Unzipping Genetics

UNZIPPING
GENETICS
2009
PROF. P. UMAHARAN
The University of the West Indies
9/1/2009
 2002 P. Umaharan – Unzipping Genetics
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INTRODUCTION TO ADVANCED GENETICS.......................................................Error! Bookmark not defined.
CHAPTER ONE...........................................................................................................................................................1
CYTOGENETICS........................................................................................................................................................1
1.1 INTRODUCTION.............................................................................................................................................1
1.1.1 The emergence of cytogenetics...............................................................................................................1
1.1.2 Chromosome theory of inheritance.........................................................................................................1
1.1.3 What is Cytogenetics?.............................................................................................................................1
1.2 Karyotyping......................................................................................................................................................4
1.2.1 Macro and Ultra Structure of the Chromosome......................................................................................4
1.2.2 Characterisation of the Genome..............................................................................................................4
1.2.3 Karyotyping............................................................................................................................................4
1.2.4 Uses of karyotyping................................................................................................................................5
1.2.5 Practical Exercise-1 – Karyotyping - I....................................................................................................6
1.3 Chromosomal Mutations-Changes In Chromosomal Structure......................................................................8
1.3.1 Deletion Or Deficiency...........................................................................................................................8
1.3.1.1 Definition and types:..........................................................................................................................8
1.3.1.2 Diagnosis:...........................................................................................................................................8
1.3.2 Duplications Or Additions....................................................................................................................10
1.3.2.1 Definition and types.........................................................................................................................10
1.3.2.2 Diagnosis..........................................................................................................................................10
1.3.2.3 Evolutionary importance of duplications.........................................................................................11
1.3.2.4 Multigene Families, Their Evolution And Evolutionary Significance.............................................13
1.3.3 Inversions..............................................................................................................................................16
1.3.3.1 Types:...............................................................................................................................................16
1.3.3.2 Diagnosis:.........................................................................................................................................17
1.3.3.3 Inheritance........................................................................................................................................19
1.3.3.4 Evolutionary significance:...............................................................................................................20
1.3.4 Translocations.......................................................................................................................................20
1.3.4.1 Types................................................................................................................................................20
1.3.4.2 Diagnosis of Translocation...............................................................................................................20
1.3.4.3 Inheritance of translocation heterozygotes.......................................................................................21
1.3.4.4 Importance of translocations: (Tutorial)..........................................................................................23
1.3.5 PRACTICAL 2 - KARYOTYPING - II...............................................................................................25
1.4 CHROMOSOMAL MUTATIONS - CHANGES IN CHROMOSOME NUMBER...........................................27
1.4.1 Euploidy................................................................................................................................................27
1.4.1.1 Types Of Euploidy...........................................................................................................................27
1.4.1.2 Origin Of Euploidy:.........................................................................................................................27
1.4.1.3 Cytological Diagnosis Of Euploidy.................................................................................................28
1.4.1.4 Phenotypic And Genetic Diagnosis..................................................................................................28
1.4.1.5 Inheritance Of Characters................................................................................................................29
1.4.1.6 Role Of Autopolyploids In Agriculture............................................................................................31
1.4.1.7 Evolutionary significance of allopolyploids (Tutorial topic)...........................................................33
1.4.1.8 History of Allopolyploid Crops........................................................................................................34
1.4.2 Practical Exercise 3- Polyploid Series In Musa Sp...............................................................................34
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CHAPTER TWO........................................................................................................................................................37
PROKARYOTIC GENETICS...................................................................................................................................37
2.1 Introduction....................................................................................................................................................37
2.2 Recombination In Prokaryotes.......................................................................................................................39
2.2.1 Conjugation...........................................................................................................................................40
2.2.1.1 Definition:........................................................................................................................................40
2.2.1.2 The F factor:.....................................................................................................................................40
2.2.1.2 Substituted-F....................................................................................................................................40
2.2.1.3 Lfr, Hfr and F’ Strains......................................................................................................................41
2.2.1.4.1 Interrupted mating technique (Wollman and Jacob, 1957).....................................................42
2.2.1.4.2 Gradient of transfer method....................................................................................................44
2.2.1.4.3 High resolution mapping (Recombination mapping).............................................................45
2.2.2 Transduction..........................................................................................................................................46
2.2.2.1 Definition:........................................................................................................................................46
2.2.2.2 Phage Life Cycles............................................................................................................................47
2.2.2.3 Lytic pathway and generalised transduction:...................................................................................49
2.2.2.4 Lysogeny and specialised transduction:...........................................................................................49
2.2.2.5 Co-transduction frequencies can be used to map bacterial chromosomes.......................................50
2.2.3 Transformation......................................................................................................................................52
2.2.4 Transposable Genetic Elements (Transposons)..................................................................................52
2.2.4.1 Types of Transpons..........................................................................................................................52
2.2.4.2 Importance Of Bacterial Transposons (Tutorial Topic)...............................................................53
2.3 GENE FINE STRUCTURE ANALYSIS..........................................................................................................54
2.3.1 Recombination And Complementation.................................................................................................54
2.3.1.1 Recombination tests.........................................................................................................................54
2.3.1.2 Complementation test (cis-trans test)..............................................................................................55
2.3.1.3 Limitations to the cis-trans test (tutorial topic)................................................................................57
2.3.1.4 Recombination Test vs Complementation Test?..............................................................................57
2.3.2 Gene Fine Structure Analysis of Phage Genetics.................................................................................58
2.3.2.1 Recombination test:..........................................................................................................................59
2.3.2.2 Complementation spot test:..............................................................................................................60
2.3.2.3 Benzer's Deletion Mapping Technique............................................................................................60
2.3.3 The Modern Concept Of A Gene And Its Evolution.............................................................................63
CHAPTER THREE....................................................................................................................................................65
MOLECULAR GENETICS......................................................................................................................................65
3.1 Molecular Nature Of The Gene......................................................................................................................65

3.1.1 Introduction...........................................................................................................................................65
3.1.2 Dna Is The Genetic Material - Evidences.............................................................................................65
3.1.3 Structure Of Dna - Dna Is A Double Helix...........................................................................................67
3.1.4 Properties Of Dna.................................................................................................................................69
3.1.5 Role Of Dna..........................................................................................................................................70
3.2 DNA REPLICATION......................................................................................................................................70
3.2.1 Dna Replication Is Semiconservative...................................................................................................70
3.2.2 Mechanism Of Replication...................................................................................................................72
3.2.2.1 Theta - Mode....................................................................................................................................72
3.2.2.2 DNA replication in Phages - Sigma mode (rolling-circle replication).............................................74
3.2.2.3. The Replication Enzyme And Its Properties....................................................................................74
3.2.2.4 The Growing Point Paradox And Semi-Discontinuous Replication................................................75
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3.2.2.5. The Replisome.................................................................................................................................76

3.2.2.6. Applications: (Tutorial Topic)..........................................................................................................78
3.2.2.6.1 Polymerase Chain Reaction (PCR)........................................................................................78
3.3 Phenotypic Function Of Dna.........................................................................................................................80
3.3.1. Historical Perspectives Of The Central Dogma....................................................................................80
3.3.1.1 One gene-one metabolic block.........................................................................................................80
3.3.1.2 One gene-one enzyme hypothesis....................................................................................................80
3.3.1.3 One gene-one polypeptide...............................................................................................................81
3.3.1.4 Colinearity of gene and protein........................................................................................................81
3.3.2 Exceptions: Non-colinearity; one gene many polypeptides..................................................................81
3.3.3 Transcription.........................................................................................................................................82
3.3.3.1 Definition.........................................................................................................................................82
3.3.3.2 RNA Polymerase Enzyme................................................................................................................82
3.3.3.3 Steps in Transcription:.....................................................................................................................83
3.3.4 Translation............................................................................................................................................84
3.3.4.1 Definition.........................................................................................................................................84
3.3.4.2 The Protein Factory..........................................................................................................................85
3.3.4.3 The Adapter Molecule (tRNA)........................................................................................................85
3.3.4.4 Steps in Translation or Protein Synthesis.........................................................................................85
3.3.4.5 Polysomes:.......................................................................................................................................87
3.3.5 The Genetic Code.................................................................................................................................87
3.3.5.1 Nature Of The Genetic Code - Experimental Evidence...................................................................88
3.3.5.2 Cracking the Genetic Code:.............................................................................................................88
3.3.5.3 How Does The Cell System Cope With Degeneracy.......................................................................90
3.3.5.3.1. Crick's Wobble Hypothesis:....................................................................................................90
3.3.5.3.2 Iso-accepting species of tRNA...............................................................................................90
3.3.6 Gene Regulation....................................................................................................................................91
3.3.6.1.1 The Lac Operon......................................................................................................................93
3.3.6.1.2 The Trp Operon......................................................................................................................96
3.3.6.1.3 Temporal Control Of Genes In Phages...................................................................................97
CHAPTER FOUR.....................................................................................................................................................111
POPULATION GENETICS.....................................................................................................................................111
4.1 Introduction..................................................................................................................................................111
4.2 Populations:.................................................................................................................................................111
4.2.1 Genetic Variation.................................................................................................................................112
4.2.1.1 Measures Of Genetic Variation......................................................................................................112
4.2.1.1.1 Concept of allele frequency.................................................................................................112
4.2.1.1.2 Concept of polymorphism...................................................................................................113
4.2.1.1.3 Concept of Average heterozygosity:....................................................................................114
4.2.1.2 Methods Of Determining The Genetic Variability Of Populatons.................................................114
4.2.1.2.1 Phenotypic study..................................................................................................................114
4.2.1.2.2 Protein electrophoresis.........................................................................................................115
4.2.1.2.3 DNA Polymorphisms:...........................................................................................................118
4.2.1.3 Usefulness Of Studying Genetic Variation.....................................................................................119
4.2.1.4 Inheritance Of Genetic Variability - The Hardy-Weinberg Model.................................................120
4.2.1.4.1 Hardy-Weinberg Model........................................................................................................120
4.2.1.4.2 Proof Of The Hardy-Weinberg Principles............................................................................121
4.2.1.4.3 Test Of H-W Equilibrium.....................................................................................................122
4.2.1.4.4 Applications Of The H-W Model.........................................................................................124
4.2.1.4.5 Extensions Of The H-W Model............................................................................................125
4.3.1 Causes Of Evolution...........................................................................................................................135
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4.3.1.1 Definition.......................................................................................................................................135
4.3.1.2 Evolutionary Forces.......................................................................................................................135
4.3.1.2.1 Migration.............................................................................................................................135
4.3.1.2.2. Neutral Mutations.................................................................................................................136
4.3.1.3 Selection.........................................................................................................................................144
4.3.1.3.1 Definition:.............................................................................................................................144
4.3.1.3.2 Phenotypic selection:............................................................................................................144
4.3.1.3.3 Fittness..................................................................................................................................144
4.3.1.3.4 Selection coefficient:............................................................................................................145
4.3.1.3.5 Directional, stabilizing and disruptive selection..................................................................145
4.3.1.3.6 Selection Changes Allelic Frequencies................................................................................145
4.3.1.3.7 Selection - Mutation Equilibrium........................................................................................148
4.3.1.3.8 Hard Selection And Soft Selection.......................................................................................149
4.4 Polymorphisms.............................................................................................................................................150
4.5 Examples Of Evolution.................................................................................................................................151
4.5.1 Plant And Animal Breeding................................................................................................................151
4.5.2. Industrial Melanism............................................................................................................................152
4.5.3 Pesticide Resistance............................................................................................................................152
4.6 PRACTICAL II: APPLICATIONS OF HARDY-WEINBERG EQUILIBRIUM - (PROBLEM SOLVING)...155
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BIOL2162 – ADVANCED GENETICS

(4 CREDITS)
The course deals with the organization, structure, function and regulation of the genetic material of
prokaryotes and eukaryotes (molecular and gross levels). The course introduces the student to the
concept of a gene and methodologies that have led to the advancement of knowledge on gene
control. The goals and detailed learning objectives are outlined in another handout.
Pre-requisite
The course requires a pass in an introductory level genetics course (BIOL 1061 OR AGRI 1011 or
any other course accepted by the department) as a pre-requisite. This course will serve as a pre-
requisite to other tertiary level courses viz: BIOL3061: Molecular Biology; BIOL3762: Plant
Biotechnology and BIOL3763: Crop Improvement.
This course is a core course for students majoring in Biology.
Lectures/ Tutorials and Practicals:
The course consists of 3 hours lectures per week. There is also a 5 hour laboratory/tutorial session
every other week. If the class size is large the class will be divided into two laboratory streams to
facilitate the conduct of practicals.
Examination:
Incourse Assessments = 40%

Three in-course tests (3 x 10) = 30%
Quizzes = 10%
Theory paper (2 hours) = 60%
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THE UNIVERSITY OF THE WEST INDIES, ST. AUGUSTINE

DEPARTMENT OF LIFE SCIENCES.
BIOL2162: ADVANCED GENETICS
Course Goals:
At the end of the course students would be able to understand
i. The cellular and molecular organisation and function (genomics) and mechanisms of recombination in prokaryotic and
eukaryotic genomes.
ii. chromosome mutations – diagnosis, cytogenetic effects, inheritance and role in evolution.
iii. DNA structure and replication; storage of genetic information in DNA; evolution of the concept of a gene and the expression
and regulation of genes; and recombination mapping, complementation mapping and deletion mapping.
iv. Genes in population; genetic structure and evolution of populations; and methods of analysing populations.
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1 Topic Learning objectives

CHAPTER 1
Eukaryotic chromosome structure/organisation  Students should be able to explain the chromosome theory of
inheritance that led to the evolution of the science of
cytogenetics; describe chromosome structure at the macro and
ultra levels; and define unineme model.
 Students should be able to define karyotyping; describe how it

is done; and write an account of its uses, with examples.
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CHAPTER 1
Eukaryotic chromosome structure/organisation (ctd…)  Students should be able to differentiate between hetero – and
euchromatin; and between facultative -, constitutive – and
condensed heterochromatin; describe staining methods to
reveal these; and be able to discuss the role of these in
regulation at the chromosomal level with a clear definition
and example of ‘position effects’
 Students should be able to write brief notes on polytene and

lampbrush chromosomes with clear definitions, detail their
role in tissue specific amplification and be able to write a note
on their use in karyotyping.
Chromosomal aberrations
deletions  Students should be able to define deletion; enumerate types of
it; describe the methods of diagnosis, both cytological and
genetic; describe its inheritance and explain its use in deletion
mapping.
duplications  Students should be able to define duplication; enumerate types

of it; detail cytological and genetic evidence with examples;
and write an essay on the evolutionary importance of
duplications with examples. Students should also be able to
define multigene families, superfamilies and pseudogenes,
with examples. Students should be able to enumerate the
evolutionary mechanisms that result in multigene families and
multigene superfamilies.
 Students should be able to write an essay on multigene
families- evolutionary flexibility it provides to the organism –
developmental flexibility, environmental flexibility and
differentiation. (with examples).
 Students should be able to define homeobox genes (homeotic
genes of Drosophila and hox genes in mammals) – origin,
evolution and function as master control genes. Role of
homeotic genes in the development of Drosophila.
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Chromosomal aberrations (ctd…)

inversions  Students should be able to define inversion; enumerate types
of it; describe with diagrams cytological transmission of peri-
and paracentric inversion; describe genetic effects and
evolutionary importance.
translocations  Students should be able to define translocations and

enumerate types of it; list methods of cytological and genetic
detection of it and be able to provide cytogenetic explanation
for the listed genetic effects with diagrams; differenciate
between alternate and adjacent segregation; write notes on (a)
sterility in translocation heterozygotes; (b) inheritance of
translocations in pest control; (d) multiple translocation
systems and their effects (e) evolutionary importance of
translocations. Cytogenetic effects of translocation.
 Epigenetic inheritance - Epigenetics, epigenome, epigenetic marks, stem cells,

Epigenetic imprinting – chromatin remodeling –
histone
code hypothesis; DNA methylation,
Epigenetic memory – mechanisms (a) chromatin based
mechanisms (b) DNA methylation mark transfer
Epigenetics – animals vs plants.
Uses of epigenetic imprinting.
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Changes in chromosome number  Students should be able to define basic chromosome number
(x) and be able to differentiate it from haploid number (n);
differentiate euploidy from aneuploidy; and define polyploidy.
Aneupliody  Students should be able to define and differentiate between

hyperploidy and hypoploidy with examples; list uses of
aneuploidy in monosomic analysis and able to write notes on
chromosome manipulation methods in plant breeding and
monosomic analysis.
Euploidy  Students should be able to define and differentiate between
autopolyploids and allopolyploids; write notes on (a) effects
i. autopolyploidy of autopolyploids (b) inheritance of autopolyploids (c)
induction of autopolyploidy; (d) sterility in triploids (e)
polyploid series in Musa spp.; differentiate between random
chromosome assortment and random chromatid assortment
allopolyploidy  Students should be able to describe how allopolyploids are

formed; discuss the role of allopolyploidy in evolution with
examples such as wheat, Brassica spp., Musa spp., tobacco
Triticale, Raphano-brassica; briefly discuss partial sterility in
allopolyploids and process of diplodization leading to the
production of amphidiplpoids. Students should be able to
write an essay on the use of polyploids in agriculture.
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CHAPTER 2
Prokaryotic genome structure/  Students should be able to clearly differentiate between the
organisation genomes of eukaryotes and prokaryotes based on (a)
organisation (b) structure (c) inhertance/transmission and (d)
recombination.
Recombination  Students should be able to define conjugation; be able to

 Conjugation and conjugation differentiate this from other methods of recombination; be
mapping able to enumerate the functions of the F factor in conjugation;
be able to differentiate a plasmid from a episome; should be
able to differentiate between F+ hfr and F’ factors with respect
to description, origin, ability to convert recipients to donor,
fate of transferred DNA, recombination frequency, probability
of recombination of any gene and their uses; differentiate
between Lfr and Hfr transfer and sexduction; be able to
describe the use of Hfr strains in genome mapping.
Conjugation mapping methods  Students should be able to describe clearly the interrupted
mating technique of genome mapping and critically discuss it
in relation to other methods; should be able to describe
evidences that led to chromosome circularity; should be able
to describe the method of ordering genes on the bacterial
genome using ‘gradient transfer’; should be able to describe
the method of high resolution mapping(recombination
mapping) in bacteria and critically discuss this in relation to
other methods of mapping. Students should be able to solve
problems on conjugation mapping.
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Transduction  Students should be able to define and be able to clearly

distinguish between generalized and specialized transduction;
between virulent phage and temperate phage; between lysis
and lysogenic modes of replication of lamda phage; and
between hfr lysate and lfr lysate. Should be able to explain
the use of co-transduction frequencies in genome mapping
and solve problems.
Transformation  Students should be able to define and list the steps in

transformation; discuss the factors that affect transformation
efficiency; and be able to calculate co-transformation
frequency and construct genetic maps.
Concept of complementation/cistron  Students should be able to clearly define complementation, be

able to distinguish it from recombination, describe how a
complementation test (cis-trans test) is carried out and discuss
the merits and limitations of this test; Students should be able
to describe the use of sexduction in complementation tests, be
able to define complementation maps and describe how they
are constructed?; and be able to define clearly the term
‘cistron’
 Students should be able to describe complementation and

recombination mapping of the rII locus in T4 phage; define
and distinguish between the terms, cistron, recon, muton,;
should be able to describe the Benzer’s deletion mapping
technique and discuss its use in gene fine structure analysis.
Modern concept of the gene  Students should be able to trace the evolution of the concept
of a gene from the Mendelian concept to the modern concept.
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CHAPTER 3
MOLECULAR GENETICS  Students should be able to list the evidences that led to the
conclusion that DNA is genetic material- Griffith’s
 DNA is the genetic material transforming principle; Avery, McLeod and McCarty’s
elucidation of DNA as the transforming principle; Hershey-
Chase experiment; reconstituted TMV experiments.
 Students should be able to describe DNA structure – double

helical nature; complementarity of strands; antiparellel nature
of strands (polarity) and be able to provide experimental
evidences for them.
 DNA replication  Students should be able to describe the various models

proposed to explain DNA replication and provide
experimental evidences for the ‘semiconservative’
mechanism. E.g. Meselson and stahl experiment.
 Students should be able to explain and provide experimental

evidence for (a) bidirectional nature of repliction, (b) unique
origin vs multiple origin of replecation, (c) semi-
discontinuous nature of replication, (d) theta mode (moving
fork) vs sigma mode of replication (rolling circle replication),
(e) primer requirement of DNA polymerases
 Students should be able to write notes on the growing point

paradox and the okazaki hypothesis.
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 Students should be able to differentiate between the

replication apparatus of the prokaryotes from that in the
eukaryotes.
 Students should be able to define the terms replicon and

replisome; and be able to describe the steps in DNA
replication and how they are carried out by the replisome;
Describe In vitro replication (Kornberg experiment) and the
PCR.
Phenotypic function of DNA  Students should be able to define transcription, describe the
steps in transcription, and its enzymology. Students should be
able to differentiate between the transcription machinery in
prokaryotes from that in eukaryotes. Students should be able
to define split genes in eukaryotes and splicing pathways
involved. Students should be able to list post transcriptional
modification that affect mRNA stability.
 Students should be able to define translation, outline the steps

in translation, and be able to describe the role of ribosomes
and t-RNA in the process.
 The genetic code  Students should be able to describe the nature of the genetic
code and experimental evidence for it – triplet code, non-
overlapping, commaless/gapless, degenerate code, university
of the genetic code. Students should be able to describe
experiments that led to the breaking of the genetic code.
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 Gene regulation in prokaryotes  Students should be able to define operons, controlling

elements – cis-acting elements and trans-acting elements; be
able to write notes on promoter, operator, regulator, enhancer
etc.; Students should be able to discuss the organisation and
function of controlling elements in lac operon of E. coli; be
able to describe the mutational analysis of Jacob and Monad
which led to the elucidation of the role of contolling elements
in lac operon and be able to discuss how the regulation of the
lac operon leads to greater efficiency at the cellular level.
Students should be able to differentiate between positive
(arabinose locus) and negative control and between inducible
and repressible (histidine locus) control, with example.
Students should be able to explain coordinated control
mechanisms – temporal control in SPO1 and T7 phage,
attenuation as a method of control.
 Introduction to gene regulation in eukaryotes.  Students should have an appreciation of the more complicated
control systems in Eukaryotes – Chromosomal,
transcriptional, post-transcriptional, translational control
mechanisms. Students should be able to define transposons
and transposition.
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MOLECULAR GENOME ORGANISATION AND

REGULATION
Eukaryotic genome organisation  Students should be able to define C-value and C-paradox, be
able to clearly differentiate between kinetic complexity,
chemical complexity and briefly outline how they are
measured.
- Complexity of the genome  Students should be able to discuss the components that make
up the non-repetitive fraction of the genome, moderately
repetitive sequences and highly repetitive sequences and their
roles.
- Gene evolution  Students should be able to discuss the theories of the
- Introns evolution of genes in light of the understanding of the
variation of structure of genes in organisms. Students should
be able to write notes on intervening sequences.
- Gene families & clusters  Students should be able to explain the evolution of multigene
families and their advantages to organisms with examples.
- Other repetitive sequences.  Students should be able to distinguish between satellite DNA,
minisatelite-DNA, SINES and LINES.
Regulation of genes in eukaryotes.  Students should be able to clearly distinguish between gene
regulation in prokaryotes and eukaryotes. Students should be
able to distinguish between control at the chromosomal,
transcriptional, translational post-translational and functional
levels. Students should be able to explain the structure of
- Eukaryotic promoters eukaryotic promoters, enhancers and their function.
- Transcription complexes  Students should be able to write notes on transcription

complexes and should be able to explain regulation in named
examples.
- Protein transport and turnover  Students should be able to explain mechanisms of protein
transport and turnover.
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CHAPTER 4
POPULATION GENETICS  Students should be able to define ‘population’, ‘gene pool’
and ‘population genetics’’; be able to characterise populations
1 Genetic diversity and its measurement based on their universal attribute – ‘genetic diversity’.
Students should be able to define and clearly differentiate
between the parameters used in studying genetic diversity –
allelic frequency, percentage polymorphic loci and average
heterozygosity. Students should be able to describe and
critically discuss the methods used in studying genetic
diversity. Students should be able to enumerate the uses of
studying genetic variation in populations. Students should be
able to discuss the statement –‘mendelian populations are the
units of evolution’
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2 Inheritance of genetic variability – the Hardy-Weinberg  Students should be able to define the Hardy-Weinberg model,
model provide a proof of it and describe the conditions under which
the model is upheld. Student should be able to clearly outline
the implications of H-W equilibrium.
3 Applications of H-W equilibrium  Students should be able to evaluate, based on allelic
frequencies, whether a population is in Hardy-Weinberg
equilibrium with respect to a character. Students should be
able to calculate the frequency of heterozygotes, under
dominance; calculate equilibrium allelic frequencies (H-W),
for X-linked genes, genes under linkage disequilibrium and
multiple alleles. Students should be able to describe how
mating systems can affect H-W equilibrium; differentiate
between the various mating methods, and calculate inbreeding
coefficients from pedigree records.
4 Causes of evolution  Students should be able define evolution, understand that
evolution is the process as well as the result; and list the
causes of evolution. Students should be able to define the
 Random genetic drift selectionist view of evolution and differentiate that from the
neutral theory of evolution. Students should be able to define
neutral, silent, synonymous and nonsynomymous mutations.
Students should be able to describe the probability of ultimate
fixation of a neutral allele, time to fixation, rate of fixation,
average time between fixation , probability of polymorphism
and the average heterozygosity, based on the neutral theory.
Students should be able to describe what is meant by the
molecular clock and be able to write notes on the uses of it;
define random genetic drift and describe its consequences.
Mutation and selection  Students should be able to describe how mutations affect the
evolution and describe equilibrium conditions for neutral
mutations. Students should be able to describe selection,
describe the various types of selection and describe the effect
of selection on allelic frequencies – selection favouring the
dominant allele, recessive allele, additive allele and the
heterozygote.
1  Stiudents should be able to define polymorphism and describe
13
conditions under which polymorphisms are maintained.

Students should be able to write notes on the following with
examples frequency dependent selection, heterozygote
advantage and inferiority, evolution of industrial melanism,
evolution of selection is applied to improve populations in
plant breeding.
2  Students should be able to describe mutation-selection
balance and be able to calculate the frequency of the mutant
phenotype and equilibrium allelic frequencies.
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CHAPTER ONE
CYTOGENETICS
1.1 INTRODUCTION
1.1.1 The emergence of cytogenetics.

-
The chromosome theory of inheritance led to the beginnings of the science of cytogenetics.
1.1.2 Chromosome theory of inheritance
Proposed by Walter Sutton and Theodor Boveri in 1907, the theory states that the chromosomes are
the units of inheritance and therefore perhaps the carriers of the genetic material. The evidence for
this came from the parallality observed between the behavior of chromosomes during meiosis and
that predicted for Mendel's 'particles' in Mendels principles.
- Members of a gene pair determining a character segregate at equal frequency into gametes
(So do members of a pair of homologous chromosomes during meiosis).
- Different gene pairs segregate independently (so do different chromomosomal pairs)
Other evidences came from the fidelity of chromosomal divisions in mitosis, constancy of
chromosome numbers maintained in subsequent generations etc.
1.1.3 What is Cytogenetics?
The chromosomes became the centre of interest following the elucidation that chromosomes are
perhaps the units of inheritance and this led to the beginnings of the science of cytogenetics.
Cytogenetics therefore deals with the study of nuclear organisation of chromosomes as well as
chromosome structure and behavior during meiosis. This also deals with the study of the
cytogenetic effects of chromosomal mutations (changes in structure and number) on phenotype,
inheritance and evolution.
The primary concern of cytogenetics is therefore to first characterise the genome and the
chromosomes within it, so that any changes to the organisation or structure can be detected and
correlated with behavioral changes or evolutionary leaps.
Chromosome
The chromosome biochemically is a nucleo-protein made up of DNA and proteins. The protein
component is a simple protein called histones. Five histone proteins (H1, H2A, H2B, H3 and H4)
make up the lattice work of the chromosomes which carries a single very long molecule of the
double helical DNA. The one chromosome-one DNA molecule relationship is referred to as the
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unineme model, which is widely accepted now. The multineme model suggested that the
chromosome is made up of several molecules of DNA.
Ultra structure of the Chromosome:- The unit of packaging in a chromosome is a nucleosome.

Nucleosome consist of a spherical core made of eight molecules of histone protein (octomer
-2H2A, 2H2B, 2H3 and 2H4) around which there are two turns of the double helical DNA (140 bp)
wrapped around. The double helical DNA of adjacent nucleosomes are connected by linker DNA.
The stability of this continuous nucleosome thread, which forms the primary structure of the
chromosome, is given by the H1 protein. The nucleosome thread or nucloeosome fibre (100Ao) is
thrown into loops and coils to form a solenoid structure (200-300 Ao) -approximately six
nucleosomes per turn. The solenoid structure can be further thrown into tertiary and quaternary
coiling to form the metaphase chromosomes during meiosis.
Characterisation of the genome
The genome can be characterised in terms of
a) the basic chromosome number ( monoploid number)

This refers to the number of chromosomes in the basic chromosome set. The basic
chromosome set refers to the unique set of chromosomes within the nucleus. eg. the basic
chromosome number in a human nucleus is 23. The basic chromosome number is generally
indicated by 'X' X is 4 in Drosophila (fruit fly), 10 in corn and 23 in Man.
b) ploidy
This refers to the number of repetitions of the basic chromosome set within the nucleus.
Such whole set repetitions of the basic chromosome set is called Euploidy. Animals are
diploids in that they have only two basic chromosome sets and cannot tolerate higher levels
of ploidy. Plants however can tolerate higher ploidy levels. eg. corn, tomato, cowpea,
pigeonpea etc are diploid, but banana is triploid, irish potato is tetraploid, sweet potato is
hexaploid etc.
Some individuals have an incomplete chromomosomal set or have supernumerary

chromosomes in some sets - these are called aneuploids and are departures from the normal
ploidy levels. Such repetitions of the basic chromosome set that are not whole number
replicates of the basic chromosome set is called aneuploidy. These abnormalities are often
associated with serious debilitating features. We shall deal with these later.
c) Characterising chromosomes within the basic chromosome set
Chromosomes can be characterised based on size, shape, unique features such as nucleolar
organiser, satellites or banding pattern or combinations of these.
SIZE: The size of the chromosome is the easiest to distinguish, and can be measured as the
length or diameter. The chromosomes are categorised into A, B, C groups etc based on
length. Length can vary between 0.5-400 uM.
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SHAPE: Shape is imparted to a chromosome by the relative location of the centromere. The
centromere is the primary constriction on the chromosome, which divides the chromosome
into arms. If the centromere is at the centre of the chromosome giving two equal arms the
chromosomes are called mesocentric or metacentric. If it is towards one side, it leads to
unequal arm lengths and is referred to an acrocentric chromosome. The degree of
acrocentricity can also vary. The centromere at an end leads to telocentric chromosomes.
Secondary constrictions arise due to the presense of gene clusters, which are a series of
sequentially repeated genes. An example is the nucleolar organiser, which is a region of
chromosome which contains a rRNA gene cluster. Associated to this gene cluster, you will
find the nucleolus, which is a spherical darkly stained body in the nucleus, which stores,
rRNA. Different organisms have varying numbers of nucleoli and associated nucleolar
organiser regions. This can also be used as a landmark to classify chromosomes. Secondary
constrictions can be found in association with satellite DNA, as well. These are tandemly
repeated non-coding sequences found in the DNA. These can also be used to distinguish
chromosomes. These constrictions are referred to as satellites.
BANDING PATTERNS: These are features of the chromosomes that can also be used to
distinguish between chromosomes. The banding pattern shows the location of constitutive
heterochromatin (darkly stained) as opposed to Euchromatin (lightly stained). The
constitutive heterochromatin is associated generally with the centromeric region,
telomeric region (associated with chromosome ends) and intercalary regions. In addition to
these patterns, during cell division, certain regions of the chromosomes coil more heavily to
reveal a characteristic pattern of chromomeres.
Quinacrine mustard staining reveals florescent banding patterns on chromosomes referred to

by cytogeneticists as Q bands. The impermanence of these florescent bands make this a
serious disadvantage.
Giemsa staining is therefore a more popular method. This produces dark bands called 'G
bands'. A variation of this technique can reveal 'R bands', which are the reverse of 'G
bands'. When it is fixed with Alkali it produces a C banding pattern, which is specific to
the heterochromatic region. Giemsa staining is hence very versatile.
Note 1: Heterochromatin
The heterochromatic regions are heavily coiled regions of the chromosome which are
functionally inert. The Euchromatic regions seem be the functionally active regions. The
heterochromatin can be subdivided into constitutive heterchromatin, facultative
heterochromatin and condensed heterochromatin. The constitutive heterochromatin is
associated with the telomeres, centromeres, intercalary regions, sattelites etc. These are the
highly repetitive non-coding sequences. Facultative heterochromatin reflects the
regulatory devices designed to adjust the dosage of certain genes. Condensed
heterochromatin is distributed differently from tissue to tissue and appears during cell
maturation. This reflects the permanent turning off of certain genes during cell
differentiation.
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Note 2: Position effects

The relative expression of certain genes in the euchromatic region can be affected by their
relative proximity to heterochromatic regions. This spreading suppressing effect of
heterchromatic regions on genes in euchromatin is referred to as position effects. The closer
the proximity to the heterochromatic region, greater is the suppressing effect. see text book
for examples.
Facultative heterochromatin and position effects demonstrate mechanisms of control of gene

expression at the chromosomal level.
1.2 Karyotyping
1.2.1 Macro and Ultra Structure of the Chromosome

Karyotype is a diagrammatic representation of all the chromosomes (at the mitotic metaphase) of a
cell arranged in homologous pairs of decreasing size, indicating characteristic distinguishing
features of the chromosomes.
1.2.2 Characterisation of the Genome

The Karyotype of a human cell is given below.
Figure 1
1.2.3 Karyotyping
Karyotyping is carried out by examination of mitotic metaphase chromosomes. In plants, growing
root tips are squashed, stained and metaphase chromosomes in transverse views are micro-
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photographed. The photograph is enlarged. The chromosomes are then traced and transferred onto a
drawing sheet in homologous pairs in descending order of size, indicating the landmark features that
enabled you to distinguish the homologous pairs.
Karyotyping is made easier in Drosophila and amphibia by the presence of special giant
chromosome structures in certain organs.
Polytene chromosomes
In the salivary glands of insects in the order of Diptera (flies, mosquitos, midges) nucleii enlarges by
extra replication of each chromosome within the nucleus through a process called enodpolyploidy
or polyteny. The replicated chromosomes stay together in stacks, resulting in thick giant
chromosomes.
In such chromosomes the chromomeres are seen as thick bands and can be visually observed in a
light microscope. In Drosophila, the four chromosomes are joined together by a chromocentre.
Each of the chromomeres in Drosophila, correspond to a gene. In Drosophila there are 5000-6000
chromomeres. The bands expand to form 'puffs' when they are active and are condensed otherwise.
Chromosomal aberrations can be detected as loops.
Lampbrush chromosomes
Found in the oocytes of some amphibia with large yorky eggs. The meiotic chromosomes reach
1000 uM in thickness with long latteral loops. Each loop arises from a single chromomere along the
double stranded DNA molecule. Loops are considered to be duplicated genetic material, which
enables amplification of the product. Some of the loops are pinched off as balbiani rings which can
also independently express the gene products. As puffs in polytene chromosomes, puffs in
lampbrush chromosomes help to overexpress material used in the cell.
1.2.4 Uses of karyotyping
1. Enables one to identify chromosomal aberrations in individuals. Chromosomal

aberrations can be in the form of ploidy changes - euploidy or aneuploidy or
structural changes to the chromosome such as loss of a segment, decreasing the
length of the chromosome or an addition of a segment that may increase the length
of an arm or an inversion of a segment that can result in difficulty in pairing in
meiosis or translocation.
Study of such chromosomal aberrations have led to the understanding of several
debilitating conditions in Man. eg. Down Syndrome.
2. Assign taxonomic status and enable the study of taxonomical relationships

Where species have been assigned close taxonomic relationships, structural as well
as banding similarities between chromosome have been found. For instance
between tomato and pepper nine out of twelve chromosomes were found to be homo
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sequential. Others have been found to contain paracentric inversions. Hence proper
karyotyping can clarify and inform classical taxonomic studies.
3. Enables understanding of evolution.
If one assumes that modifications to chromosomal structure accumulate in a

sequential process in evolutionary time. The number of accumulated differences
between species can indicate the relative degree of divergence between species
and can lead to the development of a phylogenetic tree.
1.2.5 Practical Exercise-1 – Karyotyping - I
1. An enlarged photograph of a cell in the mitotic metaphase is provided in the attached

sheet. Cut out the chromosomes and construct a karyotype. Based on the karyotype that
you have developed answer the following questions.
a) Is the specimen from a male or a female?

b) What is the basic chromosome number of the organism?
c) What is the general ploidy level of the organism?
d) What chromosomal abnormalities may be present in this individual?
2. Abyssinian oat (Avena abyssinica) appears to be a tetraploid with 28 chromosomes. The

common cultivated oat (Avena sativa) appears to be a hexaploid in this same species.
How many chromosomes does the common oat possess? What is the basic chromosome
number?
3. Make a large drawing of the Drosophila salivary gland chromosomes. How many pairs
of chromosomes are visible? Note the visible bands on the chromosomal arms. The
centromeres are attached together at a chromocenter. The giant chromosomes are thought
to be composed of 100-1000 chromatin strands (chromonemata) fused together
(polytene). The homologous pairs undergo close somatic pairing throughout the length
and cannot be distinguished.
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1.3 Chromosomal Mutations-Changes In Chromosomal Structure
Introduction
Mutations are spontaneous heritable changes that affect the genome. Mutations can be classified
as genic mutations, when they are confined to within a gene or chromosomal mutations, when
they affect part of or the entire chromosome. Chromosomal mutations in turn can be of two
kinds a) chromosomal aberrations, which are structural changes to the chromosomes b)
changes in ploidy, which are changes to the number of chromosomes. These changes can be
detected in the karyotype of the affected individual.
In the following few lectures we will learn about the various types of chromosomal mutations,
how they occur, possible ways of detecting them cytologically/ genetically, inheritance of such
aberrations, consequences of such aberrations to the organisms and how they affect the
evolutionary process.
CHANGES TO CHROMOSOMAL STRUCTURE
Changes in chromosome structure can occur as a result of deletion of a segment of the

chromosome, addition of a segment, inversion of a segment or translocation of a segment to a
non-homologous chromosome. Such changes occur very rarely in nature but can be induced to
higher levels by external factors such as high energy radiations etc.
1.3.1 Deletion Or Deficiency
1.3.1.1 Definition and types:
This refers to the loss of a chromosomal segment. There are two ways they can be produced -
terminal deletions, interstitial deletions. The latter is more the general method.
Terminal deletions occur independently by breakage of a chromosome followed by the loss of

the acentric fragment during cell division. The deletions survive only if the sticky ends heal,
successfully.
Interstitial deletion involves two homologous chromosomes and produces deletions and
duplications simultaneously. This involves breakage of the two chromosomes followed by
exchange of unequal arms. These aberrations do not result in sticky ends and hence are more
stable.
1.3.1.2 Diagnosis:
Cytology:- karyotypes or ideograms can reveal deletions as changes in the size of
chromosomes or as changes in banding pattern.
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In additions, deletions are often seen by the buckling of chromosomes

(DELETION LOOPS) during meiotic pairing of homologues. This indicates an
interstitial deletion.
Genetic:- One of the characteristic features of deletions is their inability to revert to wild
type state. All point mutations have a capacity to revert to normal wild type state.
Deletions cannot. Why?
Deletions are often lethal as a deletion homozygote and therefore behave

like lethal mutations. This indicates that through evolution the genome has been
fine-tuned to contain a specific balance of genes. The F2 generation will show a
1:2 ratio instead of a 1:2:1. Some mutation are not viable even as a deletion
heterozygote.
Deletion heterozygotes show pseudo-dominance. When you cross a wild

type with a mutant you will normally expect all progeny to contain wild type
characters, but if a deletion has occurred in a segment the mutant genes
corresponding to that deleted region may express since the wild type dominant
genes have been deleted.
Cross over suppression - When a portion of a chromosome is deleted,

buckling of homologues as we saw before affects close meiotic pairing of
homologues. This will affect the formation of chiasmata in the region of the
deletion and in the immediately adjacent regions. Hence the gene combinations
are maintained faithfully with no possibility of recombination in this region.
Partial Pollen sterility- male gametogenesis is very sensitive to deletions

in plants as a result pollen grains carrying deletions are sterile and can be detected
as shrivelled pollen. Hence a deletion heterozygote is partially pollen sterile.
Male gametogenesis, therefore serves as a screen to eliminate deletions. Ovules of
both diploid and polyploid plants are tolerant to deletions, presumably because of
the nurturing effect of the surrounding maternal tissues. Deletions are passed to
subsequent generations in plants only through female gametogenesis. Hence in
plants homozygous deletions do not occur. In animals, both, male and female
gametogenesis are not sensitive to deletions and deletions are passed on.
Homozygous deletions however do not survive. They are lost as embryo
abortions.
Some deletions produce unique phenotypes that will allow their

detection. In Drosophila spp (fruit fly) deletion of a certain chromosomal region
appears as a notched wing phenotype and operates like a dominant mutation.
Inheritance: Despite buckling or looping pairing normally occurs successfully between

homologues and therefore meiosis is normal. However due to partial pollen
sterility deletion homozygotes are not formed. In animals they are formed but are
often lethal.
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Uses: Deletions are useful in deletion mapping. Deletion mapping allows the chromosomal
location of genes. This is done by correlating character defects with chromosomal
looping or changes in banding pattern observed cytologically. Deletion maps first
provided evidence to the fact that linkage maps are infact reflections of the physical
chromosomal maps.
1.3.2 Duplications or Additions
1.3.2.1 Definition and types

This occurs when a segment of the chromosome is duplicated. Duplications may arise in
conjunction with deletions as we saw before or independently due to errors in the replication
process, by the DNA polymerase copying a chromosomal loop twice. Once duplications have
occurred, higher order duplications occur through a process called unequal crossing over. Can
you diagrammatically show how these various forms of duplication occur?
There are various types of duplications
Adjacent - tandem (abcdede.fghi)

- reverse tandem (abcdeed.fghi)
Non-adjacent - displaced homobrachial (adebcde.fghi)
- displaced heterobrachial (abcde.fdeghi)
- extra chromosome (.de)
- transposition to a homologue (klmn.opde)
Adjacent types are the most common and can lead to higher order duplications by asymmetric
pairing and unequal crossing over.
1.3.2.2 Diagnosis
Cytology: In the karyotype, they are revealed by duplicate banding patterns or if the
duplication is large by increase in chromosome length.
During meiosis, the bivalents show buckling, looping or coiling of the
duplicated segments, depending on the length and type of duplication. If the
duplication involves two chromosomes then cross pairing between chromosomes
can occur. Can you diagrammatically show how the various types of duplications
can pair as duplication heterozygotes?
Genetic: Duplications are hard to detect genetically as no function is lost. Sometimes can
be detected phenotypically. eg. bar mutation in Drosophila produces smaller eyes,
double bar mutations as a result of duplication produces slit like eyes.
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Cytological transmission/ inheritance:
There is normally nor pairing problems during meiosis as duplicated segments are looped out in
duplication heterozygotes. Hence, transmission is normal. This is why duplications have
accumulated in genomes through evolution as they do not render the individuals less fit.
However, non-tandem mutations can rarely undergo crossing over within the looped segment of
the duplication giving rise to dicentric chromosomes which behave abnormally giving rise to
some level of sterility. But this is rare.
1.3.2.3 Evolutionary importance of duplications
We saw in the previous section that duplications fuel further duplications due to asymmetric
pairing and unequal crossing over. We also saw that duplications are very rarely deleterious and
hence they tend to accumulate during evolution as they do not confer a disadvantage to the
organism. Such duplications we shall see in this section can infact be advantages to the organism
by serving as an evolutionary pump.
a) Divergence through duplication:

When a segment of a chromosome is duplicated one of the duplicated segment is free to
undergo divergence (through mutations/ shuffling) to assume new functions while the
original copy can maintain the original function. This has led to the origin of multigene
families and super families. Multigene family is a family of genes that are derived from a
single ancestral gene, but have undergone various degrees of divergence over
evolutionary time. Multigene families provide developmental flexibility as in the case of
the B-globin family, provides environmental flexibility as in the case of the trypsin family
or provides specificity to a wide range of antibodies as in the case of the immunoglobin
family or serves diverse function as in the case of the actin family. Another example is
the t-RNA genes. These have made the organism better fit for survival.
b) hybrid genes
Similarly, divergence can result due to unequal crossing over between members of a
multigene family of genes leading to hybrid genes. A deleterious example is the
thalassemia (a kind of inherited blood disorder with anaemic symptoms) This is caused
by unequal recombination between a family of B globin genes, delta, beta and gamma.
Unequal c.o between delta and beta results in part delta-part beta, resulting in lepore
haemoglobin type. Similarly unequal recombination between gamma and beta genes
results in part gamma-part beta gene (kenya haemoglobin type). Both lepore- and kenya-
haemoglobin types produce anaemic symptoms.
c) Complementary genes and polygenes

Complementary genes, few genes involved in producing a phenotype or multiple gene are
also considered to have originated initially as duplications.
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c) Cluster genes:
Duplicated genes have often served to increase the product, where large quantities of the
product is necessary for cell function. One example in the Xenopus 5S rRNA gene cluster
at the nucleolar organizer. Another example is the Sea urchin histone gene cluster;
histones are the most abundant protein in cells. In some cases, duplication of functional
domains or regulatory regions of the genes result in increased gene product.
d) Permanent heterozygosity:
Heterozygosity in many organisms is associated with heterosis, or hybrid vigour. Since
heterozygous situations would lead to segregation it cannot be fixed in an individual line.
Duplication is one of evolution’ way of bringing together heterotic allelic combinations
on a single chromosomes so that it can be fixed (or permanent feature) to the individual
and to its progeny.
e) Backup genes:
Duplication or redundancy is also a way of backing up genes to provide greater stability
to the organism.
f) Satellite DNA
Non-coding sequences in chromosome termini and intercalary regions may also aid in
pairing of chromosomes and recombination, and have served as important hypervariable
regions used in DNA fingerprinting.
NOTE: MULTIGENE FAMILY
eg: The haemoglobin gene family.

Haemoglobin is a tetrameric protein in association with a heme group. It is coded for by the
globin gene family. The human genome consists of a beta cluster consisting of five functional
genes (episilon, gammaA, gammaG, delta and beta) and a pseudogene and an alpha cluster
which consists of three functional genes (alpha1, alpha2, zeta) and two pseudogenes.
Pseudogenes are non-functional copies of the genes and are considered remnants of evolution.
Two alpha-like and two beta-like polypeptides form the tetramer.
Embryo Foetus Adult

alpha-like zeta alpha alpha
beta-like episilon gammaA,G beta, delta (rare)
The various forms of haemoglobin cater for the varying requirements of oxygen during
development. For instance, during foetal development, oxygen is provided to the foetus solely
through the mothers blood and a greater affinity to oxygen is required by the haemoglobin to
meet the demands of the foetus and this is infact the case.
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1.3.2.4 Multigene Families, Their Evolution And Evolutionary Significance
What are multigene families?
A group of genes, linked or unlinked, related in both structure and function, which probably
arose by duplication of a primordial mother gene, followed by divergence. In other words,
members of a gene family share a common ancestry, and because of this the members possess
some level of structural homology and functional similarities. Some members of the gene family
lose their function during evolution and are referred to as pseudogenes. Multigene families are
more common than it was once thought and with the numerous gene families being discovered
every year, it is becoming apparent that multigene families may be the norm than the exception
in higher eukaryotes.
Gene families can be classified based on whether all members are linked (undispersed family),
some members linked and others unlinked (dispersed family), all members are unlinked
(holodispersed family). Members who are unlinked but are on different arms of the same
chromosome are called ‘Syntenic families’.
Apart from gene families of coding sequences, there are families of related sequences that are
non-coding. Tandemly repeated non-coding sequences are called satellite sequences, while those
that are dispersed within the genome are referred to as SINES (short interspersed sequences) and
LINES (long interspersed sequences).
Multigene super family
Several sets of multigene families that share some recognisable homology implying a common
ancestry have undergone major divergence in function and have been relocated within the
genome, that they cannot be referred to as belonging to a gene family. These sets of historically
related but functionally distinct genes constitute a multigene superfamily.
One of the best characterised multigene superfamily is that of the immune system superfamily,
which includes genes that code for the immunoglobulins (Ig), major histocompatibility complex
(MHC), T-cell receptors and other cell receptors. These are related in structure and with some
functional similarities. For example, they all encode proteins having cell surface functions.
MHC codes for molecules that recognise foreign antigens. Both MHC and Ig provide for an
immune response but do it in different ways, the former provides a cellular response, while the
latter a humoral response. Hence the superfamily provides various levels of protection to the
cell.
Origin and evolution of multigene families
Divergence:- Multigene families originate by duplication of the primordial gene, followed by

sequence divergence of the duplicated copies. Duplicated genes evolve is separate ways under
the influence of mutation, natural selection and random genetic drift. Divergence over
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evolutionary time allows the duplicated genes to assume new but related functions. Some
members of the multigene family lose their function and are referred to as pseudogenes.
Homogenisation:- Members of a multigene family tend to retain more similarity than would be
expected due to concerted evolution. This is because there are mechanisms that operate to
homogenise the sequences within the family. The two homogenising mechanisms are gene
conversion and unequal crossing over, counteract the process of diversion of genes and continue
to maintain some homology among members of a gene family. The homogenisation process has
been described by some researchers as the molecular drive.
Gene conversion is a process in which nucleotide pairing between two sufficiently homologous
genes is accompanied by the excision of all or part of the nucleotide sequence of one gene and its
replacement by a replica of the nucleotide sequence from another member of the family. In other
words, the sequence in one gene “converts” the sequence of the other gene to be exactly like
itself.
In tandem multigene families the mismatch pairing between non-matching members of the gene
family during meiosis and crossing over can lead to one type of sequence in the gene family over
represented. Unequal crossing over can result in the increase or decrease in the number of genes
in the family. The net result of unequal crossovers is genetically equivalent to gene conversion,
since nucleotide sequences of some members of the gene family are replaced by nucleotide
sequence from other members.
Dispersion:- Multigene families can become dispersed either by inversion or by translocation.

Dispersion of sequences can also occur by transposition. This is often mediated through an RNA
intermediate. The dispersed copies are referred to as processed pseudogenes.
Retroviruses can undergo reverse transcription and integration into the genome. Some genomes
contain 10-100 copies of these retrovirus sequences, which are referred to as virogenes. Once
established in a genome virogenes undergo sequence evolution. Retroviruses can incorporate
host genes such as the oncogene growth factors that contribute to cancer formation.
Evolutionary significance of multigene families
The individuals of a gene family perform a somewhat different role in the lives of the organism
and accounts for the complex levels of differentiation, developmental flexibility and
environmental flexibility of highly evolved organisms.
a) Developmental flexibility: The most obvious example of different members of the family
participating in different periods of development are the  and -globin genes. These genes
produce different forms of the haemoglobin in different periods in the life of the human -
embryo, foetus, and adult. (give details). Another example is the distinction between foetal
and adult forms of the muscle acetylcholine receptor of cattle.
b) Differentiation: The homeobox gene superfamily illustrates yet another aspect of family
function – regulation of development by encoding binding proteins that are involved in the
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regulation of transcription of proteins, with important functions in differentiation. All

homeobox genes share a 180-bp homeodomain (HD), which is highly conserved. Once they
are encoded they start a cascade of effects culminating in a fully developed adult. These can
be considered master switches that determine spatial and temporal patterns in development in
Eukaryotes.
For example, Drosophila has two homeobox gene clusters in Chromosome 3R, BX-C
(Bithorax), ANT-C (Antenapedia). The proteins of these families act as transcription factors,
which control an array of genes involved in development. Mutations in Ubx domain
(consisting of 6 loci) of BX-C show transformation in thoracic segmentation, nervous
system, musculature. Mutations in ANT-C family (at least four genes known) results in
changes in the head and anterior thoracic segments. (refer to powerpoint presentations).
Similarly collagen gene family in human can produce 23 different types of collagen. These
are involved in cell to cell adhesion, maintain integrity to the vertebrate organs and various
other supportive function, as well as wound healing. These are transcriptionally controlled
during various stages of development and differentiation by different transacting proteins.
This shows that differentiation of multicellular organisms is as a result of many multigene
families.
c) Environmental flexibility: Organisms to be able to survive in a diverse environmental

influences should have the flexibility of using different substrates or food depending on the
availability, deal with different forms of invasion of pathogens, deals with a diverse array of
environmental toxins etc. Multigene families provide the organism with such flexibility.
For example, Immune system super family as we saw can mount diverse levels of immune
responses against invading organisms. The major families Ig, MHC and T-cell receptor
complex are directly involved as the major components of the vertebrate immune system.
The members of these families are dispersed over eight chromosomes in humans. The MHC,
Ig and T-cell families have different but related functions. They provide for an immune
response but do it in different ways. In the immunoglobin system the B lymphocytes
circulate in the blood plasma and secrete antibodies, which attack carriers of foreign
antigens. The MHC system acts with T-type lymphocytes and macrophages, which do not
secrete antibodies but have receptors on the cell membrane. The T-Cell system is not well
understood, it functions like the Ig system but does not secrete antibodies.
The P450 family encodes the cytochrome 450 enzymes, which are involved in oxidative
metabolism of a wide variety of compounds such as prostaglandins, steroids, fatty acids,
various plant products, drugs, and toxic compounds in the environment such as carcinogens
and pesticides. It is widely dispersed family with genes present in seven chromosomes in
eight clusters. The P450 enzymes have a myriad of monoxygenase activities ranging from
oxidation of simple aliphatic compounds to oxidative deamination, to sulfoxide formation
and hydroxylation of aromatic compounds among others. These enzymes allow the liver to
detoxify many different toxic compounds. These are inducible enzymes induced by these
toxicants. Some of these enzymes participate in the synthesis and degradation of steroids and
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catabolism of fatty acids. This allows organisms to survive in toxic environments and to
ingest a wide array of food that can be detoxified.
Similarly the trypsin- and the amylase gene families allow the utilisation of a wide array of
compounds. The human alpha amylase family for instance contains five active genes and
one pseudogene, two of these genes encode pancreatic amylase and three amylase salivary
enzymes of parotid glands.
1.3.3 Inversions
Rearrangement of genetic material involving a change in the gene order. Inversions can be
generated by simultaneous break at two points in a chromosome followed by an incorrect reunion.
There are two different kinds of inversions relative to the position of the centromere.
1.3.3.1 Types:
 Paracentric inversions - Centromere not included in the inversion.
 Pericentric inversions - where inversions span the centromere.
(Generally pericentric is less frequently found than paracentric)
Figure 2
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1.3.3.2 Diagnosis:
Cytological detection:
- Karyotype may show marked changes in banding pattern in the inverted region of the
chromosome. In Drosophila salivary gland chromosomes inversions are seen as loops in
the karyotype, due to linear pairing of chromosomes.
- Changes in arm ratios. As shown in the above figure pericentric inversions may result in a
change in the arm ratios (paracentric inversions are more difficult to detect this way.
- Inversions can be detected in the meiotic prophase by the presence of inversion loops in
paired homologues.
Genetic effects:
a) Viability of inversion homozygotes:
Since there is no net gain or loss in genetic material brought about by inversions, both
inversion homozygotes and heterozygotes are viable.
b) Cross over suppression:

This refers to the suppression of crossovers and thus recombination of genes within the
inverted region. Reasons:- If the inverted region is small, close pairing of the inverted
region is difficult and hence the inverted region is looped-out in the bivalent, preventing
crossing overs within the inverted region. If the inverted region is large inversion loops
can form allowing pairing within the inverted region. However, crossover in the invertion
loop (rare), in a paracentric inversion heterozygote, has the effect of connecting
homologous centomeres in a dicentric bridge, as well as producing an acentric piece of
chromosome. Thus as the chromosomes separate during anaphase-I, the disjoining
centromeres will remain linked by means of a bridge, which may break at a random point,
resulting in unbalanced chromosomes. The acentric fragment cannot align itself or move
and is lost, leading to deficiencies. These lead to gene imbalances within the genetic
constitution of gametes and in plants such gametes are sterile. In the animal kingdom,
fertilisation of a nucleus carrying the broken bridge would produce defective zygotes which
die because they have unbalanced set of genes. Thus when pairing occurs in the inverted
region crossover products do not survive resulting in cross over suppression.
The net effect of pericentric inversion is the same as that of paracentric inversions, but for
different reasons. Here, since centromeres are contained in the inverted region, disjunction
of crossover chromosomes occurs in the normal fashion, without the creation of a bridge.
However, a crossover within the inversion produces chromatids that contain duplications or
deficiencies for different genes of the chromosome. Fertilisation of such gametes result in
zygote mortality caused by imbalance of genes.
In conclusion, inversion heterozygosity results in crossover suppression by (i) inhibiting the

process of chromosome pairing in the vicinity of the inversion and by (ii) selectively
eliminating the products of crossovers. For instance, if inversion is small close pairing
between opposing inverted regions is difficult and hence c.o. is inhibited. If inverted region
is long inverted regions can form loops which may allow c.o, but only to produce
recombinant gametes that are unbalanced and sterile.
17
Figure 3
18
Note: In plants the unbalanced gametes are sterile. In Drosophila, meiotic planes are parallel and
the dicentrics and acentrics that lag are included in the polar nuclei therefore the egg
nucleus (non-recombinant) remains fertile, even when crossover occurs.
c) Position effects:
This refers to the suppressing influence of heterochromatic regions of the chromosome on
the expression of genes adjacent to the heterochromatic region. Generally, the suppressing
effect decreases as you move away from the heterchomatic region. Inversions may bring
new genes under the influence of the heterochomatic region or remove some away from it
thereby affecting their levels of expression.
d) Genetic co-adaptation
A chromosome with an inversion has a certain segment of its genetic loci in reverse of the
normal order. Since inversions prevent recombination in that region, each gene represents
a sort of "super gene" and natural selection accumulates beneficially interacting alleles
within each inversion. The beneficially interacting alleles are said to show genetic
coadaptation.
1.3.3.3 Inheritance
Transmission of paracentric inversions
When the inverted region is small, the inverted region is looped out during meiotic pairing and
therefore there is no crossover in the inverted region. This results in normal fertile (non-
recombinant) gametes. If the inversions are long enough pairing within the loops can occur, but
once no crossing over occurs products will be non-recombinant but fertile.
Single c.o = recombinant chromosomes are dicentric and acentric chromosomes which produce
unbalanced gametes, that are non-functional. In Drosophila the unviable recombinants are included
in the polar nuclei, so that the egg nucleus is non-recombinant.
Double c.o = d.c.o are rarer than single c.o. Occurs only if the inversion zone is very long. Two
strand d.c.o may diminish the apparent suppression of cross over, as they can produce
balanced normal gametes. Three strand D.C.O produces two functional gametes (of which
one is recombinant) and two non-functional gametes (dicentric + acentric), while four
strand d.c.o produce two dicentrics and two acentrics and therefore all meiotic products are
non-functional.
 Crossing over is rare and double crossovers are rarer. Hence the level of sterility is low.
Transmission of pericentric inversions
Single c.o = produces duplications and defficiencies, such unbalanced gametes are
often not functional leading to sterility.
D.C.O = Only two strand d.c.o produces non-defficient and non-duplicated gametes.
19
1.3.3.4 Evolutionary significance:

Pericentric inversions are rare and when they occur they are protected from cross over by
lack of pairing. Even if pairing occurs such pericentrics are semi-sterile as a heterozygote
but fertile as a homozygote. This will lead to the evolution of a mutually fertile distinct
group of individuals (inversion homozygotes) that do not breed well with the rest of the
species (normal). These distinct interbreeding groups will diverge into different species, in
evolutionary time.
1.3.4 Translocations
1.3.4.1 Types
Transfer of a section of one chromosome to a non-homologous chromosome is known as

translocation. Translocations can be classified into the following types.
Simple translocations: This involves a single break in one chromosome and transfer of this
segment to another non-homologous chromosome. This is rare because of the presumed presence
of unsticky telomeres on the unbroken chromosome.
Shift translocation: Translocation involves three breaks. A segment released by two breaks in
one chromosome attaches between a break in a non-homologous chromosome.
Reciprocal translocations: A segment from one choromosome is exchanged with a segment from
another non-homologous chromosome. Most common type.
1.3.4.2 Diagnosis of Translocation

Cytological detection:
 Cross shaped configurations in transverse section of metaphase-I of meiosis

 Change in size and shape (centromere position) of chromosomes, changes in banding
patterns detected in the karyotype.
 Small chromosomes resulting from reciprocal translocation between two telocentric
chromosomes can be easily lost, thus changing the number of chromosomes.
Genetic effects:
 Partial sterility in plants, due to meiotic abnormalities. Reduced viability of progeny in the
animal kingdom. Crossing over further increases sterility.
 Changes in linkage relationship between genes and associated position effects.

 Position effects: Movement of genes closer or away from heterochromatic regions
can affect their levels of expression.
 Change in linkage groups: Chromosomal location (therefore linkage group) of a
gene/s can change by being translocated to another chromosome.
 Independent assortment of non-homologous chromosomes is seriously violated.
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1.3.4.3 Inheritance of translocation heterozygotes

Translocation homozygotes - pair and segregate normally to produce viable balanced gametes
Translocation heterozygote - form a connected group of non-homologous chromosomes tied

together by translocated sections- a quadrivalent. (Cross
configuration)
The movement to poles takes two configurations - in alternate segregation a zigzag configuration is
formed while in adjacent segregation a 'O' shaped configuration is formed.
Figure 4:
21
a) In alternate segregation opposite non-homologous centromeres move towards the opposite pole
in a zigzag fassion so that the translocated and non-translocated chromosomes are in opposite poles
and therefore in different gametes. All gametes are balanced.
b) In Adjacent-I segregation, an 'O' configuration is formed and adjacent non-homologous

chromosomes move to the opposite pole. Each gamete contains a translocated and a normal
chromosome with deficiences and duplications. These gametes are sterile in plants.
c) Adjacent-II segregation is uncommon as compared to the previous methods. Again a 'O'

configuration is formed. Here, both homologous adjacent chromosomes go to opposite poles and
into different gametes. All gametes contain duplication and defficiencies.
Note: This leads to partial sterility. Generally alternate and adjacent segregations occur 50 % of
the times. So that 50% of the gametes are sterile. In barley and wheat, however, evolution
has selected types that undergo high percent of alternate segregation with no c.o., therefore
translocation heterozygotes are highly fertile.
 In plants - unbalanced gametes are sterile. Hence gametophytes act as screens, eliminating
unbalanced deleterious combinations.
 In animals - gametes are functional and unbalanced combinations result in zygote
inviability (translocation inviability). If unbalances are small and do not affect essential
loci, zygotes may survive, although they may be seriously affected. Complementation of
duplications and defficiencies in the male and female gametes occationally lead to normal
zygotes.

Table 1
♂ alternate adjacent-I adjacent-II
♀ 12 1' 2' 1 2' 1' 2 1 1' 1' 1
alternate 1 2 normal transhet
1' 2' transhet transhom
adjacent-I 1 2' transhet
1' 2 transhet
adjacent-II 1 1' transhet
1' 1 transhet
Note: Independent assortment is affected as only gametic products of alternate segregation

survive. eg. Assume that there is a translocation between Chromosome-1 & 2 - Say
Chromosome 1 has a gene for flower colour (white/purple) and Chromosome-2 a gene for
seed shape (wrinkled/ round). If you cross pprr x P'P'R'R' - the former a normal plant,
while the latter is a translocation homozygote (prime denotes those genes are on a
chromosome that is involved in a translocation). The F1 will be a translocation
22
heterozygote of a genetic constitution, P'pR'r. We would expect a test cross ratio of 1:1:1:1,
if there were independent assortment. But only gametes from alternate segregation would
survive (P'R' ; pr) and therefore a 1:1 ratio is obtained. Note no recombinants have been
formed. This shows that genes residing on translocated chromosomes do not show
independent assortment, whether they are involved in the translocation or not.
Note: Crossing over in translocation heterozygotes is rare, due to lack of close pairing in the cross
configuration. If c.o occurs in the arms it would have no effect on the results. Interstitial
c.o are very very rare. But if they occur
alternate - all gametes containing c.o products are unbalanced, rest balanced.
adjacent-I- all gametes containing c.o products are balanced, rest unbalanced.
adjacent-II: all gametes imbalanced
1.3.4.4 Importance of translocations: (Tutorial)
1. Evolutionary significance of translocations:

Discuss the various genetic effects of translocations on the evolutionary process. For
instance, mating within translocation homozygotes will produce fertile gametes, while
mating with the normal population will result in translocation heterozygotes which will
produce a large proportion of sterile gametes. Hence, translocation homozygotes will form
a distinct mutually interbreeding genetic stock, which will eventually lead to speciation.
2. Role in Agriculture
In agriculture the occurrence of translocations in certain crops can reduce yields
considerably due to the number of unbalanced gametes/zygotes that form.
3. Role of Translocations in Pest Control.

Insect control: Translocation homozygotes of a pest can be bred in the laboratory and
released when a pest is found in epidemic proportions in the field. Discuss how this will
lead to a drastic reduction in the pest population.
Hint: 50% of the offsprings of crosses between insects carrying the translocation and wild
types would die. Similarly 10/16 of the progeny of crosses between translocation bearing
insects would die.
4. Role in Plant Breeding

Chromosome manipulation techniques, serve to move linkage groups from an alien species
by translocation onto a desirable genetic background.
5. Familial Down Syndrome in humans.

Illustrate how Down syndrome can be transmitted through family lines.
Down syndrome can arise in the progeny of an individual heterozygous for a translocation
involving chromosome 21. The heterozygous person is phenotypically normal and is called
a carrier. During meiosis and adjacent-1 segregation will produce gametes carrying
duplicated parts of chromosome 21 and deficiency for some part of the other chromosome
involved in the translocation. This gamete will lead to down syndrome individuals. Half of
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the normal children of carriers will be carriers. One in three children will have a possibility
of having down syndrome.
6. Cancers that are caused as a result of translocations:

Discuss the role of positional effects on expression of cancerous genes with
examples.Burkitts Lymphoma, a cancer of human antibody producing cells called B cells,
is caused by translocation of a cancer producing gene to a position next to a highly
expressed region that normally enhances antibody production. This activates the cancer
producing gene.
7. Multiple translocation systems in Oenothera (Evening Primrose)

Discuss the genetic influences of a multiple translocation system. Has 7 pairs of
chromosomes, of which 6 pairs are involved in translocation. During meiosis only one pair
forms a bivalent, while the rest of the chromosomes due to cross pairing form a ring
structure. If each of the arms of the chromosomes can be numbered numerically. The
structure would be as shown below:
1.2:1.2 3.4:4.12:12.11:11.9:9.6:6.5:5.8:8.14:14.13:13.10:10.9:9.3
Alternate segregation is exclusively observed as a result a zigzag configuration is formed,
where each complex of six chromosomes behaves as a linkage group. One group is called
velans (non-translocated), the other is called glaudens (translocated). Each of the groups
contain recessive lethal mutations and hence in a homozygous form they do not survive
when selfed. Only half, translocation heterozygotes survive. Outcrossing is hence favoured
in the system. This system has very restricted possibility of undergoing recombination.
8. Rapid evolution – The story of the Wallabies.
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1.3.5 PRACTICAL 2 - KARYOTYPING - II
OBSERVING MITOTIC CHROMOSOMES IN ROOT-TIP SQUASHES
In the last practical, you were given an enlargement of a photomicrograph of a cell in mitotic
metaphase and you went on to characterize the karyotype of the particular organism. In the
laboratory session today, you will learn how to collect growing root-tips, fix them in a fixative
solution and stain them with Feulgen, a DNA stain. You will also learn to identify mitotic
metaphase in the transverse view, where all the chromosome lie on the equatorial plane and
therefore can be identified and counted readily.
1. Collecting growing root-tips
Root-tips of different crops are known to undergo mitosis in different times of the day and
hence it is important that growing root-tips (white in colour) be collected at different times
to ensure that all stages of the divisions are represented.
2. Fixation:
The root-tips are sectioned, washed and then immersed immediately in a fixative solution.
The fixative solution used normally is FAA (Formadehyde:Acetic-Acid:Ethanol), which
kills the cell and maintains the chromosomes at the particular stage of cell division as they
are going through the division. Additionally, it serves as a preservative. Some fixative
solutions are known to enhance the visibility of chromosomes.
3. Feulgen staining
The root-tips provided to you are already fixed and hence you can begin the process of
staining. The Feulgen stain is DNA specific and intercalates between the double helix. The
hydrolysis breaks the hydrogen bonds that holds together the helical molecule and thereby
loosens the helix, allowing intercalation of more molecules of the stain. Therefore, the
degree of staining of DNA can be improved by hydrolysis with 1N HCl. The HCl also
helps in softening the often brittle root tissue so that it can be pressed under the coverslip to
produce a good cell spread. Prolonged heating can result in denaturation of cell contents.
Heating in a water bath for 3-5 minutes is normally sufficient.
Having hydrolysed the root-tips, they are then rinsed in distilled water and transferred into
a staining dish containing the Feulgen stain. The time needed to stain depends on the
species studied. Often they are left as long as required for the stain to be absorbed.
Improper hydrolysis would require a longer time to stain.
Wash away the excess stain and mount in 45% acetic-acid. Place the coverslip and gently
tap with the back of a pencil, or press with your thumb to get an even spread of cells.
25
4. Observation
The tip of the root, which contains the root cap and the quiescent region is normally
removed before mounting on a slide. Look under low power to ensure that you have
obtained proper staining and that the cells are properly distributed.
Now go on to higher power gradually (up to X100) and look for cells in mitotic metaphase.
Make the following observations
1. How many nucleoli do you observe in the interphase cells? This gives an
indication of the number of nucleolar organizers.
2. Determine the number of chromosomes in the metaphase chromosomes. You may

have to look for metaphase in the transverse view to be able to see a cell with
chromosomes well distributed throughout it. Sometimes it is easy to count the
heavily stained region of the centromere (heterochromatic region) when the
chromosomes are clumped together. How well you can karyotype depends on how
well distributed the chromosomes are in mitotic metaphase. You may have to make
several squashes before you come across a truly good prep, which can be
photographed and used in karyotyping.
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1.4 CHROMOSOMAL MUTATIONS - CHANGES IN CHROMOSOME NUMBER
1.4.1 Euploidy
In the last few classes we discussed chromosomal changes that affect the structure of the
chromosome. Chromosomal mutations can sometimes also involve changes in chromosome
number. Such changes are referred to as changes in ploidy. The number of unique chromosomes in
a basic set is referred to as the monoploid number or the basic chromosome number (x).
Chromosomal mutations that result in whole number multiples of the monoploid number are
referred to as euploidy. Similarly, changes in chromosomal number that affect only part of the
chromosomal set (sometimes affecting only a single chromosome in a monoploid set) are referred to
as aneuploidy. Aneuploids can be further subdivided into hyperploids, when the change in number
involves addition of one or a few chromosomes to the basic set (or) hypoploids, when there is a loss
of one or a few chromosomes from the basic set. We will deal with these later.
1.4.1.1 Types Of Euploidy
AUTOPOLYPLOIDY: Unusual duplication of the entire basic set of an organism results in

autopolyploidy. eg. 2x, 3x, 4x, 5x, 6x etc. The multiple basic sets
found in an organism are derived essentially from a single species.
ALLOPOLYPLOIDY: Unusual duplication of variable chromosomal sets brought together into an

organism through interspecific hybridisation is referred to as
allopolyploidy. In this case, the multiple basic sets found in an
organism are derived from different species e.g. 2x + 2x' or 4x + 4x'
(where x, and x' are basic sets from different but related species).
1.4.1.2 Origin Of Euploidy:
Autopolyploids: Autopolyploids arise in nature by cytological accidents and artificially by

induction.
a) Misadventures in meiosis : Non-disjunction during meiosis results in unreduced gametes

being produced. Mating of such unreduced gametes (2x gametes) with normal gametes (x)
can result in 3x plants.
b) Chimeras with tetraploid sectors can result from diploid tissue by failure of mitosis.
The occurrence of which is sometimes seen in stock-scion unions of graftings or seen in
response to heat/ cold shocks.
c) Autopolyploids can be artificially induced by applying an alkaloid 'colchicine' to
meristematic tissue. This inhibits the formation of mitotic spindles, preventing the
separation of the duplicated chromosomes into daughter cells, thus resulting in ploidy.
Colchicine is usually applied to the meristematic points of seedlings.
27
Allopolyploids: Allopolyploids arise in nature by rare interspecific hybridisation followed by

spontaneous allopolyploidy, resulting in fertile allopolyploids.
1.4.1.3 Cytological Diagnosis Of Euploidy
Autopolyploids:
Karyotypes would reveal extra sets of chromosomes.
Meiotic metaphase I would reveal quadrivalents. Depending on the number and position of
chiasmata within the quadrivalents can result in the formation of various forms of ring
structures and chains during anaphase I.
Karyotypes would reveal extra sets of chromosomes which can be differentiated from the
original set by changes in banding pattern, size and shape of chromosomes etc.
Meiosis in evolved amphidiploids is normal, with normal bivalent formation, but in new
allopolyploids some level of quadrivalent formation and associated sterility can be seen.
Since the differentiated chromosomal complements in the allopolyploids are not
homologous, neither are they heterologous as they come from a related species. A basic set
derived from a related species is only somewhat different from the original set and is
referred to as homeologous.
1.4.1.4 Phenotypic And Genetic Diagnosis
Autopolyploids:
Partial sterility/sterility: Tetraploids produce bivalents or quadrivalents, the latter may

undergo unequal dysjunction resulting in unbalanced gametes, which are sterile. Over
evolutionary time, however, since nature would have been constantly selecting for increased
fertility levels, tetraploids tend to form bivalents more regularly through the accumulation of
asynaptic genes or may undergo quadrivalent formation with balanced 2:2 segregation to
yield fertile gametes. This results in reduced sterility levels. Triploids and other uneven
ploidy levels are sterile. This is because dysjunction is erratic and occurs by the formation
of univalents, divalents or trivalents. The probability of a normal balanced gamete occurring
in these circumstances is extremely rare and is given by the following equation
P = (1/2)x-1 ....... where x = basic chrom. no.
If x = 20; P = 9.5 x 10-7... a vanishingly small chance!!
Gigantism: Autopolyploids, because of their duplicated genome, would have extra copies of
genes. The cell size and stomatal size and fruit size are hence larger. The plants in general
28
show gigantism and show vegetative vigour. The partial sterility further allows greater
allocation of resources into vegetative growth. eg. The triploid banana is taller and bears
larger bunches, tetraploid grapes and watermelons are much larger, tetraploid pastures
produce more dry matter (40 t/ha as opposed 15-20 t/ha by their diploid counterparts).
Recessive alleles are hidden. Since you have four alleles per locus in a tetraploid the
recessive alleles are often hidden. Hence there is low genetic load. Difficult however to
breed for homozygous recessive genotypes.
eg: potato (4x); banana (3x, 4x); sweet potato (6x); sugarbeet (3x); pyrethrum (3x,4x);
clover (4x); Napier grass (2x; 4x)
Allopolyploids
Permanent hybridity: Brings novel features of two different species into one, which results
in genetic buffering and evolutionary flexibility. Most of our crop plants have an
allopolyploid history. eg. wheat; tobacco; sugarcane; arabica coffee; banana; brassicas; oats;
cotton; peanuts; triticale; finger millet; Okra etc.
Very low levels of sterility or full fertility: New allopolyploids are somewhat sterile. But
through nature's selection over an evolutionary time scale the allopolyploids evolve to form
bivalents regularly with a result of little or no sterility. This process is called 'diplodisation',
a long term diploid differentiation process through adaptive adjustment of duplicate loci and
accumulation of differences between the homeologous chromosomes so that they do not
cross pair very well. Diploidisation is a result of (a) asynaptic genes that have evolved
which prevent homeologous pairing (b) the differences between homeologous chromosome
increasing over evolutionary time scales until the chromosomes are heterologous. Either
way homeologous pairing is reduced and hence the allopolyploids function like diploids and
hence are fully fertile,
1.4.1.5 Inheritance Of Characters
Autotetraploids:
Genotypes of autotetraploids can be classified depending on the number of dominant genes into -
nulliplex aaaa; simplex Aaaa; duplex AAaa; triplex AAAa; and quadriplex AAAA.
As we saw earlier, in autotetraploids, bivalents and quadrivalents that undergo a 2-2 segregation are
the ones that would give fertile gametes. Quadrivalents that undergo 1-3 or 4-0 segregation can
result in unbalanced and therefore sterile gametes. Hence the degree of sterility of an autotetraploid
depends on the frequency with which the various conformations are formed during prophase and the
segregation pattern of homologues.
Assume a tetraploid has evolved to form quadrivalents regularly and undergo 2-2 segregation and is
therefore fertile. The inheritance of a gene depends on whether the gene is
29
a) centromeric: which means that the gene is tightly linked to the centromere so that there is no
cross overs between the centromere and the gene. Such genes show random chromosome
assortment. The two chromatids carrying a gene are held together by the centromere and
therefore go to one pole during meiosis-I and therefore into separate gametes during
meiosis-II. Hence genes on a particular chromosome cannot go into the gametes together.
eg: In a duplex, the gametic ratio would be 4Aa:1AA:1aa. The probability of a nulliplex
(aaaa) = 1/6 x 1/6 = 1/36. Therefore the phenotypic ratio would be 1 recessive:35dominant
Figure 5.
b) Distal gene: When the gene is very distal to the centromere chromatids can be exchanged
between chromosomes in many ways (the cross over frequency = 50%), which results in
random chromatid assortment. The probability of one recessive allele going into a
gamete is decided randomly and would be 4/8 for a duplex genotype. The probability of a
second 'a' allele going would be 3/7. Therefore the probability of an aa gamete = 4/8 x 3/7 =
12/56 = 3/14. The probability of a nulliplex would be 12/56 x 12/56 = 9/196 = 1/22. The
phenotypic ratio would therefore be 1 recessive : 21 dominant
c) If the gene is incompletely linked to the centromere. Some random chromosome

assortment and some random chromatid assortment would occur resulting in intermediary
ratios.
Conclusion: A autotetraploid can either form regular bivalents or regular quadrivalents with 2-2
segregation to produce fertile gametes. If regular bivalent formation has evolved the genes will
show random chromosome assortment. If regular quadrivalents formation and 2-2 segregation were
to have evolved then whether segregation would show random chromosome assortment or random
30
chromatid assortment or little of both depending on the position of the gene in relation to the
centromere.
1.4.1.6 Role of Autopolyploids in Agriculture
In the early part of the century it was thought that increasing ploidies in crop species, where it had
been missed by nature, would revolutionize agriculture.
In general this strategy has not lived up to its promise as newly created ploidies resulted in varying
levels of sterility and associated reduction in seed yield in many seed crops. However, there has
been promising results especially in crops grown for vegetative parts or for fruits. Some examples
1. Rye (2x = 14)

Rye is a rare example of a seed crop which has benefitted from artificial induction of
autotetraploidy (4x=2n=28). The autotetraploid varieties have larger kernals, higher seed
protein (superior baking quality), stiff straw and can emerge under adverse conditions.
However the varieties were not taken up by farmers since the tall straw and larger kernals
meant that existing machineries had to be redesigned. Further these varieties had lower
tillering (branching) ability than their diploid counterparts.
2. Triploids produce seedless fruits.

In many crops triploids have been created by crossing autotetraploid varieties with diploid
varieties. These triploid varieties are seedless, as trivalents formed during meiosis show
erratic segregation and hence fully sterile gametes.
eg. seedless watermelons; Seedless fruits are hollow and are often of irregular shape.
Pollination is necessary for podset and hence diploid varieties have to be interplanted to
serve as pollen parents.
eg. triploid fish are released to clear waterways of water weeds. The fish cannot multiply
and overpopulate the waterways because they are sterile.
3. Fruit crops:
In grapes, tetraploids produce larger fruits with fewer seeds. However the clusters are poorly
filled. In apples the triploids produce larger seedless fruits. But need diploid pollinator
varieties for fruitset. Autotetraploid bananas however produce smaller irregular bunches
and have taller but weaker pseudostems and foliage and hence suffer from wind damage.
4. Forage crops:
Has been an attraction in forage crops. Greater yields, better ratoons were reported in
tetraploid red clover (4x=28). However, the lower seed set has increased seed cost.
5. Ornamentals:
The ornamental industry has benefitted by producing varieties with larger blooms, longer
blooming times and improved shelf life.
31
6. Tuber crops:
Triploid sugar beets have performed better than diploids, but haven't supplanted the diploids
since the yield advantage is not very great and perhaps not worth the increased cost of
producing the triploids.
Table 2
x= Crops
2 Haploppapus
3 Crepis
4 -
5 Oxalis
6 Spinach, Vicia faba, V. sativa
7 Wheat, oats, barley rye, pea , jute, Brassica nigra,
8 Alfalfa, Amaranthus
9 Cassava, sugarbeet, papaya, B. oleracea, Raphanus sativus
10 Maize, peanut, sorghum, cacao, cola, B. campestris
11 Banana, coffee, watermelon, pigeonpea, Eucalyptus, cowpea
12 Rice, potato, tobacco, tomato, conifers,
13 Cotton, Acacia
14 Mabonia
15 Sweet potato, carnation, tea
16 -
17 Sunflower, Bougainvilla, apple, pears
18 Tripsacum, Hibiscus
19 Magnolia, grapes
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1.4.1.7 Evolutionary significance of allopolyploids (Tutorial topic)
Interspecific hybridisation followed by allopolyploidy has played and important role in the ancestry
of many important cultivated crops. e.g. wheat, cotton, oats, Brassica spp., tobacco, sugarcane,
banana, okra etc.
The allopolyploids are fully fertile among themselves but produce sterile progeny with the parental
species. This serves as a reproductive isolation mechanism allowing them to follow a different
evolutionary path from their ancestral species. This allows divergence and acquisition of a distinct
taxonomical identity - speciation. Wheat and Brassica species are excellent examples of
allopolyploidy leading to speciation. Although the initially formed allopolyploids show some level
of sterility, they became fully fertile by a process called 'diplodisation', over an evolutionary time
scale. Such species would regularly form bivalents during meiosis which will result in balanced
gametes.
Genetic variation is the raw material that fuels evolution. Greater the variability within an
interbreeding population, greater the resource pool from which individuals can acquire gene
combinations for survival. The allopolyploids have a much greater array of genetic variation as they
combine genes derived from two different (but related) species. Over evolutionary time there is
adaptive evolution of these duplicate loci, which results in better fit organisms.
Further being polyploids, they are more vigorous, and better fit as a population since deleterious
recessive alleles are masked. The allopolyploids thus are generally more vigorous, suffer less with
undesirable genetic loads and show greater evolutionary flexibility than their diploid counterparts.
Cytogenetic analysis reveals that many crops previously thought to be diploids may indeed have at
some time in evolution undergone allopolyploidy and diplodisation.
e.g. Read up on the evolutionary history of Brassica spp., wheat, cotton. You will be looking at
the evolutionary history of banana in a laboratory session.
Artificial Allopolyploids
With the realization that many allopolyploid crops have become successful in nature, plant breeders
decided to produce artificial allopolyploids by producing interspecific hybridisation followed by
allopolyploidy. One such breeding programme aimed at producing hybrids between Durum wheat
(Triticum durum) with rye (Secale cereale). The resulting allopolyploid called TRITICALE
combines the high protein content and baking quality of wheat with the high lysine content and
ruggedness of rye. This is grown in marginal areas where wheat cannot be grown, such as in the
colder parts of Canada and drier parts of Africa, where wheat cannot grow well.
33
1.4.1.8 History of Allopolyploid Crops
Wheat (AABB) x Rye (RR)

4x = 2n = 28 2x = 2n = 14
F1 Hybrid (ABR)
3x = 21
Sterile
allopolyploidy
TRITICALE (AABBRR)
6x = 2n = 42
Fertile
Another example is Rhaphanobrassica. This is an allopolyploid between cabbage (Brassica

oleracea) and Radish (Raphanus sp.)
Brassica x Raphanus sativa

2x1 = 2n = 18 2x2 = 2n = 18
F1 hybrid (sterile)
x1 + x2 = 18
allopolyploidy
Raphanobrassica
2x1 + 2X2 = 36
It was hoped that Raphanobrassica would combine the rooting characteristic of Raphanus with the
shoot characteristic of cabbage. However it didn't turn out that way!!
1.4.2 Practical Exercise 3- Polyploid Series In Musa Sp.
In this laboratory session we will be closely looking at a naturally occurring polyploid crop (Musa
sp.), which forms an integral part of the economy of many islands in the Caribbean. The present
day banana and plantain cultivars originated from two wild species, Musa acuminata (A genome)
and Musa balbisiana (B genome). Using this as a model plant we will learn, some of the effects of
34
ploidy, the concept of optimum ploidy and identification of ploidy levels in polyploids. We will
also learn how the polyploid series in Musa evolved and trace the origin of some of the commonly
grown banana and plantain cultivars. A cultivar refers to a botanical variety that has originated and
persisted in cultivation.
ORIGIN
The 'A ' genome
Wild diploid forms (2x=22) of Musa acuminata (AA) originated in Western Malaysia, where four
of the five recognized subspecies are found. These are all generally seeded forms. Edibility is
believed to have originated independently in the various subspecies of M. acuminata as
parthenocarpic female sterile mutants that did not produce seeds. It is believed that these sterile
edible diploids by a process called female restitution produced AA gametes (22 chromosomes)
which mated with the 'A' gametes (11 chromosomes) of normal diploids to produce the natural
triploids (3x=33), which are both male and female sterile. The triploids have larger fruits and are
more vigorous, more productive and hardier than the diploids. The edible diploids and triploids
form the majority of the dessert banana cultivars grown throughout the world. The ripe bananas are
sugary and easily digestible and are eaten raw as a dessert fruit. The high yielding triploids are the
ones that are of commercial importance. There are no naturally occurring tetraploids (AAAA), but
have been produced experimentally by breeders. ICS2 is one such tetraploid bred in the West
Indies. Higher ploidies produce weaker stems.
The cultivar 'Sucrier' is a diploid (AA), grown popularly in the West Indies. Other important diploid
varieties include 'Pisang lilin' 'Paka' and 'Sikuzani. Triploids (AAA) popularly grown in the West
Indies include 'Gros Michel', the best banana type but is very susceptible to Panama disease and
hence is presently not of commercial importance. The dwarf mutant, 'Highgate' that arose from
'Gros Michel' as a mutant and is of some commercial importance. The Cavendish subgroup of
triploids (AAA) including 'Lacatan', 'Robusta' (similar to Valery in Central America) and 'Giant
Cavendish' and 'Dwarf Cavendish' have become the main banana cultivars in trade. They are
resistance to Panama disease and show tolerance to wind damage. 'Lacatan' and 'Robusta' are
important in the tropics while 'Dwarf Cavendish' is more popular in the subtropics.
Musa balbisiana (BB genome)
Musa balbisiana is believed to have evolved in India under a strong monsoonal climate, with severe
seasonal droughts. This has made them hardier, with a large variation for disease resistance and
tolerant to drought. The fruits are starchy and acidic (high Vitamin C), with variation in texture and
flavour that are not available in Musa acuminata. The seeded wild diploid forms of M. balbisiana
are however of no commercial importance. It is believed the edible M. acuminata taken by man to
areas where M. balbisiana was native may have led to natural hybridisation that produced the
35
plantain group (AB; AAB; ABB). Cultivar 'Lady's finger' (syn. Ney Poovan), belonging to the AB
group is widely distributed in South India, but lacks vigour and therefore not important
commercially. Among the AAB group cultivars 'Silk', 'Mysore' (used as shade in cocoa estates in
Trinidad), 'Pisang raja', 'Maia maoli' and 'Pome' are commercially important. The ABB group is
extremely drought tolerant and immune to leaf spot and Panama disease. These have short bunches
with dark green, angular fruits, which are starchy, which are solely used for cooking. e.g. Bluggoe.
Tetraploids of a genome constitution ABBB are also found to occur naturally.
Exercise
The polyploids can be identified cytologically or by using DNA cytometry, which measures the
DNA content. These methods, however, are tedious and expensive. It would be much easier for
breeders, if some phenotypic character that is sensitive to ploidy changes can be identified, that can
be used in the prediction of the ploidy levels. Although we know that increased ploidy generally
increases cell size and vigour, there seems to be an optimum ploidy level above which vigour and
yield seem to decrease. Different crops have different optimum ploidy levels and it is reasonable to
think that nature would have selected types that represent the optimum ploidy level. The following
exercise aims to illustrate this point, using Musa species as an example. In addition, you will carry
out a search of a number of leaf and floral characteristics to determine the best indicator of ploidy in
banana. State reasons for your recommendation. You are provided with a polyploid series
consisting of AA, AAA and AAAA genotypes.
1. Make triplicate measurements of floral parts to determine mean
a) Length of large perianth parts

b) Length of small perianth parts
c) Length of filament and anther
d) Length of style and stigma
e) Size of pollen grains
f) No. of germ pores in the pollen grains
Note that the banana inflorescence consists of female flowers which produce the fruits at the
base, hermaphrodite flowers at the middle and male flowers at the apex. The male bud
consists therefore of the male flowers.
2. Make triplicate measurements of the following vegetative characteristics
a) Thickness of leaf
b) Size of epidermal cell
c) No. of stomata/ HP field
d) Size of stomata
e) No. of plastids in the guard cells
f) distance between neighbouring veins under high power
g) distance between two large veins
36
CHAPTER TWO
PROKARYOTIC GENETICS
2.1 Introduction
In this part of the course we will be diverging from the all too familiar eukaryotic genetic systems
into the area of prokaryotic genetic systems. Prokaryotes are genetically simpler systems which are
easier to understand and therefore manipulate. Understanding gained in prokaryotic genetic
systems has proved to be vital in the development of recombinant DNA technology and
biotechnology. In addition, prokaryotes constitute some of the important pathogens that man has to
learn to conquer to survive in this world. It is therefore important to understand how bacteria
undergo recombination and evolution.
Prokaryotic genetics is attractive to the geneticist because a) it is a simpler genetic system to

understand b) the small size of prokaryotes, allow large populations to be grown on petri dishes in
the laboratory (c) their short generation time (e.g. Escherichia coli, 20 min.) enables genetic studies
to be completed in relatively short time frames and (c) screening techniques using nutritional
requirements, sensitivity to drugs, tolerance to stresses and sensitivity to bacteriophages enables
phenotypes to be discerned from large populations, relatively easily.
In order to understand the differences between a prokaryotic and a eukaryotic system it is necessary
that we systematically look at the differences at the organismal level, cellular level, genomic level,
gene level, and finally at the level of recombination and inheritance of genes. The table below
summarizes the differences, which will be elucidated in greater detail in subsequent sections of the
course.
Table 3
Level/ character Eukaryotes Prokaryotes
Organism multicellular organism with varying simple, small, unicellular organisms.
levels of differentiation and
specialization.
Cellular
nucleus True nucleus: Well defined nucleus Nucleoid: Nuclear material is
bounded by a nuclear membrane. concentrated in a nucleoid region.
organelles Organelles present. Metabolic No organelles, cytoplasm is filled with

activities are compartmentalized. enzymes and ribosomes. No
compartmentalisation.
Genome
37
size Large genome - 108-1011 bp; consists Small genome 1/1000th (E. coli 4.2 x
of 100,000 genes or more. 106); approx. 3000 genes.
organization DNA in the genome organized into Consist of a single circular double helix;
numerous linear DNA molecules there may be accessory genome -
carried on chromosomes. plasmids and/or episomes (constitute
0.5-2% of genomic DNA)
ploidy Consists of more than one genomic Single genomic complement -haploid.
complements.
DNA Complexity DNA is complexed with proteins. DNA is naked.
DNA Redundancy DNA is redundant consisting of Unique sequences.

highly repetitive, moderately
repetitive and unique sequences. The
repetitive fraction forms a large
portion.
Gene
Size large genes. In vertebrates average genes are small - average gene size is
gene size is 16 kb; genes can be as 1.2 kb
large as 100 kb.
structure Split genes: intervening non-coding Contiguous genes.

sequences (introns) split the coding Genes and proteins are colinear.
sequence into exons.
replication Multiple origin, bidirectional Single origin, bidirectional
expression Different mRNA splicing can lead to One gene - one polypeptide.
different polypeptides from a single
gene
regulation Regulation at individual gene level. Functionally related genes are organized
Mediated through regulatory proteins in regulatory units called operons and
called transcription factors. are coordinately controlled.
Recombination is through meiosis. No meiosis; recombination through

Recombination conjugation, transduction and
transformation.
Follows Mendels' principles of Mendels principles do not apply.
Inheritance inheritance.
38
2.2 Recombination In Prokaryotes
Demonstration of recombination
Early experiments showed that two autotrophic strains can produce prototrophic strains (those that
can grow in minimal medium) by recombination. Mutations were excluded as a possibility because
none of the autotroph-controls produced prototrophs.
eg. Autotrophic strains A and B, when plated together produced prototrophic bacteria (met+
bio+ thr+ leu+ thi+), indicating that bacteria were capable of recombination.
Strain A: met- bio- thr+ leu+ thi+

Strain B: met+ bio+ thr- leu- thi-
Demonstration of linkage
Lidenberg and Tatum also demonstrated that the genes were linked. After plating the above two
strains together, which will allow recombination, they transferred them into a plate with minimal
medium + biotin. This would select only strains that are met+ bio? thr+ leu+ thi+. They later tested
the colonies on minimal medium to find out how many of the lot were in fact bio+. They found that
60 of them were bio+ and 10 bio-. The excess of one over the other indicated that the genes were
linked. Can you recall why from what you already know from your introductory genetics course?
Methods of recombination in bacteria
a) Conjugation - refers to the one way transfer of DNA from a donor bacteria to a recipient
mediated through a conjugation tube. This method requires physical contact of the two strains
involved. This method of recombination is sensitive to DNAse treatment.
b) Transformation - refers to the uptake of small segments of pure naked DNA from the medium
and incorporation into the bacterial genome. This method does not require physical contact
but is sensitive to DNAase activity.
c) Transduction - The transfer of a DNA fragment from one bacteria to another mediated through a
bacteriophage. This method is neither sensitive to DNase nor does it require physical
contact.
The existence of different methods of recombination can be demonstrated through a U-tube

experiment. Can you explain how, from your understanding of the differences between the
methods?
39
2.2.1 Conjugation
2.2.1.1 Definition:
2.2.1.2 The F factor:

The ability to transfer DNA from the donor to the recipient in bacteria is conferred by the F factor,
found exclusively in the donor bacterium. The F factor is an episome, capable of an autonomous
mode of existence or coexistence within the bacterial genome by integration into it. The
autonomous-F is capable of autonomous replication and faithful transmission into daughter cells
during binary fission. When the F-factor is integrated in the bacterial chromosome, it replicates
itself with the bacterial chromosome and is transmitted along with it to daughter cells.
The autonomous-F: During conjugation, the F factor replicates and a copy is transferred from the
donor to the recipient through the conjugation tube, thus converting the
donor to a recipient. The F consists around 100 genes, which are involved in
the coding of pili and coding of molecules involved in the establishment of
contact, replication of the F-factor and mediation of transfer. The
autonomous-F thus confers to the donor bacteria (F+) the ability to effect
conjugal contact with a recipient strain (F-) and to transfer a copy of itself to
the recipient.
The integrated-F :
Integration of the F-factor into the bacterial chromosome occurs through a process called
non-homologous recombination involving a single crossover event between the bacterial
chromosome and the F-factor. The site of integration and the orientation of integration is
random with non-homologous recombination. During conjugation, the integrated-F factor,
now being part of a larger bacterial chromosome, mediates the duplication and transfer of a
copy of the entire integrated bacterial chromosome into the recipient, in a polar fashion,
beginning at the origin end. Since the origin is at the opposite end to that of the episome, the
episome would be the last to be transferred. The entire transfer process takes as much as
100 minutes and more often than not is interrupted before completion. The episome is,
hence, rarely transmitted and the conversion of the recipient into a donor is therefore also
rare. Hence, only a partial copy of the bacterial genome is transferred. Due to the polarity
of transfer the genes closer to the origin ('O') end (proximal genes) are more likely to be
transferred before conjugation is interrupted than genes further away from the origin (distal
genes). The resulting progressively lower probability of recombination of genes, further
they are from the origin, is referred to as gradient of transfer. The polar nature of transfer
stems from the orientation of integration of the F-factor into the bacterial chromosome.
2.2.1.2 Substituted-F
Sometimes the integrated-F can dissociate from the bacterial chromosome and resume
autonomous replication. This occurs through a process of self-excision from the bacterial
chromosome. During the process of self-excision mistakes sometimes occur resulting in a
40
piece of the bacterial DNA being removed with the F-factor, leaving part of the F-factor
behind on the bacterial chromosome. The resulting F-factor is referred to as the F' factor
(or) a substituted-F factor to denote that it is has some portions of the bacterial genome in it.
2.2.1.3 Lfr, Hfr and F’ Strains

Low frequency donors (Lfr Strains):
These are bacterial populations containing predominantly 'autonomous-F'. However,

integrations do occur, rarely, at a frequency of 1/1000. Such integrations are random (site
and orientation). Hence within a majority of individuals with free-F factors there will be a
small fraction of bacteria containing integrated-F. Conjugation, generally would result in the
recipients being converted to donors, as the individuals with free-F predominate in the
population and would be transferring a copy of the F-factor into the recipient. Only the rare
integrated strains would be capable of transferring incomplete copies of the bacterial
chromosome. Within this small fraction, however, we can expect a large variation in the
integration site/orientation and therefore probability of transfer of every gene is likely to be
equal as the process of integration is random. Nevertheless, the fate of the transferred DNA
is uncertain. Portions of the transferred fragments of DNA can be integrated by a process
called homologous recombination. But this requires homologous pairing and the occurrence
of an even number of crossovers, which is rare, 1/1000. Hence most of the successful
transfers are abortive with the result that integrative recombinations are rare. The
probability of recombination of any gene is 1/1000 x 1/1000 = 10 -6. Since integration is
random, the probability of recombination of any gene is the same.
High Frequency donors (HFr Strains) :
These are bacterial populations containing individuals which possess a 'single type of
integrated-F' within the bacterial genome. Each Hfr strain would hence have an F-factor
incorporated in a particular location in a particular orientation within the bacterial genome.
One can hence visualize having an infinite number of Hfr strains each having a different
location and or orientation of integration into the bacterial genome. Any one Hfr strain will
however have individuals with only one type of integration event.
Due to the polarity of transfer the direction of integration determines the direction in which
the bacterial chromosome will be transferred into the recipient strain. Due to interruptions
in mating, as we saw before, only partial transfers of the genome occur. Since most of the
transfers are abortive as they only rarely undergo successful homologous recombination, the
probability of recombination is low (10-3), but is a 1000 times more frequent than in low
frequency donors. Hence called Hfr donors.
Due to the polarity of transfer and partial transfers being the general case due to
interruptions in mating, the recipients are very rarely converted to recipients, which is also
in stark contrast to what is observed with Lfr strains.
Further, for the same reason, the probability of recombination of every gene is not equal.
The proximal genes (those near the origin) have a much greater chance of being transferred
41
and being incorporated than the more distal genes. This results in a gradient of transfer,
which means that among the recombinants, the recombinants for the more proximal genes
(to the origin) will be more frequent.
Upon transfer merozygotes or partial diploids are formed (consisting of an endogenote and
an exogenote). Only those exconjugants that undergo an even no. of crossovers between the
exogenote and endogenote resulting in the formation of successful recombinants.
F'strains and Sexduction:
A strain containing a particular F' factor is referred to as an F' strain. These can mate with F-
strains and convert recipients into donors, just as efficiently as the Lfr donors. The transfer
of the F' factor is infectious. All transfers will result in a permanent merozygote (or) a partial
diploid situation for the transferred bacterial genes. Recombination is however restricted to
only the genes carried on the F' plasmid. Since the transferred F' factor, containing some of
the bacterial genes, can replicate autonomously, they need not have to be incorporated into
the bacterial genome to be faithfully transmitted to progeny bacteria. Hence sexduction, the
transfer of the F' factor from donor to the recipient strains, does not result in abortive
recombinants. In conclusion, sexduction is an effective process in converting recipients into
donors and in transferring the genes contained in the F' factor to the recipients.
Exercise:
Can you discuss Lfr, Hfr and F' strains in terms of their ability to convert recipients into donors,
ability to effect recombination, probability of any particular gene being transferred and the fate of
the transferred genes.
Discuss the statement 'Hfr strains provide evidence for chromosome circularity in bacteria'
2.2.1.4 Conjugation Mapping Methods.
Evidences for chromosome circularity and linkage of genes in bacteria, described in the previous
section, has led to the realization that all the genes are found in a single circular chromosome in
bacteria. Many researchers have used different methods to map the genes (determine gene order
and distance between them) using various mapping techniques. Here we discuss some of the
methods involving conjugation.
2.2.1.4.1 Interrupted mating technique (Wollman and Jacob, 1957)
The map developed using this method measures the distance between genes in time units. The
following is a brief summary of the procedure.
42
a) Mating
A Hfr strain and a F- strain, selected based on contrasting characteristics, are cultured
together, in a complete medium and incubated at 37 oC, providing them the opportunity to
conjugate and produce recombinants. e.g.
Donor Hfr: thr+ leu+ azi-s T1-s lac+ gal+ Str-s

F- (Recipient): thr- leu- azi-r T1-r lac- gal- str-r
Genetic Markers:
thr+, leu+ = ability to synthesize the amino acids.

lac+, gal+ = ability to use lactose and galactose as substrate
T1-r = resistance to the T1 phage
Str-r = resistance to the antibiotic streptomycin
azi-r = ability to tolerate/ deactivate sodium azide
b) Interruption of mating
At regular intervals (1 min interval for a period of 60 minutes), aliquots of the culture is
removed and placed in a Warring blender and blended at high speed for a few seconds. This
process disrupts conjugation tubes and results in the interruption of mating. The interrupted
cultures are then plated in a complete medium with streptomycin, to eliminate the donor
bacterium. Can you explain why?
c) Replica plating
The colonies are tested by replica plating to determine the genetic markers? e.g. a replica
plate consisting of complete medium lacking leucine would determine which ones are Leu+.
Similarly the presence of various markers can be determined.
d) Compilation of results.
time marker first observed

----------------------------------------
8 0
8.5 thr+
9 leu+
11 azi-r
18 T1-r
25 lac+
50 gal+
________________________
43
e) Results and discussion:
Genes that are proximal to the origin are transferred first. In other words there is polarity in the
transfer. In this case 'thr' must be the most proximal gene.
Exercise:
Can you order the genes from the origin?

Can you identify the position of the F factor in relation to the genes?
Later the genes the lower is their probability of transfer at any given time. Why?
Can you construct a map based on the data provided with distance between genes in time units?
Note that this only produces a partial map. By using different hfr strains, each having different site
and direction of integration of the factor, you will be able to develop a complete genetic linkage
map.
Can you show the relative order of genes in Escherichia coli and the site and orientation of
attachment of the F factor in the following strains.
Hfr Strain-1: ORI - thr, pro, lac, pur, gal, his, gly, thi
Hfr Strain-2: ORI - thr, thi, gly, his, gal, pur, lac, pro
Hfr Strain-3: ORI - pro, thr, thi, gly, his, gal, pur, lac
Hfr Strain-4: ORI - pur, lac, pro, thr, thi, gly, his, gal
Hfr Strain-5: ORI - thi, thr, pro, lac, pur, gal, his, gly
Disadvantages:
a. Close genes are difficult to separate in time and appear together in replica plates and hence
are difficult to place in genetic linkage maps.
b. Only gives a rough estimate of distance between genes
c. Cannot be compared with other genetic linkage maps, which are based on recombination
units not time units.
2.2.1.4.2 Gradient of transfer method.
The technique is performed essentially similar to the above experiment, except that interruption is
allowed to happen naturally rather than by blending. Spontaneous breaks in the conjugation tube
results in the interruption of mating, creating a natural gradient of transfer.
e.g.
Hfr: str-s met+ arg+ aro+ his+

F- : str-r met- arg- aro- his-
The donor strain is eliminated (by plating in complete media consisting of streptomycin) and
selected for an early marker by leaving out methionine (met is the early marker in this case) in the
medium. Why did we select for an early marker?
Replica plating is now done to identify the presence of the genetic markers.
44
Results
Recombinants %
-----------------------------------------
met 100%
arg 60%
aro 20%
his 4%
________________________
Can you order the genes? This method only allows one to order the genes but does not tell us much
about the distances between them.
2.2.1.4.3 High resolution mapping (Recombination mapping)
Classical linkage analysis can be performed if one can eliminate the problem of gradient
transfer and create a situation where every gene has an equal chance of recombining. We
can achieve this by allowing mating between a Hfr strain and an F- strain and selecting for a
late marker. This would mean that within the selected progeny the probability of
recombination of every early gene would be equal. In other words, an equal probability
would have existed for the recombination of every early gene, within this selected
population. Now we can apply the classical approach of linkage analysis.
Methodology:
a) Mating is achieved between an Hfr and an F- strain selected to possess contrasting

phenotypes with respect to the characters being mapped. Mating is usually
achieved by co-culturing the two strains.
b) The cultures are plated in a medium that allows for the selection of strains
containing markers known to be distal to the genes being mapped. e.g We intend to
find the distance between genes met, arg and leu. Let say that previous interrupted
mating studies show that 'leu' is distal to the other two genes. In order to select for
the distal marker 'leu', you would have plated the cultures in a minimal medium
lacking leucine so that leu+ recombinants would be selected.
The cross would be Hfr Met+ arg+ leu+ x F- met- arg- leu-
c) The culture will then be subjected to a series of serial dilution (1/10 - 1/100
-1/1000) and the neat suspension plated on complete medium lacking
a) medium - leu b) medium - leu ; - argc) medium - leu ; - met
d) medium - leu ; - arg ; - met
45
Can you tell what will be the phenotypes of the bacteria selected from these media.
leu+ arg- met- = 150
leu+ arg+ met- = 110
leu+ arg+ met+ = 85
leu+ arg- met+ = 5
d) Find the gene order

Find the plate with the lowest frequency. The unrecombined gene in that plate is the
central gene. Now draw a diagram to indicate how these recombinants could have
been formed.
e) Find the distances between the genes
Use the formula RC% (RI) = sco (RI) + dco

-------------------- x 100
total
Similarly can find recombination % in Region II.
f) Draw the linkage map
Advantage:
The map is based on recombination percentage rather than time. It is more accurate as well as more
acceptable as it can be comparable to maps in other organisms.
The power of recombination mapping:

1. Large number of individuals can be evaluated rapidly and markers mapped.
2. Because very large populations can be screened very rare recombinants can be mapped.
We will later use this mapping procedure to develop the concept of a gene.
2.2.2 Transduction
2.2.2.1 Definition:
Transfer of genes from one bacterium to another mediated through a bacteriophage. Such a phage
carrying bacterial genes is called a transducing phage. Two types of transducing phages are known,
generalised and specialised.
46
2.2.2.2 Phage Life Cycles

Types Of Transduction
There are two types of transduction, generalised transduction, which involves a virulent phage (e.g.
P1 phage), is associated with the lytic life cycle; and specialised transduction, which involves a
temperate phage (e.g. lambda phage), is associated with the lysogenic life cycle. Fig-1 summarises
the life cycles of a virulent phage and a temperate phage. The temperate phage generally forms
lysogenic associations by integrating into the bacterial chromosome but can sometimes revert to the
lytic pathway, depending on the host and environmental factors. e.g. UV has been shown to induce
lysis. Virulent phages, however, do not have the capability to establish lysogeny and hence solely
follow the lytic pathway.
Figure 6
47
Differences between generalised and specialised transduction
Table 4
Differences Generalised Specialised
Phage / life cycle virulent phage - lytic cycle. temperate phage - lysogeny.
Transducing is produced by a random piece of Spontaneous reversions to the lytic

particle (TP) the bacterial DNA being form occur at a frequency of 10-5 by
erroneously wrapped within the a self-excision. During excision,
protein coat in place of the λ DNA errors occur at a freq. of 10-6,
producing the generalised TP. producing the specialised TP.
Frequency of TP 3/1000 = 0.003 10-5 x 10-6 = 10-11

If excision induced by uv/chemical =
10-6
Transducing ability is wide range, because the λ head Narrow transducing ability, since the
can hold up to 91 kb of the DNA, gene adjacent to the att-B site, i.e.
which is about 2-4% of the the gal gene is only transduced
bacterial genome (1/50) = approx. (λdg).
70 genes.
Prob. of
transduction of any Prob. is the same for all genes of The frequency is very low with λdg.
gene the genome = 0.03 x 0.0003 = 10-6. The frequency of transduction of the
Need an efficient selection system gal+ gene can be increased by using
to detect it. and HFT lysate.
Host bacterium Double lysogens can be formed in

Needs to be rec+ for recombination rec- hosts as well, since phage
to occur, since host encoded encoded enzymes effect such
enzymes effect recombination. recombination. Stable integration
would, however, require a rec+ host.
Genome mapping Useful in genome mapping since a Not useful in genome mapping.
large fragment of DNA is tran-
sferred through the generalised
phage.
48
2.2.2.3 Lytic pathway and generalised transduction:
During lysis, the virulent phage undergoes autonomous replication, packages itself into
protein coats and is liberated by lysing the bacteria. The lysed cells are revealed in a petri
plate as a plaque - a clear region within the bacterial lawn.
After infecting the bacterial cell the virulent P1 phage produces a nuclease which degrades
the bacterial genome into small fragments. During the process of packaging into protein
sheaths, mistakes occur rarely, resulting in a bacterial DNA segment being erroneously
packaged into the phage heads instead of the viral DNA. These phages containing bacterial
DNA are called generalised transducing phages. Since any DNA fragment can be packaged
erroneously, the probability of any gene being represented in a transducing particle is the
same and is approx. 10-6. The phage head has the capacity of holding approximately 1/50th
of the bacterial genome (90-100 kb), which corresponds to around 50-70 genes.
The generalised transducing phages are capable of infecting a bacterial cell and injecting the
DNA into it. The injected DNA creates a transient partial diploid situation and can
recombine into the genome at frequency of 10-6. The host bacteria should be rec+ to code for
the recombination enzymes.
2.2.2.4 Lysogeny and specialised transduction:
The alternate to the lytic cycle is a lysogenic cycle, and in a lysogenic cycle the temperate
phage integrates into the bacterial chromosome at the att-B site by homologous
recombination. The integrated phage is referred to as a prophage and the surviving bacterial
cell is called a lysogen. For example, if the integrated phage is the lambda phage the
association is called a lambda-lysogen. In the prophage, the replication genes and lysis
genes are repressed. Hence it replicates with the bacterial DNA and is passed onto daughter
cells, during binary fission, as passenger-DNA. The prophage confers to the host bacteria,
immunity to infection by the same or similar lytic bacteria. Lysogeny is hence a harmonious
relationship between the prophage and the host bacterium.
The sites of breakage and reunion in the bacterial and phage DNA are called the bacterial
attachment (att-B) and phage attachment sites (att-P), respectively. Each of these sites have
three segments of which the central segments show perfect nucleotide homology and hence
is the region where breakage and reunion occurs. The att-P is designated POP' and the att-B
designated BOB' to indicate the different segments. A phage encoded protein, integrase, is
responsible for the site specific integration event. The integrated sites become BOP' and
POB'
A lysogenic cell can replicate indefinitely without release of the phage. However, the
prophage can sometimes become induced to undergo a lytic life cycle. This phenomenon is
called phage induction. Spontaneous reversions take place at a frequency of 10 -5. This is
caused by environmental factors such as radiation and chemicals. Phage induction leads to
self-excision, catalysed by the phage enzyme integrase together with a phage protein
49
excisionase. The former is involved in the cutting and rejoining, while the latter aids the
former in locating the BOP' and POB' sites.
Excision errors (at a frequency of 10-6), in which the sites of breakage and reunion are
misplaced, sometimes occur, resulting in the formation of specialised transducing phages.
The probability of these transducing particles = 10 -5 x 10-6. These transducing phages are
sometimes called restricted transducing phages because they can carry only genes adjacent
to the att-B site, namely the gal or bio genes (in the case of the λ lysogen). Since the λ head
is finite in size, transducing particles carrying bacterial genes have to leave some of the
phage genes and hence they are defective. This results in transducing phages, λ defective
gal (λdg) or λ-plaque-forming-bio (λpbio). λdg is defective in its replication genes and has a
defective integration site and requires a helper phage to aid in replication and integration.
On the other hand λpbio would have lost its lysogenic capability, forms plaques regularly
and is hence not useful in transduction.
The λdg particles by themselves cannot effect significant transduction, because they cannot
multiply by themselves, neither can they integrate into the att-B sites of bacteria and are
hence very rare. However, along with wild type λ phage (λ+) they can form double
lysogens. If such a culture is lysed by induction (UV) and used as a gal + donor in
transduction a very high frequency of transduction is obtained. Such a lysate is called high-
frequency transduction (HFT) lysate. Infection with HFT lysate can lead to stable
transformation by crossovers flanking the gal+ gene and unstable transduction by the
formation of λ+ / λdg double lysogens. Even the unstable transductants can multiply ad
infinitum without being induced. This creates a gal-/gal+ partial diploid condition.
2.2.2.5 Co-transduction frequencies can be used to map bacterial chromosomes
If two genes are co-transduced they are within 90-100 kb from each other and are therefore
fairly closely linked.
f (co-transduction) = (1-d/L) d = distance between markers

L = length of fragment that can be carried by the phage.
eg: 60 kb = 4%
30 kb = 30%
10 kb = 70%
1 kb = 96%
The closer the genes are higher the co-transduction frequencies. If the co-transduction frequency is
50% it means that 50% of the transducing particles containing one gene also have the other one.
The central gene can be identified in three point crosses because the wild type allele of the central
gene will almost always be co transformed with the wild type alleles of the flanking markers.
50
Figure 7
51
2.2.3 Transformation
Definition: Uptake of pure naked DNA from the surrounding medium and incorporation into the
bacterial genome by recombination.
Steps: a) Cell becomes stationary.

b) Reversible binding of double stranded DNA on the cell surface.
c) Irreversible uptake of DNA into the recipient cell.
d) Conversion of the dsDNA to ssDNA
e) Incorporation into the genome.
f) Replication and segregation of recombined genes.
Competence for transformation:
Only 1% of the bacterial population are competent for transformation. Moreover, competence
occurs only at a certain stage of growth. Furthermore even within the competent population the
amount of DNA taken in is small due to restricted entry sites. The competence of bacteria can be
improved by various treatments.
Gene mapping
Transformation affords a convenient way of gene mapping. With suitable recipient cells and excess
DNA, transformation takes place at a frequency of about 1 transformed cell per 1000 cells. If two
genes are far apart they are unlikely to be transformed by the same fragment and the probability of
co-transformation would be in the order of 10-3 x 10-3 = 10-6. Hence, higher co-transduction
frequencies indicate that the genes are closely linked.
2.2.4 Transposable Genetic Elements (Transposons)
These are DNA sequences that are present in a genome in multiple copies and have the capability of
occasional movement (transposition) to new locations within the genome. They can potentially
integrate in a large number of target sites and hence are random in distribution throughout the
genome. In bacteria these sequences account for some degree of rearrangement and recombination.
2.2.4.1 Types of Transpons

IS ELEMENTS:
First discovered transposons in bacteria. They possess inverted repeat sequences at the termini and
code for a transposase protein, required for transposition and one or more proteins which affect the
rate of transposition. These do not carry any bacterial genes.
TN ELEMENTS:
These transposons have composite structures, with bacterial genes sandwiched between two
insertion sequences. Such transposable elements can shuttle several bacterial genes between host
52
bacteria by transposing into plasmids, which are capable of conjugal transfer (bom+). Eg. Tn5
contains resistant genes (neo-r; ble-r; str-r) against three different antibiotics, neomycin, bleomycin
and streptomycin.
Figure 8
2.2.4.2 Importance Of Bacterial Transposons (Tutorial Topic)
1. Multiple antibiotic resistance:

In nature sequential insertion of transposons containing different antibiotic resistance genes
into the same plasmid results in the evolution of plasmids that confer resistance to multiple
antibiotics. These multiple resistance plasmids are called R-plasmids. The evolution of R-
plasmids is promoted by regular and often improper use of antibiotics, which selects for
resistance cells, since resistant cells have a growth advantage over sensitive cells in the
presence of antibiotics. Presence of multiple antibiotics in the environment selects for
multiple drug resistance. Serious clinical complications can occur when plasmids resistant
to multiple drugs are passed onto bacterial pathogens that cause disease.
2. Transposon tagging allows one to isolate unknown genes.

e.g. Suppose we want to isolate a disease causing gene from a bacteria, a transposon
containing a neomycin resistant gene can be introduced into bacteria. We can select for
successful integrations into the bacterial genome by using neomycin in the selection
medium. These bacterial colonies can be screened for inability to cause infection. This
probably is brought about by the insertion of the transposon into the disease causing gene,
53
thus inactivating it. Now it will be easy to isolate the gene because we know the sequence
of the transposon.
2.3 GENE FINE STRUCTURE ANALYSIS

2.3.1 Recombination And Complementation
Power of recombination mapping
In the last section we saw that recombination mapping is a powerful system in prokaryotes (high
resolution mapping), which allows the determination of the distance between closely linked genes.
Lets see why it is such a powerful system. You will recall that recombination mapping involves
allowing two autotrophic strains containing contrasting genetic markers to mate e.g. (Hfr) arg + pyr-
x (F-) arg- pyr+, followed by selection for a distal marker (e.g. leu +). From this plate a neat
suspension is prepared. This is (i) diluted to 1/1000, through a dilution series and plated in a
complete medium while the undiluted suspension is plated (ii) on a minimal medium. The complete
medium will give the total no. of colonies (both parental types and recombinant types). e.g. if you
get 100 colonies. The total number would be 100 x 1000 (dilution factor) = 100,000; while the petri
dish with the minimal medium will give the number of wild type recombinants (arg +pyr+), which
would be only half of all possible recombinants (the other half being arg -pyr-). Say this is 5. Then
recombination frequency = 5 x 2 /100,000 = 0.0001 (0.01%). The power of the technique lies in the
fact that recombinant as rare as one in 100,000 can be detected by just plating twice.
Such high resolution mapping studies very soon showed that two mutants of the same gene eg. leu -
x leu-, can recombine to give a wild type recombinant (leu +), which meant that recombination can
take place within a gene. This was an astonishing experience for the researchers, then, since it was
widely believed, at that time, that recombination occurred only between genes. If genes were not
the units of recombination, then what were they?
2.3.1.1 Recombination tests.
These are tests conducted to understand whether two mutants affecting a character are the same
or different. In a recombination test, if wild type recombinants are formed the test is said to be
positive, indicating that the two mutants were different. Conversely, if no wild type recombinants
were formed the test is said to be negative, indicating that the mutants were in fact on the same point
and are hence probably the same. (We say probably because there is always some doubt, whether a
more sensitive recombination test would have recovered wild type recombinants). Positive
recombination tests also allow us to find the distance between the two distinct mutations identified.
What it doesn't tell us is whether the two distinct mutations detected belong to one gene or to two
different genes affecting the same character.
Recombination maps: Recombination maps are linkage maps indicating the position and
distances between distinct mutations. Recombination maps cannot
indicate whether mutations affecting a certain trait are on the same
gene or on different genes.
54
2.3.1.2 Complementation test (cis-trans test)
Definition: The ability of two recessive mutations to restore the wild type phenotype in the trans
position but not in the cis position, in a single cell system.
The test: The test involves bringing together of two mutations in (a) cis configuration and (b)
trans configuration in a single cell system. The cis test is a control and always
results in a wild type phenotype. If the two mutants, in the trans configuration,
result in a wild type they are said to complement each other (+ve complementation
test or +ve cis-trans test) and therefore are said to be on separate genes. On the other
hand, if the two mutants 'in trans' produce a mutant phenotype, they are said to be
non-complementary (-ve complementation test) and therefore belong to the same
gene. Trans refers to a condition, where mutations are on different DNA molecules
while cis refers to a condition where the mutations are on the same DNA molecule.
Explanation: What does the complementation test tell us? Lets say a character is determined by
two genes and that those two genes code for two enzymes, each catalyzing a single step in a two
step pathway, leading to a product. In other words, we can say that the two genes complement each
other in producing the final phenotype.
Figure 9
Now if the two mutations reside on two different genes, a) in cis configuration, then the
phenotype would be wild type, since functional enzymes can be produced by the two genes
on the other DNA molecule; again b) in trans configuration, a wild type phenotype will
result, because one of the two genes will be non-mutant in the two DNA molecules and thus
can produce the two functional enzymes, which can complement each other in a single cell
system to give the wild type phenotype.
55
On the other hand if the two mutations reside on the same gene a) in the cis
configuration, a functional enzyme can be produced by the other DNA molecule
resulting in a wildtype phenotype but b) in the trans configuration the same gene is
mutated in both DNA molecules and hence the enzymes produced by both DNA
molecules are non-functional, resulting in a mutant type.
Hence, a complementation test will allow us to conclude whether two mutations

are on the same gene (allelic) or not (non-allelic), since if the two mutations in
question were on the same gene they would have produced a mutant type in trans
configuration. Hence complementation tests provides us with a functional definition
of the gene - that the gene is the unit of function.
Method: Two mutations can be brought together in a single cell system by sexduction, thus
creating a partial diploid situation, with respect to the transferred bacterial
chromosomal segment on the F' plasmid. Hence, while recombination test uses Hfr
strains, complementation test uses F' strains.
Cistron: All mutants that give a negative cis-trans test are said to belong to one
complementation group or cistron. Cistron is considered synonymous to a gene. A
cistron is defined by the cis-trans test and is the functional unit that produces a single
polypeptide.
Complementation map: This is a map of mutations affecting a particular character, indi-

cating which of the mutations are allelic and which are non-allelic.
e.g. There are five known mutations in the histidine locus - CD16, 245, 261, D-566,
1438. The results of a cis-trans test (bringing together of two mutations at a time by
sexduction) are provided below (+ refers to +ve complementation and - refers to -ve
complementation).
CD16 245 261 D-566 1438

______________________________
CD16 - - - - -
245 - + + +
261 - + +
D566 - -
1438 -
______________________________
Complementation map:
245 261 D-566 & 1438
____ ____ ____________ Histidine locus has three genes.
CD16 _________________________ deletion spanning the three genes
56
2.3.1.3 Limitations to the cis-trans test (tutorial topic).
1. Cannot be used for dominant mutations (Why?)
2. Mutations that exhibit intragenic complementation (oligomeric proteins formed). (Why?)
3. Polar mutations (pleiotropic mutations) on a coordinately regulated operon. (Why?)
4. Mutations on cis-acting elements (ie. mutations on promoter and operator regions). (Why?)
5. Conditions that allow leakage of recombination to occur. Conditions in other words should
be restrictive to prevent recombination e.g. use of rec- host bacteria. (Why?)
2.3.1.4 Recombination Test vs Complementation Test?
Table 5 below shows the differences between complementation tests and recombination tests
Table 5: Differnces between Recombination and Complementation tests.

Recombination test Complementation test
Creation of new combination of mutations New combinations are not formed by breakage
through the breakage and reunion of DNA and reunion but brought together into a single
molecules (crossing over). cell system so that the products of the new gene
combinations can mix and complement each
other.
The progeny has new combinations of Does not involve a change in the genotype of
mutations. the individual, neither are new progeny
produced.
Tells us whether two mutations are the same or Tells us if the mutations are allelic or non-
different and if they are different the distance allelic (ie in the same gene or on different
between them. genes).
Provides definition of a mutational unit. ie the Provides functional definition of a gene.

smallest that is non-divisible by mutations.
57
2.3.2 Gene Fine Structure Analysis of Phage Genetics
The development of methods to study genetics of phage traits, led to the fine structure analysis of a
locus responsible for plaque morphology.
Inheritance of plaque morphology
Plaques are clear areas within bacterial lawns caused by several rounds of phage infection of
bacterial cells and lysis. Plaques are visible to the naked eye, within 8 hours of initial infection. The
plaque morphology can be small or large; fuzzy or sharp.
A phage cross can be carried our by co-infecting an E. coli plate with two phages at a concentration
called 'multiplicity of infection'. At this concentration double infections occur and the two phage
DNA molecules can undergo recombination within the E. coli cell.(Figure 10)
e.g. h- r+ x h+ r-
h+ = ability to infect two distinct E. coli strains

h- = ability to infect only one E. coli strain
r+ = normal plaque size

r- = rapid lysis leading to a larger plaque size
The progeny phages can be collected and the proportion of recombinant (h +r+ ; h-r-) determined by
making observations of plaque morphology in a mixed E. coli plate (both E. coli strains are plated
together, which results in a mixed lawn).
h- r+ = clear and small plaques

h+ r- = cloudy and large plaques
h+ r+ = Cloudy and small plaques
h- r- = Clear and large
Note: Clearness is produced by the h- allele, which allows infection of both bacterial strains in the
lawn; cloudiness is produced by the h+ allele, which limits infection to only one E. coli strain. r-
mutants lead to rapid lysis producing larger plaques compared to r+
Recombination percentage = No. of recombinant

--------------------------------------- x 100
total no. of plaques
58
FINE STRUCTURE ANALYSIS OF THE rII LOCUS
A scientist by the name of Benzer mapped the 'r' mutants (rapid lysis) into two loci (rI and rII) in the
T4 phage. The mutations in the rII locus were intensively studied by him, which led to his nobel
prize winning work on the fine structure analysis of the rII locus.
Mutations in the rII locus, in T4 phage, behave differentially in two E. coli strains.
- Escherichia coli (Strain B), allows both the wild type (r+) and mutant (r-) T4 phages
to grow, but different size plaques result. Wild type phages (r+) produce small,
round plaques, but r- mutants, show rapid lysis producing large, more irregular
plaques. E. coli Strain B, which allows both the wild type and mutant phages to
grow is referred to as a permissive host.
- Escherichia coli (Strain Kλ), which does not permit the growth of rII mutants, but
allows the wild type phages to grow is called a selective host or a non-permissive
host.
2.3.2.1 Recombination test:
Recombination tests in E. Coli can be carried out using the following steps:
i) Plating the mutants together at a concentration which allows double infection

('multiplicity of infection') on E. coli (Strain B).
The two mutants are mixed together with E. coli (Str-B) in top agar (42oC) and
plated on an agar plate. The agar plate is incubated overnight. Collect lysate. The
lysate containing progeny phages would have recombinants. Typically, 1 ml of
lysate contains in excess of 109 phages.
ii) The lysate is subjected to a dilution series and plated on E. coli (Str-B) and without
dilution on E. coli (Str-Kλ). Only the wild type recombinants would grow in E. coli
(Kλ), while all would be able to grow in E. coli (B).
iii) RC (%) = No. of plaques in E. coli (Kλ) x 2

---------------------------------------------- X 100
No. of plaques in E. coli (B)
Recombinant as rare as 1 in 109 can be detected. Such is the resolving power of this system.
Typically the resolving power of the system is in excess of 0.000001.
Note: The recombination test tells us whether the mutations used are the same or different.
Using this method Benzer was able to map 3000 mutations in the rII locus. Although his
system had a resolving power of 0.000001, he found that the minimum recombination
frequency in his system was 0.0001, which allowed him to conclude that genes are made up
59
of smaller units of recombination called recons. He concluded that recombination occurred

between recons and never within recons.
2.3.2.2 Complementation spot test:
In this test two mutants are spotted together on a E. coli (Kλ) lawn at a concentration that
would allow double infection. The plate is incubated at 37 oC to determine if a plaque is
formed or not. Note that double infection would bring the two phages together in a single
cell system, therefore allowing the test of complementation.
- If a plaque is formed it indicates + complementation and suggests that the mutations are on
different genes or cistrons (ie non-allelic).
- If a plaque is not formed it indicates - complementation and suggests that the mutations are
on the same gene or cistron (allelic).
Note: Benzer was able to classify the 3000 mutations as belonging to two cistrons (Cistron
rIIA and Cistron rIIB) from complementation tests.
How did Benzer do it?
To map the 3000 mutations (recombination test) by co-infecting two mutations at a time would have
required n (n-1) / 2, which is 3000 x (2999) /2 = 4.5 million tests, a virtually impossible task. How
did he achieve it in a relatively short time? would be the topic of discussion in our next section.
2.3.2.3 Benzer's Deletion Mapping Technique
Benzer used deletion mapping to rapidly map mutational sites on the rII locus. He noted that some
mutations did not recombine with many mutations - He called them multisite mutations. He argued
that such multisite mutations are deletions based on electron microscopic evidence and evidence of
non-reversion of such multisite mutations.
The procedure:
1. Benzer established several reference deletions spanning different lengths of the rII locus.
This he did by crossing the deletions with several established reference mutations. Lets say
r1-r6 are reference point mutations spanning the rII locus.
|_____|_____|_________|________|_______|______|___|
r1 r2 r3 r4 r5 r6
60
By doing recombination tests with the unknown deletions and each of the six reference
mutations one can establish the area of the rII locus that each deletion spans. Lets say that
six deletions studied gave the following results.
r1 r2 r3 r4 r5 r6
deletion-1 - - - + + +
deletion-2 - + + + + +
deletion-3 - - + + + +
deletion-4 - - - - + +
deletion-5 - - - - - +
deletion-6 - - - - - -
These results would suggest that the deletions spanned various portions of the rII locus
|_____|_____|_________|________|_______|______|___|
r1 r2 r3 r4 r5 r6
d6 ------------------------------------------------------------------
d5 ----------------------------------------------------------
d4 -----------------------------------------------
d1 ------------------------------------
d3 -------------------
d2 ----------
2. Recombination tests with reference deletions.
Having established reference deletions recombination tests are carried out between the
unknown mutation, whose position in the rII locus one is desirous of mapping, and the
reference deletions. Recall that this is done by plating the unknown mutant with a reference
deletion in E. coli (B), collecting the lysate and plating it in E. coli (Kλ). If plaques are
formed it indicates that recombination between the unknown mutant and the reference
deletion has resulted in a wild type. This indicates that the unknown mutation is outside the
deletion. Lets say that the unknown mutation rx gave the following results with the the
reference deletions.
rIIA rIIB
|-----------------------------------------------------------|---------------------|
A1 A2 A3 A4 A5 A6
rx
D1 --------------------------------------------------------------------------------- -
D2 ---------------------------------------------------------------------- -
D3 -------------------------------------------------------------- -
D4 -------------------------------------------------- -
D5 ----------------------------------------- -
D6 ---------------------------------- +
61
The results indicate that the mutation rx is present in the region A5, since it was contained in
deletion 4, but was outside deletion 5.
3. Multistage testing.
Now the unknown deletion is tested against a number of reference deletions spanning the
region A5, so that the deletion can be narrowed down to a small subregion within A5, say
A5C. Subsequently the unknown deletion would be tested against deletions that span
subsub regions of A5C and lets say rx was found to be in the subsub region A5C-4.
A5A A5B A5C A5D A5E rx

D12 ------------------------------------------------- -
D13 -------------------------------------- -
D14 --------------------------- -
D15 ---------------- +
D16 ----- +
A5C-1 A5C-2 A5C-3 A5C-4 A5C-5 rx

______________________________________________________ -
__________________________________________ -
______________________________ -
_________________ -
_____ +
4. Recombination tests with reference point mutations.
Having narrowed down the mutation to a very small region in multistage testing the
unknown mutation is now tested against all known point mutations in this small region, to
see if this is one of the known ones or a newly identified mutation. Lets say in A5C-4, there
62
exists 4 known point mutations r345, r527, r980, r1235. We now perform individual
recombination tests with these four point mutation.
r345 r527 r980 r1235

----------------------------------------
rx + - + +
The results suggest to us that rx is one and the same as r527.
Tutorial (Part 2):
Benzer in his study assigned all the mutations to locations within the rII locus. Out of the greater
than 3000 mutations he investigated he found that 1612 were non-reversion mutations, and can be
assigned to chromosomal positions without complications. He mapped the 1612 mutations to 256
mutational sites. He coined the term mutons to point mutations. He found 129 sites were non-
mutable. He found that some spots were more frequently mutated than others. He called them hot
spots. These hot spots had 60-500 mutations assigned to them. The remaining mutational sites
(approximately 200) had only 1-2 mutations assigned to them. These mutable sites were like islands
within the non-mutable sites, which led him to suggest the term recon for the unit of recombination.
By carrying out complementation tests he demonstrated that the mutations he found mapped into
two cistrons, rIIA and rIIB. Hence, the rII locus has 2 cistrons (genes).
A comparison of Benzer's mutational sites with nucleotide lengths (bp) reveals that each of the
mutational sites were less than 5 nucleotides and as small as 1 nucleotide. This indicates that the
unit of mutation and unit of recombination is a nucleotide, which is the unit of structure.
2.3.3 The Modern Concept Of A Gene And Its Evolution
The word gene has assumed a variety of meanings during the process of development of the Science
of Genetics. At different times certain aspects were attached to it and certain aspects removed. This
account traces the various concepts of the gene during the development of the Science of Genetics
and its meaning as it stands now.
1. The Mendelian Concept
During Mendel's days the location of genes or their composition was not known and the
genes were regarded as particles, hence the term particulate inheritance. These particles
were considered to be carriers of units of inheritance, from generation to generation, and
were thought to occur in pairs. These particles were hence, thought to be the unit of
inheritance, the unit of recombination and the unit of function.
2. The Chromosome Theory of Inheritance and the Beads-on-String Concept
63
During the early 20th century with the rediscovery of Mendel's work and the emergence of
the light microscopy, showed that the units of inheritance in fact were chromosomes and that
the genes that were responsible for various characters (unit of function) were arranged in a
linear order on the chromosomes. The genes were believed to be analogous to beads and the
chromosome was thought of as a string with beads. It was believed that mutations occurred
on the beads and crossing over (breakage and reunion) occurred on the pieces of string
between the beads. Hence the genes were thought to be the unit of mutation and therefore
the unit of structure and the unit of recombination (since they were thought not to be
divisible by recombination) and of course the unit of function.
3. Benzer's Theory of Recons, Mutons, and Cistrons.
During the 1940's the nobel prize winning work of Benzer led to a new concept of the gene.
He studied one locus in bacteriophage T4, called the rII locus. Mutation of this locus
produced larger irregular plaques compared to the normal small circular plaques produced
by the wild type r+. He mapped 1612 mutations into 300 points within the rII locus by
recombination tests. This clearly showed that the gene was divisible by recombination
events. He called the points of mutation, mutons and referred to them as the unit of
structure. He found through complementation tests that the 300 mutons fell into two
complementation groups, Cistron A and Cistron B. He called these functional units cistrons
and considered them analogous to the gene. Further he called the subdivisions within which
recombination cannot occur as recons.
His concept of the gene was that the gene was the functional unit. This was subdivided into
recons which were considered the unit of recombination. ie recombination can occur only
between recons. The recons were subdivided into mutons, which were the structural units.
4. Modern Concept.
The modern concept of the gene emerged from a series of discoveries starting from the
discovery of the nature of genetic material by Watson and Crick in 1954. They defined the
genetic material as DNA and the unit of structure of the DNA molecule as the nucleotide.
Hence the unit of mutation is also a nucleotide, as point mutations affect only a single
nucleotide. According to the modern concept, provided adequate progeny numbers are
screened, mutations on adjacent nucleotides can be separated by recombination. Hence the
nucleotide is also the unit of recombination. Beadle and Tatum developed the concept of one
gene - one polypeptide and clearly defined the gene as the functional unit. One gene
(cistron) therefore results in one polypeptide, which controls certain functions and is made
up of a series of nucleotides that code for a polypeptide. We will learn about this in greater
detail in the next section of the course.
Hence, as it is understood today the genes are the functional units defined by the cis-trans
test but not the structural, mutational or the recombination units.
5. Genomics concept (refer to the section on genomics in molecular genetics)
64
CHAPTER THREE
MOLECULAR GENETICS
3.1 Molecular Nature Of The Gene
3.1.1 Introduction
In the previous sections, we saw that the chromosomal theory of inheritance based on the parallel
between the behavior of chromosomes, during meiosis and mitosis, and that of Mendel's particles
suggested that chromosomes are the carriers of the genetic material. It also became apparent from
Benzer's gene fine structure analysis, in the 1940's, that the genes are not the unit of structure (unit
of mutation) or the unit of recombination of the chromosome, but are the unit of function, defined
by the cis-trans test. Although, research work by Friedreich Meischer in the 1880's (1884-1887) had
indicated that nucleoproteins consisted of a nucleic acid fraction and protein fraction (histones) and
later studies had shown that the chromosomes were indeed made up of nucleoprotein, there was
considerable debate as to whether which of these fractions (nucleic acid or protein) constituted the
genetic material. The first experimental evidences of DNA as genetic material came with some
carefully designed experiments, outlined below.
3.1.2 Dna Is The Genetic Material - Evidences.
1. Discovery of the transforming principle
Frederick Griffith (1928), in experiments with Streptococcus pneumoniae found that heat
killed S. pneumoniae from a virulent strain was able to convert a normally harmless strain
into a virulent one. He called this factor that was able to convert the harmless strain to a
virulent strain the 'transforming principle'
There are two serological types of Streptococcus pneumoniae, those that produce a type-II
capsule are serologically different from those that produce a type-III capsule. The
capsulated types produce smooth colonies and are virulent. Occasionally these can mutate
to noncapsulated forms which produce rough colonies and are avirulent.
Griffith's experiment was conducted on rats, where he demonstrated that a heat killed
virulent type-III strains (Type IIIS) were able to transform avirulent, type IIR, into virulent
forms. Details are as follows.
a) Type IIIS (heat killed) injected to rats - no death

b) Type IIR (live) injected to rats - no death
c) Type IIIS (live) injected to rats - death
d) Type IIIS (heat killed) + type IIR injected to rats - death
65
Later, in 1944, Avery, Macleod and McCarty demonstrated that the transforming principle
is DNA. They separated the heat killed type IIIS into polysaccharides, proteins and DNA.
They found that the DNA component, even after treatment with proteases to degrade all
residual protein, was able to transform type-IIR to type-IIIS. This strongly suggested that
DNA is the genetic material capable of causing a hereditary change.
2. Hershey-Chase Experiment
Bacteriophages consist of a DNA core with a protein coat. Phosphorus is not found in
protein but is an integral part of the DNA, similarly sulphur is present in proteins and never
in DNA. In separate experiments they incorporated 32P or 35S to the medium in which the
bacterial lawn and the phages (T2) were grown. This would produce T2 phage particles
with either 32P labelled DNA or those with 35S-labelled protein coats. They used these
phages, in separate experiments, to infect Escherichia coli. The empty protein sheaths
(called ghosts) attached to the bacterial cells are first sheared away by high speed blending.
The ghosts were separated from the bacterial cell by centrifugation and each fraction was
tested for its radioactivity. When the 32P label was used most radioactivity ended up in the
bacterial cell fraction, and when 35S was used the radioactivity ended up in the ghost
fraction. The results strongly suggested that the DNA that enters the bacterial cell is the
genetic material.
Figure 11
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In a similar experiment the phages were plasmolyzed to cause ruptures on the protein coat
and were treated with DNAse. The DNase would enter through the ruptures in the protein
coat and degrade the DNA. They used the resulting 'ghosts' to infect bacteria, there was no
lysis and no progeny phages. However, without DNAse treatment the phages caused lysis.
The results indicated that when there is no DNA there is no phage.
3. Reconstituted Tobacco Mosaic Virus (TMV) Experiments.
Each strain of TMV is surrounded by a protein coat with specific antigenic properties. TMV
particles were reconstituted in vitro with the RNA of strain A and the protein coat of strain
B. The reconstituted TMV particles were used to infect tobacco plants and the progeny
TMV particles were collected and analysed. TMV recovered had a type A coat. This shows
that the protein coats were coded for by RNA, and not the proteins, again confirming that
protein is not the genetic material.
Figure 12
3.1.3 Structure Of Dna - Dna Is A Double Helix
While the debate on what was the genetic material was going on scientists were better
understanding what the DNA was made up of.
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Levene elucidated the constituents of DNA as deoxyribose sugar, N-containing bases and PO 4- and
found that the DNA was made up of nucleotides, with each nucleotide consisting of a deoxyribose
sugar, N-base and a PO4- group. The PO4- group was attached to the deoxyribose sugar at the
Carbon-5, while the bases were attached to the sugar at Carbon-1. There were four kinds of bases
(H-ion acceptors), adenine, guanine, thymine and cytosine.
adenine - 6C-NH2 (amino base)

Bases purine
(two ring) guanine - 6C-C=O (keto base)
thymine - 6C-C=O (keto base)

pyrimidines
(single ring) cytosine - 6C-NH2 (amino base)
In the 1940's Chagaff, found that organisms varied in the content of the various bases, but he found
that
Purines = pyrimidines
hence A+G/T+C = 1 ; A+C/G+T = 1
6-amino bases = 6-ketobases
This suggested a paired relationship between A and T and between G and C.
Franklin and Wilkins based on X-ray diffraction data, were accumulating evidences, at the same
time, which suggested that DNA was a long, linear helical molecule with a parallel multistrand
structure.
Watson and Crick (1953) were able to put all the above evidences together through a process of
model building and come up with the most plausible three dimensional structure for the DNA -
double helix. The double helix nicely accounted for the X-ray data and tied in very nicely
Chargaff's base pairing rules.
The structure consists of two antiparallel strands made up of sugar-PO4- linkages (phosphodiester
bonds), forming the backbone of the molecule which are interconnected by H-bonds between
complementary base pairs (A-T; G-C), forming the steps. Watson and Crick showed that G and C
and A and T showed the necessary lock and key shapes to permit efficient hydrogen bonding, when
the strands ran antiparellal to each other (ie one strand runs 5'-3', while the other runs 3'-5'). There
are three H-bonds between G-C and two between A-T. The entire staircase like structure is helically
coiled to produce a double helix which maintains the same diameter (22 A o) throughout the DNA
molecule and same width of the steps (11 Ao). The steps are 3.4 Ao apart and each step is turned 36o
such that a complete turn of 360o involved 10 steps and is 34 Ao long.
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Evidence for antiparellality and complementarity of bases.
- comes from nearest neighbour analysis of in vitro DNA synthesis experiments. We will come to
this point after we deal with DNA replication.
3.1.4 Properties Of Dna
The double helix (dsDNA) can be denatured (melted) to produce ssDNA. This can be achieved by
temperature, chemicals or by lowering salt concentration. The ssDNA can anneal to produce
dsDNA when the conditions are returned to normal conditions.
a) Denaturation:
Melting Temperature (Tm):
The temperature at which DNA denatures into single stranded molecules is called the melting
temperature (Tm). The melting temperature varies between 82-95oC, depending on the GC content
and the salt concentration. Note that the DNA strands are held together by three H-bonds between
G-C and two H-bonds between A-T. Hence greater the GC content, higher the melting temperature.
Also note that lowering the salt concentration would remove the cations that shield the negative
charges in the DNA molecule and hence DNA melts at a relatively lower temperature.
Chemical denaturation:
Denaturation of dsDNA can also be accomplished by using the chemicals formamide or

dimethysulphoxide, which disrupts the H-bonds.
b) Renaturation or annealing:
Renaturation occurs 25oC below Tm. However, quenching, process of rapid cooling to very low
temperatures, keeps the DNA molecules single stranded. The annealing time depends on DNA
concentration, the salt concentration and the uniqueness of the DNA sequence.
c) Feulgen test
Feulgen test provides a unique test for DNA. DNA is subjected to warm acid hydrolysis, which
loosens the DNA helical structure and exposes the ketogroups. The ketogroups of the deoxyribose
are stained by basic feulgen (Schiff's reagent) to give a reddish purple colour.
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d) DNA fragments can be separated based on size by gel electrophoresis
DNA size is measured in bp, length or MW.

DNA fragment size can be determined by gel electrophoresis with an adjacent lane
of DNA fragments of known size (bp).
10 bp = 34 Ao Therefore, bp x 3.4 = length in Ao (10-10 m)
The molecular weight of one nucleotide is approximately 660.
Hence, bp x 660 = MW of DNA
3.1.5 Role Of DNA
DNA should fulfill two basic functions
a) Genotypic function: Should account for its precise replication and faithful
transmission from one generation to next. This means it
should be able to replicate itself accurately and transmit itself
to the future generations.
b) Phenotypic function: Should account for the storage of the enormous information
coding for various traits and account for the expression of
phenotypes and regulation of expression.
3.2 DNA REPLICATION
In this section we will look at how the DNA molecule fulfills its genotypic function of accurate
replication of itself and transmission perpetually. We will be focusing on DNA replication in
prokaryotes, but would be drawing your attention to some of the differences in Eukaryotes as we go
along.
3.2.1 DNA Replication Is Semiconservative
Watson and Crick, at the time of elucidation of the DNA structure, noted the above as a simple and
quite possible mechanism of DNA replication, self-evident from its structure. They suggested that
the DNA helix could unzip to produce single stranded molecules, which can serve as templates for
the building of the double helix by complementary base pairing. According to this model, each
daughter molecule would contain one parental nucleotide chain and one newly synthesized
nucleotide chain. Hence semi-conservative, since only one of the two parental strands is conserved
in the newly formed helix. At the time it was proposed it was considered oversimplistic because it
was not clear how a long, cumbersomly coiled DNA molecule complexed with proteins can simply
unwind, reform a new strand and rewind again at the speed at which DNA replication is known to
occur.
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The Meselson-Stahl Experiment (1958).
Their elegant experiment was able to clearly establish that DNA replication in fact followed the
Watson-Crick Model (Semi-conservative model). The Watson-Crick model was tested against two
other hypothetical models, viz.
a) conservative model.
In this model the parental double helix is conserved while the daughter strands
formed by an unknown mechanism rewind to produce a new helix (consisting of the
newly synthesized strands).
b) dispersive model.
In this model, the double helix formed would consist of segments of parental and
newly synthesized strands. This model implied that the parental helix is fragmented,
copied and formed by union of newly synthesized and parental fragments.
The method
Meselson and Stahl devised a method (CsCl2 buoyant density gradient centrifugation), which
allowed DNA to be separated based on its density. In this method, DNA and CsCl 2 (6M) are
centrifuged in an ultracentrifuge at high speeds (50,000 rpm) for many hours. The centrifugal force
establishes a CsCl2 gradient. The DNA because of the opposing forces of buoyancy and centrifugal
forces attains an equilibrium level at a particular density level. They found that DNA isolated from
E. coli, grown in a medium containing heavy 15N, had denser DNA than DNA from E. coli grown in
a '14N medium'.
The experiment
Meselson and Stahl grew E. coli in heavy 15N medium for several generations, transferred them into
an 14N medium and then after a single generation in 14N harvested the cells, isolated the DNA and
subjected it to CsCl2 centrifugation. The found a band at an intermediate level to those grown in 14N
and 15N. This suggested that the helices formed were in fact hybrid in nature, consisting of one
heavy and one light DNA strands. When they allowed growth in 14N for one additional generation
of multiplication, they found that they had the hybrid band along with an additional lighter band.
When sequentially more and more generations of growth were allowed before DNA isolation, they
found that the lighter band progressively increased in width but the hybrid band remained at the
same time. The results confirmed the semi-conservative model and not the other two. Can you
explain why?
Hint: If the conservative model was the case, one would expect to find a light and a heavy band
after one generation, not a hybrid band. If the model was dispersive, one would not expect two
distinct bands at the end of two generations but rather a hybrid band, perhaps slightly shifted
towards the lower density.(Figure 13)
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The results strongly suggested that replication of DNA follows a semi-conservative model. This
was confirmed later by another scientist (Taylor) who allowed replication to occur for two
generations in the presence of bromodeoxyuridine, followed by giemsa and florescent dye staining,
which revealed the hybrid nature of the helix.
3.2.2 Mechanism of Replication
Although, following Meselson and Stahl's experiment the semi-conservative model was widely
accepted the mode by which semi-conservative replication occurred continued to be a dilemma
since the model could not explain the speed at which replication occurred in the cell. For instance,
the largest chromosome in E. coli consists of 65 million base pairs. How does semi-conservative
model of replication take place at such rapid pace?
3.2.2.1 Theta - Mode

DNA replication in Escherichia coli: theta mode, single origin, bi-directional.
Evidence for the Theta-Mode
You will remember that bacteria have a single circular 'chromosome'. John Cairns (1963) grew E.
coli on radioactively labelled thymidine (tritiated thymidine, 3H) for several generations and at the
end of each replication cycle he isolated DNA, autoradiographed it and examined the
autoradiograms under a electron microscope. The procedure allows the newly synthesized DNA
strand incorporating the radioactive thymidine to be 'radioactive' or 'hot'. Autoradiograms are
produced by exposing the DNA smeared on a slide, on photographic film. At the end of the first
cycle, the emissions emitted from the 'hot', newly synthesized strand would reveal a circular
chromosome and as the second cycle of replication begins a theta structure is revealed (see figure,
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below)- hence referred to as the theta mode of replication. The theta structures always originate
from a single point on the chromosome, referred to as the origin of replication. E. coli therefore has
a single origin of replication.
Figure 14
Evidence for single origin, bidirectional replication (Moving fork model) - pulse-chase
experiment
In a modification of the technique, replication was allowed for one generation with a pulse exposure
with 'hot' thymidine (referred to as the pulse step) and followed by an excess of 'cold' thymidine
(referred to as the chase step or the dilution step). Such experiments were called pulse-chase
experiments. The autoradiograms during the second cycle of replication would reveal forks moving
in opposite orientation from the origin, with the relatively newly synthesised regions of DNA
revealed as relatively less intense in autoradiograms as compared to the older regions of the
synthesized strands, closer to the origin, which appear more intense. Hence the theta mode of
replication was referred to as the 'Moving Fork' replication method since two forks move (@ 60,000
nucleotide incorporations per min) bidirectionally from the single origin of replication until the
entire chromosome is copied. It takes 40 min for each replication cycle (E. coli genome size = 4.7 x
106).
Note: In Eukaryotes, DNA replication also shows the theta mode or the moving fork model, but
has multiple origins. Hence replication begins at several points along the chromosome and
proceeds bidirectionally. This was shown by similar experiments, as above. Replication
occurs at a rate of 2600 nucleotides per min originating from around 8500 origins of
replication per chromosome. Replication therefore takes place in only 5-10 hours).
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3.2.2.2 DNA replication in Phages - Sigma mode (rolling-circle replication)
Phages in addition to the theta mode show a sigma mode of replication, which accounts for the rapid
increase in progeny phages during the multiplication step during the lytic cycle. The sigma mode of
replication, however, results in a greater error rate (1 in 1000) than the theta mode. Hence has a
lower fidelity of replication.
Steps in the sigma mode:
i) Initiation: The phage has a circular double helix. The outer strand is referred to as
the +ve strand, while the inner is referred to as the -ve strand. Replication starts at a
specific nick (cut) site (sugar-phosphate bond is broken) on the +ve strand.
ii) Elongation: DNA is synthesized by addition of dNTPs (the four

deoxyribonucleotides, in the triphosphate form; N = A; T; C or G) at the 3' end of the
nick site, by the DNA polymerase. DNA synthesis proceeds in the 5'---3' direction,
copying the negative strand.
As the synthesis begins at the 3' end, the 5' end of the -ve strand is dragged out from
the circle. Therefore as the replication proceeds around the circle the 5' end rolls out
as a tail, increasing in length.
As a tail is extended, a complementary strand is produced (-ve strand), which results

in a double stranded concatemer tail. Concatemers refer to long linear molecules of
DNA formed by multiple unit length copies of the circular DNA linked end to end.
iii) Resolution of concatemers into unit lengths.

Concatemers formed are resolved into unit lengths which are packaged in protein
sheets due to the affinity of protein to the +ve outer strand of the circular double
helix.
3.2.2.3. The Replication Enzyme and its Properties
The replicating enzyme, DNA polymerase, was isolated and purified first from E. coli by Arthur
Kornberg in 1950's. There are three species of DNA polymerase enzymes in E. coli, viz. DNA
polymerase I; DNA polymerase II; and DNA polymerase III. The roles of the pol-I and pol-III in
replication have been well defined. Pol-III is the major replicating enzyme, while Pol-I plays a
secondary role as a repair enzyme. Role of Pol-II is not defined.
In vitro synthesis of DNA
Arthur Kornberg (1957) showed that DNA polymerase was able to replicate DNA in in vitro
experiments, in the presence of
74
a) Single stranded (ss) template DNA: The template DNA provides a single stranded
template to be copied by DNA polymerase.
b) Primers: These are short complementary ssDNA or RNA molecules that bind to the
template DNA and provide start sites for the DNA polymerase to bind and initiate
replication.
c) dNTPs (the four deoxynucleotides in the triphosphate form): dNTPs serve as the
building blocks, which are hooked-up into a nucleotide chain by the polymerase
activity of the DNA polymerase enzyme following the dictations of the single
stranded template DNA strand.
d) Mg++ : serves as cofactor to the replicating enzyme.
Properties of the DNA polymerases:
The properties of DNA polymerase is as follows:
i. Template requirement
ii. Primer requirement: In other words there is the requirement of free 3'-OH for
replication initiation by DNA polymerases. Therefore replication begins at the 3'
end. As a consequence of this the replicating strand always proceeds in the 5'-.3'
direction, only. DNA polymerases therefore have a 5'-3' polymerase activity.
iii. Mg++ requirement as a cofactor.
iv. Polymerase and proof reading functions : DNA polymerase III, the major
replicating enzyme, has two activities. As we saw before it has a 5'- 3' polymerase
activity, which accounts for the formation of phosphodiester bond between the 3'-
OH and the 5' PO4 group, thus linking dNTPs together into a chain. In addition, it
has a 3'- 5' exonuclease activity, which provides it the proof reading function. For
instance, if a mistake occurs during the incorporation of a nucleotide the polymerase
enzyme has the ability to degrade the synthesized chain in a 3'- 5' direction until the
mistake is removed and the chain resynthesized. This provides fidelity to the
replication process. DNA polymerase I, has a 5'-3' polymerase activity and a 5-3'
exonuclease activity (repair activity).
3.2.2.4 The Growing Point Paradox And Semi-Discontinuous Replication.
Growing Point Paradox:
Autoradiography and electron microscopy (pulse-chase experiment) show that the DNA
strands synthesized at each strand of the replicating fork extend in the same direction at the
macromolecular level. The template DNA of the replicating fork consists of a leading strand
that runs in the 3'--5' direction and a lagging strand that runs in the 5'--3' direction (see figure
below). While it is possible for the DNA polymerase to copy the 3'-5' leading strand in the
5'--3' direction, owing to its 5'-3' polymerase activity, the observation that the lagging strand
75
too was copied in the 3'-5' direction was paradoxical, in light of the known absence of 3'--5'
polymerase activity in DNA polymerases.
The growing point paradox, hence, refers to the inability of researchers to reconcile the
observation (based on autoradiography and electron microscopy) that the replication fork
moves at the macromolecular level in both DNA strands (5'-3' and 3'-5') in the same
direction with the biochemistry of DNA polymerase enzyme, the fact that DNA polymerase
only has a 5'-3' polymerase activity.
Okazaki Fragments and the Resolution of the Paradox:
Okazaki by digesting replicating DNA with DNase, an enzyme which specifically degrades
DNA, recovered small RNA fragments that were resistant to degradation by DNase. Further,
the addition of rifampicin, an inhibitor of RNA polymerase, inhibited DNA synthesis, which
suggested a role for RNA. He recognized that these RNA fragments were priming DNA
synthesis in the 5'--3' direction on the lagging strand of the replication fork in a
discontinuous manner in small stretches (now referred to as okazaki fragments). Hence
although at the macromolecular level it appears as though replication was taking place in the
3'-5' direction on the lagging strand, at the micromolecular level it was really taking place in
the 5'--3' direction in small stretches primed by RNA primers. These okazaki fragments
(approximately 2000 bases long) are later joined by DNA ligase to give a continuous DNA
strand complementary to the lagging strand.
Semi-discontinuous replication.
Replication is hence semi-discontinuous, continuous on the leading strand and

discontinuous on the lagging strand.
3.2.2.5. The Replisome
Replication is carried out by a complex system of enzymes and proteins. This multienzyme-
protein complex is called the replication apparatus or the replisome. How the replisome
carries out the process of replication is well understood in prokaryotes and is described
below.
i) Strand separation:
The DNA double helix in E. coli has about 470,000 turns and the replication
molecule must make a full rotation to unwind each of these gyres. The
topoisomerases prevent tangling by uncoiling short stretches of the double helix
within the replication fork at a time.
Topoisomerases are capable of nicking one strand of the double helix, serving as
swivel (ie. hold one end while the DNA unwinds within the replication fork) and
repairing the nicks as replication proceeds past that location. DNA gyrase is one
such topoisomerase capable of forming negative supercoils, which unwinds the
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helix. DNA helicases (replication protein) are actually involved in separating the
leading and lagging strands. Rep protein is known to have ATPase activity, which
provides the energy for the process.
The free single stranded DNA is delicate and is prone to degradation and is hence
protected by ssDNA binding proteins.
Figure 15
ii) Priming:
Primers are synthesized along the template strands. They are capable of priming
DNA synthesis by DNA polymerase, by providing free 3'-OH groups necessary for
initiation of DNA synthesis. The approximately 20 polypeptides involved in the
synthesis of primers are referred to as the primosome.
The main components of the primosome are (i) the dnaB protein which serves as the
site organiser and has the banding sites for all the other components of the
primosome. It also carries the primosome along the DNA to new sites of priming in
the 5'-3' direction as several primers are required on the lagging strand. (ii) The n'
protein attached to dnaB has ATPase activity and is capable of harnessing and
providing energy for the priming reaction. (iii) primase: The primers are synthesized
by an RNA polymerase enzyme called primase. Primase does not have a 3'-OH
requirement to catalyse the synthesis of RNA primers. The primases build 10-50
nucleotide RNA stretches, which serve as initiation points for subsequent DNA
synthesis.
iii) Elongation and Proof Reading.
This is carried on by the DNA Polymerase III (replication enzyme). On the leading
strand continuous synthesis occurs, while in the lagging strand discontinuous
synthesis occurs (5'--3' polymerase activity). Errors are corrected by the same
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enzyme, owing to its 3'-5' exonuclease activity. Hence the DNA polymerase can
move back and forth polymerizing and correcting mistakes during the elongation
process. Pol-III is a complex of seven different polypeptide chains.
iv) DNA Repair
DNA repair is performed by DNA polymerase I, the repair enzyme. This removes
the RNA primers and replaces them with DNA. Where the DNA and RNA meet
there is a single stranded nick. The DNA ligase cannot seal the nick because a
triphosphate is present on the RNA end (DNA ligase can only link 3'-OH with a 5'-
monophosphate). Pol-I binds to the nick site and removes the RNA by its 5'-3'
exonuclease activity and at the same time builds the DNA chain by its 5'-3'
polymerase activity. DNA ligase can now link the nick, to produce a continuous
DNA chain.
v) Termination
Topoisomerases are involved in the rewinding of the helix while the replication fork
moves forward.
3.2.2.6. Applications: (Tutorial Topic)
3.2.2.6.1 Polymerase Chain Reaction (PCR)
PCR uses the understanding gained in DNA replication to clone (replicate) any part of the
genome flanked by known sequences. PCR uses two primers (ss DNA,10-20 bases long)
complementary to regions flanking the gene of interest.
PCR reaction mix: The template DNA, primers, Taq polymerase (a polymerase enzyme,
capable of surviving temperatures up to 92oC, isolated from Themophillus aquaticus),
MgCl2 , dNTPs and a buffer are mixed together and placed on a thermal cycler. The thermal
cycler is a electronically controlled equipment capable of changing temperatures in a cyclic
manner (92oC 3 min; 50oC 30 sec; and 70oC 1.5 min;, each cycle repeated 40 times).
Steps in PCR
i. Strand separation (denaturation)

At the first temperature step of 92 oC, the double helix denatures to produce ssDNA,
which can serves as templates for DNA replication.
ii. Primer annealing

Then the temperatures are reduced to 50oC to allow the primers to bind to the
template DNA.
iii. Polymerization reaction.
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At the temperature of 70oC (optimum temperature for TAQ polymerase activity)

elongation of the primers occurs to produce variable length strands.
iv. The cycle is repeated again. In the second cycle DNA is denatured again, primers
bound to the templates and primers extended. This would produce unit length
strands. Further repetition of thermal cycling would increase the copy no. of the
amplified unit lengths in an exponential fashion. In 40 cycle 10 12 copies of the
sequence would be produced. The amplified sequence would contain the gene of
interest flanked by the primer sequences.
3.2.2.6.2 Nearest neighbour analysis provides evidence for DNA synthesis in the 5'-3'
direction, opposite polarity of DNA strands and complementarity of bases.
In an in vitro DNA synthesis experiment 32P labelled dCTP was used so that the synthesized
DNA strands would have incorporated the radioactively labelled dCTP*. The synthesized
strands were either digested with spleen diesterase which breaks the bond between 5C and P
group or snake venom diesterase, which breaks the bond between the 3C and P group.
a) DNA synthesis in the 5'-3' direction: If an analysis of breakdown products with spleen
diesterase shows that the radioactivity has been transferred to the nearest neighbour at the 5'
end (which could be any of the four nucleotides) or if the breakdown products with snake
venom diesterase shows that radioactivity remains only on dCTP, it would confirm that the
DNA strand elongates by addition of nucleotides at the 3' end. Opposite results with
radioactivity being transferred to nearest neighbour with snake venom diesterase but not
with spleen diesterase would suggest that nucleotides are added to the 5' end. Empirical
results showed that the former was the case.
b) Opposite polarity: In this study, the four nucleotides were labelled and used in separate in
vitro DNA synthesis experiments. The DNA was degraded with Spleen Diesterase and
nearest neighbour determined.
If opposite polarity: T G If same polarity T A
= =
C A C G
The first case was only obtained, which suggested that DNA strands have opposite polarity.
c) Complementarity of bases: In the same experiment as above, it was noted that

G C T A
= =
G C T A
This suggested that G and C are complementary and A and T are complementary.
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3.3 Phenotypic Function of DNA
In the last section we looked at how the DNA performs its genotypic function of precise replication
and perpetual transmission. In this section we will examine the phenotypic function of DNA, in
other words, how does the gene work? The phenotypic function of DNA pertains to its ability to
express phenotypes, from its location within the nucleus or organelles. The DNA, through a process
of transcription and translation is able to produce proteins, which singly or in combination with
other proteins determine the phenotypes that we see. Cellular enzymes are proteins, which are able
to catalyse biochemical pathways leading to products which can influence phenotypes. Other
proteins can have a structural role. This is referred to as the central dogma, the hypothesis that the
information stored in the DNA flows from DNA to RNA to protein. Although there are some
exceptions now known, the rule is generally valid. For instance, in some viruses, RNA is the
genetic material, not DNA.
3.3.1. Historical Perspectives Of The Central Dogma
3.3.1.1 One gene-one metabolic block
Garrod (1902) first suggested that a defect in one gene may result in a metabolic block. He
observed that the patients suffering from a condition called Alcaptonuria, caused by a single gene
mutation, had a characteristic black coloured urine, due to high levels of homogentisic acid in the
urine. He concluded that the excretion of this was because of the inability to of the defective
individual to convert it.
3.3.1.2 One gene-one enzyme hypothesis
Beadle and Tatum (1940's) through a number of elegantly designed experiments, for which they
received a nobel prize, were able to demonstrate that defective genes result in defective enzymes
which function as metabolic blocks.
Genetic characterization: They mutated ascospores of Neurospora and selected for arginine
requiring mutants. How can you do this? By chromosome mapping they found that the mutations
mapped into three different loci (arg-1, arg-2, arg-3), on three separate chromosomes.
Biochemical characterization: They biochemically characterised the mutations. The arg-1 mutations
were able to grow when supplied with ornithine, citrulline or arginine. The arg-2 mutations were
able to grow in arginine or citrulline but not with ornithine, while arg-3 mutations were only able to
grow in the presence of arginine. Based on their finding they put forward the following biochemical
pathway.
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arg-1 arg-2 arg-3
enzyme-1 enzyme-2 enzyme-3
precursor --------- ornithine -------- citrulline ----------- arginine
Their findings suggested that each gene codes for one enzyme, in a complex biochemical pathway
leading to a product. Mutation of any one of those genes would code for a defective enzyme which
is unable to catalyse one step in the pathway, thus resulting in the mutant phenotype.
3.3.1.3 One gene-one polypeptide
Vernon Ingram (1957) studied the haemoglobin protein. The haemoglobin-A (found in adults) is
made up of four polypeptides, two alpha and two beta chains. He compared the haemoglobin of a
normal person (HbA) with that of a person with sickle cell anaemia (HbS) and found that the
protein fingerprint differed only in one spot. He sequenced the spot to determine the aminoacid
sequence of that fragment using Sanger's technique of aminoacid sequencing. He found that at the
6th position of the beta chain, the aa glutamic acid found in HbA was substituted by valine in HbS.
This suggested that one mutation of a gene affects only one aminoacid of a polypeptide chain and
that many polypeptides can make up a protein. Mutation of GAA to GTA
3.3.1.4 Colinearity of gene and protein.
Tryptophan synthetase, which catalyses the conversion of indole glycerol phosphate into tryptophan
is a multimeric protein made up of two polypeptides coded for by two genes trpA and trpB. Charles
Yanofsky (1964) studied the mutations in trpA in E. coli. He ordered and mapped 16 mutations
within the trpA gene using recombination mapping that you have been exposed to before. He later
analysed the position of aa changes in respect of the mapped mutations and found that the position
of aa change within the polypeptide corresponded to the position of the mutation within the gene.
He concluded that the gene and the protein are colinear.
3.3.2 Exceptions: Non-colinearity; one gene many polypeptides
There are exceptions to the rule. For instance, in eukaryotes the coding sequences (exons) are
intervened by intervening sequences (intron) which are spliced out of the mRNA and are hence not
translated. Here, although the order of mutations may be the same they may not be colinear with
respect to exact positions. Similarly, in viruses, it is common to find genes within genes. Different
polypeptides can be formed by the same DNA sequence being read in different reading frames.
Hence, one gene may produce more than one polypeptide. Some viruses produce polyproteins,
which are many polypeptides translated together as one protein. This is later cleaved to produce
different polypeptides. These exceptions do not detract from the general case.
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Role of mRNA as an intermediary between DNA and protein
DNA-RNA
i) Correspondence between DNA and RNA base ratios in phages.

ii) ssDNA and RNA form hybrid molecules, which shows complementarity.
iii) If use DNAse to degrade DNA, there was no RNA synthesis.
iv) Evidence from in vitro synthesis of RNA - DNA template requirement for RNA synthesis.
RNA-PROTEIN
i) Cells that produce large amount of proteins also had large amounts of RNA while cells
producing less proteins had less RNA (liver, pancreas, silk glands - more RNA; heart, lungs
- less RNA)
ii) RNA rich areas are heavily stained with basic dyes. Cells subjected to RNase showed
degradation of these heavily stained areas and a corresponding drop in protein synthesis.
iii) When cells are fed with radioactive rNTP and a pulse-chase experiment carried out the
labelled RNA first shows up in the nucleus and then ends up in the cytoplasm. This
suggested that RNA is synthesized in the nucleus and transported to the cytoplasm.
iv) When cell homogenates are fractionated into nuclei mitochondria, ribosomes and
supernatant by differential centrifugation, most of the RNA was located in the ribosomal
fraction. Further, electron microscopy show polysome association of mRNA.
v) Cracking of the genetic code showed an association between RNA sequence and the aa it
coded for.
3.3.3 Transcription
We saw in the previous section, evidences that accumulated supporting the central dogma,
which suggested that the information stored in DNA flows to proteins through RNA
intermediaries. This information flow occurs through two processes, transcription and
translation.
3.3.3.1 Definition
Transcription refers to the synthesis of a mRNA molecule by the DNA dependent RNA
polymerase enzyme according to the dictations of the genetic code on the sense strand of the
double helix.
3.3.3.2 RNA Polymerase Enzyme

It is DNA dependent. In other words it requires a DNA template to initiate RNA synthesis.
However, it does not have a primer requirement. It initiates RNA synthesis by attaching
itself to a DNA sequence that is found immediately upstream of the coding sequence of the
gene, called the promoter sequence. Nucleotides are added only to the 3'-OH end and hence
the chain grows in the 5'-3' direction.
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The RNA polymerase holo enzyme has two components a) the core enzyme, which is made
up of five polypeptides (2 α, 1β, 1 β' and 1ω) and (b) a sigma factor. The core enzyme has
the DNA dependent polymerase activity, while the sigma factor provides specificity to the
holo enzyme ie. it tells the holo enzyme genes are to be transcribed.
Note: In prokaryotes, the same holo enzyme transcribes, rRNA, mRNA and t-RNA. In
eukaryotes, however, there are three RNA polymerase, each of which transcribes only one
RNA species.
RNA polymerase-I = rRNA

RNA polymerase-II = mRNA
RNA polymerase-III = tRNA
Promoter: These are upstream regulatory DNA sequences (20-200 bp long) which provides
sites for RNA polymerase binding to the upstream end of the gene to be transcribed. They
also provide sites for other regulatory protein binding. In a typical prokaryotic promoter,
there are two conserved domains to which the RNA polymerase of prokaryotes will bind,
the -10 box (plibnow box or TATA box) with a conserved sequence of TATAAT and the -35
Box with a conserved sequence domain of TTGACA. There may be other domains in the
promoter to which other regulatory proteins can bind. There is substantial variation among
promoters that determines the strength of RNA polymerase binding and therefore the
transcription rate. The promoter strength varies (104 fold) depending on variation in the -35
and -10 domains and the no. of bases between them. More closely the -35 and -10 domains
resemble the consensus sequence the stronger the promoter would be. Note: conserved
sequences or domains are those typical of a promoter determined by the majority rule
Transcription is asymmetric: RNA is transcribed from a single strand of the double helix
in each gene. The transcribed strand of DNA is called the sense strand (anticoding strand).
The sense strand would produce sense mRNA (coding mRNA = codons), which on
transcription would produce the sense protein. The untranscribed strand is called the
antisense strand (coding strand, because resembles the coding mRNA) and does not give a
functional protein. The position of the promoter in relation to the coding sequence
determines which strand would be transcribed.
3.3.3.3 Steps in Transcription:
1. Promoter recognition: Promoter recognition and specificity is provided by the sigma factor
of the RNA polymerase holo enzyme. The strength of binding of the RNA polymerase to
the promoter and therefore the transcription rate depends on the conserved domains
themselves and the distance between them as we saw before.
2. Chain initiation: The binding of the RNA polymerase to the promoter causes localised
melting of a 10 bp region near the transcription start site (+1 site), forming an open promoter
complex (transient bubble). Transcription of the 3'-5' DNA strand occurs from the +1 site.
The first ribonucleotides added are ATP or GTP.
83
3. Chain elongation: As transcription is completed in the bubble the bubble moves to the next
10 bp, synthesizing RNA in the 5'-3' direction (asymmetric transcription). As the bubble
moves the transcribed part of the DNA rewinds to produce the double helix. Once the first
RNA polymerase molecule reaches has transcribed 50-60 nucleotides, the promoter
becomes available for another RNA polymerase to initiate transcription. Often many RNA
polymerase molecules transcribe the gene at any one time.
4. Chain termination: Special sequences terminate RNA synthesis. When the RNA
polymerase reaches the termination sequence, it dissociates from the DNA and the newly
synthesized RNA molecule is released. Two kinds of termination events are known a) DNA
sequences that form secondary structures that removes the RNA polymerase from the DNA
molecule. These sequences are called self-terminating. b) DNA sequences that provide sites
for the binding of termination proteins such as the rho protein, which stops the RNA
polymerase from continuing transcription.
In E. coli the average transcribed RNA has a molecular weight of 500,000 and an average
life span of 2 min at 37oC. Hence it has to be translated quickly or else it gets degraded.
The transcribed mRNA contains a Shine-Dalgarno sequence (ribosome binding site)
upstream of the translation initiation site (AUG). This untranslated mRNA sequence targets
the mRNA to the ribosome. This is also called a leader sequence.
Note In Eukaryotes, transcription is carried out by the RNA polymerase in association with a
number of proteins called transcription factors. The RNA polymerase together with the
transcription factors form a transcription complex which carries out transcription. This
allows a greater degree of regulatory control of transcription because modification of any
one of the polypeptides involved in the transcription complex can affect RNA polymerase
binding to the promoter and therefore transcription.
In Eukaryotes, immature mRNA (primary transcript) is normally subjected to several

modifications before it is converted to mRNA. This is called RNA processing. a) capping:
the 5' end of the transcript is modified by addition of a cap - a modified guanosine is added
to the 5' end by an atypical 5'-5' linkage. b) the 3' end is modified by addition of a
polyadenosine sequence (poly-A tail), which consists of as many as 200 nucleotides. c)
introns are removed by splicing. Capping is necessary for mRNA binding to the ribosome,
whereas polyadenylation improves mRNA stability. These modifications allow another
level of regulatory control of gene expression to be exerted at the post-transcriptional level.
Prokaryotic mRNA does not undergo any modifications.
3.3.4 Translation
3.3.4.1 Definition
The process by which genetic information stored in the form of a ribonucleotide sequence is
translated following the dictations of the genetic code into a polypeptide chain.
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3.3.4.2 The Protein Factory
Translation of mRNA into a polypeptide chain takes place on a ribosome. The ribosome
hence is referred to as the protein factory. Ribosome is an RNA-protein complex consisting
of two subunits, a smaller 30S subunit and the larger 50S subunit. The smaller subunit is
made up of a 16S rRNA molecule and 21S protein molecule; whereas the larger subunit is
made up of two rRNA molecules (23S; 5S) and a 30S protein molecule.
The function of the ribosome:
i) serves as an intermediary in stabilizing the trinucleotide attachments between

mRNA and tRNA. The larger subunit stabilises the aa charged tRNA and has two
tRNA binding sites. The smaller subunit stabilises the surface of the mRNA.
ii) The ribosome moves along the mRNA bringing different codons into the t-RNA
binding sites.
iii) Provides sites for enzymes involved in translation such as aminoacyl tRNA
synthetase, peptidyl transferase, other protein factors involved in transcription and
GTP.
3.3.4.3 The Adapter Molecule (tRNA)
The transfer RNA (tRNA) serves as the adapter molecule which carries the aminoacids at
one end and attaches to the codon on the mRNA sequence by the other end. The tRNA
molecules are 70-80 nucleotides long and are subjected to several post transcriptional
modification which include, clevage, methylation of bases, addition of rare bases, addition
of an ACC sequence to the end, which gives the tRNA molecules the characteristic clover
shaped structure. The structure consists of steps which are double stranded and loops which
are not. The various t-RNA species in the cell are capable of carrying a specific aa. eg
tRNAcys can carry the aminoacid cystein to the ribosome.
3.3.4.4 Steps in Translation or Protein Synthesis
1. Activation: The tRNA molecules become charged by the addition of the specific
aminoacid to the ACC- end of the corresponding tRNA species. This occurs
in two steps
a) Activation of the AA:
aminoacyl t-RNA synthetase + AA + ATP = enzyme-AA-AMP + H2P2O7
For each AA there is a specific enzyme (20 different enzyme).
85
b) t-RNA attachment:
enzyme-AA-AMP + tRNA = AA-tRNA + AMP + enzyme
Again the tRNA is specific to each aminoacid. The aminoacid attaches to the OH- group in
adenine at the ACC- end.
2. Initiation:
a) The mRNA binds to the 16S rRNA in the small subunit of the ribosome by its ribosome
binding site (5'-GGAGG-3') located on the untranslated leader sequence. The efficiency of
translation initiation is determined by the efficiency with which mRNA can bind to the
ribosome (no of bases homology, varies from 3-6) and the length of the leader sequence.
b) At this point the large subunit of the ribosome attaches to the small subunit. Remember
the large subunit has two sites for t-RNA binding, the A site and the P site.
c) The initiation codon (AUG) registers in the aminoacyl site (A site) and the tRNA-
methionine-F attaches itself to the initiation codon at the anticodon end, by hydrogen
bonding. Three initiator proteins (initiation factors), IF1, IF2 and IF3 along with GTP are
necessary for this bond formation.
3. Elongation:
a) The ribosome moves leftwards so that the initiation codon registers in the P site
(peptidyl site) while the second codon of the mRNA registers in the A site. This
movement is mediated by a translocation factor called translocase (EFG)
b) tRNA-AA attaches to the new codon in the aminoacyl site (A site). Elongation
factors EFTs and EFTu and GTP required.
c) Peptidyl transferase catalyses the formation of peptide bonds and the t-RNA on the P
site is released.
Peptidyl transferase
aa1-tRNA1 + aa2-tRNA2 -------------------- aa1-aa2-tRNA2 + tRNA1
d) mRNA is shuttled through the ribosome bringing another new codon into the A site,
while the codon in the A site is pushed into the P site.
PT
aa3-tRNA3 + aa1-aa2-tRNA2 --------- aa1-aa2-aa3-tRNA3 + tRNA2
Polypeptide elongation can be considered a cycle of events which are repeated again
and again. The m-RNA is transcribed always in a 5'-3' direction. 1000 nucleotides
86
are translated in one second. The translated proteins are folded in another 10
seconds.
4. Termination.
Translation is stopped when a termination codon registers in the A site. Termination codon
or stop codons are UAA, UAG, or UGA. There are no tRNA species that can bind to the
stop codons. These codons interact with release factors RF1, RF2, and RF3 which cleaves
the polypeptide chain from the last tRNA. There is another release factor RR F that ejects the
last t-RNA from the ribosome.
3.3.4.5 Polysomes:
The unit of translation is never simply one ribosome traversing an mRNA molecule. After
about 25 amino acids have been joined together in a polypeptide chain, and when the AUG
initiation codon is completely free from the ribosome a second initiation complex can form.
Similarly when the second ribosome has relieved the initiation codon a third ribosome can
initiate translation. Hence in a translation unit (called a polysome) the mRNA is covered
with ribosomes at a density of approximately one ribosome per 80 nucleotides. This is how
translation occurs in both prokaryotes and eukayotes.
3.3.5 The Genetic Code
Introduction
How does the four nucleotides present in the DNA code for the 20 amino acids as well as start and
stop codons in translation? The gemetic code is the list of all codons and the amino acids that each
one encodes. Before the genetic code was determined experimentally researchers had reasoned
(deductive reasoning) that the word size of the code should be at least a triplet. Codons consisting
of pairs of bases would be insufficient. For example, with a duplex codon, four bases can only
form 42 = 16 codons, insufficient to code for the twenty amino acids and hence the code would be
ambiguous! With a triplet codon, there can be 4 3 = 64 codons, excess of codons to code for the 20
amino acids and stop codons - the code would be degenerate.
We will look at some of the experimental evidences that showed that the genetic code in fact was a
triplet code (word size), with 64 possible codons. It was therefore degenerate. Evidences also
showed that the code was commaless and non-overlapping.
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3.3.5.1 Nature Of The Genetic Code - Experimental Evidence
Mutation studies in the rIIB cistron of T4: Crick and Barnett (1961) studied the rIIB cistron of
the T4 phage. They induced mutations in the gene by growing the bacteria in a medium containing
acridine dyes (proflavin), known to induce single nucleotide pair addition or deletion mutations.
Such mutations are called frameshift mutations, because the addition of a nucleotide or a deletion of
a nucleotide can alter the reading frame of the gene. Hence, the amino acid sequence coded for
would be entirely different from the point of mutation, resulting in in large plaques in E. coli
(Strain-B) - mutant phenotype.
Crick and Barnett classified mutations as '+' or '-', depending on whether they were additions or
deletions.
- When a '+' mutation was brought together with a '-' mutation by recombination, the wildtype
phenotype was restored. Only a part of the gene was 'out of phase', a situation referred to as
'part in phase'
- When two '+' mutations were brought together or when two '-' mutations were brought
together the resulting phenotypes were mutant because the proteins were 'out of phase'
- When three '+' mutations or when three '-' mutations were brought together a wildtype
phenotype resulted.
The experiment clearly showed that the code language was in fact based on a triplet code. In
addition, they showed that a substitution mutation affects only one amino acid in the polypeptide
chain, hence the code language must be non-overlapping. They also argued that the code language
should be commaless, because '+' and '-' mutations were able to restore wildtype phenotype. This
showed that even the 'out of phase' codons were able to code for missense amino acids and not
result in nonsense codons, which would have terminated translation. The fact that all 'out of phase'
reading frames, still code for amino acids means that the genetic code should be degenerate. A
degenerate code is one in which some amino acids are encoded by more than one codon each.
3.3.5.2 Cracking the Genetic Code:
The genetic code was deciphered by in vitro protein synthesis experiments conducted by Nirenberg
and Matthaei (1961) and later by Khorana. Polypeptide synthesis can be carried out in E. coli cell
extracts obtained by breaking cells open. When radioactively labelled amino acids and poly-U
RNA were added to the system, radioactively labelled phenylalanine -PA-PA- peptide chain was
synthesised. From this result and with the knowledge that the codon was a triplet it was concluded
that UUU must be the codon for phenylalanine. Similarly, poly-A RNA produced lysine residues
and poly-C RNA produced proline residues. Khorana used repeating dinucleotide RNA chains to
determine the aminoacid sequence. These experiments lead to the cracking of the genetic code.
88
Figure 16
Note: The genetic code is degenerate. Note that leucine, serine and arginine have 6 codons each.
Proline, threonine and alanine have four codons each. Isoleucine has three and
phenylalanine, tyrosine, cystein etc have two and methionine, tryptophan have only one
codon. Note that all amino acids except methionine and tryptophan are coded for by more
than one codon. Hence degeneracy or redundancy is the general case.
Note: That synonymous codons only differ in the third base. eg glycine GGU, GGC, GGA and
GGG. Moreover, in all cases in which two codons code for the same amino acid, the third
base is either A or G (both purines) or T or C (both pyrimidines).
Note: Three codons were found to be stop signals for translation, UAA, UAG and UGA. AUG,
the initiation codon encodes methionine.
Note: The genetic code is near universal. The same genetic code is used in nuclear genes of
almost all organisms. However, there are some minor descrepancies found in certain
protozoa and in the genetic codes of organelles. eg. in the mitochondrial genome of
Drosophila, UGA is not a stop codon but codes for trp; AGA is serine not arginine.
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3.3.5.3 How Does The Cell System Cope With Degeneracy
3.3.5.3.1. Crick's Wobble Hypothesis:
The wobble hypothesis states that while the first two bases of the codon pair with the anticodon
strictly based on the Watson and Crick rule, the third anticodon pairs loosely, sometimes to more
than one nucleotide at the third position of the codon.
Crick suggested that the pairing of the codon and anticodon has a special feature, which he
described as 'wobble', which enables pairing of an anticodon (tRNA) to several codons (mRNA).
The wobble pairing is however confined to only the third base of the triplet codon pairing.
Evidences for Crick's wobble hypothesis came from i) observed degeneracy in the genetic code (ii)
the identity of the third base in the codon is often unimportant in coding amino acids (iii) the
number of distinct tRNA molecules present in the cell is less than the codons.
Wobble has since been confirmed by direct sequencing of tRNA molecules which showed that U, I
or G in the 5' position in the anticodon can pair with more than one base on the third position of the
codon. Allowed pairing due to wobble are presented in the following table. All pairs of bases that
can form hydrogen bonds are possible except base pairs between two purines, which would cause
excessive distortion in the region of pairing.
Anticodon Allowed pairing to the third

(5' position) codon position.
__________________________________________________
A U
C G
U A or G
G C or U
I A or C or U
___________________________________________________
3.3.5.3.2 Iso-accepting species of tRNA
Certain amino acids are brought to the mRNA by more than one tRNA types. These are
tRNA species that bind to the same amino acid but have different anticodons which would
allow it to recognise different anticodons. Such tRNA species are referred to as iso-
accepting tRNA species.
90
Certain amino acids are brought to the mRNA by only one tRNA species, in such cases
degeneracy is accounted for by wobble.
Serine is an example of an amino acid in which both wobble and isoaccepting tRNA species
account for the degeneracy.
Codon tRNA Anticodon

________________________________
UCU
UCC tRNAser1 AGG + wobble
UCA
UCG tRNAser2 AGU + wobble
AGU
AGC tRNAser3 UCG + wobble
__________________________________
3.3.6 Gene Regulation
In the previous section, we examined the process by which gene expression takes place. Only a
fraction of genes (10% or less) are expressed continuously throughout the life of the cell. eg.
Krebs's cycle genes. Such genes are referred to as constitutive genes. Most other genes are
regulatable genes.
Rationale for regulation.
1. Most of the genes are required to function only at a particular developmental stage or time
of the life cycle or under particular environmental conditions. Expression of such genes
constitutively would be a drain on the resources of an organism and would not be selected
for in nature. Natural selection has, hence, ensured effective ways by which genes are
controlled in a way that they are expressed only when they are needed.
2. Preprogrammed control circuits are important to ensure that genes are expressed in a
sequential manner so that an organism can follow a particular developmental path leading to
determination, or adaptations to an environment etc.
3. Coordinated control is important since a number of gene products are essential to

accomplish a particular function and have to be expressed together simultaneously to be
useful.
4. Regulatory mechanisms provide plasticity to organisms, being able to adapt to different

conditions by expressing a different array of genes.
91
A diverse array of regulatory mechanisms have evolved in organisms, all of which are effective. We
will be mainly surveying regulatory mechanisms in prokaryotes and phages, which are simple and
would help to understand control mechanisms in eukaryotes, which are rather complex.
3.3.6.1 Regulation in Prokaryotes.
Operon:
In prokaryotes, genes are regulated in a coordinated fashion. Functionally related genes are hence
organised into operon. Operon are superstructures in which a group of genes within the operon are
coordinately controlled (transcriptional control) by a common set of controlling elements. Operon
are hence the unit of regulation. As a result a polycistronic mRNA is produced which is translated
into different polypeptides.
Controlling elements: cis- trans- acting elements
There are two type of controlling elements (sequences), cis acting elements and transacting
elements. Cisacting elements exert control only when they are present on the same DNA strand as
the genes they control, often immediately upstream of the genes themselves. Examples are the
promoter element and the operator element. Cis acting elements exert control by physical proximity
to the genes. Trans acting elements are controlling elements that can exert their influence on genes
from a distal position on the same DNA molecule or from a separate DNA molecule. Trans acting
elements exert their control on genes through mobile substances.
Types of control:
Control in prokaryotes is exerted at the transcriptional level - transcriptional control. There are
basically two types of control mechanisms operative in prokaryotes.
1. Negative control: The regulatory gene produces a repressor that turns off the gene. The gene is
turned on only when conditions prevail that will remove the repressor
protein.
2. Positive control: The regulatory gene produces an activator that binds to the promoter and
turns on the gene. The gene will be turned off only when the activator is not
formed.
There are two type of negative control mechanisms.
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NEGATIVE CONTROL
_______________________________________________________________
Inducible System Repressible System

--------------- -------------------
associated with associated with
catabolic pathways anabolic pathways
eg. lac operon eg. histidine operon
comes into being when comes into being when

an inducer is in the a cosuppressor is in the system
the system.
_______________________________________________________________________
3.3.6.1.1 The Lac Operon.
The lac operon has a negative control system (inducible system) with a superimposed positive
control system. This allows the lac operon to produce the lactose degrading enzymes only when
lactose is present and glucose is absent. This ensures that the enzymes are not wastefully produced.
The lac operon consists of three structural genes that are coordinately expressed under the control of
a number of controlling elements (promoter P, operator O, and repressor gene, I). The structural
genes code for B galactosidase (Z gene), B galactoside permease (Y gene) and galactoside
transacetylase (A gene). Permease is responsible for pumping in lactose into the cell system, while
B galactosidase is responsible for cleaving lactose into glucose and galactose (by breaking the B
galactosidic bond). The function of the 'A' gene is not known. The structure of the lac operon is
shown in the figure below.
Polar mutations provided evidence for coordinated transcription (therefore coordinated control) of
all the genes in the lac operon. Mutations in the Z gene, extinguishes expression of Y and A genes.
Polar mutations are chain terminating nonsense mutations, which can not only affect the genes in
which the mutation is present but also all downstream genes in the same operon. They can also be
insertions that can cause frame shifts.
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Figure 17
Negative control:
Jacob and Monad found that the lactose degrading enzymes were produced only when lactose was
present in the medium. When they removed lactose from the system within minutes the lactose
degrading enzymes disappeared. They found that the enzymes can be induced by using a lactose
analog such as IPTG (isopropyl-B-D-thiogalactoside), which was not a substrate. This suggested
that the inducer does not have to be a substrate.
Characterisation of the I and O mutations led to the understanding of the control
mechanism
Repressor gene (I gene)
Jacob and Monad characterised a mutant that was able to constitutively produce the structural
enzymes in the presence or absence of lactose. These mutations mapped close to the lac operon but
were distinct, based on complementation tests. Complementation tests also showed that I+ was trans
dominant over I-. This suggested that the I gene was able to control the structural genes through a
movable protein product. This led them to conclude that a distinct regulatory gene (I) was able to
control expression of the lactose catabolic genes through a movable repressor protein. Mutation of
the gene produced a ineffective repressor that could not block the lactose catabolic genes, hence the
reason for constitutive synthesis.
They characterised another mutation of the I gene (Is). These mutants prevented induction by
lactose. These mutations were transdominant over I+ (Is > I+ > I-). This suggested that the mutation
had altered the stereospecific inducer binding site, such that the inducer cannot bind to the repressor
protein, anymore. Can you say why Is is dominant over I-.
The operator (O)
Operator constitutive mutations (Oc) were found which were cis dominant. Cis dominance reflects
action of an element that affects genes only adjacent to it. These mutations produce enzymes
constitutively with or without the inducer and with or without an active repressor. This suggested
that these mutations had affected the repressor binding site. Therefore the repressor cannot bind
94
efficiently. Mapping experiments showed that the operator mutations were between the Z and I
genes. DNA binding assays using the repressor molecule showed that it is immediately upstream of
the transcription initiation site.
The mechanism:
The I gene produces a repressor protein which has two binding sites one that allows it to bind to the
operator region and the other which allows it to bind to the inducer molecule. In the absence of the
inducer the repressor binds to the operator and blocks the RNA polymerase from transcribing the
lactose catabolic genes. In the presence of an inducer (lactose or IPTG) the inducer binds to the
repressor protein, which changes the allostery of the repressor protein. This new conformation
lowers the affinity of the repressor to the operator and therefore it is removed from the operator.
Hence, the operon is turned on when the inducer is present and turned off when the inducer is
absent. (note: Allostery are protein-protein interactions that changes the protein conformation and
therefore its function).
When the I gene is mutated at the operator binding site (I -), it produces an ineffective repressor that
cannot bind to the operator, which results in constitutive production of enzymes. If the I gene is
mutated in the inducer binding site (Is), the repressor protein produced can bind to the operator but
cannot bind to the inducer and hence cannot be removed. This results in complete shut down of the
operon. In other words the lac operon is turned off permanently. Is is trans dominant over I+ or I-,
because the latter two do not bind at all or bind conditionally. So when the operator becomes vacant
Is repressor protein would occupy the operator and turn off the operon permanently.
Oc is a mutation of the operator region, which changes the repressor binding site, so that the
repressor molecule cannot bind efficiently. Although enzymes are produced constitutively, if the
enzyme produced is 100% with inducers, in the absence of inducers it would be around 20%. It acts
as a cis dominant mutation. Promoter mutations also behave as cis dominant mutations.
Positive control
Although the negative control system prevents the wasteful production of lactose catabolic enzymes
in the absence of lactose. The positive control system enables the operon to be turned off when
glucose is present in the medium (whether lactose is present or not). This is useful because in the
presence of glucose it can be directly used for energy, without having to wastefully degrade lactose.
The mechanism of control is called catabolic repression.
When glucose is present in the cell, glucose catabolic products increase in the cell. These
catabolites exert a suppressing influence on cAMP levels. Hence, cAMP serves as a sensor of
glucose level, increasing with reduced glucose levels and reducing with elevated glucose levels.
The crp gene produces a protein called the Catabolite activator protein (CAP). This in the presence
of cAMP produces a CAP-cAMP complex. This complex serves as an activator molecule and binds
to the upstream end of the promoter as shown by DNA binding assays. It is believed that this
95
activator complex activates the operon by facilitating the formation of an open promoter complex,
which is important for transcription initiation. In the presence of glucose on the other hand, there
will less CAP-cAMP activator molecules and hence the transcription will be shut down, unable to
initiate transcription. Hence lac operon will be turned on only in the absence of glucose and
presence of lactose in the medium.
3.3.6.1.2 The Trp Operon
This is an example of a negative control (repressible system) with superimposed attenuation. The
trp operon consists of five coordinately controlled tryptophan biosynthetic genes, trpE, trpD, trpC,
trpB and trpA. The cis acting controlling elements include a promoter sequence, operator sequence
and a leader sequence, containing the attenuator sequence. trpR gene is a transacting controlling
element. The organization of the trp operon is shown below.
Figure 18
Negative control (repressible system)
1. The trpR gene produces an aporepressor which in the presence of tryptophan (co-repressor)
binds to it to produce the active repressor. The active repressor molecule because of its
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affinity to the operator region of the trp operon, binds to it, and shuts off transcription. Note:
Only when tryptophan is in sufficient amounts will the active repressor molecule be formed.
2. Secondly, tryptophan acts as an inhibitor to the first enzyme (coded by trpE and trpD) in the
tryptophan biosynthetic pathway.
3. Thirdly, attenuation (premature termination of transcription in a 28 bp region within the

leader sequence) occurs in the presence of even small amounts of tryptophan due to
interaction between sequences present in the leader sequence. The leader sequence consists
of a translation initiator codon (AUG) and a stop codon UGA, defining a 14 amino acid
polypeptide. There are four segments of the leader RNA (1,2,3,4) which are capable of
forming duplexes (2:3 or 3:4). Duplex formation 3:4 results in termination of transcription.
There are two adjacent trp codons at the 10 and 11 positions in segment 1 of the leader
sequence which are critical to the mechanism.
Transcription and translation takes place simultaneously in prokaryotes. Hence while the
RNA polymerase is transcribing section 4 if the ribosome is in contact with section 2 duplex
formation occurs between 3:4 which results in termination of transcription. But when
tryptophan is low, there are fewer trp charged tRNA molecules and hence the ribosome stalls
in segment 1 at the trp codons and hence doesn't reach segment 2 and therefore transcription
proceeds without termination. this mechanism has evolved to prematurely terminate
transcription when tryptophan is present.
3.3.6.1.3 Temporal Control Of Genes In Phages
There are two mechanisms by which temporal control is achieved in phages a) Altered specificity of
RNA polymerases by modification of the sigma factor b) antitermination. We will look at an
example of each of these methods of control.
SPO1 Phage
The genes are expressed in a time controlled temporal programme. Early genes that are involved in
multiplication are switched on during the first five minutes, then the middle genes responsible for
synthesis of proteins an protein assembly are turned on for 10 minutes and then the late genes that
are responsible for lysis are turned on. The early genes are transcribed by the host RNA polymerase
holoenzyme. The last gene among the early genes is a gene called g28 which codes for a sigma
factor gp28. Once this reaches sufficient concentration, because of its greater affinity to the RNA
polymerase core enzyme it replaces the host sigma factor. The new sigma factor provides an altered
specificity to the RNA polymerase holoenzyme and directs it to a the middle genes. Last among the
middle genes are g33 and g34. These also encode altered sigma factors gp23 and gp34, which have
even higher affinity to the RNA polymerase holo enzyme than gp28. So when these proteins reach
sufficient concentration they replace gp 28 and redirect the RNA polymerase core enzyme to two
sets of late genes. Mutation of the g28 gene prevented the early to middle switch.
97
lambda phage
Transcription begins at the pL promoter and proceeds leftwards until it terminates at tL1; similarly
transcription proceeds in the rightward direction from the pR promoter until it is terminated at tR1.
The early genes code for regulatory proteins. When the N protein accumulates to high enough
concentration it will serve as an antiterminator to tL1 and would allow transcription to proceed
through this terminator. Similarly accumulation of cro protein to sufficient concentration would
serve as an antiterminator to tR1. The replication and recombination genes are now transcribed.
When the Q gene reaches high enough concentration as a result of transcription of the middle genes,
it would serve as an antiterminator to tR2 and allow transcription of the lysis and protein sheath
assembly genes.
Regulation of genes in eukaryotes
Eukaryotic genes are not organised into structures such as operons, but rather have individual
promoters. The control of gene expression is mostly at the level of transcription, although
control mechanisms are also found at the chromosomal-, post-transcriptional , translational, and
posttranslational levels, which makes the regulatory mechanisms of eukaryotes much more
complex and much more tightly controlled than in Prokaryotes.
Activation of gene structure (Chromosomal control)
Facultative and Condensed heterochromatin
Chromatin, which occurs in the heterochromatic state is so tightly coiled that it is difficult for the
RNA polymerase enzyme and the transcription factors to access the genes in that region. Hence
genes in the heterochromatic region are invariably shut off. The chromatin structure should
become less coiled to activate the gene. There are three types of heterochromatin. Condensed
heterochromatin is euchromatin, which becomes heterochromatic following a certain
developmental stage, when the function of genes in that location are no more necessary to the
organism. Once turned heterochromatin it remains heterochromatic throughout the life cycle of
the organism. The facultative heterochromatin on the other hand is a regulatory devise that can
adjust the dosage of gene according to the environmental cues, and hence reversible in its states.
The other type of heterochromatin is constitutive heterochromatin, which is mainly confined to
centromeric and telomeric regions of the chromosome and remains heterochromatic through out
the life of the organism and hence does not have a regulatory role.
Position effects
In addition to their direct influence heterochromatic regions may exert an influence on genes
residing in the euchromatic region as well. The suppressing effect of the heterochromatic region
on genes that are proximal to it in the euchromatin is called position effects. The magnitude of
suppressing effect depends on the proximity of the gene to the heterochromatic region.
98
Methylation
Transcriptionally inactive regions are methylated. Approximately 70% of the CG pairs in
Eukaryotes are methylated. Methylation suppresses expression by affecting DNA-protein
interaction, and the phenomenon is referred to as ‘gene silencing’. Silent genes can be activated
by adding a methylation inhibitor, 5-azacytosine.
Initiation of Transcription (Transcriptional Control)
This is the most important stage of control in eukaryotes. Control is at the initiation of
transcription only (in prokaryotes, control occurs both at the ‘initiation of transcription’ and
‘termination of transcription’ levels). Further, the control mechanism is positive. There are two
types of controlling elements, cis-acting elements (promoters, enhancers) and transacting
elements (that produce the various transcription factors). The conserved sequences in the cis-
acting elements (promoter and enhancer) (DNA) interact with the transcription factors, which
are proteins, by DNA-protein interaction, and between themselves by looping and protein-
protein interactions, to bring about transcription (see Fig-1).
The basal transcription complex is formed by the interaction between the –25 box (TATA
BOX) of the promoter and the basal transcription factors. It is involved in the recruitment,
stabilisation and positioning of RNA polymerase-II .
The upstream transcription complex is formed by upstream transcription factors interacting

with the upstream elements (CCAAT box, and or GC Box) of the promoter. It is involved in
helping transcription initiation and upregulating transcription. This accounts for why some
promoters are stronger than others.
The response transcription factors or inducible factors interact with the response elements of
the promoter to produce the response transcription complex. This provides positive control of
gene expression. This allows genes to be controlled co-ordinately, temporally or spatially and is
responsible for turning on or off genes depending on environmental cues.
Example of positive control: For instance if a hormone such as a steroid in introduced, it binds
to receptor proteins, which change the allostery allowing them to bind to response elements/
enhancer elements, which recognise this receptor protein and coordinately upregulate all those
genes.
Transcription and termination of transcription
Transcription of mRNA genes are carried out by RNA Polymerase-II. Termination of

transcription is brought about by encountering a sequence AAUAAA, which brings about
termination 11-30 bp down stream of the sequence. The newly synthesized RNA is called the
heterogenous nuclear RNA (hnRNA). This contains transcribed regions corresponding to both
introns and exons. No control is exerted at the stage.
99
Processing of transcript and transport to cytoplasm (post-transcriptional control)
A number of post-transcriptional changes before RNA is transported to the cytoplasm. These are
i) trimming of RNA outside the coding region ii) polyadenylation of 3’ ends of RNA iii) capping
(blocking the 5’ end of the mRNA) iv) splicing out the introns to produce the mature mRNA v)
methylation of mRNA.
Trimming of RNA
The rRNA genes in eukaryotes are repeated several hundred times and clustered together in the
chromosome. The rRNA genes are separated by non-translated spacers, which have to be
trimmed out before translation. This process is not essential for mRNA genes.
Polyadenylation of 3’ ends of mRNA
Polyadenylation is peculiar for mRNA genes and does not occur in rRNA genes and tRNA genes.
One exception to the rule is the histone mRNA which is not polyadenylated. A poly-A tail often
150-200 nucleotides long is added to the 3’ end of the hnRNA by a poly-A polymerase enzyme in
the presence of AMP. The function of the poly-A tail is believed to be protecting the RNA from
degradation by ribonucleases lurking about in the cell. The signal that tells the poly-A
polymerase to add poly-As to the 3’ end is the AAUAAA, which the signal that specifies
termination.
Capping
The 5’ end of the eukaryotic hnRNA undergoes capping. The cap typically contains a methylated
guanine nucleotide linked by a triphosphate to the penultimate nucleotide (5’-5’ linkage), which
is frequently methylated also. Capping occurs while transcription occurs hence cannot be strictly
referred to as a post-transcriptional process, but rather should be referred to as a co-
transcriptional process. Caps have two roles. Firstly to protect the mRNA from degrading and
secondly to allow attachment to the ribosome.
Splicing pathway
The hnRNA has to be spliced to remove the introns to create a mature mRNA, which is ready for
translation. The process of cutting the introns out of immature hnRNA and stitching the exons
together to form the mature mRNA is called RNA splicing. The splicing signals tell the where to
cut and are normally found in the exon-intron junctions (exon/GU-intron-AG/exon). The
consensus sequence for recognition of splice site is
5’ CAG/GUAAGU-intron-YNNAG/G
A G
100
Where YN denotes a string of nine pyrimidines (U’s and C’s) and N is any base. If this is
mutated abnormal splicing occurs.
Splicing is carried out by small nuclear RNA’s (transcribed by RNA polymerse III) and small
nuclear ribonucleoproteins (snRNP’s) – called ‘snurps’. The U1 snRNA is complementary to the
5’ and 3’ splice sites. The whole splicing complex is referred to as the spliceosome. Splicing is a
two step process and occurs through a lariat shaped intermediary.
Post-transcriptional control occurs due to altering mRNA stability. But this does not appear to be
through withholding poly-adenylation or capping. Eg. Increased casein synthesis when breast
tissue is induced with prolactin. There is 20-fold increase in the production of the milk protein
casein, although the increase in mRNA synthesis was only 2 fold. The rest of the increase comes
from increased mRNA stability.
Translational control
Posttranslational control
101
102
103
104
105
106
BL27B: QUESTIONS IN MOLECULAR GENETICS
1. Describe the experimental procedures and discuss the results and conclusions of the
Meselson-Stahl Experiment.
Distinguish between either unidirectional and bidirectional synthesis of DNA or

continuous and discontinuous synthesis of DNA.
2. What evidence exist for the claim that : (answer any three)
a) DNA is the genetic material
b) The DNA molecule elongates by the addition of nucleotides to the hydroxyl group
at the 3’ carbon position and not at the 5’ position?
c) Bidirectional replication
d) The complementary strands of DNA are of opposite polarity ( anti-parallel.
e) DNA replication follows a semi-conservative mechanism.
3. Explain what you understand by transposition? Differentiate between the replicative and
conservative modes of transposition. Describe the transposable genetic elements in
Escherichia coli. Discuss the generation of multiple antibiotic resistance in bacteria.
4. What do you mean, when we say that Oc mutations in the lac system are cis-dominant?
Explain why I- mutations in the lac system are normally recessive to I+ mutations and
why are I+ mutations recessive to Is mutations.
5. Explain the fundamental differences between negative control and positive control.
Compare the arrangement of cis-acting sites in the control regions of eukaryotes and
prokaryotes.
6. Write notes on any three of the following
a) Cot analysis
b) Crick's wobble hypothesis
c) Replisome
107
d) Rolling cirlce replication

e) Colinearity of the gene
7. Describe the hypothetical models proposed for the replication of DNA. Which among
them would you consider the most probable method of replication and why?
8. The map of the lac operon is
I P O Z Y A
The promoter (P) region is the start site of transcription through the binding of the RNA
Polymerase Molecule before actual mRNA production. Mutationally altered promoters
(P-) apparently cannot bind the RNA polymerase molecule. Certain predictions can be
made about the effect of P- mutations. Use your predictions and your knowledge of the
lactose system to complete Table-1. Insert a ‘+’ where an enzyme is produced and a ‘-‘,
where no enzyme is produced.
Table-1
B galactosidase transferase
Genotype
- lactose + lactose - lactose + lactose
- + - +
I+P+O+Z+Y+/ I+P+O+Z+Y+
I-P+OcZ+Y+ / I+O+Z-Y+
I+P-OcZ-Y+ / I-P+OcZ+Y-
IsP+O+Z+Y- / I+P+O+Z-Y+
IsP+O+Z+Y+ / I-P+O+Z+Y+
I-P+OcZ+Y+ / I-P+O+Z-Y+
I-P-O+Z+Y+ / I-P+OcZ+Y-
I+P+O+Z-Y+ / I-P+O+Z+Y-
108
9. Write notes on three of the following
a) Modulation of structural genes in the lac-operon of E. coli

b) Genetic code
c) Gene transposition
d) Watson and Crick Model of DNA structure
e) Asymmetric transcription
10. If the GC content of a DNA molecule is 56%, what are the percentages of each of the
bases in this molecule?
The temperature at which a DNA sample denatures can be used to estimate the proportion
of its nucleotide pairs that are G-C. What would be the basis of this determination, and
what would a high denaturation temperature for a DNA sample indicate?
If a molecule of DNA is 2250 bp long, what would be its length in Ao . Determine its
molecular weight?
11. In humans, the disease galactosemia causes mental retardation at an early age because
lactose in milk cannot be broken down, and this failure affects brain function. How would
you provide a secondary cure for galactosemia? Would you expect this phenotype to be
dominant or recessive?
12. In humans, PKU (phenylkenuria) is a disease caused by an enzyme inefficiency at step A

in the following simplified reaction sequence and AKU (alkaptonuria) is due to an
enzyme inefficiency in step B. A person with PKU marries a person with AKU. What
phenotypes do you expect for the children?
A B
Phenylalanine -------- tyrosine ---------- CO2 + H2 O
13. In Drosophila, the autosomal recessive bw causes a dark brown eye and the unlinked
autosomal recessive st causes a bright scarlet eye. A homozygote for both genes has a
white eye. Thus we have the following correspondences between genotypes and
phenotypes.
+/+ +/+ = red eye (wild type)

+/+ bw/bw = brown eye
st/st +/+ = scarlet eye
st/st bw/bw = white eye
109
Construct a hypothetical biochemical pathway showing how the gene products interact
and why the different mutant combinations have different phenotypes?
14. Describe the expected patterns of bands in a CsCl gradient for conservative replication,
semi-conservative replication and dispersive mechanisms. Draw diagrams.
110
APPENDIX 1 – CYTOGENETICS SUMMARY.
1
APPENDIX TWO – PROKARYOTIC GENETICS-SUMMARY
2
APPENDIX THREE – EUKARYOTIC GENOME STRUCTURE AND ORGANISATION - SUMMARY.
APPENDIX FOUR – POPOULATION GENETICS – SUMMARY
3
APPENDIX – FIVE.
PROBLEM SEET 1
1
APPENDIX SIX
DNA SYNTHESIS
2
APPENDIX SEVEN
DNA MICROSATELLITES:AGENTS OF EVOLUTION?
3
APPENDIX EIGHT
TRANSPOSITION IMPACTS ON PUBLIC HEALTH

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INTRODUCTION TO ADVANCED GENETICS.......................................................Error! Bookmark not defined.

3.1 Molecular Nature Of The Gene......................................................................................................................65

3.2.2.5. The Replisome.................................................................................................................................76

BIOL2162 – ADVANCED GENETICS

This course is a core course for students majoring in Biology.

Lectures/ Tutorials and Practicals:

Incourse Assessments = 40%

THE UNIVERSITY OF THE WEST INDIES, ST. AUGUSTINE

BIOL2162: ADVANCED GENETICS

At the end of the course students would be able to understand

1 Topic Learning objectives

 Students should be able to define karyotyping; describe how it

 Students should be able to write brief notes on polytene and

duplications  Students should be able to define duplication; enumerate types

Chromosomal aberrations (ctd…)

translocations  Students should be able to define translocations and

 Epigenetic inheritance - Epigenetics, epigenome, epigenetic marks, stem cells,

Aneupliody  Students should be able to define and differentiate between

allopolyploidy  Students should be able to describe how allopolyploids are

Recombination  Students should be able to define conjugation; be able to

Transduction  Students should be able to define and be able to clearly

Transformation  Students should be able to define and list the steps in

Concept of complementation/cistron  Students should be able to clearly define complementation, be

 Students should be able to describe complementation and

 Students should be able to describe DNA structure – double

 DNA replication  Students should be able to describe the various models

 Students should be able to explain and provide experimental

 Students should be able to write notes on the growing point

 Students should be able to differentiate between the

 Students should be able to define the terms replicon and

 Students should be able to define translation, outline the steps

 Gene regulation in prokaryotes  Students should be able to define operons, controlling

MOLECULAR GENOME ORGANISATION AND

- Transcription complexes  Students should be able to write notes on transcription

conditions under which polymorphisms are maintained.

1.1.1 The emergence of cytogenetics.

1.1.2 Chromosome theory of inheritance

- Different gene pairs segregate independently (so do different chromomosomal pairs)

1.1.3 What is Cytogenetics?

Ultra structure of the Chromosome:- The unit of packaging in a chromosome is a nucleosome.

Characterisation of the genome

The genome can be characterised in terms of

a) the basic chromosome number ( monoploid number)

Some individuals have an incomplete chromomosomal set or have supernumerary

c) Characterising chromosomes within the basic chromosome set

Quinacrine mustard staining reveals florescent banding patterns on chromosomes referred to

Note 2: Position effects

Facultative heterochromatin and position effects demonstrate mechanisms of control of gene

1.2.1 Macro and Ultra Structure of the Chromosome

1.2.2 Characterisation of the Genome

1.2.4 Uses of karyotyping

1. Enables one to identify chromosomal aberrations in individuals. Chromosomal

2. Assign taxonomic status and enable the study of taxonomical relationships

3. Enables understanding of evolution.

If one assumes that modifications to chromosomal structure accumulate in a

1.2.5 Practical Exercise-1 – Karyotyping - I

1. An enlarged photograph of a cell in the mitotic metaphase is provided in the attached

a) Is the specimen from a male or a female?

2. Abyssinian oat (Avena abyssinica) appears to be a tetraploid with 28 chromosomes. The

1.3 Chromosomal Mutations-Changes In Chromosomal Structure

CHANGES TO CHROMOSOMAL STRUCTURE

Changes in chromosome structure can occur as a result of deletion of a segment of the