules.
Alignment or pairing or synapses of homologous sequences of two different
DNA molecules.
Formation of a crossover structure or Holliday junction in which DNA strand
from each molecule creates short regions of heteroduplux DNA.
Extension of heteroduplex DNA, which is called branched migration.
Lastly, resolution of crossover junction to yield end products.
This is an extremely complex process involving the action of several different
enzymes. The first event of creating breaks or nicks in DNA strands and the last
event of resolution are undertaken by various enzymes like helicase, nuclease, and

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ligases. But the event starting from pairing of DNA molecules, formation of Holliday Recombination
and its Types
junction branch migration are the central features in recombination process. These
events are undertaken by a special protein called RecA protein.
RecA protein is involved in pairing, exchange of strands and branch migration.
NOTES
It is also known as strand exchange protein.
RecA protein plays a major role in homologous recombination. It is a special
protein a completely distinct class of enzymes.
RecA protein binds quickly to single stranded DNA along the phosphate
backbone of DNA helix.
DNA is completely covered by RecA protein. Alongside RecA, a second
protein called Single Strand Binding Protein (SSBP) is also involved.
Each RecA molecule has 352 amino acids. There is one RecA monomer every
3-4 nucleotides, of DNA. The ssDNA in duplex is aligned with homologous
sequence of the other DNA molecule. Several steps occur in this process. Two
types of homologous interactions occur. The first is the formation of paranemic
joints in aligned homologous strands. The end second interaction involves formation
of plactonemic joints. RecA protein is a DNA dependent ATPase. ATP hydrolysis
is required for branch migration, in which strands are replaced and strand exchange
occurs. It exhibits polarity as branch migration proceeds in 52 to 32 direction
only.
5.3.5 Homologous Recombination in Prokaryotes and Eukaryotes
Homologous recombination in Prokaryotes and Eukaryotes is described below.
Homologous Recombination in Prokaryotes
Mechanics of molecular level of exchange is studied in detail in bacteria and phages.
At present we will restrict our discussion to the recombination mechanism where
DNA strands recognize each other by complementary strands bounded by base
pairs. In bacteria, genetic recombination occurs during conjugation, transformation,
transduction post-replication repair, during repair of double strand breaks in DNA,
integration of phage DNA with chromosomal DNA and transposons etc.
Homologous recombination can lead to gene conversion. Bacteria are haploid,
therefore do not undergo meiosis. They possess only one double stranded DNA
molecule or chromosome. There are several types of genetic recombination in
microorganisms. The most common recombination is the reciprocal exchange
between homologous DNA sequences. During genetic recombination usually only
a part of the genetic material of a donor cell is transferred to a recipient cell. The
DNA of the recipient cell and the donor pair with each other and reciprocally
exchange DNA strands by crossing over. This gives rise to a new genetic constitution
of the recipient cell with new characters. Subsequent daughter cells that contain
only recombined chromosome.

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Recombination The exchange of DNA strand is an important event in post-replication repair
and its Types
and very prominent in Escherichia coli. The strand exchange plays a very
prominent, role in repair of DNA damage. As the advancing replication fork comes
across a lesion or damaged site such as thymine dimers, it is bypassed during
NOTES replication process. The damaged protein may be cleaved which may prove to be
lethal. Repair of this lesion requires conversion of this DNA into double stranded
DNA and this is achieved by RecA protein. RecA protein plays its role in retrieving
a portion of the complementary strand from other side of the replication fork to fill
the gap. This involves branch migration by RecA protein. This proves that branch
migration is essential activity of the cell.
Homologous Recombination in Eukaryotes
Recombination of DNA takes place by mutation, exchange of DNA strands and
incorporation of DNA. In this process the genetic information is rearranged between
chromosomes that possess similar sequences. Homologous genetic recombination
occurs in eukaryotes at the time of gamete formation during long Prophase I of
Meiosis.
Each chromosome has two sister chromatids, each of which contains a
duplex DNA. The homologous chromosomes (one maternal and the other paternal)
pair with each other, pairing is known as synapsis and involves entire length of
homologous chromosomes. The recombination occurs by crossing-over. It involves
reciprocal exchange of chromosomal segments between non-sister chromatids of
a homologous pair involving breakage and subsequent reunion in a new
arrangement. Chiasma is formed at the site of crossing over. Enzymes like helicases,
endonucleases and ligases are involved.
The genetic recombination causes re-arrangement of genes producing
altogether new genotypes and phenotypes. These cause variations which lead to
evolution. In humans about 30 homologous recombination events occurs during
meiosis. The recombination events are much more in bacteria and even more in
fungi. The studies of Meiosis in Lily plants by Herbert Stern and Yasuo Hotta have
provided clinching evidence of recombination. Meiocytes of Lily flower buds divide
synchronously. It has also been discovered that Endonuclease, DNA Polymerase,
Ligase and other repair enzymes are present in Early Prophase.
5.3.6 In Vitro Homologous Recombination
The DNA shuffling is the first reported method of in vitro recombination. Here,
DNAse I is first used to randomly fragment the parental genes. Then, provided
there is sufficient sequence similarity between overlapping regions of DNA fragments
from different parents, the fragments can anneal to one another and reassemble
into a full length gene using PCR. Depending on the total gene size, the degree of
sequence similarity between parents, and desired number of crossovers (regions

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where different parental fragments are assembled), fragment sizes isolated from Recombination
and its Types
DNAse I treatment for reassembly can vary from as low as 10–50 bp to greater
than 1 kb. Random point mutation tend to occur at low rates during recombination
even with a high-fidelity polymerase, and researchers will often intentionally employ
error-prone PCR during PCR-based gene recombination to further diversify their NOTES
library. In a noteworthy, early example of DNA shuffling, a family of twenty Human
Interferon-a genes were shuffled followed by selection of antiviral and anti-
proliferation activities in murine cells, resulting in variants having 285000-fold
increased activities. The best chimeras were composed of up to five parental genes
and contained no random point mutations. In another example, shuffling of 26
homologous proteases genes generated many chimeric proteases that were
significantly improved over any of the parental enzymes for four different properties
assayed.
In vitro recombination methods are also often used in directed evolution,
even when the only genetic diversity is introduced by random mutagenesis of a
single parent gene. Here, one or more rounds of mutagenesis and screening to
isolate improved variants results in a handful of mutant genes, each carrying a
different set of point mutations. By shuffling these highly identical mutant DNA
sequences, one can readily obtain a library containing all combinations of point
mutations. Beneficial mutations can be combined and may show additive effects,
while any potentially deleterious mutations that have accumulated will be eliminated
by ‘Back-Crossing’ with the wild-type sequence.
Non-Reciprocal Recombination
It is also known as gene conversion. The fundamental law of genetics is that the
two partners contribute the equal amount of genes to the offspring’s. It means that
the offspring’s inherit the half complete set of genes from the male and half from
the female. One Diploid Cell undergoes Meiosis producing Four Haploid Cells;
therefore, the number of genes contributed by male gets halved and so the genes
of female. In higher animals like man it is not possible to analyse these genes taking
a single cell. However, in certain organisms such as fungi it is possible to recover
and analyse all the four daughter cells produced from a single cell through Meiosis.
Occasionally, three copies of maternal allele and only one copy of paternal allele is
formed by Meiosis. This indicates that one of two copies of parental alleles has
been altered to the maternal allele. This gene alteration is of non-reciprocal type
and is called gene conversion. Gene conversion is thought to be an important
event in the evolution of certain genes and occurs as a result of the mechanism of
general recombination and DNA repair (Refer Figure 5.9).

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Recombination
and its Types

NOTES

Fig. 5.9 Ideal Model of Non-Reciprocal Recombination

The above Figure 5.9 shows the non-reciprocal general recombination, it

starts when a nick is made in one of the strands, in which the following are
represented: Step (a): From this point DNA polymerase synthesizes an extra copy
of a strand and displaces the original copy as a single strand.
Step (b): This single strand starts pairing with the homologous region as in
lower duplex of DNA molecule in (b). The short unpaired strand produced in
Step (b) is degraded when the transfer of nucleotide sequence is completed.
Step (c): The results are observed (in the next cycle) when DNA replication
has separated the two non-matching strands.

Check Your Progress

4. When does homologous recombination occur?
5. Is nucleotide sequences changed at the site of exchange in heteroduplex?
Why?
6. What is strand pairing?

5.4 SITE-SPECIFIC RECOMBINATION

Site-specific recombination events are mediated by sequence-specific

recombination enzymes often encoded by viruses or transposable elements. The
site specific recombination alters the relative position of nucleotide sequences in
chromosome. The base pairing reaction depends on protein mediated recognition
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of the two DNA sequences that will combine. Very long homologous sequence is Recombination
and its Types
not required. Unlike general recombination, site specific recombination is guided
by a recombination enzyme that recognizes specific nucleotide sequences present
on one of both recombining DNA molecules. Base pairing is not involved, however,
if occurs the heteroduplex joint is only a few base pair long (Refer Figure 5.10). NOTES
It was first discovered in Phage (Lambda) by which its genome moves
into and out of the Escherichia coli chromosome. After penetration phage encoded
an enzyme, lambda or integrase which catalyses the recombination process.
Lambda integrase binds to a specific attachment site of DNA sequence on each
chromosome. It makes cuts and breaks a short homologous DNA sequences.
The integrase switches the partner strands and rejoins them to form a heteroduplex
joint of 7 bp long. The integrase resembles a DNA topoisomerase in rejoining the
strands which have previously been broken.

Fig. 5.10 Schematic Representation of Site-Specific Recombination

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Recombination 5.4.1 Types of Site-Specific Recombination
and its Types
Following are the types of site-specific recombination:
Conservative Site-Specific Recombination: Production of a very short
NOTES heteroduplex by requiring some DNA sequence that is the same on the two
DNA molecules is known as conservative site-specific recombination.
Transpositional Site-Specific (TSS) Recombination: There is another
type of recombination system known as Transpositional Site-Specific (TSS)
recombination. The TSS recombination does not produce heteroduplex
and requires no specific sequences on the largest DNA.
5.4.2 Molecular Mechanism of Site-Specific Recombination
There are several mobile DNA sequences including many viruses and transposable
elements that encode integrates. The enzyme integrates by involving a mechanism
different from ‘Phage ’ insert its DNA into a chromosome. Each enzyme of
integrates recognizes a specific DNA sequence like ‘Phage l’. K. Mizuuchi (1992)
reviewed the mecha­nism of transpositional recombination based on the studies of
Bacteriophage Mu and the other elements. The enzyme integrase was first purified
from Mu. Similar to integrase of phage , the Mu integrase also carries out of its
cutting and rejoining reactions without requirement of ATP. Also they do not require
a specific DNA sequence in the target chromosome and do not form a joint of
heteroduplex (Refer Figure 5.11).
During steps of TSS recombinational events the integrase makes a cut in
one strand at each end of the viral DNA sequences, and exposes the 32 -OH
group that protrudes out. Therefore, each of these 32 -OH ends directly invades
a phosphodiester bond on opposite strands of a randomly selected site on a target
chromosome. This facilitates to insert the viral DNA sequence into the target
chromosome, leaving two short single stranded gaps on each side of recombinational
DNA molecule. These gaps are filled in later on by DNA repair process (i.e.,
DNA polymerase) to complete the recombination process. This mechanism results
in the formation of short duplication of the adjacent target DNA sequences (short
repeats of 3 to 12 nucleotide long). Formation of short repeats is the hall-marks of
a TSS recombination.

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 Recombination
and its Types

NOTES

Fig. 5.11 Mechanism of Transpositional Site-Specific (TSS) Recombination;

SDR, Short Direct Repeats of Target DNA Sequence

5.4.3 Biological Roles of Site-Specific Recombination

The cells and viruses use conservative site-specific recombination for a wide variety
of biological functions. Many phages insert their DNA into the host chromosome
during infection using this recombination mechanism. In other cases, site-specific
recombination is used to alter gene expression. For example, inversion of a DNA
segment can allow two alternative genes to be expressed. Site-specific
recombination is also widely used to help maintain the structural integrity of circular
DNA molecules during cycles of DNA replication, homologous recombination,
and cell division.

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Recombination A comparison of site-specific recombination systems reveals some general
and its Types
themes. All reactions depend critically on the assembly of the recombinase protein
on the DNA, and the bringing together of the two recombination sites. For some
recombination reactions this assembly is very simple, requiring only the recombinase
NOTES and its DNA recognition sequences as just described for Cre. In contrast, other
reactions require accessory proteins. These accessory proteins include so-called
architectural proteins that bind specific DNA sequences and bend the DNA. They
organize DNA into a specific shape and thereby stimulate the recombination.
Architectural proteins can also control the direction of a recombination reaction,
for example, to ensure that integration of a DNA segment occurs while preventing
the reverse reaction. This type of regulation is essential for a logical biological
outcome. As can be seen from the examples discussed above, the same mechanism
of DNA recombination can be utilized in different biological contexts to bring
about integration, excision (deletion) and inversion of DNA segments. In principle,
then, one should be able to adapt site-specific recombination systems to direct
one or more of these types of DNA rearrangements in selected regions of a genome
of interest. This expectation has been fully validated. Site-specific recombination
has been utilized in promoting genetic alterations for answering fundamental questions
in biology and for developing biotechnological tools.
Some of these functions are as follows:
Lamda phage integrase promotes the integration and excision of a viral
genome into the host cell chromosome.
Tracking cell lineage during development.
Ablating a gene function during development.
Inducing the expression of a gene at a specific time in development.
Site-specific recombination in biotechnological applications.
Removal of integrated harmful viral sequences would be a potential
beneficial application of site-specific recombination.
Making the genetic screen more robust.
Structure based mutagenesis.
Changing the target specificity of Cre.
Changing the target specificity of Flp.
Since Cre and Flp have extremely simple reaction requirements, these
recombination systems have been reconstituted in a variety of organisms, such as
Bacteria, Fungi, Plants, Nematodes, Flies and Animals. Cre and Flp can be placed
under regulatable promoters for conditional or tissue-specific expression. An
important first step in applying site-specific recombination in a genetic context of
interest is the introduction of the target site or sites at the desired locale(s). Once
this has been accomplished, the rest of the experimental steps are quite
straightforward. To facilitate the rapid screen of desired variants, bacterial cells
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 Recombination
and its Types
Check Your Progress
7. How are site-specific recombination events mediated?
8. On what does base pair reaction depend? NOTES
9. Give the function of lambda integrase.
10. What happens in TSS recombinational events? Give its result.

5.5 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. The Genetic recombination (also known as genetic reshuffling) is the

exchange of genetic material between different organisms which leads to
production of offspring with combinations of traits that differ from those
found in either parent.
2. The genetic recombination involves the exchange or sharing of genetic
information between multiple parental sequences to create new ‘progeny’
genes.
3. Genetic recombination occurs when genetic material is exchanged between
two different chromosomes or between different regions within the same
chromosome.
4. Homologous recombination can occur between homologous
chromosomes or sister chromatids in meiotic cell and mitotic cells as well.
The homologous recombination is also called general recombination.
5. In the heteroduplex no nucleotide sequences are changed at the site of
exchange due to cleavage and rejoining events.
6. The RecBCD proteins act as DNA helicase because these hydrolyze ATP
and travel along DNA helix. Thus, the RecBCD proteins result in formation
of single stranded whisker at the recognition site which is displaced from
the helix. This initiates a base pairing interaction between the two
complementary sequences of DNA double helix.
7. Site-specific recombination events are mediated by sequence-specific
recombination enzymes often encoded by viruses or transposable elements.
8. The base pairing reaction depends on protein mediated recognition of the
two DNA sequences that will combine.
9. Lambda integrase binds to a specific attachment site of DNA sequence on
each chromosome. It makes cuts and breaks a short homologous DNA
sequences.
10. During steps of TSS recombinational events the integrase makes a cut in
one strand at each end of the viral DNA sequences, and exposes the 32 -
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Recombination OH group that protrudes out. Therefore, each of these 32 -OH ends directly
and its Types
invades a phosphodiester bond on opposite strands of a randomly selected
site on a target chromosome. This facilitates to insert the viral DNA sequence
into the target chromosome, leaving two short single stranded gaps on each
NOTES side of recombinational DNA molecule.

5.6 SUMMARY

Genetic recombination refers to the process of recombining genes to produce

new gene combinations that differ from those of either parent.
Genetic recombination produces genetic variation in organisms that
reproduce sexually.
Genetic recombination happens as a result of the separation of genes that
occurs during gamete formation in meiosis, the random uniting of these genes
at fertilization, and the transfer of genes that takes place between
chromosome pairs in a process known as crossing-over.
Crossing over allows alleles on DNA molecules to change positions from
one homologous chromosome segment to another.
Genetic recombination is responsible for genetic diversity in a species or
population.
Chromosomes are located within the nucleus of our cells and are formed
from chromatin (mass of genetic material consisting of DNA that is tightly
coiled around proteins called histones).
A chromosome is typically single-stranded and consists of a centromere
region that connects a long arm region (q arm) with a short arm region
(p arm).
When a cell enters the cell cycle, its chromosomes duplicate via DNA
replication in preparation for cell division.
Each duplicated chromosome is comprised of two identical chromosomes
called sister chromatids that are connected to the centromere region.
During cell division, chromosomes form paired sets consisting of one
chromosome from each parent. These chromosomes, known as homologous
chromosomes, are similar in length, gene position, and centromere location.
Genetic recombination that involves crossing over occurs during prophase
I of meiosis in sex cell production.
The duplicated pairs of chromosomes (sister chromatids) donated from
each parent line up closely together forming what is called a tetrad. A tetrad
is composed of four chromatids.
As the two sister chromatids are aligned in close proximity to one another,
one chromatid from the maternal chromosome can cross positions with a
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chromatid from the paternal chromosome. These crossed chromatids are Recombination
and its Types
called a chiasma.
Crossing over occurs when the chiasma breaks and the broken chromosome
segments get switched onto homologous chromosomes.
NOTES
The broken chromosome segment from the maternal chromosome gets joined
to its homologous paternal chromosome, and vice versa.
At the end of meiosis, each resulting haploid cell will contain one of four
chromosomes. Two of the four cells will contain one recombinant
chromosome.
In eukaryotic cells (those with a defined nucleus), crossing over can also
occur during mitosis.
Somatic cells (non-sex cells) undergo mitosis to produce two distinct cells
with identical genetic material.
Any crossover that occurs between homologous chromosomes in mitosis
does not produce a new combination of genes.
Crossing over that occurs in non-homologous chromosomes can produce
a type of chromosome mutation known as a translocation.
A translocation happens when a chromosome segment detaches from one
chromosome and moves to a new position on another non-homologous
chromosome. This type of mutation can be dangerous as it often leads to
the development of cancer cells.
Prokaryotic cells, like bacteria which are unicellular with no nucleus, also
undergo genetic recombination.
Although bacteria most commonly reproduce by binary fission, this mode
of reproduction does not produce genetic variation.
In bacterial recombination, genes from one bacterium are incorporated into
the genome of another bacterium through crossing-over.
Bacterial recombination is accomplished by the processes of conjugation,
transformation, or transduction.
In conjugation, one bacterium connects itself to another through a protein
tube structure called a pilus. Genes are transferred from one bacterium to
the other through this tube.
In transformation, bacteria take up DNA from their environment. The DNA
remnants in the environment most commonly originate from dead bacterial
cells.
In transduction, bacterial DNA is exchanged through a virus that infects
bacteria known as a bacteriophage. Once the foreign DNA is internalized
by a bacterium via conjugation, transformation, or transduction, the bacterium
can insert segments of the DNA into its own DNA. This DNA transfer is
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Recombination accomplished via crossing-over and results in the creation of a recombinant
and its Types
bacterial cell.
DNA of the donor cell recombines with recipient DNA by reciprocal
exchange of DNA strands. The recombining DNA molecules have
NOTES
homologous sequences.
The DNA molecules align or pair with each other and undergo crossing-
over and homologous recombination. The recombinant DNA has new genetic
constitution.
Bacteria can acquire DNA of other closely related bacterial species and
can become transformed. This is known as transformation.
Transformation was first demonstrated by Griffith in Diploccous
pneumoniae bacteria to confirm DNA as genetic material. Homologous
recombination is catalysed by RecA protein.
Often the transposable elements replicate to generate two copies. One copy
remains at the original site while the other jumps to the target site.
When Phage l (lambda) infects Escherichia coli bacterium, DNA of the
phage is inserted into the DNA of the bacterium. In this way phage DNA
becomes integrated into host DNA and becomes a part of the host
chromosome.
Bacterium containing a complete set of phage DNA is called lysogen. Phage
DNA is inserted at a specific site into the host DNA.
The attachment site of both DNAs possesses identical 15 bp sequence. A
phage encoded protein called integrase catalyses the integration.
A host protein called Integration Host Factor (IGF) is also involved. Integrase
is a topoisomerase enzyme which breaks, rotates and then re-joins the
strands of both molecules. In eukaryotes site-specific recombination
produces antibody diversity.
Although common, genetic recombination is a highly complex process. It
involves the alignment of two homologous DNA strands (the requirement
for homology suggests that this occurs through complementary base pairing,
but this has not been definitively shown), precise breakage of each strand,
exchange between the strands, and sealing of the resulting recombined
molecules. This process occurs with a high degree of accuracy at high
frequency in both Eukaryotic and Prokaryotic cells.
The basic steps of recombination can occur in two pathways, according to
whether the initial break is single or double stranded. In the single-stranded
model, following the alignment of homologous chromosomes, a break is
introduced into one DNA strand on each chromosome, leaving two free
ends.

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 Each end then crosses over and invades the other chromosome, forming a Recombination
and its Types
structure called a Holliday junction. The next step, called branch migration,
takes place as the junction travels down the DNA. The junction is then
resolved either horizontally, which produces no recombination, or vertically,
which results in an exchange of DNA. NOTES
In the alternate pathway initiated by double-stranded breaks, the ends at
the breakpoints are converted into single strands by the addition of 3' tails.
These ends can then perform strand invasion, producing two Holliday
junctions. From that point forward, resolution proceeds as in the single-
stranded model.
Third model of recombination, Synthesis-Dependent Strand Annealing
(SDSA), has also been proposed to account for the lack of crossover typical
of recombination in mitotic cells and observed in some meiotic cells to a
lesser degree.
The molecular processes they catalyze may rely on very short stretches of
homology between the interacting DNAs, or they may be entirely non-
homologous.
Recombination that involves very limited or no homology between the
interacting DNA sequences is termed illegitimate or non-homologous
recombination.
Sometimes a few matched base pairs are seen precisely at illegitimate
recombination junctions, and these are called microhomologies.
An event supported by homologies of 100 bp or more would typically be
classified as homologous, a match of 10 bp or fewer would be non-
homologous, and there is evidently a gray area in between.

5.7 KEY WORDS

Genetic recombination: Genetic recombination refers to the process of

recombining genes to produce new gene combinations that differ from those
of either parent.
Non-homologous recombination: Recombination between DNA
molecules of similar sequences. Our cells carry out general recombination
during meiosis.
Homologous recombination: Recombination between DNA molecules
of similar sequences.
Crossing over: Crossing over is the process of the chiasma breaks and
the exchange of broken chromosome segments onto non-sister chromatids
of the homologous chromosomes.

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Recombination Conservative recombination: In conservative recombination events, the
and its Types
number of copies of the interacting chromosomes or DNA sequences is
maintained throughout the process.

NOTES
5.8 SELF ASSESSMENT QUESTIONS AND
EXERCISES

Short Answer Questions

1. What is recombination?
2. Give the formation of heteroduplex joint during homologous recombination
with the help of diagram.
3. Explain the two Holliday junctions for homologous recombination.
4. Explain Holliday model for homologous or general recombination with the
help of diagram.
5. Give the role of RecA protein in homologous genetic recombination.
6. Distinguish between homologous recombination in Prokaryotes and
Eukaryotes.
7. What are the types of site-specific recombination?
8. Give the biological role of site-specific recombination.
Long Answer Questions
1. What is homologous recombination? Discuss the mechanism of homologous
recombination with suitable examples.
2. Discuss about the types of recombination in detail.
3. What is genetic recombination? Write in detail the mechanism of
recombination based on Holliday’s model.
4. Explain with the help of diagram McClintock and Creighton’s model showing
physical evidences of recombination.
5. Give an illustrated account of microbial genetic recombination with necessary
step-by-step mechanism.
6. Draw a well labelled diagram to show the mechanism of removal of error in
newly made strand by Mismatch Repair system.
7. What is recombination? Discuss the types and significance of recombination.
8. Describe in detail the types and mechanism of site-specific recombination?

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 Recombination
5.9 FURTHER READINGS and its Types

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing NOTES
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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Conjugation
and F-Factor
UNIT 6 CONJUGATION
AND F-FACTOR
NOTES
Structure
6.0 Introduction
6.1 Objectives
6.2 Conjugation
6.2.1 Conjugation by Escherichia coli F Factor
6.2.2 Fertility Factor or F Factor
6.2.3 Hfr Conjugation and Chromosomal Transfer
6.2.4 The F (F Prime) Factor
6.2.5 Interrupted Mating and Conjugational Mapping
6.3 Answers to Check Your Progress Questions
6.4 Summary
6.5 Key Words
6.6 Self Assessment Questions and Exercises
6.7 Further Readings

6.0 INTRODUCTION

Conjugation is merely the fusion of two compatible bacterial cells. Bringing two
genotypes together and allowing them to conjugate is the equivalent of making a
cross in Eukaryotes. The conjugation process is explained on the gut bacterium
Escherichia coli or E. coli. Conjugation and gene transfer in Escherichia coli
are driven by a circular DNA plasmid called the Fertility Factor or Sex Factor
(F), which is found in some but not all cells. Therefore to understand how to make
a cross in Escherichia coli, the properties of F have to be understood.
Cells carrying the F plasmid are designated F+, and those lacking it are F–.
The F plasmid contains approximately 100 genes, which give the plasmid several
important properties. The F plasmid can replicate its own DNA, allowing the
plasmid to be maintained in a dividing cell population. The rare cells in which the F
factor is integrated into the host chromosome can be isolated from the bacterial
population to cultivate pure strains derived from these cells. In these strains, every
cell donates chromosomal alleles during F transfer, so the frequency of recombinants
for these strains is much higher as compared to cells in the original population,
where the F factor is not integrated in most cells. Consequently, strains with an
integrated F factor are termed High frequency of recombination (Hfr) strains to
distinguish them from normal F+ strains, which contain only a few rare Hfr cells
and thus display only a low frequency of recombination for the strain as a whole.
Because they transfer chromosomal markers efficiently, hence the Hfr strains are
used for genetic mapping.

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 Fundamentally, the Bacterial conjugation is the mechanism whereby DNA Conjugation
and F-Factor
is transferred from a donor to a recipient cell through a complex mechanism that is
encoded by a transfer system.
In this unit, you will study about the conjugation, bacterial conjugation by
NOTES
Escherichia coli, F factor, structure of F factor, regulation of F factor fertility,
establishment of cell contact, DNA mobilization, transfer and separation of mating
pair, Hfr conjugation and chromosomal transfer, interrupted mating and
conjugational mapping.

6.1 OBJECTIVES

After going through this unit, you will be able to:

Discuss what bacterial conjugation is
Explain conjugation by Escherichia coli
Understand F factor - the structure of F factor and regulation of F factor
fertility
Analyse DNA mobilization, Hfr conjugation and chromosomal transfer

6.2 CONJUGATION

Conjugation is the process by which one bacterium transfers genetic material to

another through direct contact. During conjugation, one bacterium serves as the
donor of the genetic material, and the other serves as the recipient. The donor
bacterium carries a DNA sequence called the Fertility factor or F factor. The F
factor allows the donor to produce a thin, tube like structure called a pilus, which
the donor uses to contact the recipient. The pilus then draws the two bacteria
together, at which time the donor bacterium transfers genetic material to the recipient
bacterium. Typically, the genetic material is in the form of a plasmid, or a small,
circular piece of DNA. The genetic material transferred during conjugation often
provides the recipient bacterium with some sort of genetic advantage. For instance,
in many cases, conjugation serves to transfer plasmids that carry antibiotic resistance
genes.
Bacterial cells may carry besides the main chromosome, one or more small
DNA molecules in the cytoplasm called plasmids. Though there are various kinds
of plasmids, but only a few are involved in conjugation and are therefore called
conjugative plasmids. The Sex element or Fertility factor or F factor is the type
of conjugative plasmid.
The presence or F factor in different strains has given rise to two mating
types in bacteria namely, the donor which possesses the Fertility factor or F factor
and referred to as F+ strain, the second which lacks Fertility factor or F factor is
the F– strain. The F factor is itself the genetic element which is passed from donor
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Conjugation to recipient cells during conjugation. Remember that there is no conjugation between
and F-Factor
two F+ strains or between two F– strains.
6.2.1 Conjugation by Escherichia coli F Factor
NOTES Principally, the ‘Conjugation’ is referred as the fusion of two compatible bacterial
cells. Bringing two genotypes together and allowing them to conjugate is the
equivalent of making a cross in Eukaryotes. The conjugation process is explained
on the gut bacterium Escherichia coli or E. coli. Conjugation and gene transfer in
Escherichia coli are driven by a circular DNA plasmid called the Fertility factor
or Sex factor (F factor), which is found in some but not all cells. Therefore to
understand how to make a cross in Escherichia coli, the properties of F factor
have to be understood.
6.2.2 Fertility Factor or F Factor
The Fertility factor or the F factor was first named ‘F’ by one of its discoverers
Esther Lederberg. It is also called the sex factor in Escherichia coli or the F sex
factor, and also the F plasmid. The F factor permits genes to be transferred from
one bacterium carrying the factor to another bacterium lacking the factor by the
process termed as conjugation. The F plasmid belongs to a class of conjugative
plasmids that control sexual functions of bacteria with a fertility inhibition, the
‘Fin’ system.
Structure of F Factor
The most common functional segments that constitute the F factors include the
following:
OriT (Origin of Transfer): The sequence which marks the starting point of
conjugative transfer.
OriC (Origin of Replication): The sequence starting with which the plasmid
DNA will be replicated in the recipient cell.
tra-Region (Transfer Genes): Genes coding the F pilus and DNA transfer
process.
IS (Insertion Elements): These are composed of one copy of IS2, two copies
of IS3, and one copy of IS1000, the so-called ‘Selfish Genes’, the sequence
fragments which can integrate copies of themselves at different locations.
traA: Pilin, the major subunit of the pilus, the F plasmid genes.
Relation to the Genome
The episome that harbors the F factor can exist as an independent plasmid or
integrate into the bacterial cell’s genome. The following are the names for the
possible states:
Hfr Bacteria: It possess the entire F episome integrated into the bacterial genome.
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F+ Bacteria: It possess F factor as a plasmid independent of the bacterial genome. Conjugation
and F-Factor
The F plasmid contains only F factor DNA and no DNA from the bacterial genome.
F (F Prime) Bacteria: These are formed by incorrect excision from the
chromosome, resulting in F plasmid carrying bacterial sequences that are next to
NOTES
where the F episome has been inserted.
F– Bacteria: These do not contain F factor and acts as the recipients.
The F element contains about 2 percent of the cell’s total DNA. It is capable of
autonomous replication and is made up of a circular, double stranded DNA
molecule having the molecular weight approximately 35 × 106. It contains about
15 genes, out of which 8 control the formation of F pili or sex pili which are hair-
like appendages extending from the surface of F+ cells. The function of F pili is
conjugation.
Structure of Pili
The F pili (singular, pilus) originate from cell membrane and project outward beyond
the cell wall as shown in Figure 6.1. Pili are minute proteinaceous tubules that
allow the F+ cells to attach to other cells and to maintain contact with them. The
width of pili in different bacteria varies between 4 and 35 nm. The pilus is made up
of a Phosphate-Carbohydrate-Protein complex with a single Polypeptide subunit
called ‘Pilin’ of 11,000 to 12,000 Daltons.
Each pilin subunit has 2 residues of Phosphate and one of Glucose. The pili
have been isolated and analysed by electron microscopy and X-ray diffraction
techniques. The F pilus consists of a hollow cylinder 80 Å in diameter. The central
hollow core is about 20 Å. The pilin subunits are arranged in the form of four
helical chains. Figure 6.1 illustrates the Escherichia coli cell showing Pili on the
surface.

Fig. 6.1 The Escherichia coli Cell Showing Pili on the Surface

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Conjugation F Factor - Mating Process
and F-Factor
In a combination of F+ and F– cells, an F+ donor cell can communicate with an F–
recipient cell by the F pilus. Therefore, the pilus is essential for recognition of
NOTES recipient cell with which mating can take place. Figure 6.2 illustrates the transfer
of F element factor from F+ to F– cells during the conjugation process.

Fig. 6.2 Transfer of F Element Factor from F+ to F– Cells during Conjugation

After initial contact between the pilus and recipient cell is established, the
pilus serves as a protoplasmic connection between the two cells and is called the
conjugation tube. A donor cell devoid of pili cannot conjugate. The sex plasmid
passes from the donor F+ to the recipient F– cell through the conjugation tube.
Transfer takes place when DNA replicates through the rolling circle method.
A nick or incision produced by an endonuclease in one strand of the plasmid
DNA duplex produces a free 5 and a 3 end. The strand moves across the
cytoplasmic bridge with the 5 end first, into the F– cell. The second inner strand
of the plasmid DNA duplex is retained in the F+ cell and synthesizes its
complementary strand. The two cells separate after mating and are known as ex-
conjugants. Thus the originally mixed population of F+ and F– cells comes to have
all F+ cells only.

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 The transfer of sex element from F+ to F– cell has one more significant feature. Conjugation
and F-Factor
Not only can the plasmid exist in the cytoplasm as an autonomous entity, but it can
also become incorporated in the main bacterial chromosome in a frequency of
about 1 in 10,000 F+ cells.
NOTES
Integration takes place at a specific site in the host chromosome which has
homologous sequences. Such an integrated plasmid is known as episome and
promotes the transfer of the main bacterial genophore from donor F+ to recipient
F– cells during conjugation, an occurrence followed by recombination.
Cells carrying the F plasmid are termed as F+ while those lacking it are
termed as F–. The F plasmid contains approximately 100 genes, which provide
several significant properties to plasmid, such as:
1. The F plasmid is capable of replicating its own DNA that allows the
plasmid to be maintained in a dividing cell population (Refer Figure 6.3
(a)).
2. Cells that carry the F plasmid stimulate the synthesis of pili on the bacterial
cell surface. Pili allow the F+ cells to be attached to other cells and to
maintain contact with them, i.e., to conjugate (Refer Figure 6.3 (b)).
3. Fundamentally, the F+ and F– cells can conjugate. When conjugation
occurs, the F+ cells can act as F donors. The F plasmid DNA replicates
and the newly synthesized copy of the circular F molecule is transferred
to the F– recipient (Refer Figure 6.3 (c)). Though, a copy of F
permanently remains behind in the donor cell. The recipient cell is
converted into F+, since it contains a circular F genome. The transfer of
the F plasmid from F+ to F– is fast, therefore the F plasmid is capable of
spreading quickly throughout a population from strain to strain.
4. F+ cells are typically inhibited after building contact with other F+ cells,
therefore the F plasmid is not transferred from F+ to F+.

Fig. 6.3 F Plasmid Replicating its Own DNA

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Conjugation 5. Occasionally F within its genome carries one or more IS (Insertion
and F-Factor
Sequence) elements. An IS element is referred as a mobile segment of
DNA that moves from one location to another location within the host
chromosome or between the chromosome and the plasmid. The presence
NOTES of a specific IS element both in the plasmid and in the chromosome can
provide a site at which homologous crossing over can occur occasionally.
A crossover between the two circular DNAs results in the integration of
the plasmid into the bacterial chromosome, as shown in Figure 6.4.

Fig. 6.4 Crossover between the Two Circular DNAs

6.2.3 Hfr Conjugation and Chromosomal Transfer

After the discovery of F+ strains, a distinctive kind of strain was observed which
was several hundred times more fertile in crosses with F– than any known F+ strain.
This strain was isolated by Lederberg et al. (1952) and was called Hfr strain or
High frequency recombination. The Hfr strain produces about 1,000 times
more prototrophs than in the F+ × F– cross.
In the mating system of Hfr strains the main bacterial chromosome containing
an integrated F factor is transferred to F– cells. The Hfr bacteria arise spontaneously
from F+ cells at a low frequency by integration of F factor in the main chromosome.
When Hfr cells are mixed with F– cells there is conjugation and a high
frequency of transfer of only portions of the main bacterial chromosome (some
selected markers) from donor to F– recipient cells. The recipient cell remains F–.
An F+ cell is converted to Hfr when F integrates into the main chromosome
by reciprocal recombination. The process is reversible so that an Hfr cell becomes
F+ when another recombinational event causes detachment of the F factor.
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 Hayes, Wollman and Jacob conducted experiments which demonstrated Conjugation
and F-Factor
recombination during mating between Hfr and F–. They took wild type Hfr capable
of synthesizing all its organic requirements, which could also utilise the sugars
Galactose and Lactose, and was susceptible to being killed by Streptomycin. The
second strain they took was of the F– type which could not synthesise some Amino NOTES
Acids (Leucine and Threonine), nor utilise Galactose and Lactose, and was resistant
to Streptomycin.
The Hfr and mutant F– strains were mixed and grown together. For analysing
the progeny cells, samples of the cell mixture were grown on minimal medium
containing Streptomycin. Recombinants had appeared in the progeny.
Linear Chromosome Transfer by Hfr Strains
Wollman and Jacob (1956) studied kinetics of genetic transfer by the interrupted
mating technique. After mixing up two parental populations, cell samples are
withdrawn at different intervals and then stressed in a mixer so that the mating
pairs become separated and conjugation comes to an end. The mixture of cells is
diluted and plated on selective medium and the number of recombinants formed
during that interval are determined. The appearance of recombinants indicates
formation of zygotes.
The different genetic markers appear in the progeny of interrupted matings
after different time periods have been allowed before mating is disrupted by stress/
pressure. Closely linked markers appear at the same time, whereas distantly placed
markers appear at different times.
The markers Threonine and Leucine appear after about 8 minutes whereas
Gal appears after 26 minutes. The entire chromosome containing about 5 × 106 base
pairs is transferred in 90 minutes. Therefore, it is possible to map locations of
markers on the donor chromosome.
Further investigations showed that the Hfr donor cells transfer only a part of
their genome to F– cells. Moreover, there are different Hfr strains which are distinct
from each other in transferring a different part of the genome to F– cells. The
Escherichia coli genome is a closed circular loop.
In the Hfr donor cell the loop is broken at a point characteristic for that
strain. The break occurs within the F element so that a part of it is at the leading
end and is transferred to the F– cell, the other part of F is at the extreme distal end
which trails behind. Transfer takes place by injection of the linear structure into the
recipient cell.
The foremost or leading end carries with it gene loci nearest to it (Refer
Figure 6.5) until conjugation is interrupted. The transfer of DNA may be broken
off at any time due to spontaneous rupture of the connection between conjugating
cells.

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Conjugation
and F-Factor

NOTES

Fig. 6.5 Integration of F Element into Main Chromosome of F+ to Form Hfr

Figure 6.5 illustrates the integration of F element into main chromosome of

F to form Hfr. In Figure 6.5, (d) illustrates the break at specific site and transfer
+

from Donor Hfr cell.

After transfer to the F– cell the donor DNA fragment becomes incorporated
into a homologous region of the host cell chromosome. The corresponding segment
of the F– cell DNA is lost. Crossovers occur between the donor Hfr fragment and
the F– host cell chromosome. This integration is essential before donor genetic
markers can express themselves.
The presence of F factor in Hfr and F+ cells endows specific surface
properties through formation of F pilus, due to which these cells can act as donors.
In 1960 Loeb found out that certain bacteriophages could lyse only donor
Escherichia coli cells but not the recipient cells.
The phages R17 and M12 adsorb to pili present on donors but not on
recipient cells and are referred to as male-specific phages. Further, the male-
specific RNA phages are observed to adsorb along the length of the sex pilus,
whereas male-specific DNA phages adsorb to the tip of the pilus.
The surface of the recipient cell also appears to play an important role in
mating. When an F factor is present in a cell, it prevents the cell from acting as a
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recipient, so that super-infection does not occur. As this effect is due to a surface Conjugation
and F-Factor
component which depends on the F factor, the phenomenon is known as surface
exclusion.
Some of the surface proteins coded by the main bacterial chromosome
NOTES
genes are also involved in mating. The con– mutants of Escherichia coli are not
able to function as recipients and form mating pairs in conjugation. These mutants
are found to be deficient in two of the surface proteins.
6.2.4 The F (F Prime) Factor
The F element can also become integrated into the main bacterial chromosome.
Rarely an integrated F can undergo excision and become detached, carrying with
it some bacterial genes that remain attached to it. Such an F element is called an
F¢ factor.
It behaves like the F factor of F+ cells and can be transferred to F– cells.
Because F carries bacterial genes, it is able to pair with the corresponding region
in the bacterial chromosome (Refer Figure 6.6). A bacterium receiving an F factor
becomes a partial diploid for the bacterial genes carried by F . Figure 6.6 illustrates
the genomes of Escherichia coli cell with F element.

Fig. 6.6 Genomes of Escherichia coli Cell with F Element

The Transfer Genes

Thee are certain mutant strains in which transfer of F factor cannot take place. The
transfer deficient mutants have been useful for identifying the presence of transfer
(tra) genes in the F factor. Transfer genes are found to be necessary for conjugation.
About 19 tra genes have been identified so far and these are classified into 4
groups as follows:
1. The genes of first group control pilus formation and recognition of recipient
cell.
2. The genes of second group are involved in stabilisation of mating pairs.
3. The genes of the third group are essential for some metabolic changes in
DNA required for conjugation.
4. The genes of the fourth group (tra J) controls the function of all the other tra
genes.
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Conjugation 6.2.5 Interrupted Mating and Conjugational Mapping
and F-Factor
In conjugation, it was witnessed that the number of genes that are transmitted
from donor to recipient was directly proportional to the time interval for which
NOTES conjugation was permissible. Considering this feature, Francois Jacob and Ellie
Wollman developed a specific technique called ‘Interrupted Mating Technique’
for mapping the bacterial chromosome. In this technique, the donor Hfr and recipient
F strains are mixed and permitted to conjugate for a short time period. Then
samples are removed at periodic intervals and are violently agitated to break the
conjugation tube. The length of transmitted donor chromosome can then be
determined and mapped in terms of time units that are required for transfer. It is
identified that 8 minutes are required for conjugation to start and then chromosome
is transferred slowly in terms of time units, where one time unit is equal to one
minute. Therefore the complete chromosome of Escherichia coli is transferred in
about 89 minutes and therefore the bacterial chromosome is 89 time units in length.
Circular Linkage Map
When linkage maps in bacteria were prepared using several Hfr strains, then these
maps typically differ with respect to the first and the last genes of the map. Though,
if the first gene transferred is distinguished and also the direction of transfer of
subsequent genes, then the order is predetermined. There is a circular map which
cleaves at any point due to attachment of F factor to form a linear chromosome.
This linear chromosome enters the recipient cell from the end point which is far
from the site of attachment of F factor. Different experimental evidences prove
that Hfr certainly results due to the insertion of F into the chromosome. Circular
nature of linkage group was also established through the evidences for physical
circular nature of bacterial chromosome.
Linkage Information from Transformation
Transformation is accomplished through the acceptance of naked DNA that is
extracted from one strain of bacteria by another strain of bacteria. While extracting
this DNA, there may be some breakage or spiriting into small pieces. When two
genes are closer to each other, then there is possibility of being carried on the
same piece of DNA, therefore initiating double transformation. Alternatively if the
genes are widely separated, then there will be possibility of being carried on separate
DNA segments and the frequency of double transformation will be the product of
single transformation frequencies. Therefore as per the product rule of probability
for the observed frequency of double recombinants prove close linkage.
Recombination after Gene Transfer
When a chromosome segment is transferred from a donor to a recipient strain, or
through transduction or transformation, then this transferred segment must be
integrated into host genome through an exchange mechanism to produce a stable
recombinant. The recipient cell at this stage is termed a merozygote (partial diploid)
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which has the complete genome of F– termed endogenote and an incomplete Conjugation
and F-Factor
genome termed exogenote derived from F+ or Hfr. An even number of crossovers,
instead of a single or odd number of crossovers, permits incorporation of a part of
the genome from exogenote into the endogenote, one of the two products of
exchange, i.e., a linear fragment is usually lost. NOTES

Conjugation Mapping
Using different strains of F plasmids and the interrupted mating technique, the
order of genes on the chromosome can be determined.
Step 1. For a strain of Hfr, use the interrupted mating technique for determining
the order of the genes in the region of the chromosome near the plasmid insertion
point.
Step 2. By obtaining the order of the genes by means of various strains and
the interrupted mating technique (as defined in Step 1).
Step 3. By means of these orders, a map of the chromosome can be
deduced.

Check Your Progress

1. Explain the term conjugation giving example.
2. What are plasmids and conjugative plasmids?
3. What is F factor?
4. What does F element contains? How it functions?
5. How integration takes place at a specific site in the host chromosome?
6. Explain Hfr strain or High frequency recombination.
7. What is interrupted mating technique?

6.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. Conjugation is the process by which one bacterium transfers genetic material

to another through direct contact. During conjugation, one bacterium serves
as the donor of the genetic material, and the other serves as the recipient.
The donor bacterium carries a DNA sequence called the Fertility factor or
F factor. The F factor allows the donor to produce a thin, tube like structure
called a pilus, which the donor uses to contact the recipient.
Principally, the ‘Conjugation’ is referred as the fusion of two compatible
bacterial cells. The conjugation process is explained on the gut bacterium
Escherichia coli or E. coli. Conjugation and gene transfer in Escherichia
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Conjugation coli are driven by a circular DNA plasmid called the Fertility factor or Sex
and F-Factor
factor (F factor), which is found in some but not all cells.
2. Typically, the genetic material is in the form of a plasmid, or a small, circular
piece of DNA. Bacterial cells may carry besides the main chromosome,
NOTES
one or more small DNA molecules in the cytoplasm called plasmids. Though
there are various kinds of plasmids, but only a few are involved in conjugation
and are therefore called conjugative plasmids. The Sex element or Fertility
factor or F factor is the type of conjugative plasmid.
3. The Fertility factor or the F factor was first named ‘F’ by one of its discoverers
Esther Lederberg. It is also called the sex factor in Escherichia coli or the
F sex factor, and also the F plasmid. The F factor permits genes to be
transferred from one bacterium carrying the factor to another bacterium
lacking the factor by the process termed as conjugation.
4. The F element contains about 2 percent of the cell’s total DNA. It is capable
of autonomous replication and is made up of a circular, double stranded
DNA molecule having the molecular weight approximately 35 × 106. It
contains about 15 genes, out of which 8 control the formation of F pili or
sex pili which are hair-like appendages extending from the surface of F+ cells.
The function of F pili is conjugation.
5. Integration takes place at a specific site in the host chromosome which has
homologous sequences. Such an integrated plasmid is known as episome
and promotes the transfer of the main bacterial genophore from donor F+ to
recipient F– cells during conjugation, an occurrence followed by
recombination.
6. After the discovery of F+ strains, a distinctive kind of strain was observed
which was several hundred times more fertile in crosses with F– than any
known F+ strain. This strain was isolated by Lederberg et al. (1952) and
was called Hfr strain or High frequency recombination. The Hfr strain
produces about 1,000 times more prototrophs than in the F+ × F– cross.
In the mating system of Hfr strains the main bacterial chromosome containing
an integrated F factor is transferred to F– cells. The Hfr bacteria arise
spontaneously from F+ cells at a low frequency by integration of F factor in
the main chromosome.
7. In conjugation, it was witnessed that the number of genes that are transmitted
from donor to recipient was directly proportional to the time interval for
which conjugation was permissible. Considering this feature, Francois Jacob
and Ellie Wollman developed a specific technique called ‘Interrupted Mating
Technique’ for mapping the bacterial chromosome. In this technique, the
donor Hfr and recipient F strains are mixed and permitted to conjugate for
a short time period.

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 Conjugation
6.4 SUMMARY and F-Factor

Conjugation is the process by which one bacterium transfers genetic material

to another through direct contact. NOTES
During conjugation, one bacterium serves as the donor of the genetic material,
and the other serves as the recipient. The donor bacterium carries a DNA
sequence called the Fertility factor or F factor.
The F factor allows the donor to produce a thin, tube like structure called a
pilus, which the donor uses to contact the recipient. The pilus then draws
the two bacteria together, at which time the donor bacterium transfers genetic
material to the recipient bacterium.
Typically, the genetic material is in the form of a plasmid, or a small, circular
piece of DNA.
The genetic material transferred during conjugation often provides the
recipient bacterium with some sort of genetic advantage. For instance, in
many cases, conjugation serves to transfer plasmids that carry antibiotic
resistance genes.
Bacterial cells may carry besides the main chromosome, one or more small
DNA molecules in the cytoplasm called plasmids. Though there are various
kinds of plasmids, but only a few are involved in conjugation and are therefore
called conjugative plasmids. The Sex element or Fertility factor or F factor
is the type of conjugative plasmid.
Principally, the ‘Conjugation’ is referred as the fusion of two compatible
bacterial cells. Bringing two genotypes together and allowing them to
conjugate is the equivalent of making a cross in Eukaryotes. The conjugation
process is explained on the gut bacterium Escherichia coli or E. coli.
Conjugation and gene transfer in Escherichia coli are driven by a circular
DNA plasmid called the Fertility factor or Sex factor (F factor), which is
found in some but not all cells.
The Fertility factor or the F factor was first named ‘F’ by one of its discoverers
Esther Lederberg. It is also called the sex factor in Escherichia coli or the
F sex factor, and also the F plasmid.
The F factor permits genes to be transferred from one bacterium carrying
the factor to another bacterium lacking the factor by the process termed as
conjugation.
OriT (Origin of Transfer): The sequence which marks the starting point of
conjugative transfer.
OriC (Origin of Replication): The sequence starting with which the plasmid
DNA will be replicated in the recipient cell.

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Conjugation tra-Region (Transfer Genes): Genes coding the F pilus and DNA transfer
and F-Factor
process.
The F element contains about 2 percent of the cell’s total DNA. It is capable
of autonomous replication and is made up of a circular, double stranded
NOTES
DNA molecule having the molecular weight approximately 35 × 106. It
contains about 15 genes, out of which 8 control the formation of F pili or
sex pili which are hair-like appendages extending from the surface of F+ cells.
The function of F pili is conjugation.
In a combination of F+ and F– cells, an F+ donor cell can communicate with
an F– recipient cell by the F pilus. Therefore, the pilus is essential for
recognition of recipient cell with which mating can take place.
Integration takes place at a specific site in the host chromosome which has
homologous sequences. Such an integrated plasmid is known as episome
and promotes the transfer of the main bacterial genophore from donor F+ to
recipient F– cells during conjugation, an occurrence followed by
recombination.
After the discovery of F+ strains, a distinctive kind of strain was observed
which was several hundred times more fertile in crosses with F– than any
known F+ strain. This strain was isolated by Lederberg et al. (1952) and
was called Hfr strain or High frequency recombination.
The Hfr strain produces about 1,000 times more prototrophs than in the
F+ × F– cross.
In the mating system of Hfr strains the main bacterial chromosome containing
an integrated F factor is transferred to F– cells.
The Hfr bacteria arise spontaneously from F+ cells at a low frequency by
integration of F factor in the main chromosome.
When Hfr cells are mixed with F– cells there is conjugation and a high
frequency of transfer of only portions of the main bacterial chromosome
(some selected markers) from donor to F– recipient cells. The recipient cell
remains F–.
An F+ cell is converted to Hfr when F integrates into the main chromosome
by reciprocal recombination. The process is reversible so that an Hfr cell
becomes F+ when another recombinational event causes detachment of the
F factor.
In the Hfr donor cell the loop is broken at a point characteristic for that
strain. The break occurs within the F element so that a part of it is at the
leading end and is transferred to the F– cell, the other part of F is at the
extreme distal end which trails behind. Transfer takes place by injection of
the linear structure into the recipient cell.
The F element can also become integrated into the main bacterial
chromosome. Rarely an integrated F can undergo excision and become
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 detached, carrying with it some bacterial genes that remain attached to it. Conjugation
and F-Factor
Such an F element is called an F factor.
It behaves like the F factor of F+ cells and can be transferred to F– cells.
Because F carries bacterial genes, it is able to pair with the corresponding
NOTES
region in the bacterial chromosome.
The transfer deficient mutants have been useful for identifying the presence
of transfer (tra) genes in the F factor.
Transfer genes are found to be necessary for conjugation. About 19 tra
genes have been identified so far and these are classified into 4 groups.
In conjugation, it was witnessed that the number of genes that are transmitted
from donor to recipient was directly proportional to the time interval for
which conjugation was permissible. Considering this feature, Francois Jacob
and Ellie Wollman developed a specific technique called ‘Interrupted Mating
Technique’ for mapping the bacterial chromosome. In this technique, the
donor Hfr and recipient F strains are mixed and permitted to conjugate for
a short time period.

6.5 KEY WORDS

Conjugation: This is the process by which one bacterium transfers genetic

material to another through direct contact.
Plasmids: Bacterial cells may carry besides the main chromosome, one or
more small DNA molecules in the cytoplasm called plasmids.
Conjugative plasmids: The plasmids that are involved in conjugation are
called conjugative plasmids.
F factor: The Fertility factor or the F factor permits genes to be transferred
from one bacterium carrying the factor to another bacterium lacking the
factor by the process termed as conjugation.
OriT (Origin of Transfer): The sequence which marks the starting point
of conjugative transfer.
OriC (Origin of Replication): The sequence starting with which the
plasmid DNA will be replicated in the recipient cell.
tra-Region (Transfer Genes): Genes coding the F pilus and DNA transfer
process.
Hfr bacteria: It possess the entire F episome integrated into the bacterial
genome.
F+ bacteria: It possess F factor as a plasmid independent of the bacterial
genome. The F plasmid contains only F factor DNA and no DNA from the
bacterial genome.

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Conjugation F (F Prime) bacteria: These are formed by incorrect excision from the
and F-Factor
chromosome, resulting in F plasmid carrying bacterial sequences that are
next to where the F episome has been inserted.
F– bacteria: These do not contain F factor and acts as the recipients.
NOTES

6.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is conjugation?
2. What are plasmids?
3. Explain F factor.
4. Define the term mating pair.
5. What is Hfr bacteria?
6. Differentiate between F+ bacteria and F– bacteria.
7. How will you identifying the presence of transfer (tra) genes in the F factor?
8. What is circular linkage map?
Long Answer Questions
1. Briefly discuss about the significance and function of conjugation giving
appropriate examples.
2. Discuss conjugation by Escherichia coli F factor.
3. Explain the role of plasmids in bacterial conjugation.
4. What is F factor? Explain the structure of F-factor giving appropriate
examples.
5. Explain about the transfer and separation of mating pair.
6. Discuss about the Hfr conjugation and chromosomal transfer process with
the help of diagrams.
7. Briefly explain the interrupted mating and conjugational mapping.

6.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.

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140 Material
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House. Conjugation
and F-Factor
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University NOTES
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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Transformation

UNIT 7 TRANSFORMATION
NOTES Structure
7.0 Introduction
7.1 Objectives
7.2 Discovery of Transformation
7.3 Competence
7.4 Natural and Artificial Transformation
7.4.1 Natural Transformation
7.4.2 Artificial Transformation
7.5 Gene Linkage and Mapping by Transformation
7.6 Answers to Check Your Progress Questions
7.7 Summary
7.8 Key Words
7.9 Self Assessment Questions and Exercises
7.10 Further Readings

7.0 INTRODUCTION

Transformation is one of the methods of horizontal gene transfer between bacteria.

It is a process in which a recipient cell takes up naked DNA from the surrounding
medium. This DNA is integrated into the recipient genome by homologous
recombination. Basically, the donor cell lyses and fragments of its DNA are released
out in the medium. This exogenous, naked DNA is taken up by the recipient cell.
Unlike conjugation, no physical contact is required between the donor and recipient
cells during transformation.
Difference from transduction is that in this case ssDNA enters the recipient
cell cytoplasm whereas in case of transduction dsDNA enters the cell. Both Gram-
Positive and Gram-Negative Bacteria can exhibit the ability of transformation in
nature mainly under stress conditions. It was discovered in Streptococcus
pneumonia and has been extensively studied in various medically Important Gram-
Negative Bacteria species, such as Helicobacter pylori, Haemophilus
influenzae and Vibrio cholera.
In this unit, you will study about concept of transformation, concept of
transformation, mechanism of natural and artificial transformation and gene mapping
using transformation in detail.

7.1 OBJECTIVES

After going through this unit, you will be able to:

Understand the concept of transformation
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142 Material
 Explain the mechanism of natural and artificial transformation Transformation

Describe gene mapping using transformation

7.2 DISCOVERY OF TRANSFORMATION NOTES

Transformation was first reported in Streptococcus pneumoniae by Frederick

Griffith in 1928. DNA as the transforming principle was demonstrated by Avery
et al in 1944. In 1928 Griffith used two strains of Pneumococcus (Streptococcus
pneumoniae) bacteria which infect mice – a Type III-S (Smooth) which was
virulent, and a Type II-R (Rough) strain which was non-virulent. In this experiment,
bacteria from the III-S strain were killed by heat, and their remains were added to
II-R strain bacteria. While neither alone harmed the mice, the combination was
able to kill its host. Griffith was also able to isolate both live II-R and live III-S
strains of pneumococcus from the blood of these dead mice. Griffith concluded
that the Type II-R had been ‘transformed’ into the lethal III-S strain by a
‘Transforming Principle’ that was part of the dead III-S strain bacteria (Refer
Figure 7.1).
The ‘transforming principle’ Griffith observed was the DNA of the III-s
strain bacteria. While the bacteria had been killed, the DNA had survived the
heating process and was taken up by the II-R strain bacteria.

Fig. 7.1 Griffith’s Experiment Reporting Transformation

The exact nature of the transforming principle (DNA) was verified in the
experiments done by Avery, McLeod and McCarty (Refer Figure 7.2). In 1944,
Avery, Macleod, McCarty revisited the experiment by Griffith with genetic material
transforming in strands of bacteria. Using the same strain of bacteria, S strain,
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Transformation Avery and his team extracted pure DNA, pure proteins and other materials from
the bacterial cells. The materials were mixed with the R strain of bacteria. Rather
than using mice, test tubes were used to show the mixing of materials. After
examining each mixture, only the R strain mixed with the pure DNA transformed
NOTES into the S strain bacteria. Since this transformation only occurred within the pure
DNA composition, implications that DNA hold genetic material were produced.
Because of this reason, transformation is sensitive to DNAse (enzymes that cleave
DNA).

Fig. 7.2 Avery, Macleod, McCarty Experiment for Finding out the
Transforming Principal

Check Your Progress

1. When was transformation reported for the first time?
2. What conclusion did Griffith drew from his experiment?
3. When was the exact nature of the transforming principle (DNA) verified?

7.3 COMPETENCE

Competence is the ability of cell to take up naked DNA. Competence usually

arises at late log phase or stationary phase. It is often triggered by nutritional
shortages or stress conditions. Natural competence is genetically encoded. It results
from changes in cell wall of bacteria. Receptors on the cell surface are responsible
for initial binding of DNA. The number of active receptors varies from 80 for
Self-Instructional Pneumococcus and 4 in Haemophilus.
144 Material
 Competence initially arises in only small fraction of cells. These cells secrete Transformation

competence factors that act as signaling molecules or autoinducers or pheromones

to make the rest of the cells in the population competent. This is an example of the
phenomenon known as quorum sensing. Quorum sensing describes the bacterial
communication between cells that enables populations of bacteria to synchronously NOTES
regulate gene expression and therefore behavior. Competence pheromones are
short peptides that are secreted into the culture medium by dividing cells. In late
log phase, when density of bacteria is high, sufficient levels of pheromones is
achieved and this triggers competence. This mechanism is meant to ensure that
any DNA taken up will come from related bacteria as competence is only induced
when there are large number of cells of same species.
Types of Competence
There are two types of competence, i.e., as follows:
Natural Competence Artificial Competence
Naturally Transformable Bacterium (or naturally competent bacterium) can
take up DNA from the environment without requiring special treatment. Examples
of naturally competent cells include Bacillus subtilis, Streptococcus pneumonia,
Haemophilus influenza, Neisseria gonorrhoeae and Helicobacter pylori.
Reasons or evolutionary significance of natural competence in bacteria
include:
Selective advantage of genetic diversity.
DNA uptake as a source of Nucleotides (DNA as food). The recipient
cells require nucleotides for their own DNA and RNA synthesis during
starvation conditions, so they become naturally component and take up
exogenous DNA and utilize their nucleotides for their own synthesis.
Some naturally competent bacteria also secrete nucleases into their
surroundings, and all bacteria can take up the free nucleotides these
nucleases generate from environmental DNA.
Repair of DNA damage. The selective advantage of a new strand of
DNA to promote homologous recombinatorial repair of damaged DNA.
The problem of DNA damage is most pronounced during periods of
oxidative stress that occur during crowding or starvation conditions.
Artificial Competence can also be induced in laboratory making the recipient
cells passively permeable to DNA, by exposing it to conditions that do not normally
occur in nature.

Check Your Progress

4. Define competence.
5. When competence arises?
6. What binds DNA?
7. How many types of competence are there?
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Transformation
7.4 NATURAL AND ARTIFICIAL
TRANSFORMATION

NOTES Transformation is one of three basic mechanisms for genetic exchange in bacteria.
Transformation may be either a natural process that is, one that has evolved in
certain bacteria or it may be an artificial process whereby the recipient cells are
forced to take up DNA by a physical, chemical, or enzymatic treatment. In both
cases, exogenous DNA (DNA that is outside the host cell), is taken into a recipient
cell where it is incorporated into the recipient genome, changing the genetic makeup
of the bacterium. Natural and artificial transformation is described in detail below.
7.4.1 Natural Transformation
Gram-Positive and Gram-Negative Bacteria differ in the structure of their cell
envelope, thus there are some differences in the mechanisms of DNA uptake in
these cells.
The basic steps of transformation include the following (Refer Figure 7.3)
Binding of double-stranded DNA to the outer cell surface of bacterium.
Movement of DNA across the cell wall and outer membrane (no outer
membrane in gram positive bacterium).
Degradation of one of the DNA strands.
Translocation of the remaining single strand of DNA into cytoplasm of the
cell across inner membrane.
Once in the cell, the single-stranded transforming DNA might synthesize the
complementary strand and re-establish itself as a plasmid, stably integrate
into the chromosome, or get degraded.
The uptake of DNA is generally non-sequence specific, although in some species
the presence of specific DNA uptake sequences may facilitate efficient DNA
uptake.

Fig. 7.3 Overview of DNA Uptake in Bacteria

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Mechanism of Natural Transformation in Bacillus subtilis Transformation

Various competence factors are involved in uptake of DNA in Gram-Positive

Bacterium, Bacillus subtilis. Some of these include Pilin Complex (ComGC),
DNA Binding Protein (ComEA), Nuclease (N), Channel Protein (ComEC) and NOTES
DNA Translocase that moves the DNA into the Cytoplasm (ComFA) (Refer Figure
7.4).
In Gram-Positive Bacteria pseudopilus system is responsible for DNA
uptake. It acts as a piston, moving up and down in the thick peptidoglycan cell
wall layer thereby binding to extracellular dsDNA and pulling it towards the cell
membrane where DNA uptake proteins reside. In Bacillus subtilis, major
pseudopaline complex ComGC is able to assemble into a polymeric structure that
is essential for transformation. Next, dsDNA bind to ComEA, DNA binding protein
which shuffles the DNA to the inner membrane transporter protein, ComEC.
Transport across the inner membrane is accomplished by ComEC concomitantly
with the degradation of one strand of dsDNA by nuclease producing ssDNA.
Next, this ssDNA binds to ComFA that moves it into the cytoplasm. Inside the
cytoplasm, this ssDNA is bound by various SSB (Single Strand Binding) Proteins
for protection against degradation. Finally, it gets integrated into the recipient
genomic DNA by homologous recombination catalysed by RecA Proteins.

Fig. 7.4 Competence in Bacillus subtilis

Mechanism of Natural Transformation in Streptococcus pneumoniae

Streptococcus pneumonia is an important human pathogen. Natural transformation
was discovered in this species. It involves internalization of exogenous DNA and
its incorporation into the genome. Environmental factors trigger the competence.
Once competent, transformation is mediated by a series of competence factors or
proteins.

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Transformation Steps in transformation of Streptococcus include the following (Refer Figure
7.5):
Exogenous dsDNA attaches to the Pilus (ComGC).
NOTES It is passed to the DNA Receptor Protein ComEA.
ComEA directs it to EndA Nuclease which degrades one strand of the
dsDNA resulting in ssDNA. This ssDNA is transferred across the
membrane via ComEC pore.
After entry into the cell, ssDNA is coated with SSBs which protect it
from nuclease degradation.
Next, recombinase DprA also binds the ssDNA and recruits
recombinase RecA onto ssDNA.
RecA can then polymerize on the ssDNA, and promotes strand exchange.

Self-Instructional Fig.7.5 Competence in Streptococcus pneumonia

148 Material
7.4.2 Artificial Transformation Transformation

Most of the cells are not naturally competent. In addition, natural competence and
transformation are efficient for only linear molecules, such as chromosomal DNA
but not for circular plasmid molecules. Thus, prokaryotic as well as eukaryotic NOTES
cells need to be made competent artificially in laboratory. This is an essential step
in recombinant DNA technology, mainly to incorporate plasmids carrying gene of
interest into the host cells. Unlike natural competence, artificial competence is not
encoded by genes of the bacteria.
Methods of Artificial Competence in Bacteria
There are various methods to prepare competent cells in the laboratory. Few of
them include:
Calcium Chloride Mediated Transformation
Freeze Thaw Method
Electroporation
Calcium Chloride Method: It is the most common chemical method of making
cells competent. Treatment with Calcium ions make some Bacteria competent.
The Calcium ions increase the permeability of cell membrane making transient
pores which facilitate DNA entry. Basically, role of Calcium ions is to neutralize
the negative charges present on the surface of DNA and LipoPolySaccharide
(LPS) to avoid electrostatic repulsion between the two. Positively charged Calcium
ions (Ca2+) attract both the negatively charged DNA backbone (Phosphate) and
the negatively charged groups in the LPS inner core. Other divalent cations, such
as MnCl2 and KCl can also increase the efficiency of transformation. DiMethyl
SulfOxide (DMSO) can also increase the efficiency by generating ionic shield.
Once competent cells are prepared, the plasmid DNA is then added to the
cells by heat shock method, where chilled cells are heated to a higher temperature
of 42oC for short time (generally two minutes). A sudden increase in temperature
further creates pores in the plasma membrane of the bacteria and allows for plasmid
DNA to enter the bacterial cell. After heat shock, the mixture is immediately placed
on ice for 1-2 minutes before adding culture medium for growing. Intact plasmid
DNA molecules replicate in bacterial host cells. To help the bacterial cells recover
from the heat shock cells are briefly incubated with non-selective growth media.
As the cells recover, plasmid genes are expressed. Bacterial colonies selected
using antibiotic selection techniques (Refer Figure 7.6).

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Transformation

NOTES

Fig. 7.6 Steps of Bacterial Transformation by Calcium Chloride Method

In case of artificial transformation, transformation efficiency can also be

calculated. Transformation efficiency is defined as the number of Colony Forming
Units (CFUs) which would be produced by transforming 1 g of plasmid into a
given volume of competent cells.
Freeze Thaw Method: In the freeze thaw method, actively growing cells
are pelleted down by centrifugation at 5000 rpm at room temperature. Pellet is
washed. Cells are then frozen in liquid nitrogen and stored at -70oC. Competent
cells are thawed on ice and DNA is added. Cells are then incubated for recovery.
Transformed cells are selected by antibiotic selection.
Electroporation: Electroporation is a method of transformation that allows
the introduction of foreign DNA into host cells via the application of high-voltage
electric pulses.
Cells are placed in buffer and put into electroporator. DNA is added and
subjected to a high-voltage electrical pulse of defined magnitude and length. The
cells are then allowed to recover and are finally selected by antibiotic selection
technique.

Check Your Progress

8. Is transformation natural or artificial?
9. How does uptake of DNA occur in bacteria pseudopilus system?
10. What are the methods of artificial competence in bacteria?
11. Define the term electroporation.
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 Transformation
7.5 GENE LINKAGE AND MAPPING BY
TRANSFORMATION

Genetic linkage is the tendency of DNA sequences that are close together on NOTES
a chromosome to be inherited together. Genes that are located on the same
chromosome are described as linked genes. If genes are very close together, they
are more likely to be transmitted as a block. The distance between genes is
designated as map units.
Transformation is used to map genes where mapping by conjugation or
transduction is not possible. Transformation experiments can be used to determine
the following:
Whether the genes are linked.
Order of genes on the genetic map.
Map distance between the genes.
In case transformation experiments, donor DNA is purified, fragmented
and added to competent recipient bacteria. Donor and recipient both have
detectable differences in phenotype and therefore genotype. If the DNA fragment
undergoes homologous recombination with the recipient’s chromosome, a new
phenotype may be produced. Transformants are detected by testing for phenotypic
changes (Refer Figure 7.7).

Fig. 7.7 Gene Mapping Experiments using Transformation

Gene linkage and order can also be determined by cotransformation, i.e.,

the ability of two genetic markers to be transformed together. If two donor genes
are located close together on the chromosome, then there is a greater chance that
they will be carried on the same piece of transforming DNA and hence will cause
a double transformation or cotransformation. Conversely, if genes are widely
separated on the chromosome, then most likely they will be carried on separate
transforming segments and the frequency of double transformants will equal the
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Transformation product of the single-gene transformation frequencies. Thus, it is possible to test
for close linkage. For example, if a+ b+ donor DNA is used to transform a– b–
recipient cells, then, if a and b are closely linked, the proportion of a+ b+
cotransformants, should exceed the product of the proportions of
NOTES single a+ and b+ transformants. Relative map distances of closely linked genes can
be deduced from cotransformation percentages in an approach similar to that of
cotransduction frequencies.

Check Your Progress

12. What is genetic linkage?
13. What are linked genes?
14. How can the distance between genes designated?
15. Give the uses of transformation experiments.

7.6 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. Transformation was first reported in Streptococcus pneumoniae by

Frederick Griffith in 1928.
2. Griffith concluded that the type II-R had been ‘transformed’ into the lethal
III-S strain by a ‘Transforming Principle’ that was part of the dead III-S
strain bacteria.
3. The exact nature of the transforming principle (DNA) was verified in the
experiments done by Avery, McLeod and McCarty.
4. Competence is the ability of cell to take up naked DNA.
5. Competence usually arises at late log phase or stationary phase.
6. Receptors on the cell surface are responsible for initial binding of DNA.
The number of active receptors varies from 80 for Pneumococcus and 4 in
Haemophilus.
7. There are two types of competence, i.e., as follows:
Natural Competence
Artificial Competence
8. Transformation may be either a natural process that is, one that has evolved
in certain bacteria or it may be an artificial process whereby the recipient
cells are forced to take up DNA by a physical, chemical, or enzymatic
treatment.
9. In Gram-Positive Bacteria pseudopilus system is responsible for DNA
uptake. It acts as a piston, moving up and down in the thick peptidoglycan
cell wall layer thereby binding to extracellular dsDNA and pulling it towards
the cell membrane where DNA uptake proteins reside.
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 10. There are various methods to prepare competent cells in the laboratory. Transformation
Few of them include:
Calcium Chloride Mediated Transformation
Freeze Thaw Method NOTES
Electroporation
11. Electroporation is a method of transformation that allows the introduction
of foreign DNA into host cells via the application of high-voltage electric
pulses.
12. Genetic linkage is the tendency of DNA sequences that are close together
on a chromosome to be inherited together.
13. Genes that are located on the same chromosome are described as linked
genes.
14. The distance between genes is designated as map units.
15. Transformation experiments can be used to determine the following:
Whether the genes are linked.
Order of genes on the genetic map.
Map distance between the genes.

7.7 SUMMARY
Transformation was first reported in Streptococcus pneumoniae by
Frederick Griffith in 1928. DNA as the transforming principle was
demonstrated by Avery et al., in 1944.
In 1928 Griffith used two strains of Pneumococcus (Streptococcus
pneumoniae) Bacteria which infect mice – a Type III-S (Smooth) which
was virulent, and a Type II-R (Rough) strain which was non-virulent.
Griffith was also able to isolate both live II-R and live III-S strains of
pneumococcus from the blood of these dead mice.
Griffith concluded that the Type II-R had been ‘transformed’ into the lethal
III-S strain by a ‘Transforming Principle’ that was part of the dead III-S
strain bacteria.
The ‘transforming principle’ Griffith observed was the DNA of the III-s
strain bacteria. While the bacteria had been killed, the DNA had survived
the heating process and was taken up by the II-R strain bacteria.
The exact nature of the transforming principle (DNA) was verified in the
experiments done by Avery, McLeod and McCarty.
In 1944, Avery, Macleod, McCarty revisited the experiment by Griffith
with genetic material transforming in strands of bacteria.
Competence is the ability of cell to take up naked DNA. Competence
usually arises at late log phase or stationary phase. It is often triggered by
nutritional shortages or stress conditions. Self-Instructional
Material 153
Transformation Natural competence is genetically encoded. It results from changes in cell
wall of bacteria.
Receptors on the cell surface are responsible for initial binding of DNA.
The number of active receptors varies from 80 for Pneumococcus and 4 in
NOTES Haemophilus.
Competence initially arises in only small fraction of cells. These cells secrete
competence factors that act as signaling molecules or autoinducers or
pheromones to make the rest of the cells in the population competent.
Competence pheromones are short peptides that are secreted into the culture
medium by dividing cells. In late log phase, when density of bacteria is high,
sufficient levels of pheromones is achieved and this triggers competence.
Naturally Transformable Bacterium (or naturally competent bacterium) can
take up DNA from the environment without requiring special treatment.
Transformation is one of three basic mechanisms for genetic exchange in
bacteria.
Transformation may be either a natural process that is, one that has evolved
in certain bacteria or it may be an artificial process whereby the recipient
cells are forced to take up DNA by a physical, chemical, or enzymatic
treatment.
In both cases, exogenous DNA (DNA that is outside the host cell), is taken
into a recipient cell where it is incorporated into the recipient genome,
changing the genetic makeup of the bacterium.
Gram-Positive and Gram-Negative Bacteria differ in the structure of their
cell envelope, thus there are some differences in the mechanisms of DNA
uptake in these cells.
The uptake of DNA is generally non-sequence specific, although in some
species the presence of specific DNA uptake sequences may facilitate
efficient DNA uptake.
Various competence factors are involved in uptake of DNA in Gram Positive
Bacterium, Bacillus subtilis. Some of these include Pilin Complex
(ComGC), DNA Binding Protein (ComEA), Nuclease (N), Channel Protein
(ComEC) and DNA Translocase that moves the DNA into the Cytoplasm
(ComFA).
In Gram-Positive Bacteria pseudopilus system is responsible for DNA
uptake. It acts as a piston, moving up and down in the thick peptidoglycan
cell wall layer thereby binding to extracellular dsDNA and pulling it towards
the cell membrane where DNA uptake proteins reside.
Streptococcus pneumonia is an important human pathogen. Natural
transformation was discovered in this species.
Most of the cells are not naturally competent. In addition, natural competence
and transformation are efficient for only linear molecules, such as
chromosomal DNA but not for circular plasmid molecules.
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 In the freeze thaw method, actively growing cells are pelleted down by Transformation

centrifugation at 5000rpm at room temperature.

Electroporation is a method of transformation that allows the introduction
of foreign DNA into host cells via the application of high-voltage electric
pulses. NOTES
Genetic linkage is the tendency of DNA sequences that are close together
on a chromosome to be inherited together.
Genes that are located on the same chromosome are described as linked
genes.
If genes are very close together, they are more likely to be transmitted as a
block.
The distance between genes is designated as map units.
Transformation is used to map genes where mapping by conjugation or
transduction is not possible.
In case transformation experiments, donor DNA is purified, fragmented
and added to competent recipient bacteria.
Donor and recipient both have detectable differences in phenotype and
therefore genotype.
If the DNA fragment undergoes homologous recombination with the
recipient’s chromosome, a new phenotype may be produced.
Gene linkage and order can also be determined by cotransformation, i.e.,
the ability of two genetic markers to be transformed together.

7.8 KEY WORDS

Transformation: It is type of HGT in bacteria involving uptake of free

DNA from the environment.
Transforming principle: Component which is able to cause transformation
of the recipient cell. DNA is the transforming principle.
Competence: It is the ability of cell to take up naked DNA.
Quorum sensing: It is the regulation of gene expression in response to
fluctuations in cell-population density.
Cotransformation: It is the ability of transformation of two closely placed
genetic markers together.
Transformation efficiency: Transformation efficiency is defined as the
number of Colony Forming Units (CFUs) which would be produced by
transforming 1 µg of plasmid into a given volume of competent cells.

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Transformation
7.9 SELF ASSESSMENT QUESTIONS AND
EXERCISES

NOTES Short Answer Questions

1. Define the term transformation.
2. What is competence?
3. Write the differences between natural and artificial competence.
4. Enlist the competence factors involved in natural transformation of
Streptococcus.
5. What is gene linkage?
6. What is cotransformation and it application?
Long Answer Questions
1. Discuss about transformation in detail. Also write about the discovery of
transformation.
2. Write a note on types of transformation.
3. Diagrammatically explain the mechanism of competence in Bacillus subtilis.
4. What is natural transformation? Explain the Mechanism of natural
transformation in Streptococcus pneumonia.
5. Explain artificial transformation and methods of artificial competence.
6. Elaborate a note on gene linkage. How can transformation be used to
determine linkage of genes?
7. Elaborate on the various methods of artificial transformation.
8. Describe the experiment of discovery of transformation.
9. A transformation experiment is carried out using a donor strain that has
three chromosomal antibiotic resistance mutations, namely strR , rifR , and
nalR. These mutations confer resistance to the Antibiotics Streptomycin,
Rifampicin and Nalidixic Acid. The recipient strain is sensitive to all 3
antibiotics. The results are:

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What do you conclude about the positions of the genes?
156 Material
 Transformation
7.10 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing NOTES
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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Transduction
and its Types
UNIT 8 TRANSDUCTION
AND ITS TYPES
NOTES
Structure
8.0 Introduction
8.1 Objectives
8.2 Bacteriophage and its Types
8.3 Transduction: Discovery and Types
8.3.1 Types of Transduction
8.3.2 Generalized Transduction
8.3.3 Specialized Transduction
8.3.4 Transduction Mapping
8.4 Answers to Check Your Progress Questions
8.5 Summary
8.6 Key Words
8.7 Self Assessment Questions and Exercises
8.8 Further Readings

8.0 INTRODUCTION

Horizontal Gene Transfer (HGT) is the transfer of genetic material from one bacteria
to another. It is mediated by three methods, viz., conjugation, transduction and
transformation. Conjugation has been discussed in detail in the previous unit. Here,
we will study transduction. Transduction is the process by which DNA is transferred
from one bacteria to another via a bacteriophage (virus that infects bacteria). Such
a virus particle carrying bacterial DNA is called a transducing particle. Unlike
conjugation, transduction does not require physical contact between the two cells
neither it is inactivated by DNase as in case of transformation.
Transduction is a type of horizontal gene transfer in bacteria leading to transfer
of transfer of genes from one bacteria to another via bacteriophages. It is of two
type, viz., generalized and specialized. In case of generalized transduction, any
part of the host bacterial genome can be transferred. It is mediated by lytic phages,
such as T4 Phage and results from error in packaging of the genome in phage
head. In case of specialized transduction, only specific regions of the host genome,
i.e., the genes flanking the site of integration of the phage genome can be transferred.
It is mediated by lysogenic phages such as lambda phage and results from error in
excision of the phage genome from the host genome. The genes transferred via
transduction can recombine in the recipient cell via homologous recombination.
Transduction can be used as a means of genetic mapping. Cotransduction frequency
of two genetic markers can be calculated. Closer the genes, more is their probability
of being transduced together, higher will be their cotransduction frequency. This
basis is used to infer the linkage between genes. Three factor crosses can further
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be used to decipher the order of the gene. Besides mapping, other applications of Transduction
and its Types
transduction include their use as cloning vectors and for hybridization studies.
In this unit, you will study about types of life cycle of bacteriophages, concept
of genetic transfer in bacteria by bacteriophages, types of transduction, uses of
NOTES
specialized transduction and gene mapping using bacteriophages in detail.

8.1 OBJECTIVES

After going through this unit, you will be able to:

Discuss the types of life cycle of bacteriophages
Explain the concept of genetic transfer in bacteria by bacteriophages
Understand the types of transduction
Analyse the uses of specialized transduction
Discuss gene mapping using bacteriophages

8.2 BACTERIOPHAGE AND ITS TYPES

Transduction is virus mediated bacterial DNA transfer. Virus that infect bacteria
are called bacteriophages. Although each virus has unique aspects to its life cycle,
a general pattern of replication is observable. The typical virus life cycle consists
of five steps, namely attachment to the host cell, entry into the host cell, synthesis
of viral nucleic acid and proteins within the host cell, self-assembly of virions
(progeny virus) within the host cell, and release and maturation of virions from the
host cell.
On the basis of their replication cycle bacteriophages are of two types, i.e.,
as follows (Refer Figure 8.1):
Lytic Bacteriophages
Lysogenic Bacteriophages
Lytic Bacteriophages
Lytic bacteriophages are those that break open or lyse the host bacterial cell
immediately after replication of the virion. As the bacterial cell is Lysed then the
phage Progeny can find new hosts to infect. The phages that carry out Lytic Cycle
are called Virulent or Lytic Phages. An example of a Lytic Bacteriophage is Phage
T4, which infects Escherichia coli.
Lysogenic Bacteriophages
Lysogenic phages are the one that do not result in immediate lysis of the host
bacterial cell. Instead their viral genome gets stably integrated into the host genome
forming a prophase. This prophage DNA which has the viral DNA incorporated
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Transduction can be passed down to the daughter cells when the bacteria divides. The phage
and its Types
remains dormant thus does not harm the host. However, under unfavorable and
stressful conditions, such as depletion of nutrients or UV exposure, the phage
genome excises out of the host genome and the phage switches to Lytic cycle.
NOTES They initiate their genome replication, assemble and result in lysis of host cell to
release out. The phage that carry out Lysogenic cycle are called temperate phages.
An example of such a bacteriophage is Lambda ( ) phage, which infects
Escherichia coli.

Fig. 8.1 Lytic versus Lysogenic Cycle of Bacteriophage

Check Your Progress

1. How many types of bacteriophages are there?
2. Define lytic bacteriophages.
3. What is the difference between virulent and temperate bacteriophage?
4. What are lysogenic phages?

8.3 TRANSDUCTION: DISCOVERY AND TYPES

Transduction was discovered by Norton Zinder and Joshua Lederberg at

the University of Wisconsin–Madison in 1952 using Salmonella typhimurium.
They used two Salmonella auxotrophic strains LA-22 and LA-2. Auxotrophic
strain is a mutant that cannot grow on minimal medium and requires certain
supplements whereas prototrophic strains are wild type as it will grow in minimal
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medium or medium lacking the supplement. In this experiment, Salmonella strain Transduction
and its Types
LA-2 was auxotroph for methionine and histidine (met- his-) and strain LA-22 was
auxotroph for phenylalanine and tryptophan (phe- trp-). They mixed the two strains
together in the U-tube, and when the mixture was plated on minimal medium, they
recovered prototrophic cells (Refer Figure 8.2). This observation showed that NOTES
there is some Filterable Agent (FA) that transferred the Phenylalanine and
Tryptophan marker from strain LA-2 to LA-22 making it prototrophic.

Fig. 8.2 Lederberg-Zinder Experiment of Bacterial Transduction

Three observations were used to identify the Filterable Agent (FA):

The FA was produced by the LA-2 cells only when they were grown in
association with LA-22 cells.
The addition of DNase, which enzymatically digests DNA, did not render
the FA ineffective. Therefore, the FA is not exogenous DNA, ruling out
transformation.
The FA could not pass across the filter of the Davis U-tube when the pore
size was reduced below the size of bacteriophages.
Aided by these observations and aware that temperate phages could lysogenize
Salmonella, researchers proposed that the genetic recombination event was
mediated by bacteriophage P22 and they named this process as transduction.
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Transduction 8.3.1 Types of Transduction
and its Types
There are two types of transduction depending on which part of bacterial DNA is
transferred to the recipient cell. The two forms are:
NOTES Generalized Transduction: Any piece of the bacterial genome can be
transferred.
Specialized Transduction: Only specific pieces of the bacterial
chromosome can be transferred.
8.3.2 Generalized Transduction
Generalized transduction is the process by which any part of bacterial DNA can
be transferred from donor to recipient bacteria via a bacteriophage. It is a rare
event with 1 in 10,000 phages being transducing. It is a random event as any part
of the bacterial genome can be transferred to the recipient cell. Lytic bacteriophages
carry out generalized transduction. Generalized transducing phage have a packaging
error and thus by mistake pack segment of bacterial DNA in place of viral DNA.
Bacteriophage pack their genome by headful packaging mechanism so any part of
bacterial host genome which is similar in size to its own genome can be packaged
leading to formation of a transducing phage. Thus, generalized transducing phage
produce some particles that contain only DNA obtained from the host bacterium,
rather than phage DNA. Examples of generalized transducing phage include
Escherichia coli P1 and Salmonella typhimurium P22.
Requirements for generalized transducing phage are:
Phage must not degrade the host DNA completely.
Less pac specificity during packaging.
Package by the headful rule (based only on size of the DNA).
The transducing phage loaded with bacterial DNA continues to infect another
bacterial cell.
Fate of DNA that enters the recipient cell:
The DNA can be degraded.
It can circularize to become a plasmid.
If it matches with a homologous region of the recipient cell’s chromosome,
it can be exchanged by bacterial recombination.
I. Escherichia coli Phage P1 - Generalized Transducing Phage
During infection of Escherichia coli by phage P1, a phage-encoded nuclease is
made that causes fragmentation of bacterial DNA. A single fragment of bacterial
DNA comparable in size to P1 DNA is packaged into a phage particle instead of
P1 DNA. The positions of the nuclease cuts in the host chromosome are fairly
random, so a transducing particle may contain a fragment derived from any region

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of the host. When a transducing particle adsorbs to a bacterium, the DNA carried Transduction
and its Types
within the particle head is injected into the cell and becomes available for double
crossing over with the homologous region of the bacterial chromosome. For
example, Phage P1 can transfer Leucine marker converting leu- recipient strain of
Escherichia coli into leu+ (Refer Figure 8.3). NOTES

Fig. 8.3 Generalized Transduction by Escherichia coli Phage P1

II. Abortive Transduction

• Transduction requires that the recipient cell be Rec+ for recombination to
occur. No transductants are observed with RecA– or RecBC recipients.
The donor cell can be either Rec+ or Rec– . RecA protein is required for
single strand displacement and RecBC protein unwinds the helix.
• Abortive transduction is an event in which transducing DNA fails to be
incorporated into the recipient chromosome.
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Transduction • The bacterial DNA remains in the cytoplasm and does not replicate but is
and its Types
transmitted to one progeny cell following each division.
• Only a single cell, partially diploid for the transduced genes is produced.
NOTES 8.3.3 Specialized Transduction
In case of specialized transduction, only specific parts of host genome can be
transferred to the recipient cell. It takes place via temperate phages, i.e., phages
that undergo both lysogenic and lytic cycle. It is produced during induction of
Lysogen. As we discussed earlier, integrated phage DNA can excise out from the
bacterial genome and follow lytic cycle. Specialized transducing phages are
produced when there is error in excision. Host genes that flank the integrated
phage DNA are also excised out by mistake leaving some of its own genes. Thus,
transducing phage produces particles containing both phage and bacterial genes
linked in a single DNA molecule.
Example of a sspecialized transducing phage is Lambda ( ) phage. It gets
integrated between the Galactose (Gal) and Biotin (Bio) Operon. Due to error in
excision or abnormal out looping, it can carry Genes of Gal or Bio Operon leaving
behind some of its own genes. As some of its own genes have been replaced by
bacterial genes, these phages are defective and cannot reproduce. The dgal or gal
carries Bacterial Gal Genes and lacks tail fiber genes and thus cannot make Progeny
or Plaques (Refer Figure 8.4). Similarly, dbio or bio carries the Biotin Genes
and lacks integration, exision genes thus cannot integrate.

Fig. 8.4: Formation of Specialized Transducing Phage - dgal

Types of Specialized Transduction

Depending on the frequency of transducing phages obtained after lysis of the host
cells, specialized transduction is of two types:
Low Frequency Transduction (LFT)
High Frequency Transduction (HFT)

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Low Frequency Transducing (LFT) Lysate Transduction
and its Types
A lysate formed from a single Lysogen in which wrong excision occurs only
infrequently is called a LFT lysate.
LFT Lysates are the Lysates containing mostly normal phages and just a NOTES
few specialized transducing phages.
LFT is a result of rare incorrect excision events from a single Lysogen produce
transducing particles at about 10-6 to 10-7 of the total phage particles in the
Lysate.
High Frequency Transducing (HFT) Lysate
Lysates containing a relatively large number of specialized transducing
phages.
They are created by co-infecting a host cell with a helper phage (normal
phage) and a transducing phage, i.e., Dilysogen (Refer Figure 8.5).
In case of transducing phage certain genes are replaced by bacterial genes
so it cannot replicate. However, co-infection with helper phage allows the
transducing phage to replicate as it provides functional counterparts of genes
that are lacking in transducing phage, thus increasing the number of transducing
phages in the Lysate.
The Dilysogens yield Lysates half of whose phage are transducing particles.

Fig. 8.5: Dilysogen with dgal and helper Integrated into the Escherichia
coli Genome
Both LFT and HFT also contain phage that are not transducing particles for
LFT Lysates these will be 99.99% of the total particles in the population and for
HFT Lysates these will be 50% of the total particles in the population.
Uses of Specialized Transducing Phages
Can be used as cloning vehicle as they can transfer specific bacterial genes
into the recipient cell.
The gal can serve as a source of Gal DNA for base-sequencing analysis.
Specialized transducing phages have also served as hybridization probes
for identifying specific mRNA molecule.
8.3.4 Transduction Mapping
• Only a small amount of chromosome, a few genes, can be transferred by
transduction. The closer the two genes are to each other, the more likely Self-Instructional
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Transduction they are to be transduced by the same phage. Thus, ‘Cotransduction
and its Types
Frequency’ is the key parameter used in mapping genes by transduction.
Cotransduction frequency is defined as the ratio of transductants that co-
inherit both markers divided by the total number of transductants.
NOTES
• Linkages are expressed as cotransduction frequency. Generalized
transduction can be used to derive linkage information about bacterial genes
in cases in which genes are close enough that the phage can pick them up
and transduce them as a single piece of DNA.
• Transduction studies can also determine the precise order of these genes.
• Consider three genes ‘a’, ‘b’ and ‘c’ for which the frequencies of
cotransduction are:
a-b : 90%
a-c : 33%
b-c : 32%
The values indicate that ‘b’ is closer to ‘a’ than to ‘c’, but the order cannot
still be deduced as it can either be ‘a-b-c’ or ‘b-a-c’. Only order ‘a-c-b’ can be
excluded.
Three factor crosses can be used to determine the order of closely linked
markers with respect to a third gene. Principle of this method is that the probability
of appearance of a genotype requiring four exchanges for its formation is much
smaller than the probability of one formed by two exchanges. The results of
reciprocal crosses are compared. A reciprocal cross is cross with the phenotype
of each sex reversed as compared with the original cross.
If for Cross 1 the frequency of ‘+ + +’ is observed to be much less than that
of Cross 2 then, the order must be ‘a-b-c’.
Another Method for Mapping
• Transduction between Donor Strain A+B+C- to recipient that is A-B-C+.
• One phenotype A+ is checked by plating on medium lacking A and 100 A+
colonies are tested further for B and C genotypes by replica plating.

Genotypes Observed Number of Colonies

A+B+C+ 5

A+B+C 19

A+B C+ 49

A+B C 27

100

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 1. Calculate Cotransduction Frequencies as follows: Transduction
and its Types
A+B+ (5 + 19) / 100 = 0.24
A+C- (19 + 27) / 100 = 0.46
Thus, C is nearer to A than B is to A. NOTES
Order can be A-C-B or C-A-B
2. Rule to Analyze the Data: The rarest recombination class is that achieved
by the maximum number of exchanges.
3. Rarest class is A+B+C+ and thus Order 1 (A-C-B) is the correct or
accurate order.

Check Your Progress

5. What is generalized transduction?
6. Which specific part of genome can be transferred during specialized
transduction?
7. Why does HFT Lysate have large number of specialized transducing
phages?

8.4 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. On the basis of their replication cycle bacteriophages are of two types, i.e.,
as follows:
Lytic Phage
Lysogenic Phage
2. Lytic bacteriophages are those that break open or lyse the host bacterial
cell immediately after replication of the virion.
3. Virulent bacteriophage carries out lytic cycle of replication whereas temperate
phage can carry out both lysogenic as well as lytic cycle.
4. Lysogenic phages are the one that do not result in immediate lysis of the
host bacterial cell.
5. It is called generalized transduction because any random part of the host
bacterial genome can be transferred to the recipient cell.
6. Only the host genes flanking the site of integration of the phage genome can
be transferred via specialized transduction.
7. HFT Lysate has large number of specialized transducing phages because
the genome of the helper phage gets integrated along with the transducing
phage genome forming a Dilysogen. Helper phage aids in transducing phage
replication.
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Transduction
and its Types 8.5 SUMMARY

Transduction is virus mediated bacterial DNA transfer. Virus that infect

NOTES bacteria are called bacteriophages.
Although each virus has unique aspects to its life cycle, a general pattern of
replication is observable.
The typical virus life cycle consists of five steps, namely attachment to the
host cell, entry into the host cell, synthesis of viral nucleic acid and proteins
within the host cell, self-assembly of virions (progeny virus) within the host
cell, and release and maturation of virions from the host cell.
Lytic bacteriophages are those that break open or lyse the host bacterial
cell immediately after replication of the virion.
As the bacterial cell is lysed, the phage progeny can find new hosts to
infect.
The phages that carry out lytic cycle are called virulent or lytic phages.
Lysogenic phages are the one that do not result in immediate lysis of the
host bacterial cell.
The phage that carry out lysogenic cycle are called temperate phages. An
example of such a bacteriophage is Lambda ( ) phage, which infects
Escherichia coli.
Transduction was discovered by Norton Zinder and Joshua Lederberg at
the University of Wisconsin–Madison in 1952 using Salmonella
typhimurium.
Auxotrophic strain is a mutant that cannot grow on minimal medium and
requires certain supplements whereas prototrophic strains are wild type as
it will grow in minimal medium or medium lacking the supplement.
Generalized transduction is the process by which any part of bacterial DNA
can be transferred from donor to recipient bacteria via a bacteriophage.
Generalized transducing phage have a packaging error and thus by mistake
pack segment of bacterial DNA in place of viral DNA.
Generalized transducing phage produce some particles that contain only
DNA obtained from the host bacterium, rather than phage DNA.
The transducing phage loaded with bacterial DNA continues to infect another
bacterial cell.
During infection of Escherichia coli by phage P1, a phage-encoded nuclease
is made that causes fragmentation of bacterial DNA.
A single fragment of bacterial DNA comparable in size to P1 DNA is
packaged into a phage particle instead of P1 DNA.

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 When a transducing particle adsorbs to a bacterium, the DNA carried within Transduction
and its Types
the particle head is injected into the cell and becomes available for double
crossing over with the homologous region of the bacterial chromosome.
In case of specialized transduction, only specific parts of host genome can
NOTES
be transferred to the recipient cell. It takes place via temperate phages, i.e.,
phages that undergo both Lysogenic and Lytic cycles.
Specialized transducing phages are produced when there is error in excision.
Integrated phage DNA can excise out from the bacterial genome and follow
Lytic cycle.
Specialized transducing phages are produced when there is error in excision.
Host genes that flank the integrated phage DNA are also excised out by
mistake leaving some of its own genes.
Transducing phage produces particles containing both phage and bacterial
genes linked in a single DNA molecule.
In case of transducing phage certain genes are replaced by bacterial genes
so it cannot replicate.
Only a small amount of chromosome, a few genes can be transferred by
transduction.
The closer the two genes are to each other, the more likely they are to be
transduced by the same phage.
Cotransduction frequency is the key parameter used in mapping genes by
transduction.
Cotransduction frequency is defined as the ratio of transductants that co-
inherit both markers divided by the total number of transductants.

8.6 KEY WORDS

Bacteriophage: Bacteriophage is a virus that infects the bacteria.

Lytic cycle: Viral life cycle involving entry of viral genome, replication,
assembly and release by lysis of the host cell.
Lysogenic cycle: Viral life cycle involving integration of the viral genome
into the bacterial host genome, followed by its proliferation.
Prophage: A prophage is a bacteriophage genome integrated into the
circular bacterial genome.
Generalized transduction: Transfer of any part of the bacterial genome
from donor to recipient cells via a Lytic bacteriophage.
Specialized transduction: Transfer of only specific parts of bacterial
genome from donor to recipient via a Lysogenic phage.
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Transduction Cotransduction frequency: The cotransduction frequency is the ratio of
and its Types
transductants that co-inherited both genes divided by the total number of
transductants.

NOTES
8.7 SELF ASSESSMENT QUESTIONS AND
EXERCISES

Short Answer Questions

1. What is a bacteriophage?
2. How was transduction discovered?
3. What are virulent and temperate bacteriophages?
4. What is generalized transduction?
5. Write in brief about abortive transduction.
6. What are the uses of specialized transducing phages?
7. What is a Dilysogen? State its importance in HFT.
Long Answer Questions
1. Explain the types of transduction.
2. Tabulate the differences between generalized and specialized transduction.
3. Describe any one generalized transducing phage.
4. Write a note on Escherichia coli Phage P1 generalized transducing phage.
5. What is specialized transduction? Explain its types and give its uses.
6. What is the basis of gene mapping using transduction?
7. Differentiate between LFT and HFT Lysate.

8.8 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.

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Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial Transduction
and its Types
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
NOTES
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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Gene Concept and
Lactose System BLOCK - III
OPERON CONCEPT AND EXTRA
CHROMOSOMAL MATERIALS
NOTES

UNIT 9 GENE CONCEPT AND

LACTOSE SYSTEM
Structure
9.0 Introduction
9.1 Objectives
9.2 Gene Concept
9.2.1 Gene Expression in Eukaryotes
9.3 Regulation of Bacterial Gene Expression
9.4 Lactose System - Coordinate Regulation
9.5 Lac Components
9.6 Answers to Check Your Progress Questions
9.7 Summary
9.8 Key Words
9.9 Self Assessment Questions and Exercises
9.10 Further Readings

9.0 INTRODUCTION

A gene is the basic physical and functional unit of heredity. Genes are made up of
DNA. Some genes act as instructions to make molecules called proteins. However,
many genes do not code for proteins. In humans, genes vary in size from a few
hundred DNA bases to more than 2 million bases. Gene expression is the process
by which information from a gene is used in the synthesis of a functional
gene product. These products are often proteins, but in non-protein
coding genes, such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes,
the product is a functional RNA. The DNA of prokaryotes is organized into a
circular chromosome, supercoiled within the nucleoid region of the cell cytoplasm.
Proteins that are needed for a specific function, or that are involved in the same
biochemical pathway, are encoded together in blocks called operons. For example,
all of the genes needed to use lactose as an energy source are coded next to each
other in the lactose (or lac) operon, and transcribed into a single mRNA.
Regulation of gene expression, or gene regulation, includes a wide range of
mechanisms that are used by cells to increase or decrease the production of
specific gene products (protein or RNA). Sophisticated programs of gene
expression are widely observed in biology, for example to trigger developmental
pathways, respond to environmental stimuli, or adapt to new food sources. Virtually
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172 Material
to RNA processing, and to the post-translational modification of a protein. Often, Gene Concept and
Lactose System
one gene regulator controls another, and so on, in a gene regulatory network.
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases
the versatility and adaptability of an organism by allowing the cell to express protein
when needed. NOTES
In biology, an operon is a functioning unit of DNA containing a cluster of
genes under the control of a single promoter. The genes are transcribed together
into an mRNA strand and either translated together in the cytoplasm, or undergo
splicing to create monocistronic mRNAs that are translated separately, i.e., several
strands of mRNA that each encode a single gene product. The result of this is that
the genes contained in the operon are either expressed together or not at all.
Several genes must be co-transcribed to define an operon.
Originally, operons were thought to exist solely in Prokaryotes, but since
the discovery of the first operons in Eukaryotes in the early 1990s, more evidence
has arisen to suggest they are more common than previously assumed. In general,
expression of Prokaryotic Operons leads to the generation of polycistronic mRNAs,
while Eukaryotic Operons lead to monocistronic mRNAs. The Lac Operon
(Lactose Operon) is an operon required for the transport and metabolism of lactose
in Escherichia coli and many other enteric bacteria. Although glucose is the
preferred Carbon source for most bacteria, the lac operon allows for the effective
digestion of lactose when glucose is not available through the activity of beta-
galactosidase. Gene regulation of the lac operon was the first genetic regulatory
mechanism to be understood clearly, so it has become a foremost example of
prokaryotic gene regulation.
In this unit, you will study about the gene concept, regulation of bacterial
gene expression, lactose system, coordinate regulation, Lac components, positive
and negative regulations, and catabolite repression.

9.1 UNIT OBJECTIVES

After going through this unit, you will be able to:

Understand the gene concept
Explain about the regulation of bacterial gene expression
Discuss the significant features of lactose system
Elucidate on coordinate regulation and Lac components
Analyse the positive and negative regulation, and catabolite repression

9.2 GENE CONCEPT

Genes are the small segments present on DNA. They encode for proteins which
describes an individual’s phenotype. They are characterized by the presence of
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Gene Concept and start codon in the beginning and stop codon at the end. There are few genes which
Lactose System
do not code for functional proteins and many are hypothetical due to their unknown
functions. Interestingly, a gene may code for more than one proteins in eukaryotes
by the phenomenon known as alternative splicing. In short, genes are the basic
NOTES unit of hereditary in all the organisms from simpler like prokaryotes to highly
complex like eukaryotes. As we all must know by now that DNA consists of
Nucleic Acids, Sugars and Phosphate, i.e., Nucleotides which are of four types:
Adenine (A), Guanine (G), Cytosine (C) and Thymine (T). These nucleotide are
arranged one after the other in a genome and encode for proteins which perform
functions responsible for a phenotype. A codon is made up of three nucleotides
which encodes for an amino acid. There are 64 possible Codons and 20 Amino
acids, each amino acid is encoded by more than one codon. Also, three start and
one stop codon is there which initiate or terminates the expression processes.
The term ‘Gene’ was coined by Johannsen in 1909 and he named hereditary
units of Mendel as ‘Genes’. Subsequently many concepts and views emerged on
genes calling them as the hereditary component, thread like structures, etc. Basically,
a gene encodes for a single polypeptide has a start codon in the beginning and
stop codon at the end. Following points are under consideration of modern gene
concept:
Both male and female parents pass on their genes to the offspring, only
cytoplasmic inheritance is pass on from mother to offspring.
DNA is arranged in chromosomes or linkage groups and in diploid organisms,
genes are paired. The phenotype is the result of the behaviour resultant of
their combination whether dominant or recessive, co-dominance, incomplete
dominance, etc.
Genes present on same chromosomes are generally transmitted together,
called as linked genes.
Gene has a position on chromosomes called locus. Due to chromosomal
aberrations, locus may change which can affect phenotypic expression.
Gamete formation leads to segregation of genes and haploid gametes have
only one gene of its type.
Non-linked genes assort differently and independently.
Gene Expression Control in Prokaryotes
Prokaryotes are the simplest among the organisms by being single celled and
simpler life processes. However, similar to eukaryotes, they perform the essential
functions like eating, digestion, respiration, osmoregulation, excretion, reproduction,
etc. For this, or any activity, proteins or enzymes are required which are obtained
by the process of transcription and translation of the genetic material. These
processes do not occur continuously, instead are being under the control of other
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genetic factors which make balance by keeping transcription and translation switch Gene Concept and
Lactose System
on or off. Hence, the mechanism to control the expression of genes can be
considered as positive or negative on the basis of whether it turn on and enhance
the gene expression or turn off and repress. This is important as in different processes
different proteins are required. Some proteins or enzymes are antagonist in NOTES
behaviour so, the control mechanisms help in deciding which process should take
place. We shall now focus on the different types of gene expression control in
prokaryotes.
The mechanism of gene expression was first observed in 1900 when the
bacteria was grown under the presence of lactose showed a production of an
enzyme which was not observed in the absence of lactose. This gives an idea that
certain products are utilised by the bacteria when the machinery of its breakdown
is turned on. As in presence of lactose it is being utilised but in its absence, bacteria
still can obtain carbon source from different sources. These enzymes are called as
adaptive or facultative. More appropriately, adaptive enzymes are called as
inducible, whose production is induced by the presence of a factor or substrate,
commonly called as inducer. This is an example of positive control of gene
expression. Similarly, if an enzyme or protein production is repressed by some
compound that enzyme is called as repressible and the compound which repressed
is called as repressor. Under negative control or repression, the expression of a
gene remain continued till the repressor molecule get activated and repressed the
expression. There are some genes whose expression is continuous and not
dependent upon the circumstances or environment. These are called constitutive
enzymes. Let us discuss the positive and negative control in detail below.
Negative Control of Gene Expression and Lac Operon
Research were started in the year 1940s on lactose metabolism and many scientists
did significant contribution. These include, Jacques Monad, Joshua Lederberg,
Francois Jacob and Andre L’ Wolf. It was evident that in the presence of lactose,
enzyme which cause lactose breakdown increases in concentration from few
molecules to thousand molecules. However, Lac the absence of lactose decreases
the production of lactose metabolising enzyme. Thus, this enzyme is inducible and
lactose is a substrate as well as an inducer here. It was determined that lactose
metabolism is under the control of three structural genes which are arranged together
in a cluster in genome of prokaryotes (Refer Figure 9.1). These are:
LacZ Gene: It encodes for beta-galactosidase, an enzyme which convert
lactose (disaccharide) to the monomer units, glucose and galactose
(monosaccharides).
LacY Gene: This gene encodes for permease which helps in the entry of
lactose through the cell wall of bacteria.
LacA Gene: This gene encodes for transacetylase which known to have
probable role in exporting out as by product of lactose breakdown.
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Gene Concept and Repressor Promoter Operator Leader Structural genes
Lactose System A)
lacI lacP lacO lacL lacZ lacY lacA

NOTES Repressor Lactose RNA polymerase

Repressor Promoter Operator Leader Structural genes

B) lacI lacP lacO lacL lacZ lacY lacA

Repressor Promoter Operator Leader Structural genes

C) lacI lacP lacO lacL lacZ lacY lacA

mRNA transcript

Fig. 9.1 The Lac Operon

The above Figure 9.1 shows the lac operon, in which; (A) components of lac
operon; (B) transcription is blocked in the absence of lactose as the repressor
binds to the operator; (C) transcription of structural genes into mRNA transcripts
in the presence of lactose. Lactose binds allosterically to the repressor and
inactivate it
In order to identify their functional role, researchers produced the various
mutants of these three genes one by one to observe the effects. They found that
lacZ and lacY plays a key role and if any one of them is not functional then an
organism cannot metabolise lactose. Also, these genes, if present, are always reside
one after the other in a cluster as lacZ-lacY-lacA. This operon is called as lactose
operon or lac operon. Along with three structural genes, it also has a promoter,
operator and a repressor gene called as lacI. Promoter and operator together
form a regulatory region. LacI produces a repressor molecule which is allosteric
in nature. Jacob and Monad worked and suggested that the repressor molecule
interacts allosterically with operator to control the gene expression. It do so by
affecting the binding of RNA polymerase, thus slow or shut down the transcription.
But, in the presence of lactose, lactose molecule binds to the repressor leading to
the confirmations change allosterically. As a result, the repressor get deactivated
and unable to bind to the operator region. Hence, the operon continued transcription
and translation. This justifies the role of lactose as an inducer and substrate as its
presence initiates its conversion into glucose and galactose. This operon and
regulatory mechanisms of prokaryotes are dependent on the presence of lactose
and inactivation of repressor molecule hence, this mechanism is an example of
negative control of gene expression in prokaryotes.
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 There are two different type of mutations which can explain these conditions Gene Concept and
Lactose System
in detail, I- and OC. I- is a mutation which inactivate or produces a mutant repressor,
non-functional and OC is the constitutive mutation in operator region which lead to
constitutive expression of structural genes. In both the cases, the lac operon is
NOTES
turned on Nd continuous expression of genes which facilitate the lactose breakdown
takes place. Following are the combinations of gene mutations and lactose present/
absent and their corresponding results of switching on/off the operon.
Tryptophan Operon and Repressible Gene System
Unlike lac operon where the presence of lactose was required to switch on the
transcription, tryptophan operon works differently. In 1953, Monad and colleagues
identified the tryptophan operon and its functioning. In prokaryotes, enzymes
necessary for the production of amino acids are present, which activates in the
absence of external source of amino acids (in a media or environment). When,
there is scarcity of tryptophan, the operon get activated and continued. The
repressor molecule do not bind to the operator during this time. But, when tryptophan
is present and can be obtained from the environment, it act as a co-repressor.
Tryptophan binds to the repressor molecule and activate it allosterically, afterwards,
repressor-trp complex binds to the operator and switched off the gene expression
of trp operon. Hence, the control of this operon is different from lac operon as trp
is acting as a co-repressor while lactose is an inducer.
Structurally, trp operon consists of five genes whose product is important
for trp biosynthesis. These are trpA, trpB, trpC, trpD and trpE. TrpP is a promoter
and trpO is an operator where RNA polymerase binds and initiates transcription
(Refer Figure 9.2). There is 5’leader sequence following trpP which is of 162
nucleotide long which consists of a site called attenuator. Following this, genes are
arranged in form of trpE, trpD, trpC, trpB and trpA. Both leader peptide and
attenuator have sequences which induced the formation of terminator loops in the
presence of trp. The leader sequence consists of two triplet sequence of UUG
which encodes for trp amino acid. Upstream to these sequence, AUG codon is
present which encode for start codon. Hence transcription is initiated from here.
In the presence of trp, tRNAtrp is abundant and thus participate in forming termination
loops. In the absence or scarcity of trp, termination loops do not form, instead,
anti-termination hairpin is produced together by leader sequence and attenuator
which allow RNA polymerase to surpass and continue transcription. This model
of trp operon is present in Escherichia coli. In Bacillus subtilis, there is another
way to control the expression of trp operon. Trp RNA-binding Attenuation Protein
(TRAP) is a molecule present in the cell. When trp is present, it binds to trp and
form a complex. TRAP HAS 11 subunits and each subunit binds to the trp. When
fully saturated, it binds to 5’ leader peptide which consists of 11 triplet codons of
either UAG or GAG, separated by spacer nucleotides. As a result, it disturb the
production of anti-termination hairpin, instead terminator loop is formed which
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Gene Concept and prevent the expression of trp operon. In the absence or less concentration of trp,
Lactose System
TRAP remain unsaturated and do not interfere with the production of anti-
termination hairpin, thus transcription proceeds.

NOTES
Promoter Leader
trpP L
Repressor Operator Attenuator
A)
trpR trpO trpE trpD trpC trpB trpA

Tryptophan
Repressor

B)

trpR O L A trpE trpD trpC trpB trpA

P

mRNA transcript

C) trpE trpD trpC trpB trpA

trpR P O L A

Transcription is
blocked

Fig. 9.2 The trp Operon

In the above Figure 9.2 shows the trp operon, in which (A) the components of trp
operon. (B) in the absence of trp, repressor molecule cannot bind to the operator
hence, transcription occur. (C) in the presence of trp, it act as a co-repressor,
binds to the repressor and blocks the transcription.
Arabinose (ara) Operon: Positive and Negative Control
Arabinose operon is a good example of both positive and negative control of gene
expression. It performs the metabolic action of arabinose and consists of three
structural genes, araB, araA and araD. Their transcription is controlled by a
regulatory protein AraC which is encoded by araC gene. This protein interacts
with two regions of the operon, araI and araO2. AraI is a inducer region which
promotes the transcription of structural genes. AraO2 is the operator region
responsible for resisting the expression of ara operon.
The inducer site consists of Catabolite Activating Protein (CAP protein).
Functionally, in the presence of arabinose and cAMP, AraC get activated,
dimerised and binds to the inducer site. Also, CAP binds to cAMP and form a
complex of CAP-cAMP which interact with CAP region. This initiates the
transcription of structural genes. In the absence of arabinose, or cAMP, the dimer
of AraC binds to the inducer as well as araO2 region which facilitates the looping
of the DNA molecule at the region so that the dimers interact. This loop formation
prevents the expression of genes. Thus, ara operon and interaction with AraC
shows both positive and negative control of gene expression.

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9.2.1 Gene Expression in Eukaryotes Gene Concept and
Lactose System
Eukaryotes or multicellular organisms are complex and differ in regulating gene
expression as compared with prokaryotes. The differences are as follows:
Eukaryotic DNA is much longer than prokaryotic DNA so, the packaging NOTES
of Eukaryotic DNA is much more complex by using histones around which
DNA is wrapped in a chromatin structure. One of the control of gene
expression is the packaging of eukaryotic DNA. The tight or closed
packaging is responsible for no expression or DNA is not accessible for
transcription while, the open configuration or loose chromatin folding make
DNA accessible for gene expression.
In Eukaryotes, DNA is arranged in many chromosomes rather one or two.
Their transcription and translation is controlled differently. These
chromosomes reside in the nucleus bounded by double layered nuclear
membrane.
In Eukaryotes, transcription and translation takes place in different
compartments. Transcription takes place inside the nucleus whereas
translation proceeds in cytoplasm.
Also, it is required that the mRNA so produced from transcription should
undergo three different processes of post transcriptional modification, i.e.,
5’ methylguanosine capping, splicing and addition of poly Adenine (A) tail.
After this, the mature transcript proceeds for translation.
The half-life of Eukaryotic mRNA is much more than Prokaryotic as
Prokaryotes harbour wide environmental changes and require to produce
proteins faster and decay them soon when not needed.
Each cell of eukaryotes consists of same DNA molecule but they have
specialised tissues, organs, etc., for particular function. Hence, few genes
are expressed only in one tissue or organ which bear related function. Rest
genome stay non-functional in that tissue. Similarly, different genes are
activated in different organs.
mRNA of eukaryotes is more stable than Prokaryotes, hence translation
controls are more active and applied.
This suggests that in case of Eukaryotes, the gene expression control is at the
stage of:
Transcription
Post Transcriptional Modifications
Transfer of mRNA to Cytoplasm
Stability of mRNA
Translation Control by selecting mRNA for Translation
Post Translational Modifications
Let us discuss the control of gene expression in Eukaryotes in detail.
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Gene Concept and Transcriptional Control of Gene Expression
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There are three regions which plays an important role in modulating the transcription
levels or switching it ON or OFF, promoters, enhancer and silencers. These are
NOTES called as cis regulators as they control the gene regulation by being present on the
same chromosome. Trans regulators, on the other hand, are the ones which control
gene regulation by being present on another chromosome. These are generally
transcription factors and modulators. Promoter region is situated upstream to the
genes for initiating transcription. These are the sites recognised by transcriptional
machinery. They are up to 100 bp long and consists of the repeat of TATA
nucleotides, i.e., Thiamine (T) and Adenine (A). Hence, these are also referred as
TATA box. It may also contain other boxes like CAAT, GC, etc. TATA box is
situated 25-35 bp above the gene and consensus ATAT region of 7-8 bp long.
CAAT BOX is situated 70 -80 bp above the gene and GC box with consensus
sequence of GGGCGG is situated 110 bp above the gene. Mutations in promoter
regions have shown the effect on the rate of transcription.
Enhancers are another type of cis regulators, controls gene expression by
residing either upstream or downstream, even thousand bp away from the gene or
within the gene. Enhancers interact with promoters, transcriptional factors,
regulatory proteins, etc., to enhance the rate of transcription. If enhancers are
placed apart from the gene, they interact by bending of the DNA molecule in a
way to come closer to the regulatory region. They have a tendency to enhance
transcription by many folds. It has been shown that if enhancer is placed at some
new location in the genome, it enhances the expression of other genes too. Also, if
an unrelated gene is placed near the enhancer or at a place where a gene was
already present, it get enhanced in expression by the activated enhancer. In yeast,
regions similar to enhancers are found upstream to the genes they regulate called
Upstream Activator Sequences (UAS). They are only found upstream and not
downstream to a gene they regulate hence, differ from enhancers.
Apart from these, chromatin remodeling is also important way to control
gene expression at transcriptional level. This refers to change in organisation of
DNA with histone and non-histone protein. The loose interaction of proteins with
the DNA make it accessible to transcriptional factors. This condition is called as
euchromatin when DNA is transcriptionally active. When DNA is tightly bound
with proteins then it stay transcriptionally inaccessible and called as heterochromatin.
This inactivation can also achieved by methylation or acetylation of DNA
nucleotides.
Chromatin remodeling make promoters and genes free from histone
molecules. One example is SWI/SWF, a nucleoside remodeling complex which
has 11 subunits. One subunit allow binding with non-specific DNA another is an
ATPase. This is directed to specific targets by the presence of leucine zippers,
binding to acetylated and methylated DNA. This can make changes like sliding
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the nucleosome or twisting the nucleosome to pull out DNA molecule so that Gene Concept and
Lactose System
make it accessible for transcription.
Another way is histone modifications. This modifications is catalysed by the
enzymes called as Histone AcetylTransferase (HAT). It adds an acetate group to NOTES
basic amino acid of histone tail. This interaction decrease the attraction between
basic amino acids and acidic DNA molecule making it accessible to transcriptional
factors. Reversing this reaction, Histone Deacetylase (HDAC) removes acetyl
group from histone proteins. Along with acetylation, methylation and phosphorylation
are also other mode of modifications. Also, transcriptional factors interact with
other factors or co-activators which modulate their activity.
Methylation of DNA is an important way to reduce the gene expression.
The methyl group protrudes out from the DNA molecule and affects the binding of
RNA polymerase and transcription factors. Methylation occur more often at CG
repeats. To determine the presence of methylated residues, a restriction enzyme
HpaII can be used which recognises CCGG region. If second C is methylated,
the enzyme cannot make a cut in recognition sequence, evident of methylation.
Hence, methylation and gene expression are opposite as more the methylation,
lesser will be gene expression. One example is X chromosome inactivation in
human females for dosage compensation with human males. The inactivated X
chromosome show high level of methylation than active X chromosome. Also,
methylation can be observed in genes sequences which remain silent in tissues or
organs where they do not have any function to perform.
Regulation at Post-Transcriptional Modification
As mentioned in above section, we mentioned that there are three types of post
transcriptional modifications, 5’-methyl guanosine cap, splicing and 3’poly A tail.
Among these, regulation of gene expression can be attained at the level of alternative
splicing by which different proteins can be manufactured and on the basis of stability
of mRNA.
In alternative splicing, a single transcript can give rise to multiple proteins
depends on the requirements of the cell. This is achieved as few exons become
introns and introns become exons during splicing of the regions from the primary
transcripts. In humans, hundred thousands of proteins are encoded by nearly 30,000
genes because of alternative splicing mechanism. It maintain the regulation and
also provide direction to the transcript for production of a particular protein. By
this way, gene regulation can also be controlled after the transcription.
mRNA stability is defined as the turnover of mRNA or its half life till it
undergone translation. Also, RNA interference is a method identified to control
the translation of a mRNA. There are short RNA of nearly 21 nucleotides called
as siRNA (small interfering RNA) which participate in cleavage of mRNA. The
process starts when a double stranded RNA (nearly 70 bp long) binds to a protein
bearing RNAse activity called as dicer. It cleaves the RNA in short RNA called as
siRNA. Then, the both helix of double stranded siRNA unwinds and the anti- Self-Instructional
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Gene Concept and sense helix binds with a protein called as RISC (RNA-Induced Silencing
Lactose System
Complex). This helps recognising the mRNA which has a complementary region
to the siRNA, interact with those and cleave them. This machinery also plays an
important role to fight against viruses and bacteria.
NOTES Another example is miRNA (micro RNA), which are 70-110 bp long and
binds with dicer and target mRNA. The interaction is not exact and even if the 19-
24 nucleotides paired, the target mRNA is cleaved and degraded. Thus, its
translation is blocked. Phage are those viruses which infect and harbour the DNA
of bacteria and archaea and may modulate the eukaryotic genome also. They
enter in the cell and through lysogenic cycle they are capable to incorporate their
genome into the genome of host organism. RNA interference is initially evolved as
a mechanism to protect against the phage or virus attack (it can be lytic also) to
protect the species and maintain its survival. To prevent, miRNA and siRNA which
consists of foreign sequences or random sequences encounter phage genome and
destroy them. In bacteria, there are specific regions called as CRISPR (Clustered
Regularly Interspaced Short Palindromic Repeats) elements which consists of
repeatedly present spacers and repeat regions. When a phage infects a bacteria,
it keep the memory of that encounter by keeping a region of short nucleotide
stretch (20-24 bp) integrated in its genome surrounded by repeat regions called
as spacer. On the repeat attack, the bacteria produces mRNA of the
complementary spacer and perform the degradation of phage genome by the
process similar to RNA interference.
There is another type of regulation is identified from the RNA molecules
called as RNA Directed DNA Methylation (RdDM). These RNA molecules binds
to the complementary DNA and initiates its methylation. Thus, regulating their
expression by methylating the target DNA.
There are proteins called as RNA-Binding Proteins or RBPs which can
bind to the untranslated regions, or UTRs of mRNA. UTRs are the region which
cannot be translated and help in mRNA localization, stability, and protein translation.
Region present before the protein-coding region is 52 UTR, whereas after the
coding region is 32 UTR. Binding of proteins at these regions can greatly affect
the stability of mRNA as it may enhance or decrease.
Translation and Post-Translational Control of Expression
Translation is a process by which a mature mRNA is converted into designated
protein molecule in eukaryotes. Regulation of this process is achieved by sending
a message for proceeding or not for translation. One such example is synthesis of
- and -tubulin which are subunits of microtubules. Colchicine treatment to the
cell can induce the disassembly of microtubules and accumulate the - and -
tubulin in the cell. This shut down the production of - and -tubulin in the cell.
Oppositely, treatment with a drug called vinblastine causes microtubule disassembly
but initiates the production of - and -tubulin in the cell. This is because vinblastine
precipitates these subunits and make them unavailable to the cell which in turn
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induces their production in the cell. This regulation is takes place at translational Gene Concept and
Lactose System
level called as autoregulation.
After translational, a protein undergoes proper binding, folding of the motifs
in Golgi complex and its transportation. When a cell or extracellularly, a protein is
NOTES
required then its processing takes place without any delay and smoothly by the
cell but if not then a well formed protein or enzyme is processed to inactivated by
the process of phosphorylation, acetylation, methylation, dephosphorylation, etc.
By this way, an enzyme get deactivated but not permanently as reversing the
condition make the enzyme available again for the reaction.

Check Your Progress

1. Explain the term gene. How are they characterized?
2. Who coined the term gene? How coding is done by genes?
3. When gene expression was first observed? What are adaptive, facultative
and repressible enzymes?
4. How the presence and absence of lactose effects the production of lactose
metabolising enzyme? Which three structural genes determine the lactose
metabolism?
5. Explain about cis regulators and trans regulators that control the gene
regulation.
6. What is translation process by which a mature mRNA is converted into
designated protein molecule? How it is regulated?

9.3 REGULATION OF BACTERIAL GENE

EXPRESSION

Cell is defined as the basic unit of life. It grows and divides in different phases of
cycle. Cell cycle consists of mainly four phases, Growth phase 1 (G1), Synthesis
phase (S), Growth phase 2 (G2) and Mitosis (M) or Meiosis. In G1 phase, cell
prepares for the next stage, i.e., DNA synthesis or replication in S phase. In S
phase, chromosomal DNA is replicated. This is followed by G2 phase where cell
continue to grow and increase the amount of organelles, other molecules so that in
next stage it can be divided. In M phase, the chromosomes get condensed
(prophase) followed by aligning at equatorial pole (metaphase). Then, the sister
chromatids get separated to different poles by the action of kinetochores
(anaphase). After this, division takes place in (telophase) and cytoplasm is also
equally divided (cytokinesis).
The cell cycle progression is continued in this way till the cell is signaled to
undergone to the progression arrest from internal factors. In such cases, cell
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Gene Concept and undergoes programmed cell death by activating different factors in a cascade and
Lactose System
triggering their own death called as apoptosis. Also, during tissue injury, cell also
suffer a sudden death which is called as necrosis. We shall discuss these in later
sections of this unit. It is important to note here that all the cells are programmed to
NOTES
death when the signal arrives and they cannot be immortal. This is achieved by the
regulation of cell cycle by the action of internal factors or external trigger. Although,
if a cell is failed to regulate the cell cycle, the cell undergo uncontrollable growth
and divisions which can lead to lethal conditions like benign tumor or malignant
tumor. This raised a good point that cell cycle regulation is very important. In next
sections, we shall focus that how regulation is maintained.
Cell Cycle Regulation
During cell cycle progression, if a cell signalled to stop dividing and growing, it
arrest in stage G1. Then it enters in a G0 stage where no growth or division takes
place, instead, a cell stay and perform its functions normally. Most differentiated
cells stay in G0 stage indefinitely and do not enters in cell cycle again. However, if
signalled from the external environments then they may enter in the cell cycle again.
During the loss of this regulation, a cell proceeds with all the four stages of cell
cycle one after the other continuously and cannot stop growing and dividing.
In a cell cycle, there are three points where cell monitors and respond to its
internal equilibrium before moving to the next stage. These are between G1 and S
phase G1/S, G2 and M phase G2/M and M phase. These are also called as
checkpoints as they check the progression and can arrest the cell for proceeding.
Literally, a checkpoint is a stage in the eukaryotic cell cycle during which cell
determines internal and external signals and decides whether or not to move forward
to the next phase. Different check points monitors differently and are explained as
below:
G1/S Check Point: It checks the size of the cell, growth factors, nutrients
and determine any damage to DNA molecule which should be repaired
before it proceeds to the next stage. Size of the cell should be enough large
which can support for division later. Another notable factor is that whether
a cell is reside on proper extracellular matrix which supports its division.
The cycle remain halted for longer periods till the cellular machinery corrects
the faults and signal for the progression. Initially, if a cell has normal size and
no defects in DNA then, it continue to the next phase of cycle. It is also
important because at this point cell decides that whether to or not proceed
for the division. If this check point is crossed then cell has to cross the entire
cycle. If the fault is not corrected then the cell enters in G0 phase and
remain non-dividing.

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 G2/M Check Point: In this check point, physiological conditions of the Gene Concept and
Lactose System
cell is monitored. The cell is analyzed for the complete DNA replication and
whether DNA is damaged. If so, then cell cycle stay arrested at this point
and cannot be proceeded to the next phase which is the mitotic division of
NOTES
the cell. Hence, these checkpoints prevent the spread of any damage or
mistake in the DNA which may affect the physiology of the organism.
M Check Point: It is third checkpoint and it is monitored during the time
of mitosis. It checks the formation of spindle fibers and their proper
attachment with the kinetochores which helps in pulling the sister chromatids
towards the opposite poles. If spindle fibers are not formed properly or the
attachment is not proper then the mitosis stage is arrested.
If the DNA damage is not corrected at the checkpoints or the errors are not
possible to remove then cell trigger the programmed cell death to ensure that
damaged or faulty DNA or cell should not proliferate.
Role of Cyclins and Cyclin-Dependent Kinases (CDKs) in Cell Cycle
Regulation
We discussed about the different checkpoints of cell cycle progression. In this
section we will focus on the molecules which mainly control the arrest at the
checkpoints. The regulation of cell-cycle is performed by two type of proteins:
Cyclins: Cyclins are one of the important cell cycle regulators. Four basic types
of cyclins are found in humans and other eukaryotes. These are G1, G1/S, S and
M cyclins. According to their names, each cyclin is related to particular phase or
transition of the cell cycle. The S cyclin promotes the DNA replication during S
phase. A cyclin’s concentration also varies throughout the cell cycle, i.e., their
concentration stays low throughout the cell cycle but increases during the phase or
transition at which they required. The M cyclin is present in highest concentration
during M phase. S cyclin is at highest concentration during S and G2 phase also.
G1 cyclin is an exception as it stays in a similar concentration throughout the cycle
as needed at many points. Hence, cyclins are synthesized during different stages
of cell cycle and subsequently destroyed also to maintain the progression. These
cyclins are also named as cyclin A, cyclin B, cyclin D and cyclin E.
Cyclin-Dependent Kinases (CDKs): To perform functions of activating or
deactivating many proteins inside the cell, cyclins bind to a specialized group of
proteins called as Cyclin Dependent Kinases (CDKs). Without cyclins they remain
inactive but binding with cyclins activates them making it a functional enzyme and
allowing it to modify target proteins. As the name suggests, CDKs are the kinases
which phosphorylates or attach phosphate groups to target proteins. These target
proteins are generally specific to specific kinases. This affect the activity of target
protein by making them more or less active. Cyclins not only activate CDKs but
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Gene Concept and also directs them to a specified set of proteins for phosphorylation so that the cell
Lactose System
corrects the errors. Concentration of CDKs remain constant throughout the cell
cycle but it is the concentration of cyclins which make them active and inactive.
CDKs itself must also be phosphorylate to get activated.
NOTES
Cyclins are synthesized during different stages of cell cycle and subsequently
destroyed also to maintain the progression. Cyclins bind to CDK molecules and
form a cyclin/CDK complex which is specific for a particular check point. The
main function of this complex is to phosphorylate (due to the presence of kinase)
the other proteins and activate the changes necessary for helping the cell for
progression. Cyclins and CDKs are evolutionarily conserved and are found in
many different types of species, from yeast to humans. The details of the system
vary as, yeast has just one CDK molecule, while humans and other mammals
have multiple CDKs for different stages of the cell cycle.
Maturation Promoting Factor (MPF): In 1980s, MPF, was discovered as a
Cdk bound to its M cyclin partner. During M phase, M cyclin accumulates, it
binds to MPF which add phosphate group to different proteins in nuclear envelope,
resulting its breakdown. It also promote chromosome condensation and other
events of M phase. After its work is done, MPF promotes its own destruction by
activating a complex called as Anaphase-Promoting Complex/Cyclosome (APC/
C). It is a protein complex that degrade M cyclins during in anaphase. Destruction
of M cyclins pushes the cell to enter in mitosis. This complex also causes destruction
of the proteins which held sister chromatids together thus, allowing them to separate
during anaphase and move to opposite poles. APC/C do not phosphorylate its
target proteins instead, it adds a small protein tag called as Ubiquitin (Ub). This
target can now sent to the proteasome in the cell, which is a recycle bin of the cell,
and degraded. During metaphase, this complex also sends signals to
destroys cohesin, the protein glue that holds sister chromatids together. This is
done by first adding ubiquitin tag to a protein called securing which cause its
degradation. Securin binds and inactivates a protein called separase. Hence,
separase is now become active, it cleaves the cohesion and allows the sister
chromatids to separate.
CDK and Cyclin Complexes a Different Cell Cycle Stages
As mentioned above, different CDK and cyclin complexes regulate at different
checkpoints (Refer Figure 9.3). These complexes are:
In G1 phase: CDK4 or CDK6 with cyclin D
In G1/S phase: CDK2 with cyclin E
In S phase: CDK2 with cyclin A
In G2/M phase: CDK1 (CDC2) with cyclin B or A
In M phase: CDK1 with cyclin B or A
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 Cyclin B/A + CDK1
Gene Concept and
Lactose System

M
G2
NOTES

Cell Cycle
G1

S
Cyclin D + CDK4/6

Cyclin A +CDK2

Cyclin E + CDK2

Fig. 9.3 Various Stages of Cell Cycle and Corresponding Cyclin and CDK Complexes
which Keep a Check on Cell Cycle Progression

In G1 phase, the complex of CDK4/cyclin D activates expression of those genes

whose products required during DNA replication during S phase. It also activates
a protein called as pRB which initiates the expression of number of genes that
encode for cyclins E and A, Cdk1, and proteins involved in replication. In G1/S
phase, cyclin E form a complex with CDK2 and initiates replication. Similarly, as
cell cycle progress, cyclin A forms a complex with CDK2 and promotes replication.
Then, during transition from G2 to M phase, CDK1/ cyclin A and CDK1/cyclin B
complexes phosphorylate variety of proteins which are necessary for mitosis, such
as cytoskeletal proteins, histones, and proteins of the nuclear envelope.

9.4 LACTOSE SYSTEM - COORDINATE

REGULATION

Operon
The Basic Concept Structural Gene = Gene that Codes for a Polypeptide.
A regulated cluster of adjacent structural genes with related functions.
Has a single promoter region, so an RNA polymerase will transcribe all
structural genes on an all-or-none basis.
Transcription produces a single polycistronic mRNA with coding sequences
for all enzymes in a metabolic pathway.
Polycistronic mRNA
A large mRNA molecule that is a transcript of several genes. Contains stop
and start codons for the translation of each polypeptide.
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Gene Concept and Operator
Lactose System
A DNA segment between an operon’s promoter and structural genes, which
controls access of RNA polymerase to structural genes.
NOTES Acts as an ON/OFF switch for movement of RNA polymerase and
transcription of the operon’s structural genes.
Repressor
Specific protein that binds to an operator and blocks transcription of the
operon.
Blocks attachment of RNA polymerase to the promoter.
Repressors are encoded by regulatory genes.
Regulatory Genes
Genes that code for repressor or regulators of other genes.
Are often located some distance away from the operons they control.
Are involved in switching on or off the transcription of structural genes.
Corepressor
A molecule, usually a metabolite that binds to a repressor protein, causing
the repressor to change into its active conformation.
Only the repressor-corepressor complex can attach to the operator and
turn off the operon.
Inducer
An inducer is a molecule that regulates gene expression. An inducer can
bind to protein repressors or activators.
Inducers function by disabling repressors.
The gene is expressed because an inducer binds to the repressor.
The binding of the inducer to the repressor prevents the repressor from
binding to the operator. RNA polymerase can then begin to transcribe operon
genes.
Repressible Versus Inducible Enzymes
Repressible Enzymes: Enzymes which have their synthesis inhibited by a
metabolite (for example, tryptophan).
Inducible Enzymes: Enzymes which have their synthesis stimulated or induced
by specific metabolites (for example, lac operon).

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 Gene Concept and
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NOTES

Positive control of a regulatory system occurs only if an activator molecule interacts

directly with the genome to turn on transcription.
CRP (cAMP Receptor Protein)
A protein that binds within an operon’s promoter region and enhances the
promoter’s affinity for RNA polymerase.
When glucose is missing, the cell accumulates cyclic AMP (cAMP), a
nucleotide derived from ATP. cAMP activates CAP so that it can bind to
the lac promoter.
When glucose concentration rises, glucose catabolism decreases the
intracellular concentration of cAMP. Thus, cAMP releases CRP.
Catabolite Repression
Repression of a variety of unrelated catabolic enzymes when cells are grown
in a medium containing glucose.
Enhancer/Silencer
Regulatory regions on eukaryotic DNA that bind activator/repressor proteins
controlling single gene expression.

9.5 LAC COMPONENTS

An operon is a cluster of functionally-related genes that are controlled by a shared

operator. Operons consist of multiple genes grouped together with a promoter
and an operator. Operons are present in prokaryotes (bacteria and archaea), but
are absent in eukaryotes. In some situations multiple operons are controlled by
the same regulatory protein; in these cases the operons form a regulon. Operons
were first identified as a mode of gene expression control in 1961 by François
Jacob and Jacques Monod.
Operon Structure
Operons are regions of DNA that contain clusters of related genes. They are
made up of a promoter region, an operator, and multiple related genes. The
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Gene Concept and operator can be located either within the promoter or between the promoter and
Lactose System
the genes. RNA polymerase initiates transcription by binding to the promoter region.
The location of the operator is important as its regulation either allows or prevents
transcription of the genes into mRNA.
NOTES
Operon Function
An operon is a complete package for gene expression and synthesis of
polypeptides. By combining the related genes, all polypeptides required for a
specific function are synthesized in response to a single stimulus. For example, the
bacterium Escherichia coli contains a number of genes clustered into operons
and regulons: the Lac operon which is involved in lactose degradation, the Trp
operon which is involved in tryptophan biosynthesis, and the His operon which is
involved in histidine biosynthesis. These operons are turned on when the gene
products are needed.
Positive and Negative Control
Operons can be under negative or positive control. Negative control involves
turning off the operon in the presence of a repressor; this can be either repressible
or inducible. A repressible operon is one that is usually on but which can be
repressed in the presence of a repressor molecule. The repressor binds to the
operator in such a way that the movement or binding of RNA polymerase is blocked
and transcription cannot proceed. An inducible operon is one that is usually off. In
the absence of an inducer the operator is blocked by a repressor molecule. When
the inducer is present it interacts with the repressor protein, releasing it from the
operator and allowing transcription to proceed. Repressible operons are generally
involved in anabolic pathways, or the synthesis of an essential component, while
inducible operons are generally involved in catabolic pathways, or the breakdown
of a nutrient. Positive control of an operon is when gene expression is stimulated
by the presence of a regulatory protein.
Lac Operon
The Lac operon is the classic operon example, and is responsible for the
degradation of the milk protein lactose. The Lac operon is an inducible operon; in
the absence of lactose the operator is blocked by a repressor protein. The operon
is made up of a promoter with operator, and three genes (lacZ, lacY, and lacA)
which encode -galactosidase, permease, and transacetylase. The three genes
are involved in the breakdown of lactose into its metabolites: -galactosidase
breaks lactose down into glucose and galactose, while the other two proteins aid
in the metabolic process. The expression of the Lac operon is controlled by the
regulatory gene lacI, located immediately adjacent to the promoter region. LacI
encodes an allosteric repressor protein that keep the Lac operon ‘OFF’.
In order for the Lac operon to be turned on, an inducer molecule must
inactivate the repressor protein. The inducer molecule in this system is allolactose,
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190 Material
an isomer of lactose. When lactose and its isomer are present in the cell, allolactose Gene Concept and
Lactose System
will bind to allosteric sites on the repressor protein, changing its conformation and
rendering it inactive. As the repressor protein detaches from the operator, RNA
polymerase can bind to the promoter, transcription can occur, and the three lactose
degradation genes can be synthesized (Refer Figure 9.4). NOTES

Fig. 9.4 Structure of the Lac Operon and the Adjacent lacR Repressor Gene

The Lac operon is also under positive gene regulation. While the removal of
the repressor protein in the presence of lactose is required for synthesis of the
lacZ, lacY, and lacA genes, the gene expression will remain low. The level of gene
expression is controlled by the amount of the preferred energy source, glucose, in
the cell. This control is regulated by an allosteric regulatory protein, Catabolite
Activator Protein (CAP). When glucose levels in the cell are low, the organic
molecule cyclic AMP is in high concentration. Cyclic AMP activates CAP by binding
to the allosteric sites, causing CAP to attach to the Lac operon promoter. Unlike
the repressor proteins, binding of CAP to the Lac operon stimulates gene expression.
When the cell glucose levels increase, the cyclic AMP levels in the cell decrease,
and the activator protein will disassociate from the promoter. Transcription will
return to low levels, or will turn off if the repressor protein reattaches (Refer Figure
9.5).

Fig. 9.5 Lac Operon When Both Glucose and Lactose are Present

The Figure 9.6 depicts the Lac operon and how its gene expression is
under both positive and negative control.
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Gene Concept and The lac operon exhibits both systems. It is a negative control system because
Lactose System
expression is typically blocked by an active repressor (the lac repressor) that
turns off transcription. The lac repressor binds to the operator region and negatively
controls (prevents) transcription.
NOTES
However, when CAP (Catabolite Activating Protein) binds upstream of this
operator region near the promoter and transcription increases, this is an example
of a positive control system. We see this positive control of transcription happen
when glucose levels decline.

Fig. 9.6 Positive and Negative Controls in Lac Operon

Positive Regulation of the Lac Operon

A bacterium’s environment is too complex for its genes to be controlled by
one signal.
Other factors besides lactose affect the expression of the lac genes, such as
the availability of glucose.
Glucose, is Escherichia coli preferred energy source. Other sugars can
serve as the main or sole nutrient, but extra steps are required to prepare
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 them for entry into glycolysis, necessitating the synthesis of additional Gene Concept and
Lactose System
enzymes. Clearly, expressing the genes for proteins that metabolize sugars,
such as lactose or arabinose is wasteful when glucose is abundant.
Expression of the Lac Operon when Both Glucose and Lactose are Present NOTES
A regulatory mechanism known as catabolite repression restricts expression
of the genes required for catabolism of lactose, arabinose, and other sugars
in the presence of glucose, even when these secondary sugars are also
present.
The effect of glucose is mediated by cAMP, as a co-activator, and an activator
protein known as cAMP receptor protein, or CRP (the protein is sometimes
called CAP, for catabolite gene activator protein).
CRP is a homodimer (subunit Mr 22,000) with binding sites for DNA and
cAMP.
Binding is mediated by a helix-turnhelix motif within the protein’s DNA-
binding domain. When glucose is absent, CRP-cAMP binds to a site near
the lac promoter and stimulates RNA transcription 50-fold.
CRP-cAMP is therefore a positive regulatory element responsive to glucose
levels.
CRP-cAMP has little effect on the lac operon when the Lac repressor is
blocking transcription, and dissociation of the repressor from the lac operator
has little effect on transcription of the lac operon unless CRP-cAMP is
present to facilitate transcription; when CRP is not bound, the wild-type lac
promoter is a relatively weak promoter.
The open complex of RNA polymerase and the promoter does not form
readily unless CRP-cAMP is present.
CRP interacts directly with RNA polymerase through the polymerase’s á
subunit.
The effect of glucose on CRP is mediated by the cAMP interaction. CRP
binds to DNA most avidly when cAMP concentrations are high.
In the presence of glucose, the synthesis of cAMP is inhibited and efflux of
cAMP from the cell is stimulated. As [cAMP] declines, CRP binding to
DNA declines, thereby decreasing the expression of the lac operon.
Strong induction of the lac operon therefore requires both lactose (to
inactivate the lac repressor) and a lowered concentration of glucose (to
trigger an increase in [cAMP] and increased binding of cAMP to CRP.
CRP and cAMP are involved in the coordinated regulation of many operons,
primarily those that encode enzymes for the metabolism of secondary sugars,
such as lactose and arabinose.
A network of operons with a common regulator is called a regulon.
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Gene Concept and
Lactose System

NOTES

Fig. 9.7 The Catabolite Activator Protein (CAP) Binds to the Promoter of the Lac
Operon and Stimulates Transcription.

CAP must complex cAMP before binding to the promoter of the lac operon.
The binding of cAMP–CAP to the promoter activates transcription by facilitating
the binding of RNA polymerase. Levels of cAMP are inversely related to glucose:
low glucose stimulates high cAMP; high glucose stimulates low cAMP

Check Your Progress

7. What is cell cycle progression?
8. Explain the cyclin process.
9. Give the features of operator, repressor and regulatory genes.
10. What is operon?
11. Explain about the term Lac operon.
12. What is catabolite repression?
13. How the effect of glucose is mediated by cAMP?

9.6 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. Genes are the small segments present on DNA. They encode for proteins
which describes an individual’s phenotype. They are characterized by the
presence of start codon in the beginning and stop codon at the end. There
are few genes which do not code for functional proteins and many are
hypothetical due to their unknown functions.
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194 Material
2. The term Gene was coined by Johannsen in 1909 and he named hereditary Gene Concept and
Lactose System
units of Mendel as ‘Genes’.
A gene may code for more than one proteins in Eukaryotes by the
phenomenon known as alternative splicing. Therefore, the genes are the
NOTES
basic unit of hereditary in all the organisms from simpler like Prokaryotes to
highly complex like Eukaryotes. As we all must know by now that DNA
consists of Nucleic Acids, Sugars and Phosphate, i.e., Nucleotides which
are of four types: Adenine (A), Guanine (G), Cytosine (C) and Thymine
(T). These nucleotide are arranged one after the other in a genome and
encode for proteins which perform functions responsible for a phenotype.
3. The mechanism of gene expression was first observed in 1900 when the
bacteria was grown under the presence of lactose showed a production of
an enzyme which was not observed in the absence of lactose. Certain
products are utilised by the bacteria when the machinery of its breakdown
is turned on. In presence of lactose it is being utilised but in its absence,
bacteria still can obtain carbon source from different sources. These enzymes
are called as adaptive or facultative. More appropriately, adaptive enzymes
are called as inducible, whose production is induced by the presence of a
factor or substrate, commonly called as inducer. This is an example of positive
control of gene expression. Similarly, if an enzyme or protein production is
repressed by some compound that enzyme is called as repressible and the
compound which repressed is called as repressor. Under negative control
or repression, the expression of a gene remain continued till the repressor
molecule get activated and repressed the expression.
4. It was evident that in the presence of lactose, enzyme which cause lactose
breakdown increases in concentration from few molecules to thousand
molecules. However, Lac the absence of lactose decreases the production
of lactose metabolising enzyme. Thus, this enzyme is inducible and lactose
is a substrate as well as an inducer here. It was determined that lactose
metabolism is under the control of three structural genes which are arranged
together in a cluster in genome of prokaryotes. These are:
LacZ gene: It encodes for beta-galactosidase, an enzyme which convert
lactose (disaccharide) to the monomer units, glucose and galactose
(monosaccharides).
LacY gene: This gene encodes for permease which helps in the entry of
lactose through the cell wall of bacteria.
LacA gene: This gene encodes for transacetylase which known to have
probable role in exporting out as by product of lactose breakdown.
5. There are three regions which plays an important role in modulating the
transcription levels or switching it ON or OFF, promoters, enhancer and
silencers. These are called as cis regulators as they control the gene regulation
by being present on the same chromosome.
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Gene Concept and Trans regulators, on the other hand, are the ones which control gene regulation
Lactose System
by being present on another chromosome. These are generally transcription
factors and modulators. Promoter region is situated upstream to the genes
for initiating transcription. These are the sites recognised by transcriptional
NOTES machinery. They are up to 100 bp long and consists of the repeat of TATA
nucleotides, i.e., Thiamine (T) and Adenine (A).
6. Translation is a process by which a mature mRNA is converted into
designated protein molecule in eukaryotes. Regulation of this process is
achieved by sending a message for proceeding or not for translation. One
such example is synthesis of - and -tubulin which are subunits of
microtubules. Colchicine treatment to the cell can induce the disassembly
of microtubules and accumulate the - and -tubulin in the cell.
7. During cell cycle progression, if a cell signalled to stop dividing and growing,
it arrest in stage G1. Then it enters in a G0 stage where no growth or
division takes place, instead, a cell stay and perform its functions normally.
Most differentiated cells stay in G0 stage indefinitely and do not enters in
cell cycle again. However, if signalled from the external environments then
they may enter in the cell cycle again. During the loss of this regulation, a
cell proceeds with all the four stages of cell cycle one after the other
continuously and cannot stop growing and dividing.
In a cell cycle, there are three points where cell monitors and respond to its
internal equilibrium before moving to the next stage. These are between G1
and S phase G1/S, G2 and M phase G2/M and M phase. These are also
called as checkpoints as they check the progression and can arrest the cell
for proceeding.
8. Cyclins are one of the important cell cycle regulators. Four basic types of
cyclins are found in humans and other eukaryotes. These are G1, G1/S, S
and M cyclins. According to their names, each cyclin is related to particular
phase or transition of the cell cycle. The S cyclin promotes the DNA
replication during S phase. A cyclin’s concentration also varies throughout
the cell cycle, i.e., their concentration stays low throughout the cell cycle
but increases during the phase or transition at which they required. The M
cyclin is present in highest concentration during M phase. S cyclin is at
highest concentration during S and G2 phase also. G1 cyclin is an exception
as it stays in a similar concentration throughout the cycle as needed at many
points. Hence, cyclins are synthesized during different stages of cell cycle
and subsequently destroyed also to maintain the progression. These cyclins
are also named as cyclin A, cyclin B, cyclin D and cyclin E.
9. Operator
A DNA segment between an operon’s promoter and structural genes,
which controls access of RNA polymerase to structural genes.
Acts as an on/off switch for movement of RNA polymerase and
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196 Material
 Repressor Gene Concept and
Lactose System
Specific protein that binds to an operator and blocks transcription of
the operon.
Blocks attachment of RNA polymerase to the promoter. NOTES
Repressors are encoded by regulatory genes.
Regulatory Genes
Genes that code for repressor or regulators of other genes.
Are often located some distance away from the operons they control.
Are involved in switching on or off the transcription of structural genes.
10. An operon is a cluster of functionally-related genes that are controlled by a
shared operator. Operons consist of multiple genes grouped together with
a promoter and an operator. Operons are present in prokaryotes (bacteria
and archaea), but are absent in eukaryotes. In some situations multiple
operons are controlled by the same regulatory protein; in these cases the
operons form a regulon. Operons were first identified as a mode of gene
expression control in 1961 by François Jacob and Jacques Monod.
11. The Lac operon is the classic operon example, and is responsible for the
degradation of the milk protein lactose. The Lac operon is an inducible
operon; in the absence of lactose the operator is blocked by a repressor
protein. The operon is made up of a promoter with operator, and three
genes (lacZ, lacY, and lacA) which encode b-galactosidase, permease,
and transacetylase.
12. A regulatory mechanism known as catabolite repression restricts expression
of the genes required for catabolism of lactose, arabinose, and other sugars
in the presence of glucose, even when these secondary sugars are also
present.
13. The effect of glucose is mediated by cAMP, as a co-activator, and an
activator protein known as cAMP receptor protein, or CRP (the protein is
sometimes called CAP, for Catabolite gene Activator Protein).

9.7 SUMMARY

Genes are the small segments present on DNA. They encode for proteins
which describes an individual’s phenotype. They are characterized by the
presence of start codon in the beginning and stop codon at the end.
There are few genes which do not code for functional proteins and many
are hypothetical due to their unknown functions.
A gene may code for more than one proteins in Eukaryotes by the
phenomenon known as alternative splicing. In short, genes are the basic
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Gene Concept and unit of hereditary in all the organisms from simpler like Prokaryotes to highly
Lactose System
complex like Eukaryotes. As we all must know by now that DNA consists
of Nucleic Acids, Sugars and Phosphate, i.e., Nucleotides which are of
four types: Adenine (A), Guanine (G), Cytosine (C) and Thymine (T). These
NOTES nucleotide are arranged one after the other in a genome and encode for
proteins which perform functions responsible for a phenotype.
A codon is made up of three nucleotides which encodes for an amino acid.
There are 64 possible codons and 20 amino acids, each amino acid is
encoded by more than one codon. Also, three start and one stop codon is
there which initiate or terminates the expression processes.
The term Gene was coined by Johannsen in 1909 and he named hereditary
units of Mendel as ‘Genes’. Basically, a gene encodes for a single polypeptide
has a start codon in the beginning and stop codon at the end.
Gene has a position on chromosomes called locus. Due to chromosomal
aberrations, locus may change which can affect phenotypic expression.
The mechanism of gene expression was first observed in 1900 when the
bacteria was grown under the presence of lactose showed a production of
an enzyme which was not observed in the absence of lactose. This gives an
idea that certain products are utilised by the bacteria when the machinery of
its breakdown is turned on.
In presence of lactose the produced enzyme is being utilised but in its
absence, bacteria still can obtain carbon source from different sources. These
enzymes are called as adaptive or facultative.
The adaptive enzymes are called as inducible, whose production is induced
by the presence of a factor or substrate, commonly called as inducer. This
is an example of positive control of gene expression.
If an enzyme or protein production is repressed by some compound that
enzyme is called as repressible and the compound which repressed is called
as repressor. Under negative control or repression, the expression of a
gene remain continued till the repressor molecule get activated and repressed
the expression.
There are some genes whose expression is continuous and not dependent
upon the circumstances or environment.
It was evident that in the presence of lactose, enzyme which cause lactose
breakdown increases in concentration from few molecules to thousand
molecules. However, Lac the absence of lactose decreases the production
of lactose metabolising enzyme. Thus, this enzyme is inducible and lactose
is a substrate as well as an inducer here.
It was determined that lactose metabolism is under the control of three
structural genes which are arranged together in a cluster in genome of
prokaryotes.
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198 Material
LacZ gene encodes for beta-galactosidase, an enzyme which convert lactose Gene Concept and
Lactose System
(disaccharide) to the monomer units, glucose and galactose
(monosaccharides).
LacY gene encodes for permease which helps in the entry of lactose through
NOTES
the cell wall of bacteria.
LacA gene encodes for transacetylase which known to have probable role
in exporting out as by product of lactose breakdown.
In prokaryotes, enzymes necessary for the production of amino acids are
present, which activates in the absence of external source of amino acids
(in a media or environment). When, there is scarcity of tryptophan, the
operon get activated and continued. The repressor molecule do not bind to
the operator during this time.
When tryptophan is present and can be obtained from the environment, it
act as a co-repressor. Tryptophan binds to the repressor molecule and
activate it allosterically, afterwards, repressor-trp complex binds to the
operator and switched off the gene expression of trp operon. Hence, the
control of this operon is different from lac operon as trp is acting as a co-
repressor while lactose is an inducer.
Structurally, trp operon consists of five genes whose product is important
for trp biosynthesis. These are trpA, trpB, trpC, trpD and trpE. TrpP is a
promoter and trpO is an operator where RNA polymerase binds and initiates
transcription.
Trp RNA-binding Attenuation Protein (TRAP) is a molecule present in the
cell. When trp is present, it binds to trp and form a complex.
Arabinose operon is a good example of both positive and negative control
of gene expression. It performs the metabolic action of arabinose and
consists of three structural genes, araB, araA and araD. Their transcription
is controlled by a regulatory protein AraC which is encoded by araC gene.
In eukaryotes, DNA is arranged in many chromosomes rather one or two.
Their transcription and translation is controlled differently. These
chromosomes reside in the nucleus bounded by double layered nuclear
membrane.
In eukaryotes, transcription and translation takes place in different
compartments. Transcription takes place inside the nucleus whereas
translation proceeds in cytoplasm.
There are three regions which plays an important role in modulating the
transcription levels or switching it on or off, promoters, enhancer and
silencers. These are called as cis regulators as they control the gene regulation
by being present on the same chromosome.
Trans regulators are the ones which control gene regulation by being present
on another chromosome. These are generally transcription factors and
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Gene Concept and modulators. Promoter region is situated upstream to the genes for initiating
Lactose System
transcription. These are the sites recognised by transcriptional machinery.
They are up to 100 bp long and consists of the repeat of TATA nucleotides,
i.e., Thiamine (T) and Adenine (A).
NOTES
Methylation of DNA is an important way to reduce the gene expression.
The methyl group protrudes out from the DNA molecule and affects the
binding of RNA polymerase and transcription factors. Methylation occur
more often at CG repeats.
In humans, hundred thousands of proteins are encoded by nearly 30,000
genes because of alternative splicing mechanism. It maintain the regulation
and also provide direction to the transcript for production of a particular
protein. By this way, gene regulation can also be controlled after the
transcription.
There is another type of regulation is identified from the RNA molecules
called as RNA Directed DNA Methylation (RdDM). These RNA molecules
binds to the complementary DNA and initiates its methylation. Thus,
regulating their expression by methylating the target DNA.
There are proteins called as RNA-Binding Proteins or RBPs which can
bind to the untranslated regions, or UTRs of mRNA. UTRs are the region
which cannot be translated and help in mRNA localization, stability, and
protein translation. Binding of proteins at these regions can greatly affect
the stability of mRNA as it may enhance or decrease.
Translation is a process by which a mature mRNA is converted into
designated protein molecule in eukaryotes. Regulation of this process is
achieved by sending a message for proceeding or not for translation. One
such example is synthesis of - and -tubulin which are subunits of
microtubules. Colchicine treatment to the cell can induce the disassembly of
microtubules and accumulate the - and -tubulin in the cell.
During cell cycle progression, if a cell signalled to stop dividing and growing,
it arrest in stage G1. Then it enters in a G0 stage where no growth or
division takes place, instead, a cell stay and perform its functions normally.
Most differentiated cells stay in G0 stage indefinitely and do not enters in
cell cycle again. However, if signaled from the external environments then
they may enter in the cell cycle again. During the loss of this regulation, a
cell proceeds with all the four stages of cell cycle one after the other
continuously and cannot stop growing and dividing.
In a cell cycle, there are three points where cell monitors and respond to its
internal equilibrium before moving to the next stage. These are between G1
and S phase G1/S, G2 and M phase G2/M and M phase. These are also
called as checkpoints as they check the progression and can arrest the cell
for proceeding.
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200 Material
G1/S check point checks the size of the cell, growth factors, nutrients and Gene Concept and
Lactose System
determine any damage to DNA molecule which should be repaired before
it proceeds to the next stage.
G2/M check point, the physiological conditions of the cell is monitored.
NOTES
The cell is analyzed for the complete DNA replication and whether DNA is
damaged.
Cyclins are one of the important cell cycle regulators. Four basic types of
cyclins are found in humans and other eukaryotes. These are G1, G1/S, S
and M cyclins.
According to their names, each cyclin is related to particular phase or
transition of the cell cycle. The S cyclin promotes the DNA replication
during S phase.
A cyclin’s concentration also varies throughout the cell cycle, i.e., their
concentration stays low throughout the cell cycle but increases during the
phase or transition at which they required.
The M cyclin is present in highest concentration during M phase. S cyclin is
at highest concentration during S and G2 phase also.
G1 cyclin is an exception as it stays in a similar concentration throughout the
cycle as needed at many points.
The cyclins are synthesized during different stages of cell cycle and
subsequently destroyed also to maintain the progression. These cyclins are
also named as cyclin A, cyclin B, cyclin D and cyclin E.
To perform functions of activating or deactivating many proteins inside the
cell, cyclins bind to a specialized group of proteins called as Cyclin Dependent
Kinases (CDKs). Without cyclins they remain inactive but binding with
cyclins activates them making it a functional enzyme and allowing it to modify
target proteins.
The CDKs are the kinases which phosphorylates or attach phosphate groups
to target proteins. These target proteins are generally specific to specific
kinases. This affect the activity of target protein by making them more or
less active.
An inducer is a molecule that regulates gene expression. An inducer can
bind to protein repressors or activators.
Repressible enzymes have their synthesis inhibited by a metabolite (for
example, tryptophan).
Inducible enzymes have their synthesis stimulated or induced by specific
metabolites (for example, lac operon).
An operon is a cluster of functionally-related genes that are controlled by a
shared operator. Operons consist of multiple genes grouped together with
a promoter and an operator.
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Gene Concept and Operons are present in prokaryotes (bacteria and archaea), but are absent
Lactose System
in eukaryotes. In some situations multiple operons are controlled by the
same regulatory protein; in these cases the operons form a regulon.
Operons were first identified as a mode of gene expression control in 1961
NOTES
by François Jacob and Jacques Monod.
An operon is a complete package for gene expression and synthesis of
polypeptides. By combining the related genes, all polypeptides required for
a specific function are synthesized in response to a single stimulus.
Operons can be under negative or positive control.
Negative control involves turning off the operon in the presence of a
repressor; this can be either repressible or inducible. A repressible operon
is one that is usually on but which can be repressed in the presence of a
repressor molecule.
Positive control of an operon is when gene expression is stimulated by the
presence of a regulatory protein.
The Lac operon is the classic operon example, and is responsible for the
degradation of the milk protein lactose. The Lac operon is an inducible
operon; in the absence of lactose the operator is blocked by a repressor
protein.
The operon is made up of a promoter with operator, and three genes (lacZ,
lacY, and lacA) which encode -galactosidase, permease, and
transacetylase.
The expression of the Lac operon is controlled by the regulatory gene lacI,
located immediately adjacent to the promoter region. LacI encodes an
allosteric repressor protein that keep the Lac operon ‘OFF’.
A bacterium’s environment is too complex for its genes to be controlled by
one signal. Other factors besides lactose affect the expression of the lac
genes, such as the availability of glucose.
A regulatory mechanism known as catabolite repression restricts expression
of the genes required for catabolism of lactose, arabinose, and other sugars
in the presence of glucose, even when these secondary sugars are also
present.
The effect of glucose is mediated by cAMP, as a co-activator, and an activator
protein known as cAMP receptor protein, or CRP (the protein is sometimes
called CAP, for Catabolite gene Activator Protein).
CRP-cAMP is therefore a positive regulatory element responsive to glucose
levels.

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202 Material
 Gene Concept and
9.8 KEY WORDS Lactose System

Genes: These are the small segments present on DNA. They encode for
proteins which describes an individual’s phenotype. They are characterized NOTES
by the presence of start codon in the beginning and stop codon at the end.
Locus: Gene has a position on chromosomes called locus. Due to
chromosomal aberrations, locus may change which can affect phenotypic
expression.
LacZ gene: It encodes for beta-galactosidase, an enzyme which convert
lactose (disaccharide) to the monomer units, glucose and galactose
(monosaccharides).
LacY gene: This gene encodes for permease which helps in the entry of
lactose through the cell wall of bacteria.
LacA gene: This gene encodes for transacetylase which known to have
probable role in exporting out as by product of lactose breakdown.
G1/S check point: It checks the size of the cell, growth factors, nutrients
and determine any damage to DNA molecule which should be repaired
before it proceeds to the next stage.
G2/M check point: In this check point, physiological conditions of the cell
is monitored, the cell is analyzed for the complete DNA replication and
whether DNA is damaged.
Inducer: An inducer is a molecule that regulates gene expression, it can
bind to protein repressors or activators.
Repressible enzymes: Enzymes which have their synthesis inhibited by a
metabolite (for example, tryptophan).
Inducible enzymes: Enzymes which have their synthesis stimulated or
induced by specific metabolites (for example, lac operon).
Operon: An operon is a cluster of functionally-related genes that are
controlled by a shared operator.

9.9 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is the significance of gene?
2. How the regulation of bacterial gene expression is done?
3. Define the terms - operon, repressor, inducer, polycistronic mRNA and
regulatory genes giving examples.
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Material 203
Gene Concept and 4. Write a short note on CRP (cAMP receptor protein).
Lactose System
5. What do you understand by positive and negative control of operons?
6. How is cell cycle regulation done? Why it is important?
NOTES 7. What is the relation between cyclins and CDKs?
8. What is lactose system?
9. Explain the Lac components.
10. Differentiate between positive and negative regulation.
11. What is catabolite repression?
Long Answer Questions
1. Briefly discuss the significance and concept of gene giving appropriate
examples.
2. Explain the significant features of regulation of bacterial gene expression.
3. Differentiate between repressible enzymes and inducible enzymes.
4. Describe the structure and functions of operons.
5. What do you understand by the expression of the lac operon when both
glucose and lactose are present?
6. Explain the role of cyclins and CDKs in cell cycle regulation. What if only
one cyclin or CDK loss their function?
7. Briefly discuss about the lactose system and Lac operon.
8. Explain the following terms giving appropriate examples:
(a) Inducer
(b) Enhancer
(c) Operon Structure
(d) Lac Components
(e) Positive and Negative Regulation
(f) Catabolite Repression

9.10 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.

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204 Material
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H. Gene Concept and
Lactose System
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
NOTES
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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Tryptophan Operon
and Arabinose Operon
UNIT 10 TRYPTOPHAN OPERON
AND ARABINOSE OPERON
NOTES
Structure
10.0 Introduction
10.1 Objectives
10.2 Tryptophan Operon
10.3 Arabinose Operon and its Regulation
10.4 Answers to Check Your Progress Questions
10.5 Summary
10.6 Key Words
10.7 Self Assessment Questions and Exercises
10.8 Further Readings

10.0 INTRODUCTION

The tryptophan operon is the regulation of transcription of the gene responsible

for biosynthesis of tryptophan from chorismate. Tryptophan operon consists of
structural gene and regulatory gene. The regulatory gene include Promoter,
Repressor, Operater and Leader sequence while the structural gene include TrpE,
TrpD, TrpC, TrpB and TrpA. Fundamentally, the Trp operon is an operon in
which a group of genes is used or transcribed together that codes for the components
for production of tryptophan. The ‘Trp’ operon is present in many bacteria, but
was first characterized in Escherichia coli. The operon is regulated so that when
tryptophan is present in the environment, the genes for tryptophan synthesis are
not expressed. It was an important experimental system for learning about gene
regulation, and is commonly used to teach gene regulation. Attenuation is a second
mechanism of negative feedback in the Trp operon. The repression system targets
the intracellular Trp concentration whereas the attenuation responds to the
concentration of charged tRNAtrp. Attenuation is possible in prokaryotes (which
have no nucleus), the ribosomes begin translating the mRNA while RNA polymerase
is still transcribing the DNA sequence. This allows the process of translation to
affect transcription of the operon directly.
Arabinose is a 5 Carbon sugar that can be used as an alternative Carbon
and energy source by the bacteria Escherichia coli. The enzymes necessary for
the metabolism of this sugar are encoded by the araBAD operon. The operon
encodes a single polycistronic mRNA with three open reading frames. Each open
reading frame encodes one of the enzymes of the catabolic pathway for Arabinose.
The regulator protein AraC is sensitive to the level of arabinose and plays a dual
role as both an activator in the presence of arabinose and a repressor in the absence
of arabinose to regulate the expression of araBAD.
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206 Material
 In this unit, you will study about the Tryptophan operon, attenuation Tryptophan Operon
and Arabinose Operon
mechanism in the Trp operon, Arabinose operon and its regulation mechanism in
detail.
NOTES
10.1 OBJECTIVES

After going through this unit, you will be able to:

Understand what Tryptophan operon is
Explain the attenuation mechanism
Discuss about the Arabinose operon and its regulation mechanism

10.2 TRYPTOPHAN OPERON

The tryptophan operon is the regulation of transcription of the gene responsible

for biosynthesis of tryptophan from chorismate. Tryptophan operon consists of
structural gene and regulatory gene. The regulatory gene include Promoter,
Repressor, Operater and Leader sequence while the structural gene include TrpE,
TrpD, TrpC, TrpB and TrpA. Fundamentally, the Trp operon is an operon in
which a group of genes is used or transcribed together that codes for the components
for production of tryptophan. The ‘Trp’ operon is present in many bacteria, but
was first characterized in Escherichia coli. The operon is regulated so that when
tryptophan is present in the environment, the genes for tryptophan synthesis are
not expressed. It was an important experimental system for learning about gene
regulation, and is commonly used to teach gene regulation.
The operon operates by a negative repressible feedback mechanism. The
repressor for the trp operon is produced upstream by the trpR gene, which is
constitutively expressed at a low level. Synthesized trpR monomers associate into
dimers. When tryptophan is present, these tryptophan repressor dimers bind to
tryptophan, causing a change in the repressor conformation, allowing the repressor
to bind to the operator. This prevents RNA polymerase from binding to and
transcribing the operon, so tryptophan is not produced from its precursor. When
tryptophan is not present, the repressor is in its inactive conformation and cannot
bind the operator region, so transcription is not inhibited by the repressor.
The tryptophan operon is the regulation of transcription of the gene
responsible for biosynthesis of tryptophan from chorismate.

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Tryptophan Operon
and Arabinose Operon

NOTES

Fig. 10.1 Trp Operon

Tryptophan operon consists of structural gene and regulatory gene.

I. Regulatory gene are Promoter, Repressor, Operator, and Leader
sequence.
II. Structural gene are TrpE, TrpD, TrpC, TrpB and TrpA
1. trpE: It Encodes the Enzyme Anthranilate Synthase I
2. trpD: It Encodes the Enzyme Anthranilate Synthase II
3. trpC: It Encodes the Enzyme N-5’-Phosphoribosyl Anthranilate
Isomerase and Indole-3-Glycerolphosphate Synthase
4. trpB: It Encodes the Enzyme Tryptophan Synthase-B sub unit
5. trpA: It Encode the Enzyme Tryptophan Synthase-A sub unit
Tryptophan operon is regulated by following mechanism:
1. Repression

(i) When Tryptophan is Absent in Cell

Repressor gene (trpR) encodes the repressor protein which is originally inactive.
In the absence of tryptophan, transcription of structural gene occur for the
biosynthesis of tryptophan from chorismate (Refer Figure 10.2)..

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208 Material
 Tryptophan Operon
and Arabinose Operon

NOTES

Fig. 10.2 Tryptophan Absent, Repressor Inactive, Operon on (b) Tryptophan Absent,
Repressor Inactive, Operon Off

(ii) When Tryptophan is High in Cell

When tryptophan is high in cell then it binds with repressor protein and change its
confirmation so that it become active and bind to the operator near promoter.
Binding of repressor protein to operator overlaps the promoter, so RNA
polymerase cannot bind to the promoter. Hence transcription is halted.
Since tryptophan is already high in cell, no transcription of structural gene is
required for biosynthesis of tryptophan. This is also known as negative regulation.
2. Attenuation
Attenuation is a second mechanism of negative feedback in the Trp operon. The
repression system targets the intracellular Trp concentration whereas the attenuation
responds to the concentration of charged tRNAtrp. Attenuation is possible in
prokaryotes (which have no nucleus), the ribosomes begin translating the mRNA
while RNA polymerase is still transcribing the DNA sequence. This allows the
process of translation to affect transcription of the operon directly.
In bacteria, transcription and translation occurs simultaneously. The
translation starts before transcription completes. In this attenuation mechanism,
rate of translation determines whether transcription continues or terminates.
Therefore the attenuation mechanism is only found in bacteria but not in eukaryotic
cell.

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Material 209
Tryptophan Operon Leader sequence (trpL) play important role in attenuation. Leader sequence
and Arabinose Operon
contains such a nucleotide sequence that mRNA transcribed from it contains four
specific region. Region 1, Region 2, Region 3 and Region 4. Region 3 is
complementary to both Region 2 and Region 4.
NOTES
If region 3 and region 4 base pair with each other, they form a loop like
structure called attenuator and it function as transcriptional termination. If pairing
occur between Region 3 and Region 2, then no such attenuator form so that
transcription continues.
Region 1 is the most important region that determines whether to form loop
between Region 2-Region 3 or Region 3-Region 4. The Region 1 consists of
sequence of 14 Codons, out of which two codons are Tryptophan Codon (Codon
10 and 11) (Refer Figure 10.3).

Fig. 10.3 Attenuation

When tryptophan is high in cell then tRNA carrying tryptophan encodes

Codon 10 and 11, such that ribosome encloses the Region 2 which is near to the
tryptophan codon. Hence region 3 base pair with Region 4 to form attenuator as
Region 2 is not available for pairing. Consequently, transcription is halted.
When tryptophan is low or absent in cell, then translation stops at the position
of Tryptophan Codon. Such that loop between Region 2 and Region 3 forms.
Transcription continues.
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210 Material
Feedback Mechanism of Trp Operon Tryptophan Operon
and Arabinose Operon
1. When tryptophan is high in cell then transcription of structural gene does
not occur.
2. When tryptophan is absent or very low then transcription continues. NOTES
3. If chorismate is high in cell then it favors the transcription of structural gene.
4. If chorismate is low or absent in cell then it inhibit transcription.

10.3 ARABINOSE OPERON AND ITS REGULATION

Arabinose is a 5 Carbon sugar that can be used as an alternative Carbon and

energy source by the bacteria Escherichia coli. The enzymes that are essential
for the metabolism of this sugar are encoded by the araBAD operon. The operon
encodes a single polycistronic mRNA with three open reading frames. Each open
reading frame encodes one of the enzymes of the Catabolic Pathway for Arabinose.
Expression of the araBAD operon is highly regulated. Key to its regulation is the
composition of sugars in the environment, for example, the operon shows a classic
pattern of substrate level regulation. When arabinose is absent from the environment
then this operon is not expressed, therefore it is essential that to express this
arabinose operon it must be present in the environment. This is an adaptive
mechanism that ensures the enzymes required to catabolize arabinose and are
only produced when arabinose is present in the environment.
The araBAD operon also exhibits the phenomena of catabolite repression.
High levels of glucose in the environment prevent the expression of the araBAD
operon even if arabinose is present. This is believed to be adaptive because
bacteria can extract energy more efficiently from glucose than from arabinose.
Therefore if glucose is present in the environment, the enzymes for metabolism of
arabinose are not produced.
The L-Arabinose operon, also called the ara or araBAD operon, is an
operon required for the breakdown of the five Carbon sugar the L-arabinose, in
Escherichia coli. The L-arabinose operon contains three structural genes - araB,
araA, araD, collectively known as araBAD, which encode for three metabolic
enzymes that are required for the metabolism of L-arabinose. AraB (Ribulokinase),
AraA (an Isomerase), AraD (an Epimerase) produced by these genes catalyse
conversion of L-arabinose to an intermediate of the pentose phosphate pathway,
D-Xylulose-5-Phosphate.
Figure 10.4 illustrates the structure of the araBAD operon. The three open
reading frames, araB, araA and araD, are denoted by the boxes. The cis elements
regulate transcription. In addition to the promoter (PBAD), there are three critical
regulatory elements araI1, araI2 and araO2 that are involved in substrate level
regulation.
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Tryptophan Operon
and Arabinose Operon

Fig. 10.4 Structure of the araBAD Operon

NOTES AraC is the regulatory protein that mediates substrate level regulation of
transcription.  It is a sequence specific binding protein that binds to the sequences
at araI1, araI2 and araO2.  Because AraC functions as a homodimer, it always
binds two of these regulatory elements at the same time. AraC also has an Arabinose
Binding Pocket.  The binding of Arabinose to AraC alters is allosteric conformation,
making the AraC protein more flexible. This increase flexibility is significant to the
regulation of the operon. The AraC also has a domain that interacts with RNA
polymerase, helping to recruit RNA polymerase to PBAD and thereby promoting
transcription of araBAD.
In the presence of arabinose, the AraC homodimer has a more flexible
structure which allows the AraC homodimer to simultaneously bind the nearby
adjacent sites araI1 and araI2, as shown in Figure 10.5. Binding of AraC to araI1/
araI2 allows it to actively recruit RNA polymerase to PBAD and thereby promotes
transcription.

Fig. 10.5 AraC Homodimer Simultaneously Binding the Nearby

Adjacent Sites araI1 and araI2

In the absence of Arabinose, the AraC homodimer has a ridged structure.

This ridged structure interferes in binding to the closely adjacent sites araI1 and
araI2. As an alternative the ridged AraC binds to araI1, and araO2. For a single
homodimer of AraC to bind to these two distant DNA site requires DNA looping.
The binding of AraC to these two sites prevents transcription of the araBAD
operon in two different ways. First, since AraC is bound to araO2 instead of
araI2, it is not able to promote recruitment of RNA polymerase. Second, the loop
of DNA hinders the finding of the RNA polymerase to PBAD, as well as the binding
of CAP to the cap binding site.
Figure 10.6 illustrates the map of the Ara region, in which the B, A, and D
genes together with the I and O sites constitute the Ara operon.

Fig. 10.6 Map of the Ara Region

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212 Material
 The specific structural genes, araB, araA and araD encode the metabolic Tryptophan Operon
and Arabinose Operon
enzymes for breaking down Arabinose are transcribed as a multigenic mRNA.
Transcription is activated at araI, termed as the initiator region, which contains
both an operator site and a promoter. When the araC gene, when bound to
arabinose, encodes an activator protein that activates transcription of the Ara NOTES
operon, possibly by facilitating RNA polymerase bind to the promoter that is
located within the araI region. An additional activation event is typically mediated
by the similar CAP–cAMP catabolite repression system that is responsible for the
regulation of lac operon expression.
When the arabinose is present then both the CAP–cAMP complex and the
AraC arabinose complex essentially bind to the initiator region so that the RNA
polymerase binds to the promoter and transcribe the Ara operon (Refer Figure
10.7). When the arabinose is not present then the AraC protein assumes a dissimilar
or different conformation and represses the Ara operon by binding both to araI
and to a second operator region AraO, thus forming a loop in order to prevent
transcription. Consequently, the AraC protein has two conformations, first that
acts as an activator and the second that acts as a repressor. Figure 10.7 illustrates
the control mechanism of the Ara operon.

Fig. 10.7 Control Mechanism of the Ara Operon

Check Your Progress

1. Explain the term tryptophan operon giving its types.
2. What happens when tryptophan is high in cell?
3. What is attenuation?
4. Explain about Arabinose operon giving example.
5. What happens when arabinose is absent from the environment?
6. How araBAD operon exhibits the phenomena of catabolite repression?
7. What is L-Arabinose operon?
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Material 213
Tryptophan Operon
and Arabinose Operon 10.4 ANSWERS TO CHECK YOUR PROGRESS
QUESTIONS

NOTES 1. The tryptophan operon is the regulation of transcription of the gene

responsible for biosynthesis of tryptophan from chorismate. Tryptophan
operon consists of structural gene and regulatory gene. The regulatory gene
include Promoter, Repressor, Operater and Leader sequence while the
structural gene include TrpE, TrpD, TrpC, TrpB and TrpA. Fundamentally,
the Trp operon is an operon in which a group of genes is used or transcribed
together that codes for the components for production of tryptophan. The
‘Trp’ operon is present in many bacteria, but was first characterized in
Escherichia coli. The operon is regulated so that when tryptophan is present
in the environment, the genes for tryptophan synthesis are not expressed.
2. When tryptophan is high in cell then it binds with repressor protein and
change its confirmation so that it become active and bind to the operator
near promoter.
Binding of repressor protein to operator overlaps the promoter, so RNA
polymerase cannot bind to the promoter. Hence transcription is halted.
Since tryptophan is already high in cell, no transcription of structural gene is
required for biosynthesis of tryptophan. This is also known as negative
regulation.
3. Attenuation is a second mechanism of negative feedback in the Trp operon.
The repression system targets the intracellular Trp concentration whereas
the attenuation responds to the concentration of charged tRNAtrp. Attenuation
is possible in prokaryotes (which have no nucleus), the ribosomes begin
translating the mRNA while RNA polymerase is still transcribing the DNA
sequence. This allows the process of translation to affect transcription of
the operon directly.
4. Arabinose is a 5 Carbon sugar that can be used as an alternative Carbon
and energy source by the bacteria Escherichia coli. The enzymes that are
essential for the metabolism of this sugar are encoded by the araBAD operon.
The operon encodes a single polycistronic mRNA with three open reading
frames. Each open reading frame encodes one of the enzymes of the
Catabolic Pathway for Arabinose. Expression of the araBAD operon is
highly regulated. Key to its regulation is the composition of sugars in the
environment, for example, the operon shows a classic pattern of substrate
level regulation.
5. When arabinose is absent from the environment then this operon is not
expressed, therefore it is essential that to express this arabinose operon it
must be present in the environment. This is an adaptive mechanism that
ensures the enzymes required to catabolize arabinose and are only produced
Self-Instructional when arabinose is present in the environment.
214 Material
 6. The araBAD operon exhibits the phenomena of catabolite repression. High Tryptophan Operon
and Arabinose Operon
levels of glucose in the environment prevent the expression of the araBAD
operon even if arabinose is present. This is believed to be adaptive because
bacteria can extract energy more efficiently from glucose than from
arabinose. Therefore if glucose is present in the environment, the enzymes NOTES
for metabolism of arabinose are not produced.
7. The L-Arabinose operon, also called the ara or araBAD operon, is an
operon required for the breakdown of the five Carbon sugar the L-arabinose,
in Escherichia coli. The L-arabinose operon contains three structural genes
- araB, araA, araD, collectively known as araBAD, which encode for three
metabolic enzymes that are required for the metabolism of L-arabinose.
AraB (Ribulokinase), AraA (an Isomerase), AraD (an Epimerase) produced
by these genes catalyse conversion of L-arabinose to an intermediate of the
pentose phosphate pathway, D-Xylulose-5-Phosphate.

10.5 SUMMARY

The tryptophan operon is the regulation of transcription of the gene

responsible for biosynthesis of tryptophan from chorismate.
Tryptophan operon consists of structural gene and regulatory gene. The
regulatory gene include Promoter, Repressor, Operater and Leader sequence
while the structural gene include TrpE, TrpD, TrpC, TrpB and TrpA.
Fundamentally, the Trp operon is an operon in which a group of genes is
used or transcribed together that codes for the components for production
of tryptophan.
The ‘Trp’ operon is present in many bacteria, but was first characterized in
Escherichia coli. The operon is regulated so that when tryptophan is present
in the environment, the genes for tryptophan synthesis are not expressed.
The tryptophan operon is the regulation of transcription of the gene
responsible for biosynthesis of tryptophan from chorismate.
The operon operates by a negative repressible feedback mechanism. The
repressor for the trp operon is produced upstream by the trpR gene, which
is constitutively expressed at a low level.
Synthesized trpR monomers associate into dimers. When tryptophan is
present, these tryptophan repressor dimers bind to tryptophan, causing a
change in the repressor conformation, allowing the repressor to bind to the
operator.
Repressor gene (trpR) encodes the repressor protein which is originally
inactive. In the absence of tryptophan, transcription of structural gene occur
for the biosynthesis of tryptophan from chorismate.

Self-Instructional
Material 215
Tryptophan Operon When tryptophan is high in cell then it binds with repressor protein and
and Arabinose Operon
change its confirmation so that it become active and bind to the operator
near promoter.
Binding of repressor protein to operator overlaps the promoter, so RNA
NOTES
polymerase cannot bind to the promoter. Hence transcription is halted.
Since tryptophan is already high in cell, no transcription of structural gene is
required for biosynthesis of tryptophan. This is also known as negative
regulation.
Attenuation is a second mechanism of negative feedback in the Trp operon.
The repression system targets the intracellular Trp concentration whereas
the attenuation responds to the concentration of charged tRNAtrp.
Attenuation is possible in prokaryotes (which have no nucleus), the ribosomes
begin translating the mRNA while RNA polymerase is still transcribing the
DNA sequence. This allows the process of translation to affect transcription
of the operon directly.
In bacteria, transcription and translation occurs simultaneously. The
translation starts before transcription completes. In this attenuation
mechanism, rate of translation determines whether transcription continues
or terminates. Therefore the attenuation mechanism is only found in bacteria
but not in eukaryotic cell.
When tryptophan is high in cell then tRNA carrying tryptophan encodes
Codon 10 and 11 such that ribosome encloses the Region 2 which is near
to the Tryptophan Codon. Hence Region 3 base pair with Region 4 to form
attenuator as Region 2 is not available for pairing. Consequently, transcription
is halted.
Arabinose is a 5 Carbon sugar that can be used as an alternative Carbon
and energy source by the bacteria Escherichia coli.
The enzymes that are essential for the metabolism of this sugar are encoded
by the araBAD operon. The operon encodes a single polycistronic mRNA
with three open reading frames. Each open reading frame encodes one of
the enzymes of the Catabolic Pathway for Arabinose.
Expression of the araBAD operon is highly regulated. Key to its regulation
is the composition of sugars in the environment, for example, the operon
shows a classic pattern of substrate level regulation.
When arabinose is absent from the environment then this operon is not
expressed, therefore it is essential that to express this arabinose operon it
must be present in the environment. This is an adaptive mechanism that
ensures the enzymes required to catabolize arabinose and are only produced
when arabinose is present in the environment.

Self-Instructional
216 Material
The araBAD operon also exhibits the phenomena of catabolite repression. Tryptophan Operon
and Arabinose Operon
High levels of glucose in the environment prevent the expression of the
araBAD operon even if arabinose is present. This is believed to be adaptive
because bacteria can extract energy more efficiently from glucose than from
arabinose. NOTES
Therefore if glucose is present in the environment, the enzymes for metabolism
of arabinose are not produced.
The L-Arabinose operon, also called the ara or araBAD operon, is an
operon required for the breakdown of the five Carbon sugar the L-
Arabinose, in Escherichia coli.
The L-arabinose operon contains three structural genes - araB, araA, araD,
collectively known as araBAD, which encode for three metabolic enzymes
that are required for the metabolism of L-arabinose. AraB (Ribulokinase),
AraA (an Isomerase), AraD (an Epimerase) produced by these genes
catalyse conversion of L-arabinose to an intermediate of the pentose
phosphate pathway, D-Xylulose-5-Phosphate.
AraC is the regulatory protein that mediates substrate level regulation of
transcription.  It is a sequence specific binding protein that binds to the
sequences at araI1, araI2 and araO2.  Because AraC functions as a
homodimer, it always binds two of these regulatory elements at the same
time.
AraC has an Arabinose Binding Pocket.  The binding of Arabinose to AraC
alters is allosteric conformation, making the AraC protein more flexible. This
increase flexibility is significant to the regulation of the operon.
The AraC has a domain that interacts with RNA polymerase, helping to
recruit RNA polymerase to PBAD and thereby promoting transcription of
araBAD.
AraC is the regulatory protein that mediates substrate level regulation of
transcription.
In the presence of arabinose, the AraC homodimer has a more flexible
structure which allows the AraC homodimer to simultaneously bind the
nearby adjacent sites araI1 and araI2.
In the absence of Arabinose, the AraC homodimer has a ridged structure.
This ridged structure interferes in binding to the closely adjacent sites araI1
and araI2. As an alternative the ridged AraC binds to araI1, and araO2.
When the arabinose is present then both the CAP–cAMP complex and the
AraC arabinose complex essentially bind to the initiator region so that the
RNA polymerase binds to the promoter and transcribe the Ara operon.

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Material 217
Tryptophan Operon
and Arabinose Operon 10.6 KEY WORDS

Tryptophan operon: The tryptophan operon is the regulation of transcription

NOTES of the gene responsible for biosynthesis of tryptophan from chorismate, the
tryptophan operon consists of structural gene and regulatory gene.
Attenuation: It is a second mechanism of negative feedback in the Trp
operon, the repression system targets the intracellular Trp concentration
whereas the attenuation responds to the concentration of charged tRNAtrp.
Arabinose: Arabinose is a 5 Carbon sugar that can be used as an alternative
Carbon and energy source by the bacteria Escherichia coli.
AraC: It is the regulatory protein that mediates substrate level regulation of
transcription.

10.7 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is tryptophan operon?
2. Differentiate between regulatory and structural genes.
3. What happens when tryptophan is absent in cells?
4. Explain the term attenuation giving example.
5. What is Arabinose operon?
6. How the Arabinose operon is regulated?
7. What is araBAD?
Long Answer Questions
1. Briefly discuss about the tryptophan operon and its types giving examples.
2. Explain the mechanism required for the regulation of tryptophan operon.
3. What is attenuation? Explain giving examples.
4. Discuss the significant features of Arabinose operon.
5. Explain in detail how the regulation of Arabinose operon is done with the
help of diagrams.
6. “AraC is the regulatory protein that mediates substrate level regulation of
transcription”. Discuss.

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218 Material
 Tryptophan Operon
10.8 FURTHER READINGS and Arabinose Operon

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing NOTES
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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Material 219
Plasmids: Types
and its Properties
UNIT 11 PLASMIDS: TYPES
AND ITS PROPERTIES
NOTES
Structure
11.0 Introduction
11.1 Objectives
11.2 Plasmids: Types and Properties
11.2.1 Types of Plasmids
11.2.2 Properties and Characteristics
11.3 Answers to Check Your Progress Questions
11.4 Summary
11.5 Key Words
11.6 Self Assessment Questions and Exercises
11.7 Further Readings

11.0 INTRODUCTION

Plasmid, in microbiology, an extrachromosomal genetic element that occurs in

many bacterial strains. Plasmids are circular DeoxyriboNucleic Acid (DNA)
molecules that replicate independently of the bacterial chromosome. They are not
essential for the bacterium but may confer a selective advantage. One class of
plasmids, Colicinogenic (or Col) factors, determines the production of proteins
called Colicins, which have antibiotic activity and can kill other bacteria. Another
class of plasmids, R factors, confers upon bacteria resistance to antibiotics.
Some Col factors and R factors can transfer themselves from one cell to another
and thus are capable of spreading rapidly through a bacterial population. A plasmid
that is attached to the cell membrane or integrated into the bacterial chromosome
is called an episome.
Plasmids are extremely valuable tools in the fields of molecular biology and
genetics, specifically in the area of genetic engineering. They play a critical role in
such procedures as gene cloning, recombinant protein production, and gene
therapy research. In such procedures, a plasmid is cut at a specific site using
enzymes called restriction endonucleases. A foreign DNA element is then spliced
into the plasmid. The resulting circular structure, a recombinant DNA molecule, is
then introduced into bacterial cells. The autonomous replication of the plasmid
within the bacterial cells makes it possible to produce large numbers of copies of
the recombinant DNA molecule for experimental manipulation or commercial
purposes. Plasmids are well suited to genetic engineering in other ways.
Their antibiotic resistance genes, for example, prove useful in identifying those
bacterial cells that have taken up the recombinant DNA molecule in a high
background of untransformed cells.
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 In this unit, you will study about plasmids, its different types - F, R and Col Plasmids: Types
and its Properties
plasmids, properties of plasmids - sex factors, drug resistant, Colicinogenic,
Agrobacterium Ti and broad host range plasmid in detail.

NOTES
11.1 OBJECTIVES

After going through this unit, you will be able to:

Understand about plasmids
Discuss the types of plasmids
Explain the properties of plasmids

11.2 PLASMIDS: TYPES AND PROPERTIES

A plasmid is a small DNA molecule within a cell that is physically separated

from chromosomal DNA and can replicate independently. They are most commonly
found as small circular, double-stranded DNA molecules in bacteria; however,
plasmids are sometimes present in archaea and eukaryotic organisms. In nature,
plasmids often carry genes that benefit the survival of the organism, such as by
providing antibiotic resistance. While the chromosomes are big and contain all the
essential genetic information for living under normal conditions, plasmids usually
are very small and contain only additional genes that may be useful in certain
situations or conditions. Artificial plasmids are widely used as vectors in molecular
cloning, serving to drive the replication of recombinant DNA sequences within
host organisms. In the laboratory, plasmids may be introduced into a cell
via transformation. Figure 11.1 shows the image of a plasmid.

Fig. 11.1 Plasmid

Plasmids were discovered by William Hayes and Joshua Lederberg (1952)

in bacterial cells. They are small, double stranded, closed circular, symbiotic DNA
molecules that occur naturally in bacteria, outside the bacterial chromosome. They
are regarded as extra chromosomal DNA containing extrachromosomal genome.
A bacterial cell may possess one or many copies of one or more plasmids. They
are inherited from parent bacterial cell to daughter cell and have capability to self-
replication in the cytoplasm of bacterial cell. The circular molecule of DNA can be
broken to yield a linear molecule which passes from one bacterial cell to another.
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Plasmids: Types There are many kinds of plasmids which differ from one another in size and in
and its Properties
composition of genes. They vary in size from 2 kilobars to more than 400 kilobars.
The major components of a plasmid comprise of an origin site (the replication
origin) or a specific porion of their genome that serves as start signal for self-
NOTES replication. Under natural conditions, each plasmid replicates to produce 20-30
copies per cell. This number can be artificially increased. In presence of certain
antibiotics the number can be increased to about 1000 copies.
Another valuable feature of plasmids is the presence of specific restriction
sites where the enzyme restriction endonuclease makes a cut so that a foreign
DNA segment may be jointed to the plasmid. This property helps plasmids to act
as trusted cloning vehicles during gene transfer in genetic engineering. Plasmids
are considered replicons, units of DNA capable of replicating autonomously within
a suitable host. However, plasmids, like viruses, are not generally classified as life.
Plasmids are transmitted from one bacterium to another (even of another species)
mostly through conjugation. This host-to-host transfer of genetic material is one
mechanism of horizontal gene transfer, and plasmids are considered part of
the mobilome. Unlike viruses, which encase their genetic material in a protective
protein coat called a capsid, plasmids are ‘naked’ DNA and do not encode genes
necessary to encase the genetic material for transfer to a new host.
However, some classes of plasmids encode the conjugative ‘sex’ pilus
necessary for their own transfer. The size of the plasmid varies from 1 to over 200
kbp, and the number of identical plasmids in a single cell can range anywhere from
one to thousands under some circumstances. The relationship between microbes
and plasmid DNA is neither parasitic nor mutualistic, because each implies the
presence of an independent species living in a detrimental or commensal state with
the host organism. Rather, plasmids provide a mechanism for horizontal gene transfer
within a population of microbes and typically provide a selective advantage under
a given environmental state. Plasmids may carry genes that provide resistance to
naturally occurring antibiotics in a competitive environmental niche, or the proteins
produced may act as toxins under similar circumstances, or allow the organism to
utilize particular organic compounds that would be advantageous when nutrients
are scarce.
Vectors
In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially
carry foreign genetic material into another cell, where it can be replicated and/
or expressed (for example, plasmid, cosmid, Lambda Phages). A vector containing
foreign DNA is termed recombinant DNA. The four major types of vectors
are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most
commonly used vectors are plasmids. Common to all engineered vectors are
an origin of replication, a multi-cloning site, and a selectable marker.

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 Viral Vectors: Viral vectors are generally genetically engineered viruses Plasmids: Types
and its Properties
carrying modified viral DNA or RNA that has been rendered noninfectious, but
still contain viral promoters and also the transgene, thus allowing for translation of
the transgene through a viral promoter. However, because viral vectors frequently
are lacking infectious sequences, they require helper viruses or packaging lines for NOTES
large-scale transfection.
Viral vectors are often designed for permanent incorporation of the insert
into the host genome, and thus leave distinct genetic markers in the host genome
after incorporating the transgene. For example, retroviruses leave a characteristic
retroviral integration pattern after insertion that is detectable and indicates that the
viral vector has incorporated into the host genome.
Artificial Chromosomes: Artificial chromosomes are manufactured
chromosomes in the context of Yeast Artificial Chromosomes (YACs), Bacterial
Artificial Chromosomes (BACs), or Human Artificial Chromosomes (HACs). An
artificial chromosome can carry a much larger DNA fragment than other vectors.
YACs and BACs can carry a DNA fragment up to 300,000 nucleotides long.
Three structural necessities of an artificial chromosome include an origin of
replication, a centromere, and telomeric end sequences. Artificially constructed
plasmids may be used as vectors in genetic engineering. These plasmids serve as
an important tools in genetics and biotechnology labs, where they are commonly
used to clone and amplify (make many copies of) or express particular genes.
A wide variety of plasmids are commercially available for such uses. The
gene to be replicated is normally inserted into a plasmid that typically contains a
number of features for their use. These include a gene that confers resistance to
particular antibiotics (Ampicillin is most frequently used for bacterial strains),
an origin of replication to allow the bacterial cells to replicate the plasmid DNA,
and a suitable site for cloning (referred to as a multiple cloning site). In molecular
cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign
genetic material into another cell, where it can be replicated and/or expressed (for
example, plasmid, cosmid, Lambda Phages). A vector containing foreign DNA is
termed recombinant DNA. The four major types of vectors are plasmids, viral
vectors, cosmids, and artificial chromosomes. Of these, the most commonly used
vectors are plasmids. Common to all engineered vectors are an origin of replication,
a multi cloning site, and a selectable marker. The vector itself is generally
a DNA sequence that consists of an insert (transgene) and a larger sequence that
serves as the ‘backbone’ of the vector. The purpose of a vector which transfers
genetic information to another cell is typically to isolate, multiply, or express the
insert in the target cell.
All vectors may be used for cloning and are therefore cloning vectors, but
there are also vectors designed especially for cloning, while others may be designed
specifically for other purposes, such as transcription and protein expression. Vectors
designed specifically for the expression of the transgene in the target cell are
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Plasmids: Types called expression vectors, and generally have a promoter sequence that drives
and its Properties
expression of the transgene. Simpler vectors called transcription vectors are only
capable of being transcribed but not translated - they can be replicated in a target
cell but not expressed, unlike expression vectors. Transcription vectors are used
NOTES to amplify their insert. The manipulation of DNA is normally conducted
on Escherichia coli vectors, which contain elements necessary for their
maintenance in Escherichia coli.
However, vectors may also have elements that allow them to be maintained
in another organism such as yeast, plant or mammalian cells, and these vectors are
called shuttle vectors. Such vectors have bacterial or viral elements which may be
transferred to the non-bacterial host organism, however other vectors termed
intragenic vectors have also been developed to avoid the transfer of any genetic
material from an alien species.
Generally, the bacterial plasmids are 1 to 5% of the chromosomal DNA in
size. Plasmids vary widely in size. The smaller plasmids have molecular weights
ranging from 4 - 5 106 Daltons, while the larger ones have molecular weights of
25 to 95 106 Daltons. Plasmids not only vary in size, but also in copy number
which denotes the number of copies of a specific plasmid in a cell. Copy number
is discontinuously variable, i.e., some plasmids generally the smaller ones — have
a high copy number, while the larger plasmids have characteristically a low copy
number. Those having a high copy number are known as relaxed plasmids and
those having low copy number are called stringent plasmids. Whatever may be the
copy number, plasmids are generally distributed equally in the daughter cell during
cell division. Rarely, a plasmid-free cell may arise spontaneously at a frequency of
about 1 in 104 cells. Plasmid free cells may also be produced artificially by the use
of mutagens. The process is commonly called curing of plasmids. Usually, the
low-copy number large plasmids have one or two copies per cell and are easier to
be cured. The smaller plasmids, in contrast, may have 10 to 100 copies per cell.
So far as the biological functions of plasmids are concerned, they are not
indispensable constituents of the bacteria. This is proved by the fact that the bacteria
cured of plasmids can grow normally without any difficulty. However, the genes
carried on the plasmid DNA confer special properties to the host bacteria and
such properties may become advantageous under special environmental conditions.
For example, bacteria carrying the R-plasmids (resistance plasmids) can
survive when the environment contains inhibitory concentrations of one or more
antibiotics. Obviously, the R-plasmid-less bacteria are destroyed under such
conditions. Another example is provided by the plasmids of some species of
Pseudomonas which carry genes for production of enzymes catalyzing degradation
of complex hydrocarbons.
Bacteria carrying such plasmids are capable of using such unusual substrates
for growth and, obviously, enjoy special advantage over others lacking them. The
F plasmid gives the power to carry out a type of sexual reproduction to bacteria
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Again, some bacteria, like Escherichia coli, Pseudomonas and Lactobacillus, Plasmids: Types
and its Properties
etc., produce special type of proteins, called bacteriocins which are coded by
plasmid genes. These proteins are able to kill other closely related bacteria and,
thereby, they can eliminate competition for food and space. Thus it is seen that
even though plasmids are not absolutely essential for the life of bacteria under NOTES
normal conditions of growth, their presence may become valuable and
advantageous for the host under special conditions, or may even prove critical for
survival as in case of the R-plasmids.
The R-plasmids with the help of the resistance genes produce proteins which
can inactivate or destroy specific antibiotics. Besides the advantageous properties
attributable to the plasmids, these extra-chromosomal genetic elements have played
an important role in the development of recombinant DNA technology. In this
technology, the plasmids are used as vectors for transferring a gene of interest
from one organism to another organism. Such transfer of a gene is possible, not
only from one bacterium to another, but also from eukaryotic organism to a
bacterium, or vice versa. A segment of DNA containing the specific gene is isolated
from a suitable donor and inserted by recombinant DNA technology into a plasmid.
The recombinant plasmid is next introduced into a suitable host cell where the
gene is expressed producing the gene product. In this way, several human genes
producing therapeutically important proteins have been introduced into bacteria.
Also, some bacterial genes have been transferred to eukaryotic hosts, like plants,
and some viral genes have been transferred to yeasts. In most of such gene transfers,
plasmids play a key role as vectors or carriers.
11.2.1 Types of Plasmids
Following are the types of plasmids:
F Plasmid
The F plasmid, also known as the fertility factor or sex factor, determines the sex
of Escherichia coli bacteria. The cells containing this plasmid are designated as
F+ and those without it as F–. F+ bacteria are considered as male, because they
can act as donor of not only the plasmid, but also chromosomal genes to the F–
cells which act as recipient and are, therefore, considered as female.
The process of transfer takes place by conjugation of the F+ cell with the F–
cell. The F plasmid is a conjugative plasmid.
We know that a characteristic feature of the F plasmid is that it can either
remain as an independent entity replicating separately along with the chromosomal
DNA, or it can be inserted into the chromosome as its integral part. When an F
plasmid is integrated into the Escherichia coli chromosome, the bacterial cell
changes from P to an Hfr strain (High frequency of recombination).
There are many sites on the Escherichia coli chromosome where the F
plasmid can be integrated. Depending on the site, each integration gives rise to a
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Plasmids: Types different Hfr-strain. In F+ F– conjugation, the plasmid alone is transmitted, but in
and its Properties
Hfr F– conjugation, chromosomal genes are transmitted and rarely also the F
plasmid.
The F plasmid is a large self-transmissible plasmid having a double-stranded
NOTES
circular DNA molecule. Its molecular weight is 63 106 Daltons and it contains
about genes controlling the transfer of the plasmid from the donor to the recipient.
A mutation in any of the essential genes results in the loss of transmissibility of the
plasmid.
Just as an F plasmid can be integrated into the chromosome of the host cell,
so it can though on rare occasions, be separated or excised from the Escherichia
coli chromosome in the free state to form the circular plasmid. It has been observed
that the excision process is sometimes imperfect in the sense that some parts of the
Escherichia coli chromosome adjoining the linearly inserted F plasmid are included
in the excised F DNA and at the same time, parts of the plasmid DNA are retained
in the Escherichia coli chromosome.
The F plasmids containing parts of the chromosomal DNA are designated
as F1 plasmids. When an F plasmid loses some of its essential genes during the
excision process, the plasmid is rendered incapable of independent existence and
is, ultimately, eliminated during cell division.
When an F plasmid is transmitted by conjugation to an F recipient, it can
transfer the chromosomal genes carried by it. Thereby, the recipient becomes
diploid in respect of these transferred genes (because it now contains one copy of
its own and another copy of the same gene transmitted by the F plasmid). Thus,
exchange of chromosomal genes may occur through F1 plasmids. This has been
described as sexduction (Refer Figure 11.2).

Fig. 11.2 F Plasmid

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R-Plasmids Plasmids: Types
and its Properties
R-plasmids conferring resistance to various drugs individually or multiple resistance
to several antibacterial agents were first discovered in Japan in the 1950s in the
gastroenteritis causing Shigella dysenteriae. Since then these plasmids have been NOTES
found in Escherichia coli and other enteric bacteria. Such plasmids have proved
a great threat to the medical science.
The large R-plasmids having molecular weights ranging between
30 106 Daltons are self- transmissible by conjugation with other bacteria. They
are, therefore, conjugative plasmids, like the F plasmid.
Smaller R-plasmids having molecular weights of about 5 to 6 106 Daltons
are non-transmissible. Most of the self-transmissible large plasmids like R100 of
Shigella conferring multiple drug resistance are co-integrates of two DNA segments
joined to each other by covalent linkage to form a single double-stranded circular
molecule.
One DNA segment is called the Resistance Transfer Factor (RTF), while
the other segment contains the drug-resistance genes. The RTF is mainly involved
in the transfer function of the R-plasmid and contains a number of genes (the
transfer genes) and some others controlling replication of the plasmid in the host
cell.
The resistance genes located in the other segment elaborate enzymes for
destruction of the antibacterial drugs, like Penicillins, Streptomycin,
Chloramphenicol, Tetracyclines, Kanamycin, Sulfonamides, etc.
In some drug-resistant bacteria, such as Salmonella typhimurium strain
29, the resistance genes are located not in the same plasmid, but in separate plasmids
of different size. This is sometimes known as plasmid aggregation.
The transposable elements that complex transposons may carry genes for
drug resistance. Such elements can be integrated into plasmids giving rise to a
drug-resistance plasmid. Thus, R plasmids may be made up of a collection of
transposons, each of which may carry one or more genes for antibiotic resistance.
For example, Tn 5 carrying a gene for Kanamycin resistance may be inserted into
the plasmid R100 of Shigella making the plasmid able to resist the antibiotic.
Besides drug resistance, plasmids may also make bacterial hosts resistant
to the toxic effects of heavy metals. Plasmid coded resistance to Nickel, Cobalt,
Mercury, Arsenic and Cadmium has been reported in different species belonging
to the genera Pseudomonas, Escherichia, Salmonella and Staphylococcus.
Col-Plasmids
The Col-plasmids are present in different strains of Escherichia coli and they
contain genes controlling synthesis of a class of proteins called Colicines. Colicines
are able to inhibit the growth of related bacteria which lack a Col-plasmid (Cor).

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Plasmids: Types Several different types of Col-plasmids have been discovered, each of which
and its Properties
produces Colicines having a different mode of inhibition of susceptible bacteria.
For example, Col B induces a damage of the cytoplasmic membrane of the target
bacteria and Col E2 and Col E3 cause degradation of nucleic acids.
NOTES Like R-plasmids, Col-plasmids may be self-transmissible or non-self
transmissible. Large Col plasmids, like Col I and Col V-K94 having molecular
weights of 60 106 Daltons or above are self- transmissible. They have a small
copy number, usually 1 to 3 copies per cell. Small Col-plasmids, like Col-El, have
molecular weight weighs of about 4 to 5 106 Daltons.
They have a high copy number, usually 10 to 30 copies per cell. They are
self-non-transmissible, but may be mobilized with the help of F plasmid. This
means that when an F+ cell contains also a Col El plasmid and conjugates with an
F cell, the Col El plasmid can be transferred to the recipient through the mating
bridge constructed by the F-plasmid. Obviously, an F ColEl+ cell is unable to
mobilize the Col-plasmid to another cell, because it is unable to build a mating
bridge.
In contrast, the large Col-plasmids are self-transmissible, because they have
the genes for building the conjugation apparatus themselves and do not depend on
the F plasmid for transfer to other cells. Like F and large R-plasmids, the large
Col-plasmids are also conjugative plasmids.
Colicins belong to a general class of proteins, called bacteriocins. Many
bacteria have been found to elaborate bacteriocins which are able to kill other
related or even unrelated bacteria. Such proteins are coded by genes present in
bacteriocin ogenic plasmids.
Bacteriocins produced by different bacteria are sometimes given different
names, like pyocin produced by Pseudomonas aeruginosa, megasine elaborated
by Bacillus megaterium, nisin by lactobacilli, etc. In general, bacteriocins exert
their antibacterial action by binding to the cell wall of the target cells and by inhibiting
one of the vital metabolic processes, like replication of nucleic acids, transcription,
protein synthesis or energy metabolism.
Bacteriocins produced by enteric bacteria help to maintain a healthy
ecological balance in the human colon. Other bacteriocins produced by bacteria
under natural environmental conditions probably function by eliminating competitors.
Nisin produced by lactic acid bacteria has been commercially used for preservation
of food and dairy products.
Degradative Plasmids
Degradation or dissimilation of organic compounds in course of mineralization is
often controlled by plasmid-borne genes in many microorganisms. Such plasmids
with genes coding for enzymes that catabolize complex organic molecules are
known as degradative or dissimilation plasmids. For example, in species of
Pseudomonas, both chromosomal and plasmid genes produce enzymes for
break­down of complex compounds.
Some of the plasmid genes code for enzymes which degrade such unusual
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228 Material
hydrocarbons of crude petroleum. With the help of these enzymes, the bacteria Plasmids: Types
and its Properties
can utilize these compounds as source of carbon and energy.
As a result, bacteria possessing such degradative plasmids stand a much
better chance of survival under conditions where only such unusual compounds
are available. Normal bacteria without such plasmid-coded enzymes would perish NOTES
under similar conditions.
The capability of organisms carrying degradative plasmids to metabolize
unusual diverse complex compounds suggests the possibility of employing them as
means of bioremediation of the polluted environment. The development of genetic
engineering techniques has encouraged scientists to develop genetically improved
strains of bacteria containing plasmids capable of degradation of an array of
complex compounds, such as those occurring in crude petroleum.
A synthetic strain of Pseudomonas has been developed by Ananda Mohan
Chakraborty of the University of Illinois, USA offering prospects of practical use
in removing oil-spills in the oceans, caused by leakage of crude petroleum from
tankers. Oil-spills prove a great danger to marine life, both plants and animals.
Ti Plasmid of Agrobacterium
Ti plasmid is a tumour-inducing large extra-chromosomal double stranded circular
DNA which is present in Agrobacterium tumefaciens, a plant-pathogenic bacterium
causing the crown-gall disease in many dicotyledonous species. Crown-gall is a
tumour produced at the collar region of plants by agrobacteria which possess the
Ti plasmid. Bacteria lacking the plasmid are non-virulent.
Ti plasmid is about 200 kilo base-pair long circular DNA. Only a small part
of this large molecule, a 30 kilo base-pair long fragment is responsible for tumour
formation. This fragment is called the T DNA (T stands for transformation). When
Agrobacterium infects a susceptible host plant, the Ti plasmid is released in the
host cell and a copy of the T DNA is integrated into the genome of the host plant.
The integrated T DNA then stimulates cellular atrophy producing eventually
a tumour, called a crown gall. The T DNA insertion in plant host genome is the first
instance of an inter-kingdom genetic exchange by natural means.
A notable feature of T DNA is that once it is incorporated into the host
genome, the presence of the pathogenic organism is no longer necessary for
induction of tumour. Thus, a close parallelism with cancer induction in animal cell is
observed. The T DNA segment of the Ti plasmid contains genes controlling
synthesis of phytohormones, like indole acetic acid and cytokinins, as well as
several other compounds, called opines. Opines, such as octopine and nopaline
are used as growth substrates by agrobacteria.
The rest of the Ti plasmid contains several genes controlling virulence (Vir
genes). These genes control T DNA transfer to the host. Other genes of the plasmid
control functions relating to bacterial conjugation, DNA replication and catabolism
of opines synthesised by gene products of the T DNA segment.
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Plasmids: Types The T DNA acts as a mobile unit like a transposon, but it does not have a
and its Properties
gene, like transposase to mediate its own mobilization. Its mobilization is effected
by genes located in the Ti plasmid, but outside T-DNA. The 30 kilo base long T
DNA is flanked on either side by 25 base pair imperfect direct repeats forming T
NOTES DNA borders.
The Vir genes of Ti plasmid are involved in the generation of a transferable
copy of T DNA and its transfer to plant cell through the cell membrane and the
nuclear membrane, as well as through the bacterial and plant cell walls. T DNA is
transferred as a single-stranded copy.
The copy is separated from T DNA segment, capped at the 52 -end by a
protein coded by a Vir gene (Vir D2) and covered by a large number of protein
molecules coded by another Vir gene (Vir E2). This T complex is transported to
the plant cell through a membrane pore produced by another Vir gene. The T
complex (ss-DNA + Proteins) is about 3.6 m long and less than 2 nm thick.
A gross structure of the Ti plasmid and generation of the T complex have
been diagrammatically represented (Refer Figure 11.3).
The ability of Agrobacterium tumefaciens to transfer its Ti plasmid to
many dicotyledonous plants (but not monocotyledonous ones) opened up the
possibility of introducing foreign genes into the hosts using the Ti plasmid as a
vehicle (vector).
This has been practically employed to insert a gene of interest into the T
DNA segment by recombinant DNA technology. The tumour-inducing genes and
other unnecessary genes of T DNA are removed and replaced by the gene chosen
for insertion. Several foreign genes have been introduced into a variety of hosts to
produce transgenic plants.
Among the notable achievements are productions of transgenic plants
resistant to the herbicide glyphosate and to feeding insects. Glyphosate resistance
gene was isolated from Salmonella and the insect-resistance gene from Bacillus
thuringiensis which synthesize an insecticidal protein. Another interesting
achievement though not of practical significance was production of bioluminescent
tomato plants by introducing the gene controlling bioluminescence in fire fly.

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Eukaryotic Plasmids Plasmids: Types
and its Properties
Plasmids occur rarely in Eukaryotic cells. Some plasmids have been found in yeast
(Saccharomyces cerevisiae) and in several plants. The only RNA plasmid
discovered till now has been found in yeast. It is a double-stranded RNA having a NOTES
molecular weight of 15 106 Daltons. It contains 10 genes including one coding
for a bacteriocin-like protein. The protein can kill other yeast cells lacking the
plasmid. This yeast plasmid has been designated as killer particle.
Yeast also contains small DNA plasmids with high copy number. They are
located in the nucleus and like the chromosomal DNA are associated with basic
proteins — histones. Some yeast DNA plasmids have been genetically engineered
in such a way that they are capable of multiplication in both Escherichia coli and
yeast.
One such engineered yeast plasmid is Yep which can function as a shuttle
vector. This plasmid has been used in transfer of useful genes from other organisms
into yeast cells via Escherichia coli for production of valuable therapeutically
important proteins. A successful application of the Yep plasmid is the transfer of
the gene coding the coat glycoprotein of Hepatitis B Virus to yeast.
The transgenic yeast can express the gene successfully with production of
the viral glycoprotein. The glycoprotein has been used for preparation of Hepatitis
B Vaccine for human application. Shuttle vectors are especially useful in transferring
Eukaryotic genes, because such genes are often not successfully expressed in
bacterial hosts.
Besides yeast, DNA plasmids have been discovered in several plants, like
maize and sorghum, as also in several fungi. These plasmids are made of usually
linear double stranded DNA molecules whereas all bacterial plasmids are circular.
Replication of Plasmids
In the non-dividing plasmids, the double-stranded DNA exists as a right-handed
super-helical coil having 400-600 base pairs per turn of the coil. During replication,
the plasmids can multiply autonomously, although replication requires the host cell
enzymes. That is why plasmids can multiply only within host cells.
Each plasmid has its own origin of replication. Some plasmids also have
genes which code for proteins necessary for their own multiplication. This is proved
by the fact that a temperature sensitive mutant of F plasmid (F+ ts, i.e., temperature
sensitive replicon) is unable to replicate at 42°C, although it can function normally
at 37°C.
Different Aspects of Plasmid Replication are briefly discussed below:
Non-Transmissible Plasmids: Replication of plasmid DNA starts at the
site of origin and may proceed either bi-directionally as in case of bacterial
chromosome, or may proceed unidirectionally depending on the nature of plasmid.
In bidirectional replication, replication terminates when the two replication forks
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Plasmids: Types meet each other. In unidirectional replication, termination occurs when the
and its Properties
replication fork reaches the site of origin. In both cases, the circularity of the
plasmid DNA is maintained throughout the process.
Self-Transmissible Plasmids: In case of conjugative plasmids, like F
NOTES
plasmid or R-plasmid, replication occurs by the rolling-circle model. The supercoiled
DNA undergoes a nick in one of the strand resulting in relaxation of the super
coiled state to form an open circle. The enzyme catalyzing the nick remains attached
to the 52 -P end of the relaxed molecules. Such a single-stranded nick becomes
necessary for transfer of a copy of the plasmid during conjugation to the mating
partner.
By rolling circle replication, the donor cell retains its double-stranded plasmid,
while a single-stranded copy is transferred through the mating bridge to the recipient
cell, where a complimentary strand is synthesized and ligated to form a double-
stranded copy of the plasmid.
Control of Copy Number: The large plasmids are characterized by low
copy number (one to few) and small plasmids by high copy number (10 to 100).
The copy number is controlled by an inhibitor coded by the plasmid DNA itself.
The inhibitor concentration in the bacterial cell determines the rate of initiation of
plasmid replication.
When a cell containing two large plasmids divides to produce two daughter
cells, each having one plasmid, the inhibitor concentration in these cells is the same
as that of the mother cell. Now, the daughter cells grow in size to attain maturity
resulting in lowering of the inhibitor concentration in the cytoplasm.
As a consequence, DNA synthesis is initiated in the plasmid leading to its
replication producing two copies. As each plasmid copy possesses an inhibitor
gene, the production of inhibitor doubles and the inhibitor concentration becomes
high enough to stop plasmid DNA synthesis and further replication. Thus, the
copy number is restricted to two per cell.
A similar mechanism of control of copy member is believed to operate in
case of high copy number plasmids also. However, in this case, the inhibitor
concentration must reach a higher threshold level to stop initiation of plasmid DNA
synthesis in comparison to that of low copy number plasmids.
Plasmid Amplification: Another important point of plasmid replication is
that chromosomal DNA synthesis and plasmid DNA synthesis are independent of
each other, though, in both, DNA synthesis is followed by replication. Thus it is
possible to stop chromosomal DNA synthesis and replication without affecting
plasmid DNA synthesis and replication.
Such situation can be practically created by adding chloramphenicol to a
bacterial culture. This antibiotic specifically inhibits prokaryotic protein synthesis.
When it is added to a growing bacterial culture, chromosomal DNA synthesis is
inhibited, but plasmid DNA synthesis and replication continue at the cost of the
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232 Material
 The net result is that each bacterial cell contains large number of plasmid Plasmids: Types
and its Properties
copies. This is known as plasmid amplification. When a specific gene which has
been transferred (cloned) to a plasmid requires to be isolated, plasmid amplification
becomes a useful tool, because of high plasmid DNA concentration in the total
cellular DNA. NOTES
Transfer of Non-Self Transmissible Plasmids: There are some plasmids
which do not possess genes for self-transmission, but can be transferred to other
cells with the help of a self-transmissible plasmid when both plasmids occur in the
same cell. They are known as mobilizable plasmids.
Such plasmids possess genes for proteins needed for nicking its own DNA
at the site of origin of replication, but lack in genes needed for building the
conjugation tube. When they coexist with a self-transmissible plasmid, like F or R,
the latter can build the mating bridge through which a copy of the mobilizable
plasmid produced by rolling-circle replication is transferred to a recipient cell.
A different type of mobilization occurs when a donor cell having a self-
transmissible plasmid conjugates with a recipient having a mobilizable plasmid. In
this type of conjugation, both the donor and the recipient acquire a copy of both
types of plasmids by a process of retro transfer. First, the self- transmissible plasmid
replicates by rolling-circle model and a single-stranded copy is transferred through
the mating bridge to the recipient, where it forms a complimentary strand leading
to the formation of a copy of the self-transmissible plasmid in the usual way.
The mobilizable plasmid in the recipient cell then replicates and a single-
stranded copy is transferred to the other cell which now acts as the recipient of the
mobilizable plasmid. Finally, the two cells separate and each has a copy of the
self-transmissible plasmid and a copy of the mobilizable plasmid
Incompatibility of Plasmids
Generally, two closely related plasmids cannot coexist in a bacterial cell. In the
population of progeny cells derived from a cell containing two such plasmids, the
proportion of cells having only one of the two plasmids increases with every cell
division. This is known as plasmid incompatibility.
On the other hand, two different unrelated plasmids, for example F plasmid
and ColEl can exist together without any difficulty, because these plasmids belong
to two different incompatibility groups. Whereas, two F plasmids cannot coexist
in the same cell.
One mechanism by which a plasmid already resident in a cell prevents the
entry of a second similar plasmid into the same cell is by surface exclusion. For
example, an F plasmid of Escherichia coli does not allow entry of another F
plasmid by inhibiting it from leaving the cell where it is already located. The effect
is mediated at the surface of the cell whereby the F DNA cannot come out of the
cell.

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Plasmids: Types A different mechanism operates when a cell already has two closely related
and its Properties
plasmids, say X and Y. We know that the copy number of plasmids is controlled
by specific inhibitors coded by the plasmid itself.
As X and Y are two closely related plasmids, it would be expected that
NOTES
their inhibitors would also be closely similar and that replication of both the plasmids
would be regulated by the inhibitor produced either by X or Y.
During replication, X and Y may be selected at random, so that, during first
replication of the plasmid, a cell initially containing one copy of each plasmid may
produce two copies of either X or Y, so that the cell has now two copies of either
X or Y and one copy of the un-replicated plasmid, i.e., 2X + Y or X + 2Y. In the
second round of plasmid replication, each cell will contain 4 plasmids, but depending
on which plasmid is replicated, the combination may be X + 3Y, 2x + 2Y or 3X +
Y.
Now the cell divides to produce two daughter cells, each with 2 plasmids
and the plasmid combinations of the daughter cells may be X + X, X + Y or Y + Y.
Thus the probability of progeny cells having either two X plasmids or two Y plasmids
is equal to those having two different plasmids, i.e., X + Y. In other words, the
probability of elimination of one plasmid is 50%. Such probability increases with
more cell generations.
Plasmid Library
A plasmid library is a gene library which contains a collection of bacterial cultures,
each of which contains a plasmid, but plasmid of one culture differs from that of
another in having a separate DNA fragment of a genome of an organism. The total
genome isolated from an organism is fragmented and the fragments are inserted
(cloned) separately into individual plasmids.
These recombinant plasmids are then introduced into suitable host bacteria.
Thus each bacterial culture contains a plasmid with a fragment. The total collection
of cultures would be expected to contain the entire genome of an organism and
would constitute a gene library of the particular organism.
Agrobacterium tumefaciens is a plant pathogen with the capacity to deliver
a segment of oncogenic DNA carried on a large plasmid called the tumor-inducing
or Ti plasmid to susceptible plant cells. Agrobacterium tumefaciens belongs to
the class Alphaproteobacteria, whose members include other plant pathogens
(Agrobacterium rhizogenes), plant and insect symbionts (Rhizobium
spp. and Wolbachia spp., respectively), human pathogens (Brucella
spp., Bartonella spp., Rickettsia spp.), and non-pathogens (Caulobacter
crescentus, Rhodobacter sphaeroides). Many species of Alphaproteobacteria
carry large plasmids ranging in size from ~100 kilobases (kbs) to nearly 2
megabases (Mbs). These large replicons typically code for functions essential for
cell physiology, pathogenesis or symbiosis. Most of these elements rely on a
conserved gene cassette termed RepABC for replication and partitioning, and
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maintenance at only one or a few copies per cell. The subject of this review is the Plasmids: Types
and its Properties
~200 kb Ti plasmids carried by infectious strains of Agrobacterium tumefaciens.
We will summarize the features of this plasmid as a representative of
the repABC family of megaplasmids. We will also describe novel features of this
plasmid that enable Agrobacterium tumefaciens cells to incite tumor formation NOTES
in plants, sense and respond to an array of plant host and bacterial signal molecules,
and maintain and disseminate the plasmid among populations of agrobacteria.
This natural genetic engineer has been adapted to spawn an entire industry of
plant biotechnology and review its potential for use in future therapeutic applications
of plant and non-plant species (Refer Figure 11.4).

Fig. 11.4 Types of Plasmid Integration into a Host Bacteria

11.2.2 Properties and Characteristics

In order for plasmids to replicate independently within a cell, they must possess a
stretch of DNA that can act as an origin of replication. The self-replicating unit, in
this case the plasmid, is called a replicon. A typical bacterial replicon may consist
of a number of elements, such as the gene for plasmid-specific Replication initiation
protein (Rep), repeating units called iterons, DNAA boxes, and an adjacent AT-
rich region. Smaller plasmids make use of the host replicative enzymes to make
copies of themselves, while larger plasmids may carry genes specific for the
replication of those plasmids. A few types of plasmids can also insert into the host
chromosome, and these integrative plasmids are sometimes referred to
as episomes in Prokaryotes.
Plasmids almost always carry at least one gene. Many of the genes carried
by a plasmid are beneficial for the host cells, for example enabling the host cell to
survive in an environment that would otherwise be lethal or restrictive for growth.
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Plasmids: Types metal, while others may produce virulence factors that enable a bacterium to
and its Properties
colonize a host and overcome its defences, or have specific metabolic functions
that allow the bacterium to utilize a particular nutrient, including the ability to degrade
recalcitrant or toxic organic compounds. Plasmids can also provide bacteria with
NOTES the ability to fix nitrogen. Some plasmids, however, have no observable effect on
the phenotype of the host cell or its benefit to the host cells cannot be determined,
and these plasmids are called cryptic plasmids.
Naturally occurring plasmids vary greatly in their physical properties. Their
size can range from very small mini-plasmids of less than a 1 kilobase pairs (kbp),
to very large megaplasmids of several megabase pairs (Mbp). At the upper end,
little can differentiate between a megaplasmid and a minichromosome. Plasmids
are generally circular, but examples of linear plasmids are also known. These linear
plasmids require specialized mechanisms to replicate their ends.
Plasmids may be present in an individual cell in varying number, ranging
from one to several hundreds. The normal number of copies of plasmid that may
be found in a single cell is called the Plasmid copy number, and is determined by
how the replication initiation is regulated and the size of the molecule. Larger plasmids
tend to have lower copy numbers. Low copy number plasmids that exist only as
one or a few copies in each bacterium are, upon cell division, in danger of being
lost in one of the segregating bacteria. Such single-copy plasmids have systems
that attempt to actively distribute a copy to both daughter cells. These systems,
which include the parABS system and parMRC system, are often referred to as
the partition system or partition function of a plasmid.

Check Your Progress

1. What are plasmids?
2. Where are artificial plasmids used?
3. Write in short about vector.
4. What are F plasmid?
5. Define R-plasmids.
6. Write a short note on non-transmissible plasmids.

11.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. A plasmid is a small DNA molecule within a cell that is physically separated

from chromosomal DNA and can replicate independently. They are most
commonly found as small circular, double-stranded DNA molecules
in bacteria, however, plasmids are sometimes present
in archaea and eukaryotic organisms. In nature, plasmids often carry genes
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 that benefit the survival of the organism, such as by providing antibiotic Plasmids: Types
and its Properties
resistance.
2. Artificial plasmids are widely used as vectors in molecular cloning, serving
to drive the replication of recombinant DNA sequences within host
NOTES
organisms.
3. In molecular cloning, a vector is a DNA molecule used as a vehicle to
artificially carry foreign genetic material into another cell, where it can
be replicated and/or expressed (for example, plasmid, cosmid, Lambda
Phages). A vector containing foreign DNA is termed recombinant DNA.
The four major types of vectors are plasmids, viral vectors, cosmids,
and artificial chromosomes. Of these, the most commonly used vectors are
plasmids. Common to all engineered vectors are an origin of replication,
a multi cloning site, and a selectable marker.
4. The F plasmid, also known as the fertility factor or sex factor, determines
the sex of Escherichia coli bacteria. The cells containing this plasmid are
designated as F+ and those without it as F–. F+ bacteria are considered as
male, because they can act as donor of not only the plasmid, but also
chromosomal genes to the F– cells which act as recipient and are, therefore,
considered as female.
5. R-plasmids conferring resistance to various drugs individually or multiple
resistance to several antibacterial agents were first discovered in Japan in
the 1950s in the gastroenteritis causing Shigella dysenteriae. Since then
these plasmids have been found in Escherichia coli and other enteric
bacteria. Such plasmids have proved a great threat to the medical science.
6. Replication of plasmid DNA starts at the site of origin and may proceed
either bi-directionally as in case of bacterial chromosome, or may proceed
unidirectionally depending on the nature of plasmid. In bidirectional
replication, replication terminates when the two replication forks meet each
other. In unidirectional replication, termination occurs when the replication
fork reaches the site of origin. In both cases, the circularity of the plasmid
DNA is maintained throughout the process.

11.4 SUMMARY

A plasmid is a small DNA molecule within a cell that is physically separated

from chromosomal DNA and can replicate independently.
In nature, plasmids often carry genes that benefit the survival of the organism,
such as by providing antibiotic resistance.
Artificial plasmids are widely used as vectors in molecular cloning, serving
to drive the replication of recombinant DNA sequences within host
organisms.
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Plasmids: Types A bacterial cell may possess one or many copies of one or more plasmids.
and its Properties
They are inherited from parent bacterial cell to daughter cell and have
capability to self-replication in the cytoplasm of bacterial cell.
The circular molecule of DNA can be broken to yield a linear molecule
NOTES
which passes from one bacterial cell to another. There are many kinds of
plasmids which differ from one another in size and in composition of genes.
Another valuable feature of plasmids is the presence of specific restriction
sites where the enzyme restriction endonuclease makes a cut so that a foreign
DNA segment may be jointed to the plasmid.
Plasmids are transmitted from one bacterium to another (even of another
species) mostly through conjugation. This host-to-host transfer of genetic
material is one mechanism of horizontal gene transfer, and plasmids are
considered part of the mobilome.
Plasmids provide a mechanism for horizontal gene transfer within a population
of microbes and typically provide a selective advantage under a given
environmental state.
In molecular cloning, a vector is a DNA molecule used as a vehicle to
artificially carry foreign genetic material into another cell, where it can
be replicated and/or expressed (for example, plasmid, cosmid, Lambda
phages).
A vector containing foreign DNA is termed recombinant DNA. The four
major types of vectors are plasmids, viral vectors, cosmids, and artificial
chromosomes.
Viral vectors are generally genetically engineered viruses carrying modified
viral DNA or RNA that has been rendered noninfectious, but still contain
viral promoters and also the transgene, thus allowing for translation of the
transgene through a viral promoter.
Artificial chromosomes are manufactured chromosomes in the context
of Yeast Artificial Chromosomes (YACs), Bacterial Artificial
Chromosomes (BACs), or Human Artificial Chromosomes (HACs).
The four major types of vectors are plasmids, viral vectors, cosmids,
and artificial chromosomes. Of these, the most commonly used vectors are
plasmids
The purpose of a vector which transfers genetic information to another cell
is typically to isolate, multiply, or express the insert in the target cell.
Vectors designed specifically for the expression of the transgene in the target
cell are called expression vectors, and generally have a promoter sequence
that drives expression of the transgene.
Transcription vectors are used to amplify their insert. The manipulation of
DNA is normally conducted on Escherichia coli vectors, which contain
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Plasmids vary widely in size. The smaller plasmids have molecular weights Plasmids: Types
and its Properties
ranging from 4 - 5 106 Daltons, while the larger ones have molecular
weights of 25 to 95 106 Daltons.
Copy number is discontinuously variable, i.e., some plasmids generally the
NOTES
smaller ones — have a high copy number, while the larger plasmids have
characteristically a low copy number.
A plasmid free cell may arise spontaneously at a frequency of about 1 in
104 cells. Plasmid free cells may also be produced artificially by the use of
mutagens. The process is commonly called curing of plasmids.
Bacteria carrying such plasmids are capable of using such unusual substrates
for growth and, obviously, enjoy special advantage over others lacking them.
The F plasmid gives the power to carry out a type of sexual reproduction to
bacteria making it possible to exchange genetic materials leading to genetic
recombination.
The R-plasmids with the help of the resistance genes produce proteins which
can inactivate or destroy specific antibiotics.
A segment of DNA containing the specific gene is isolated from a suitable
donor and inserted by recombinant DNA technology into a plasmid.
The recombinant plasmid is next introduced into a suitable host cell where
the gene is expressed producing the gene product.
The F plasmid, also known as the fertility factor or sex factor, determines
the sex of Escherichia coli bacteria.
The cells containing this plasmid are designated as F+ and those without it
as F–.
F+ bacteria are considered as male, because they can act as donor of not
only the plasmid, but also chromosomal genes to the F– cells which act as
recipient and are, therefore, considered as female.
R-plasmids conferring resistance to various drugs individually or multiple
resistance to several antibacterial agents were first discovered in Japan in
the 1950s in the gastroenteritis causing Shigella dysenteriae.
The RTF is mainly involved in the transfer function of the R-plasmid and
contains a number of genes (the transfer genes) and some others controlling
replication of the plasmid in the host cell.
The Col-plasmids are present in different strains of Escherichia coli and
they contain genes controlling synthesis of a class of proteins called Colicines.
Colicines are able to inhibit the growth of related bacteria which lack a Col-
plasmid (Cor).
Degradation or dissimilation of organic compounds in course of mineralization
is often controlled by plasmid borne genes in many microorganisms.
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Plasmids: Types Ti plasmid is a tumour inducing large extra-chromosomal double stranded
and its Properties
circular DNA which is present in Agrobacterium tumefaciens, a plant
pathogenic bacterium causing the crown gall disease in many dicotyledonous
species.
NOTES
Ti plasmid is about 200 kilo base pair long circular DNA. Only a small part
of this large molecule, a 30 kilo base pair long fragment is responsible for
tumour formation.
Plasmids occur rarely in Eukaryotic cells. Some plasmids have been found
in yeast (Saccharomyces cerevisiae) and in several plants.
The protein can kill other yeast cells lacking the plasmid. This yeast plasmid
has been designated as killer particle.
In the non-dividing plasmids, the double-stranded DNA exists as a right-
handed super-helical coil having 400-600 base pairs per turn of the coil.
Each plasmid has its own origin of replication. Some plasmids also have
genes which code for proteins necessary for their own multiplication.
The total genome isolated from an organism is fragmented and the fragments
are inserted (cloned) separately into individual plasmids.
Plasmids may be classified in a number of ways. Plasmids can be broadly
classified into conjugative plasmids and non-conjugative plasmids.
Conjugative plasmids contain a set of transfer or tra genes which promote
sexual conjugation between different cells.
In the complex process of conjugation, plasmid may be transferred from
one bacterium to another via sex pili encoded by some of the tra genes.
Non-conjugative plasmids are incapable of initiating conjugation, hence they
can be transferred only with the assistance of conjugative plasmids.
Plasmids can also be classified into incompatibility groups. A microbe can
harbour different types of plasmids, but different plasmids can only exist in
a single bacterial cell if they are compatible.
Incompatible plasmids (belonging to the same incompatibility group)
normally share the same replication or partition mechanisms and can thus
not be kept together in a single cell.

11.5 KEY WORDS

Plasmid: A plasmid is a small DNA molecule within a cell that is physically

separated from chromosomal DNA and can replicate independently.
Relaxed plasmids: Plasmids having a high copy number are known as
relaxed plasmids
Stringent plasmids: Plasmids having low copy number are called stringent
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240 Material
 Plasmids: Types
11.6 SELF ASSESSMENT QUESTIONS AND and its Properties

EXERCISES

Short Answer Questions NOTES

1. What are plasmids?

2. Name the types of plasmids.
3. Draw a well-labelled diagram of a plasmid.
4. What is a viral vector?
5. What is F plasmid?
6. Draw a well-labelled diagram of Ti plasmid.
7. What are the different aspects of plasmid replication?
8. Give some general properties of plasmids.
Long Answer Questions
1. What are plasmids? Discuss the types of plasmids giving examples.
2. Elaborate a note on artificial chromosomes.
3. Draw a well-labelled diagram to show F plasmid.
4. Write a detailed note on degradative plasmids.
5. Discuss about Ti plasmid of Agrobacterium.
6. Explain the replication of plasmids.
7. Write a note on incompatibility of plasmids.
8. Draw a well-labelled diagram of types of plasmid integration into a host
bacteria.
9. Discuss about the properties of plasmids.

11.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
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Plasmids: Types Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
and its Properties
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
NOTES
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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 Detection, Purification
BLOCK - IV and Transfer of Plasmid
DNA, and Plasmid
TRANSPOSABLE ELEMENTS AND EPIGENETICS Replication

NOTES
UNIT 12 DETECTION,
PURIFICATION AND
TRANSFER OF PLASMID
DNA, AND PLASMID
REPLICATION
Structure
12.0 Introduction
12.1 Objectives
12.2 Plasmid DNA
12.3 Detection and Purification of Plasmid DNA
12.3.1 Isolation and Purification of Plasmid DNA
12.4 Transfer of Plasmid DNA
12.5 Replication of Plasmid
12.6 Plasmid Copy Number
12.7 Answers to Check Your Progress Questions
12.8 Summary
12.9 Key Words
12.10 Self Assessment Questions and Exercises
12.11 Further Readings

12.0 INTRODUCTION

A plasmid is a small DNA molecule within a cell that is physically separated from
chromosomal DNA and can replicate independently. They are most commonly
found as small circular, double-stranded DNA molecules in bacteria; however,
plasmids are sometimes present in Archaea and Eukaryotic organisms. In nature,
plasmids often carry genes that benefit the survival of the organism, such as by
providing antibiotic resistance. While the chromosomes are big and contain all the
essential genetic information for living under normal conditions, plasmids usually
are very small and contain only additional genes that may be useful in certain
situations or conditions.
The term ‘plasmid’ was coined by Joshua Lederberg in 1592. Originally
evolved from bacteria, plasmids are extra-chromosomal genetic elements that are
present in most species of Archae, Eukarya and Eubacteria that have ability to
replicate independently. Plasmids are circular double stranded DNA molecule that
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Detection, Purification are distinct from the cells chromosomal DNA. The structure and function of a
and Transfer of Plasmid
DNA, and Plasmid bacterial cell is directed by the genetic material contained within the chromosomal
Replication DNA. In some cases plasmids are generally not essential for the survival of the
host bacterium. Plasmids specify traits that allow the host to persist in environments
NOTES that would otherwise be either lethal or restrictive for growth. For example, antibiotic
resistance and protein expression. Antibiotic resistance genes are typically encoded
by the plasmid, which allows the bacteria to survive in an antibiotic containing
environment, thereby providing the bacterium with a competitive advantage over
antibiotic-sensitive species. In addition, the plasmids as a tool can be modified to
express the protein of interest, for example production of human insulin using
recombinant DNA technology.
Every gene manipulation procedure requires genetic material like DNA and
RNA. Fundamentally, the nucleic acids occur naturally in association with proteins
and lipoprotein organelles. The dissociation of a nucleoprotein into nucleic acid
and protein moieties and their subsequent separation, are the essential steps in the
isolation of all species of nucleic acids. Isolation of nucleic acids is followed by
quantitation of nucleic acids which is normally done by either spectrophotometric
or by using fluorescent dyes to determine the average concentrations and purity of
DNA or RNA present in a mixture. Isolation of the genetic material (DNA) from
cells (bacterial, viral, plant or animal) involves three basic steps, namely the rupturing
of cell membrane to release the cellular components and DNA, separation of the
nucleic acids from other cellular components and purification of nucleic acids.
In this unit, you will study about the detection and purification of plasmid
DNA, transfer of plasmid DNA, replication of plasmid, control of copy number,
plasmid amplification, curing and incompatibility.

12.1 OBJECTIVES

After going through this unit, you will be able to:

Discuss what plasmid DNA is
Explain the detection and purification of plasmid DNA
Analyse the transfer of plasmid DNA
Define replication of plasmid
Understand control of copy number and plasmid amplification
Elaborate on curing and incompatibility

12.2 PLASMID DNA

A plasmid is a small DNA molecule within a cell that is physically separated from
chromosomal DNA and can replicate independently. They are most commonly
found as small circular, double-stranded DNA molecules in bacteria; however,
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plasmids are sometimes present in Archaea and Eukaryotic organisms. In nature, Detection, Purification
and Transfer of Plasmid
plasmids often carry genes that benefit the survival of the organism, such as by DNA, and Plasmid
providing antibiotic resistance. While the chromosomes are big and contain all the Replication
essential genetic information for living under normal conditions, plasmids usually
are very small and contain only additional genes that may be useful in certain NOTES
situations or conditions. Artificial plasmids are widely used as vectors in molecular
cloning, serving to drive the replication of recombinant DNA sequences within
host organisms. In the laboratory, plasmids may be introduced into a cell via
transformation.
Plasmids are considered replicons, units of DNA capable of replicating
autonomously within a suitable host. However, plasmids, like viruses, are not
generally classified as life. Plasmids are transmitted from one bacterium to another
(even of another species) mostly through conjugation. This host-to-host transfer
of genetic material is one mechanism of horizontal gene transfer. Unlike viruses,
which encase their genetic material in a protective protein coat called a capsid,
plasmids are ‘naked’ DNA and do not encode genes necessary to encase the
genetic material for transfer to a new host. However, some classes of plasmids
encode the conjugative sex pilus essential for their own transfer. The size of the
plasmid varies from 1 to over 200 kbp and the number of identical plasmids in a
single cell can range anywhere from one to thousands under some circumstances.
In addition, the plasmids as a tool can be modified to express the protein of
interest, for example production of human insulin using recombinant DNA
technology.
Characteristics of Plasmids
Plasmids present in the bacterium differ in their physical properties, such as in size
(kbp), geometry and copy number.
Plasmid Size: Plasmids range in size from 1 kbp (kilo base pair) to 1000 (kilo
base pair) mega-plasmids that are many hundred base pairs in size.
Plasmid Geometry: Even though most plasmids possess a circular geometry,
there are now many examples of plasmids that are linear in most of the bacteria.
Plasmid DNA may appear in one of the five conformations nicked open circular
DNA which has one strand cut. The undisturbed circular DNA is completely intact
with both strands uncut, but has been enzymatically relaxed. The linear DNA has
free ends, while the supercoiled DNA is fully intact with both strands uncut.
Plasmid Copy Numbers: Copy number is the significant feature and refers to the
average or expected number of copies per host cell. Plasmids have either low,
medium or high copy number. After knowing that to which category plasmid belongs
to is very significant to start an experiment. When working with a low copy number
plasmid which is associated with a low yield may therefore require more cultures
to be done. Alternatively, if the yield obtained from a high copy plasmid is poor,
then troubleshooting is essential. In bacterium with high copy number plasmids,
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Detection, Purification during cell division the plasmids get segregate randomly in the daughter cells,
and Transfer of Plasmid
DNA, and Plasmid whereas bacterium with low copy numbers, during cell division and partition the
Replication plasmids divided equally in the daughter cells. An advantage of high copy number
is the greater stability of the plasmid when random partitioning, i.e., the partitioning
NOTES of plasmids into daughter cells occurs at cell division.
Every gene manipulation procedure requires genetic material like DNA and
RNA. Fundamentally, the nucleic acids occur naturally in association with proteins
and lipoprotein organelles. The dissociation of a nucleoprotein into nucleic acid
and protein moieties and their subsequent separation, are the essential steps in the
isolation of all species of nucleic acids. Isolation of nucleic acids is followed by
quantitation of nucleic acids which is normally done by either spectrophotometric
or by using fluorescent dyes to determine the average concentrations and purity of
DNA or RNA present in a mixture. Isolation of the genetic material (DNA) from
cells (bacterial, viral, plant or animal) involves three basic steps, namely the rupturing
of cell membrane to release the cellular components and DNA, separation of the
nucleic acids from other cellular components and purification of nucleic acids.

12.3 DETECTION AND PURIFICATION OF

PLASMID DNA

Plasmids are important vehicles for rapid adaptation of bacterial populations to

changing environmental conditions. Among the various methods available for the
preparation of plasmid DNA for rapid screening, a protocol involving the use of
an alkaline solution to lyse the cells, salt precipitation to remove cell debris and
chromosomal DNA and application to bind DNA column to eliminate proteins
and other contaminants has been widely used. Gel electrophoresis is considered
as the popular method that can be easily performed resolution technique in nucleic
acid research. Gel Electrophoresis using Agarose, is considered as highly purified
linear polysaccharide derived from agar and is widely used in the detection and
characterization of plasmids, and also the linear DNA fragments. Plasmids of sizes
ranging from less than one kilo base (kb) to over a few hundred kb can be easily
resolved by conventional Agarose Gel Electrophoresis.
Plasmid purification is a technique used to isolate and purify plasmid DNA
from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is
a small, circular, double-stranded DNA that is used as a carrier of specific DNA
molecules. When introduced into a host organism via transformation, a plasmid
will be replicated, creating numerous copies of the DNA fragment under study.
A plasmid preparation is a method of DNA extraction and purification for
plasmid DNA. Various methods have been developed to purify plasmid DNA
from bacteria. All the purification methods involve following three steps:
Step 1: Growth of the Bacterial Culture.

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 Step 2: Harvesting and Lysis of the Bacteria. Detection, Purification
and Transfer of Plasmid
Step 3: Purification of Plasmid DNA. DNA, and Plasmid
Replication
The purification of plasmid DNA from bacterial cells is a significant step
specifically in the cloning process. During plasmid purification, the bacterial cells NOTES
are lysed, freeing DNA and other cellular components from the cell wall. Cellular
components are then removed, and the DNA containing lysate is processed to
further remove contaminants to separate the plasmid DNA from the genomic DNA.
12.3.1 Isolation and Purification of Plasmid DNA
The isolation of plasmid DNA from bacteria is a critical technique in molecular
biology and is an essential step in many procedures, such as cloning, DNA
sequencing, transfection, and gene therapy. These manipulations require the isolation
of high purity plasmid DNA. The purified plasmid DNA can be used for immediate
use in all molecular biology procedures, such as digestion with restriction enzymes,
cloning, PCR, transfection, in vitro translation, blotting and sequencing.
Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA
or other cell components, such as proteins by breaking the cells. Bacteria containing
the plasmid of interest is first grown, and then permitted to lyse with an alkaline
lysis buffer consisting of a detergent Sodium Dodecyl Sulfate (SDS) and a strong
base Sodium Hydroxide (NaOH). The detergent splits the phospholipid bilayer of
membrane and the alkali denatures the proteins which are involved in maintaining
the structure of the cell membrane. This involves series of steps, such as agitation,
precipitation, centrifugation, and the removal of supernatant. The cellular debris is
removed and the plasmid is isolated and purified.
Principle
Purification of plasmid DNA from bacterial DNA is based on the differential
denaturation of chromosomal and plasmid DNA that uses the alkaline lysis for
separating the two. The basic steps of plasmid isolation are disruption of the cellular
structure to create a lysate, separation of the plasmid from the chromosomal DNA,
cell debris and other insoluble material.
Bacteria are lysed with a lysis buffer solution containing Sodium Dodecyl
Sulfate (SDS) and Sodium Hydroxide (NaOH). Throughout this step, the disruption
of most cells is done, i.e., the chromosomal as well as plasmid DNA are denatured
and the resulting Lysate is cleared by means of centrifugation, filtration or magnetic
clearing. Subsequent neutralization with Potassium Acetate helps only the covalently
closed plasmid DNA to reanneal and to remain solubilized. Most of the
chromosomal DNA and proteins precipitate in a complex are formed with Potassium
and SDS, which is removed by centrifugation.
The bacteria is resuspended in a resuspension buffer (50mM Tris-Cl, 10
mM EDTA, 100 g/ ml RNase A, pH 8.0) and then treated by 1% SDS (w/v) /
Alkaline Lysis buffer (200mM NaOH) to liberate the plasmid DNA from the
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Detection, Purification Escherichia coli host cells. Neutralization buffer (3.0 M Potassium Acetate, pH
and Transfer of Plasmid
DNA, and Plasmid 5.0) neutralizes the resulting Lysate for creating appropriate or suitable conditions
Replication to bind the plasmid DNA to the Silica membrane column. Subsequently, the
precipitated Protein, Genomic DNA, and Cell Debris are pelleted through a
NOTES centrifugation step and the supernatant is loaded onto a column. Contamination,
such as the salts, metabolites, and soluble macromolecular cellular components
are removed using the simple washing with Ethanolic Wash buffer (1.0 M NaCl,
50mM MOPS, pH 7.0, Isopropanol (v/v) 15 %). Finally, the ‘Pure Plasmid DNA’
is eluted under low ionic strength conditions with slightly Alkaline buffer (5 mM
Tris / HCl, pH 8.5).
Culture Media
Yield and quality of plasmid DNA extremely depends on the type of culture media
being used. Most of the plasmid purification are optimized with cultures grown in
standard Luria Bertani (LB) medium.
To prepare the LB medium dissolve 10 g Tryptone, 5 g Yeast Extract, and
10 g NaCl in 800 ml Distilled Water. Adjust the pH to 7.0 with 1 N NaOH.
Adjust the volume to 1 liter by adding distilled water and sterilize by autoclaving.
The cell culture should be incubated at 37°C with constant shaking (200–250
rpm) preferably 12–16 hours overnight. Alternatively, rich media like 2 x YT (Yeast
/ Tryptone), TB (Terrific Broth), or CircleGrow can be used.
To find the optimal culture conditions, the culture medium and incubation
times have to be optimized individually for each host strain / plasmid construct
combination.
Lysate and Neutralization
Lysis formulas can differ depending on which extract DNA/RNA/Plasmid is
required. All methods of lysing bacteria will yield plasmid solutions that is
contaminated with chromosomal DNA and RNA. Centrifugation removes the
majority of chromosomal DNA that forms a pellet, while the plasmid DNA remains
soluble. The treatment with RNase will eliminate contaminating RNA.
In general, lysis buffers contain a high concentration of chaotropic salts.
Chaotropes have two important roles in nucleic acid extraction. First is they
destabilize Hydrogen bonds, van der Waals forces and hydrophobic interactions,
leading to destabilization of proteins, including nucleases. Second is they disrupt
the association of nucleic acids with water, hence providing optimal conditions for
their transfer to Silica.
Separation and removal of the plasmids from the bacterial cell is performed
by resuspension of 1-5 mL of culture in a resuspension buffer (50mM Tris-Cl, 10
mM EDTA, 100 µg/ ml RNase A, pH 8.0) and pellet cells in a microcentrifuge at
11000 x g for 30 seconds.

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 Lysate is achieved by adding 250 L of lysis buffer with neutralization buffer, Detection, Purification
and Transfer of Plasmid
as it helps in complete precipitation of SDS, Protein, and Genomic DNA. DNA, and Plasmid
Incomplete neutralization will lead to reduced yield. Do not shake the released Replication
plasmid DNA too much or too strongly because it will damage the DNA.
NOTES
Binding and Washing in Silica Membrane
After centrifuging the Lysate through Silica membrane, the desired nucleic acids
are bound to the columned and impurities, such as Protein and Polysaccharides
should be removed. Principally, the plant samples contain Polysaccharides and
Pigments, while for blood samples contain the membrane which may be slightly
brown or yellow in colour. The washing steps will remove such impurities. Typically,
there are two washing steps, though it varies depending on the sample type. The
first washing step includes a low concentration of Chaotropic Salts to remove
residual Proteins and Pigments and is always followed with an Ethanol Wash to
remove the Salts.
Columns containing the Silica resin selectively binds to DNA/RNA. The
DNA of interest can be isolated due to its ability to bind Silica in the presence of
high concentrations of Chaotropic Salts. These salts are then removed with an
Alcohol Based Wash and the DNA is eluted using a low-ionic-strength solution,
such as TE buffer or Water. The binding of DNA to Silica appears to be determined
by dehydration and Hydrogen bond formation, which competes against weak
electrostatic repulsion. Hence, a high concentration of Salt will help in the
determination of DNA adsorption onto Silica, and a low concentration will release
the DNA.
Elution
The elution buffer volume and method is specifically adapted to the subsequent
application for achieving higher yield and / or concentration that uses the standard
method. Elution buffer is used to wash the Unbound Proteins at first and at a
greater concentration it releases the Desired Protein from the ligand. It is significant
that the elution buffer mechanism works fast without changing the function or activity
of the Desired Protein. For maximal DNA elution, the buffer is allowed to stand in
the membrane for a few minutes before centrifugation. Elution Buffer AE (5 mM
Tris/HCl, pH 8.5) can also be replaced by TE buffer or Water. Using a weakly
buffered slightly alkaline buffer containing no EDTA is preferred especially if the
plasmid DNA is intended for sequencing reactions.
Analytical Gel Analysis
It is recommended to remove and save the aliquots during the purification
procedure. If the plasmid DNA is of low yield or low in quality, then the samples
can be analyzed by Agarose Gel Electrophoresis to determine at what stage of the
purification procedure the problem occurred.

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Detection, Purification Procedure: It includes the following steps.
and Transfer of Plasmid
DNA, and Plasmid Step 1: Harvesting Bacterial and Resuspended Cells
Replication
1. Choose a single colony from a freshly streaked selective plate and inoculate
NOTES a starter culture of 2–5 ml LB medium containing the appropriate selective
antibiotic. Incubate for approximately 8 hours at 37°C with vigorous shaking
(approximately 300 rpm).
2. Dilute the starter culture 1/500 to 1/1000 into 3 ml selective LB medium.
Grow at 37°C for 12–16 hours with vigorous shaking (approximately 300
rpm).
3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 minutes and
remove as much of the supernatant as possible. Resuspend the bacterial
pellet in 0.1-0.5 ml of resuspension buffer (50mM Tris-Cl, 10 mM EDTA,
100 g/mL RNase A, pH 8.0). The bacteria should be resuspended
completely by vortexing or pipetting up and down until no cell clumps remain.
Step2: Cell Lysis
4. Add 0.25 ml of lysis buffer, mix thoroughly by vigorously inverting the sealed
tube 4–6 times, and incubate at room temperature (15–25°C) for 5 minutes.
Do not vortex, as this will result in shearing of genomic DNA. The lysate
should appear viscous. Do not allow the lysis reaction to proceed for more
than 5 minutes.
Step 3: Neutralization
5. Add 0.3 mL of neutralization buffer, mix immediately and thoroughly by
vigorously inverting 4–6 times, and incubate on ice for 5 minutes.
Precipitation is enhanced by using chilled neutralization buffer and incubating
on ice. After addition of neutralization buffer, a fluffy white material forms
and the Lysate becomes less viscous. The precipitated material contains
Genomic DNA, Proteins, Cell Debris, and KDS. The Lysate should be
mixed thoroughly to ensure even Potassium Dodecyl Sulphate precipitation.
If the mixture still appears viscous, more mixing is required to completely
neutralize the solution. A homogeneous colorless suspension indicates that
the SDS has been effectively precipitated.
Step 4: Loading Lysate on Column
6. Before loading the column, carefully remove the supernatant and then transfer
it to a collection tube containing the column and centrifuge at 13,000 rpm
for 1 minute.
7. Discard the flow-through liquid and remove supernatant containing plasmid
DNA promptly. After centrifugation, the supernatants should be clear.
8. If the supernatant is not clear, a second, shorter centrifugation should be
carried out to avoid applying any suspended or particulate material to the
column. Suspended material, which can cause the sample to appear turbid,
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250 Material
Step 5: Binding and Washing Detection, Purification
and Transfer of Plasmid
9. Add 0.7 ml of wash buffer to the column placed in the collection tube and DNA, and Plasmid
Replication
centrifuge for 10 minutes at 13000 rpm for 1 minute. Equilibrate by applying
1 mL equilibration buffer (750 mM NaCl, 50 Mm MOPS, pH 7.0, 15 %
NOTES
Isopropanol) and allow the column to empty by gravity flow. Flow of buffer
will begin automatically by reduction in surface tension due to the presence
of detergent in the equilibration buffer.
10. Apply the supernatant from Step 6 to the column and allow it to enter the
resin by gravity flow.
Step 6: Plasmid Elution
11. Elute DNA with 0.8 mL elution buffer (1.23 M NaCl, 50 mm Tris-Cl, pH
8.5, 15 %v Isopropanol). Collect the elute in a 1.5 mL or 2 mL
microcentrifuge tube.
12. Precipitate DNA by adding 0.7 volumes (0.56 mL per 0.8 mL of elution
volume) of room temperature Isopropanol to the eluted DNA. Mix and
centrifuge immediately at 10,000 rpm for 30 minutes in a microcentrifuge.
Cautiously decant the supernatant. All solutions should be at room
temperature with the objective of minimize salt precipitation.
13. Wash DNA pellet with 1 mL of 70% Ethanol and centrifuge at 10,000 rpm
for 10 minutes.
14. Carefully decant the supernatant without disturbing the pellet.
15. The 70% Ethanol removes precipitated salt and replaces Isopropanol with
the more volatile Ethanol, making the DNA easier to re-dissolve.
16. Air-dry the pellet for 5–10 minutes, and re-dissolve the DNA in a suitable
volume of buffer (e.g., TE buffer, pH 8.0, or 10mM Tris-Cl, pH 8.5). Re-
dissolve the DNA pellet by rinsing the walls for recovering all the DNA.
Determination of Yield
For the determination the yield, DNA concentration must be determined by means
of UV spectrophotometry at 260 nm and also by quantitative analysis on an Agarose
Gel. To quantitate the nucleic acid concentration, dilute the plasmid DNA 1 : 100
or 1 : 50, this typically depends on the plasmid copy number, in TE buffer and
measure the absorbance (optical density) at 260 nm (A260) and 280 nm (A280).
The TE buffer must be used as the blank. This measurement can be done using the
direct calculation of the nucleic acid concentration using the formula:
[DNA] ( g/mL) = A260 × Dilution Factor × 50
Where, 50 is the Extinction Coefficient of DNA. The ratio A260/A280
provides a reasonable or realistic estimate of the purity of the preparation.

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Detection, Purification
and Transfer of Plasmid 12.4 TRANSFER OF PLASMID DNA
DNA, and Plasmid
Replication
Plasmids are referred as extra chromosomal circular DNA molecules that exist in
NOTES most bacterial species and in some species of Eukaryotes. Under normal conditions,
a specific plasmid is dispensable to its host cell. Many plasmids contain plasmid
genes that are essential in certain specified environments, for example ‘R’ plasmids
carry genes that provide resistance to numerous antibiotics such that the cell
containing this plasmid can resist antibiotic action. Figure 12.1 illustrates the process
of horizontal gene transfer.

Fig. 12.1 Process of Horizontal Gene Transfer

Fundamentally, all known plasmids are supercoiled circular DNA except

the one exceptional, i.e., the killer plasmid of Yeast, a RNA molecule. The molecular
weight ranges from 106 – 108. The number of copies of a Plasmid Per Cell ranges
from 1–2 for low copy number plasmids and to 10–60 for high copy number
plasmids.
Plasmid DNA can be isolated from bacteria using the culturing plasmid
containing bacteria. Detergent is added to it to get Lysed bacteria. Then the Lysate
is centrifuged. The smaller plasmid DNA remains in the supernatant. Cesium
Chloride (CsCl) and Ethidium Bromide is added to the supernatant. The CsCl
forms a density gradient. The supercoiled DNA with high density can be identified
by Ethidium Bromide and easily removed.
Transfer of Plasmid DNA
The bacterium Escherichia coli possesses 2 mating types, i.e., donors or male
and recipients or female. The determinant of male feature is the ‘F’ factor or sex
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plasmid or donor, therefore a male cell is designated as F+. Female lacks ‘F’ Detection, Purification
and Transfer of Plasmid
plasmid and designated as F–. When the culture of male and female is mixed, the DNA, and Plasmid
conjugation takes place between the male and female pairs forming a conjugation Replication
bridge. Pairing induces looped rolling circle replication of ‘F’, the recipient cell
contains the ‘F’ plasmid. During transfer both ‘Donor’ and ‘Recipient’ cells show NOTES
DNA synthesis. In ‘Donor’, the synthesis replaces single strand to be transferred,
the synthesis in the recipient converts the transferred single strand to double stranded
DNA.
‘F’ plasmid has an ability to integrate into the bacterial chromosome.
Principally, the ‘F’ fuses with chromosome increasing its size. The cell is now
called Hfr cell or High frequency recombination cell. When a culture of Hfr is
mixed with F– culture, the conjugation occurs. Replication starts in the Hfr cell and
is transferred to F– cell. The direction of replication is specific, i.e., small portion of
‘F’ is transferred first and then the major part is transferred. Female receives both
small and large fragments of the male chromosome. The Hfr is produced when ‘F’
integrates firmly into the chromosome. The plasmid containing both ‘F’ genes and
chromosomal genes is called as ‘F’ plasmid.

12.5 REPLICATION OF PLASMID

An essential feature of bacterial plasmids is their ability to replicate as autonomous

genetic elements in a controlled way within the host. Therefore, they are used for
exploring the mechanisms involved in the DNA replication and for analyzing the
different strategies that couple DNA replication to other critical procedures in the
cell cycle.
Plasmid replication process is divided into three stages, namely initiation,
elongation, and termination. The inability of DNA polymerases to initiate de novo
replication for creating the essential independent generation of a primer.
This is explained, in circular plasmids, through following two main strategies:
Opening of the strands followed by RNA priming (theta and strand
displacement replication).
Cleavage of one of the DNA strands to generate a 32 -OH end (rolling-
circle replication).
Initiation is catalyzed regularly through one or a few plasmid-encoded initiation
Proteins that are capable of recognizing the plasmid-specific DNA sequences and
determining the point from which replication starts, i.e., the origin of replication. In
some cases, these Proteins also participate directly in the generation of the primer.
These initiators are also involved in the role of Pilot Proteins that guide the assembly
of the host Replisome at the Plasmid Origin.
Elongation of plasmid replication is carried out basically through DNA
Polymerase III Holoenzyme and in some cases using DNA Polymerase I at an
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Detection, Purification early stage, with the participation of other Host Proteins that form the Replisome.
and Transfer of Plasmid
DNA, and Plasmid Termination of replication includes the precise requirements and implications for
Replication reinitiation.
The initiation stage performs an additional role, as at this stage the mechanisms
NOTES
controlling replication function or operate. The key objective of this control is to
maintain a fixed concentration of plasmid molecules in a growing bacterial population,
i.e., duplication of the plasmid pool paced with duplication of the bacterial
population. The molecules that are directly involved in this control can be,
RNA (Antisense RNA)
DNA Sequences (Iterons)
Antisense RNA and Proteins involved in the Mechanism.
Characteristically, the control elements maintain an average frequency of
one plasmid replication through ‘per plasmid copy per cell cycle’ and to ‘sense’
and correct deviations from this average. The plasmid replication and its control is
based on the results of analyses performed using pure cultures under steady-state
growth conditions. Thus setting significant parameters required to understand the
maintenance of these genetic elements in mixed populations and under environmental
conditions.
As already discussed the plasmids are extra chromosomal DNA elements
with characteristic copy numbers within the host. These replicons occur in species
from the three representatives of the living world, namely, the domains Archaea,
Bacteria, and Eukarya. Plasmids may constitute a substantial amount of the total
genetic content of an organism, representing more than 25% of the genetic material
of the cell in some members of the Archaea. They can, therefore, incorporate and
deliver genes by recombination or transposition, thus supporting genetic exchanges
in bacterial populations. Since plasmids can be hosted or introduced into new
hosts through various mechanisms, hence they are considered as a group of extra
chromosomal DNA which is shared between populations. The genetic information
are carried by plasmids, and their effect in the microbial communities, and the
potential of these elements perform as natural cloning vectors that have stimulated
exploration into plasmids not only from the fundamental but also from the clinical,
biotechnological, and environmental aspects.
Following are the three key factors that contribute to the development of
plasmid investigation or exploration:
The genetic organization of these elements is apparently simple.
They can be easily isolated and manipulated in vitro.
Because plasmids are dispensable, hence their manipulation does not
appear, in principle, to have adverse consequences to the hosts.
The characteristic feature that expresses plasmids is that they can replicate
in an autonomous and self-controlled method. The plasmid replication analysis
and its control, such as the existence of antisense RNAs, have contributed to the
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unraveling of mechanisms of DNA replication, macromolecular interactions, and Detection, Purification
and Transfer of Plasmid
control of gene expression. Some plasmids have the ability to pass across the so- DNA, and Plasmid
called genetic barriers among different living organisms and thus pretending questions Replication
about general mechanisms that govern the replication and the communication
between plasmid replication components and the host machinery involved in DNA NOTES
replication.
Despite their autonomous replication, the plasmids extensively use the
replication machinery of the host, and therefore plasmid replication facilitates the
exploration of the mechanisms involved in chromosome replication.
Origins of Replication
Plasmid origins of replication can be defined as follows:
The minimal cis acting region that can support autonomous replication of
the plasmid.
The region where DNA strands are melted to initiate the replication process.
The base(s) at which leading-strand synthesis starts.
Replication origins contain sites that are required for interactions of plasmid encoded
proteins and/or host encoded proteins.
General Features: With some exceptions, initiation of plasmid DNA replication
requires a specific plasmid-encoded Rep initiator protein. This is reflected by the
presence, at the origin of replication, of specific sequences with which the Rep
protein interacts. Additional features found in many origins of theta-replicating
plasmids include an adjacent AT-rich region containing sequence repeats, where
opening of the strands and assembly of host initiation factors occur, and one or
more sites where the host DNAA initiator protein binds. Multiple methylation
sequences that are present in the origin of replication of the Escherichia coli
chromosome, oriC, can also be found at the origin of replication of plasmids, such
as P1 and pSC101. Methylation is not essential for replication, because its primary
role is in post-replication.
Iteron Containing Origins: In many situations, the origin of replication contains
directly repeated sequences, termed iterons, which are the binding sites for the
plasmid-encoded Rep Proteins having control properties. The iterons are not only
essential for replication but are also the key elements for the control of plasmid
replication. Among plasmids which restrict their establishment to a single or a few
species of Enterobacteria, iterons have been described for several replicons like
P1, F, pSC101, etc. Iterons can be adjacent or separated by intervening sequences.

12.6 PLASMID COPY NUMBER

Plasmids must regulate their copy number, average number of plasmid copies per
cell, to ensure that they do not excessively burden the host or become lost during
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Detection, Purification cell division. Plasmids may be either high copy number plasmids or low copy
and Transfer of Plasmid
DNA, and Plasmid number plasmids; the regulation mechanisms between these two types are often
Replication significantly different. Biotechnology applications may involve engineering plasmids
to allow a very high copy number. For example, pBR322 is a low copy number
NOTES plasmid (~20 copies per cell) from which several very high copy number cloning
vectors (~1000 copies per cell) have been derived.
Regulation
High copy number plasmids, also called relaxed plasmids, require a system to
ensure that replication is inhibited once the number of plasmids in the cell reaches
a certain threshold. Relaxed plasmids are generally regulated through one of two
mechanisms, namely Antisense RNA or Iteron Binding Groups. Low copy number
plasmids, also called stringent plasmids, require tighter control of replication.
ColE1 Derived Plasmids: Antisense RNA
In ColE1 derived plasmids, replication is primarily regulated through a small
plasmid-encoded RNA called RNA I. A single promoter initiates replication in
ColE1: the RNA II promoter. The RNA II transcript forms a stable RNA-DNA
hybrid with the DNA template strand near the origin of replication, where it is then
processed by RNaseH to produce the 3' OH primer that DNA Polymerase I uses
to initiate leading strand DNA synthesis. RNA I serves as a major plasmid-encoded
inhibitor of this process whose concentration is proportional to plasmid copy
number. RNA I is exactly complementary to the 5' end of the RNA II (because it
is transcribed from the opposite strand of the same region of DNA as RNA II).
RNA I and RNA II first form a weak interaction which is stabilized by a Protein
called Rop (Repressor of primer) and a double-stranded RNA-I/RNA-II, RNA
duplex is formed. This altered shape prevents RNA II from hybridizing to the
DNA and being processed from RNaseH to produce the primer necessary for
initiation of plasmid replication. More RNA I is produced when the concentration
of the plasmid is high, and high concentration of RNA I inhibits replication, resulting
in regulation of copy number.
R1 and ColIb-p9 Plasmids: Antisense RNA
Most plasmids require a plasmid-encoded protein, usually called Rep, to separate
the strands of DNA at the origin of replication (oriV) to initiate DNA replication.
Rep binds to specific DNA sequences in oriV which are unique to a plasmid type.
The synthesis of Rep protein is controlled in order to limit plasmid replication and
therefore regulate copy number. In R1 plasmids RepA can be transcribed from
two different promoters. It is made from the first promoter until the plasmid reaches
its copy number, upon which the protein CopB represses this primary promoter.
RepA expression is also regulated post-transcriptionally from the secondary
promoter by an Antisense RNA called CopA. CopA interacts with its RNA target
in the RepA mRNA and forms a RNA-RNA duplex. The resultant double stranded
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RNA is cleaved by RNase III, preventing synthesis of RepA. The higher the
256 Material
concentration of the plasmid, the more CopA RNA is produced and the less RepA Detection, Purification
and Transfer of Plasmid
protein can be synthesized, increasing inhibition of plasmid replication. DNA, and Plasmid
Replication
Col1b-P9: Antisense RNA
Replication of the low-copy-number ColIb-P9 depends upon Rep, which is NOTES
produced by expression of the repZ gene. The repZ expression requires formation
of a pseudoknot in the mRNA. Basically, repZ is repressed by a small antisense
Inc RNA, which binds to repZ mRNA, forms an Inc RNA-mRNA duplex, and
prevents formation of the pseudoknot to inhibit repZ translation into Rep.
pSC101: Iteron Plasmid
Iteron plasmids, including F and RK2-related plasmids, have oriV regions containing
multiple (~3-7) repeats of 17-22 bp iteron sequences. The pSC101 represents a
simple model of an iteron plasmid. Iteron plasmids control copy number through
two combined methods, suitable for low copy number stringent plasmids. One
method is control of RepA synthesis. RepA is the only plasmid-encoded protein
required for replication in pSC101. RepA protein represses its own synthesis by
binding to its own promoter region and blocking transcription of itself
(Transcriptional Autoregulation). Thus, the more RepAis made, the more its synthesis
is repressed, and subsequently limiting plasmid replication. The coupling hypothesis
proposes that the second method is coupling of plasmids through the Rep Protein
and Iteron Sequences. When the plasmid concentration is high, RepA plasmids
bound to iterons form dimers in between two plasmids, ‘handcuffing’ them at the
origin of replication and inhibiting replication.
Incompatibility
Plasmids can be incompatible if they share the same replication control mechanism.
Under these circumstances, both plasmids contribute to the total copy number
and are regulated together. They are not recognized as distinct plasmids. As such,
it becomes much more likely that one of the plasmids may be out-copied by the
other and lost during cell division, the cell is ‘cured’ of the plasmid. This is particularly
likely with low copy number plasmids. Plasmids can also be incompatible due to
shared partitioning systems.
Incompatibility Groups
1. Not all plasmids can live together.
2. Plasmids that are able to coexist in the same cell do not interfere with each
other’s replication.
3. A single cell can have as many Inc group plasmids as it can tolerate and
replicate.
Detection and purification of plasmid DNA. Transfer of plasmid DNA. Replication
of plasmid. Control of copy number, plasmid amplification, curing and
incompatability. Self-Instructional
Material 257
Detection, Purification
and Transfer of Plasmid
DNA, and Plasmid Check Your Progress
Replication
1. Explain the term plasmid.
NOTES 2. Why plasmids are considered replicons?
3. How are plasmids transmitted?
4. What is plasmid copy numbers? Explain with example.
5. What is plasmid purification? How it is done?
6. How the isolation of plasmid DNA is done from bacteria?
7. How is plasmid DNA transferred?
8. Explain the term plasmid replication.
9. How the control elements maintain an average frequency of one plasmid
replication?
10. When plasmids can be incompatible?

12.7 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. A plasmid is a small DNA molecule within a cell that is physically separated

from chromosomal DNA and can replicate independently. They are most
commonly found as small circular, double-stranded DNA molecules in
bacteria; however, plasmids are sometimes present in Archaea and
Eukaryotic organisms. In nature, plasmids often carry genes that benefit the
survival of the organism, such as by providing antibiotic resistance. Plasmids
present in the bacterium differ in their physical properties, such as in size
(kbp), geometry and copy number.
2. Plasmids are considered replicons, units of DNA capable of replicating
autonomously within a suitable host.
3. Plasmids are transmitted from one bacterium to another (even of another
species) mostly through conjugation. This host-to-host transfer of genetic
material is one mechanism of horizontal gene transfer. Unlike viruses, which
encase their genetic material in a protective protein coat called a capsid,
plasmids are ‘naked’ DNA and do not encode genes necessary to encase
the genetic material for transfer to a new host. However, some classes of
plasmids encode the conjugative sex pilus essential for their own transfer.
4. Copy number is the significant feature and refers to the average or expected
number of copies per host cell. Plasmids have either low, medium or high
copy number. After knowing that to which category plasmid belongs to is
very significant to start an experiment. When working with a low copy
number plasmid which is associated with a low yield may therefore require
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 more cultures to be done. Alternatively, if the yield obtained from a high Detection, Purification
and Transfer of Plasmid
copy plasmid is poor, then troubleshooting is essential. In bacterium with DNA, and Plasmid
high copy number plasmids, during cell division the plasmids get segregate Replication
randomly in the daughter cells, whereas bacterium with low copy numbers,
during cell division and partition the plasmids divided equally in the daughter NOTES
cells. An advantage of high copy number is the greater stability of the plasmid
when random partitioning, i.e., the partitioning of plasmids into daughter
cells occurs at cell division.
5. Plasmid purification is a technique used to isolate and purify plasmid DNA
from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A
plasmid is a small, circular, double-stranded DNA that is used as a carrier
of specific DNA molecules. When introduced into a host organism via
transformation, a plasmid will be replicated, creating numerous copies of
the DNA fragment under study.
A plasmid preparation is a method of DNA extraction and purification for
plasmid DNA. Various methods have been developed to purify plasmid
DNA from bacteria. All the purification methods involve following three
steps:
Step 1: Growth of the Bacterial Culture.
Step 2: Harvesting and Lysis of the Bacteria.
Step 3: Purification of Plasmid DNA.
During plasmid purification, the bacterial cells are lysed, freeing DNA and
other cellular components from the cell wall. Cellular components are then
removed, and the DNA containing lysate is processed to further remove
contaminants to separate the plasmid DNA from the genomic DNA.
6. The isolation of plasmid DNA from bacteria is a critical technique in molecular
biology and is an essential step in many procedures, such as cloning, DNA
sequencing, transfection, and gene therapy. These manipulations require
the isolation of high purity plasmid DNA. Alkaline lysis is a method used in
molecular biology, to isolate plasmid DNA or other cell components, such
as proteins by breaking the cells. Bacteria containing the plasmid of interest
is first grown, and then permitted to lyse with an alkaline lysis buffer consisting
of a detergent Sodium Dodecyl Sulfate (SDS) and a strong base Sodium
Hydroxide (NaOH). The detergent splits the phospholipid bilayer of
membrane and the alkali denatures the proteins which are involved in
maintaining the structure of the cell membrane. This involves series of steps,
such as agitation, precipitation, centrifugation, and the removal of supernatant.
The cellular debris is removed and the plasmid is isolated and purified.
7. ‘F’ plasmid has an ability to integrate into the bacterial chromosome.
Principally, the ‘F’ fuses with chromosome increasing its size. The cell is
now called Hfr cell or High frequency recombination cell. When a culture of
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Material 259
Detection, Purification Hfr is mixed with F– culture, the conjugation occurs. Replication starts in
and Transfer of Plasmid
DNA, and Plasmid the Hfr cell and is transferred to F– cell. The direction of replication is specific,
Replication i.e., small portion of ‘F’ is transferred first and then the major part is
transferred. Female receives both small and large fragments of the male
NOTES chromosome. The Hfr is produced when ‘F’ integrates firmly into the
chromosome. The plasmid containing both ‘F’ genes and chromosomal
genes is called as ‘F’ plasmid.
8. An essential feature of bacterial plasmids is their ability to replicate as
autonomous genetic elements in a controlled way within the host. Therefore,
they are used for exploring the mechanisms involved in the DNA replication
and for analyzing the different strategies that couple DNA replication to
other critical procedures in the cell cycle. Plasmid replication process is
divided into three stages, namely initiation, elongation, and termination. The
inability of DNA polymerases to initiate de novo replication for creating the
essential independent generation of a primer.
9. Characteristically, the control elements maintain an average frequency of
one plasmid replication through ‘per plasmid copy per cell cycle’ and to
‘sense’ and correct deviations from this average. The plasmid replication
and its control is based on the results of analyses performed using pure
cultures under steady-state growth conditions. Thus setting significant
parameters required to understand the maintenance of these genetic elements
in mixed populations and under environmental conditions.
10. Plasmids can be incompatible if they share the same replication control
mechanism. Under these circumstances, plasmids contribute to the total
copy number and are regulated together. They are not recognized as distinct
plasmids.
One of the plasmids may be out-copied by the other and lost during cell
division, the cell is ‘cured’ of the plasmid. This is particularly likely with low
copy number plasmids. Plasmids can also be incompatible due to shared
partitioning systems.

12.8 SUMMARY

A plasmid is a small DNA molecule within a cell that is physically separated

from chromosomal DNA and can replicate independently. They are most
commonly found as small circular, double-stranded DNA molecules in
bacteria; however, plasmids are sometimes present in Archaea and
Eukaryotic organisms.
In nature, plasmids often carry genes that benefit the survival of the organism,
such as by providing antibiotic resistance.
While the chromosomes are big and contain all the essential genetic
information for living under normal conditions, plasmids usually are very
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small and contain only additional genes that may be useful in certain situations Detection, Purification
and Transfer of Plasmid
or conditions. DNA, and Plasmid
Replication
Artificial plasmids are widely used as vectors in molecular cloning, serving
to drive the replication of recombinant DNA sequences within host
NOTES
organisms. In the laboratory, plasmids may be introduced into a cell via
transformation.
Plasmids are considered replicons, units of DNA capable of replicating
autonomously within a suitable host. However, plasmids, like viruses, are
not generally classified as life.
Plasmids are transmitted from one bacterium to another (even of another
species) mostly through conjugation. This host-to-host transfer of genetic
material is one mechanism of horizontal gene transfer.
Unlike viruses, which encase their genetic material in a protective protein
coat called a capsid, plasmids are ‘naked’ DNA and do not encode genes
necessary to encase the genetic material for transfer to a new host.
The size of the plasmid varies from 1 to over 200 kbp and the number of
identical plasmids in a single cell can range anywhere from one to thousands
under some circumstances.
Plasmids present in the bacterium differ in their physical properties, such as
in size (kbp), geometry and copy number.
Plasmids range in size from 1 kbp (kilo base pair) to 1000 (kilo base pair)
mega-plasmids that are many hundred base pairs in size.
Plasmid DNA may appear in one of the five conformations nicked open
circular DNA which has one strand cut. The undisturbed circular DNA is
completely intact with both strands uncut, but has been enzymatically relaxed.
Copy number is the significant feature and refers to the average or expected
number of copies per host cell. Plasmids have either low, medium or high
copy number.
Among the various methods available for the preparation of plasmid DNA
for rapid screening, a protocol involving the use of an alkaline solution to
lyse the cells, salt precipitation to remove cell debris and chromosomal
DNA and application to bind DNA column to eliminate proteins and other
contaminants has been widely used.
Gel electrophoresis is considered as the popular method that can be easily
performed resolution technique in nucleic acid research.
Gel Electrophoresis using Agarose, is considered as highly purified linear
polysaccharide derived from agar and is widely used in the detection and
characterization of plasmids, and also the linear DNA fragments.
Plasmids of sizes ranging from less than one kilo base (kb) to over a few hundred
kb can be easily resolved by conventional Agarose Gel Electrophoresis.
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Detection, Purification Plasmid purification is a technique used to isolate and purify plasmid DNA
and Transfer of Plasmid
DNA, and Plasmid from genomic DNA, proteins, ribosomes, and the bacterial cell wall.
Replication
A plasmid is a small, circular, double-stranded DNA that is used as a carrier
of specific DNA molecules. When introduced into a host organism via
NOTES
transformation, a plasmid will be replicated, creating numerous copies of
the DNA fragment under study.
A plasmid preparation is a method of DNA extraction and purification for
plasmid DNA. Various methods have been developed to purify plasmid
DNA from bacteria.
The isolation of plasmid DNA from bacteria is a critical technique in molecular
biology and is an essential step in many procedures, such as cloning, DNA
sequencing, transfection, and gene therapy. These manipulations require
the isolation of high purity plasmid DNA.
The purified plasmid DNA can be used for immediate use in all molecular
biology procedures, such as digestion with restriction enzymes, cloning,
PCR, transfection, in vitro translation, blotting and sequencing.
Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA
or other cell components, such as proteins by breaking the cells. Bacteria
containing the plasmid of interest is first grown, and then permitted to lyse
with an alkaline lysis buffer consisting of a detergent Sodium Dodecyl Sulfate
(SDS) and a strong base Sodium Hydroxide (NaOH).
The detergent splits the phospholipid bilayer of membrane and the alkali
denatures the proteins which are involved in maintaining the structure of the
cell membrane. This involves series of steps, such as agitation, precipitation,
centrifugation, and the removal of supernatant. The cellular debris is removed
and the plasmid is isolated and purified.
Yield and quality of plasmid DNA extremely depends on the type of culture
media being used. Most of the plasmid purification are optimized with
cultures grown in standard Luria Bertani (LB) medium.
The elution buffer volume and method is specifically adapted to the
subsequent application for achieving higher yield and / or concentration that
uses the standard method.
Fundamentally, all known plasmids are supercoiled circular DNA except
the one exceptional, i.e., the killer plasmid of Yeast, a RNA molecule. The
molecular weight ranges from 106 – 108. The number of copies of a Plasmid
Per Cell ranges from 1–2 for low copy number plasmids and to 10–60 for
high copy number plasmids.
‘F’ plasmid has an ability to integrate into the bacterial chromosome.
Principally, the ‘F’ fuses with chromosome increasing its size. The cell is
now called Hfr cell or High frequency recombination cell. When a culture
of Hfr is mixed with F– culture, the conjugation occurs.
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Replication starts in the Hfr cell and is transferred to F– cell. The direction Detection, Purification
and Transfer of Plasmid
of replication is specific, i.e., small portion of ‘F’ is transferred first and then DNA, and Plasmid
the major part is transferred. Female receives both small and large fragments Replication
of the male chromosome.
NOTES
The Hfr is produced when ‘F’ integrates firmly into the chromosome. The
plasmid containing both ‘F’ genes and chromosomal genes is called as ‘F’
plasmid.
An essential feature of bacterial plasmids is their ability to replicate as
autonomous genetic elements in a controlled way within the host. Therefore,
they are used for exploring the mechanisms involved in the DNA replication
and for analyzing the different strategies that couple DNA replication to
other critical procedures in the cell cycle.
Plasmid replication process is divided into three stages, namely initiation,
elongation, and termination. The inability of DNA polymerases to initiate de
novo replication for creating the essential independent generation of a primer.
Characteristically, the control elements maintain an average frequency of
one plasmid replication through ‘per plasmid copy per cell cycle’ and to
‘sense’ and correct deviations from this average.
The plasmid replication and its control is based on the results of analyses
performed using pure cultures under steady-state growth conditions. Thus
setting significant parameters required to understand the maintenance of
these genetic elements in mixed populations and under environmental
conditions.
Plasmids must regulate their copy number, average number of plasmid copies
per cell, to ensure that they do not excessively burden the host or become
lost during cell division.
Plasmids may be either high copy number plasmids or low copy number
plasmids; the regulation mechanisms between these two types are often
significantly different.
Biotechnology applications may involve engineering plasmids to allow a
very high copy number. For example, pBR322 is a low copy number plasmid
(~20 copies per cell) from which several very high copy number cloning
vectors (~1000 copies per cell) have been derived.
Plasmids can be incompatible if they share the same replication control
mechanism. Under these circumstances, plasmids contribute to the total
copy number and are regulated together. They are not recognized as distinct
plasmids.
One of the plasmids may be out-copied by the other and lost during cell
division, the cell is ‘cured’ of the plasmid. This is particularly likely with low
copy number plasmids. Plasmids can also be incompatible due to shared
partitioning systems.
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Detection, Purification
and Transfer of Plasmid 12.9 KEY WORDS
DNA, and Plasmid
Replication
Plasmid: A plasmid is a small DNA molecule within a cell that is physically
NOTES separated from chromosomal DNA and can replicate independently.
Plasmid size: Plasmids range in size from 1 kbp (kilo base pair) to 1000
(kilo base pair) mega-plasmids that are many hundred base pairs in size.
Plasmid copy numbers: Copy number is the significant feature and refers
to the average or expected number of copies per host cell. Plasmids have
either low, medium or high copy number.
‘F’ plasmid: It has an ability to integrate into the bacterial chromosome.
Incompatibility: Plasmids can be incompatible if they share the same
replication control mechanism, under these circumstances, plasmids
contribute to the total copy number and are regulated together.

12.10 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is plasmid DNA?
2. What is plasmid copy number?
3. Explain the significance of purification of plasmid DNA.
4. How the transfer of plasmid DNA is done?
5. What is replication of plasmid?
6. How the copy number is controlled?
7. What is plasmid incompatibility?
Long Answer Questions
1. Briefly discuss about the significance and function plasmid DNA.
2. Explain the detection and purification process of plasmid DNA giving
appropriate examples.
3. Discuss the transfer mechanism of plasmid DNA giving appropriate
examples.
4. What is replication of plasmid? Explain giving significance and examples.
5. Briefly discuss the concept control of copy number.
6. Explain plasmid curing and incompatibility process.

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 Detection, Purification
12.11 FURTHER READINGS and Transfer of Plasmid
DNA, and Plasmid
Replication
Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing NOTES
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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Transposable Elements

UNIT 13 TRANSPOSABLE
ELEMENTS
NOTES
Structure
13.0 Introduction
13.1 Objectives
13.2 Transposable Elements
13.2.1 Types of Transposable Elements
13.3 Genetic Organization and Mechanism of Transposition
13.3.1 Tn3 Transposon
13.3.2 Tn5 Transposon
13.3.3 Bacteriophage Mu
13.3.4 Insertion Sequence–Tn7 and IS911
13.3.5 Integrons and Retrotransposons
13.4 Answers to Check Your Progress Questions
13.5 Summary
13.6 Key Words
13.7 Self Assessment Questions and Exercises
13.8 Further Readings

13.0 INTRODUCTION

A Transposable Element (TE) or Transposon or Jumping Gene is a DNA sequence

that can change its position within a genome, sometimes creating or reversing
mutations and altering the cell’s genetic identity and genome size. Transposition
often results in duplication of the same genetic material. Transposable Elements or
TEs make up a large fraction of the genome and are responsible for much of the
mass of DNA in a Eukaryotic cell. Although TEs are selfish genetic elements,
many are significant in genome function and evolution. Transposons are also very
useful to researchers as a means to alter DNA inside a living organism.
There are at least two classes of TEs, namely Class I TEs or
Retrotransposons generally function via Reverse Transcription, while Class II TEs
or DNA Transposons encode the Protein Transposase, which they require for
insertion and excision, and some of these TEs also encode other Proteins. TEs,
both active and inactive, occupy approximately half the human genome and a
substantially greater fraction of some plant genomes. These movable elements are
ubiquitous in the biosphere, and are highly successful in propagating themselves.
Some TEs are also viruses, for instance, some retroviruses can integrate into a
host genome to form endogenous retroviruses. Certainly, some viruses may be
derived from natural transposable elements and vice versa. Since viruses move
between individuals, at least some transposable elements can move between
genomes (between individuals) as well as within an individual’s genome.
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 Transposition is related to replication, recombination and repair. The process Transposable Elements

of moving from one place to another involves a type of recombination, insertions

of transposable elements can cause mutations, and some transpositions are
replicative, generating a new copy while leaving the old copy intact. However, this
ability to move is a unique property of transposable elements. NOTES
Bacteriophage Mu, also known as mu phage or mu bacteriophage, is a
mulikevirus (the first of its kind to be identified) of the family Myoviridae which has
been shown to cause genetic transposition. It is of specific importance as its
discovery in Escherichia coli by Larry Taylor was among the first observations of
insertion elements in a genome. Transposons are mobile genetic elements which
can move anywhere in a genome by two mechanisms ‘copy and paste
(Retrotransposons)’, and ‘cut and paste (DNA Transposons)’ and code for many
necessary enzymes. Integrons are genetic elements able to acquire and rearrange
Open Reading Frames (ORFs) embedded in gene cassette units and convert them
to functional genes by ensuring their correct expression.
In this unit, you will study about the transposable elements, types of
transposable elements, genetic organization and mechanism of transposition of
Tn5, Tn3 and related transposons, bacteriophage Mu, Tn7 and IS911, integrons
and retrotransposons.

13.1 OBJECTIVES

After going through this unit, you will be able to:

Understand what transposable elements are
Explain the types of transposable elements
Discuss the structure, genetic organization and mechanism of transposition
of Tn5, Tn3 and related transposons
Analyse the bacteriophage Mu, Tn7 and IS911
Define integrons and retrotransposons

13.2 TRANSPOSABLE ELEMENTS

A Transposable Element (TE) or Transposon or Jumping Gene is a DNA sequence

that can change its position within a genome, sometimes creating or reversing
mutations and altering the cell’s genetic identity and genome size. Transposition
often results in duplication of the same genetic material. Transposable Elements or
TEs make up a large fraction of the genome and are responsible for much of the
mass of DNA in a Eukaryotic cell. Although TEs are selfish genetic elements,
many are important in genome function and evolution. Transposons are also very
useful to researchers as a means to alter DNA inside a living organism.

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Transposable Elements There are at least two classes of TEs, namely Class I TEs or
Retrotransposons generally function via Reverse Transcription, while Class II TEs
or DNA Transposons encode the Protein Transposase, which they require for
insertion and excision, and some of these TEs also encode other Proteins. Figure
NOTES 13.1 illustrates a bacterial DNA transposon.

Fig. 13.1 A Bacterial DNA Transposon

TEs, both active and inactive, occupy approximately half the human genome
and a substantially greater fraction of some plant genomes. These movable elements
are ubiquitous in the biosphere, and are highly successful in propagating themselves.
Some TEs are also viruses, for instance, some retroviruses can integrate into a
host genome to form endogenous retroviruses. Certainly, some viruses may be
derived from natural transposable elements and vice versa. Since viruses move
between individuals, at least some transposable elements can move between
genomes (between individuals) as well as within an individual’s genome.
Transposition is related to replication, recombination and repair. The process
of moving from one place to another involves a type of recombination, insertions
of transposable elements can cause mutations, and some transpositions are
replicative, generating a new copy while leaving the old copy intact. However, this
ability to move is a unique property of transposable elements.
Properties and Effects of Transposable Elements or TEs
The defining property of transposable elements is their mobility, i.e., they are genetic
elements that can move from one position to another in the genome. In addition to
the common property of mobility, transposable elements show considerable
diversity. Some move by DNA intermediates, and others move by RNA
intermediates. Much of the mechanism of transposition is distinctive for these two
classes, but all transposable elements effectively insert at staggered breaks in
chromosomes. Some transposable elements move in a replicative manner, whereas
others are non-replicative, i.e., they move without making a copy of themselves.
Transposable elements are major forces in the evolution and rearrangement
of genomes (Refer Figure 13.2). Some transposition events inactivate genes, since
the coding potential or expression of a gene is disrupted by insertion of the
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transposable element. A classic example is the ‘r allele’ (Rugosus) of the gene Transposable Elements

encoding a starch branching enzyme in peas is nonfunctional due to the insertion of

a transposable element. This allele causes the wrinkled pea phenotype in
homozygotes originally studied by Mendel. In other cases, transposition can activate
nearby genes by bringing an enhancer of transcription (within the transposable NOTES
element) adequately close to a gene to stimulate its expression. If the target gene is
not usually expressed in a certain cell type, then this activation can lead to pathology,
such as activation of a proto-oncogene causing a cell to become cancerous. In
other cases, no obvious phenotype results from the transposition. A particular
type of transposable element can activate, inactivate or have no effect on nearby
genes, depending on exactly where it inserts, its orientation and other factors.

Fig. 13.2 Possible Effects of Movement of a Transposable Element

in the Expression of the Target Gene

In Figure 13.2, the transposable element is shown as rectangle and the

Target Gene (X) is composed of multiple exons. The angled arrow indicates the
start site for transcription.
Transposable elements can cause deletions or inversions of DNA. When
transposition generates two copies of the same sequence in the same orientation,
recombination can delete the DNA between them. If the two copies are in the
opposite orientations, recombination will invert the DNA between them.
As part of the mechanism of transposition, additional DNA sequences can
be mobilized. DNA located between two copies of a transposable element can be
moved together with them when they move. In this manner, transposition can move
DNA sequences that are not normally part of a transposable element to new
locations. Certainly, the ‘host’ sequences can be acquired by viruses and propagated
by infection of other individuals. This may be a natural means for evolving new
strains of viruses. One of the most striking examples is the acquisition and
modification of a proto-oncogene, such as cellular c-src, by a retrovirus to generate
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Transposable Elements a modified, transforming form of the gene, called v-src. These and related
observations provided insights into the progression of events that turn a normal
cell into a cancerous one. They also point to the continual acquisition and possibly
deletion of information from host genomes as a natural part of the evolution of
NOTES viruses.
13.2.1 Types of Transposable Elements
Transposable Elements or TEs represent one of several types of mobile genetic
elements. TEs are assigned to one of two classes according to their mechanism of
transposition, which can be described as either copy and paste ‘Class I TEs’ or
cut and paste ‘Class II TEs’.
Retrotransposon
Class I TEs are copied in two stages. First, they are transcribed from DNA to
RNA, and the RNA produced is then reverse transcribed to DNA. This copied
DNA is then inserted back into the genome at a new position. The reverse
transcription step is catalyzed by a reverse transcriptase, which is often encoded
by the TE itself. The characteristics of retrotransposons are similar to retroviruses,
such as HIV (Human Immunodeficiency Virus).
Retrotransposons are commonly grouped into three main orders as follows:
• Retrotransposons, with Long Terminal Repeats (LTRs), which encode
reverse transcriptase, similar to retroviruses.
• Retroposons, Long Interspersed Nuclear Elements (LINEs, LINE-1s,
or L1s), which encode reverse transcriptase but lack LTRs, and are
transcribed by RNA Polymerase II.
• Short Interspersed Nuclear Elements (SINEs) do not encode reverse
transcriptase and are transcribed by RNA Polymerase III.
Retroviruses can also be considered TEs. For example, after conversion of
Retroviral RNA into DNA inside a host cell, the newly produced Retroviral DNA
is integrated into the genome of the host cell. These integrated DNAs are termed
proviruses. The provirus is a specialized form of Eukaryotic Retrotransposon,
which can produce RNA intermediates that may leave the host cell and infect
other cells. The transposition cycle of retroviruses has similarities to that of
prokaryotic TEs, suggesting a distant relationship between the two.
DNA Transposons
The ‘Cut and Paste’ transposition mechanism of Class II TEs does not involve an
RNA intermediate. The transpositions are catalyzed by several transposase
enzymes. Some transposases non-specifically bind to any target site in DNA,
whereas others bind to specific target sequences. The transposase makes a
staggered cut at the target site producing sticky ends, cuts out the DNA transposon
and ligates it into the target site. A DNA polymerase fills in the resulting gaps from
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the sticky ends and DNA ligase closes the Sugar-Phosphate backbone. This results Transposable Elements

in target site duplication and the insertion sites of DNA transposons may be identified
by short direct repeats (a staggered cut in the target DNA filled by DNA polymerase)
followed by inverted repeats (which are important for the TE excision by
transposase). NOTES
The ‘Cut and Paste TEs’ may be duplicated if their transposition takes
place during S phase of the cell cycle, when a donor site has already been replicated
but a target site has not yet been replicated. Such duplications at the target site can
result in gene duplication, which plays an important role in genomic evolution.
Not all DNA transposons transpose through the cut and paste mechanism.
In some situations, a replicative transposition is observed in which a transposon
replicates itself to a new target site, for example Helitron.
Class II TEs comprise less than 2% of the human genome, making the rest
Class I.
Figure 13.3 illustrates the structure of DNA transposons and mechanism of
transposition. Figure 13.3 (A) illustrates the structure of DNA transposons (Mariner
type), the two Tandem Inverted Repeats (TIR) flank the transposase gene. Two
short Tandem Site Duplications (TSD) are present on both sides of the insert,
while the Figure 13.3 (B) illustrates the mechanism of transposition in which two
transposases recognize and bind to TIR sequences, join together and promote
DNA double-strand cleavage. The DNA-transposase complex then inserts its
DNA cargo at specific DNA motifs elsewhere in the genome, creating short TSDs
upon integration.

Fig. 13.3 (A) Structure of DNA Transposons (Mariner Type), (B) Mechanism of
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Transposable Elements Autonomous and Non-Autonomous
Transposition can be classified as either ‘Autonomous’ or ‘Non-Autonomous’ in
both Class I and Class II TEs. Autonomous TEs can move by themselves, whereas
NOTES non-autonomous TEs require the presence of another TE to move. This is often
because dependent TEs lack transposase for Class II or reverse transcriptase for
Class I.
Activator (Ac) element is an example of an autonomous TE, and Dissociation
(Ds) elements is an example of a non-autonomous TE. Without Ac, Ds is not able
to transpose.
Instances of TEs
• The first TEs were discovered in maize (Zea mays) by Barbara McClintock
in 1948, for which she was later awarded a Nobel Prize. She noticed
chromosomal insertions, deletions, and translocations caused by these
elements. These changes in the genome could, for example, lead to a change
in the color of corn kernels. About 85% of the maize genome consists of
TEs. The Ac/Ds system described by McClintock are Class II TEs.
Transposition of Ac in Tobacco has been demonstrated by B. Baker.
• In the pond microorganisms, Oxytricha, TEs play a critical role that when
removed, the organism fails to develop.
• One family of TEs in the fruit fly Drosophila melanogaster are called P
elements. They seem to have first appeared in the species only in the middle
of the twentieth century; within the last 50 years, they spread through every
population of the species.
• Transposons in bacteria usually carry an additional gene for functions other
than transposition, often for antibiotic resistance. In bacteria, transposons
can jump from chromosomal DNA to plasmid DNA and back, allowing for
the transfer and permanent addition of genes such as those encoding antibiotic
resistance. Bacterial transposons of this type belong to the ‘Tn’ family. When
the transposable elements lack additional genes, they are known as insertion
sequences.
• The most common transposable element in humans is the ‘Alu’ sequence. It
is approximately 300 bases long and can be found between 300,000 and
one million times in the human genome. Alu alone is estimated to make up
15–17% of the human genome.
• Mariner like elements are another prominent class of transposons found in
multiple species, including humans. The Mariner transposon was first
discovered by Jacobson and Hartl in Drosophila melanogaster. This Class
II TE is known for its uncanny ability to be transmitted horizontally in many
species. There are an estimated 14,000 copies of Mariner in the human
genome comprising 2.6 million base pairs. The first Mariner element
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272 Material
 • Mu phage transposition is the best-known example of replicative Transposable Elements

transposition.
• Yeast, Saccharomyces cerevisiae, genomes contain five distinct
retrotransposon families, namely Ty1, Ty2, Ty3, Ty4 and Ty5.
NOTES
• A Helitron is a TE found in Eukaryotes that is thought to replicate by a
rolling-circle mechanism.
• In human embryos, two types of transposons combined to form noncoding
RNA that catalyzes the development of stem cells. During the early stages
of a fetus’s growth, the embryo’s inner cell mass expands as these stem
cells enumerate. The increase of this type of cells is crucial, since stem cells
later change form and give rise to all the cells in the body.
• In peppered moths, a transposon in a gene called cortex caused the moths’
wings to turn completely black. This change in coloration helped moths to
blend in with ash and soot-covered areas during the Industrial Revolution.
Genetic Diseases and TEs
TEs are mutagens and their movements are often the causes of genetic diseases.
They can damage the genome of their host cell in different ways, such as:
• A transposon or a retrotransposon that inserts itself into a functional gene
will most likely disable that gene.
• After a DNA transposon leaves a gene, the resulting gap will probably not
be repaired correctly.
• Multiple copies of the same sequence, such as Alu sequences, can hinder
precise chromosomal pairing during mitosis and meiosis, resulting in unequal
crossovers, one of the main reasons for chromosome duplication.
Diseases often caused by TEs include Hemophilia A and B, severe combined
immunodeficiency, Porphyria, predisposition to Cancer, and Duchenne muscular
dystrophy. LINE1 (L1) TEs that reside on the human Factor VIII have been
shown to cause Haemophilia and insertion of L1 into the APC gene causes Colon
Cancer, confirming that TEs play an important role in disease development.
Transposable element dysregulation can cause neuronal death in Alzheimer’s disease
and similar tauopathies. Additionally, many TEs contain promoters which drive
transcription of their own transposase. These promoters can cause aberrant
expression of linked genes, causing disease or mutant phenotypes.
Rate of Transposition, Induction and Defense
One study estimated the rate of transposition of a particular retrotransposon, the
Ty1 element in Saccharomyces cerevisiae. Using several assumptions, the rate of
successful transposition event per single Ty1 element came out to be about once
every few months to once every few years. Some TEs contain heat-shock like
promoters and their rate of transposition increases if the cell is subjected to stress,
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Transposable Elements thus increasing the mutation rate under these conditions, which might be beneficial
to the cell.
Cells defend against the proliferation of TEs in a number of ways. These
include piRNAs and siRNAs, which silence TEs after they have been transcribed.
NOTES
If organisms are mostly composed of TEs, one might assume that disease
caused by misplaced TEs is very common, but in most cases TEs are silenced
through epigenetic mechanisms like DNA methylation, chromatin remodeling and
piRNA, such that little to no phenotypic effects nor movements of TEs occur as in
some wild-type plant TEs. Certain mutated plants have been found to have defects
in methylation-related enzymes, Methyl Transferase, which cause the transcription
of TEs, thus affecting the phenotype.
One hypothesis suggests that only approximately 100 LINE1 related
sequences are active, despite their sequences making up 17% of the human
genome. In human cells, silencing of LINE1 sequences is triggered by an RNA
interference (RNAi) mechanism. Surprisingly, the RNAi sequences are derived
from the 5' UnTranslated Region (UTR) of the LINE1, a long terminal which
repeats itself. Supposedly, the 5' LINE1 UTR that codes for the sense promoter
for LINE1 transcription also encodes the antisense promoter for the miRNA that
becomes the substrate for siRNA production. Inhibition of the RNAi silencing
mechanism in this region showed an increase in LINE1 transcription.
Evolution of TEs
TEs are found in almost all life forms, and the scientific community is still exploring
their evolution and their effect on genome evolution. It is unclear whether TEs
originated in the last universal common ancestor, arose independently multiple
times, or arose once and then spread to other kingdoms by horizontal gene transfer.
While some TEs confer benefits on their hosts, most are regarded as selfish DNA
parasites. In this way, they are similar to viruses. Various viruses and TEs also
share features in their genome structures and biochemical abilities, leading to
speculation that they share a common ancestor.
Because excessive TE activity can damage exons, many organisms have
acquired mechanisms to inhibit their activity. Bacteria may undergo high rates of
gene deletion as part of a mechanism to remove TEs and viruses from their genomes,
while Eukaryotic organisms typically use RNA interference to inhibit TE activity.
Nevertheless, some TEs generate large families often associated with speciation
events. Evolution often deactivates DNA transposons, leaving them as introns
(Inactive Gene Sequences). In vertebrate animal cells, nearly all 100,000 + DNA
Transposons Per Genome have genes that encode Inactive Transposase
Polypeptides.
The first synthetic transposon designed for use in vertebrate (including human)
cells, the ‘Sleeping Beauty Transposon System’, is a ‘Tc1/Mariner like
Transposon’. Its dead or ‘fossil’ versions are spread widely in the Salmoid genome
Self-Instructional and a functional version was engineered by comparing those versions. The Sleeping
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Beauty Transposon System has been used extensively as an insertional tag for Transposable Elements

identifying Cancer Genes. Human Tc1-like transposons are divided into ‘Hsmar1’
and ‘Hsmar2’ subfamilies. Although both types are inactive, one copy of Hsmar1
found in the SETMAR gene is under selection as it provides DNA-binding for the
histone-modifying protein. Many other human genes are similarly derived from NOTES
transposons. Hsmar2 has been reconstructed multiple times from the fossil
sequences.
Interspersed repeats within genomes are created by transposition events
accumulating over evolutionary time. Because interspersed repeats block gene
conversion, they protect novel gene sequences from being overwritten by similar
gene sequences and thereby facilitate the development of new genes. TEs may
also have been co-opted by the vertebrate immune system as a means of producing
antibody diversity. The V(D)J recombination system operates by a mechanism
similar to that of some TEs.
TEs can contain many types of genes, including those conferring antibiotic
resistance and ability to transpose to conjugative plasmids. Some TEs also contain
integrons, genetic elements that can capture and express genes from other sources.
These contain integrase, which can integrate gene cassettes. There are over 40
antibiotic resistance genes identified on cassettes, as well as virulence genes.
Transposons do not always excise their elements precisely, sometimes
removing the adjacent base pairs, this phenomenon is called ‘exon shuffling’.
Shuffling two unrelated exons can create a novel gene product or, more likely, an
intron.

13.3 GENETIC ORGANIZATION AND MECHANISM

OF TRANSPOSITION

The phenomenon of moving genetic segments from one location to the other in a
genome is known as transposition. There are two types of transposition, replicative
and conservative transposition. The replicative transposition involves the events of
both replication and recombination processes generating the two daughter copies
of the original transposable elements, one remaining at the parental site and the
other at the target site.
In addition, the conservative transposition does not involve replication. Simply
the elements move to a new site. When the target site is present within a gene both
types of transposition takes place. The frequency of transposition varies among
different elements.
The overall rate of transposition is 105 -104 per element per generation.
Different IS elements contain different number of bases.
The bacterial transposon Tn3 has been extensively studied. Analysis of DNA
sequences and its junction with target DNA provides some clue to the mechanism
of transposition. Self-Instructional
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Transposable Elements Movement of transposons occurs only when the enzyme transposase
recognises and cleaves at either 52 or 32 of both ends of transposon, and catalyses
at either 52 or 32 of both ends of transposon and catalyses a staggered cut at the
target site. Depending on transposon, a duplication of 3-12 bases of target DNA
NOTES occurs at the site where insertion is to be done. One copy remains at each end of
the transposon sequence.
Types of Transposable Elements
The maize Ac transposable element is only one of several types found in nature.
Transposable elements can be divided into two major classes based on method of
transposition:
Retrotransposons (Class 1)
Use reverse transposase to make RNA intermediate for transposition.
Encode an integrase and reverse transcriptase for transposition.
Found in viruses.
Transposons (Class 2)
DNA fragments transpose directly from DNA segment to DNA segment as follows:
Producing a DNA copy that transposes (Replicative Transposition).
Cut/paste into a new locus (Conservative Transposition).
The features include:
Encode a transposase for transposition.
Can carry additional genes.
Found in Eukaryotes and Prokaryotes.
Transposase and integrase proteins carry a ribonuclease-like catalytic domain and
can use the same target site to catalyse both DNA cleavage and DNA strand
transfer. However, transposases and integrases are only active when assembled
into a synaptic complex (transpososome) on the DNA. The transpososome
provides a scaffold to support the transposition reactions, changing its conformation
to accommodate the different steps in transposition.
Transposase Families
Five families have been classified till now, although this number will most likely
grow as new transposases are characterised. These families use distinct catalytic
mechanisms for break/rejoining of DNA. For example, some transposases cut/
transfer/paste original DNA, while others copy DNA into the target site.

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These families include the following: Transposable Elements

DDE Transposases: These transposases carry a triad of conserved amino acids,

namely aspartate (D) and glutamate (E), which are required for the coordination
of a metal ion required for catalysis, although the DDE chemistry can be integrated
NOTES
into the transposition cycle in differing ways. These employ a cut and paste
mechanism of the original transposon. This family includes the maize Ac transposon,
as well as the Drosophila P element, Bacteriophage Mu, Tn5 and Tn10, Mariner,
IS10, and IS50.
Tyrosine (Y) Transposases: These also use a cut and paste mechanism of
transposition, but employ a site-specific tyrosine residue. The transposon is excised
from its original site (which is repaired); the transposon then forms a closed circle
of DNA, which is integrated into a new site by a reversal of the original excision
step. These transposons are usually found only in Bacteria, and include Kangaroo,
Tn916, and DIRS1.
Serine (S) Transposases: These transposases use a cut and paste (cut out/
paste in) mechanism of transposition involving a circular DNA intermediate, which
is similar to that of tyrosine transposases, only they employ a site-specific serine
residue. These transposons are usually found only in Bacteria, and include Tn5397
and IS607.
Rolling Circle (RC) or Y2 Transposases: These employ either a copy in
mechanism, where they copy a single strand directly into the target site by DNA
replication, so that the old (template) and new (copied) transposons both have
one newly synthesized strand. These transposons usually employ host DNA
replication enzymes. Examples include IS91 and Helitrons.
Reverse Transcriptases/Endonucleaseses (RT/En): Retrotransposons can
vary in their mechanism of transposition. Some use the RT/En method, employing
an endonuclease to nick the target site DNA, the nick serving as a primer for
reverse transcription of an RNA copy by the reverse transcriptase enzyme.
Examples include LINE-1 and TP-Retrotransposons.
13.3.1 Tn3 Transposon
The Tn3 transposon is a 4957 base pair mobile genetic element, found in
prokaryotes. It encodes following three proteins:
• -Lactamase, an enzyme that confers resistance to -Lactam Antibiotics
and is encoded by the gene Bla.
• Tn3 Transposase encoded by Gene tnpA.
• Tn3 Resolvase encoded by Gene tnpR.
Initially discovered as a repressor of transposase, resolvase also plays a
significant role in facilitating Tn3 replication (Sherratt 1989). The transposon
is flanked by a pair of 38 bp inverted repeats.

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Transposable Elements Mechanism of Replication
The mechanism of replication involves the following steps.
Step 1 – Replicative Integration
NOTES
This first stage is catalysed by transposase.
The plasmid containing the transposon (the donor plasmid) fuses with a
host plasmid (the target plasmid). In the process, the transposon and a short section
of host DNA are replicated. The end product is a ‘cointegrate’ plasmid containing
two copies of the transposon.
Shapiro (1978) proposed the following mechanism for this process:
1. Four single-strand cleavages occur – one on each strand of the donor
plasmid and one on each strand of the target plasmid.
2. The donor and target plasmids are ligated together, but there are two
single-stranded regions, due to the positioning of the original cleavages.
3. DNA replication makes the single-stranded regions double stranded,
using the existing strand as a template. It is in this stage that the transposon
is replicated.
Figure 13.4 illustrates the replicative integration process.

Fig. 13.4 Replicative Integration

Step 2 – Resolution
To separate the host and target molecules Tn3 resolvase executes site-specific
recombination between the old and new copy of transposon at a specific site
called ‘Res’, which is present in each copy of the transposon. Res is 114 bp long
and it consists of 3 sub-sites, namely Site I, Site II and Site III. Each of these sites
is of different lengths (28, 34 and 25 bp, respectively) and they are unevenly
spaced with 22 bp separating Site I and Site II and only 5 bp between Site II and
Site III. The sites consist of 6 bp inverted repeat motifs flanking a central sequence
of variable length. These motifs act as binding sites for resolvase, so that each site
binds a resolvase dimer but with varying affinity and probably a slightly different
protein-DNA complex architecture. All three sub-sites are essential for
recombination.
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 At recombination, two directly repeated res sites with resolvase dimers Transposable Elements

bound to each sub-site, come together to form a large complex structure called
the Synaptosome. Resolvase bound to Site II and Site III initiates the assembly of
this complex. In this structure, exact architecture of which is still unclear, two Res
sites are intertwined in such a way as to juxtapose two copies of Site I, allowing NOTES
resolvase dimers bound to each site to form a tetramer. Again, it is the interaction
between the resolvase dimers bound at accessory sites (Site II and Site III) and
resolvase at Site I that causes the two dimers to synapse and form a tetramer.
After the tetramer is formed it becomes activated and the top and bottom DNA
strands are simultaneously cleaved in the middle of the Site I with a 2 bp overhang.
The strand exchange ensues by as yet unknown mechanism with a resulting net
rotation of 180°.
The strand exchange is then followed by the religation (Stark et al., 1992).
Recombination between two directly repeated Res sites separates, or resolves,
the ‘cointegrate’ into two original molecules, each one now containing a copy of
the ‘Tn3 transposon’.
After resolution these two molecules remain linked as a simple two-node
catenane which can be easily separated in vivo by a Type II topoisomerase (Grindley
2002). Wild type resolvase system absolutely requires a supercoiled substrate
and that the recombination sites are oriented in a direct repeat on the same DNA
molecule. However, a number of ‘deregulated’ or ‘hyperactive’ mutants that have
lost the requirement for the accessory sites have been isolated. These mutants are
capable of catalysing recombination between two copies of Site I only, which
basically reduces the recombination site size from 114 bp to only 28 bp.
Furthermore, these mutants have no supercoiling or connectivity requirements
(Arnold et al., 1999) and have been shown to work in mammalian cells.
Hyperactive resolvase mutants have so far proven useful in creating resolvases
with altered sequence specificity but also in structural work.
The entire resolvase recombination reaction can be reproduced in vitro,
requiring only resolvase, a substrate DNA and multivalent cations, using either
wild type protein or hyperactive mutants.
Hyperactive resolvase mutants, if further developed, could become an
alternative to Cre and FLP, the most commonly used recombination systems in
molecular biology to date.
13.3.2 Tn5 Transposon
Tn5 was one of the first transposons to be identified. As a result of Tn5’s early
discovery and its simple macromolecular requirements for transposition, the Tn5
system has been a very productive tool for studying the molecular mechanism of
DNA transposition. These studies are of broad value because they offer insights
into DNA transposition in general, because DNA transposition is a useful model
with which to understand other types of Protein-DNA interactions, such as retroviral
DNA integration and the DNA cleavage events involved in immunoglobulin gene Self-Instructional
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Transposable Elements formation, and because Tn5 derived tools are useful adjuncts in genetic
experimentation.
Transposons are a class of genetic elements that can move from one site in
a cell’s genome to another independently of the cell’s general recombination system.
NOTES
Little is known about the mechanism of transposition of compound transposons
such as Tn5, but it is thought that a transposon-encoded protein (a transposase)
must recognize the outer ends of the element and, together with host factors,
catalyse the transfer of the internal DNA into a new site in a manner that may
involve replication. It has previously been shown that the synthesis of an IS50R-
encoded Protein (Protein 1) is an essential requirement for Tn5 transposition.
Here we demonstrate that a structure containing only the outer 186 base pairs
(bp) of both inverted repeats is capable of being efficiently complemented to
transpose in Escherichia coli, provided IS50R is located close by on the same
replicon. In addition, Bal31-generated deletions indicate that 16-18 bp of the
outer end of IS50L are required for transposition. This 16-18 bp sequence contains
the 8-9 bp small inverted repeat present at each end of IS50 plus a 9 bp sequence
which is homologous to an interrelated sequence present in four copies in the
chromosomal origin of replication in a variety of Gram-Negative Bacteria. This
sequence organization suggests that the ends of Tn5 may function to provide a
recognition site for the Tn5 transposase adjacent to a sequence recognized by the
host replication system.
The bacterial transposon Tn5 encodes two proteins, the transposase and a
related protein, the transposition inhibitor, whose relative abundance determines,
in part, the frequency of Tn5 transposition. The synthesis of these proteins is
programmed by a complex set of genetic regulatory elements. The host DNA
methylation function, dam, inhibits transposase promoter recognition and indirectly
enhances the transposition inhibitor promoter. The inhibitor lacks the N-terminal
55 amino acids of the transposase, suggesting that this sequence plays a key role
in the transposition process. An intact N-terminal sequence is required for the
transposase’s recognition of the 19 bp end DNA sequences. This is the first critical
step in the transposition process. Transposase-end DNA interaction is itself
regulated by an intricate series of reactions involving several host proteins, such as
DnaA, Dam, and Fis. The transposase is a unique protein in that it acts primarily in
cis and inhibits its own activity in trans. Models to explain these properties are
described. Finally circumstantial evidence suggests that transposition occurs
preferentially from newly replicated DNA that has yet to be partitioned to progeny
cells.
13.3.3 Bacteriophage Mu
Transposable phage Mu has played a historic role in the development of the mobile
DNA element field.
Bacteriophage Mu, also known as mu phage or mu bacteriophage, is a
mulikevirus (the first of its kind to be identified) of the family Myoviridae which
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has been shown to cause genetic transposition. It is of particular importance as its Transposable Elements

discovery in Escherichia coli by Larry Taylor was among the first observations of
insertion elements in a genome. This discovery opened up the world to an
investigation of transposable elements and their effects on a wide variety of
organisms. While Mu was specifically involved in several distinct areas of research NOTES
including Escherichia coli, maize, and HIV, the wider implications of transposition
and insertion transformed the entire field of genetics.
Phage Mu is nonenveloped, with a head and a tail. The head has an
icosahedral structure of about 54 nm in width. The neck is knob-like, and the tail
is contractile with a base plate and six short terminal fibers. The genome has been
fully sequenced and consists of 36,717 nucleotides, coding for 55 proteins.
Mu phage was first discovered Larry Taylor at UC Berkeley in the late
1950s. His work continued at Brookhaven National Laboratory, where he first
observed the mutagenic properties of Mu; several colonies of Hfr Escherichia
coli which had been lysogenized with Mu seemed to have a tendency to develop
new nutritional markers. With further investigation, he was able to link the presence
of these markers to the physical binding of Mu at a certain loci. He likened the
observed genetic alteration to the ‘controlling elements’ in maize, and named the
phage ‘Mu’, for mutation. This, however, was only the beginning. Over the next
sixty years, the complexities of the phage were fleshed out by numerous researchers
and labs, resulting in a far deeper understanding of mobile DNA and the mechanisms
underlying transposable elements.
Some genetic elements, for example bacteriophage Mu, combine the
properties of both viruses and transposons.
Bacteriophage Mu represents a hybrid gene creature that exists as both a
transposable element and a bacteriophage, a virus that infects bacteria. Mu
integrates into its Escherichia coli host by transposition. Mu may exist as a prophage
or enter into the lytic cycle. Mu undergoes replicative transposition when it replicates
and produces many copies of itself that ultimately destroy the host. However, Mu
does have a mechanism in place to ensure that it does not integrate into its own
genome and destroy itself. This is called transposition immunity and relies on the
binding of specific Mu Proteins.
13.3.4 Insertion Sequence–Tn7 and IS911
Insertion element, also known as an IS, an Insertion Sequence element, or an IS
element, is a short DNA sequence that acts as a simple Transposable Element
(TE). Insertion sequences have two major characteristics: they are small relative
to other transposable elements, generally around 700 to 2500 bp in length, and
only code for proteins implicated in the transposition activity. They are thus different
from other transposons, which also carry accessory genes, such as antibiotic
resistance genes. These proteins are usually the transposase which catalyses the
enzymatic reaction allowing the IS to move, and also one regulatory protein which
either stimulates or inhibits the transposition activity. The coding region in an insertion Self-Instructional
Material 281
Transposable Elements sequence is usually flanked by inverted repeats. For example, the well-known
IS911 (1250 bp) is flanked by two 36 bp inverted repeat extremities and the
coding region has two genes partially overlapping orfA and orfAB, coding the
transposase (OrfAB) and a regulatory protein (OrfA). A particular insertion
NOTES sequence may be named according to the form ISn, where n is a number, for
example IS1, IS2, IS3, IS10, IS50, IS911, IS26, etc. Although insertion
sequences are usually considered in the context of Prokaryotic genomes, but certain
Eukaryotic DNA sequences belonging to the family of Tc1/Mariner transposable
elements may be considered to be the insertion sequences.
In addition to occurring autonomously, insertion sequences may also occur
as parts of composite transposons, in a composite transposon, two insertion
sequences flank one or more accessory genes, such as an antibiotic resistance
gene, such as Tn10 and Tn5. Nevertheless, there exist another sort of transposons,
called unit transposons that do not carry insertion sequences at their extremities,
such as Tn7.
A complex transposon does not rely on flanking insertion sequences for
resolvase. The resolvase is part of the ‘Tn’ genome and cuts at flanking inverted
repeats.
Transposition frequency of IS elements is dependent of multiple parameters,
including culture growth phase, medium composition, oxygen tension, growth scale,
and structural conformation of target sites, for example curvature, presence of
certain motifs, DNA composition.
13.3.5 Integrons and Retrotransposons
Integrons are genetic mechanisms that allow bacteria to adapt and evolve rapidly
through the stockpiling and expression of new genes. These genes are embedded
in a specific genetic structure called gene cassette (a term that is lately changing to
integron cassette) that generally carries one promoterless ORF together with a
recombination site (attC). Integron cassettes are incorporated to the attI site of
the integron platform by site-specific recombination reactions mediated by the
integrase.
Integrons were initially discovered on conjugative plasmids through their
role in antibiotic resistance. Indeed, these mobile integrons, as they are now known,
can carry a variety of cassettes containing genes that are almost exclusively related
to antibiotic resistance. Further studies have come to the conclusion that integrons
are chromosomal elements, and that their mobilisation onto plasmids has been
fostered by transposons and selected by the intensive use of antibiotics. The function
of the majority of cassettes found in chromosomal integrons remains unknown.
An integron is minimally composed of:
Gene Encoding for a Site-Specific Recombinase: intI, belonging to the integrase
family.

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Proximal Recombination Site: attI, which is recognized by the integrase and at Transposable Elements

which gene cassettes may be inserted.

Promoter: Pc, which directs transcription of cassette-encoded genes.
Gene Cassettes: Additionally, an integron will usually contain one or more gene NOTES
cassettes that have been incorporated into it. The gene cassettes may encode
genes for antibiotic resistance, although most genes in integrons are uncharacterized.
An attC sequence (also called 59-be) is a repeat that flanks cassettes and enables
cassettes to be integrated at the attI site, excised and undergo horizontal gene
transfer.
Occurrence: Integrons may be found as part of mobile genetic elements, such as
plasmids and transposons. Integrons can also be found in chromosomes.
Retrotransposons
Retrotransposons, also called Class I transposable elements or transposons via
RNA intermediates, are genetic elements that can amplify themselves in a genome
and are ubiquitous components of the DNA of many Eukaryotic organisms. These
DNA sequences use a ‘copy and paste’ mechanism, whereby they are first
transcribed into RNA, then converted back into identical DNA sequences using
reverse transcription, and these sequences are then inserted into the genome at
target sites.
Retrotransposons form one of the two subclasses of transposons, where
the others are DNA transposons, which does not involve an RNA intermediate.
Retrotransposons are particularly abundant in plants, where they are often
a principal component of nuclear DNA. In maize, 49–78% of the genome is made
up of retrotransposons. In wheat, about 90% of the genome consists of repeated
sequences and 68% of transposable elements. In mammals, almost half the genome
(45% to 48%) is transposons or remnants of transposons. Around 42% of the
human genome is made up of retrotransposons, while DNA transposons account
for about 2–3%.
Types: Retrotransposons, also known as Class I transposable elements,
consist of two subclasses, the Long Terminal Repeat (LTR-retrotransposons) and
the non-LTR retrotransposons. Classification into these subclasses is based on
the phylogeny of the reverse transcriptase, which goes in line with structural
differences, such as presence/absence of long terminal repeats as well as number
and types of open reading frames, encoding domains and target site duplication
lengths. LTR Retrotransposons have direct LTRs that range from ~100 bp to over
5 kb in size. In plant genomes, LTR retrotransposons are the major repetitive
sequence class, such as able to constitute more than 75% of the maize genome.
The Non-LTR Retrotransposons consist of two sub-types, long interspersed
elements (LINEs) and short interspersed elements (SINEs). They can also be
found in high copy numbers, as shown in the plant species. Non-Long Terminal
Repeat (LTR) retroposons are widespread in Eukaryotic genomes. LINEs possess
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Transposable Elements two ORFs, which encode all the functions needed for retrotransposition. These
functions include reverse transcriptase and endonuclease activities, in addition to
a nucleic acid-binding property needed to form a ribonucleoprotein particle. SINEs,
on the other hand, co-opt the LINE machinery and function as nonautonomous
NOTES retroelements. While historically viewed as ‘junk DNA’.

Check Your Progress

1. Explain the term transposable element.
2. What are the classes of transposable element?
3. Explain the defining property of transposable elements.
4. What is transposition? Explain giving example.
5. What is Tn3 transposon? Which three proteins it encodes?
6. Explain insertion sequence element giving examples.

13.4 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. A Transposable Element (TE) or Transposon or Jumping Gene is a DNA

sequence that can change its position within a genome, sometimes creating
or reversing mutations and altering the cell’s genetic identity and genome
size. Transposition often results in duplication of the same genetic material.
Transposable Elements or TEs make up a large fraction of the genome and
are responsible for much of the mass of DNA in a Eukaryotic cell. Although
TEs are selfish genetic elements, many are important in genome function
and evolution. Transposons are also very useful to researchers as a means
to alter DNA inside a living organism.
2. There are at least two classes of TEs, namely Class I TEs or
Retrotransposons generally function via Reverse Transcription, while Class
II TEs or DNA Transposons encode the Protein Transposase, which they
require for insertion and excision, and some of these TEs also encode other
Proteins. Transposable Elements or TEs represent one of several types of
mobile genetic elements. TEs are assigned to one of two classes according
to their mechanism of transposition, which can be described as either copy
and paste ‘Class I TEs’ or cut and paste ‘Class II TEs’.
3. The defining property of transposable elements is their mobility, i.e., they
are genetic elements that can move from one position to another in the
genome. In addition to the common property of mobility, transposable
elements show considerable diversity. Some move by DNA intermediates,
and others move by RNA intermediates.

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 4. The phenomenon of moving genetic segments from one location to the other Transposable Elements

in a genome is known as transposition. There are two types of transposition,

replicative and conservative transposition. The replicative transposition
involves the events of both replication and recombination processes
generating the two daughter copies of the original transposable elements, NOTES
one remaining at the parental site and the other at the target site. In addition,
the conservative transposition does not involve replication. Simply the
elements move to a new site. When the target site is present within a gene
both types of transposition takes place. The frequency of transposition varies
among different elements. The overall rate of transposition is 105 -104 per
element per generation. Different IS elements contain different number of
bases. The bacterial transposon Tn3 has been extensively studied. Analysis
of DNA sequences and its junction with target DNA provides some clue to
the mechanism of transposition.
5. The Tn3 transposon is a 4957 base pair mobile genetic element, found in
prokaryotes. It encodes following three proteins:
• -Lactamase, an enzyme that confers resistance to -Lactam Antibiotics
and is encoded by the gene Bla.
• Tn3 Transposase encoded by Gene tnpA.
• Tn3 Resolvase encoded by Gene tnpR.
6. Insertion element, also known as an IS, an Insertion Sequence element, or
an IS element, is a short DNA sequence that acts as a simple Transposable
Element (TE). Insertion sequences have two major characteristics: they are
small relative to other transposable elements, generally around 700 to 2500
bp in length, and only code for proteins implicated in the transposition activity.
A particular insertion sequence may be named according to the form ISn,
where n is a number, for example IS1, IS2, IS3, IS10, IS50, IS911, IS26,
etc. There exist another sort of transposons, called unit transposons that do
not carry insertion sequences at their extremities, such as Tn7.

13.5 SUMMARY

A Transposable Element (TE) or Transposon or Jumping Gene is a DNA

sequence that can change its position within a genome, sometimes creating
or reversing mutations and altering the cell’s genetic identity and genome
size.
Transposition often results in duplication of the same genetic material.
Transposable Elements or TEs make up a large fraction of the genome and
are responsible for much of the mass of DNA in a Eukaryotic cell.
Although TEs are selfish genetic elements, many are important in genome
function and evolution. Transposons are also very useful to researchers as a
means to alter DNA inside a living organism. Self-Instructional
Material 285
Transposable Elements There are at least two classes of TEs, namely Class I TEs or
Retrotransposons generally function via Reverse Transcription, while Class
II TEs or DNA Transposons encode the Protein Transposase, which they
require for insertion and excision, and some of these TEs also encode other
NOTES Proteins.
Transposition is related to replication, recombination and repair. The process
of moving from one place to another involves a type of recombination,
insertions of transposable elements can cause mutations, and some
transpositions are replicative, generating a new copy while leaving the old
copy intact. However, this ability to move is a unique property of
transposable elements.
The defining property of transposable elements is their mobility, i.e., they
are genetic elements that can move from one position to another in the
genome.
In addition to the common property of mobility, transposable elements show
considerable diversity. Some move by DNA intermediates, and others move
by RNA intermediates.
Transposable elements can cause deletions or inversions of DNA. When
transposition generates two copies of the same sequence in the same
orientation, recombination can delete the DNA between them. If the two
copies are in the opposite orientations, recombination will invert the DNA
between them.
Transposable Elements or TEs represent one of several types of mobile
genetic elements. TEs are assigned to one of two classes according to their
mechanism of transposition, which can be described as either copy and
paste ‘Class I TEs’ or cut and paste ‘Class II TEs’.
Transposition can be classified as either ‘Autonomous’ or ‘Non-
Autonomous’ in both Class I and Class II TEs. Autonomous TEs can move
by themselves, whereas non-autonomous TEs require the presence of
another TE to move.
The first synthetic transposon designed for use in vertebrate (including human)
cells, the ‘Sleeping Beauty Transposon System’, is a ‘Tc1/Mariner like
Transposon’. Its dead or ‘fossil’ versions are spread widely in the Salmoid
genome and a functional version was engineered by comparing those
versions.
The Sleeping Beauty Transposon System has been used extensively as an
insertional tag for identifying Cancer Genes.
The phenomenon of moving genetic segments from one location to the other
in a genome is known as transposition. There are two types of transposition,
replicative and conservative transposition.

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The replicative transposition involves the events of both replication and Transposable Elements

recombination processes generating the two daughter copies of the original

transposable elements, one remaining at the parental site and the other at
the target site.
NOTES
The conservative transposition does not involve replication. Simply the
elements move to a new site. When the target site is present within a gene
both types of transposition takes place. The frequency of transposition varies
among different elements.
The overall rate of transposition is 105 -104 per element per generation.
Different IS elements contain different number of bases.
The bacterial transposon Tn3 has been extensively studied. Analysis of DNA
sequences and its junction with target DNA provides some clue to the
mechanism of transposition.
DDE transposases carry a triad of conserved amino acids, namely aspartate
(D) and glutamate (E), which are required for the coordination of a metal
ion required for catalysis, although the DDE chemistry can be integrated
into the transposition cycle in differing ways. This family includes the maize
Ac transposon, as well as the Drosophila P element, Bacteriophage Mu,
Tn5 and Tn10, Mariner, IS10, and IS50.
Tyrosine (Y) transposases use a cut and paste mechanism of transposition,
but employ a site-specific tyrosine residue. The transposon is excised from
its original site (which is repaired); the transposon then forms a closed circle
of DNA, which is integrated into a new site by a reversal of the original
excision step. These transposons are usually found only in Bacteria, and
include Kangaroo, Tn916, and DIRS1.
Serine (S) transposases use a cut and paste (cut out/paste in) mechanism of
transposition involving a circular DNA intermediate, which is similar to that
of tyrosine transposases, only they employ a site-specific serine residue.
These transposons are usually found only in Bacteria, and include Tn5397
and IS607.
Rolling Circle (RC) or Y2 transposases employ either a copy in mechanism,
where they copy a single strand directly into the target site by DNA
replication, so that the old (template) and new (copied) transposons both
have one newly synthesized strand. These transposons usually employ host
DNA replication enzymes. Examples include IS91 and Helitrons.
The Tn3 transposon is a 4957 base pair mobile genetic element, found in
prokaryotes.
Tn5 was one of the first transposons to be identified. As a result of Tn5’s
early discovery and its simple macromolecular requirements for transposition,
the Tn5 system has been a very productive tool for studying the molecular
mechanism of DNA transposition.
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Transposable Elements Bacteriophage Mu, also known as mu phage or mu bacteriophage, is a
mulikevirus (the first of its kind to be identified) of the family Myoviridae
which has been shown to cause genetic transposition.
Bacteriophage Mu represents a hybrid gene creature that exists as both a
NOTES
transposable element and a bacteriophage, a virus that infects bacteria. Mu
integrates into its Escherichia coli host by transposition.
Insertion element, also known as an IS, an Insertion Sequence element, or
an IS element, is a short DNA sequence that acts as a simple Transposable
Element (TE). Insertion sequences have two major characteristics: they are
small relative to other transposable elements, generally around 700 to 2500
bp in length, and only code for proteins implicated in the transposition activity.
A particular insertion sequence may be named according to the form ISn,
where n is a number, for example IS1, IS2, IS3, IS10, IS50, IS911, IS26,
etc.
There exist another sort of transposons, called unit transposons that do not
carry insertion sequences at their extremities, such as Tn7.
Integrons are genetic mechanisms that allow bacteria to adapt and evolve
rapidly through the stockpiling and expression of new genes.
Retrotransposons, also called Class I transposable elements or transposons
via RNA intermediates, are genetic elements that can amplify themselves in
a genome and are ubiquitous components of the DNA of many Eukaryotic
organisms. These DNA sequences use a ‘copy and paste’ mechanism,
whereby they are first transcribed into RNA, then converted back into
identical DNA sequences using reverse transcription, and these sequences
are then inserted into the genome at target sites.

13.6 KEY WORDS

Transposable Element (TE): Also termed as Transposon or Jumping

Gene is a DNA sequence that can change its position within a genome,
sometimes creating or reversing mutations and altering the cell’s genetic
identity and genome size.
DDE transposases: These transposases carry a triad of conserved amino
acids, namely aspartate (D) and glutamate (E), which are required for the
coordination of a metal ion required for catalysis, although the DDE chemistry
can be integrated into the transposition cycle in differing ways.
Tyrosine (Y) transposases: These also use a cut and paste mechanism of
transposition, but employ a site-specific tyrosine residue.
Serine (S) transposases: These transposases use a cut and paste (cut
out/paste in) mechanism of transposition involving a circular DNA
intermediate, which is similar to that of tyrosine transposases, only they
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 Tn3 transposon: The Tn3 transposon is a 4957 base pair mobile genetic Transposable Elements

element, found in prokaryotes.

Bacteriophage Mu: Also known as mu phage or mu bacteriophage, is a
mulikevirus (the first of its kind to be identified) of the family Myoviridae
NOTES
which has been shown to cause genetic transposition.
Integrons: These are genetic mechanisms that allow bacteria to adapt and
evolve rapidly through the stockpiling and expression of new genes.

13.7 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is transposable elements?
2. Explain the types of transposable elements.
3. What is genetic organization?
4. When the mechanism of transposition of Tn5 and Tn3 transposons are used?
5. Define Bacteriophage Mu.
6. Explain insertion sequence Tn7 and IS911.
7. Differentiate between integrons and retrotransposons.
Long Answer Questions
1. Briefly discuss about the significance and functions of transposable elements
giving diagrams and examples.
2. Explain the types of transposable elements.
3. Discuss the structure, genetic organization and mechanism of transposition
of Tn5 and Tn3 transposons.
4. Explain the significant features of Bacteriophage Mu.
5. Explain briefly insertion sequence with reference to Tn7 and IS911.
6. Discuss the significance and features of integrons and retrotransposons.

13.8 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
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Transposable Elements Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.
NOTES
Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.
Pierce, Benjamin A. 2017. Genetics: A Conceptual Approach, 6th Edition. New
York: W.H. Freeman and Company.
Hartwell, Leland, Leroy Hood, Michael Goldberg, Ann E. Reynolds and Lee
Silver. 2010. Genetics: From Genes to Genomes (Hartwell, Genetics),
4th Edition. New York: McGraw-Hill Education.
Klug, William S., Michael R. Cumming, Charlotte A. Spencer and Michael A.
Palladino. 2016. Concepts of Genetics, 10th Edition. London: Pearson
Education.
De Robertis, E.D.P. and E.M.F. De Robertis (Jr.). 2010. Cell and Molecular
Biology. Philadelphia: Lippincott Williams & Wilkins.
Young, M. M. 1992. Plant Biotechnology. Oxford: Pergamon Press.

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 Epigenetics

UNIT 14 EPIGENETICS
Structure NOTES
14.0 Introduction
14.1 Objectives
14.2 Epigenetics
14.2.1 Definitions of Epigenetics
14.2.2 Molecular Basis of Epigenetic
14.2.3 Epigenetics Mechanisms
14.2.4 Functions and Consequences
14.2.5 Epigenetics in Bacteria
14.3 Answers to Check Your Progress Questions
14.4 Summary
14.5 Key Words
14.6 Self Assessment Questions and Exercises
14.7 Further Readings

14.0 INTRODUCTION

Epigenetics is the study of heritable phenotype changes that do not involve

alterations in the DNA sequence. Derived from the Greek prefix ‘epi-’ means
‘over, outside of, around’, i.e., in epigenetics implies features that are ‘on top of’
or ‘in addition to’ the traditional genetic basis for inheritance. Principally, the word
‘epigenetic’ literally means ‘in addition to changes in genetic sequence’. The term
has evolved to include any process that alters gene activity without changing the
DNA sequence, and leads to modifications that can be transmitted to daughter
cells, although experiments show that some epigenetic changes can be reversed.
Many types of epigenetic processes have been identified—they include methylation,
acetylation, phosphorylation, ubiquitylation, and sumolyation. Epigenetic processes
are natural and essential to many organism functions, but if they occur improperly,
there can be major adverse health and behavioral effects.
Epigenetics most often denotes changes that affect gene activity and
expression, but can also be used to describe any heritable phenotypic change.
Such effects on cellular and physiological phenotypic traits may result from external
or environmental factors, or be part of normal development. The standard definition
of epigenetics requires these alterations to be heritable, in the progeny of either
cells or organisms. The term also refers to the changes themselves, functionally
relevant changes to the genome that do not involve a change in the nucleotide
sequence. Examples of mechanisms that produce such changes are DNA
methylation and histone modification, each of which alters how genes are expressed
without altering the underlying DNA sequence. Gene expression can be controlled

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Epigenetics through the action of repressor proteins that attach to silencer regions of the DNA.
These epigenetic changes may last through cell divisions for the duration of the
cell’s life, and may also last for multiple generations even though they do not involve
changes in the underlying DNA sequence of the organism, as an alternative, non-
NOTES
genetic factors cause the organism’s genes to behave or ‘express themselves’
differently.
Epigenetic changes modify the activation of certain genes, but not the genetic
code sequence of DNA. The microstructure (not code) of DNA itself or the
associated chromatin proteins may be modified, causing activation or silencing.
This mechanism enables differentiated cells in a multicellular organism to express
only the genes that are necessary for their own activity. Epigenetic changes are
preserved when cells divide. Most epigenetic changes only occur within the course
of one individual organism’s lifetime, however, these epigenetic changes can be
transmitted to the organism’s offspring through a process called transgenerational
epigenetic inheritance. Moreover, if gene inactivation occurs in a sperm or egg cell
that results in fertilization, this epigenetic modification may also be transferred to
the next generation.
In this unit, you will study about the epigenetics, its definition, molecular
basis, mechanisms, functions and epigenetics in bacteria.

14.1 OBJECTIVES

After going through this unit, you will be able to:

Discuss the significance of epigenetics
Explain the definition, molecular basis, mechanisms and functions of
epigenetics
Describe the epigenetics in bacteria

14.2 EPIGENETICS

Epigenetics is the study of heritable phenotype changes that do not involve

alterations in the DNA sequence. Derived from the Greek prefix ‘epi-’ means
‘over, outside of, around’, i.e., in epigenetics implies features that are ‘on top of’
or ‘in addition to’ the traditional genetic basis for inheritance.
Principally, the word ‘epigenetic’ literally means ‘in addition to changes in
genetic sequence’. The term has evolved to include any process that alters gene
activity without changing the DNA sequence, and leads to modifications that can
be transmitted to daughter cells, although experiments show that some epigenetic
changes can be reversed. Many types of epigenetic processes have been

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identified—they include methylation, acetylation, phosphorylation, ubiquitylation, Epigenetics

and sumolyation. Epigenetic processes are natural and essential to many organism
functions, but if they occur improperly, there can be major adverse health and
behavioral effects.
NOTES
Epigenetics most often denotes changes that affect gene activity and
expression, but can also be used to describe any heritable phenotypic change.
Such effects on cellular and physiological phenotypic traits may result from external
or environmental factors, or be part of normal development. The standard definition
of epigenetics requires these alterations to be heritable, in the progeny of either
cells or organisms. The term also refers to the changes themselves, functionally
relevant changes to the genome that do not involve a change in the nucleotide
sequence. Examples of mechanisms that produce such changes are DNA
methylation and histone modification, each of which alters how genes are expressed
without altering the underlying DNA sequence. Gene expression can be controlled
through the action of repressor proteins that attach to silencer regions of the DNA.
These epigenetic changes may last through cell divisions for the duration of the
cell’s life, and may also last for multiple generations even though they do not involve
changes in the underlying DNA sequence of the organism, as an alternative, non-
genetic factors cause the organism’s genes to behave or ‘express themselves’
differently.
One example of an epigenetic change in eukaryotic biology is the process
of cellular differentiation. During morphogenesis, totipotent stem cells become the
various pluripotent cell lines of the embryo, which in turn become fully differentiated
cells. In other words, as a single fertilized egg cell – the zygote – continues to
divide, the resulting daughter cells change into all the different cell types in an
organism, including neurons, muscle cells, epithelium, endothelium of blood vessels,
etc., by activating some genes while inhibiting the expression of others.
Historically, some phenomena not necessarily heritable have also been
described as epigenetic. For example, the term epigenetic has been used to describe
any modification of chromosomal regions, especially histone modifications, whether
or not these changes are heritable or associated with a phenotype. The consensus
definition now requires a trait to be heritable for it to be considered epigenetic.
Figure 14.1 illustrates the epigenetic mechanisms.

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Epigenetics

NOTES

Fig. 14.1 Epigenetic Mechanisms

14.2.1 Definitions of Epigenetics

The term epigenetics in its contemporary usage emerged in the 1990s, but for
some years has been used with somewhat variable meanings.
A consensus definition of the concept of epigenetic trait as a, ‘Stably heritable
phenotype resulting from changes in a chromosome without alterations in the DNA
sequence’ was formulated at a Cold Spring Harbor meeting in 2008, although
alternate definitions that include non-heritable traits are still being used.
The term epigenesis has a generic meaning of ‘extra growth’, and has been
used in English since the 17th century.
Waddington’s Definition on Canalisation
From the generic meaning, and the associated adjective epigenetic, British
embryologist C. H. Waddington coined the term ‘Epigenetics’ in 1942 as pertaining
to epigenesis, in parallel to Valentin Haecker’s ‘phenogenetics’. Epigenesis in the
context of the biology of that period referred to the differentiation of cells from
their initial totipotent state during embryonic development.
When Waddington coined the term ‘Epigenetics’, the physical nature of
genes and their role in heredity was not known. He used it instead as a conceptual
model of how genetic components might interact with their surroundings to produce
a phenotype, he used the phrase ‘epigenetic landscape’ as a metaphor for biological
development. Waddington held that cell fates were established during development
in a process he called canalisation much as a marble rolls down to the point of
lowest local elevation. Waddington suggested visualising increasing irreversibility
of cell type differentiation as ridges rising between the valleys where the marbles
(analogous to cells) are travelling.
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 In recent times, Waddington’s notion of the epigenetic landscape has been Epigenetics

rigorously formalized in the context of the systems dynamics state approach to the
study of ‘Cell Fate’. Cell Fate determination is predicted to exhibit certain dynamics,
such as attractor-convergence, where the attractor can be an equilibrium point,
limit cycle or strange attractor or oscillatory. NOTES

Contemporary Definitions
Robin Holliday defined epigenetics as, ‘The study of the mechanisms of temporal
and spatial control of gene activity during the development of complex organisms’.
Thus, in its broadest sense, epigenetic can be used to describe anything other than
DNA sequence that influences the development of an organism.
More recent usage of the word in biology follows stricter definitions. It is,
as defined by Arthur Riggs and colleagues as, ‘The study of mitotically and/or
meiotically heritable changes in gene function that cannot be explained by changes
in DNA sequence’.
The term has also been used, however, to describe processes which have
not been demonstrated to be heritable, such as some forms of histone modification,
there are therefore attempts to redefine the term ‘Epigenetics’ in broader terms
that would avoid the constraints of requiring heritability. For example, Adrian Bird
defined epigenetics as, ‘The structural adaptation of chromosomal regions so as
to register, signal or perpetuate altered activity states’.
This definition would be inclusive of transient modifications associated with
DNA repair or cell cycle phases as well as stable changes maintained across
multiple cell generations, but exclude others, such as templating of membrane
architecture and prions unless they impinge on chromosome function. Such
redefinitions however are not universally accepted and are still subject to debate.
The NIH ‘Roadmap Epigenomics Project’, ongoing as of 2016, uses the
following definition:
‘For purposes of this program, epigenetics refers to both heritable changes
in gene activity and expression, in the progeny of cells or of individuals, and also
stable, long term alterations in the transcriptional potential of a cell that are not
necessarily heritable’.
In 2008, a consensus definition of the epigenetic trait, a ‘Stably heritable
phenotype resulting from changes in a chromosome without alterations in the DNA
sequence’, was made at a Cold Spring Harbor meeting.
The similarity of the word to ‘Genetics’ has generated many parallel usages.
The term ‘Epigenome’ is a parallel to the word ‘Genome’, referring to the overall
epigenetic state of a cell, and epigenomics refers to global analyses of epigenetic
changes across the entire genome. The phrase ‘Genetic Code’ has also been adapted
– the ‘Epigenetic Code’ has been used to describe the set of epigenetic features
that create different phenotypes in different cells from the same underlying DNA
sequence.
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Epigenetics Additionally, the term ‘Epigenetic Code’ could represent the total state of
the cell, with the position of each molecule accounted for in an epigenomic map, a
diagrammatic representation of the gene expression, DNA methylation and histone
modification status of a particular genomic region. More typically, the term is used
NOTES in reference to systematic efforts to measure specific, relevant forms of epigenetic
information such as the histone code or DNA methylation patterns.
14.2.2 Molecular Basis of Epigenetic
Epigenetic changes modify the activation of certain genes, but not the genetic code
sequence of DNA. The microstructure (not code) of DNA itself or the associated
chromatin proteins may be modified, causing activation or silencing. This mechanism
enables differentiated cells in a multicellular organism to express only the genes
that are necessary for their own activity. Epigenetic changes are preserved when
cells divide. Most epigenetic changes only occur within the course of one individual
organism’s lifetime, however, these epigenetic changes can be transmitted to the
organism’s offspring through a process called transgenerational epigenetic
inheritance. Moreover, if gene inactivation occurs in a sperm or egg cell that results
in fertilization, this epigenetic modification may also be transferred to the next
generation.
Specific ‘Epigenetic’ processes include Paramutation, Bookmarking,
Imprinting, Gene Silencing, X Chromosome inactivation, Position effect, DNA
Methylation reprogramming, Transvection, Maternal Effects, the progress of
Carcinogenesis, effects of Teratogens, Regulation of Histone modifications and
Heterochromatin, and technical limitations affecting Parthenogenesis and Cloning.
DNA Damage
DNA damage can also cause epigenetic changes. The DNA damage is very frequent,
occurring on average about 60,000 times a day per cell of the human body. These
damages are largely repaired, but at the site of a DNA repair, epigenetic changes
can remain. In particular, a double strand break in DNA can initiate unprogrammed
epigenetic gene silencing both by causing DNA methylation as well as by promoting
silencing types of histone modifications.
In addition, the enzyme Parp1 (poly(ADP)-ribose polymerase) and its
product poly(ADP)-ribose (PAR) accumulate at sites of DNA damage as part of
a repair process. This accumulation, in turn, directs recruitment and activation of
the chromatin remodelling protein ALC1 that can cause nucleosome remodelling.
Nucleosome remodelling has been found to cause, for instance, epigenetic silencing
of DNA repair gene MLH1. DNA damaging chemicals, such as Benzene,
Hydroquinone, Styrene, Carbon Tetrachloride and Trichloroethylene, cause
considerable Hypomethylation of DNA, some through the activation of oxidative
stress pathways.

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 Foods are known to alter the epigenetics of rats on different diets. Some Epigenetics

food components epigenetically increase the levels of DNA repair enzymes, such
as MGMT and MLH1 and p53. Other food components can reduce DNA
damage, such as Soy Isoflavones. In one study, markers for oxidative stress, such
as modified nucleotides that can result from DNA damage, were decreased by a NOTES
3-week diet supplemented with Soy. A decrease in oxidative DNA damage was
also observed after the consumption of Anthocyanin-Rich Bilberry, Vaccinium
myrtillius L.
Techniques to Study Epigenetics
Epigenetic research uses a wide range of molecular biological techniques to further
understanding of epigenetic phenomena, including chromatin immunoprecipitation,
together with its large-scale variants ChIP-on-chip and ChIP-Seq, fluorescent in
situ hybridization, methylation sensitive restriction enzymes, DNA adenine
methyltransferase identification (DamID) and bisulfite sequencing. Furthermore,
the use of bioinformatics methods has a role in computational epigenetics.
14.2.3 Epigenetics Mechanisms
Several types of epigenetic inheritance systems have a significant role as cell
memory, however not all of these are universally accepted to be examples of
epigenetics. Following are some universally accepted mechanisms of epigenetics.
Covalent Modifications
Covalent modifications of DNA, for example Cytosine Methylation and
Hydroxymethylation or of Histone Proteins, such as Lysine Acetylation, Lysine
and Arginine Methylation, Serine and Threonine Phosphorylation, and Lysine
Ubiquitination and Sumoylation, play central roles in many types of epigenetic
inheritances. Therefore, the word ‘Epigenetics’ is sometimes used as a synonym
for these processes. However, this can be misleading. Chromatin remodelling is
not always inherited, and not all epigenetic inheritance involves chromatin
remodelling. DNA associates with histone proteins to form chromatin.
Because the phenotype of a cell or individual is affected by which of its
genes are transcribed, heritable transcription states can give rise to epigenetic
effects. There are several layers of regulation of gene expression. One way that
genes are regulated is through the remodelling of chromatin. Chromatin is the
complex of DNA and the histone proteins with which it associates. If the way that
DNA is wrapped around the histones changes, gene expression can change as
well. Chromatin remodelling is accomplished through the following two main
mechanisms:
Mechanism 1: The first method is post translational modification of the amino
acids that make up histone proteins. Histone proteins are made up of long chains
of amino acids. If the amino acids that are in the chain are changed, the shape of
the histone might be modified. DNA is not completely unwound during replication.
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Epigenetics It is possible, then, that the modified histones may be carried into each new copy
of the DNA. Once there, these histones may act as templates, initiating the
surrounding new histones to be shaped in the new manner. By altering the shape
of the histones around them, these modified histones would ensure that a lineage-
NOTES specific transcription program is maintained after cell division.
Mechanism 2: The second method is the addition of methyl groups to the DNA,
mostly at CpG sites, to convert Cytosine (C) to 5-Methylcytosine. 5-Methylcytosine
performs much like a regular Cytosine (C), pairing with a guanine in double-stranded
DNA. However, some areas of the genome are methylated more heavily than
others, and highly methylated areas tend to be less transcriptionally active, through
a mechanism not fully understood. Methylation of Cytosine’s can also persist from
the germ line of one of the parents into the zygote, marking the chromosome as
being inherited from one parent or the other genetic imprinting.
Mechanisms of heritability of histone state are not well understood, however,
much is known about the mechanism of heritability of DNA methylation state during
cell division and differentiation. Heritability of methylation state depends on certain
enzymes, such as DNMT1 that have a higher affinity for 5-Methylcytosine than
for Cytosine (C). If this enzyme reaches a ‘hemimethylated’ portion of DNA,
where 5-Methylcytosine is in only one of the two DNA strands, the enzyme will
methylate the other half.
Although histone modifications occur throughout the entire sequence, the
unstructured N-termini of histones (called histone tails) are particularly highly
modified. These modifications include Acetylation, Methylation, Ubiquitylation,
Phosphorylation, Sumoylation, Ribosylation and Citrullination. Acetylation is the
most highly studied of these modifications. For example, Acetylation of the K14
and K9 Lysines of the tail of histone H3 by Histone AcetylTransferase (HAT)
enzymes is generally related to transcriptional competence.
One mode of thinking is that this tendency of acetylation to be associated
with ‘active’ transcription is biophysical in nature. Because it normally has a
positively charged Nitrogen at its end, Lysine can bind the negatively charged
phosphates of the DNA backbone. The acetylation event converts the positively
charged amine group on the side chain into a neutral amide linkage. This removes
the positive charge, thus loosening the DNA from the Histone. When this occurs,
complexes like SWI/SNF and other transcriptional factors can bind to the DNA
and allow transcription to occur. This is the ‘cis’ model of epigenetic function. In
other words, changes to the histone tails have a direct effect on the DNA itself.
Another model of epigenetic function is the ‘trans’ model. In this model,
changes to the histone tails act indirectly on the DNA. For example, Lysine
Acetylation may create a binding site for chromatin-modifying enzymes or
transcription machinery. This chromatin remodeler can then cause changes to the
state of the chromatin. Certainly, a bromodomain – a protein domain that specifically
binds Acetyl-Lysine – is found in many enzymes that help activate transcription,
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including the SWI/SNF complex. It may be that acetylation acts in this and the Epigenetics

previous way to aid in transcriptional activation.

The idea that modifications act as docking modules for related factors is
borne out by histone methylation as well. Methylation of Lysine 9 of Histone H3
NOTES
has long been associated with constitutively transcriptionally silent chromatin
(constitutive heterochromatin). It has been determined that a chromodomain (a
domain that specifically binds methyl-lysine) in the transcriptionally repressive
protein HP1 recruits HP1 to K9 methylated regions. One example that seems to
refute this biophysical model for methylation is that tri-methylation of histone H3
at lysine 4 is strongly associated with transcriptional activation. Tri-methylation in
this case would introduce a fixed positive charge on the tail.
It has been shown that the histone lysine methyltransferase (KMT) is
responsible for this methylation activity in the pattern of histones H3 and H4. This
enzyme utilizes a catalytically active site called the SET domain. The SET domain
is a 130-amino acid sequence involved in modulating gene activities. This domain
has been demonstrated to bind to the histone tail and causes the methylation of the
histone.
Differing histone modifications are likely to function in differing ways;
acetylation at one position is likely to function differently from acetylation at another
position. Also, multiple modifications may occur at the same time, and these
modifications may work together to change the behavior of the nucleosome. The
idea that multiple dynamic modifications regulate gene transcription in a systematic
and reproducible way is called the histone code, although the idea that histone
state can be read linearly as a digital information carrier has been largely debunked.
One of the best-understood systems that orchestrates chromatin-based silencing
is the SIR protein based silencing of the yeast hidden mating type loci HML and
HMR.
DNA methylation frequently occurs in repeated sequences, and helps to
suppress the expression and mobility of ‘transposable elements’. Because 5-
methylcytosine can be spontaneously deaminated (replacing nitrogen by oxygen)
to thymidine, CpG sites are frequently mutated and become rare in the genome,
except at CpG islands where they remain unmethylated. Epigenetic changes of
this type thus have the potential to direct increased frequencies of permanent genetic
mutation. DNA methylation patterns are known to be established and modified in
response to environmental factors by a complex interplay of at least three
independent DNA methyltransferases, DNMT1, DNMT3A, and DNMT3B, the
loss of any of which is lethal in mice. DNMT1 is the most abundant methyltransferase
in somatic cells, localizes to replication foci, has a 10–40 fold preference for
hemimethylated DNA and interacts with the Proliferating Cell Nuclear Antigen
(PCNA).
By preferentially modifying hemimethylated DNA, DNMT1 transfers
patterns of methylation to a newly synthesized strand after DNA replication, and
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Epigenetics therefore is often referred to as the ‘maintenance’ methyltransferase. DNMT1 is
essential for proper embryonic development, imprinting and X-inactivation. To
emphasize the difference of this molecular mechanism of inheritance from the
canonical Watson-Crick base-pairing mechanism of transmission of genetic
NOTES information, the term ‘Epigenetic Templating’ was introduced. Furthermore, in
addition to the maintenance and transmission of methylated DNA states, the same
principle could work in the maintenance and transmission of histone modifications
and even cytoplasmic (structural) heritable states.
Histones H3 and H4 can also be manipulated through demethylation using
histone lysine demethylase (KDM). This recently identified enzyme has a catalytically
active site called the Jumonji domain (JmjC). The demethylation occurs when
JmjC utilizes multiple cofactors to hydroxylate the methyl group, thereby removing
it. JmjC is capable of demethylating mono-, di-, and tri-methylated substrates.
Chromosomal regions can adopt stable and heritable alternative states
resulting in bistable gene expression without changes to the DNA sequence.
Epigenetic control is often associated with alternative covalent modifications of
histones. The stability and heritability of states of larger chromosomal regions are
suggested to involve positive feedback where modified nucleosomes recruit enzymes
that similarly modify nearby nucleosomes. A simplified stochastic model for this
type of epigenetics is found here.
It has been suggested that chromatin-based transcriptional regulation could
be mediated by the effect of small RNAs. Small interfering RNAs can modulate
transcriptional gene expression via epigenetic modulation of targeted promoters.
RNA Transcripts
Sometimes a gene, after being turned on, transcribes a product that (directly or
indirectly) maintains the activity of that gene. For example, Hnf4 and MyoD enhance
the transcription of many liver specific genes and muscle specific genes, respectively,
including their own, through the transcription factor activity of the proteins they
encode. RNA signalling includes differential recruitment of a hierarchy of generic
chromatin modifying complexes and DNA methyltransferases to specific loci by
RNAs during differentiation and development. Other epigenetic changes are
mediated by the production of different splice forms of RNA, or by formation of
double-stranded RNA (RNAi). Descendants of the cell in which the gene was
turned on will inherit this activity, even if the original stimulus for gene-activation is
no longer present. These genes are often turned on or off by signal transduction,
although in some systems where syncytia or gap junctions are important, RNA
may spread directly to other cells or nuclei by diffusion. A large amount of RNA
and protein is contributed to the zygote by the mother during oogenesis or via
nurse cells, resulting in maternal effect phenotypes. A smaller quantity of sperm
RNA is transmitted from the father, but there is recent evidence that this epigenetic
information can lead to visible changes in several generations of offspring.
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MicroRNAs Epigenetics

MicroRNAs (miRNAs) are members of non-coding RNAs that range in size from
17 to 25 nucleotides. The miRNAs regulate a large variety of biological functions
in plants and animals. So far, in 2013, about 2000 miRNAs have been discovered NOTES
in humans and these can be found online in a miRNA database. Each miRNA
expressed in a cell may target about 100 to 200 messenger RNAs (mRNAs) that
it downregulates. Most of the downregulation of mRNAs occurs by causing the
decay of the targeted mRNA, while some downregulation occurs at the level of
translation into protein.
It appears that about 60% of human protein coding genes are regulated by
miRNAs. Many miRNAs are epigenetically regulated. About 50% of miRNA
genes are associated with CpG islands that may be repressed by epigenetic
methylation. Transcription from methylated CpG islands is strongly and heritably
repressed. Other miRNAs are epigenetically regulated by either histone
modifications or by combined DNA methylation and histone modification.
mRNA
In 2011, it was demonstrated that the methylation of mRNA plays a critical role in
human energy homeostasis. The obesity-associated FTO gene is shown to be
able to Demethylate N6-Methyladenosine in RNA.
sRNAs
sRNAs are small (50–250 nucleotides), highly structured, non-coding RNA
fragments found in bacteria. They control gene expression including virulence genes
in pathogens and are viewed as new targets in the fight against drug-resistant
bacteria. They play an important role in many biological processes, binding to
mRNA and protein targets in prokaryotes. Their phylogenetic analyses, for example
through sRNA–mRNA target interactions or protein binding properties, are used
to build comprehensive databases. sRNA-gene maps based on their targets in
microbial genomes are also constructed.
Prions
Prions are infectious forms of proteins. In general, proteins fold into discrete units
that perform distinct cellular functions, but some proteins are also capable of forming
an infectious conformational state known as a prion. Although often viewed in the
context of infectious disease, prions are more loosely defined by their ability to
catalytically convert other native state versions of the same protein to an infectious
conformational state. It is in this latter sense that they can be viewed as epigenetic
agents capable of inducing a phenotypic change without a modification of the
genome.
Fungal prions are considered by some to be epigenetic because the infectious
phenotype caused by the prion can be inherited without modification of the genome.
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Epigenetics PSI+ and URE3, discovered in yeast in 1965 and 1971, are the two best studied
of this type of prion. Prions can have a phenotypic effect through the sequestration
of protein in aggregates, thereby reducing that protein’s activity. In PSI+ cells, the
loss of the Sup35 protein (which is involved in termination of translation) causes
NOTES ribosomes to have a higher rate of read-through of stop codons, an effect that
results in suppression of nonsense mutations in other genes. The ability of Sup35
to form prions may be a conserved trait. It could confer an adaptive advantage by
giving cells the ability to switch into a PSI+ state and express dormant genetic
features normally terminated by stop codon mutations.
Structural Inheritance
In ciliates, such as Tetrahymena and Paramecium, genetically identical cells show
heritable differences in the patterns of ciliary rows on their cell surface.
Experimentally altered patterns can be transmitted to daughter cells. It seems existing
structures act as templates for new structures. The mechanisms of such inheritance
are unclear, but reasons exist to assume that multicellular organisms also use existing
cell structures to assemble new ones.
Nucleosome Positioning
Eukaryotic genomes have numerous nucleosomes. Nucleosome position is not
random, and determine the accessibility of DNA to regulatory proteins. This
determines differences in gene expression and cell differentiation. It has been shown
that at least some nucleosomes are retained in sperm cells where most but not all
histones are replaced by protamines. Thus nucleosome positioning is to some
degree inheritable. Recent studies have uncovered connections between
nucleosome positioning and other epigenetic factors, such as DNA methylation
and hydroxymethylation.
14.2.4 Functions and Consequences
Developmental epigenetics can be divided into predetermined and probabilistic
epigenesis. Predetermined epigenesis is a unidirectional movement from structural
development in DNA to the functional maturation of the protein. ‘Predetermined’
here means that development is scripted and predictable. Probabilistic epigenesis
on the other hand is a bidirectional structure-function development with experiences
and external molding development.
Somatic epigenetic inheritance, particularly through DNA and histone
covalent modifications and nucleosome repositioning, is very important in the
development of multicellular eukaryotic organisms. The genome sequence is static
(with some notable exceptions), but cells differentiate into many different types,
which perform different functions, and respond differently to the environment and
intercellular signalling. Thus, as individuals develop, morphogens activate or silence
genes in an epigenetically heritable fashion, giving cells a memory. In mammals,
most cells terminally differentiate, with only stem cells retaining the ability to
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differentiate into several cell types, ‘totipotency’ and ‘multipotency’. In mammals, Epigenetics

some stem cells continue producing new differentiated cells throughout life, such
as in neurogenesis, but mammals are not able to respond to loss of some tissues,
for example, the inability to regenerate limbs, which some other animals are capable
of. Epigenetic modifications regulate the transition from neural stem cells to glial NOTES
progenitor cells, for example, differentiation into oligodendrocytes is regulated by
the deacetylation and methylation of histones. Unlike animals, plant cells do not
terminally differentiate, remaining totipotent with the ability to give rise to a new
individual plant. While plants do utilise many of the same epigenetic mechanisms
as animals, such as chromatin remodelling, it has been hypothesised that some
kinds of plant cells do not use or require ‘cellular memories’, resetting their gene
expression patterns using positional information from the environment and
surrounding cells to determine their fate.
Epigenetic changes can occur in response to environmental exposure – for
example, mice given some dietary supplements have epigenetic changes affecting
expression of the agouti gene, which affects their fur color, weight, and propensity
to develop cancer.
Transgenerational
Epigenetic mechanisms were a necessary part of the evolutionary origin of cell
differentiation. Although epigenetics in multicellular organisms is generally thought
to be a mechanism involved in differentiation, with epigenetic patterns ‘reset’ when
organisms reproduce, there have been some observations of transgenerational
epigenetic inheritance, for example the phenomenon of paramutation observed in
maize. Although most of these multigenerational epigenetic traits are gradually lost
over several generations, the possibility remains that multigenerational epigenetics
could be another aspect to evolution and adaptation. As mentioned above, some
define epigenetics as heritable.
Two important ways in which epigenetic inheritance can be different from
traditional genetic inheritance, with important consequences for evolution, are that
rates of epimutation can be much faster than rates of mutation and the epimutations
are more easily reversible. In plants, heritable DNA methylation mutations are
100,000 times more likely to occur compared to DNA mutations. An epigenetically
inherited element, such as the PSI+ system can act as a ‘stop-gap’, good enough
for short-term adaptation that allows the lineage to survive for long enough for
mutation and/or recombination to genetically assimilate the adaptive phenotypic
change. The existence of this possibility increases the evolvability of a species.
More than 100 cases of transgenerational epigenetic inheritance phenomena
have been reported in a wide range of organisms, including prokaryotes, plants,
and animals. For instance, mourning cloak butterflies will change color through
hormone changes in response to experimentation of varying temperatures.

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Epigenetics The filamentous fungus Neurospora crassa is a prominent model system
for understanding the control and function of cytosine methylation. In this organism,
DNA methylation is associated with relics of a genome defense system called RIP
(Repeat-Induced Point) mutation and silences gene expression by inhibiting
NOTES transcription elongation.
The yeast prion PSI is generated by a conformational change of a translation
termination factor, which is then inherited by daughter cells. This can provide a
survival advantage under adverse conditions. This is an example of epigenetic
regulation enabling unicellular organisms to respond rapidly to environmental stress.
Prions can be viewed as epigenetic agents capable of inducing a phenotypic change
without modification of the genome.
Direct detection of epigenetic marks in microorganisms is possible with
single molecule real time sequencing, in which polymerase sensitivity allows for
measuring methylation and other modifications as a DNA molecule is being
sequenced.
14.2.5 Epigenetics in Bacteria
While epigenetics is of fundamental importance in eukaryotes, especially metazoans,
it plays a different role in bacteria. Most importantly, eukaryotes use epigenetic
mechanisms primarily to regulate gene expression which bacteria rarely do.
However, bacteria make widespread use of post-replicative DNA methylation
for the epigenetic control of DNA-protein interactions. Bacteria also use DNA
adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal.
DNA adenine methylation is important in bacteria virulence in organisms, such as
Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In
Alphaproteobacteria, methylation of Adenine (A) regulates the cell cycle and
couples gene transcription to DNA replication. In Gammaproteobacteria, Adenine
(A) methylation provides signals for DNA replication, chromosome segregation,
mismatch repair, packaging of bacteriophage, transposase activity and regulation
of gene expression.
There exists a genetic switch controlling Streptococcus pneumoniae (the
pneumococcus) that allows the bacterium to randomly change its characteristics
into six alternative states that could pave the way to improved vaccines. Each
form is randomly generated by a phase variable methylation system. The ability of
the pneumococcus to cause deadly infections is different in each of these six states.
Similar systems exist in other bacterial genera.

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 Epigenetics

Check Your Progress

1. Explain the term epigenetics. Which processes it includes?
2. Give the C. H. Waddington and Robin Holliday definitions for epigenetics. NOTES
3. How DNA damage can cause epigenetic changes?
4. What are prions?
5. Give examples of transgenerational epigenetic inheritance phenomena.

14.3 ANSWERS TO CHECK YOUR PROGRESS

QUESTIONS

1. Epigenetics is the study of heritable phenotype changes that do not involve

alterations in the DNA sequence. Derived from the Greek prefix ‘epi-’
means ‘over, outside of, around’, i.e., in epigenetics implies features that
are ‘on top of’ or ‘in addition to’ the traditional genetic basis for inheritance.
Principally, the word ‘epigenetic’ literally means ‘in addition to changes in
genetic sequence’. The term has evolved to include any process that alters
gene activity without changing the DNA sequence, and leads to modifications
that can be transmitted to daughter cells, although experiments show that
some epigenetic changes can be reversed. Many types of epigenetic
processes have been identified—they include methylation, acetylation,
phosphorylation, ubiquitylation, and sumolyation. Epigenetic processes are
natural and essential to many organism functions, but if they occur improperly,
there can be major adverse health and behavioral effects.
2. British embryologist C. H. Waddington coined the term ‘Epigenetics’ in
1942 as pertaining to epigenesis, in parallel to Valentin Haecker’s
‘phenogenetics’ and defined ‘Epigenesis’ in the context of the biology of
that period referred to the differentiation of cells from their initial totipotent
state during embryonic development.
Robin Holliday defined epigenetics as, ‘The study of the mechanisms of
temporal and spatial control of gene activity during the development of
complex organisms’. Thus, in its broadest sense, epigenetic can be used to
describe anything other than DNA sequence that influences the development
of an organism.
3. DNA damage can also cause epigenetic changes. The DNA damage is
very frequent, occurring on average about 60,000 times a day per cell of
the human body. These damages are largely repaired, but at the site of a
DNA repair, epigenetic changes can remain. In particular, a double strand

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Epigenetics break in DNA can initiate unprogrammed epigenetic gene silencing both by
causing DNA methylation as well as by promoting silencing types of histone
modifications.
4. Prions are infectious forms of proteins. In general, proteins fold into discrete
NOTES
units that perform distinct cellular functions, but some proteins are also
capable of forming an infectious conformational state known as a prion.
Although often viewed in the context of infectious disease, prions are more
loosely defined by their ability to catalytically convert other native state
versions of the same protein to an infectious conformational state. It is in
this latter sense that they can be viewed as epigenetic agents capable of
inducing a phenotypic change without a modification of the genome.
5. More than 100 cases of transgenerational epigenetic inheritance phenomena
have been reported in a wide range of organisms, including prokaryotes,
plants, and animals. For instance, mourning cloak butterflies will change
color through hormone changes in response to experimentation of varying
temperatures.
The filamentous fungus Neurospora crassa is a prominent model system
for understanding the control and function of cytosine methylation. In this
organism, DNA methylation is associated with relics of a genome defense
system called RIP (Repeat-Induced Point) mutation and silences gene
expression by inhibiting transcription elongation.
The yeast prion PSI is generated by a conformational change of a translation
termination factor, which is then inherited by daughter cells. This can provide
a survival advantage under adverse conditions. This is an example of
epigenetic regulation enabling unicellular organisms to respond rapidly to
environmental stress. Prions can be viewed as epigenetic agents capable of
inducing a phenotypic change without modification of the genome.

14.4 SUMMARY

Epigenetics is the study of heritable phenotype changes that do not involve

alterations in the DNA sequence.
Derived from the Greek prefix ‘epi-’ means ‘over, outside of, around’, i.e.,
in epigenetics implies features that are ‘on top of’ or ‘in addition to’ the
traditional genetic basis for inheritance.
Principally, the word ‘epigenetic’ literally means ‘in addition to changes in
genetic sequence’. The term has evolved to include any process that alters
gene activity without changing the DNA sequence, and leads to modifications
that can be transmitted to daughter cells, although experiments show that
some epigenetic changes can be reversed.

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Many types of epigenetic processes have been identified—they include Epigenetics

methylation, acetylation, phosphorylation, ubiquitylation, and sumolyation.

Epigenetic processes are natural and essential to many organism functions,
but if they occur improperly, there can be major adverse health and behavioral
effects. NOTES
A consensus definition of the concept of epigenetic trait as a, ‘Stably heritable
phenotype resulting from changes in a chromosome without alterations in
the DNA sequence’ was formulated at a Cold Spring Harbor meeting in
2008, although alternate definitions that include non-heritable traits are still
being used.
The term epigenesis has a generic meaning of ‘extra growth’, and has been
used in English since the 17th century.
British embryologist C. H. Waddington coined the term ‘Epigenetics’ in
1942 as pertaining to epigenesis, in parallel to Valentin Haecker’s
‘phenogenetics’ and defined ‘Epigenesis’ in the context of the biology of
that period referred to the differentiation of cells from their initial totipotent
state during embryonic development.
Robin Holliday defined epigenetics as, ‘The study of the mechanisms of
temporal and spatial control of gene activity during the development of
complex organisms’. Thus, in its broadest sense, epigenetic can be used to
describe anything other than DNA sequence that influences the development
of an organism.
The term ‘Epigenome’ is a parallel to the word ‘Genome’, referring to the
overall epigenetic state of a cell, and epigenomics refers to global analyses
of epigenetic changes across the entire genome. The phrase ‘Genetic Code’
has also been adapted – the ‘Epigenetic Code’ has been used to describe
the set of epigenetic features that create different phenotypes in different
cells from the same underlying DNA sequence.
Epigenetic changes modify the activation of certain genes, but not the genetic
code sequence of DNA. The microstructure (not code) of DNA itself or
the associated chromatin proteins may be modified, causing activation or
silencing. This mechanism enables differentiated cells in a multicellular
organism to express only the genes that are necessary for their own activity.
Epigenetic changes are preserved when cells divide.
DNA damage can also cause epigenetic changes. The DNA damage is
very frequent, occurring on average about 60,000 times a day per cell of
the human body.
Covalent modifications of DNA, for example Cytosine Methylation and
Hydroxymethylation or of Histone Proteins, such as Lysine Acetylation,
Lysine and Arginine Methylation, Serine and Threonine Phosphorylation,

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Epigenetics and Lysine Ubiquitination and Sumoylation, play central roles in many types
of epigenetic inheritances.
DNA methylation frequently occurs in repeated sequences, and helps to
suppress the expression and mobility of ‘transposable elements’.
NOTES
sRNAs are small (50–250 nucleotides), highly structured, non-coding RNA
fragments found in bacteria. They control gene expression including virulence
genes in pathogens and are viewed as new targets in the fight against drug-
resistant bacteria.
Prions are infectious forms of proteins. In general, proteins fold into discrete
units that perform distinct cellular functions, but some proteins are also
capable of forming an infectious conformational state known as a prion.
Somatic epigenetic inheritance, particularly through DNA and histone
covalent modifications and nucleosome repositioning, is very important in
the development of multicellular eukaryotic organisms.
Epigenetic modifications regulate the transition from neural stem cells to
glial progenitor cells, for example, differentiation into oligodendrocytes is
regulated by the deacetylation and methylation of histones.
More than 100 cases of transgenerational epigenetic inheritance phenomena
have been reported in a wide range of organisms, including prokaryotes,
plants, and animals. For instance, mourning cloak butterflies will change
color through hormone changes in response to experimentation of varying
temperatures.
The filamentous fungus Neurospora crassa is a prominent model system
for understanding the control and function of cytosine methylation. In this
organism, DNA methylation is associated with relics of a genome defense
system called RIP (Repeat-Induced Point) mutation and silences gene
expression by inhibiting transcription elongation.
Eukaryotes use epigenetic mechanisms primarily to regulate gene expression
which bacteria rarely do. However, bacteria make widespread use of post-
replicative DNA methylation for the epigenetic control of DNA-protein
interactions.
Bacteria also use DNA adenine methylation (rather than DNA cytosine
methylation) as an epigenetic signal. DNA adenine methylation is important
in bacteria virulence in organisms, such as Escherichia coli, Salmonella,
Vibrio, Yersinia, Haemophilus, and Brucella.

14.5 KEY WORDS

Epigenetics: It is the study of heritable phenotype changes that do not

involve alterations in the DNA sequence.
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 MicroRNAs (miRNAs): These are members of non-coding RNAs that Epigenetics

range in size from 17 to 25 nucleotides, the miRNAs regulate a large variety

of biological functions in plants and animals.
Prions: These are infectious forms of proteins, basically, proteins fold into
NOTES
discrete units that perform distinct cellular functions, but some proteins are
also capable of forming an infectious conformational state known as a prion.

14.6 SELF ASSESSMENT QUESTIONS AND

EXERCISES

Short Answer Questions

1. What is epigenetics?
2. Give definitions of epigenetics.
3. What are the molecular basis of epigenetics?
4. Explain the mechanisms and functions of epigenetics.
5. What is epigenetics in bacteria?
Long Answer Questions
1. Briefly discuss about the significance and functions of epigenetics giving
appropriate examples.
2. Explain the various definitions of epigenetics.
3. Discuss the role and significance of the molecular basis, mechanisms and
functions of epigenetics.
4. Explain the epigenetics process in bacteria.

14.7 FURTHER READINGS

Lewin, Benjamin. 1999. Genes VII. New York: Oxford University Press.
Freifelder, David. 2008. Microbial Genetics. New Delhi: Narosa Publishing
House.
Jeyanthi, G. P. 2009. Molecular Biology. Chennai: MJP Publishers.
Freifelder, David. 2004. Microbial Biology. New Delhi: Narosa Publishing House.
Kornberg, Arthur and Tania A. Baker. 1992. DNA Replication. New York: W.H.
Freeman & Company.
Singer, Maxine and Paul Berg. 1991. Genes & Genomes. California: University
Science Books.

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Epigenetics Cronan, John E., David Freifelder and Stanley R. Maloy. 2008. Microbial
Genetics. New Delhi: Narosa Publishing House.
Berg, Jeremy M., John L. Tymoczko and Lubert Stryer. 2010. Biochemistry, 7th
Edition. New York: W.H. Freeman & Company.
NOTES
Verma, P. S. and A. K. Agarwal. 2004. Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. New Delhi: S. Chand and Company.
Emmanuel, C., S Ignacimuthu and Dr S Vincent. 2006. Applied Genetics: Recent
Trends and Techniques. Tamil Nadu: MJP Publishers.
Brown, Terence A. 1998. Genetics: A Molecular Approach. England: Stanley
Thornes.

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