Life Sciences Fundamentals and Practice - II

Life Sciences
Fundamentals and Practice II
Sixth edition
Pranav Kumar | Usha Mina

Life Sciences
Fundamentals and Practice II
Sixth edition
Pranav Kumar
Former faculty,
Department of Biotechnology
Jamia Millia Islamia (JMI),
New Delhi, India
Usha Mina
Associate Professor,
School of Environmental Sciences,
Jawaharlal Nehru University (JNU),
New Delhi, India
Pathfinder Publication
New Delhi, India
Pranav Kumar
Former faculty,
Department of Biotechnology,
Jamia Millia Islamia (JMI),
New Delhi, India
Usha Mina
Associate Professor,
School of Environmental Sciences,
Jawaharlal Nehru University (JNU),
New Delhi, India
Life Sciences : Fundamentals and Practice
Sixth edition
ISBN: 978-81-906427-7-4 (paperback)
Copyright © 2017 by Pathfinder Publication, all rights reserved.
This book contains information obtained from authentic and highly

regarded sources. Reasonable efforts have been made to publish reliable data
and information, but the author and the publisher cannot assume responsibility
for the validity of all materials or for the consequences of their use.
No part of this book may be reproduced by any mechanical, photographic, or
electronic process, or in the form of a phonographic recording, nor it may be
stored in a retrieval system, transmitted, or otherwise copied for public or
private use, without written permission from the publisher.
Publisher : Pathfinder Publication

Production editor : Ajay Kumar
Copy editor : Jomesh Joseph
Illustration and layout : Pradeep Verma
Cover design : Monu
Marketing director : Arun Kumar
Production coordinator : Murari Kumar Singh
Pathfinder Publication
A unit of Pathfinder Academy Private Limited, New Delhi, India.
pathfinderpublication.in
iii
Preface
Life Sciences have always been a fundamental area of science. The exponential increase in
the quantity of scientific information and the rate, at which new discoveries are made, require
very elaborate, interdisciplinary and up-to-date information and their understanding. This sixth
edition of Life sciences, Fundamentals and practice includes extensive revisions of the previous
edition. We have attempted to provide an extraordinarily large amount of information from
the enormous and ever-growing field in an easily retrievable form. It is written in clear and
concise language to enhance self-motivation and strategic learning skill of the students and
empowering them with a mechanism to measure and analyze their abilities and the confidence
of winning. We have given equal importance to text and illustrations. The sixth edition has
a number of new figures to enhance understanding. At the same time, we avoid excess
details, which can obscure the main point of the figure. We have retained the design elements
that have evolved through the previous editions to make the book easier to read. Sincere
efforts have been made to support textual clarifications and explanations with the help of flow charts,
figures and tables to make learning easy and convincing. The chapters have been supplemented
with self-tests and questions so as to check one’s own level of understanding. We hope you will
find this book interesting, relevant and challenging.
Acknowledgements
Our students were the original inspiration for the first edition of this book, and we remain continually
grateful to all of them, because we learn from them how to think about the life sciences and how
to communicate knowledge in most meaningful way. We thank, Neeraj Tiwari, Diwakar Kumar
Singh, Rahul Shukla and Ajay Kumar, reviewers of this book, whose comment and suggestions
were invaluable in improving the text. Any book of this kind requires meticulous and painstaking
efforts by all its contributors. Several diligent and hardworking minds have come together to
bring out this book in this complete form. This book is a team effort, and producing it would be
impossible without the outstanding people of Pathfinder Publication. It was a pleasure to work
with many other dedicated and creative people of Pathfinder Publication during the production of
this book, especially Pradeep Verma and Rajnish Kumar Gupta.
Pranav Kumar
Usha Mina
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Contents
Chapter 1
Genetics
1.1 Mendel’s principles 1
1.1.1 Mendel’s laws of inheritance 3
Law of segregation 3
Law of independent assortment 5
1.1.2 Incomplete dominance and codominance 7
1.1.3 Multiple alleles 8
1.1.4 Lethal alleles 10
1.1.5 Penetrance and expressivity 11
1.1.6 Probability 11
1.2 Chromosomal basis of inheritance 14
1.3 Gene interaction 15
1.3.1 Dominant epistasis 17
1.3.2 Recessive epistasis 18
1.3.3 Duplicate recessive epistasis 19
1.3.4 Duplicate dominant interaction 19
1.3.5 Dominant and recessive interaction 19
1.3.6 Genetic dissection to investigate gene action 21
1.3.7 Pleiotropy 22
1.4 Genetic linkage and gene mapping 22
1.4.1 Genetic mapping 26
1.4.2 Gene mapping from two point cross 28
1.4.3 Gene mapping from three point cross 29
1.4.4 Interference and coincidence 31
1.5 Tetrad analysis 32
1.5.1 Analysis of ordered tetrad 34
1.5.2 Analysis of unordered tetrad 35
1.6 Sex chromosomes and sex determination 36
1.6.1 Sex chromosome 36
1.6.2 Sex determination in animals 37
Sex determination in humans 39
Genic balance theory of sex determination in Drosophila 40
1.6.3 Sex determination in plants 41
1.6.4 Mosaicism 41
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1.6.5 Sex-linked traits and sex-linked inheritance 41
1.6.6 Sex-limited traits 43
1.6.7 Sex-influenced traits 43
1.6.8 Pedigree analysis 43
1.7 Quantitative inheritance 47
1.7.1 Quantitative trait locus analysis 51
1.7.2 Heritability 51
1.8 Extranuclear inheritance and maternal effect 52
1.8.1 Maternal effect 55
1.9 Cytogenetics 57
1.9.1 Human karyotype 57
1.9.2 Chromosome banding 58
1.9.3 Variation in chromosome number 59
1.9.4 Chromosome aberrations 63
1.9.5 Position effect 68
1.10 Genome 69
1.10.1 Genome complexity 70
1.10.2 Transposable elements 73
1.10.3 Gene 81
1.10.4 Introns 82
1.10.5 Acquisition of new genes 84
1.10.6 Fate of duplicated genes 85
1.10.7 Gene families 86
1.10.8 Human nuclear genome 87
1.10.9 Organelle genome 88
1.10.10 Yeast S. cerevisiae genome 89
1.10.11 E. coli genome 89
1.11 Eukaryotic chromatin and chromosome 90
1.11.1 Packaging of DNA into chromosomes 92
1.11.2 Histone modification 95
1.11.3 Heterochromatin and euchromatin 97
1.11.4 Polytene chromosomes 100
1.11.5 Lampbrush chromosomes 101
1.11.6 B-chromosomes 102
1.12 DNA replication 102
1.12.1 Semiconservative replication 102
1.12.2 Replicon and origin of replication 104
1.12.3 DNA replication in E. coli 106
Topoisomerase 107
1.12.4 Telomere replication 117
1.12.5 Rolling circle replication 118
1.12.6 Replication of mitochondrial DNA 119

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1.13 Recombination 119
1.13.1 Homologous recombination 120
1.13.2 Site-specific recombination 125
1.14 DNA repair 127
1.14.1 Direct repair 127
1.14.2 Excision repair 127
1.14.3 Mismatch repair 129
1.14.4 Recombinational repair 130
1.14.5 Repair of double strand DNA break 132
1.14.6 SOS response 133
1.15 Transcription 134
1.15.1 Transcription unit 135
1.15.2 Prokaryotic transcription 135
Process of transcription 137
1.15.3 Eukaryotic transcription 141
1.15.4 Role of activator and co-activator 146
1.15.5 Long-range regulatory elements 147
1.15.6 DNA binding motifs 149
1.16 RNA processing 151
1.16.1 Processing of eukaryotic pre-mRNA 151
5’-capping 151
Splicing of GU-AG intron 154
1.16.2 Processing of pre-rRNA 160
1.16.3 Processing of pre-tRNA 163
1.17 mRNA degradation 164
1.18 Regulation of gene transcription 165
1.18.1 Operon model 166
1.18.2 Tryptophan operon system 172
1.18.3 Riboswitches 176
1.19 Bacteriophage lambda: A transcriptional switch 177
1.20 Regulation of transcription in eukaryotes 180
1.20.1 Influence of chromatin structure on transcription 180
1.20.2 DNA methylation and gene regulation 182
1.20.3 Post-transcriptional gene regulation 184
1.21 RNA interference 185
1.22 Epigenetics 188
1.23 Genetic code 189
1.24 Protein synthesis 194
1.24.1 Incorporation of selenocysteine 206
1.24.2 Cap snatching 206
1.24.3 Translational frameshifting 207
1.24.4 Antibiotics and toxins 207

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1.24.5 Post-translational modification of polypeptides 208
1.25 Mutation 211
1.25.1 Mutagen 216
1.25.2 Types of mutation 218
1.25.3 Fluctuation test 222
1.25.4 Replica plating experiment 223
1.25.5 Ames test 224
1.25.6 Complementation test 225
1.26 Developmental genetics 226
1.26.1 Genetic control of embryonic development in Drosophila 227
1.26.2 Genetic control of vulva development in C. elegans 232
Chapter 2
Recombinant DNA technology
2.1 DNA cloning 239
2.2 Enzymes for DNA manipulation 241
2.2.1 Template-dependent DNA polymerase 241
2.2.2 Nucleases 241
2.2.3 End-modification enzymes 245
2.2.4 Ligases 247
2.2.5 Linkers and adaptors 247
DNA and RNA purification 248
2.3 Vectors 250
2.3.1 Vectors for E. coli 251
Cosmids 254
Cloning vectors based on M13 phage 254
Phagemid vectors 255
2.3.2 Cloning vectors for yeast, S. cerevisiae 256
2.3.3 Vectors for plants 257
Plasmid based vector 257
Viral vectors 260
2.3.4 Vectors for animals 261
2.4 Introduction of DNA into the host cells 261
2.4.1 In bacterial cells 261
2.4.2 In plant cells 261
2.4.3 In animal cells 264
Transfection 265
Transduction (Virus-mediated transfection) 266
2.5 Selectable and screenable marker 267
2.6 Selection of transformed bacterial cells 268
2.7 Recombinant screening 269

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2.8 Expression vector 271
2.8.1 Expression system 272
2.8.2 Fusion protein 273
2.9 DNA library 273
Colony and plaque hybridization 276
2.10 Polymerase chain reaction 276
2.11 DNA sequencing 280
Chain termination method 280
Genome sequencing 282
Chromosome walking 283
2.12 Genome mapping 284
2.12.1 Genetic marker 284
2.12.2 Types of DNA markers 285
2.12.3 Physical mapping 289
2.12.4 Radiation hybrids 291
2.13 DNA profiling 292
2.14 Genetic manipulation of animal cells 293
2.14.1 Transgenesis and transgenic animals 293
2.14.2 Gene knockout 295
2.14.3 Formation and selection of recombinant ES cells 297
2.15 Nuclear transfer technology and animal cloning 298
2.16 Gene therapy 299
2.17 Transgenic plants 304
2.17.1 General procedure used to make a transgenic plant 304
2.17.2 Antisense technology 307
2.17.3 Molecular farming 308
2.18 Plant tissue culture 309
2.18.1 Cellular totipotency 309
2.18.2 Tissue culture media 309
2.18.3 Types of cultures 312
Callus cultures 312
Protoplast cultures 312
Cell-suspension cultures 313
Organ culture 313
Meristem culture 313
Embryo culture 314
Haploid culture 314
Somatic embryogenesis 315
Organogenesis 316
2.18.4 Somaclonal and gametoclonal variation 316
2.18.5 Somatic hybridization and cybridization 317
2.18.6 Applications of cell and tissue culture 318

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2.19 Animal cell culture 320
2.19.1 Primary cultures 320
2.19.2 Cell line 320
2.19.3 Growth cycle 322
2.19.4 Culture media 323
Chapter 3
Plant Physiology
3.1 Plant-water relationship 329
3.1.1 Diffusion and osmosis 329
3.1.2 Chemical potential of water and water potential 331
3.1.3 Mass flow 333
3.2 Absorption and radial movement of water 333
3.2.1 Absorption of water 333
3.2.2 Soil water 335
3.2.3 Radial movement of water from root surface to the tracheary element 335
3.2.4 Root pressure 336
3.3 Ascent of sap 336
3.3.1 Xylem anatomy 337
3.3.2 Mechanism of ascent of sap 337
3.4 Transpiration 338
3.4.1 Mechanism of stomatal opening and closing 339
3.4.2 Factors influencing transpiration 341
3.4.3 Guttation 341
3.5 Absorption and radial movement of mineral nutrients 342
3.6 Mineral nutrition 343
Types of essential elements 344
Roles of essential elements and deficiency symptoms 345
3.6.1 Liebig’s law of the minimum 347
3.6.2 Nitrogen cycle 347
3.6.3 Nitrogen assimilation 348
3.6.4 Biological nitrogen fixation 350
3.7 Translocation in the phloem 353
Phloem loading and unloading 355
3.7.1 Allocation and partitioning of photoassimilates 356
3.8 Plant hormones 356
Types of plant hormones 357
3.8.1 Auxin 357
3.8.2 Gibberellins 361
3.8.3 Cytokinins 363
3.8.4 Abscisic acid 365

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3.8.5 Ethylene 366
3.8.6 Brassinosteroids 367
3.8.7 Strigolactones 367
3.8.8 Jasmonates 368
3.8.9 Hormones signaling pathway 368
3.9 Photomorphogenesis 373
3.9.1 Phytochrome 373
3.9.2 Cryptochrome 376
3.9.3 Phototropin 377
3.9.4 Photoperiodism 378
3.9.5 Florigen 380
3.10 Vernalization 381
3.11 Flowering genes 381
Role of floral organ identity genes 383
3.12 Plants movements 384
Tropic movements 384
Nastic movements 386
3.13 Seed dormancy and Germination 387
3.14 Plant development 388
3.14.1 Pollination and Self-incompatibility 392
3.14.2 Asexual reproduction 394
Apomixis 394
3.14.3 Embryogenesis 395
Root apical meristems 397
Shoot apical meristem 398
3.15 Plant secondary metabolites 400
3.15.1 Terpenes 400
3.15.2 Phenolics 402
3.15.3 Glycosides 405
3.15.4 Alkaloids 406
Chapter 4
Human Physiology
4.1 Tissues 411
Epithelial tissue 411
Connective tissue 415
Nervous tissue 418
Muscular tissues 419
4.1.1 Organ systems of the human body 420
4.2 Nervous Systems 421
4.2.1 Histology of nervous tissue 422

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Neurons 422
Neuroglia 424
4.2.2 Structural organization of CNS 425
Blood-brain barrier 426
4.2.3 Major parts of the brain 427
Limbic system 429
4.2.4 Spinal cord 430
Reflex and reflex arc 433
4.2.5 Peripheral nervous system 433
4.2.6 Autonomic nervous system 435
Somatic system 438
4.3 Sensory organs 439
4.3.1 Eye 439
Operation of photoreceptors 443
4.3.2 Ear 445
4.4 Endocrine System 448
Major hormones producing endocrine glands and organs 449
4.4.1 Hypothalamus 449
4.4.2 Pituitary gland 451
4.4.3 Pineal gland 453
4.4.4 Thyroid gland 453
4.4.5 Parathyroid gland 454
4.4.6 Thymus gland 454
4.4.7 Pancreas 454
4.4.8 Adrenal glands 457
4.4.9 Gonadal hormone 459
4.4.10 Hormones from kidney, heart, placenta and gastrointestinal tract 459
4.4.11 General mechanisms of hormone action 461
4.4.12 Hormones and diseases 462
4.5 Respiratory System 465
4.5.1 Respiratory organs 465
4.5.2 Mechanics and breathing 469
4.5.3 Respiratory volumes and capacities 471
4.5.4 Exchange of oxygen and carbon dioxide 472
4.5.5 Transport of oxygen and carbon dioxide 475
4.5.6 Control of respiration 478
4.5.7 Chemoreceptor 479
4.5.8 Disorders of respiratory system 480
4.6 Cardiovascular System 481
4.6.1 Blood 481
4.6.2 Heart 487
4.6.3 Blood vessels 494

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4.6.4 Circulatory routes 498
4.6.5 Lymphatic system 501
4.6.6 Intracellular and extracellular fluid 502
4.6.7 Cardiovascular disorders 502
4.7 Digestive System 503
4.7.1 Gastrointestinal tract 503
Mouth 504
Pharynx 506
Esophagus 506
Stomach 506
Small intestine 509
Large intestine 510
Gastrointestinal motility 511
4.7.2 Accessory digestive organs 512
Salivary glands 512
Liver 512
Gallbladder 513
Pancreas 514
4.7.3 Digestion of foods 515
4.7.4 Absorption of foods 518
4.7.5 Regulation of digestive function 520
4.8 Excretory System 521
4.8.1 Structure of the kidneys 522
4.8.2 Nephron 524
4.8.3 Urine formation 527
4.8.4 Atrial Natriuretic peptide 534
4.8.5 Countercurrent exchange 537
4.9 Reproductive System 538
4.9.1 Male reproductive system 538
4.9.2 Female reproductive system 541
4.9.3 Female reproductive cycle 544
4.10 Embryonic development 547
4.10.1 Fertilization 547
4.10.2 A generalized pattern of early development 550
4.11 Regeneration 554
Chapter 5
Ecology
5.1 What is Ecology? 561
Level of organization 561
5.2 Environment 562

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Soil 562
Air and Atmosphere 563
Light 564
Temperature 564
5.3 Adaptation and Acclimatization 564
Plants adaptation to water stress 565
Animals adaptation to thermal stress 565
5.4 Shelford’s law of tolerance 566
5.5 Ecological species concept 567
5.6 Habitat and niche 567
5.7 The ecosystem concept 569
5.7.1 Ecosystem components 569
5.7.2 Ecosystem function 570
5.7.3 Productivity 571
5.7.4 Energy flow 572
5.7.5 Energy flow model 574
5.7.6 Transfer efficiencies 574
5.7.7 Concept of the trophic level 575
5.7.8 Food chains 576
Autotroph and detritus-based ecosystem 577
Autochthonous and Allochthonous 577
5.7.9 Ecological pyramid 578
5.7.10 Nutrient cycling 579
Carbon cycle 580
Nitrogen cycle 581
Phosphorus cycle 582
Sulfur cycle 582
5.7.11 Decomposition 583
5.7.12 Ecosystem services 583
5.7.13 Controls on ecosystem function 584
5.7.14 Types of Ecosystems 584
Aquatic ecosystem 585
Marine ecosystem 585
Estuary 586
Freshwater ecosystem 586
Wetlands 588
5.8 Biomes 589
5.9 Population ecology 592
5.9.1 Population characteristics 592
5.9.2 Population growth 595
Population regulation 599
5.9.3 r-strategists and K-strategists 601

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5.10 Biotic community 603
5.10.1 Ecological characteristics 604
Species composition 604
Keystone species 604
Species diversity 604
Diversity index 605
Disturbance and species diversity 606
5.10.2 Island biogeography 606
5.10.3 Nature and structure of community 608
5.10.4 Ecological interdependence and interactions 609
Positive interaction 609
Negative interaction 610
Effect of competition 611
Lotka-Volterra model 613
Predation 617
5.11 Succession 618
5.11.1 Types of succession 620
5.11.2 Mechanism of succession 621
5.11.3 Model of succession 623
5.11.4 Hydrarch and Xerarch succession 624
5.12 Biodiversity 626
5.12.1 Levels of biodiversity 626
5.12.2 Gradients and Magnitude of biodiversity 627
5.12.3 Uses of biodiversity 627
5.12.4 Threats to biodiversity 628
5.12.5 Extinction of species 629
IUCN red list categories and criteria 630
5.12.6 Conservation of biodiversity 631
In-situ conservation strategies 632
Biodiversity Hotspots 633
5.12.7 Biogeographic classification of India 634
5.13 Behavioural ecology 635
Altruism 635
Calculation of the coefficient of relatedness 636
Reciprocal altruism 638
Mating behaviour 638
Optimal foraging theory 639
Imprinting 640
5.14 Environmental pollution 640
5.14.1 Air pollution 640
5.14.2 Types of air pollutants 641
5.14.3 Criteria air pollutants 641

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5.14.4 Greenhouse effect 644
Global warming 645
5.14.5 Stratospheric ozone 646
5.14.6 Acid rain 647
5.14.7 Water pollution 648
Causes of water pollution 648
Biochemical Oxygen Demand and Chemical Oxygen Demand 649
Bioaccumulation, bioconcentration and biomagnification 649
Eutrophication 649
5.14.8 Soil pollution 650
5.15 Bioremediation 651
Bioremediation strategies 651
Phytoremediation 652
Chapter 6
Evolution
6.1 Origin of Life 657
6.2 Theories of evolution 662
6.2.1 Lamarckism 662
6.2.2 Darwinism 663
6.3 Evidences of evolution 667
6.4 Natural selection 669
Evidences of natural selection 669
Modes of natural selection 670
Sexual selection 671
6.5 Pattern of evolution 672
Adaptive radiation 673
Red Queen hypothesis 673
6.6 Population genetics 673
6.6.1 Calculation of allelic frequencies 674
6.6.2 Hardy-Weinberg Law 675
Extension of Hardy-Weinberg equilibrium 677
Assumptions underlying Hardy–Weinberg equilibrium 679
6.6.3 Inbreeding 680
Wahlund effect 684
Effective population size 684
6.7 Species and speciation 685
Concept of species 685
Reproductive isolation 686
Haldane’s rule 687
Speciation 687
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6.8 Evolutionary forces involved in speciation 689
6.9 Pattern of evolutionary changes 691
6.10 Nature of evolution 692
6.11 Molecular phylogeny 693
Molecular clock 694
Phylogenetic tree 696
Answers of self test 704
Index 705
Chapter 01
Genetics
All living organisms reproduce. Reproduction results in the formation of offspring of the same kind. However, the
resulting offspring need not and, most often, does not totally resemble the parent. Several characteristics may differ
between individuals belonging to the same species. These differences are termed variations. The mechanism of
transmission of characters, resemblances as well as differences, from the parental generation to the offspring, is
called heredity. The scientific study of heredity, variations and the environmental factors responsible for these, is
known as genetics (from the Greek word genno = give birth). The word genetics was first suggested to describe
the study of inheritance and the science of variation by prominent British scientist William Bateson.
Genetics can be divided into three areas: classical genetics, molecular genetics and evolutionary genetics. In classical
genetics, we are concerned with Mendel’s principles, sex determination, sex linkage and cytogenetics. Molecular
genetics is the study of the genetic material: its structure, replication and expression, as well as the information
revolution emanating from the discoveries of recombinant DNA techniques. Evolutionary genetics is the study of
the mechanisms of evolutionary change or changes in gene frequencies in populations (population genetics).
Classical genetics
1.1 Mendel’s principles
Gregor Johann Mendel (1822–1884), known as the Father of Genetics, was an Austrian monk. In 1856, he published
the results of hybridization experiments titled Experiments on Plant Hybrids in a journal ‘The proceeding of the
Brunn society of natural history’ and postulated the principles of inheritance which are popularly known as Mendel’s
laws. But his work was largely ignored by scientists at that time. In 1900, the work was independently rediscovered
by three biologists - Hugo de Vries of Holland, Carl Correns of Germany and Erich Tschermak of Austria. Mendel
did a statistical study (he had a mathematical background). He discovered that individual traits are inherited as
discrete factors which retain their physical identity in a hybrid. Later, these factors came to be known as genes.
The term was coined by Danish botanist Wilhelm Johannsen in 1909. A gene is defined as a unit of heredity that
may influence the outcome of an organism’s traits.
Mendel’s experiment
Mendel chose the garden pea, Pisum sativum, for his experiments since it had the following advantages.
1. Well-defined discrete characters
2. Bisexual flowers
3. Predominant self fertilization
4. Easy hybridization
5. Easy to cultivate and relatively short life cycle
2 Genetics
Characters studied by Mendel
The characteristics of an organism are described as characters (or traits). Traits studied by Mendel were clear cut
and discrete. Such clear-cut, discrete characteristics are known as Mendelian characters. Mendel studied seven
characters/traits (all having two variants) and these are:
Dominant Recessive
1. Stem length Tall Dwarf
2. Flower position Axial Terminal
3. Flower color Violet White
Seed coat color Grey White
4. Pod shape Inflated Constricted
5. Pod color Green Yellow
6. Cotyledon color Yellow Green
7. Seed form Round Wrinkled
Flower color is positively correlated with seed coat colors. Seeds with white seed coats were produced by plants
that had white flowers and those with gray seed coats came from plants that had violet flower.
Allele
Each gene may exist in alternative forms known as alleles, which code for different versions of a particular inherited
character. We may also define alleles as genes occupying corresponding positions on homologous chromosomes
and controlling the same characteristic (e.g. height of plant) but producing different effects (tall or short). The
term homologous refers to chromosomes that carry the same set of genes in the same sequence, although they
may not necessarily carry identical alleles of each gene.
Wild-type versus Mutant alleles
Prevalent alleles in a population are called wild-type alleles. These alleles typically encode proteins that are made
in the right amount and function normally. Alleles that are present at less than 1% in the population and have been
altered by mutation are called mutant alleles. Such alleles usually result in a reduction in the amount or function
of the wild-type protein and are most often inherited in a recessive fashion.
Dominant and Recessive alleles
A dominant allele masks or hides expression of a recessive allele and it is represented by an uppercase letter. A
recessive allele is an allele that exerts its effect only in the homozygous state and in heterozygous condition its
expression is masked by a dominant allele. It is represented by a lowercase letter.
Homozygous and Heterozygous
Each parent (diploid) has two alleles for a trait — they may be:
Homozygous, indicating they possess two identical alleles for a trait.
Homozygous dominant genotypes possess two dominant alleles for a trait (TT).
Homozygous recessive genotypes possess two recessive alleles for a trait (tt).
Heterozygous genotypes possess one of each allele for a particular trait (Tt).
Genetics 3
E e Heterozygous alleles
d d Homozygous recessive alleles
C C Homozygous dominant alleles
B B
Alleles of different genes
A A
T t Alleles of the same gene
Figure 1.1 Homologous chromosome.
Genotype and Phenotype
To distinguish physical appearance from the genetic constitution, two different terms are used in genetics i.e.
genotype and phenotype. The genotype is defined as the genetic constitution of an individual for any particular
character or trait. The genotype of an individual is usually expressed by a symbol e.g. tt, Tt or TT etc. The phenotype
is defined as the physical appearance of an individual for any particular trait. The phenotype of an individual is
dependent on its genetic constitution.
1.1.1 Mendel’s laws of inheritance

On the basis of hybridization experiment on Pisum sativum, Mendel proposed the principles of inheritance known
as Mendel’s Laws:
Law of segregation
On the basis of the monohybrid cross (a cross involving only one trait), Mendel formulated the law of segregation.
This law states that each individual possesses two factors (later termed as genes) for a particular character. At the
time of formation of gametes each member of the pair of genes separates from each other so that each gamete
carries only one factor (gene) i.e. gametes are always pure (law of purity of gametes). It also explains that
hereditary factors are discrete and don’t blend when present together. Law of segregation applies only to diploid
organisms that form haploid gamete to reproduce sexually.
Explanation
Let’s use Mendel’s cross of tall and dwarf pea plants as an example. The letters T and t are used to represent
the alleles of the gene that determine plant height; by conventions the uppercase letter represents the dominant
allele and the recessive allele is represented by the same letter in lowercase. For the P cross, both parents are
true breeding plants; the tall plant is homozygous for the tall allele ‘T’, while the dwarf plant is homozygous for
the dwarf allele ‘t’. Mendel tracked each trait through two generations. P generation is the parental generation in a
breeding experiment. When true breeding plants were crossed to each other, this is called a P cross and offspring
comprise the first filial or F1 generation. When the members of the F1 generation were crossed, this produced the
F2 generation or second filial generation. A cross between true breeding tall and dwarf plants of the P generation
yield phenotypically tall plants. Now understand the reason why all the plants were tall in F1 generation which were
obtained by crossing of pure tall with pure short plants. To determine the kind and frequencies of various types
of offsprings expected, we usually use Punnett squares (used to predict the outcome of simple genetic crosses,
proposed by R. Punnett). The genetic constitution of gametes of one sex is kept on top of the squares and those
of other sex on one side. The genetic constitution of all possible zygote is then entered in squares of the grid.
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10 Genetics
The classification of Rh-positive and Rh-negative individuals is determined by the presence or absence of the
D antigen on the surface of RBCs. If D antigen is present on a person’s RBCs, the person is Rh-positive; if it is
absent, the person is Rh-negative. Rh-negative condition arises either due to lack of the RhD protein or due to
a series of changes in the RhD protein, which in turn change the phenotype of the D antigen. The Rh-positive
condition is more common.
Just like the ABO alleles, each biological parent donates one of their two Rh alleles to their child. A person who
is Rh-negative (genotype of Rh–/Rh–) can only pass an Rh– allele to son or daughter. A person who is Rh-positive
(genotype could be either Rh+/Rh+ or Rh+/Rh–) can pass either an Rh+ or Rh– allele to son or daughter. Rh-positive
pheotype is dominant over Rh-negative pheotype. If both of a child’s parents are Rh negative, the child will definitely
be Rh-negative. Otherwise the child may be Rh positive or Rh-negative, depending on the parent’s specific genotypes.
The Rh antigen is of particular significance when Rh-negative mothers give birth to Rh-positive babies. The fetal
and maternal bloods are normally kept separate across the placenta and so the Rh-negative mother is not usually
exposed to the Rh antigen of the fetus during the pregnancy. However, during the delivery of the first child, there is
a possibility of exposure of the maternal blood to small amounts of the Rh-positive blood from the foetus. In such
cases, the mother starts preparing antibodies against the Rh antigen in her blood. However, this does not occur
always because the exposure may be minimal and because Rh-negative women vary in their sensitivity to the Rh
antigen. If the woman does produce antibodies against the Rh antigen, these antibodies can cross the placenta in
subsequent pregnancies and cause hemolysis of the Rh-positive red blood cells of the fetus. Therefore, the baby
could be born anemic with a condition called erythroblastosis fetalis (or hemolytic disease of the newborn).
Erythroblastosis fetalis can be prevented by injecting the Rh-negative mother with an antibody prepared against
the Rh antigen (called anti-Rh antibodies) within 72 hours after the birth of each Rh-positive baby. This is a type
of passive immunization in which the injected antibodies inactivate the Rh antigens and thus prevent the mother
from becoming actively immunized to them.
Table 1.2 Crosses between individuals which are heterozygous for given number of gene pairs
Monohybrid Dihybrid Trihybrid General

n= 1 n=2 n=3 rule
Different kinds of gametes by F1 heterozygotes 2 4 8 2n
Number of different F2 phenotypes1 2 4 8 2n
Number of different F2 genotypes 3 9 27 3n
Total number of F2 combinations 4 16 64 4n
Total F2 genotypes that are homozygous 2 4 8 2n
Total F2 genotypes that are heterozygous 1 5 19 3n – 2n

1
If there is complete dominance.
1.1.4 Lethal alleles

Certain genes are absolutely essential for survival. The alleles created by mutations in these genes are called lethal
alleles. The phenotypic manifestation of these alleles is the death of the organism. Lethal alleles may be recessive
or dominant. Recessive lethal alleles are lethal when present in homozygous conditions whereas dominant lethal
alleles show lethal effects even in heterozygous conditions. Dominant lethal alleles are very rare. Lethal alleles fall
into four categories:
● Early onset: Lethal alleles which result in early death of an organism, during embryogenesis.
● Late onset: Lethal genes which have delayed effect so that the organism can live for some time but eventually
succumb to the disease.
Genetics 11
● Conditional: Lethal alleles which kill organism under certain environmental conditions only. For example, a
temperature sensitive lethal allele may kill organism at high temperature, but not at low temperature.
● Semilethal: Lethal alleles which kill only some individuals in the population but not all.
1.1.5 Penetrance and expressivity

The percentage of individuals that shows a particular phenotype among those capable of showing it, is known as
penetrance. Let us take an example of polydactyly in human, which is produced by a dominant gene. Homozygous
recessive genotype does not cause polydactyly. However, some heterozygous individuals are not polydactylous.
If suppose 20% of heterozygous individuals do not show polydactyly, this means that the gene has a penetrance
of 80%. Degree of expression of a trait is controlled by a gene. A particular gene may produce different degrees
of expression in different individuals. This is known as expressivity. Different degrees of expression in different
individuals may be due to variation in the allelic constitution of the rest of the genome or to environmental factors.
Thus, the terms penetrance and expressivity quantify the modification of gene expression by varying environment
and genetic background; they measure respectively the percentage of cases in which the gene is expressed and
the level of expression.
Phenocopy
A phenotype that is not genetically controlled but looks like a genetically controlled one is called phenocopy. It is
an environmentally induced phenotype that resembles the phenotype determined by the genotype. An example of
a phenocopy is Vitamin-D-resistant rickets. A dietary deficiency of vitamin D, for example, produces rickets that
is virtually indistinguishable from genetically caused rickets.
1.1.6 Probability
The chance that an event will occur in the future is called the event’s probability. For example, if you flip a coin,
the probability is 0.50, or 50%, that the head side will be showing when it lands. The probability depends on the
number of possible outcomes. In this case, there are two possible outcomes (head and tail), which are equally
likely. This allows us to predict that there is a 50% chance that a coin flip will produce head. The general formula
for the probability is:
Number of times an event occurs

Probability =
Total number of events
Phead = 1 head/(1 head + 1 tail) = 1/2 = 50%
A probability calculation allows us to predict the likelihood that an event will occur in the future. The accuracy of
this prediction, however, depends to a great extent on the size of the sample.
In genetic problems, we are often interested in the probability that a particular type of offspring will be produced.
For example, when two heterozygous tall pea plants (Tt) are crossed, the phenotypic ratio of the offspring is
3 tall : 1 dwarf. This information can be used to calculate the probability for either type of offspring:
Number of individuals with a given phenotype

Probability =
Total number of individuals
Ptall = 3 tall/(3 tall + 1 dwarf) = 3/4 = 0.75 = 75% and
Pdwarf = 1 dwarf/(3 tall + 1 dwarf) = 1/4 = 0.25 = 25%
The probability of obtaining a tall plant is 75% and a dwarf plant 25%. When we add together the probabilities of
all the possible outcomes (tall and dwarf), we should get a sum of 100% (here, 75% + 25% = 100%).
There are two basic laws of probability that are used for genetic analysis. The first law, the multiplicative law
(or product rule), states that the chance of two or more independent events occurring together is the product of the
Genetics 17
Table 1.3 Comparison between dominance and epistasis
Dominance Epistasis
Allelic suppression. Non-allelic suppression.
It involves a single pair of alleles. It involves two pairs of alleles.
A gene suppresses the expression of its allele. A gene suppresses the expression of its non-allele.
The effect of a recessive allele is suppressed. Epistatic allele suppresses the effect of both dominant
and recessive non-allele.
The effect is only due to dominant allele. It may be due to dominant or recessive allele.
Now the term epistasis has come to be synonymous with almost any type of gene interaction that involves the
masking or modifying of one of the gene effects. When epistasis is operative between two gene loci, the number
of phenotypes appearing in the offspring will be less than four (normal F2 phenotypic classes in case of dihybrid
crosses is four, 9 : 3 : 3 : 1). Such bigenic (two genes) epistatic interactions may be of several types.
1.3.1 Dominant epistasis

When the dominant allele of one gene masks the effects of either allele of the second gene, it is termed as dominant
epistasis. When the dominant allele at one locus, for example, the A allele produces a certain phenotype regardless
of the allelic condition of the other locus, then the A locus is said to be epistatic to the B locus. Furthermore, since
the dominant allele A is able to express itself in the presence of either B or b, this is a case of dominant epistasis.
Only when the genotype of the individual is homozygous recessive at the epistatic locus (aa), can the alleles of the
hypostatic locus (B or b) be expressed. Thus the genotypes A-B- and A-bb produce the same phenotype, whereas
aaB- and aabb produce 2 additional phenotypes. The classical 9 : 3 : 3 : 1 ratio becomes modified into a 12 : 3 : 1 ratio.
Explanation: Let us take the following case, in which F2 phenotypic ratio is 12 Purple : 3 Red : 1 White.
Parent 1 Parent 2
AA bb aa BB
(Purple) (Red)
F1
AaBb
(Purple)
F2
12 Purple : 3 Red : 1 White
In this example, two non-allelic genes are interacting because the F2 phenotypic classes obtained is less than 4.
This phenotypic ratio can be explained by following consideration:
Enzyme A
Purple product
White substance
Red product
Enzyme B
In this case, enzymes A and B compete for the same substrate. Enzyme A, which converts the substrate to a pur-
ple product, has much higher affinity for substrate than enzyme B, which converts the substrate to a red product.
The difference in the affinity for the substrate is so marked that enzyme B can only work effectively if no enzyme
A is present. So,
18 Genetics
Purple 12
AABB(1), AABb(2), AaBB(2), AaBb(4), These have at least one functional allele A and convert all the substrates to
AAbb(1), Aabb(2) purple product.
Red 3
aaBB(2), aaBb(1) Lack any functional enzyme A, but have a functional enzyme B, which
converts the substrate to a red product.
White 1
aabb(1) Have no functional enzymes and cannot synthesize any colored pigment.
1.3.2 Recessive epistasis

In the case of recessive epistasis, in a pair of non-allelic genes, one produces its phenotypic effect independently in
a dominant state, but another cannot produce a phenotypic effect independently. However, the latter can produce
its effect when they are together in dominant state. For example, A and B are two non-allelic genes and A can
produce a phenotypic effect independently in dominant state, but second gene B cannot produce a phenotypic effect
independently. In this case, the recessive genotype aa suppresses the expression of alleles at the B locus. But,
in the presence of dominant allele at the A locus, the alleles of the B locus express. Thus the genotypes A-B- and
A-bb produce two additional phenotypes. The 9 : 3 : 3 : 1 ratio becomes a 9 : 3 : 4 ratio.
Explanation: Let us take the following case, in which F2 phenotypic ratio is 9 Purple : 3 Red : 4 White.
Parent 1 Parent 2
AA bb aa BB
(Red) (White)
F1
AaBb
(Purple)
F2
9 purple : 3 red : 4 white
In this example, the biochemical pathway would again be a simple chain, but the product of enzyme A would be
red in color.
Enzyme A Enzyme B
White substance Red product Purple product
Purple 9
AABB(1), AaBB(2), AABb(2), AaBb(4) Have at least one functional copy of both A and B and therefore can
synthesize the purple pigment.
Red 3
AAbb(2), Aabb(1) Have only functional enzyme A and produce red pigment but do not
aaBB(2), aaBb(1) convert it to purple pigment.
Have no functional enzyme A and so cannot synthesize the red product
that is the substrate for enzyme B and will remain white.
White 4
aabb(1) Have no functional enzymes and cannot synthesize the purple pigment.
Genetics 19
1.3.3 Duplicate recessive epistasis

If two non-allelic genes are involved in a specific pathway and functional products from both are required for
expression, then one homozygous recessive allele at either allelic pair would result in the mutant phenotype. In
such case, the genotype aaBB, aaBb, AAbb and aabb produce one phenotype and genotype AABB, AaBB, AABb,
AaBb produce another phenotype (9 : 7). Because both dominant alleles complement each other for the correct
phenotype, these non-allelic genes are called complementary genes. Hence, this interaction is also termed as
complementary gene interaction.
1.3.4 Duplicate dominant interaction

If the alleles of both gene loci produce the same phenotype without cumulative effect, the 9 : 3 : 3 : 1 ratio is
modified into 15 : 1 ratio. Duplicate gene interaction allows dominant alleles of either duplicate gene to produce
the wild-type phenotype. Only organisms with homozygous recessive of both genes have a mutant phenotype.
The mechanism by which wheat kernel color is determined is an example of duplicate gene action. In wheat, kernel
color is dependent upon a biochemical reaction that converts a colorless precursor substance into a colored product,
and this reaction can be performed with the product of either gene A or gene B. Thus, having either an A allele or
a B allele produces color in the kernel, but a lack of either allele will produce a white kernel that is devoid of color.
So, if two plants with genotype AaBb are crossed with each other, the genotype AABB, AABb, AaBB, AaBb, AAbb,
Aabb, aaBB and aaBb produce the color phenotype and the genotype aabb produce no color. In this cross, whenever
a dominant allele is present at either locus, the biochemical conversion occurs, and a colored kernel results. Thus,
only the double homozygous recessive genotype produces a phenotype with no color, and the resulting phenotypic
ratio of color to noncolor is 15 : 1.
Enzyme A
(Product of gene A)
Precursor Product
(Colorless) (Colored)
Enzyme B
(Product of gene B)
1.3.5 Dominant and recessive interaction

Dominant and recessive interaction is similar to dominant epistasis but occurs when a dominant allele of one gene
completely suppresses the phenotypic expression of alleles of another gene. This type of epistasis is sometimes
called dominant suppression, because the deviation from 9 : 3 : 3 : 1 is caused by a single allele that produces a
dominant phenotype.
For example, in Primula plant, the pigment malvidin creates blue-colored flowers. Synthesis of malvidin is controlled
by gene A, yet production of this pigment can be suppressed by non-allelic gene B. In this case, the B gene is
dominant to the A gene, so plants with the genotype AaBb will not produce malvidin because of the presence of
the B gene. So, if two plants with genotype AaBb are crossed with each other, the genotype AABB, AABb, AaBB,
AaBb, aaBB, aaBb and aabb produce the white color and the genotype AAbb and Aabb produce blue color. In this
case, the presence of the B gene suppresses the production of malvidin.
Product of gene A
Precursor Malvidin
(Colorless) × (Colored)
Product of gene B
Genetics 41
1.6.3 Sex determination in plants

Sexually reproducing plant species may be ‘sexually monomorphic’ or ‘sexually polymorphic. In sexually monomorphic
condition, individual plants have both sexes – whether present within single flower (hermaphrodite) or in separate
male and female flowers (monoecious). A minority of plant species are ‘sexually polymorphic’, including dioecious
species. Dioecious species are the ones showing animal-like sexual dimorphism, with female plants bearing unisexual
flowers containing only carpels and male plants bearing unisexual flowers containing only stamens. Many, but
not all, dioecious plants have a non-identical pair of chromosomes associated with the sex determination. Of the
species with non-identical sex chromosomes, a large proportion have an XY system. For example, the dioecious
plant Melandrium album has 22 chromosomes per cell: 20 autosomes plus 2 sex chromosomes, with XX females
and XY males.
1.6.4 Mosaicism
Mosaicism is a condition in which cells within the same individual have a different genetic makeup. Individuals
showing mosaicism are referred to as mosaics. Mosaicism can be caused by DNA mutations, epigenetic alterations of
DNA, chromosomal abnormalities (change in chromosome number and structure) and the spontaneous reversion of
inherited mutations. Mosaicism can be associated with changes in either nuclear or mitochondrial DNA. An individual
with two or more cell types, differing in chromosome number or structure is either a mosaic or a chimera. If the
two cell types originated from a single zygote, the individual is a mosaic, and when originated from two or more
zygotes that subsequently fused, the individual is a chimera.
Mosaicism can exist in both somatic cells (somatic mosaicism) and germ line cells (germline mosaicism). As
their names imply, somatic and germ line mosaicism refer to the presence of genetically distinct groups of cells
within somatic and germ line tissues, respectively. If the event leading to mosaicism occurs during development,
it is possible that both somatic and germ line cells will become mosaic. In this case, both somatic and germ line
tissue populations would be affected, and an individual could transmit the mosaic genotype to his or her offspring.
Conversely, if the triggering event occurs later in life, it could affect either a germ line or a somatic cell population.
If the mosaicism occurs only in a somatic cell population, the phenotypic effect will depend on the extent of the
mosaic cell population; however, there would be no risk of passing on the mosaic genotype to offspring. On the
other hand, if the mosaicism occurs only in a germ line cell population, the individual would be unaffected, but the
offspring could be affected.
How is somatic mosaicism generated? There are many possible reasons, including somatic mutations, epigenetic
changes in DNA, alterations in chromosome structure and/or number, and spontaneous reversal of inherited
mutations. In all of these cases, a given cell and those cells derived from it could exhibit altered function.
1.6.5 Sex-linked traits and sex-linked inheritance

In an XY-chromosomal system of sex determination, both X and Y-chromosomes are sex chromosomes. In general,
genes on sex chromosomes are described as sex linked genes. However, the term sex linked usually refers to loci
found only on the X-chromosome; the term Y-linked is used to refer to loci found only on the Y-chromosome, which
control holandric traits (traits found only in males).
Cytogeneticists have divided the X and Y-chromosomes of some species into homologous and non-homologous
regions. The latter is called differential regions. These differential regions contain genes that have no counterparts
on the other sex chromosome. Genes in the differential regions are said to be hemizygous (half zygous). Genes
in the differential region of the X show an inheritance pattern called X-linkage; those in the differential region of
the Y show Y-linkage. Genes in the homologous region show what might be called X-and-Y linkage.
Another important feature of sex linked genes in XY-chromosomal system of sex determination is that females have
two X-chromosomes, they can have normal homozygous and heterozygous allelic combinations. But males, with only
one copy of the X-chromosome can be neither homozygous nor heterozygous. Hence the term hemizygous is used
Genetics 43
In X-linked inheritance, the pattern of inheritance for loci on the heteromorphic sex chromosome differs from the
pattern for loci on the homomorphic autosomal chromosomes, because sex chromosome alleles are inherited in
association with the sex of offspring. Alleles on a male’s X-chromosome go to his daughters, but not to his sons,
because the presence of his X-chromosome normally determines that his offspring is a daughter. Since the father
passes a trait to his daughters, who passes it to their sons. Hence, this pattern of inheritance is known as criss-
cross pattern of inheritance. In Drosophila, eye color has nothing to do with sex determination, so we see that
genes on the sex chromosomes are not necessarily related to sexual function. The same is true in humans, for
whom pedigree analysis has revealed many X-linked genes, of which few could be constructed as being connected
to sexual function.
1.6.6 Sex-limited traits

Sex hormones influence the action of certain genes. In some cases, a given genotype is so dependent on the
presence of these hormones that its expression is limited to one sex. The result is a sex-limited trait, which is
expressed in only one sex, although the genes are present in both sexes. Sex-limited traits are usually determined
by autosomal genes and primarily concerned with the secondary sexual characters. In humans, for example,
breast development is a trait that is normally limited to female, whereas beard growth is limited to males.
1.6.7 Sex-influenced traits

The sex-limited trait is an extreme example of how the expression of a gene can be controlled by hormones. In
other less extreme cases of sex controlled characteristics, only the dominance relationship of the two alleles is
affected. Characteristics of this type are known as sex-influenced traits (or sex-conditioned), in which an allele is
dominant in one gender, but recessive in the opposite gender. In human, pattern baldness provides an example of
a sex-influenced trait. Pattern baldness is characterized by the premature loss of hair from the front and top of the
head. It is more common in males than in females. Women who have the genotype for pattern baldness typically
show only thinning of hair rather than a complete loss. The gene that causes pattern baldness is inherited as an
autosomal trait. When a male is heterozygous for the baldness allele, he will become bald.
Phenotype
Genotype
Male Female
BB Bald Bald
Bb Bald Non-bald
bb Non-bald Non-bald
In contrast, a heterozygous female will not be bald. Women who are homozygous for the baldness allele will develop
the trait. Sex influence nature of pattern baldness appears to be related to the levels of the male sex hormones.
1.6.8 Pedigree analysis

A pedigree is a family tree or chart made of symbols and lines that represent a person’s genetic family history.
In pedigree, symbols represent people and lines represent genetic relationships. The pedigree is a visual tool for
documenting the biological relationship in families and determine the mode of inheritance (dominant, recessive etc.)
of genetic diseases. Pedigrees are most often constructed by medical geneticists or genetic counselors. A sample
pedigree is given below:
Genetics 57
1.9 Cytogenetics
A chromosome is an organized structure of DNA and protein that is found in the nucleus of a eukaryotic cell. The
study of the structure, function and abnormalities of chromosome is called cytogenetics, a discipline that combines
cytology with genetics.
1.9.1 Human karyotype

The number, sizes and shapes of the metaphase chromosomes constitute the karyotype or karyogram, which is
distinctive for each species. The useful karyotypic characteristics are: chromosome size, chromosome number, sex
chromosomes, centromere position, nucleolar organizer position, heterochromatin pattern, secondary constriction
and banding patterns. Karyotype consisting of a photograph or diagram of all the metaphasic chromosomes arranged
in homologous pairs according to decreasing length and position of centromere is described as idiogram.
Table 1.6 Symbol used in describing a karyotype
Symbol Meaning
p (petit) Short arm
q (queue) Long arm
13p Short arm of chromosome 13
13q Long arm of chromosome 13
del Deletion
del(2) Deletion in chromosome 2
dup Duplication
dup(1) Duplication in chromosome 1
inv Inversion
inv(4) Inversion in chromosome 4
t Translocation
t(2;5) Reciprocal translocation between a chromosome 2 and a chromosome 5
tel Telomere
cen Centromere
+ or – Indicate gain or loss of part of chromosome
2q– Deletion of the long arm of chromosome 2
Tijo and Levan (1956) of Sweden found that human cells have 23 pairs or 46 chromosomes. Of the 23 pairs, 22
are perfectly matched in both males and females, and are called autosomes. The remaining pair, the sex chro-
mosomes, consists of two similar chromosomes in females and two dissimilar chromosomes in males. In human,
females are designated XX and males XY. The largest autosome is number 1, and the smallest is number 21.
Denver system
According to ‘Denver system’ of classification, the 22 pairs of human chromosomes are placed in seven groups as;
Group Position of centromere Idiogram number
I (A) Metacentric or submetacentric 1, 2, 3
II (B) Submetacentric 4, 5
III (C) Submetacentric 6, 7, 8, 9, 10, 11, 12 and X
IV (D) Acrocentric 13, 14 and 15
V (E) Metacentric or submetacentric 16, 17 and 18
VI (F) Metacentric 19 and 20
VII (G) Metacentric 21, 22 and Y
58 Genetics
Male Female
1 2 3 4 5 1 2 3 4 5
6 7 8 9 10 6 7 8 9 10
11 12 13 14 15 11 12 13 14 15
16 17 18 19 20 16 17 18 19 20
21 22 XY 21 22 XX
Figure 1.37 The karyotype of a human male and female.
1.9.2 Chromosome banding

Chromosome banding is a cytological procedure of differential staining of mitotic chromosome along the longitudinal
axis. The differential staining reactions reflect the heterogeneity and complexity of the chromosome along its
length. The molecular mechanisms involved in producing the various banding patterns are not precisely defined.
Chromosome painting is different from banding. It refers to the hybridization of fluorescently labeled chromosome-
specific, composite probe pools to chromosome.
The most common methods of dye-based chromosome banding are G- (Giemsa), R- (reverse), C- (centromere) and
Q- (quinacrine) banding. Bands that show strong staining are referred to as positive bands (dark bands); weakly
staining bands are negative bands (light bands). Features of commonly used banding techniques are described in
the table 1.7.
Table 1.7 Chromosome banding techniques
Technique Procedure Banding pattern
Mild proteolysis with trypsin followed by staining Dark bands are AT-rich (low gene density)
G-banding with Giemsa (G stands for Giemsa).
Light bands are GC-rich (high gene density)
Heat denature followed by staining with Giemsa. Dark bands are GC-rich
R-banding Reverse of G-banding and R stands for Reverse.
Light bands are AT-rich
Stain with Quinacrine mustard (a fluorescent Dark bands are AT-rich

Q-banding stain). Q stands for Quinacrine.
Light bands are GC-rich
Denature with barium hydroxide and then stain Dark bands contain constitutive heterochromatin
C-banding with Giemsa. C stands for Constitutive
heterochromatin.
Genetics 59
Regions, bands and sub-bands
A region is an area that lies between two landmarks. Regions are divided into bands. A band is that part of a
chromosome that is distinctly different from the adjacent area by virtue of being lighter or darker in staining intensity.
Each band is approximately 5 to 10 megabase pairs of DNA that may include hundreds of genes. The bands and
the regions to which they belong are identified by numbers, with the centromere serving as the point of reference
for the numbering scheme. In designating a particular band, four items are required: the chromosome number,
the arm symbol, the region number and the band number within that region. A band within a region is numbered
in sequence with band 1 being nearest to the centromere.
Arm Region Band Subband

2
2
1
14.3
p 4 14.2
14.1
1 3
2
1
Centromere
1
1
2
21.1
1
21.2
2 21.3
2
q
31.1
31.2
1
31.3
2
3
3
4
5
6
Figure 1.38 The arm of each chromosome is denoted with a ‘p’ or ‘q’. Each arm is further divided into regions.
Numbering begins at the centromere and moves out toward the telomere (end). Each region is further divided into light
and dark bands which are also numbered from centromere. Each band may be even further subdivided into subbands,
which are denoted after a decimal point.
How do geneticists indicate the location of a gene? Geneticists use a standardized way of describing a gene’s
cytogenetic location. The combination of numbers and letters provides a gene’s address on a chromosome. This
address is made up of several parts. For example, if we write the location of gene as 13q14. Orally, it is referred
to as ‘13q one-four’ (not as ‘13q fourteen’). It means that gene is located in band 4 in region 1 of the long arm
of chromosome 13. Band can further be divided into sub-bands. By convention, a decimal point is placed before
any sub-band number. Sub-bands are numbered sequentially from centromere outward. For example, 13q14.2
represents sub-band 2 of 13q14.
1.9.3 Variation in chromosome number

Each species has a characteristic number of chromosomes. An organism may contain one set (termed monoploid, x),
two sets (termed diploid, 2x) or more than two sets (termed polyploid, >2x ) of chromosomes. The number of
sets is called the ploidy. Organisms with multiples of the basic chromosome set are referred to as euploid. There
is a difference between monoploid and haploid. The haploid value (n) is the number of chromosomes present in
a gamete whereas monoploid value (x) is the number of chromosomes in a single set. In case of monoploid and
diploid organisms, both n and x are same whereas in polyploid organisms x and n differ.
60 Genetics
For example, humans are diploid. A human somatic cell contains 46 chromosomes (2 sets of chromosomes). Here,
monoploid and haploid values are same (x = n = 23). In hexaploid bread wheat (Triticum aestivum), monoploid
and haploid values are not same. The somatic cells are hexaploid, with six sets of chromosomes, 6x = 42 (i.e. x is 7)
whereas gametes are haploid, with total number of chromosomes (n) is 21.
1
1 1
1 1 1
2 2
2 2 2
2
3 3 3
3 3
3
Monoploid (1 set) Diploid (2 sets) Triploid (3 sets)

x=3 2x = 6 3x = 9
Most higher eukaryotes are diploid, with two sets of chromosomes. However, not all organisms are diploids; some
organisms are polyploid that they contain more than two sets of chromosomes. Polyploidy is more common in
plants than in animals. Polyploidization is a frequent mode of diversification and speciation in plants. In animals,
polyploidy is very common in amphibians and reptiles.
Polyploidy is of two types: autopolyploidy and allopolyploidy. Autopolyploidy is the polyploidy condition resulting
from the multiplication of the same genome. Autopolyploids may be triploid (3x), tetraploid (4x), pentaploid (5x),
hexaploid (6x) and so forth. There is often a correlation between ploidy level and the size of the organism. The
higher the ploidy level, the larger the size. Polyploids with odd numbers of chromosome sets are sterile. For example,
autotriploids are characteristically sterile. The problem lies in pairing at meiosis. The synapsis can take place only
between two of the three homologous chromosomes.
An autotetraploid contains four basic sets of chromosomes. Because four is an even number, autotetraploids can
have a normal meiosis. Autotetraploids arise by the doubling of a 2x complement to 4x. This doubling can occur
spontaneously, but it can also be induced artificially through the application of chemical agents such as colchicine
(an alkaloid extracted from the autumn crocus). In colchicine-treated cells, an S-phase of the cell cycle occurs,
but no chromosome segregation during anaphase. As the treated cell enters telophase, a nuclear membrane forms
around the entire doubled set of chromosomes. Thus, treating diploid cells for one cell cycle leads to tetraploids,
with exactly four copies of each type of chromosome.
Two diploid cells
Without colchicine 2x
Mitosis in a diploid cell, 2x = 4
2x
4x
With colchicine
One tetraploid cell
Figure 1.39 The use of colchicine to generate a tetraploid from a diploid. During metaphase and anaphase, colchicine
disrupts spindle-fiber formation, preventing the migration of daughter chromosomes after the split of centromere.
Allopolyploidy is a polyploid condition formed by crossing different species and doubling the chromosomes of the
hybrid. An example of natural allopolyploid is bread wheat, Triticum aestivum (6x = 42). One man-made example
of allopolyploid is an allotetraploid Raphanobrassica, developed by Russian geneticist G. D. Karpechenko. Karpech-
enko worked with the radish (Raphanus sativus, 2x = 18, x = 9) and cabbage (Brassica oleracea, 2x = 18, x = 9).
Each of these species has 18 chromosomes and they are related closely enough to allow intercrossing.
Genetics 69
Molecular genetics
1.10 Genome
Genome is the sum total of all genetic material of an organism which store biological information. The nature of
the genome may be either DNA or RNA. All eukaryotes and prokaryotes always have a DNA genome, but viruses
may either have a DNA genome or RNA genome. The eukaryotic genome consists of two distinct parts: Nuclear
genome and organelles (mitochondrial and chloroplast) genome. The nuclear genome consists of linear dsDNA.
In a few lower eukaryotes, double-stranded circular plasmid DNA (for example, 2-micron circle in yeast) is also
present within the nucleus.
The amount of DNA present in the genome of a species is called a C-value, which is characteristic of each species.
The value ranges from <106 bps as in smallest prokaryote, Mycoplasma to more than 1011 bps for eukaryotes such
as amphibians. The genomes of higher eukaryotes contain a large amount of DNA.
Flowering plants
Mammals
Reptiles
Birds
Amphibians
Fish
Echinoderms
Insects
Worms
Algae and fungi
6 7 8 9 10 11
10 10 10 10 10 10
Size of eukaryotic haploid genome (base pairs)
Figure 1.48 The DNA content of the haploid genome of a range of phyla. The range of values within a phylum is
indicated by the shaded area.
The DNA content of the organism’s genome is related to the morphological complexity of lower eukaryotes, but
varies extensively among the higher eukaryotes. In lower eukaryotic organisms like yeast, amount of DNA increases
with increasing complexity of organisms. However, in higher eukaryotes there is no correlation between increased
genome size and complexity. This lack of correlation between genome size and genetic complexity refers to
C-value paradox. For example, a man is more complex than amphibians in terms of genetic development, but
some amphibian cells contain 30 times more DNA than human cells. Moreover, the genomes of different species
of amphibians can vary 100-fold in their DNA contents.
70 Genetics
Table 1.9 Genome size in some eukaryotes
Organism Genome size (Mb)
S. cerevisiae (yeast) 12
A. thaliana (mustard plant) 120
D. melanogaster (fruit fly) 170
H. sapiens (human) 3,300
H. vulgare (barley) 5,300
1.10.1 Genome complexity

Genome complexity is the total length of different sequences of DNA. It can be measured through the renaturation
kinetics of denatured DNA. Renaturation of DNA occurs through complementary base pairing. Renaturation of
DNA depends on the random collision of the complementary strands, and follows second-order kinetics. A DNA
renaturation (reassociation) reaction is described by the Cot1/2. If large DNA is sheared into uniform fragments and
allowed to renature, then the rate of renaturation of denatured DNA is expressed as
dC
= - kC2
dt
where k is the second-order rate constant. C is the concentration of single-stranded DNA at time t and the second
order rate equation for two complementary strands coming together is given by the rate of decrease in C.
Starting with a concentration, C0, of completely denatured DNA at t = 0, the amount of single-stranded DNA
remaining at some time t is
C 1
=
C0 (1 + k .C0 .t)
The time for half of the DNA to renature (when C/C0 = 0.5) is defined as t = t1/2. Then,
1
0.5 = and thus 1 + k .C0 .t1/2 = 2, yielding
(1 + k .C0 .t1/2 )
1
C0 .t1/2 =
k
The product of C0 × t1/2 is called the Cot1/2. It is inversely proportional to the rate constant. Since the Cot1/2 is the
product of the concentration and time required to proceed halfway, a greater Cot1/2 implies a slower reaction. The
renaturation of DNA usually is followed in the form of a Cot curve. A graph of the fraction of single-stranded DNA
reannealed (1 – C/C0) as a function of Cot on a semilogarithmic plot is referred to as a Cot curve.
5 6
Genome size 1 3500 1.7×10 4.2×10 bp
100%
Poly U:polyA MS2 T4 E.coli
Fraction
reassociated
0 –6 –4 –2 2 Figure 1.49
10 10 10 1 10
Cot curve of dsDNA
–6
2×10 8×10
–3
3×10
–1
9 Cot1/2 from the indicated source.
102 Genetics
Maternal chromosome
Paternal chromosome
Chromomere
Enlarged section of
a chromosome
Chromatin loop
Chromatin
loop
Sister chromatids
Chromomere
Figure 1.79 Lampbrush chromosome structure. Most of the DNA in each chromosome remains highly condensed in
the chromomeres. Each of the two chromosomes shown consists of two closely apposed sister chromatids. This four
stranded structure is characteristic of diplotene stage of meiosis.
1.11.6 B-chromosomes
The B-chromosomes (also referred to as supernumerary or accessory chromosomes) are additional (extra)
chromosomes that are present in some individuals in some species. In eukaryotic cells normal chromosomes are
termed as A-chromosomes. Most B-chromosomes are mainly or entirely heterochromatic and genetically inert. They
are thought to be selfish genetic elements with no defined functions. The evolutionary origin of B-chromosomes is
not clear, but presumably they must have been derived from heterochromatic segments of normal A-chromosomes.
1.12 DNA replication

Transmission of chromosomal DNA from generation to generation is crucial to cell propagation. This can only be
achieved when chromosomal DNA is accurately replicated, providing two copies of the entire genome for faithful
distribution into each daughter cell.
1.12.1 Semiconservative replication

It is crucial that the genetic material is reproduced accurately. When Watson and Crick worked out the double-helix
structure of DNA in 1953, they recognized that the complementary nature of the two strands - A paired with T
and G paired with C - might play an important role in its replication. Because the two polynucleotide strands are
joined only by hydrogen bonds, they are able to separate without requiring breakage of covalent bonds. If the two
strands of a parental double helix of DNA are separated, the base sequence of each parental strand could serve
Genetics 107
DNA helicase and primase

DNA polymerase requires single-stranded DNA as a template and cannot synthesize a chain without an RNA or DNA
primer annealed to the template. Thus, two other enzymes enable polymerase to work on a duplex DNA. One is a
DNA helicase, which opens up the duplex at the replication fork to provide a single-stranded template. The primary
replicative helicase (DnaB) binds to and moves on the lagging-strand template in the 5’→3’ direction unwinding the
duplex as it goes. This helicase action requires ATP hydrolysis. The separated strands are inhibited from subsequently
reannealing by a single-strand-binding protein (SSB protein), which binds to both separated strands. The E. coli SSB
is a tetramer and binds cooperatively in sequence independent manner. The other is a primase, which synthesizes
a short RNA primers (<15 nucleotides) to prime DNA chain elongation. The term primosome is used to denote a
complex between primase and helicase, sometimes with other accessory proteins.
Topoisomerase
During replication, topoisomerases are required to relieve the positive supercoiling that arises from DNA unwinding
mediated by helicases. In E. coli this role is typically fulfilled by DNA gyrase. By contrast, eukaryotes rely primarily
on Topo IB for relaxation of positive supercoils.
DNA gyrase is the first type II topoisomerase discovered by Martin Gellert from E. coli. It is present in prokaryotes
and some eukaryotes such as Plasmodium falciparum. DNA gyrase has the ability to remove positive supercoiling
and introduce negative supercoiling into DNA using the free energy from ATP hydrolysis. It is a tetramer of two
different subunits. The GyrA subunit cuts and rejoins the DNA and the GyrB subunit is responsible for providing
energy by ATP hydrolysis. DNA gyrase is inhibited by quinolone antibiotics, such as nalidixic acid and their fluorinated
derivatives such as norfloxacin and ciprofloxacin, which bind to the GyrA protein. Novobiocin also inhibits gyrase
by binding to the GyrB protein and preventing it from binding ATP.
Topoisomerase
A DNA topoisomerase is a nuclease that breaks a phosphodiester bond in a DNA strand. This reaction is reversible,
and the phosphodiester bond reforms as the enzyme leaves. The first DNA topoisomerase was discovered by James
Wang in 1971 from E. coli. There are several types of topoisomerases present in eukaryotes and prokaryotes. All
topoisomerases can be classified into two classes– type I and type II, depending on whether they cleave one or two
strands of DNA, respectively. Type I topoisomerases cleave one DNA strand and pass other strand through the break
before resealing it, while type II topoisomerases cleave both DNA strands and pass another double strand through
the break followed by resealing of the double strand break. Enzymes with an odd Roman numeral after their name
(for example, topo I and topo V) fall into the type I class, whereas those with an even Roman numeral after their
name are type II. Type I topoisomerases do not require ATP for activity; the reaction is driven by the energy stored
in the supercoiled DNA. So far, the only exception is reverse gyrase, which introduces positive supercoils with the
aid of ATP hydrolysis. Type II topoisomerases also do not require an external source of energy for the cleavage and
religation during reaction, but they do utilize ATP hydrolysis to drive conformational changes in the protein during
the reaction cycle.
All topoisomerases contain a nucleophilic tyrosine, which they use to promote strand cleavage. The tyrosyl oxygen
attacks and breaks phosphodiester bond and at the same time forming a covalent phosphotyrosine bond. Rejoining
of the DNA strand occurs by a second transesterification reaction, which is basically the reverse of the first.
Type I topoisomerases operate by forming a transient phosphotyrosine covalent bond with one end of the broken
DNA strand, either the 5’ or the 3’ end, followed by passage of the unbroken strand through the break, and ultimately
resealing of the break. These topoisomerases can be further divided into two subfamilies: Type IA and Type IB topoi-
somerases. During DNA hydrolysis, type IA topoisomerases covalently bind 5’-phosphate, whereas type IB enzymes
form a covalent bond with 3’-phosphate. Type IA topoisomerases pass a single-stranded DNA segment through a
transient break in a second single DNA strand. On the contrary, type IB topoisomerases nick one DNA strand, allowing
one duplex end to rotate with respect to the other around the remaining phosphodiester bond.
Genetics 119
1.12.6 Replication of mitochondrial DNA

Small and mostly circular mitochondrial and chloroplast DNA use a slightly different process of replication.
Replication of circular double stranded mitochondrial DNA starts at a specific origin. But duplex DNA uses different
origin sequences to initiate replication of each DNA strand. Initially, only one of the two parental strands is used
as a template for synthesis of a new strand. Synthesis proceeds for only a short distance, displacing the original
complementary strand, which remains single-stranded. This pattern of replication generates a displacement or
D loop (hence, termed as displacement replication). A single D loop is found as an opening of 500–600 bases in
mammalian mitochondria. Some mitochondrial DNAs possess several D loops which reflects the presence of multiple
origins. Replication of the complementary strand is initiated when its origin is exposed by the movement of the first
replication fork. The similar mechanism is employed in chloroplast DNA. Mammalian mitochondrial DNA is replicated
by the DNA polymerase γ. The replisome machinery is formed by DNA polymerase, TWINKLE and mitochondrial
SSB proteins. TWINKLE is a helicase, which unwinds short stretches of dsDNA in the 5’ to 3’ direction.
Leading strand origin (OH)
DNA synthesis initiated

at origin of replication H strand
L strand
on H strand
Synthesis of new L strand

creates D loop by displacing
parental strand
D loop expands
Lagging strand
origin (OL)
When displaced strand passes

origin of replication on L strand,
synthesis of new H strand starts
Figure 1.96 Replication of mammalian mitochondrial DNA. Replication starts at a specific origin in the circular duplex
DNA. Initially only one of the two parental strands (the H strand in mammalian mitochondrial DNA) is used as a template
for synthesis of a new strand. Synthesis proceeds for only a short distance, displacing the original partner (L) strand,
which remains single-stranded. There are separate origins for L and H strand.
1.13 Recombination
Genomes are dynamic entities that change as a result of mutations and recombinations. Recombination is a large-
scale rearrangement of a DNA molecule that involves the breakage and reunion of DNA. It was first recognized as
the process responsible for crossing-over during meiosis of eukaryotic cells, and was subsequently implicated in
the integration of the transferred DNA into bacterial genomes after conjugation, transduction or transformation.
Genetic recombination events fall into two general classes:
120 Genetics
Homologous recombination
Recombination which involves the exchange of homologous segments between any two homologous DNA molecules
(or segments of the same molecule) that share an extended homology.
Site-specific recombination
Recombination between two dsDNA molecules that have only short regions of nucleotide sequence similarity.
A different type of event called transposition, which is related to the processes of recombination, allows one DNA
sequence to be inserted into another without relying on sequence homology. It provides a means by which certain
elements move from one chromosomal location to another.
1.13.1 Homologous recombination

Homologous recombination (also termed as general recombination) is the most important version of recombination
in nature, being responsible for meiotic crossing-over in eukaryotes and the integration of acquired DNA by the
process of conjugation, transduction and transformation into bacterial genomes. It involves a reciprocal exchange
of sequences of DNA.
Holliday model for homologous recombination

An appealing scheme for homologous recombination was proposed by Robin Holliday in 1964. The Holliday model
(also known as heteroduplex model) describes recombination between two homologous double-stranded molecules,
those with identical or nearly identical sequences. But, it is equally applicable to two different molecules that share
a limited region of homology, or a single molecule that recombines with itself because it contains two separate
regions that are homologous with one another.
A B
5’ 3’
3’ 5’
3’ 5’
5’ 3’
a b
Endonuclease nicking
A B
a b
Strand displacement
A B
a b
Ligation
A B
Figure 1.97
Holliday
The Holliday model for homologous
junction
recombination. Single-strand nicks
a b are introduced at the same position
Branch migration
on both parental molecules. The
A B nicked strands then exchange by
complementary base pairing, and
ligation produces a crossed-strand
intermediate called a Holliday junction.
a b
Genetics 127
1.14 DNA repair

Although the genetic variation is important for evolution, the survival of the individual demands genetic stability
also. Maintaining genetic stability requires not only an extremely accurate mechanism for replicating DNA, but also
mechanisms for repairing the many accidental lesions that occur continually in DNA. Most such spontaneous changes
in DNA are temporary because they are immediately corrected by a set of processes that are collectively called as
DNA repair. Without repair systems, a genome would not be able to maintain its essential cellular functions. Most
cells possess four different categories of DNA repair system: Direct repair, Excision repair, Mismatch repair and
Recombination repair.
1.14.1 Direct repair

Direct repair systems act directly on damaged nucleotides, converting each one back to its original structure. But
only a few types of damaged nucleotide can be repaired directly. One very common type of UV radiation mediated
damages, pyrimidine dimers, are repaired by a light-dependent direct system called photoreactivation. In
E. coli, the process involves the enzyme called DNA photolyase. When stimulated by light with a wavelength
between 300 and 500 nm, the enzyme binds to pyrimidine dimers and converts them back to the original monomeric
nucleotides. Photoreactivation is a widespread but not universal type of repair.
5 5
T T UV-B
6 6
Photolyase + blue light

P P P P P P
Adjacent thymines Thymine dimer
Another example is the repair of O6-methylguanine, which forms in the presence of alkylating agents and is a
common and highly mutagenic lesion. It tends to pair with thymine rather than cytosine during replication. Direct
repair of O6-methylguanine is carried out by O6-methylguanine DNA methyltransferase (an alkyl transferase), which
catalyzes the transfer of the methyl group of O6-methylguanine to a specific Cys residue in the same protein.
6
P CH3 P Alkyl P P
transferase
S G C S S G C S
P P P P
1.14.2 Excision repair

Excision repair involves the excision of a segment of the polynucleotide containing a damaged site, followed by
resynthesis of the correct nucleotide sequence by a DNA polymerase. These pathways fall into two categories:
Base-excision repair
Base excision repair involves removal of a damaged nucleotide base, excision of a short piece of the polynucleotide
and resynthesis with a DNA polymerase. It is used to repair many minor damage like alkylation and deamination
resulting from exposure to mutagenic agents. Enzyme DNA glycosylase initiates the repair process. A DNA
128 Genetics
glycosylase does not cleave phosphodiester bonds, instead cleave the N-glycosidic bonds, liberating the altered
base and generating an apurinic or an apyrimidinic site, both called AP sites. The resulting AP site is then repaired
by an AP endonuclease repair pathway. All cells have endonucleases that attack the sites left after the spontaneous
loss of single purine or pyrimidine residues. The AP endonucleases are vital to the cell, because spontaneous
depurination is a relatively frequent event. These enzymes introduce chain breaks by cleaving the phosphodiester
bonds at AP sites. This bond cleavage initiates an excision-repair process with the help of three enzymes – an
exonuclease, DNA polymerase I and DNA ligase.
Deaminated C
U
5’ 3’
3’ 5’
G
Uracil DNA Glycosylase

U
5’ 3’ DNA helix with

3’ 5’ missing base
AP endonuclease and phosphodiesterase

remove sugar phosphate
5’ 3’ DNA helix with single

5’ nucleotide gap
3’
G
DNA polymerase I plus DNA ligase
C
5’ 3’
3’ 5’
G
Figure 1.104 Base excision repair.
Nucleotide excision repair

Nucleotide excision repair is similar to base excision repair, but is not preceded by the removal of a damaged base and
can act on more substantially damaged areas of DNA. This repair system includes the breaking of a phosphodiester
bond on either side of the lesion, on the same strand, resulting in the excision of an oligonucleotide. This excision
leaves a gap that is filled by repair synthesis, and a ligase seals the breaks.
The uvr system of excision repair in E. coli is the best-studied example. It involves the removal of relatively short,
usually 12 nucleotides in length. The key enzyme is made up of three subunits, products of the uvrA, uvrB and
uvrC genes, and is called the ABC excinuclease. ABC excinuclease binds to DNA at the site of a lesion. First UvrA
and UvrB attach to the DNA at the damaged site. UvrA recognizes the damage. Departure of UvrA allows UvrC to
bind, forming UvrBC dimer which cleaves the damaged strand at the eighth phosphodiester bond on the 5’ side of
the lesion and at the fourth or fifth phosphodiester bond on the 3’ side. UvrD is a helicase (also called helicase II)
that helps to unwind the DNA to allow release of the single strand between the two cuts. The resulting gap is filled
in by DNA polymerase I and sealed by ligase.
In humans, Xeroderma pigmentosum and Cockayne syndrome are caused by genetically defective nucleotide excision
repair. Xeroderma pigmentosum, a rare inherited disease, is mainly characterized by the inability of skin cells to
repair UV-induced DNA lesions (pyrimidine dimer). Individuals suffering from this autosomal recessive condition
are extremely sensitive to sunlight.
Genetics 129
Pyrimidine dimer
T T
5’ 3’
3’ 5’
UvrAB binds
A
B
5’ 3’
3’ 5’
C
A
C B
5’ 3’
3’ 5’
UvrBC cuts the polynucleotide either
side of the thymine dimer and removes
oligonucleotide by DNA helicase II
5’ 3’
3’ 5’
DNA polymerase and DNA ligase
T T
5’ 3’
3’ 5’
Figure 1.105 Nucleotide excision repair (A-UvrA, B-UvrB, C-UvrC).
1.14.3 Mismatch repair

The mismatch repair system can detect mismatches that occur in DNA replication. Enzyme systems involved in
mismatch repair are as follows:
1. Recognize mismatched base pairs.
2. Determine which base in the mismatch is the incorrect one.
3. Excise the incorrect base and carry out repair synthesis.
The repair must be made in the daughter polynucleotide because it is in this newly synthesized strand that the
error has occurred; the parent polynucleotide has the correct sequence. How does the repair process know which
strand is which? When mismatch errors occur during replication in E. coli, it is possible to distinguish the original
strand of DNA. Immediately after replication of methylated DNA, only the original parental strand carries the
methyl groups.
During the period while the newly synthesized strand awaits the introduction of methyl groups, the two strands
can be distinguished. In E. coli, the answer is that the daughter strand is, at this stage, undermethylated and
can be distinguished from the parent polynucleotide, which has a full complement of methyl groups. E. coli
DNA is methylated because of the activities of the DNA adenine methylase (Dam), which converts adenines to
6-methyladenines in the sequence 5’-GATC-3’, and the DNA cytosine methylase (Dcm), which converts internal
cytosines to 5-methylcytosines in 5’-CCAGG-3’ and 5’-CCTGG-3’. These methylations are not mutagenic, the
modified nucleotides having the same base-pairing properties as the unmodified versions. There is a delay between
DNA replication and methylation of the daughter strand, and it is during this window of opportunity that the repair
system scans the DNA for mismatches and makes the required corrections in the undermethylated, daughter strand.
Genetics 185
1.21 RNA interference

RNA interference (abbreviated RNAi) is an evolutionarily conserved mechanism of gene regulation that is induced
by small silencing RNA in a sequence-specific manner. In 1998, Fire and Mello first established this in C. elegans.
Historically, RNA interference was known by other names, including post transcriptional gene silencing (PTGS),
transgene silencing and quelling. RNAi has been observed in all eukaryotes, from yeast to mammals. RNA interference
has an important role in post-transcriptional gene regulation, transposon regulation and defending cells against
viruses. Two types of small silencing RNA molecules – small interfering RNA (siRNA) and microRNA (miRNA) – are
central to RNA interference.
siRNAs mediated RNAi

In the siRNAs mediated RNAi pathway, the dsRNAs are processed into siRNAs duplexes comprised of two ~21
nucleotides long strands with two nucleotides overhangs at the 3’ ends by an enzyme called Dicer. Dicer is a
~200 kDa multidomain, an RNase III family enzyme that functions in processing dsRNA to siRNA. The Dicer
includes an ATPase/RNA helicase domain, catalytic RNase III domains, and dsRNA binding domain. Dicer and a
dsRNA binding protein (together form the RISC loading complex) then load the RNA duplex into RISC. The siRNA
is thought to provide target specificity to RISC through base pairing of the guide strand with the target mRNA.
Only one of the two strands, which is known as the guide strand, directs the gene silencing. The other anti-guide
strand or passenger strand is degraded during RISC activation. The active components of an RNA-induced silencing
complex (RISC) are endonucleases called argonaute proteins, which cleave the target mRNA strand complementary
to their bound siRNA.
Long dsRNA
Dicer
Guide strand
siRNA duplex
Passenger strand
RISC
loading complex
pre-RISC
RISC Guide strand
Target cleavage
Figure 1.164 dsRNA precursors are processed by Dicer to generate siRNA duplexes containing guide and passenger
strands. RISC-loading complex loads the duplex into RISC. The passenger strand is later destroyed and the guide
strand directs RISC to the target RNA.
miRNAs mediated RNAi
miRNAs (microRNAs) are small, non-coding RNA molecules encoded in the genomes of plants, animals and their
viruses. These highly conserved, 20–25 mer RNAs appear to regulate gene expression post-transcriptionally by
binding to the 3’-untranslated regions (3’-UTR) of specific mRNAs. Victor Ambros and colleagues identified the
188 Genetics
Table 1.30 Types of small silencing RNAs
Types Organism Length Features
miRNA Animals, plants, protists 20–25 Dicer/Drosha-dependent
Exo-siRNA Animals, plants, fungi, protists ~21 Dicer-dependent

siRNA
Endo-siRNA Animals, plants, fungi, protists ~21 Dicer-dependent
piRNA Metazoans 24–30 Dicer-independent
Noncoding RNA
There are large numbers of functional RNAs that are transcribed but did not encode proteins. These functional RNAs
are called noncoding RNAs (ncRNAs). Noncoding RNAs perform a variety of biological functions. They regulate gene
expression at the levels of transcription, RNA processing and translation. They protect genomes from foreign nucleic
acids. They can guide DNA synthesis or genome rearrangement. Most noncoding RNAs operate as RNA-protein com-
plexes, including ribosomes, snRNPs, snoRNPs, telomerase, miRNAs and lncRNAs.
Group I and II introns: Catalytic RNAs (ribozymes), catalyze RNA splicing.
RNase P RNAs: Ribozymes, catalyze removal of 5’ leader sequence from pre-tRNAs.
Hammerhead and hepatitis delta virus: Ribozymes, induce RNA cleavage to form 2’,3’-cyclic phosphate and 5’-OH
termini; also catalyze the reverse reaction and RNA ligation.
gRNA (guide RNA): Base pairs with an RNA target, orienting bound proteins to carry out a site-specific cleavage,
ligation or modification reaction.
Xist (X-inactive-specific transcript RNA): Coats one X-chromosome in mammalian female, triggering hetero-chrom-
atization and transcriptional repression.
Telomerase RNA: Provides template for telomeric DNA synthesis and scaffolds protein assembly.
snoRNA (small nucleolar RNA): Essential for pre-rRNA processing or modification by serving as a guide RNA to direct
methylation or pseudouridylation of complementary sequence in rRNA.
siRNA (small interfering RNA): Product of dicer cleavage of dsRNA; when complexed with an AGO protein, induces
cleavage of a perfectly-complementary target RNA.
scaRNA (small Cajal body-associated RNA): Function similar to snoRNAs, but located in the Cajal body to guide
modification of snRNAs.
Riboswitch: RNA element within an mRNA that switch between two conformations upon exposure to a small-molecule
ligand or other stimulus and inhibits or promotes gene expression at the level of transcription, translation, or RNA
splicing.
piRNA (PIWI-associated RNA): RNA that directs the modification of chromatin to repress transcription; best charac-
terized in the male germline.
lncRNA (long noncoding RNA): Autonomously transcribed RNA that does not encode a protein; often capped and
polyadenylated; can be nuclear, cytoplasmic or both.
1.22 Epigenetics
Although all cells in an organism contain essentially the same DNA, cell types and functions differ because of
qualitative and quantitative differences in their gene expression. Epigenetics refers to both heritable and non-
heritable changes in gene expression that are not caused by changes in DNA sequence. The epigenetic processes
that stably alter gene expression patterns are thought to include:
1. cytosine methylation,
2. posttranslational modification of histone proteins and remodelling of chromatin and
3. RNA-based mechanisms.
Genetics 189
Methylation of the 5’-position of cytosine residues is a reversible covalent modification of DNA, resulting in produc-
tion of 5-methyl-cytosine. In general, DNA methylation is associated with gene repression. As DNA methylation
patterns can be maintained following DNA replication and mitosis, this epigenetic modification is also associated
with inheritance of the repressed state.
Posttranslational modification of histone proteins on transcription is complex and constantly expanding. Three
general principles are thought to be involved:
1. It directly affects the structure of chromatin, regulating its higher order conformation and thus acting in cis to
regulate transcription;
2. It disrupts the binding of proteins that are associated with chromatin (trans effect);
3. It attracts certain effector proteins to the chromatin (trans effect).
RNA-based mechanisms of epigenetic regulation are less well understood than mechanisms based on DNA
methylation and histones. A number of non-coding RNAs (Small non-coding RNAs as well as Long non-coding RNAs)
play important roles in modifying the sequence, structure, or expression of mRNAs and thereby also changes the
protein expression from these genes.
1.23 Genetic code

General features of genetic code
● The genetic code is a triplet code called a codon.
● How many nucleotides in DNA are needed to specify each amino acid in a protein? We know that the infor-
mation in DNA must reside in the sequence of the four nucleotides that constitute the DNA: A, T, G and C. A
doublet code involving two adjacent nucleotides would not be adequate, as four kinds of nucleotides taken
two at a time can generate only 42 = 16 different combinations. But with three nucleotides per word, the
number of different words that can be produced with an alphabet of just four letters is 43 = 64. This number
is more than sufficient to code for 20 different amino acids. Such mathematical arguments led biologists to
suspect the existence of a triplet code. Later Francis Crick, Sydney Brenner, and their colleagues provided
genetic evidence for the triplet nature of the code by studying the mutagenic effects of the chemical proflavin
on bacteriophage T4.
● Certain codons contain start and stop signals to initiate and terminate translation. The initiation codon is
usually AUG, which specifies methionine. In few mRNA, GUG or UUG also acts as initiation codon. Out of 64
codons, three do not code for any amino acids and called a stop or termination codons (UAA, UAG, and UGA).
● The code is unambiguous, meaning that each triplet specifies only a single amino acid.
● No internal punctuation (commas) is used in the code. Thus, the code is said to be commaless. Once the trans-
lation of mRNA begins, the codons are read one after the other with no breaks between them.
● The code is degenerate, meaning that a given amino acid can be specified by more than one triplet codon.
This is the case for 18 of the 22 amino acids. The different codons for a given amino acid are said to be syn-
onymous. For example, UUU and UUC are synonyms for phenylalanine, whereas serine is encoded by the
synonyms UCU, UCC, UCA, UCG, AGU and AGC.
Table 1.31 Amino acids and their synonymous codons
Amino acids Number of synonymous codon
Leu, Ser, Arg 6
Gly, Pro, Ala, Val, Thr 4
Ile 3
Phe, Tyr, Cys, His, Gln, Glu, Asn, Asp Lys 2
Met, Trp 1
206 Genetics
Proofreading
The fidelity of protein synthesis depends on the accuracy of the two mechanisms: the linking of each amino acid
to its corresponding (or cognate) tRNA molecule and the base-pairing of the codons in mRNA to the anticodons
in tRNA. Two fundamentally different proofreading mechanisms are used in the cell. Both are active processes.
One mechanism called chemical proofreading is used to improve the accuracy of amino acid attachment to tRNA.
This is carried out by aminoacyl tRNA synthetases, which recognize an incorrect amino acid attached to its tRNA
molecule and remove it by hydrolysis. There are two stages at which chemical proofreading may occur during
formation of aminoacyl-tRNA. Mostly, when a synthetase binds the incorrect amino acid and forms incorrect or
noncognate aminoacyl-adenylate then binding of the cognate tRNA is required for proofreading. After binding of the
cognate tRNA, the incorrect aminoacyl-adenylate may be hydrolyzed. Some synthetases use chemical proofreading
at a later stage. The wrong amino acid is actually transferred to tRNA, is then recognized as incorrect by its structure
in the tRNA binding site, and so is hydrolyzed and released.
Some aminoacyl tRNA synthetases are unable to distinguish cognate from noncognate amino acids in the synthetic
reactions alone. These synthetases have two active sites – the synthetic (or activation) site and the editing (or
hydrolytic) site. At editing site, hydrolysis of misactivated amino acids and misacylated tRNAs occurs.
A second mechanism called kinetic proofreading, is used to improve the fidelity of codon-anticodon pairing. Once
tRNA molecules have acquired an amino acid, they form a complex with an elongation factor (EF). This complex
pairs with the appropriate codon in an mRNA molecule. The bound elongation factor allows correct codon-anticodon
pairing to occur, but prevents the amino acid from being incorporated into the growing polypeptide chain. The
initial codon recognition, however, triggers the elongation factor to hydrolyze its bound GTP, whereupon the factor
dissociates from the ribosome without its tRNA, allowing protein synthesis to proceed. The elongation factor, thereby,
introduces a short delay between codon-anticodon base-pairing and polypeptide chain elongation, which provides
an opportunity for the bound tRNA molecule to exit from the ribosome. An incorrect tRNA molecule forms a smaller
number of codon-anticodon hydrogen bonds than a correct one; it, therefore, binds more weakly to the ribosome
and is more likely to dissociate during this period. Thus the delay introduced by the elongation factor causes most
incorrectly bound tRNA molecules to leave the ribosome without being used for protein synthesis.
1.24.1 Incorporation of selenocysteine

Amino acid selenocysteine (Sec) resembles cysteine, but contains selenium instead of sulfur. Selenocysteine
is encoded by UGA. However, UGA is one of the stop codons. Apparently, UGA is normally read as ‘stop’, but is
occasionally translated to give selenocysteine. The choice between ‘stop’ and selenocysteine depends on a special
recognition sequence called selenocysteine insertion sequence. Selenocysteine has its own tRNA and a special protein
factor to escort charged tRNA-Sec to the ribosome. In fact, selenocysteine-tRNA is initially charged with serine.
Then the attached serine is enzymatically modified to form selenocysteine. When bacteria use selenocysteine, the
selenocysteine insertion sequence forms a stem and loop structure in the mRNA molecule just after the UGA. SelB
protein recognizes both charged tRNA-Sec and the stem and loop. Thus selenocysteine bound to tRNA is delivered
to the right place.
Serine Ser Sec

Sec specific tRNA tRNA tRNA
Seryl tRNA ligase Sec synthase
1.24.2 Cap snatching

Some viruses such as influenza virus perform a unique cap-snatching process. Cap snatching is a transcription
initiation process during which a nucleotide sequence between 10 to 13 in size is cleaved from the 5’ end of host
mRNAs by an endonuclease activity present in the viral RNA-dependent RNA polymerase. The capped nucleotide
sequence removed from host mRNAs is subsequently used as a primer for transcription of the viral genome, which
ultimately leads to the synthesis of capped viral mRNAs.
Genetics 207
1.24.3 Translational frameshifting

Translational frameshifting is a mechanism by which the translational machinery (ribosomes) shifts the frame in
which it decodes the mRNA. The result is that the mRNA does not encode the protein by a continuous run of three
nucleotide codons, known as an open reading frame (ORF). Rather, the information encoding the protein comes
from two distinct ORFs. Frameshifting is a stochastic process, meaning that each translating ribosome has a certain
probability of undergoing the shift, but that only a fraction of the ribosomes does so. Classes of signals have been
identified that direct a fraction of elongating ribosomes to shift reading frame by one base in the 5’ (–1) or 3’ (+1)
direction.
In general, it is believed that the occurrence and frequency of translational frameshifting are determined predominantly
by two elements of mRNA: a slippery sequence and a downstream RNA structure. Various slippery sequences have
been identified from different retroviruses and from other viruses. A typical slippery sequence is a heptanucleotide
X XXY YYZ (where X ≠ C, Y = A or U and Z ≠ G). There are two kinds of downstream RNA structures associated
with frameshifting, stem-loop and pseudoknot. However, not all stem-loop or pseudoknot structures can induce
ribosomal frameshifting. Thus, the definitive characterization of specific RNA structures and how these RNA structures
mediate ribosomal frameshifting remain to be defined.
Ser Thr Phe Leu Asn Gly Phe Ala

5’ ... ... UCA ACG UUU UUA AAC GGG UUU GCG ORF1
Arg Val Cys

5’ ... ... UC AAC GUU UUU AAA CGG GUU UGC G ORF2
–1 frameshift
Figure 1.183 Translational frameshift in SARS coronavirus.
Ribosomes that frameshift produce a translational fusion of the two overlapping ORFs, whereas those that do not
frameshift continue normal in frame decoding and terminate at the end of the first ORF. Each of these products thus
shares a common N terminal region. Many viruses use programmed translational frameshifting to ensure synthesis
of the correct ratios of virus-encoded proteins required for proper viral particle assembly and maturation. The
phenomenon was first described in year 1985 as the way in which the Gag-Pol polyprotein of the retrovirus Rous
Sarcoma Virus (RSV) is expressed from the overlapping gag and pol ORFs. It has been demonstrated that when
ribosomes translate the unspliced genomic RNA of retroviruses, 95% of translation yields Gag proteins while only
about 5% of translation produces Gag-Pol proteins through –1 ribosomal frameshifting.
1.24.4 Antibiotics and toxins

Protein synthesis is a target of a wide variety of naturally occurring antibiotics and toxins. Mechanism of action of
some common antibiotics and toxins, which inhibit protein synthesis in prokaryotes and eukaryotes, are described
below:
Streptomycin Streptomycin, a basic trisaccharide, binds with the 30S subunit of the bacterial ribosome and
causes misreading of mRNA at relatively low concentrations.
Chloramphenicol Chloramphenicol binds to the 50S ribosomal subunit and blocks peptide bond formation
through inhibition of peptidyl transferase, but does not affect the cytosolic protein synthesis
in eukaryotes.
Tetracycline Tetracycline binds to the 30S ribosomal subunit and interferes with aminoacyl-tRNA binding.
Erythromycin Binds to the 50S ribosomal subunit and inhibits peptide chain elongation.
Fusidic acid Fusidic acid binds to EF-G and blocks translocation.

208 Genetics
Cycloheximide Cycloheximide blocks the peptidyl transferase of 80S ribosome but not that of 70S bacterial
(and mitochondrial and chloroplast) ribosomes.
Puromycin Puromycin is a secondary metabolite of Streptomyces alboniger that blocks protein biosynthesis.
Puromycin is a structural analogue of the 3’ end of aminoacyl transfer RNA, but differs from
tRNA insofar as the aminoacyl residue is linked to the ribose via an amide bond rather than
an ester bond. Puromycin, like aminoacyl-tRNA, binds to the A site of the ribosome peptidyl-
transferase center. When the A site is occupied by puromycin, peptidyl-transferase links the
peptide residues of the peptidyl-tRNA in the ribosomal P site covalently to puromycin. Since
the amide bond cannot be cleaved by the ribosome, no further peptidyl transfer takes place,
and the peptidyl-puromycin complex falls off the ribosome.
H3C CH3
NH2 N
N N
N N
tRNA
—
O P O C N HO C N
O N O N
O
O OH HN OH
C O C O
H2N C H H2N C H
CH2 CH2
O
tRNA-phenylalanine
CH3
Puromycin
Diphtheria toxin Diphtheria toxin, an exotoxin of Corynebacterium diphtheriae infected with a specific temperate
phage (Corynephage β), stops the protein synthesis in eukaryotes by inactivating the elongation
factor eEF2. Inactivation of elongation factor eEF2 occurs due to ADP-ribosylation, which is
catalyzed by A fragment of toxin.
Ricin A toxic protein of the castor bean (Ricinus communis) that inactivates the 60S subunit of
eukaryotic ribosomes by depurinating a specific adenosine in 28S rRNA.
1.24.5 Post-translational modification of polypeptides

Chemical modification
Primary translation products often undergo a variety of modification reactions, involving the addition of chemical
groups, which are attached covalently to the polypeptide. This can involve simple chemical modification like
hydroxylation and phosphorylation of the side chains of single amino acids or the addition of different types of
carbohydrate or lipid group.
Genetics 209
Table 1.34 Examples of post-translational chemical modifications
Modification Amino acids that are modified
Acetylation Lys
Methylation Lys
Phosphorylation Ser, Thr, Tyr, Asp, His, Lys
Hydroxylation Pro, Lys
Carboxylation Glu (form γ-carboxyglutamic acid)
O-linked glycosylation Ser, Thr
N-linked glycosylation Asn
Acylation Ser, Thr, Cys
Myristoylation Gly
Palmitoylation Cys
Farnesylation Cys
Biotinylation Lys
ADP ribosylation At the nitrogen atom of His, Arg, Asn and Lys or at carboxyl group of Glu.
Proteolytic cleavage
Proteolytic cleavage of polypeptide chains after synthesis is a common occurrence with certain classes of proteins.
For example, proteolytic enzymes in the digestive tract are produced in inactive forms that are generally termed
as zymogens. After selective proteolysis, these enzymes are converted into active forms. One best known example
of proteolytic cleavage is the conversion of preproinsulin into insulin. First, removal of the signal peptide from
the newly synthesized peptide, preproinsulin, generates the proinsulin precursor molecule. Finally, the removal of
C-peptide moiety of proinsulin gives insulin.
C chain C chain
A chain
Removal of Removal of S S
A chain A chain
C signal peptide C C chain N C
S S S S
S S S S
N N N C
Signal B chain B chain B chain
peptide
Preproinsulin Proinsulin Insulin
Figure 1.184 Formation of human insulin from preproinsulin.
Protein splicing
Occasionally, the primary translation product of a gene contains one or more short amino acid sequences, called
inteins that excise themselves from the nascent polypeptide. The sequences that are represented in mature
polypeptide are termed exteins. Inteins occur in both eukaryotic and prokaryotic polypeptides. An intein has the
ability to catalyze its own removal from primary translation products. The autocatalytic process of protein splicing
involves bond transfer reactions and no input of energy is required. According to the most accepted theory, the
standard protein splicing mechanism consists of following four steps:
Genetics 211
Second step: Transesterification

In this step, the side-chain of the first residue of the C-extein attacks the ester (or thioester) bond at the amino
end of the intein. Here too the attack is by a polar side chain of a Ser, Thr (both –OH) or Cys (–SH). This leads to
a transesterification and formation of thioester or ester bond between N-extein and C-extein.
Third step: Asn cyclization

Cyclization of the Asn side chain leads to cleavage of the peptide bond between the intein and the C-extein
(C-terminal splice junction). This reaction removes intein from the ligated exteins, which are linked together via
the ester bond.
Fourth step: O–N shift

This step of protein splicing is spontaneous. The reverse N–O or N–S shift takes place and peptide bond formation
occurs between N- and C-exteins.
Some inteins show sequence specific endonuclease activity also. Such inteins cut DNA in the intein-minus gene
at a specific point and allow a copy of a DNA sequence coding intein to integrate. This event is similar to intron
homing and termed as intein homing.
1.25 Mutation
Genome is not a static entity. It is dynamic in nature. It is subject to different types of heritable genetic changes.
A heritable genetic change in the genetic material of an organism that gives rise to alternate forms of any gene
is called mutation. The process by which mutations is produced is called mutagenesis. An organism exhibiting
a novel phenotype as a result of the presence of a mutation is referred to as a mutant. In a broad sense, the
term mutations include all types of heritable genetic changes of an organism not explainable by recombination of
preexisting genetic variability. Mutation may include change in chromosome number, chromosomal aberrations
and changes in chemistry of genes. But here we have described ‘mutation’ in terms of change in chemistry of gene
which is known as gene mutation.
General characteristics of mutation

● Mutations are generally recessive, but dominant mutations also occur.
● Mutations are generally harmful to the organisms.
● Mutations are random, occur at any time and in any cell of an organism.
● Mutations are recurrent i.e. the same mutation may occur again and again.
Role of mutation
● Ultimate source of all genetic variation and it provides the raw material for evolution.
● Mutation results into the formation of alleles. Without mutation, all genes would exist in only one form.
● Organisms would able to evolve and adapt to environmental change.
Molecular basis of gene mutation

Mutations arise in two ways: Some mutations are spontaneous that occur without treatment of the organism with
an exogenous mutagen. Mutagen is an agent that leads to an increase in the frequency of occurrence of mutations.
Spontaneous mutations account for the ‘background rate’ of mutation and are presumably the ultimate source
of natural genetic variation that is seen in populations. Spontaneous mutations can occur because of replication
errors, spontaneous lesions and transposition of transposable elements during the normal growth of the cell. Other
mutations called induced mutations arise because a mutagen has reacted with the parent DNA, causing a structural
change that affects the base-pairing capability of the altered nucleotide.
212 Genetics
Error in DNA replication
Each of the common bases in DNA can spontaneously undergo a transient rearrangement of bonding. A proton shift
in nitrogenous base forms one of its rare tautomeric form (termed a tautomeric shift). Formation of the tautomer
of any base alters its base pairing properties.
The more stable keto forms of thymine and guanine and amino forms of adenine and cytosine may infrequently
undergo tautomeric shifts to less stable enol and imino forms, respectively. The bases would be expected to exist
in their less stable tautomeric forms for only very short period of time.
Common (stable) Rare
O OH
H C CH3 C CH3
4 4
N3 5 C N3 5 C
Thymine
6 6
C2 1 C C2 1 C
O N H O N H
H H
Keto form Enol form
NH2 NH
C H C H
4 4
N3 5 C HN 3 5 C
Cytosine 6 6
C2 1 C C2 1 C
O N H O N H
H H
Amino form Imino form
NH2 NH
C N C N
6 6
N1 5C 7 HN 1 5C 7
Adenine 8 CH 8 CH
2 4 9 2 4 9
C 3 C C 3 C
H N H N
N H N H
Amino form Imino form
O OH
C N C N
HN 1 6 6
5C 7 N1 5C 7
Guanine 8 CH 8 CH
2 4 9 2 4 9
C 3 C C 3 C
H2N N H2N N
N H N H
Keto form Enol form
Figure 1.186 Tautomeric forms of the four common bases in DNA. The shifts of hydrogen atoms between the number
3 and number 4 positions of the pyrimidines and between the number 1 and number 6 positions of the purines change
the base-pairing potential of the bases.
However, if a base existed in the rare form at the moment that it was being replicated or being incorporated into
a nascent DNA chain, a mutation might result. When the bases are present in their rare imino or enol states, they
can form adenine-cytosine and guanine-thymine base pairs. The net effect of such an event, and the subsequent
replication required to segregate the mismatched base pair, is an AT to GC or a GC to AT base pair substitution.
222 Genetics
In sickle cell hemoglobin (Hemoglobin S) sixth amino acid i.e. glutamic acid (a negatively charged amino acid) from
the amino terminal end of the β-chain is replaced by valine (no charge at neutral pH). This changes the shape of
hemoglobin. Mutation that changes a codon in a gene to one of the three termination codon (UAA, UGA or UAG) is
described as nonsense mutation. Nonsense mutation results in a shortened protein because the translation of the
mRNA stops at this new termination codon.
Silent and neutral mutation
Not all mutations in DNA lead to a detectable change in the phenotype. Mutations without apparent effect are
called silent mutation. If a mutation in which the new codon specifying the same amino acid as the unmutated
codon then it is a case of silent or synonymous mutation. Because it has no effect on the coding function of the
genome: the mutated gene codes for exactly the same protein as the unmutated gene. For example, the change of
ACG into CGG, both code for an arginine. Codon specifies different but functionally equivalent amino acid and not
alter protein function called neutral mutation (for example, AAA → AGA : changing basic lysine to basic arginine).
Conditional mutations
Not all mutations reliably produce a mutant phenotype, regardless of environmental conditions. A conditional mutant
allele expresses a mutant phenotype only in a certain environmental condition, called the restrictive condition, but
produces a wild-type phenotype in some different environmental condition, called the permissive condition. For
example, some conditional mutants are called temperature-sensitive mutants which give wild-type phenotype at
low temperature (the permissive temperature), but exhibit mutant phenotype at high temperature (the restrictive
temperature).
Loss- and gain- of function mutations
In principle, mutation of a gene might cause a phenotypic change in either of two ways:
● Loss of function (null) mutation : the product may have reduced or no function.
● Gain of function mutation : the product may have increased or new function.
Because mutation events introduce random genetic changes, most of the time they result in loss of function.
Generally, loss of function mutations are found to be recessive. In a wild type diploid cell, there are two wild type
alleles of a gene, both making normal gene product. In heterozygotes, the single wild type allele may be able to
provide enough normal gene product to produce a wild type phenotype. In such cases, loss of function mutations
are recessive. However, some loss of function mutations are dominant. In such cases, the single wild type allele in
the heterozygote cannot provide the enough amount of gene product needed for the cells to be wild type. Gain of
function mutations usually cause dominant phenotypes, because the presence of a normal allele does not prevent
the mutant allele from behaving abnormally.
1.25.3 Fluctuation test

The fluctuation test was invented by Luria and Delbruck in 1943 to determine the randomness of mutation in bacteria.
They grew a series of E. coli cultures in different flasks and then added T1 bacteriophage to each one. Most of the
bacteria were killed by the phage, but a few T1 resistant mutants were able to survive. Luria and Delbruck measured
the number of mutants resistant to bacteriophage T1 in a large number of replicate cultures of E. coli. If mutants
occur after the culture is exposed to the phage, then little variation should occur among cultures in the number
of mutants. However, if mutants arise at random during nonselective growth of cells, each culture would contain
different number of resistant mutant. The numbers depend on how early during the growth period the first mutant
cells arose. But the consequence of that mutation would depend on when during the growth of the population the
mutation occurred. Thus a mutation during the early generations gives rise to a large clone of mutant cells, whereas
a late mutation gives rise to a few mutant cells. Among a large set of identical cultures of dividing cells, the few
cultures in which the mutation happened in the early generations have a large number of mutants, whereas the
majority of the cultures have none or a few mutants. This is what Luria and Delbruck observed.
Genetics 223
E. coli : Wild type
Normal receptor
Lysis
T1
Mutant type
Mutant receptor
T1 cannot bind
Figure 1.193 When bacteriophage T1 infects wild-type E. coli, it binds to a receptor in the outer membrane, protein
TonB. After phage replication, the E. coli cell is lysed and new phages are released. A mutation in the tonB gene results
in an altered receptor to which T1 can no longer bind and so the cells survive.
Their test is known as the fluctuation test because it measures the degree of fluctuation in the number of mutants
found in replicate cultures. They proved that mutations occur before selection. The fluctuation test is also useful
in determining mutation rates during nonselective growth.
1.25.4 Replica plating experiment

Replica plating experiment suggests that the resistant cells are selected by the environmental agent rather than
produced by it (nonadaptive nature of mutation). The technique was developed by Joshua and Esther Lederberg
in 1952. A population of bacteria was plated on nonselective medium that is, containing no phages and from each
cell a colony grew. This plate was called the master plate. A sterile piece of velvet was pressed down lightly on the
surface of the master plate, and the velvet picked up cells wherever there was a colony.
Replica plating
Master plate Replica plate Replica plate

(nonselective medium) (nonselective medium) (selective medium)
After incubation
Figure 1.194 Replica plating. For the detection of mutants, cells are transferred on to successive plates containing
either a selective medium or a non-selective medium. Colonies form on the non-selective plate in the same pattern
as on the master plate. Only mutant cells can grow on the selective plate; the mutant colonies that are formed derive
from colonies on the master plate that are mutant.
224 Genetics
In this way, the velvet picked up a colony ‘imprint’ from the whole plate. On touching the velvet to replica plates
containing selective medium (that is, containing T1 phages), cells clinging to the velvet are inoculated onto the
replica plates in the same relative positions as those of the colonies on the original master plate. As expected, rare
Tomr mutant colonies were found on the replica plates, but the multiple replica plates showed identical patterns
of resistant colonies. If the mutations had occurred after exposure to the selective agents, the patterns for each
plate would have been as random as the mutations themselves. The mutation events must have occurred before
exposure to the selective agent.
Replica plating has become an important technique of microbial genetics. It is useful in screening for mutants that
fail to grow under the selective regime. The position of an absent colony on the replica plate is used to retrieve the
mutant from the master. For example, replica plating can be used to screen auxotrophic mutants in precisely this
way. In general, replica plating is a way of retaining an original set of strains on a master plate while simultaneously
subjecting replicas to various kinds of tests on different media or under different environmental conditions.
1.25.5 Ames test

The Ames test, named for its developer, Bruce Ames, is a method to test chemicals for their cancer-causing
properties. The use of the Ames test is based on the assumption that any substance that is mutagenic may also
turn out to be a carcinogen; that is, to cause cancer.
The assay is based on the reversion of mutations in the histidine (his) operon in the genetically altered tester strains
of bacterium Salmonella typhimurium. The his operon encodes enzymes required for the biosynthesis of the amino
acid histidine. Strains with mutations in the his operon are histidine auxotrophs — they are unable to grow without
added histidine. However, this mutation can be reversed, a back mutation, with the gene regaining its function.
These revertants are able to grow on a medium lacking histidine. The tester strains are specially constructed to have
both frameshift and point mutations in the genes required to synthesize histidine, which allows for the detection
of mutagens acting via different mechanisms. The tester strains also carry mutations in the genes responsible
for lipopolysaccharide synthesis, making the cell wall of the bacteria more permeable, and in the excision repair
system to make the test more sensitive.
The Ames test can detect mutagens that work directly to alter DNA. In humans, however, many chemicals are
promutagens, agents that must be activated to become true mutagens. Activation, involving a chemical modification,
often occurs in the liver as a consequence of normal liver activity on unusual substances. Bacteria such as
S. typhimurium do not produce the enzymes required to activate promutagens, so promutagens would not be
detected by the Ames test unless they were first activated. An important part of the Ames test also involves mixing
the test compound with enzymes from rat liver that convert promutagens into active mutagens. These potentially
activated promutagens are then used in the Ames test. If the liver enzymes convert the agent to a mutagen, the
Ames test will detect it, and it will be labeled as a promutagenic agent.
Problem
In the Ames test, auxotrophic strains of Salmonella that are unable to produce histidine are mixed with a rat liver extract
and a suspected mutagen. The cells are then plated on a medium without histidine. The plates are incubated to allow any
revertant bacteria (those able to produce histidine) to grow. The number of colonies is a measure of the mutagenicity of
the suspected mutagen. Why is the rat liver extract included?
Solution
Most mutagens cannot act unless they are converted to electrophile by liver enzymes called mixed-function oxidase,
which include the cytochromes P-450s. The rat liver extract in the Ames test contains enzymes for converting suspected
mutagens to compounds that would be physiologically relevant mutation-causing agents in a mammal.
Genetics 225
1.25.6 Complementation test

If two recessive mutations arise independently and both have the same phenotype, how do we know whether they
are both mutations of the same gene? The complementation test allows us to determine whether two mutations,
both of which produce a similar phenotype are in the same gene i.e. whether they are alleles or represent mutations
in separate genes, whose proteins are involved in the same function. In genetics, complementation occurs when two
strains of an organism with different homozygous recessive mutations that produce the same phenotype produce
offspring with the wild-type phenotype when mated or crossed. Complementation will occur only if the mutations
are in different genes.
In a diploid organism the complementation test of allelism (allelism test) is performed by intercrossing homozygous
recessive mutants two at a time and observing whether or not the progeny have a wild-type phenotype. If the two
recessive mutations are in separate genes and are not alleles of one another, then following the cross, all F1 progeny
are heterozygous for both genes. Complementation is said to occur. Because each mutation is in a separate gene
and each F1 progeny is heterozygous at both loci, the normal products of both genes are produced.
Case 1 : Mutations are in separate genes (Trans heterozygote)
Gene 1 Gene 2 Gene 1 Gene 2
Mutant Wild type Wild type Mutant

× Homologs
Mutant Wild type Wild type Mutant
Gene 1 Gene 2
Mutant Wild type
Wild type Mutant
One normal copy of each gene is present.

Complementation occurs
Case 2 : Mutations are in different locations within the same gene (Cis heterozygote)
Gene 1 Gene 2 Gene 1 Gene 2
Mutant Wild type Mutant Wild type

× Homologs
Mutant Wild type Mutant Wild type
Gene 1 Gene 2
Mutant Wild type
Mutant Wild type
Gene 1 is mutant in all cases, while Gene 2 is normal.
No complementation occurs
Figure 1.195 Complementation analysis.

Chapter 02
Recombinant DNA technology
Recombinant DNA technology (also known as genetic engineering) is the set of techniques that enable the DNA
from different sources to be identified, isolated and recombined so that new characteristics can be introduced
into an organism. The invention of recombinant DNA technology—the way in which genetic material from one
organism is artificially introduced into the genome of another organism and then replicated and expressed by that
other organism—was largely the work of Paul Berg, Herbert W. Boyer, and Stanley N. Cohen, although many other
scientists made important contributions to the new technology as well. Paul Berg developed the first recombinant
DNA molecules that combined DNA from SV40 virus and lambda phage. Later in 1973, Herbert Boyer and Stanley
Cohen develop recombinant DNA technology, showing that genetically engineered DNA molecules may be cloned
in foreign cells.
One important aspect in recombinant DNA technology is DNA cloning. It is a set of techniques that are used to
assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word
cloning refers to the fact that the method involves the replication of a single DNA molecule starting from a single
living cell to generate a large population of cells containing identical DNA molecules.
2.1 DNA cloning

DNA cloning is the production of a large number of identical DNA molecules from a single ancestral DNA molecule.
The essential characteristic of DNA cloning is that the desired DNA fragments must be selectively amplified resulting
in a large increase in copy number of selected DNA sequences. In practice, this involves multiple rounds of DNA
replication catalyzed by a DNA polymerase acting on one or more types of template DNA molecule. Essentially two
different DNA cloning approaches are used: Cell-based and cell-free DNA cloning.
Cell-based DNA cloning

This was the first form of DNA cloning to be developed, and is an in vivo cloning method. The first step in this
approach involves attaching foreign DNA fragments in vitro to DNA sequences which are capable of independent
replication. The recombinant DNA fragments are then transferred into suitable host cells where they can be
propagated selectively.
The essence of cell-based DNA cloning involves following steps:
Construction of recombinant DNA molecules

Recombinants are hybrid DNA molecules consisting of autonomously replicating DNA segment plus inserted elements.
Such hybrid molecules are also called chimera. Recombinant DNA molecules are constructed by in vitro covalent
attachment (ligation) of the desired DNA fragments (target DNA) to a replicon (any sequence capable of independent
DNA replication). This step is facilitated by cutting the target DNA and replicon molecules with specific restriction
endonucleases before joining the different DNA fragments using the enzyme DNA ligase.
Recombinant DNA technology 241
Cell-free DNA cloning

The polymerase chain reaction (PCR) is a newer form of DNA cloning which is enzyme mediated and is conducted
entirely in vitro. PCR (developed in 1983 by Kary Mullis) is a revolutionary technique used for selective amplification
of specific target sequence of nucleic acid by using short primers. It is a rapid, inexpensive and simple method of
copying specific DNA sequence.
2.2 Enzymes for DNA manipulation

The enzymes used in the recombinant DNA technology fall into four broad categories:
2.2.1 Template-dependent DNA polymerase

DNA polymerase enzymes that synthesize new polynucleotides complementary to an existing DNA or RNA template
are included in this category. Different types of DNA polymerase are used in gene manipulation.
DNA polymerase I (Kornberg enzyme) has both the 3’-5’ and 5’-3’ exonuclease activities and 5’-3’ polymerase activity.
Reverse transcriptase, also known as RNA-directed DNA polymerase, synthesizes DNA from RNA.
Reverse transcriptase was discovered by Howard Temin at the University of Wisconsin, and independently by David
Baltimore at about the same time. The two shared the 1975 Nobel Prize in Physiology or Medicine.
Taq DNA polymerase is a DNA polymerase derived from a thermostable bacterium, Thermus aquaticus. It operates
at 72°C and is reasonably stable above 90°C and used in PCR. It has a 5’ to 3’ polymerase activity and a 5’ to 3’
exonuclease activity, but it lacks a 3’ to 5’ exonuclease (proofreading) activity.
2.2.2 Nucleases
Nucleases are enzymes that degrade nucleic acids by breaking the phosphodiester bonds that link one nucleotide to
the next. Ribonucleases (RNases) attack RNA and deoxyribonucleases (DNases) attack DNA. Some nucleases will
only attack single stranded nucleic acids, others will only attack double-stranded nucleic acids and a few will attack
either kind. Nucleases are of two different kinds – exonucleases and endonucleases. Exonucleases remove nucleotides
one at a time from the end of a nucleic acid whereas endonucleases are able to break internal phosphodiester bonds
within a nucleic acid. Any particular exonuclease attacks either the 3’-end or the 5’-end but not both.
Mung bean nuclease

The mung bean nuclease is an endonuclease specific for ssDNA and RNA. It is purified from mung bean sprouts.
It digests single-stranded nucleic acids, but will leave intact any region which is double stranded. It requires Zn2+
for catalytic activity.
S1 nuclease
The S1 nuclease is an endonuclease purified from Aspergillus oryzae. This enzyme degrades RNA or single stranded
DNA, but does not degrade dsDNA or RNA-DNA hybrids in native conformation. Thus, its activity is similar to mung
bean nuclease, however, the enzyme will also cleave a strand opposite a nick on the complementary strand.
RNase A
RNase A is an endonuclease, which digests ssRNA at the 3’ end of pyrimidine residues.
RNase H
It is an endonuclease which digests the RNA strand of an RNA-DNA heteroduplex. The enzyme does not digest ss
or dsDNA.
248 Recombinant DNA technology
n
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at
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30 ng
re rs
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9
3.0
2.0
BamHI BamHI BamHI 1.0

0.9
400 900
0.8
0.4
Figure A
Figure B
Why are there so many bands in the original ligation mixture?
Solution
The complexity of the original ligation pattern arises because any two BamHI ends can join together. Thus, the 0.4 kb
fragments can join together to generate a set of fragments with sizes 0.8 kb, 1.2 kb, 1.6 kb, 2.0 kb and so forth. Similarly,
the 0.9 kb fragments will produce a set of fragments with sizes 1.8 kb, 2.7 kb, 3.6 kb and so forth. Finally, combinations
of the two fragments generate a third set of fragments with sizes 1.3 kb, 1.7 kb, 2.1 kb, 2.2 kb and so forth.
DNA and RNA purification

DNA purification
DNA isolation and purification involve disruption and lysis of the cells followed by the removal of proteins and
other contaminants, and finally the recovery of the DNA. The cells are lysed using a detergent and cellular
debris removed by centrifugation. The remaining soluble material is then mixed with phenol or 1:1 mixture of
phenol and chloroform. These organic solvents precipitate proteins, but leave the nucleic acids (DNA and RNA)
in aqueous solution. Aqueous solution of nucleic acids can then be removed. If the protein content in cell extract
is very high, then protease such as pronase or proteinase K is used to cleave proteins before phenol extraction.
RNA is removed from the preparation by treatment with RNase.
Cell extract DNA, RNA, protein
Mix with phenol,

Centrifugation Centrifugation
Aqueous layer (DNA+RNA)
Coagulated proteins
Phenol
Cell debris
Figure 2.2 Removal of proteins by organic extraction and enzyme digestion.
After purification, nucleic acid present in the aqueous solution can be concentrated using ethanol (termed ethanol
precipitation). Ethanol precipitation involves the precipitation of DNA by using absolute ethanol in the presence
of monovalent cations and at a temperature of –20°C or less.
The purification of mRNA involves two basic steps: 1. Biochemical separation of total cellular RNA from DNA
and protein using a strong protein denaturant to inhibit cellular RNases, and 2. Isolation of poly A tail mRNA
using an oligo dT affinity matrix. A common method used to isolate mRNA from tissue culture cells is outlined
in the following figure 2.4. Guanidinium thiocyanate is a protein denaturant that lyses the cells and inhibits
cellular RNases.
Aqueous
phase
Guanidinium + Phenol
thiocyanate + CHCl3
Centrifuge
Removal of
Cell culture aqueous phase
Isopropanol
Separation of mRNA
Aqueous
Centrifuge RNA pellet with oligo dT affinity
phase
matrix
Figure 2.4 Isolation and purification of mRNA from tissue culture cells using guanidinium thiocyanate and oligo dT
cellulose. This purification method is based on the finding that RNA preferentially partitions to the aqueous phase in a
solution containing guanidinium thiocyanate at pH 4 in the presence of phenol and chloroform. Under these conditions,
proteins partition to the organic phase and most of the large DNA fragments trapped in the interphase. Poly A+ mRNA
is purified from total cellular RNA using polyadenylated and therefore, oligo dT affinity ligand.
2.3 Vectors
The term vector refers to the DNA molecules that act as transporting vehicle which carries foreign DNA into a host
cell for the purpose of cloning and expression. Cloning vectors are used to clone foreign DNA whereas expression
vectors are engineered so that any foreign DNA can be transcribed in RNA and translated into protein. A viral DNA
or plasmid is generally used as a vector.
The important features of a cloning vector are as follows:
1. Ability to replicate in host cells.

All cloning vectors have origin of replication for autonomous replication within the host cell. The origin of
replication is a specific sequence in DNA from where replication starts. When foreign DNA is linked to vector
containing origin of replication then along with vector replication, foreign (desirable) DNA also starts replicating
within the host cell.
2. Unique restriction enzyme sites for insertional cloning.

All cloning vectors have features that allow a foreign DNA to be conveniently inserted into the vector. This
may be a multiple cloning site (also called polylinker site) which contains many unique restriction sites. The
restriction sites in the polylinker site are first cleaved by restriction enzymes, and a target gene is then ligated
into the vectors using DNA ligase.
3. Genetic marker to select for host cells containing the vector.

Genetic marker is a gene that allow the selection of transformed from nontransformed cell and recombinant
containing transformed cell from non-recombinant containing transformed cell. Marker genes belong to two
broad categories: selectable markers and screenable markers. A selectable marker gene encodes a product
that allows the growth of one type of cells under specific conditions that kill or restrict the growth of other
2.8 Expression vector

An expression vector contains regulatory elements allowing the expression of any foreign DNA it carries. A foreign
gene present on expression vector can be efficiently transcribed and translated by the host cell. The simplest
expression vectors, transcription vectors, allow transcription, but not a translation of cloned foreign DNA. Typical
protein expression vectors allow both the transcription and translation of cloned DNA, and thus facilitate the
production of recombinant protein.
All protein expression vectors carry a transcription unit containing the sequences required for efficient gene
expression. These comprise transcription regulatory sequences, RNA processing signals and sequence for protein
synthesis and targeting. For transcription, a promoter site and a terminator site are necessary. Transcription of the
desired gene begins at the promoter site and ends at the terminator site. Promoter is the most critical component
of an expression vector since it is the site where RNA polymerase binds. It also regulates the rate of transcription.
An expression vector should carry a strong promoter so that the highest possible rate of gene expression could be
achieved. Regulation of promoter is another important factor to be considered during construction of an expression
vector. Two important ways of regulating a promoter in E. coil are:
Induction: Where transcription of a gene is switched on by the addition of a chemical.
Repression: Where gene transcription is switched off upon addition of a regulatory chemical.
Promoter
Regulatory
gene Ribosome binding site
Start
codon
Coding sequence
Origin
Stop
codon
C-terminal tag
Selectable
marker Transcription
terminator
Figure 2.18 The basic architecture of an E. coli expression vector. It contains the features: origin of replication,
promoter, regulatory gene (repressor), selectable marker and transcription terminator. Ribosome binding site (Shine-
Dalgarno sequence), multiple cloning site and N- or C-terminal tags. N- or C-terminal tags offer several potential
advantages such as improved expression and solubility, improved detection and purification.
Most frequently used promoters for an E. coli expression vector:

The lac promoter: It regulates transcription of lacZ gene coding for β-galactosidase. It can be induced by
isopropylthiogalactoside (IPTG). Fusing the lac promoter sequences to target gene will result in
the lactose- (or IPTG-) dependent expression of that gene. However, the lac promoter suffers
from a number of problems. First, the lac promoter is fairly weak and, therefore, cannot drive
very high levels of protein production, and second the lac genes are transcribed to a significant
level in the absence of induction.
The trp promoter: It regulates transcription of a cluster of genes involved in tryptophan biosynthesis. It is repressed
by tryptophan and easily induced by 3-β-indoleacrylic acid.
The tac promoter: It is a hybrid of trp and lac promoter, but is stronger than either of them. It is induced by IPTG.
The λPL promoter: It is a very strong promoter responsible for transcription of λDNA molecule in E. coli. It is repressed
by a product of λcI gene called λ repressor. Expression vector with λPL promoter is used with
mutant E. coli host that synthesizes a temperature sensitive form of the λ repressor protein. At
low temperature (<30°C), this mutant λ repressor protein is able to repress the λPL promoter;
at higher temperature the protein is inactivated resulting in transcription of the cloned gene.
2.8.1 Expression system

The heterologous protein production involves suitable expression system. Prokaryotic and eukaryotic systems are
the two general categories of expression systems. There is no universal expression system for heterologous protein
production. Heterologous (meaning ‘derived from a different organism’) protein is not synthesized by the host cell.
It is a protein from a different species or different cell types. All expression systems have some advantages as well
as some disadvantages that should be considered in selecting which one to use.
Prokaryotic expression systems
E. coli is, by far, the most widely employed host, provided the post-translational modifications of the product are
not essential. It combines high growth rates along with the ability to express high levels of heterologous proteins.
Strains used for recombinant production have been genetically manipulated so that they are generally regarded as
safe for large-scale fermentation. Purification has been greatly simplified by producing recombinant fusion proteins
which can be affinity-purified, e.g. glutathione-S-transferase and maltose-binding fusion proteins. Despite the
development of advanced expression vectors, there are still numerous difficulties associated with the production
of protein from foreign genes cloned in E. coli. These problems can be grouped into two categories:
Problems due to the nature of sequence of the foreign gene:

• Presence of introns in foreign genes
• Presence of termination signals
• Codon bias:
Genes in both prokaryotes and eukaryotes show a non-random usage of synonymous codons. The systematic
analysis of codon usage patterns in E. coli led to the following observations:
1. There is a bias for one or two codons for almost all degenerate codon families.
2. Certain codons are most frequently used by all different genes irrespective of the abundance of the protein;
for example, CCG is the preferred triplet encoding proline.
3. Highly expressed genes exhibit a greater degree of codon bias than do poorly expressed ones.
4. The frequency of use of synonymous codons usually reflects the abundance of their cognate tRNAs. These
observations imply that heterologous genes enriched with codons that are rarely used by E. coli may not
be expressed efficiently in E. coli.
Problems due to the prokaryotic host, E. coli
Processing of proteins
Prokaryotes do not carry out the same kind of post-translational modifications such as glycosylation and phosphorylation
as eukaryotes do. This affects a protein’s activity or stability, or at least its response to antibodies.
Folding
In prokaryotic expression systems, most protein products of cloned eukaryotic genes become insoluble aggregates
called inclusion bodies due to incorrect folding and are very difficult to recover as functional proteins. Target proteins
expressed in E. coli may be mis-folded for a variety of reasons, including the exposure of hydrophobic residues
that are normally in the core of the protein, the lack of its normal interaction partners and inappropriate or missing
post-translational modifications.
Eukaryotic expression systems
Problems associated with obtaining active recombinant proteins from genes cloned in prokaryotic host can be
mitigated by using eukaryotic expression system. Eukaryotic systems for the expression of protein include: yeast,
mammalian cells and baculovirus cells (insect). Advantages of eukaryotic protein expression systems include very
high levels of expression. The proteins are easy to purify using special tags which are included into the vectors
including His, Myc and other tags. The disadvantages of the systems include the fact that eukaryotic cells do grow
slower than prokaryotic cells.
2.12 Genome mapping

Genome mapping is a method used to identify the locations of genetic markers (which can be genes and other
DNA sequences) and the relative distances between genetic markers on genome. There is a difference between a
genome map and a genome sequence. A genome sequence spells out the order of every nucleotide in the genome,
while a map simply identifies a series of landmarks in the genome. A genome map is less detailed than a genome
sequence. There are three kinds of maps - genetic, cytological (or cytogenetic) and physical map.
The genetic map gives the relative position of genetic markers according to the frequency of recombination,
expressed in term of centimorgans (cM). Genetic maps illustrate the order of genetic markers on a chromosome
and the relative distances between those markers.
The cytological map depicts the locations of genetic markers in a chromosome relative to visible landmarks. In
most cases, each chromosome has a characteristic banding pattern, which may be either naturally present, (e.g. in
polytene chromosomes of Drosophila) or more commonly generated by specific staining protocols (e.g. in case of
human chromosomes); the genetic markers are mapped cytologically relative to these band locations. Fluorescent
in situ hybridization (FISH) is widely used to map the cytological locations of genes and other DNA sequences
within large eukaryotic chromosomes.
The physical maps describe the absolute distance between two genetic markers in term of base pairs.
2.12.1 Genetic marker

A gene or DNA sequence having a known location on a chromosome and associated with a particular trait or gene
is used as a genetic marker. Genes were the first markers to be used to prepare the first genetic maps of fruit fly.
Genetic markers used in genetics and plant breeding can be classified into two categories: classical markers and
DNA markers.
Classical markers include morphological markers, cytological markers and biochemical markers.
Morphological markers: Morphological (or visible) markers are usually visually characterized phenotypic traits or
characters such as flower color, seed shape, growth habits or pigmentation. However, morphological markers are
very limited, and many of these markers are not associated with important economic traits (e.g. yield and quality).
These markers are also influenced by environmental factors or the developmental stages.
Cytological markers: Cytological markers are the unique structural features of chromosomes such as bands,
secondary constrictions. These chromosome features are used not only for characterization of normal chromosomes
and detection of chromosomal mutation, but also widely used in mapping and linkage group identification. However,
direct use of cytological markers has been very limited in genetic mapping.
Biochemical markers are gene products that can be detected easily by electrophoresis and specific staining. Enzyme
variants such as isozymes and allozymes are commonly used as biochemical markers. Allozymes are enzymes
encoded by different alleles of a gene but have the same catalytic activity or function. Allozymes can be separated
by electrophoresis and other separating techniques on the basis of differences in molecular size, shape and electrical
charge. Isozymes are different from allozymes. Isozymes are enzymes that perform the same catalytic function,
but are encoded by different nonallelic genes located at different loci. Allozymes reflect the products of different
alleles of a gene rather than different nonallelic genes located at different loci. Biochemical markers are also called
as protein markers. The major disadvantages of biochemical markers are that they are limited in number.
A DNA marker is defined as a particular segment of DNA that is representative of the differences at the genome
level. DNA marker is also called as molecular marker. Strictly speaking, protein markers and DNA markers are
both molecular markers, but the current uses of the term is limited to DNA markers. DNA markers should not be
considered as normal genes, as they usually do not have any biological effect, and instead can be thought of as
constant landmarks in the genome. They are identifiable DNA sequences, found at specific locations of the genome,
and transmitted by the standard laws of inheritance from one generation to the next. An ideal DNA marker should
have the following criteria:
1. High level of polymorphism,

2. Even distribution across the whole genome,
3. Provide adequate resolution of genetic differences,
4. Co-dominance in expression (so that heterozygotes can be distinguished from homozygotes),
5. Have linkage to distinct phenotypes,
6. Genome-specific in nature.
2.12.2 Types of DNA markers

Various types of DNA markers have been described in the literature. They can be broadly divided into two classes
based on the method of their detection: Hybridization-based (such as RFLP) and PCR based (such as RAPD, AFLP,
SSLP). PCR-based techniques can further be subdivided into two subcategories: arbitrarily primed PCR-based
techniques or sequence nonspecific techniques (such as RAPD, AFLP) and sequence targeted PCR-based techniques
(such as SSLP, SNP). The molecular markers can also be classified on the basis of sequence variation (e.g. RFLP)
and length variation (e.g. SSR). DNA markers may be described as codominant or dominant. This description is
based on whether markers can discriminate between homozygotes and heterozygotes. Codominant markers indicate
differences in size whereas dominant markers are either present or absent.
P1 P2 F1 P1 P2 F1
AA aa Aa BB bb Bb
(a) (b)
Figure 2.27 Comparison between (a) codominant and (b) dominant markers. Codominant markers can clearly
discriminate between homozygotes and heterozygotes whereas dominant markers do not. Genotypes at two marker
loci (A and B) are indicated below the gel diagrams.
RFLPs
RFLP (Restriction Fragment Length Polymorphisms) is the most widely used hybridization-based molecular marker.
RFLP markers were first used in 1975 to identify DNA sequence polymorphisms for genetic mapping. RFLPs arise
because mutations can create or destroy the sites recognized by specific restriction enzymes, leading to variations
between individuals in the length of restriction fragments produced from identical regions of the genome. Although
two individuals of the same species have almost identical genomes, they will always differ at a few nucleotides
due to point mutation and insertion/deletion. Some of the differences in DNA sequences at the restriction sites can
result in the gain, loss or relocation of a restriction site.
A single base change within a restriction site is a readily detectable genetic marker because the mutated site is no
longer cleaved by the enzyme in question. Two chromosomes that differ by such a mutation are then distinguishable
on the basis of a restriction fragment length polymorphism (RFLP), which arises because a particular cleavage site
is present in only one of the two DNA molecules. A mutation that gives rise to an RFLP, thus represents a genetic
marker. RFLPs have only two alleles: the site is present or absent. The maximum heterozygosity is 0.5. The RFLP
markers are codominantly inherited and highly reproducible.
Ovum Mammary gland cells

of 6-year-old ewe
Induce G0 phase
Nucleus
Enucleated
oocyte Fusion and
activation
Renucleated
oocyte
In vitro Implant
embryo
culture
Figure 2.37 Cloning sheep by nuclear transfer. The nucleus of an ovum is removed with a pipette. Cells from the
mammary epithelium of an adult are grown in culture, and the G0 state is induced by inhibiting cell growth. A G0 cell
and an enucleated ovum are fused, and the renucleated ovum is grown in culture or in ligated oviducts until an early
embryonic stage before it is implanted into a foster mother, where development proceeds to term.
2.16 Gene therapy

Gene therapy is a technique for correcting defective genes responsible for disease development. Gene therapy
typically aims to supplement a defective mutant allele with a functional one. Scientist may use one of several
approaches for correcting defective or abnormal genes:
• A normal gene may be inserted into a nonspecific location within the genome (gene addition). This is the most
common approach.
• An abnormal gene can be replaced by a normal gene through homologous recombination (gene replacement).
• An abnormal gene can be repaired through selective reverse mutation, which returns the gene to its normal
function.
Gene therapy may be germ-line or somatic cell gene therapy. Current gene therapy is exclusively somatic gene
therapy which involves the introduction of genes into somatic cells of an affected individual. Germ-line gene therapy
involves the permanent transmissible modification of the genome of a gamete, a zygote or an early embryo. The
prospect of human germline gene therapy is currently not sanctioned.
Gene therapy may be classical and nonclassical gene therapy. In classical gene therapy genes are delivered to
appropriate target cells with the aim of obtaining the optimal expression of the introduced genes. The idea of
nonclassical gene therapy is to inhibit the expression of genes associated with the pathogenesis, or to correct a
genetic defect for restoring the normal gene expression.
Potential use of somatic gene therapy

The potential use of this therapy is to cure genetic diseases. The first case of gene therapy occurred in 1990, at the
NIH in Bethesda, Maryland. On that occasion, a four-year-old patient with a severe combined immuno- deficiency
(due to adenosine deaminase enzyme deficiency) received an infusion of white blood cells that had been genetically
modified to contain the gene that was non-functional in his genome. Since then, gene therapy has been studied
and experimentally tested for several medical conditions.
DNA
mRNA
Antisense
siRNA
oligonucleotide
Ribozyme
RISC
RNase H
No protein No protein No protein

synthesis synthesis synthesis
Figure 2.42 Comparison of different antisense strategies. Antisense-oligonucleotides block translation of the mRNA
or induce its degradation by RNase H, while ribozymes possess catalytic activity and cleave their target RNA. RNA
interference approaches are performed with siRNA molecules that are bound by the RISC and induce degradation of
the target mRNA.
2.17.3 Molecular farming

It is an application of genetic engineering in which genes, primarily of human or animal origin are introduced into
plants or farm animals for cost effective production of therapeutic products such as antibodies, blood products,
cytokines, growth factors, hormones, recombinant enzymes and human and veterinary vaccines. Therapeutic
compounds so produced are also known as biopharmaceuticals (pharmaceuticals from biological organisms).
The organisms in which genes coding for the target therapeutically active compound introduced are often referred
to as expression system. Expression system studied so far include bacteria, yeast, plant viruses, animal cell culture,
transgenic plants and transgenic animals. Initially bacteria were the most widely used expression systems but
due to the complexity of the most therapeutic proteins to be produced and simplicity of the bacterial system, new
expression systems were explored. As of now the plants are the preferred and most widely used expression system
in comparison to other systems. The first recombinant pharmaceutical protein produced in the plant was human
serum albumin, first produced in 1990 in transgenic tobacco and potato plants.
Table 2.9 Examples of some pharmaceutical recombinant human proteins expressed in plant systems
Tobacco, sunflower (plants) Growth hormone
Tobacco, potato (plants) Serum albumin
Tobacco (plants) Epidermal growth factor
Rice (plants) Alpha-interferon
Tobacco (cell culture) Erythropoietin
Tobacco (plants) Haemoglobin
Tobacco (cell culture) Interleukins-2 and 4
Tobacco (root culture) Placental alkaline phosphatase

2.18 Plant tissue culture

The field of plant tissue culture is based on the fact that plants can be separated into their component parts (organs,
tissues or cells), which can be manipulated in vitro and then grown back into complete plants. Plant cells or tissues
will continue to grow if supplied with the appropriate nutrients and conditions. The culture of plant cells, tissues
and organs such as roots, shoot tips and leaves in artificial nutrient media aseptically under defined physical and
chemical conditions is referred to as plant tissue culture. ‘Tissue culture’ is commonly used as a broad term to
describe all types of plant cultures, namely callus, cell, protoplast, anther, meristem, embryo and organ cultures.
Plant cells - Unique features

A plant cell is a eukaryotic cell and shares similar features with the typical eukaryote cell. However some features
are uniquely present in plant cells. Their distinctive features include:
• A cell wall outside the cell membrane which is composed of cellulose, hemicellulose, pectin and in many cases
lignin.
• A large central vacuole enclosed by a membrane known as the tonoplast which maintains the cell’s turgor,
controls movement of molecules between the cytosol and sap, stores useful material and digests waste proteins
and organelles.
• Specialized cell-cell communication through plasmodesmata, pores in the primary cell wall through which the
plasmalemma and endoplasmic reticulum of adjacent cells are continuous.
• Plastids such as chloroplasts which contain chlorophyll for photosynthesis, amyloplasts for starch storage,
elaioplasts for fat storage and chromoplasts for the synthesis and storage of pigments.
• A specialized peroxisome called glyoxysome for the operation of glyoxylate cycle.
• Cytokinesis by formation of a phragmoplast and cell plates.
• Absence of centrioles in MTOC that are present in animal cells.
• Plant cells are totipotent, which means that, in principle, every cell contains all genetic information to grow a
new plant.
2.18.1 Cellular totipotency

Totipotency is the ability of a single cell to divide and produce all the differentiated cells in an organism. In a
multicellular organism, a cell after regulated division undergoes for cell differentiation. It is a process of specializing
cell’s functions. Isolated cells from differentiated tissues are generally non-dividing and quiescent; to show totipotency
the differentiation process has to be reversed (called de-differentiation) and repeated again (called re-differentiation).
A differentiated cell reverting to an undifferentiated state is termed de-differentiation, whereas the ability of a
dedifferentiated cell to form a whole organism or organs is termed redifferentiation. Theoretically, all living cells
can revert to an undifferential status through de-differentiation process. However, the more differentiated a cell
has been, the more difficult it will be to induce its de-differentiation. In plants, even highly mature or differentiated
cells have the ability to regress to a meristematic state as long as they are viable and show totipotency. This
phenomenon of totipotency is an amazing developmental plasticity that sets plant cells apart from most of their
animal counterparts. In animals the differentiation is irreversible.
2.18.2 Tissue culture media

The success of tissue culture depends on the composition of the growth medium and culture conditions such as
temperature, pH, light and humidity. Growth and morphogenesis of plant in vitro are largely governed by the
composition of the culture media. Media compositions are formulated considering the requirements of a particular
culture system. Culture media used for the in vitro culture of plant cells are composed of four basic components:
There is a series of distinct advantages to producing a valuable secondary product in plant cell culture, rather than
in vivo in the whole crop plant.
These include the following:

• Production can be more reliable, simpler and more predictable.
• Isolation of the phytochemicals can be rapid and efficient, when compared with extraction from complex whole
plants.
• Interfering compounds that occur in the field-grown plant can be avoided in cell cultures.
• Tissue and cell cultures can yield a source of defined standard phytochemicals in large volumes.
Table 2.10 List of industrially important plant secondary metabolites produced through tissue culture
Product Plant source Uses
Azadirachtin Azadirachta indica Insecticidal
Berberine Coptis japonica Antibacterial
Digoxin Digitalis lanata Cardiac medicine
Diosgenin Dioscorea deltoidea Antifertility
Taxol Taxus baccata Anticarcinogenic
Codeine Papaver sp. Analgesic
2.19 Animal cell culture

Cells in animals exist in an organized tissue matrix which require for their controlled growth and differentiation.
These cells from intact organisms may be isolated, maintained and grown in vitro in culture media aseptically
containing a suitable mixture of nutrients and growth factors. This process is called animal cell culture.
2.19.1 Primary cultures

A primary cell culture is prepared by inoculating cells directly from tissues of an organism into culture media (that
is, without cell proliferation in vitro). With the exception of some cells derived from tumors, most primary cell
cultures have a limited lifespan. After a certain number of divisions, cells undergo the process of senescence and
stop dividing. In these cells, the limited proliferation capacity reflects a progressive shortening of the cell’s telomeres,
the repetitive DNA sequences and associated proteins that cap the ends of each chromosome.
The primary cell culture is of two types depending on the kind of cells in culture – attachment culture and suspension
culture. Attachment culture involves the adherent or anchorage dependent cells. To survive and grow, most cells
require a surface to which they can attach, thus they are anchorage dependent. Without the surface attachment,
these cells cannot survive. These adherent cells are usually derived from tissues of organs such as kidney, where
they are immobile and embedded in connective tissue. Suspension culture involves non-adherent or anchorage
independent cells which do not require attachment for growth or do not attach to the surface of the culture vessels. It
is a culture in which cells will multiply when suspended in growth medium. Lymphocytes are anchorage independent
cells commonly grown in suspension culture.
2.19.2 Cell line

When a primary cell culture is subcultured, it becomes a cell line. It is a propagated culture after the first subculture.
Subculture (or passage) refers to the transfer of cells from one culture vessel to another. The cell lines may be finite
cell line or infinite cell line. A finite cell line (or normal cell line) is a line of cells that will undergo only a finite number
of divisions in cell culture and eventually undergoes senescence. It has a limited number of possible subcultures or
Chapter 03
Plant Physiology
3.1 Plant-water relationship

Water is essential for life. The most abundant substance of the living cell is water. It accounts for about 70% of
a cell’s weight. It is essential for all physiological activities of the plant. It provides the medium in which most
substances remain dissolved. Water (H2O) is made up of two hydrogen atoms and one oxygen atom, with a total
atomic mass of 18 daltons. It is a polar molecule. Although water is electrically neutral, it has a partial positive
charge on each hydrogen and a partial negative charge on oxygen.
Water acts as an excellent solvent. It dissolves more substances than any other liquid. This is because it has very
high value of dielectric constant, which is a measure of the capacity to neutralize the attraction between electrical
charges. Because of this property, water is an especially powerful solvent for electrolytes and polar molecules such
as sugars.
Water has a high specific heat (the amount of energy required to raise the temperature of a unit mass of a
substance by 1°C is called its specific heat). The high specific heat of liquid water is caused by the arrangement
of its molecules, which allows the hydrogen and oxygen atoms to vibrate freely, almost as if they were free ions.
Thus, they can absorb large quantities of energy without much temperature increase. That’s why plants can resist
large fluctuations in temperature.
Water has a high heat of vaporization (the energy necessary to go from a liquid to a gas). 586 cal are required to
convert 1 g of water at 20°C to 1 g of water vapour at 20°C. Thus, evaporation from leaves cools the plant.
The extensive hydrogen bonding in water gives rise to the property known as cohesion. Cohesion gives water a
high tensile strength which is the ability to resist stretching (tension) without breaking. Cohesion among water
molecules also accounts for surface tension.
3.1.1 Diffusion and osmosis

Diffusion is the random movement of molecules along the concentration gradient (from an area of higher concentration
to an area of lower concentration) by their own kinetic energy. It is a spontaneous and passive process. The rate
of diffusion depends on several factors such as concentration difference, size of molecules and temperature. The
rate of diffusion of molecules down a concentration gradient is given by the Fick’s law:
æ DC ö
J = -D ç ÷
è Dx ø
Where J is the flux per unit area, D is the diffusion coefficient (usually expressed as cm2/sec) and ΔC is the difference
in concentration between two regions separated by a distance Δx. The negative sign accounts for the fact that
diffusion is toward the lower concentration.
330 Plant Physiology
Osmosis is a specialized case of diffusion that involves the passive transport of water (i.e. solvent). In osmosis,
water moves through a semipermeable membrane from a region of its higher concentration to a region of its lower
concentration. Semipermeable membrane selectively allows the passage of a solvent while restricting the movement
of solutes. Plasma membranes of plant cells are selectively permeable not semipermeable membrane because they
allow the movement of solvent (water) as well as solutes. The selective permeability is due to the presence of the
discriminating barrier of the lipid bilayer and the specific transport proteins.
Semipermbeable
membrane
Dilute solution Concentrated solution

Low solute concentration High solute concentration
High solvent concentration Low solvent concentration
Direction of flow of water (solvent)
Figure 3.1 Osmosis is the diffusion of solvent (water) across a semipermeable membrane.
In osmosis, selective diffusion of solvent is driven by the internal energy of the solvent molecules. It is convenient
to express the available energy per unit volume in terms of osmotic pressure. It is defined as the pressure required
to completely stop the entry of water into an osmotically active solution across a semipermeable membrane. It is
also defined as the minimum pressure needed to stop osmosis. It is measured in atmospheres or bars (1 bar equal
to 100 kilopascals). The osmotic pressure is directly proportional to the difference in the concentration of the total
number of solute molecules on each side of the membrane.
Water diffuses across

Semipermbeable membrane the membrane and raises
Osmotic pressure
the level of solution B
required to prevent
net water flow
Low solute High solute

concentration concentration
CA CB
Solution A Solution B Solution A Solution B Solution A Solution B
Direction of flow of water No flow of water
Figure 3.2 Osmotic pressure. Solutions A and B are separated by a semipermeable membrane. If the total concen-
tration of solutes in solution B is greater than solution A, water will tend to flow across the membrane from solution A
to solution B. The osmotic pressure between the solutions is the minimum pressure that would have to be applied to
solution B to stop osmosis. From the van’t Hoff equation, osmotic pressure is given by π = RT(CB–CA), where R is the
gas constant and T is the absolute temperature.
Tonicity
Tonicity is the measure of the osmotic pressure gradient of two solutions separated by a semipermeable membrane.
There are three types of tonicity that one solution can have relative to another: hypertonic, hypotonic and isotonic.
Plant Physiology 335
Root Root hair Soil particle
Figure 3.4 Root hairs make intimate contact with soil particles and greatly amplify the surface area that can be used
for water absorption by the plant. The soil is a mixture of particles, water, dissolved solutes and air. Water is adsorbed
to the surface of the soil particles. As water is absorbed by the plant, the soil solution recedes into smaller pockets,
channels, and crevices between the soil particles.
3.2.2 Soil water

Soil is a complex medium. It is a mixture of inorganic and organic materials, water, dissolved solutes and air. The
water content in soil depends on soil type. The water-holding capacity of soil is described in terms of field capacity.
It represents the maximum amount of water that soil can hold after allowing free drainage. Clay soil or soil with a
high humus content have a large field capacity.
The water stored in the soil is taken up by the plant roots or evaporated from the topsoil into the atmosphere. If
no additional water is supplied to the soil, it gradually dries out. The dryer the soil becomes, the more tightly the
remaining water is retained and the more difficult it is for the plant roots to extract it. At a certain stage, the uptake
of water is not sufficient to meet the plant's needs. The amount of water soil contains at which plants wilt beyond
recovery is called the permanent wilting percentage. The amount of water held by the soil between field capacity
and the permanent wilting point is the plant- available water or water that is available for uptake by plants.
3.2.3 Radial movement of water from root surface to the tracheary element
Once water has been absorbed into the root hairs or epidermal cells, it must traverse the cortex in order to reach
the xylem elements. A root in cross-section would have an epidermis, cortex, endodermis, pericycle, xylem and
phloem. Transpiration develops hydrostatic tension in the xylem of the root which draws water from the soil through
the intervening tissues of the cortex. The path through which water moves into xylem cells of roots occurs through
both apoplast (the continuous system of cell walls and intercellular spaces) and symplast (the continuous system
of cell protoplasts interconnected by plasmodesmata).
Apoplastic movement
Apoplastic pathway essentially involves diffusion and bulk flow of water through intercellular spaces between
cells and the walls of the cells. It is always extended from root hairs or other epidermal cells to the endodermis,
where the waterproof band of suberin on the radial walls (called the casparian strip) forces substances to enter
endodermal cells across their plasma membrane. Suberin acts as a barrier to water and solute movement. The
endodermis, the innermost layer of cells in the root cortex, surrounds the stele. From endodermal cell movements
occur through the symplast. Apoplastic pathway is the major pathway for the transport of water from the epidermis
to a tracheary element of root.
Symplastic movement
The symplastic system is the system of interconnected protoplasts. In this system, cytoplasm of neighbouring cells
are connected through plasmodesmata. In symplastic movement, the water travels through the cells. It occurs from
the root hair cell to the endodermis and across it all the way to dead xylem cells. Water enters the outermost cells
through the cell membrane and then moves from cell to cell through the plasmodesmata. Symplastic movement
is relatively slower and may be aided by cytoplasmic streaming.
Most of the water flows radially in the roots through the apoplast since the cortical cells are loosely packed, and hence
offer no resistance to water movement. However, the inner boundary of the cortex, the endodermis, is impervious
to water because of the presence of casparian strip. Water molecules are unable to penetrate the layer, so they are
directed to enter the cells. The water then moves through the symplast and again crosses a membrane to reach
the cells of the xylem. The radial movement of water through the root is ultimately symplastic in the endodermis.
This is the only way water and other solutes can enter the vascular cylinder. Once inside the xylem, water is again
free to move between cells as well as through them.
The apoplast is
the continuum
of cell walls and Apoplast
extracellular
spaces
Vacuole
The symplast is
the continuum of
Symplast
cytosol connected
by plasmodesmata
Casparian strip
Epidermis Cortex Endodermis Stele
Figure 3.5 The radial path of water movement through a root.
3.2.4 Root pressure

If the stem of a well-watered herbaceous plant is cut off just above the soil, xylem sap will exude from the cut
surface. Exudation of sap indicates the presence of a positive pressure in the xylem. The magnitude of this pressure
can be measured by attaching a manometer to the cut surface. This pressure is known as root pressure (term coined
by Stephen Hales) because the force that gives rise to the exudation originate in the root. The root pressure can
be as high as 0.5 MPa. Roots generate positive hydrostatic pressure by absorbing ions from the dilute soil solution
and transporting them into the xylem. The buildup of solutes in the xylem sap leads to a decrease in the xylem
water potential. This lowering of the xylem water potential provides a driving force for water absorption, which in
turn leads to a positive hydrostatic pressure in the xylem.
Root pressure is most likely to occur when soil water potentials are high and transpiration rates are low. When
transpiration rates are high, water is taken up so rapidly into the leaves and lost to the atmosphere that a positive
pressure never develops in the xylem.
3.3 Ascent of sap

Water and minerals from the soil enter the plant through the epidermis of roots, radially cross the root cortex, and
pass into the xylem. From there the xylem sap, the water and dissolved minerals in the xylem, moves upward. It
is vitally important for a plant to transport water and minerals from the soil to its uppermost leaves. The upward
movement of minerals and water against gravitational force from root to aerial parts of the plant through xylem
is called as ascent of sap.
3.3.1 Xylem anatomy

Xylem is a complex tissue consisting of living and non-living cells. The conducting cells in the xylem are typically
non-living and include two types of tracheary elements – tracheids and vessel elements. Both of these cell types
have thick, lignified secondary cell walls and are dead at maturity. Vessel elements are present only in angiosperm
and small group of gymnosperms. In addition to tracheary elements, the xylem tissue also contains parenchyma
cells (storage function) and fibers (mechanical function).
Tracheids are elongated and spindle-shaped cells. The walls of these cells are heavily lignified with openings in the
walls called pits. Water flows between tracheids by means of numerous pits in their lateral walls. Pits are microscopic
regions where the secondary wall is absent and the primary wall is thin. Pits of one tracheid are typically located
opposite to the pits of an adjoining tracheid forming pit pairs. The porous layer between pit pairs, consisting of
two primary walls and a middle lamella, is called the pit membrane. Vessel elements are shorter and wider than
tracheids and have perforated end walls that form a perforation plate at each end of the cell. Like tracheids,
vessel elements have pits on their lateral walls. The perforated end walls allow vessel members to be stacked end
to end to form a larger conduit called a vessel. Because of their open end walls, vessels provide a very efficient
low-resistance pathway for water movement. In rooted plants, transport of water and minerals through xylem is
essentially unidirectional, from roots to the stems.
3.3.2 Mechanism of ascent of sap

Unlike animals, plants do not have a heart or a circulatory system to move water from the soil to the leaves. Despite
this, the upward flow of water through the xylem occurs at fairly high rates. How is this movement accomplished?
A longstanding question is whether water is pushed or pulled through the plant.
As we mentioned previously some roots develop root pressure, a positive hydrostatic pressure in their xylem.
However, root pressure is typically less than 0.1 MPa and disappears when the transpiration rate is high, so it is
clearly inadequate to move water up a tall tree. It is also not a universal phenomenon. Instead, water at the top
of a tree develops a large tension (a negative hydrostatic pressure), which pulls water through the xylem.
Most researchers agree that water is mainly pulled through the plant, and that the driving force for this process is
transpiration from the leaves. Transpiration generates a negative pressure in the xylem of leaves, which pulls the
water upward. The most accepted theory for upward movement of water in the xylem is the cohesion-tension
theory (also known as transpiration pull). This theory was proposed by Dixon and Jolly. Some physical properties
of water support the formation of water column in xylem vessel and their upward movement are described below:
1. Cohesion: it is mutual attraction between water molecules.
2. Adhesion: it is attraction of molecules to the hydrophilic walls of tracheary elements.
3. Surface tension: water molecules are attracted to each other in the liquid phase more than to water in the gas
phase.
These properties give water high tensile strength (i.e. an ability to resist a pulling force) and high capillarity (i.e. the
ability to rise in a thin tube). In plants, capillarity is aided by the small diameter of the tracheary elements - the
tracheids and vessel elements.
Due to the fact that transpiration ‘pulls’ the sap from the soil to the leaves, water in the xylem is in a state of tension.
In this state, negative pressures in the xylem water may cause cavitation (embolisms) in the xylem. Cavitation is
the appearance of a gas bubble within the liquid phase. It is more common in wide vessels than in tracheids and
can occur during drought stress or when xylem sap freezes in winter. Such cavitation can block water transport
and lead to severe water deficits in the leaf. However, the impact of xylem cavitation on the plant is minimized by
several means. The principal mechanism for minimizing the effect of cavitation is a structural one. The end walls
of vessels and the pores prevent the bubble from spreading from tube to tube.
3.6.4 Biological nitrogen fixation

Nitrogen is present in many forms in the nature. The atmosphere contains about 78% (by volume) molecular
nitrogen. Acquisition of nitrogen from the atmosphere requires the breaking of an exceptionally stable triple
covalent bond between two nitrogen atoms to produce ammonia (NH3) or nitrate (NO3–). Conversion of molecular
nitrogen to nitrate or ammonia is termed as nitrogen fixation, which can be accomplished by both industrial and
natural processes. Natural processes of nitrogen fixation are of two types – Biological nitrogen fixation and Non-
biological nitrogen fixation (including nitrogen fixation by lightning and photochemical reactions). Approximately
90% of nitrogen fixation is biological nitrogen fixation, in which prokaryotic organisms fix molecular nitrogen into
ammonia. It is a reductive biosynthetic process. Few prokaryotic organisms (termed as nitrogen fixing organisms
or diazotroph) are capable of biological nitrogen fixation only. Eukaryotic organisms are unable to fix nitrogen.
The biological reaction of nitrogen fixation generates at least one mole of H2 in addition to two moles of NH3 for
each mole of nitrogen molecule. Hence, total eight electrons are required in reduction of one mole of nitrogen to
two moles of NH3.
— +
8e + 8H
N2 2NH3 + H2
However, the actual reduction of nitrogen molecule without formation of hydrogen molecule requires only six electrons:
N N + —
NH NH + —
H2N NH2 + —
2NH3
2H + 2e 2H + 2e 2H + 2e
The biological process of nitrogen fixation is catalyzed by an enzyme complex called nitrogenase complex. There
are three different forms of nitrogenase that differ in their requirement for molybdenum, vanadium, or iron as a
critical metallic component. Most of the nitrogenases that have been studied contain a Mo cofactor. Nitrogenase
consists of two proteins: a dinitrogenase reductase and dinitrogenase. The dinitrogenase reductase (also called the
Fe protein) is a dimer of identical 30 kDa subunits bridged by a 4Fe-4S cluster. Dinitrogenase is a tetramer with
two copies of two different subunits. It contains both Fe and Mo. Because molybdenum is present in this cluster,
the dinitrogenase component is also called the molybdenum-iron protein (MoFe protein). The MoFe cofactor is the
site of nitrogen fixation. The genes involved collectively in the synthesis of nitrogenase and the catalytic process of
N2 fixation are called nif genes. Accessory genes are called fix genes, and they are also necessary for the function
and regulation of nitrogenase in aerobic nitrogen-fixing bacteria.
Since nitrogen fixation is a reductive process so it requires electron donor. In most nitrogen-fixing microorganisms,
reduced ferredoxin, acts as a donor for electrons. Ferredoxin is a small (14 to 24 kDa) protein containing an Fe-S
group. At least 16 molecules of ATP are required for reduction of one molecule of N2.
16 ATP
Dinitrogenase Dinitrogenase
8 Ferredoxin reductase reductase Dinitrogenase
Reduced N2
Oxidized Reduced Oxidized +
— — — 2H
8e 8e 8e
H2
+
2NH4
Dinitrogenase Dinitrogenase
8 Ferredoxin Dinitrogenase
reductase reductase
Reduced Oxidized 16 ADP Oxidized Reduced
Figure 3.12 Nitrogen fixation by nitrogenase complex.
The nitrogenase complex is very sensitive to oxygen. It is irreversibly inactivated by oxygen. Hence, the fixation of
N2 must occur under anaerobic conditions. For anaerobic prokaryotic organisms, there is no problem. Facultative
prokaryotic organisms such as purple photosynthetic bacteria fix N2 only in anaerobic conditions. In aerobic organism
such as cyanobacteria, anaerobic conditions are created in specialized cells called heterocysts. Heterocysts are
thick-walled cells which lack photosystem II, the oxygen-producing photosystem of chloroplasts, so they do not
generate oxygen. Heterocysts appear to represent an adaptation for nitrogen fixation. Cyanobacteria that lack
heterocysts can fix nitrogen only under anaerobic conditions. Symbiotic nitrogen-fixing prokaryotes such as
Rhizobium, maintain a very low concentration of free oxygen in root nodules of leguminous plants by producing
leghemoglobin, a homolog of hemoglobin. Leghemoglobin is present in the cytoplasm of infected nodule cells at
high concentrations.
Nitrogen fixing prokaryotes

Nitrogen-fixing prokaryotes can be divided into those that carry out biological nitrogen fixation in a symbiotic
relationship with a eukaryote and those that fix nitrogen in a free-living state. Most of the nitrogen-fixing prokaryotes
are free-living in the soil.
Free-living nitrogen-fixing prokaryotes: Aerobic nitrogen-fixing bacteria such as Azospirillum and Azotobacter.
Anaerobic nitrogen-fixing bacteria such as Rhodospirillum (photosynthetic) and Clostridium (non-photosynthetic).
Symbiotic nitrogen-fixing prokaryotes: Symbiotic nitrogen-fixing prokaryotes may or may not form nodules.
Nodule forming symbiotic nitrogen-fixing prokaryotes: The most common form of symbiotic association results
in the formation of enlarged and multicellular structures called nodules on the root (or occasionally the stem) of
the host plant. In the case of Leguminous plants (such as peas, beans, clover, alfalfa and soybean), the symbiont
is a bacterium of genera Rhizobium, Bradyrhizobium and Azorhizobium. Although most leguminous plants form
nodules on their roots, a few leguminous plants bear nodules on their stems. One common example of a stem-
nodulated leguminous plant is Sesbania. Nodule formation also occurs in certain non-leguminous plants such as
Alnus, Myrica and Casuarina. The symbiont in these non-leguminous nodules is actinomycetes of genus Frankia.
Both Rhizobium and Frankia live freely in the soil, but fix nitrogen only when in symbiotic association with an
appropriate host plant.
Non-nodule forming symbiotic nitrogen-fixing prokaryotes: Some symbiotic associations do not form nodules. For
example, anabaena azollae, a cyanobacterium, lives in pores on the fronds of a water fern called Azolla.
Table 3.3 Examples of nitrogen fixing bacterial and archaeal genus and their lifestyle
Genus Phylogenetic affiliation Lifestyle
Nostoc, Anabaena Bacteria Free-living, aerobic, photolithotrophic
Pseudomonas, Azotobacter Bacteria Free-living, aerobic, chemotroph
Thiobacillus Bacteria Free-living, aerobic, chemotroph
Methanococcus Archaea Free-living, anaerobic, chemotroph
Chromatium, Chlorobium Bacteria Free-living, anaerobic, phototroph
Desulfovibrio, Clostridium Bacteria Free-living, anaerobic, chemotroph
Rhizobium, Frankia Bacteria Symbiotic, aerobic, chemotroph
Nodule formation in leguminous plants by Rhizobium species

Rhizobium is gram-negative motile rod-shaped proteobacteria that can grow free-living in soil or can infect leguminous
plants and establish a symbiotic existence. Infection of the roots of a legume with species of Rhizobium genera
leads to the formation of root nodules.
Chemotactic movement of free-living soil bacteria towards roots is mediated by chemicals such as flavonoids,
homoserine, secreted by the roots. A specific adhesion protein called rhicadhesin is present on the surfaces of
Rhizobium species. Rhicadhesin, a calcium-binding protein, plays role in plant-bacterium attachment. Root exudates
also induce the nodulation (nod) genes responsible for nodule formation. Bacteria secrete NodD, which is a protein
that recognizes the flavonoids secreted by the plants. Interaction with the flavonoids activates NodD, then NodD
returns to the rhizobium to induce the transcription of nodABC genes as well as many other nod genes.
The nod genes are classified as common nod genes or host-specific nod genes. The common nod genes – nodA, nodB
and nodC - are found in all rhizobial strains; the host-specific nod genes-such as nodP, nodQ and nodH; or nodF,
nodE and nodL-differ among rhizobial species and determine the host range. Three of the nod genes (nodA, nodB
and nodC) encode enzymes that are required for synthesis of nodulation factors or nod factors. Nod factors are
lipo-chitooligosaccharide, the derivatives of chitin. Nod factors of all studied rhizobia are composed of a β-1,4-linked
N-acetyl-D-glucosamine. The non-reducing terminal sugar moiety is substituted with a fatty acid whose structure
varies between different rhizobial species. NodA (product of gene nodA) is an N-acyltransferase that catalyzes the
addition of a fatty acyl chain. NodB (product of gene nodB) is a chitin-oligosaccharide deacetylase that removes
the acetyl group from the terminal nonreducing sugar. NodC (product of gene nodC) is a chitin-oligosaccharide
synthase that links N-acetyl-D-glucosamine monomers.
Nod factors induce root hair curling and trigger plant cell division. The process of nodule development begins with
the infection of a root hair by a Rhizobium bacterium. During the infection process, bacteria that are attached to
the root hairs release Nod factors that induce curling of the root hair cells and also sends mitogenic signals that
stimulate cell division in cell cortex. Cell divisions lead to formation of root nodule. Nod factors also induce several
nodule-specific plant genes called nodulin genes. During development of the root nodules the nodulin genes are
differentially expressed.
1 Attachment of the Rhizobia 2 Excretion of Nod factors by

to root hairs in response to the bacterium. Nod factor
chemical attractants released promotes root hair curling
by plant. and plant cortical cell division.
3 Formation of the infection thread 4 The infection thread extends and

from Golgi secretory vesicles of root branches until it reaches target cells,
cells. The infection thread reaches where vesicles composed of plant
the end of the cell, and its membrane membrane that enclose bacterial cells
fuses with the plasma membrane are released into the cytosol.
of the root hair cell.
Figure 3.13 The infection process during nodule formation.

a. Absence of ethylene b. Presence of ethylene
Copper atoms
Ethylene
ER lumen Active Inactive

ethylene receptor ethylene receptor
ER membrane
Cytosol Activated histidine Inactive histidine

kinase domain kinase domain
CTR1 Active CTR1 Inactive
Polyubiquitination and EIN3

Transcription factor EIN3
Proteasomal degradation
Ethylene responsive Ethylene responsive

genes off genes on
Figure 3.25 A current view of the ethylene signaling pathway. (a) In the absence of ethylene, both the receptors
and CTR1 are active, causing the ubiquitylation and destruction of the EIN3 protein, the gene regulatory protein in the
nucleus that is responsible for the transcription of ethylene-responsive genes. (b) The binding of ethylene inactivates
the receptors and disrupts the interaction between the receptors and CTR1. The EIN3 protein is not degraded and can
therefore activate the transcription of ethylene responsive genes.
3.9 Photomorphogenesis
Light has profound effects on the growth and development of plants. Light is vital for photosynthesis, but is also
necessary for plant growth and development. It can be observed in seedlings grown in the dark. Seedlings grown
in the dark have a pale, unusually tall and spindly appearance known as etiolated growth. The light-mediated
changes in plant growth and development, independent of photosynthesis, are called photomorphogenesis (from
the Latin word meaning light from begins). Light acts as a signal to initiate and regulate photomorphogenesis.
There are three major photoreceptors involved in these responses - phytochrome, cryptochrome and phototropin.
3.9.1 Phytochrome
Phytochrome is a photomorphogenetic pigment that absorbs red and far-red light and causes photomorphogenesis.
It also absorbs blue light. Phytochromes have been found in most plants where it regulates many growth and
developmental processes such as photoperiodic induction of flowering, chloroplast development (not including
chlorophyll synthesis), leaf senescence, leaf abscission, seed germination, stem elongation etc. Phytochrome is
a family of chromoproteins with a small covalently-bound pigment molecule. Phytochrome proteins occur as a
dimer of two ~125 kDa polypeptides, each with a covalently-attached pigment molecule. The pigment is called
the chromophore. It is a linear tetrapyrrole termed phytochromobilin and similar in structure to mammalian
bile pigments, bilirubin. Phytochromobilin is synthesized in the plastids and its precursor is δ-aminolevulinic acid.
Together, the apoprotein (polypeptide chain) and its chromophore make up the holoprotein. Assembly of apoprotein
with its chromophore is autocatalytic and occurs spontaneously.
in the companion cells of the phloem of leaves and stems during the light period. The product of CONSTANS
gene, CO protein, activates the transcription of FT gene. FT protein moves from the leaves to the apical meristem.
Once in the apical meristem, the FT protein enters the nucleus and forms a complex with FLOWERING D (FD), a
basic leucine zipper (bZIP) transcription factor that is expressed in the meristem. The complex of FT and FD then
activates floral meristem identity genes. Since the 1930s, there have been many unsuccessful attempts to isolate
and characterize. Thus florigen remained a physiological concept rather than a chemical entity. However, the FT
protein exhibits all the properties that would be expected of florigen.
3.10 Vernalization
Plants have evolved the ability to alter their developmental programme in response to environmental stimuli. A
major switch in the developmental programme is the transition to flowering. In many plant species, the timing of
this transition is determined by seasonal changes that are sensed by the plant. Photoperiod and temperature are two
of the main environmental cues that plants monitor to determine the correct time to flower. Vernalization describes
the promotion of flowering after exposure to cold (0-5°C). Vernalization (term vernalization is derived from the
Latin word vernus, meaning of the spring) results in the acquisition of competence to flower. After vernalization,
plants do not necessarily initiate flowering, but acquire the competence to do so. It is reversible and can be lost
as a result of exposure to devernalizing conditions, such as high temperature. The vernalized state can also be
maintained through tissue culture.
Studies involving grafting and localized cooling have shown that the apical meristem is the site of cold perception
during vernalization, and that vernalization causes the meristem to become competent to flower. Thus, dividing
cells (or perhaps cells in which DNA replication is occurring) are a prerequisite for vernalization. After grafting
experiment in case of henbane plant, G. Melcher postulated the existence of a hypothetical compound vernalin for
vernalization stimulus. When Melcher grafted a vernalized henbane plant to another non-vernalized plant that has
never experienced low temperature, it flowered. It suggests that some substance found in the vernalized plant is
transported to non-vernalized plant and that is responsible for the induction of flowering in the latter plant. The
substance is now termed as vernalin. Attempts to isolate and identify vernalin have failed. Whether the vernalin
is same as florigen or a precursor of florigen is not known. Later, Anton Lang found that gibberellins applied to
certain biennials induced them to flower without a low temperature treatment. It means gibberellins can substitute
vernalization.
Mechanism of floral induction in vernalized plants

Genetic and physiological studies of the vernalization pathway in Arabidopsis have identified some of the genes that
are involved in this process. Vernalization involves epigenetic changes in the expression of gene, FLC (flowering
locus C). FLC encodes a MADS-box transcription factor that represses flowering. It is expressed predominantly in
mitotically active regions. In Arabidopsis, FLC works by directly repressing the expression of FT gene in leaves and
SOC1 and FD genes at the shoot apical meristem.
In response to vernalization, the amount of FLC mRNA and protein is reduced. The reduction in FLC expression by
vernalization involves chromatin remodeling of FLC that requires the VIN3 (vernalization insensitive 3) protein. The
products of two other genes, VRN1 (VERNALIZATION1) and VRN2, are also responsible for repression of FLC gene
but in a very different manner. Its products are needed for maintenance but not for initiation of FLC silencing. In
this way vernalization promotes flowering by reducing FLC expression.
3.11 Flowering genes

Genetic analysis have identified two classes of genes that regulate floral development: floral meristem identity
and floral organ identity genes. The transition from shoot vegetative meristem to floral meristem requires floral
meristem identity genes that both specify the floral organs and cause the termination of the production of stem
cells. This group of genes includes LEAFY (LFY), APETALA1 (AP1) and CAULIFLOWER (CAL), which are expressed
Pre-chilling: Low temperature can release fully hydrated seeds from dormancy. Some seeds can be induced to
germinate by a period of pre-chilling (stratification). It requires a period of low temperature (0–10°C) in order to
germinate.
Seed germination
By definition, seed germination incorporates those events that commence with the uptake of water by the quiescent
dry seed and terminate with the elongation of the embryonic axis. It is the resumption of growth of the embryo
of the mature seed. Seed germination depends on both internal and external conditions. The most important
external factors include temperature, water, oxygen and sometimes light or darkness. Germination of many seeds
is influenced by light. Seeds that are stimulated to germinate by light are described as positively photoblastic;
seeds whose germination is inhibited by light are said to be negatively photoblastic. The germination of positively
photoblastic seeds (such as lettuce, Lactuca sativa, seed) is regulated by phytochrome. Red light irradiation
induces the germination of lettuce seeds, and far-red irradiation given after red light cancels the effect of red light.
Phytochrome regulates lettuce seed germination via the control of the endogenous level of gibberellin.
Based on the fate of the cotyledons, two kinds of seed germination occur – epigeal and hypogeal germination.
Epigeal germination is characteristic of bean and pine seeds and is considered evolutionarily more primitive than
hypogeal germination. During germination, the cotyledons are raised above the ground where they continue to
provide nutritive support to the growing points.
Hypogeal germination is characteristic of pea seeds and all grasses. During germination, the cotyledons or
comparable storage organs remain beneath the soil while the plumule pushes upward and emerges above the
ground. In hypogeal germination, the epicotyl is the rapidly elongating structure.
3.14 Plant development

Angiosperms (flowering plants) are the most advanced terrestrial plants. The development of a flowering plant, like
that of an animal, begins with the division of a fertilized egg to form an embryo with a polarized organization: the
apical part of the embryo will form the shoot, the basal part, the root, and the middle part, the stem.
Sexual life cycle of angiosperms

In flowering plants, the reproductive organs are in the flower. Meiosis and fertilization are two essential processes in
the sexual cycle of flowering plants. Sexual reproduction consists of two generations — sporophytic and gametophytic.
Gametophyte is characterized by nutritionally independent extremely short haploid phase. The sporophytic generation
begins when an egg nucleus unites with a sperm nucleus, producing an embryo, and the second sperm nucleus
fuses with the polar nuclei (secondary nucleus) producing triploid endosperm and continues with the development
of seed, seedling, mature plant and flowers. Sporophytic tissues contain a diploid chromosome number. The flower
contains spore-forming organs called anthers and ovaries. Anthers and the ovaries produce haploid (n) microspores
and megaspores, respectively. Thus, alternation of sporophytic and gametophytic generations occurs during the
life cycle of flowering plants and this form of life cycle is termed as haplodiplontic life cycle.
Sporophyte generation
Sporophyte bears spore-producing organs and produces spores by the process of sporogenesis (micro-and
megasporogenesis).
Microsporogenesis is the formation of the microspores. The microspores give rise to the male gametophyte. The
anther of the microsporophyll or stamen bears the microsporangia or pollen sacs, the function of which is to produce
the microspores or pollen grains.
Anther is the fertile portion of the stamen. A typical anther is tetrasporangiate type. Each anther lobe has two
microsporangia (pollen sac). In each lobe, some hypodermal cells become more prominent and constitute the
Chapter 04
Human Physiology
Like all multicellular animals, human body is composed of different types of cells. Groups of cells similar in structure
and function are organized into tissues. Different tissues grouped together into a structural and functional unit called
organs. An organ system is a group of organs that function together to carry out the principal activities of the body.
4.1 Tissues
A tissue is a group of similar cells that usually have a common embryonic origin and functions together to carry out
specialized activities. On the basis of structure and function, animal tissues can be classified into four basic types:
1. Epithelial tissue
2. Connective tissue
3. Nervous tissue
4. Muscular tissue
1. Epithelial tissue
An epithelial tissue or epithelium consists of cells that form membranes, which cover and line the body surfaces
and glands, which are derived from these membranes. Epithelial cells arranged in continuous sheets, in either
single or multiple layers. Because the cells are closely packed and are held tightly together by many cell junctions,
there is little intercellular space between cells. Three types of cell junctions are found in the epithelium and other
tissues. These cell junctions are called as tight, anchoring (adherens junction and desmosome) and gap junctions.
Epithelial tissue has its own nerve supply, but is avascular; that is, it lacks its own blood supply. The blood vessels
that bring in nutrients and remove wastes are located in the adjacent connective tissue. Exchange of substances
between epithelium and connective tissue occurs by diffusion. Epithelial tissue plays many roles such as protection,
filtration, secretion, absorption and excretion. Because epithelial tissue subjected to wear and tear and injury, it
has high capacity for renewal.
Epithelial tissue may be divided into two types:

A. Covering and lining epithelium forms the outer covering of the skin and some internal organs. It also forms
the inner lining of blood vessels, ducts and body cavities, and the interior of the respiratory, digestive, urinary
and reproductive systems.
B. Glandular epithelium makes up the secreting portion of glands such as the thyroid gland, adrenal glands and
sweat glands.
A. Covering and lining epithelium
The covering and lining epithelial tissue is further classified according to the two characteristics like the arrangement
of cells into layers and the shapes of the cells.
412 Human Physiology
According to the arrangement of cells into layers
The cells are arranged in one or more layers depending on the functions. It may be:
Simple epithelium
It is a single layer of cells.
Pseudostratified epithelium
It is a single layer of cells but appears to have multiple layers of cells because the cell nuclei lie at different levels
and not all cells reach the apical surface.
Stratified epithelium
It consists of two or more layers of cells.
According to the shapes of the cells

The shapes of the cells may be: squamous, cuboidal and columnar.
Squamous cells are arranged like floor tiles and are thin.
Cuboidal cells are as tall as they are wide and are shaped like cubes or hexagons.
Columnar cells are much taller than they are wide, like columns.
Combining the both characteristics i.e. arrangement of cells into layers and cell shapes, the covering and lining
epithelia are of following types:
Simple epithelium
a. Simple squamous epithelium

Description: Single layer of flat cells; centrally located nucleus.
Location: Lines the heart, blood vessels, lymphatic vessels, air sacs of lungs, Bowman’s capsule of kidneys and
inner surface of the tympanic membrane (eardrum); forms epithelial layer of serous membranes, such as the
peritoneum.
Function: Filtration, diffusion, osmosis and secretion in serous membranes.
b. Simple cuboidal epithelium

Description: Single layer of cube-shaped cells; centrally located nucleus.
Location: Covers the surface of ovary, lines the anterior surface of the capsule of the lens of the eye, forms the
pigmented epithelium at the posterior surface of the eye, lines the kidney tubules and smaller ducts of many
glands, and makes up the secreting portion of some glands such as the thyroid gland and the ducts of some
glands such as the pancreas.
Function: Secretion and absorption.
Basement
membrane
Simple squamous epithelium
Nucleus
Simple cuboidal epithelium
c. Simple columnar epithelium (nonciliated and ciliated)

Nonciliated
Description: Single layer of column-like cells with nuclei near to the base. It contains two types of cells –
columnar epithelial cells with microvilli at their apical surface and goblet cells. Microvilli, fingerlike cytoplasmic
projections, increase the surface area of the plasma membrane. Goblet cells are modified columnar epithelial
cells that secrete mucus, a slightly sticky fluid, at their apical surfaces. Before it is released, mucus accumulates
in the upper portion of the cell, causing it to bulge out and making the whole cell resemble a goblet. Secreted
4.1.1 Organ systems of the human body

The human body contains a number of systems that work together to maintain homeostasis. The digestive,
cardiovascular, respiratory and urinary systems perform processing and transporting functions that maintain the
normal conditions of the body. The skeletal system and muscular system support the body and permit movement.
The nervous system receives sensory input from sensory receptors and directs the muscles and glands to respond
to outside stimuli. The endocrine system produces hormones, some of which influence the functioning of the
reproductive system, which allows humans to make more of their own kind. The skin and its accessory organs
comprise the integumentary system. A short description of some important systems are given below:
Nervous system
Components: Brain, spinal cord, nerves and special sense organs, such as eyes and ears.
Function: Generates action potentials (nerve impulses) to regulate body activities; detects change in the body’s
internal and external environment, interprets the changes, and responds by causing muscular contractions or
glandular secretions.
Endocrine system
Components: Hormone-producing glands (pineal gland, hypothalamus, thyroid gland, parathyroid glands, adrenal
glands, pancreas, ovaries and testes) and hormone-producing cells in several other organs.
Functions: Regulates body activities by releasing hormones, which are chemical messengers transported in the
blood from an endocrine gland to a target organ.
Cardiovascular system
Components: Blood, heart and blood vessels.

Functions: Heart pumps blood through blood vessels; blood carries oxygen and nutrients to cells and carbon dioxide
and wastes away from cells and helps regulate acid-base balance, temperature and water content of body fluids;
blood components help defend against disease and mend damaged blood vessels.
Respiratory system
Components: Lungs and air passageways such as the pharynx, larynx (voice box), trachea and bronchial tubes
leading into and out of them.
Functions: Transfers oxygen from inhaled air to the blood and carbon dioxide from the blood to exhaled air; helps
regulate acid-base balance of body fluids; air flowing out of lungs through vocal cords produces sounds.
Digestive system
Components: Organs of the gastrointestinal tract, a long tube that includes the mouth, pharynx (throat), esophagus,
stomach, small and large intestines, and anus; also includes accessory organs that assist in digestive processes,
such as the salivary glands, liver, gallbladder and pancreas.
Functions: Achieves physical and chemical breakdown of food; absorbs nutrients; eliminates solid wastes.
Urinary system
Components: Kidneys, ureters, urinary bladder and urethra.

Functions: Produces, stores and eliminates urine; eliminates wastes and regulates volume and chemical composition
of blood; helps maintain the acid – base balance of body fluids; maintains body’s mineral balance; helps regulate
the production of red blood cells.
Reproductive system
Components: Gonads (testes in males and ovaries in females) and associated organs (uterine tubes, uterus and
vagina in females and epididymis, ductus deferens and penis in males).
Functions: Gonads produce gametes (sperm or oocytes) that unite to form a new organism; gonads also release
hormones that regulate reproduction and other body processes; associated organs transport and store gametes.
Human Physiology 421
4.2 Nervous Systems

Nervous system coordinates and controls the activities of the animals. Together, with the endocrine system,
nervous system maintains the homeostasis. Besides helping to maintain homeostasis, it is also responsible for our
perceptions, behaviors and memories, and controls all voluntary movements.
There are two main subdivisions of the nervous system:
1. Central nervous system (CNS): It includes the brain and the spinal cord.
2. Peripheral nervous system (PNS): It consists of nerve fibers that carry information between the CNS and the
other parts of the body (the periphery). Components of the PNS include cranial nerves and their branches,
spinal nerves and their branches, ganglia and sensory receptors.
The PNS is further subdivided into afferent and efferent divisions. The afferent division includes afferent nerve
fibres that carries information from tissues and organs to the CNS. Instructions from the CNS are transmitted
through efferent nerve fibres of the efferent division to effector organs (muscles or glands) that carry out the
orders to bring about the desired effect.
The efferent division is divided into the somatic nervous system and autonomic nervous system.
The somatic nervous system consists of the nerve fibers that transmit impulses from the CNS to skeletal muscles
and comprise voluntary branch of the peripheral efferent division. The autonomic nervous system consists of
the nerve fibers that transmits impulses from the CNS to the smooth muscles, cardiac muscles and glands of the
body and comprise involuntary branch of the peripheral efferent division. The autonomic nervous system is further
classified into the sympathetic nervous system and the parasympathetic nervous system.
In addition to the CNS and PNS, there is an extensive network of nerve in the wall of the digestive tract which
forms the enteric nervous system. Digestive activities are controlled by the autonomic nervous system and the
enteric nervous system, as well as by hormones. The enteric nervous system can act independently of the rest of
the nervous system but is also influenced by autonomic nervous system. Sometimes the enteric nervous system
is considered a third component of the autonomic nervous system, one that supplies the digestive organs only.
CNS
Brain and spinal cord
Input to Output from

CNS CNS to periphery
Afferent division PNS Efferent division
Sensory receptors
Autonomic Somatic
nervous system nervous system
Sympathetic Parasympathetic Skeletal

nervous system nervous system muscles
Smooth muscle, Cardiac muscle,

Exocrine glands
Figure 4.3 Organization of the nervous system. Subdivisions of the PNS are the SNS (somatic nervous system) and
the ANS (autonomic nervous system).
forms a rim (limbus is Latin for rim) around the corpus callosum on the medial face of the cerebral hemispheres is
called the limbic system. The prominent components of this region are the cingulate gyrus, which lies above the
corpus callosum, the hippocampus, which lies in the medial temporal lobe and the amygdala. There is no universal
agreement on the total list of structures, which comprise the limbic system. Along with the hypothalamus, it is
involved in the regulation of sexual behaviour, emotion (e.g. excitement, pleasure, anger and fear), long-term
memory and motivation. The limbic system is sometimes called the ‘emotional brain’ because it plays a primary
role in a range of emotions, including pleasure, pain, affection, fear and anger.
4.2.4 Spinal cord

The spinal cord extends from the base of the brain through a large opening in the skull called the foramen
magnum and into the vertebral canal formed by openings in the vertebrae. Paired spinal nerves emerge from the
spinal cord through spaces formed between the adjacent vertebrae. There are thirty-one pairs of spinal nerves.
The spinal nerves are named according to the region of the vertebral column from which they emerge. There
are 8 pairs of cervical nerves (C1 to C8), 12 thoracic nerves (T1 to T12), 5 lumbar nerves (L1 to L5), 5 sacral
nerves (S1 to S5) and 1 coccygeal nerve (Co1). Each spinal nerve is a mixed nerve composed of sensory and
motor nerve fibers.
1
2
3
4 Cervical nerves
5
6
7
8
1
2
3
4
5
6
7 Thoracic nerves
8
9
10
11
12
3 Lumbar nerves
Cauda
equina 4
1
2 Sacral nerves
3
4
5
1 Coccygeal nerves
Figure 4.11 Spinal nerves. The 31 pairs of spinal nerves are named according to the region of the vertebral column
from which they emerge. Because the spinal cord is shorter than the vertebral column, spinal nerve roots must descend
along the cord before emerging from the vertebral column at the corresponding intervertebral space, especially those
beyond the level of the first lumbar vertebra (L1). Collectively these rootlets are called the cauda equina, literally
“horse’s tail.”
Table 4.5 Comparison of the somatic and the autonomic nervous system
Feature Somatic nervous system Autonomic nervous system
Effector organs Skeletal muscles Cardiac muscle, smooth muscle and glands
Control Voluntary control Involuntary control
Effect of nerve impulse on effector Excitatory only Either excitatory or inhibitory
Neurotransmitter Acetylcholine Acetylcholine, epinephrine and

norepinephrine
Neuron Myelinated Myelinated (preganglionic neuron) or

unmyelinated (postganglionic neuron)
Number of neurons (from CNS to effector) One somatic motor neuron Usually two autonomic motor neurons
4.3 Sensory organs

Sensory organs of the body are the windows of the brain because they keep the brain aware of what is going on in
the external world. There are five major senses: sight, hearing, taste, smell and touch. There are organs connected
with these senses that take in information that is sent to the brain so that the body can act on it - olfaction (the
sense of smell), taste or gustation (the detection of compounds and ions by the tongue), vision (the detection of
light), hearing (the detection of sound or pressure waves in the air) and touch (the detection of changes in pressure,
temperature, and other factors by the skin).
Sensory organs Senses Stimuli
Skin Touch Pain, cold, pressure
Nose Smell (olfaction) Chemicals
Tongue Taste (gustation) Chemicals
Ear Hearing Sound
Eye Vision Light
The sensory organs (or sensory system) detect changes in the environment and send appropriate signals to the
CNS. In the CNS, these signals are processed and combined with other information to yield a perception that may
trigger a change in response. By these means, our sensory organs allow us to detect changes in our environments
and to adjust our behavior appropriately.
4.3.1 Eye
The eyes are complex sense organs. They gather information about the environment; and the brain interprets this
information to form an image of what appears within the field of vision. Each eye has a layer of receptors, a lens
system that focuses light on these receptors, and a system of nerves that conducts impulse from the receptors
to the brain. The eye is often compared to a camera, with the cornea acting as the lens, the pupillary diameter
functioning like the aperture of the camera, and the retina serving as the film.
Anatomy of the eye

Each eye is a spherical, fluid-filled structure. Anatomically, the wall of the eye consists of three layers: fibrous tunic,
vascular tunic and retina. The fibrous tunic is the outer protective layer of the eyeball and consists of the anterior
cornea and posterior sclera. The sclera (the white of the eye), is a layer of dense connective tissue made up mostly
of collagen fibers and fibroblasts. It provides shape and protects inner parts. Through the sclera no light can pass.
It is modified anteriorly to form the transparent cornea, through which light rays enter the eye.
The cornea is a transparent coat that covers the colored iris. Because it is curved, the cornea helps focus light
onto the retina. Its outer surface consists of non-keratinized stratified squamous epithelium. The middle coat of
the cornea consists of collagen fibers and fibroblasts, and the inner surface is simple squamous epithelium.
4.4.3 Pineal gland

The pineal gland (or epiphysis) is a small endocrine gland attached to the roof of the third ventricle of the forebrain.
The gland consists of masses of neuroglia and secretory cells called pinealocytes. The pineal gland secretes melatonin,
an amine hormone derived from serotonin that itself is derived from the amino acid tryptophan. Melatonin is the
hormone of darkness. Melatonin secretion increases up to 10-fold during the darkness of night and then falls to
low levels during the light of day. Melatonin appears to contribute to the setting of the body’s biological clock. As
more melatonin is liberated during darkness than light, this hormone is thought to promote sleepiness. Melatonin
is also a potent antioxidant that may provide some protection against damaging oxygen free radicals. Melatonin
levels are higher in children and decline with age into adulthood, but there is no evidence that changes in melatonin
secretion correlate with the onset of puberty and sexual maturation.
The pineal gland of fish and amphibians is located near the skin and functions to detect light. In birds, it is located
on the brain, but receives direct light stimulus through the skull. In mammals, it is located within the brain and,
therefore, cannot receive light stimulation directly. Light from the eyes stimulates the gland via the optic nerve.
4.4.4 Thyroid gland

Thyroid gland is the largest endocrine gland in the body. It is located on either side of the trachea. It is composed
of right and left lateral lobes, one on either side of the trachea, that is connected by a fibrous connective tissue,
isthmus. It produces hormones like thyroid hormones and calcitonin.
The thyroid gland consists of numerous spherical hollow sacs called thyroid follicles and parafollicular cells. Thyroid
follicles are lined with a simple cuboidal epithelium composed of follicular cells. The interior of the follicles contains
colloid, a protein-rich fluid. The follicular cells produce two hormones: thyroxine (also called tetraiodothyronine
or T4 because it contains four atoms of iodine) and triiodothyronine (or T3), which contains three atoms of iodine.
T3 and T4 together are known as thyroid hormones.
The parafollicular cells (or C-cells ) lie between follicles and secrete a hormone known as calcitonin (or thyrocalcitonin).
Colloid
Thyroid
cartilage
Thyroid
gland
Trachea
C-cells secrete Follicular cells of thyroid follicle

calcitonin secrete thyroid hormone
Figure 4.25 Follicular cells enclose the follicle lumen, which is filled with protein-rich colloid. The follicular cells
synthesize the thyroid hormones. C-cells (or parafollicular cells) are located outside the follicles; they produce the
peptide hormone calcitonin.
Production and role of thyroid hormone

The follicular cells of thyroid follicles actively accumulate iodide from the blood and secrete it into the colloid. In
the colloid, iodide is oxidized into iodine (2I– = I2) and attached to a tyrosine residue of thyroglobulin protein.
Thyroglobulin is also synthesized and secreted by follicular cells.
The attachment of one iodine to tyrosine produces monoiodotyrosine (MIT); the attachment of two iodines produces
diiodotyrosine (DIT). Within the colloid, enzymes modify the structure of MIT and DIT and couple them together.
When two DIT molecules that are appropriately modified are coupled together, a molecule of tetraiodothyronine
(T4 or thyroxine), is produced. The combination of one MIT with one DIT forms triiodothyronine (T3). Upon stimulation
by TSH, the thyroid hormones, bound to thyroglobulin, are taken into the follicular cells. Hydrolysis reactions within
the follicular cells release the free T4 and T3, which are secreted.
The major hormone secreted by the thyroid gland is thyroxine (or T4). It travels in the blood attached to carrier
proteins (primarily to thyroxine-binding globulin). The thyroid also secretes a small amount of triiodothyronine
or T3. The carrier proteins have a higher affinity for T4 than for T3. Approximately 99.96% of the T4 in the blood
remain attached to carrier proteins in the plasma. Free T4 and T3 enter target cells. Once the free T4 passes into
the target cell cytoplasm, it is enzymatically converted into T3. Hence it is the T3 rather than T4 that is active within
the target cells.
The actions of thyroid hormones are mediated by their binding to nuclear receptors. Receptors for thyroid hormone
are, like all the steroid hormone receptors, act as transcription factors that regulate gene expression in target cells.
Unlike some steroid receptors (such as glucocorticoids), thyroid hormone receptors exist in the nucleus, not the
cytoplasm, and may remain bound to DNA in the absence of hormone binding.
Thyroid hormones play an important role in the regulation of the basal metabolic rate. It increases the basal
metabolic rate in most tissues (exceptions include brain, spleen and testes) by stimulating the use of cellular oxygen.
When the basal metabolic rate increases, cellular metabolism of carbohydrates, lipids, and proteins increases. As
cells produce and use more ATP, more heat is generated, and body temperature rises. This phenomenon is called
the calorigenic effect.
A second major effect of thyroid hormones is to stimulate synthesis of Na+-K+ ATPase. Together with human growth
hormone and insulin, thyroid hormones accelerate body growth, particularly the growth of the nervous and skeletal
systems.
Calcitonin is a peptide hormone secreted by parafollicular cells of the thyroid gland that are distinct from the
thyroid follicles. Thyroid cells produce calcitonin in response to high calcium levels in the blood. It decreases plasma
calcium concentration by decreasing mobilization of calcium from bones; therefore promotes osteoblastic activity.
4.4.5 Parathyroid gland

The parathyroid glands are four small glands present on the back side of the thyroid gland, one pair each in the
two lobes of the thyroid gland. They secrete parathyroid hormone (PTH) or collip’s hormone, which increases levels
of calcium in the blood. Its secretion is regulated by the calcium level in the blood, (not hypothalamic or pituitary
hormones). Bone tissue acts as a storage reservoir for calcium and PTH stimulates the removal of calcium from
the bone to increase levels in the blood; therefore it stimulates osteoclastic activity. It increases the reabsorption
of calcium by the renal tubules of kidney so that less is lost in urine but at the same time it stimulates the loss of
phosphates in the urine. It also stimulates kidney to secrete calcitriol which, in turn, increases calcium absorption
from the digested food in the gut. PTH is thus a hypercalcemic hormone, i.e. it increases the blood calcium levels.
4.4.6 Thymus gland

The thymus is located behind the sternum between the lungs. It grows during childhood, but gradually decreases
in size after puberty. The hormones produced by the thymus—thymosin, thymic humoral factor, thymic factor
and thymopoietin—promote the maturation of T-cells (a type of white blood cell that destroys microbes and foreign
substances) and may retard the aging process. Thymus hormones called thymosins stimulate the development and
differentiation of T-lymphocytes or T-cells. They play a role in regulating the immune system by stimulating other
kinds of immune cells as well. It is also responsible for growth during childhood.
4.4.7 Pancreas
Pancreas is a composite gland which acts as both exocrine and endocrine gland. The exocrine cells of pancreas
are arranged in clusters called acini. The acini produce digestive enzymes, which flow into the gastrointestinal
4.5 Respiratory System

During aerobic respiration, oxygen is utilised by the organisms to oxidise respiratory substrates like glucose to
release energy for performing various activities. Carbon dioxide is also released during the catabolic reactions. It
is, therefore, essential that oxygen has to be continuously provided to the cells and carbon dioxide produced by
the cells have to be released out. This process of gaseous exchange i.e. intake of oxygen from the atmosphere and
release of carbon dioxide produced by the cells is called respiration.
The process of respiration includes following steps:
1. Pulmonary ventilation or breathing: The exchange of air between the atmosphere and the alveoli of the lungs. In this
process, atmospheric air is drawn in (inspiration) and carbon dioxide rich alveolar air is released out (expiration).
2. External (or pulmonary) respiration: Exchange of oxygen and carbon dioxide between air in the alveoli and the
blood within the pulmonary capillaries by the process of diffusion (termed pulmonary gas exchange).
3. The blood transports oxygen and carbon dioxide between the lungs and the tissues.
4. Internal (or tissue) respiration: Exchange of oxygen and carbon dioxide between the tissue cells and the
blood within the systemic capillaries by the process of diffusion (termed systemic gas exchange) and cellular
respiration. The cellular respiration refers to the intracellular metabolic processes in which oxidation of respiratory
substrates is carried out.
4.5.1 Respiratory organs

The respiratory system is responsible for the exchange of gases– oxygen and carbon dioxide between the atmospheric
air, blood and tissue cells. In addition to functioning in gaseous exchange, the respiratory system also helps to
regulate blood pH and contains receptors for the sense of smell. It also filters inspired air, produces vocal sounds
(phonation) and excretes small amounts of water and heat.
The respiratory system of human consists of the nose, pharynx (throat), larynx (voice box), trachea (windpipe),
bronchi and lungs.
Nasal cavity
Pharynx
Esophagus Larynx
Trachea
Parietal pleura
Pleural cavity
Visceral pleura
Right Heart
Lung Left
Lung
Abdominal cavity
Figure 4.30 The respiratory airways include the nasal passages, pharynx, larynx, trachea, bronchi, and bronchioles.
Lungs are paired organs located in the thoracic cavity enclosed by the pleural membrane. The right lung has three
lobes and the left lung has two lobes.
hydrogen ion concentration in the brain extracellular fluid that bathes them. Oxygen, in contrast, does not have
a significant direct effect on the respiratory center of the brain in controlling respiration. Instead, it acts almost
entirely on peripheral chemoreceptors.
Peripheral chemoreceptors
Most of the peripheral chemoreceptors are in the carotid bodies. However, a few are also in the aortic bodies. These
receptors are especially important for detecting changes in oxygen in the blood, although they also respond to a
lesser extent to changes in carbon dioxide and hydrogen ion concentrations. When the oxygen concentration in
the arterial blood falls below normal, the chemoreceptors become strongly stimulated.
An increase in either carbon dioxide concentration or hydrogen ion concentration also excites the chemoreceptors.
However, the effects of both these factors in the central chemoreceptors itself are so much more powerful than
their effects mediated through the peripheral chemoreceptors that, for practical purposes, the effects of carbon
dioxide and hydrogen ions through the peripheral chemoreceptors do not need to be considered.
4.5.8 Disorders of respiratory system

Chronic obstructive pulmonary disease (COPD)
COPD refers to any disorder that causes airflow obstruction primarily due to the narrowing of airways. The main
symptoms include shortness of breath and cough with sputum production. The major COPDs are asthma, chronic
bronchitis and emphysema.
Asthma
Asthma is a chronic lung disease characterized by inflammation in airway. Inflammation makes the airways swollen
and very sensitive to a variety of stimuli. Airway obstruction may be due to smooth muscle spasms in the walls of
smaller bronchi and bronchioles, edema of the mucosa of the airways, increased mucus secretion, and/or damage to
the epithelium of the airway. Symptoms include difficult breathing, coughing, wheezing, chest tightness, tachycardia,
fatigue, moist skin and anxiety.
Emphysema
Emphysema is a disorder characterized by abnormal enlargement of the air spaces due to destruction of the walls
of the alveoli. The lungs also become fibrotic and less elastic. With less surface area for gas exchange, O2 diffusion
across the damaged respiratory membrane is reduced. It also causes an increased amount of air trapped in the
lungs at the end of exhalation. Emphysema is generally caused by a long-term irritation; cigarette smoke, air
pollution, and occupational exposure to industrial dust are the most common irritants.
Bronchitis
Bronchitis is inflammation of the bronchi in the lungs. It is of two types: acute and chronic bronchitis. Acute bronchitis
(also known as a chest cold) usually has a cough that lasts around three weeks. It is short term inflammation of
the bronchi of the lungs. In more than 90% of cases the cause is a viral infection. Chronic bronchitis is defined as
a productive cough that lasts for three months or more per year for at least two years. Cigarette smoking is the
leading cause of chronic bronchitis.
Pneumonia
Pneumonia is an inflammation of the alveoli of lung. Typical signs and symptoms include a cough, chest pain,
fever and dyspnea (breathlessness). Its symptoms can vary from mild to severe. Many factors affect how serious
pneumonia is, such as the type of germ causing the infection and your age and overall health. Pneumonia is usually
caused by infection with viruses or bacteria and less commonly by other microorganisms. Bacteria are the most
common cause of pneumonia. Streptococcus pneumoniae is the most common cause of bacterial pneumonia.
Tuberculosis
Tuberculosis is an infectious disease usually caused by the bacterium Mycobacterium tuberculosis. Although several
Mycobacterium species can cause tuberculosis, M. tuberculosis is the principal causative agent. Tuberculosis
generally affects the lungs. Once the bacteria are inside the lungs, they multiply and cause inflammation, which
stimulates neutrophils and macrophages to migrate to the area and engulf the bacteria to prevent their spread. If
the immune system is not impaired, the bacteria remain dormant for life, but impaired immunity may enable the
bacteria to escape into blood and lymph to infect other organs. In many people, symptoms—fatigue, weight loss,
lethargy, anorexia, a low-grade fever, night sweats, cough, dyspnea, chest pain, and hemoptysis—do not develop
until the disease is advanced.
4.6 Cardiovascular System

In a multicellular organism, all living cells have to be provided with nutrients, oxygen and other essential substances.
Also, the waste or harmful substances produced, have to be removed continuously for healthy functioning of tissues.
It is therefore, essential to have efficient mechanisms for the transport of these substances to the cells and from
the cells. The cardiovascular system (also called circulatory system or blood vascular system) is responsible for
transporting gases, nutrients, hormones and cellular waste products throughout the body. It consists of three
interrelated components: blood, the heart and blood vessels.
4.6.1 Blood
Blood is a connective tissue composed of blood plasma (a liquid extracellular matrix) and formed elements (which
are cells and cell fragments). Blood is slightly alkaline (pH ranging from 7.3 to 7.4). It constitutes 20-30% of
extracellular fluid, amounting to 8% of the total body mass. The blood volume is 5 to 6 liters in an average–sized
adult male and 4 to 5 liters in an average–sized adult female.
Functions of blood
Blood performs following important functions:
1. Transportation of gases (oxygen and carbon dioxide), nutrients, hormones, heat and wastes.
2. Regulation of pH, body temperature and water content of cells.
3. Protection against blood loss through clotting and against disease through phagocytic activity with blood cells
and antibodies.
Components of blood
Blood has two components:
1. Blood plasma, a liquid extracellular matrix.
2. Formed elements, which are blood cells and cell fragments.
Blood plasma
Blood is about 45% formed elements and 55% blood plasma. When the formed elements are removed from blood,
a straw colored liquid called blood plasma (or simply plasma) is left. The separation can be either achieved by just
leaving the blood undisturbed for some time leading to settling down of the cells or by centrifuging them. Blood
plasma is 90-92% water and 8-10% solutes, most of which (~7% by weight) are proteins. Some of the proteins
in blood plasma are also found elsewhere in the body but those confined to blood are called plasma proteins.
Hepatocytes synthesize most of the plasma proteins, which include the albumins (~54% of plasma proteins),
globulins (~38%) and fibrinogen (~7%). Fibrinogen is an important clotting factor produced by the liver. During
the process of clot formation, soluble fibrinogen is converted into insoluble threads of fibrin. Thus, the fluid from
clotted blood, called serum, does not contain fibrinogen, but it is otherwise identical to plasma.
Plasma also contains small amounts of minerals (like sodium, calcium, magnesium, bicarbonate, chloride, etc.),
glucose, amino acids, lipids, etc.
Like other cells, the hepatocytes receive fresh arterial blood via the hepatic artery, which supplies oxygenated
blood. The hepatic portal vein is responsible for directing blood from parts of the gastrointestinal tract to the liver
(a vein that carries blood from one capillary network to another is called a portal vein). Substances absorbed in
the small intestine travel first to the liver for processing before continuing to the heart. Following diagram shows
the basic scheme of the hepatic portal system:
Interstitial fluid and Lymph

Most components of blood plasma filter through blood capillary walls to form interstitial fluid. After interstitial fluid
passes into lymphatic vessels, it is called lymph. Vessels that transport the lymph called lymphatic vessels. The
major difference between interstitial fluid and lymph is location: Interstitial fluid (tissue fluid) is found between
cells, and lymph is located within lymphatic vessels and lymphatic tissue. Its composition is just like plasma except
it lacks RBCs. It is colorless due to absence of hemoglobin. It transfers materials from blood to the body cells and
vice-versa, therefore act as ‘middle man’.
Interstitial fluid surrounds and bathes the cells. This fluid is continually being replaced by fresh fluid from blood
in the circulatory system. Body cells take up nutrients from the interstitial fluid and empty wastes into it. By
maintaining a constant pH and ionic concentration of the blood, the pH and ionic concentration of the interstitial
fluid is also stabilized.
4.6.5 Lymphatic system

The cardiovascular system is considered as a closed system because all of its tubes are connected with one another
and none is simply open ended. In another sense, however, the system is open – to diffusion through the walls of
the capillaries. This process is a necessary part of the functioning of the circulatory system, but it poses difficulties
in maintaining the integrity of that system.
Systemic circulation Pulmonary circulation

Lymph node
Initial
Lymph vessel lymphatics
Valve Blood
capillaries
Veins
Heart Arteries
Lymph node
Figure 4.56 Schematic diagram showing the relationship of the lymphatic system of the cardiovascular system.
Lymphatic vessels drain excess fluid from the tissues and return it to the cardiovascular system. The construction of
the larger lymphatic vessels is similar to that of cardiovascular veins, including the presence of valves. The enlargement
shows that lymphatic vessels, like cardiovascular veins, have valves to prevent backward flow.
Difficulties arise because diffusion from the capillaries is accompanied by the loss of large quantities of liquid from
the cardiovascular system. When blood passes through the capillaries, it loses more water to the body than it
reabsorbs from it. In a human being, about 3 liters of fluid leave the cardiovascular system in this way each day, a
quantity amounting to more than half the body’s total supply of about 5.6 liters of blood. To counteract the effects
of this process, the body uses a second open circulatory system called the lymphatic system. The lymphatic system
consists of lymphatic vessels and the lymphoid organs. The elements of the lymphatic system gather liquid from
the body and return it to cardiovascular circulation. Open–ended lymph capillaries gather up fluids and carry them
through a series of progressively larger vessels to two large lymphatic vessels, which resemble veins. The lymphatic
vessels contain a series of one-way valves like those of veins, which permit movement in only one direction.
These two lymphatic vessels drain into the veins through one-way valves. Heart does not pump fluid through the
lymphatic system. Instead, like veins, fluid is driven through lymphatic vessels when these vessels are squeezed
by the movements of the body’s muscles. Thus, the lymphatic system is an accessory route by which interstitial
fluid can be returned to the blood.
4.6.6 Intracellular and extracellular fluid

The intracellular fluid is the fluid that is present in the cytoplasm of all cells of the body. The intracellular fluid is
separated from the extracellular fluid by the cell plasma membrane. The extracellular fluid is further subdivided
into the intravascular and the extravascular. The intravascular fluid is the fluid that is present in all of body blood
vessels. This fluid is referred to as the plasma. The extravascular fluid is further subdivided into: interstitial fluid
and transcellular fluid. Interstitial fluid is the fluid that directly bathes the cells and tissues in the body. Transcellular
fluid is found in small amounts in different body regions and, in total, comprises a very small portion. This fluid
is generally separated from the plasma by an additional epithelial layer in addition to the capillary endothelium.
Cerebrospinal fluid (the fluid bathing the brain and the spinal cord), intraocular fluids (aqueous and vitreous humors),
inner ear fluids (endolymph and perilymph), pericardial fluid, peritoneal fluid, synovial fluids (in joints), as well as
some other fluids are components of the transcellular fluid in the body.
4.6.7 Cardiovascular disorders

Cardiovascular disease is a general term that describes a disease of the heart or blood vessels. There are four main
types of cardiovascular disease. They are coronary heart disease, stroke (a problem with the circulation of blood
in the blood vessels of the brain), peripheral arterial disease (circulation disorders that affect blood vessels outside
of the heart and brain) and aortic disease.
Coronary artery disease (also called coronary heart disease)
Coronary Artery Disease (CAD) refers to a pathological condition that causes narrowing of coronary arteries so
that blood flow to the heart is reduced. CAD, often referred to as atherosclerosis, affects the coronary arteries,
that supply blood to the heart muscle. The atherosclerosis is a progressive, degenerative arterial disease results
from the accumulation of atherosclerotic plaques in coronary arteries, which leads to a reduction in blood flow to
the myocardium. Atherosclerosis is the most common form of arteriosclerosis. An atherosclerotic plaque consists
of a lipid-rich core covered by an abnormal overgrowth of smooth muscle cells. It is made up of fat, cholesterol,
calcium and other substances found in the blood.
Heart failure
Heart failure occurs when heart can’t pump enough blood to meet the body’s needs. As a result, the heart cannot
pump enough oxygen and nutrients to meet the body’s needs. Heart failure does not mean the heart has stopped
working. Rather, it means that the heart’s pumping power is weaker than normal. It is not the same as cardiac arrest
(when the heart stops beating) or a heart attack (when the heart muscle is suddenly damaged by an inadequate
blood supply). Heart failure is caused by many conditions that damage the heart muscle. It is sometimes called
congestive heart failure because congestion of the lungs is one of the main symptoms of this disease.
4.7 Digestive System

All living organisms require food for energy and organic matters for growth. Food is organic compound and usually
of plant or animal origin. The major components of our food are carbohydrates, proteins and fats. It also contains
vitamins and minerals in small quantities. The process of conversion of complex food substances to simple absorbable
forms is called digestion and is carried out by our digestive system by mechanical and biochemical methods. The
digestive system also absorbs water, vitamins and minerals, and eliminates wastes from the body.
The human digestive system includes the gastrointestinal tract (or alimentary canal or digestive tract) and the
associated glands. The gastrointestinal tract is a continuous tube that extends from the mouth to the anus. The
organs of the gastrointestinal tract include the mouth, pharynx, esophagus, stomach, small intestine and large
intestine. The associated glands include salivary glands, liver, gallbladder and pancreas.
The digestive system performs six basic digestive processes:

Ingestion Taking food into the mouth.
Secretion Release of acid, enzymes and bile into the lumen of the gastrointestinal tract.
Motility Refers to propulsive and mixing movements which cause propulsion and mixing of food through the
gastrointestinal tract.
Digestion Mechanical and chemical breakdown of food.
Absorption Assimilation of digested products from the gastrointestinal tract into the blood and lymph.
Defecation The elimination of feces from the gastrointestinal tract.
4.7.1 Gastrointestinal tract

The gastrointestinal tract is an organ system responsible for transporting and digesting food, absorbing digested
products and expelling waste. It is a large, muscular tube that extends from the mouth to the anus. Organs of the
gastrointestinal tract include the mouth, pharynx, esophagus, stomach, small intestine and large intestine. The
length of the alimentary canal is about 5–7 meters. In the gastrointestinal tract, several muscle rings known as
sphincters present, which restrict the flow of contents to optimize digestion and absorption.
Layers of the gastrointestinal tract

The gastrointestinal tract from the esophagus to the anal canal is composed of four layers (or tunics). Each tunic
contains a dominant tissue type that performs specific functions in the digestive process. The four tunics of the
gastrointestinal tract, from the inside out, are the mucosa (or mucous membrane), submucosa, muscularis propria
(smooth muscle layer) and serosa (serous membrane).
Mucosa: The mucosa is the inner lining of the lumen of the gastrointestinal tract. It is the absorptive and major
secretory layer. It is a mucous membrane composed of;
1. a layer of epithelium,
2. a layer of loose connective tissue known as the lamina propria and
3. a thin layer of smooth muscle known as muscularis mucosa.
Submucosa: A layer of dense irregular connective tissue that surrounds the mucosa. It has large blood vessels and
lymphatic vessels. A nerve network known as the submucosal plexus (Meissner’s plexus) lies within the submucosa
(plexus means ‘network’).
Muscularis propria (or muscularis externa): It is dominated by smooth muscle cells in two layers - an inner circular
layer and outer longitudinal layer - that play an essential role in mechanical processing and in the movement of
materials along the gastrointestinal tract. Between inner circular layer and an outer longitudinal layer another nerve
network known as the myenteric plexus (Auerbach’s plexus), lies.
Serosa (serous membranes): The outer connective tissue covering of the digestive tract is the serosa, which secretes
a watery, slippery fluid that lubricates and prevents friction between the digestive organs and the surrounding viscera.
Table 4.18 Characteristics of the major digestive enzymes
Substrates Optimum
Enzyme Site of action Source Products
Carbohydrate pH
Salivary amylase Mouth Salivary glands Starch 6.7 Maltose, maltotriose and α-dextrins
Pancreatic amylase Small intestine Pancreatic acinar cells Starch 6.7–7.0 Maltose, maltotriose and α-dextrins
Maltase Small intestine Intestinal cells Maltose 5.0–7.0 Glucose
Sucrase Small intestine Intestinal cells Sucrose 5.0–7.0 Glucose and fructose
Lactase Small intestine Intestinal cells Lactose 5.8–6.2 Glucose and galactose
Protein
Pepsin Stomach Chief cells of gastric glands Proteins ~2.0 Peptides
Trypsin Small intestine Pancreatic acinar cells Proteins 7.0–9.0 Peptides
Chymotrypsin Small intestine Pancreatic acinar cells Proteins 8.0 Peptides
Carboxypeptidase Small intestine Pancreatic acinar cells Proteins 8.0 Amino acid and peptides
Aminopeptidase Small intestine Intestinal cells Proteins 8.0 Amino acid and peptides
Lipid
Gastric lipase Stomach Chief cells of gastric glands Triglycerides ~6.0 Fatty acids and monoglycerides
Pancreatic lipase Small intestine Pancreatic acinar cells Triglycerides 8.0 Fatty acids and monoglycerides
Nucleic acid
Nucleosidases and Small intestine Intestinal cells Nucleotides 7.5 Nitrogenous bases, pentoses and
phosphates phosphatases
4.8 Excretory System

The process by which metabolic waste products are eliminated (totally or partially) from the body of animals is
called excretion. It is different from egestion, which is the discharge of undigested food materials from the body.
Nitrogenous wastes (such as ammonia, urea and uric acid) are the most important metabolic waste excreted by
the animals. These wastes are produced from the metabolism of amino acids and nucleic acids and are very toxic
to cellular environment.
The nature of the nitrogen-containing wastes (ammonia, urea and uric acid) and their excretion varies among
the different species, depending on the availability of water. Ammonia is the most toxic form and requires large
amount of water for its elimination, whereas uric acid, being the least toxic, can be removed with a minimum loss
of water. Most animals excrete a mixture of excretory products, but one nitrogenous product usually predominates.
The human excretory system consists of two kidneys, two ureters, one urinary bladder and one urethra. The
kidney is a urine-forming organs. Each kidney is supplied with blood by a renal artery and from this blood, urine
is produced. Urine exits each kidney through a duct called the ureter. Both ureters drain into a common urinary
bladder. The urinary bladder temporarily stores urine. During urination, urine is discharged from the bladder
through a tube called the urethra.
Renal artery Path of blood flow
Segmental arteries
Interlobar arteries Inferior vena cava
Arcuate arteries Renal vein
Interlobular arteries Interlobar veins
Afferent arterioles Arcuate veins
Glomerular capillaries Interlobular veins
Efferent arterioles Peritubular capillaries Peritubular venules
The peritubular capillaries surrounds the renal tubules. A specialized portion of the peritubular capillary system
called the vasa recta which descend into the medulla in parallel with the loops of Henle and then loop back along
with the loops of Henle and return to the cortex. Thus, there are two sets of capillaries in the kidneys – the
glomerular capillaries and the peritubular capillaries. Within each nephron, the two sets of capillaries are connected
to each other by an efferent arteriole. Blood from the peritubular capillaries is drained into the venous system.
The peritubular capillaries eventually reunite to form peritubular venules. Venous system starts with peritubular
venules and continues as interlobular veins, arcuate veins, interlobar veins and finally renal veins. Blood leaves
the kidney through a single renal vein that exits at the renal hilum and carries venous blood to the inferior vena
cava. Note that no segmental veins are present in the kidneys; the interlobar veins merge in the renal sinus to
form the large renal vein.
4.8.2 Nephron
Nephrons are the functional units of the kidneys. Each kidney consists of about 1 million nephrons, which are bound
together by connective tissue. A nephron consists of tubules and associated small blood vessels. The nephron’s
tubular component is a hollow, fluid-filled tube formed by a single layer of epithelial cells.
Each nephron consists of two parts: a renal corpuscle (or malpighian body), where blood plasma is filtered, and
a renal tubule into which the filtered fluid passes.
Capillary endothelium
Basement membrane
Secondary
process
(pedicel)
Capillary
Podocyte
cell body
Primary
process
Filtration slit Foot

and slit diaphragm processes
of podocyte
Figure 4.71 A cross-section of the filtration membrane. A substance that is filtered passes first through a fenestra
in the capillary endothelium. Next, it passes across the glomerular basement membrane, which consists of a network
of collagen fibrils and other structural proteins. Finally, the substance passes through the filtration slits that are found
between the podocytes.
the long loops of Henle of juxtamedullary nephrons. The descending limb of the loop of Henle carries tubular fluid
from the renal cortex deep into the medulla, and the ascending limb carries it in the opposite direction. Since
countercurrent flow through the descending and ascending limbs of the long loop of Henle establishes the osmotic
gradient in the renal medulla, the long loop of Henle is said to function as a countercurrent multiplier. The kidneys
use this osmotic gradient to excrete concentrated urine.
4.8.5 Countercurrent exchange

Countercurrent exchange is the process by which solutes and water are passively exchanged between the blood
of the vasa recta and interstitial fluid of the renal medulla as a result of countercurrent flow. The vasa recta also
consist of descending and ascending limbs that are parallel to each other and to the loop of Henle. Just as tubular
fluid flows in opposite directions in the loop of Henle, blood flows in opposite directions in the ascending and
descending parts of the vasa recta. Since countercurrent flow between the descending and ascending limbs of the
vasa recta allows for exchange of solutes and water between the blood and interstitial fluid of the renal medulla,
the vasa recta is said to function as a countercurrent exchanger.
Cortex 100
100
300 300
Filtrate is very dilute
Medulla H2O Na+ (100 mOsm), it is
hypoosmotic to the
interstitial fluid.
H2O Cl
—
400 400 200

+ –
H2O Na+ Na and Cl are pumped
out of the filtrate. This
—
increases the osmolality of
H2O Cl interstitial fluid.
600 600 400
H2O
Na+
H2O Cl—
900 900 700
H2O
Filtrate reaches its highest
concentration at the bend
of the loop.
1200 1200
Figure 4.82 As water and solutes are reabsorbed, the loop first concentrates the filtrate, then dilutes it.
Osmolality and osmolarity

The total number of osmotically active particles in a solution is measured in osmoles. One osmole (Osm) is equal to 1
mole (6.02×1023) of osmotically active solute particles. Therefore, a solution containing 1 mole of glucose in one liter
has a concentration of 1 Osm/L. If a molecule dissociates into two ions in solution, such as sodium chloride ionizing to
give chloride and sodium ions, then a solution containing 1 mol/L will have an osmolar concentration of 2 Osm/L. In
general, the osmole is large unit to express osmotic activity of solutes. The term milliosmole (mOsm) is commonly used.
The osmolal concentration of a solution is called osmolality when the concentration is expressed as osmoles per
kilogram of water; it is called osmolarity when it is expressed as osmoles per liter of solution.
4.9 Reproductive System

The reproductive system of sexually reproducing animal consists of:
• Primary sex organs (called gonads) which produce gametes and hormones.
• Secondary sex organs which participate in reproduction but not form gametes.
• Accessory sex organs cause differences in the appearance of two sexes.
The primary reproductive organs, or gonads, consist of the ovaries and testes. These organs are responsible for
producing the egg and sperm cells and hormones. These hormones function in the maturation of the reproductive
system, the development of sexual characteristics, and have important roles in regulating the normal physiology of
the reproductive system. All other organs, ducts, and glands in the reproductive system are considered secondary,
or accessory, reproductive organs.
4.9.1 Male reproductive system

The male reproductive system consists of glands with their ducts and supporting structures
1. The glands include a pair of testes, a pair of seminal vesicles, a pair of bulbourethral (Cowper’s) glands, and
one prostate gland.
2. Ducts of testes include a pair of epididymis, a pair of vas deferens, a pair of ejaculatory ducts, and one urethra.
3. Supporting structures are divided into: Internal – a pair of spermatic cords and External - scrotum and penis.
Testes
The male gonads, testes, begin their development high in the abdominal cavity, near the kidneys. During the last
two months before birth, or shortly after birth, they descend through the inguinal canal into the scrotum, a pouch
that extends below the abdomen, posterior to the penis. Although this location of the testes, outside the abdominal
cavity, may seem to make them vulnerable to injury, it provides a temperature about 3°C below normal body
temperature. This lower temperature is necessary for the production of viable sperm. The incomplete descent of
testes on one or both sides in a newborn is called cryptorchidism, the testes remaining in the abdominal cavity or
inguinal canal.
Each testes is an oval structure. A tough, white fibrous connective tissue capsule, the tunica albuginea, surrounds
each testes and extends inward to form septa that partition the organ into lobules. There are about 250 lobules in
each testes. Each lobule contains 1 to 4 highly coiled seminiferous tubules that converge to form a single straight
tubule, which leads into the rete testes. Short efferent ducts exit the testes. Interstitial cells (cells of Leydig),
which produce male sex hormones, are located between the seminiferous tubules within a lobule.
Functions of testes
1. Production and storage of viable sperms.
2. Synthesis and secretion of the androgenic hormone, testosterone. Both these functions are under anterior
pituitary and hypothalamic control.
Ducts of the testes

Pressure generated by the fluid secreted by Sertoli cells pushes sperm and fluid along the lumen of the seminiferous
tubules and then into a series of very short ducts called straight tubules. The straight tubules lead to a network of
ducts in the testis called the rete testis. From the rete testis, sperm move into a series of coiled efferent ducts in
the epididymis that empty into a single tube called the ductus epididymis.
Epididymis: The epididymis is a comma-shaped organ about 4 cm (1.5 in.) long that lies along the posterior border
of each testis. Functionally, the epididymis is the site of sperm maturation, the process by which sperm acquire
motility and the ability to fertilize an ovum. This occurs over a period of about 14 days.
Ductus deferens: Within the tail of the epididymis, the ductus epididymis becomes less convoluted, and its diameter
increases. Beyond this point, the duct is known as the ductus deferens or vas deferens.
The dilated terminal portion of the ductus deferens is the ampulla.

Functionally, the ductus deferens conveys sperm during sexual arousal from the epididymis toward the urethra by
peristaltic contractions of the muscular coat.
Spermatic cord: The spermatic cord is the supporting structure of the male reproductive system that ascends out of
the scrotum. It consists of the ductus (vas) deferens as it ascends through the scrotum, the testicular artery, veins
that drain the testes and carry testosterone into circulation (the pampiniform plexus), autonomic nerves, lymphatic
vessels, and the cremaster muscle. The spermatic cord and ilioinguinal nerve pass through the inguinal canal.
Ejaculatory ducts: Each ejaculatory duct is about 2 cm (1 in.) long and is formed by the union of the duct from
the seminal vesicle and the ampulla of the ductus deferens.
Urethra: In males, the urethra is the shared terminal duct of the reproductive and urinary systems; it serves as a
passageway for both semen and urine. About 20 cm (8 in.) long, it passes through the prostate, the deep muscles
of the perineum, and the penis.
Accessory glands
The accessory glands of the male reproductive system are the seminal vesicles, prostate gland and the bulbourethral
glands.
Seminal vesicles: The paired seminal vesicles are saccular glands posterior to the urinary bladder. Each gland has
a short duct that joins with the ductus deferens at the ampulla to form an ejaculatory duct, which then empties into
the urethra. The fluid from the seminal vesicles is viscous and contains fructose, which provides an energy source
for the sperm; prostaglandins, which contribute to the mobility and viability of the sperm; and proteins that cause
slight coagulation reactions in the semen after ejaculation.
Prostate glands: The prostate gland is a structure located just inferior to the urinary bladder. The secretions of the
prostate are thin, milky colored, and alkaline. They function to enhance the motility of the sperm.
Bulbourethral glands: The paired bulbourethral (Cowper’s) glands are small, about the size and located near the
base of the penis. In response to sexual stimulation, the bulbourethral glands secrete an alkaline mucus-like fluid.
This fluid neutralizes the acidity of the residual urine in the urethra, helps to neutralize the acidity of the vagina,
and provides some lubrication for the tip of the penis during intercourse.
Seminal fluid: Seminal fluid, or semen is the fluid that is ejaculated at the time of orgasm. It is a slightly alkaline
mixture of sperm cells and secretions from the accessory glands. Secretions from the seminal vesicles make up
about 60 percent of the volume of the semen, with most of the remainder coming from the prostate gland.
Hormones control
The hypothalamus has ultimate control of the testes’ sexual function because it secretes a hormone called
gonadotropin-releasing hormone, or GnRH, that stimulates the anterior pituitary to secrete the gonadotropic
hormones. There are two gonadotropic hormones—follicle-stimulating hormone (FSH) and luteinizing hormone
(LH)—in both males and females. In males, FSH promotes the production of sperm in the seminiferous tubules,
which also release the hormone inhibin. LH in males is sometimes given the name interstitial cell-stimulating
hormone (ICSH) because it controls the production of testosterone by the interstitial cells, which are found in the
spaces between the seminiferous tubules. All these hormones are involved in a negative feedback relationship
that maintains the fairly constant production of sperm and testosterone. Testosterone, the main sex hormone in
males, is essential for the normal development and functioning of the organs. Testosterone also brings about and
maintains the male secondary sex characteristics that develop at the time of puberty.
Spermatogenesis
Sperms are produced by spermatogenesis within the seminiferous tubules. Immature germ cells, called spermatogonia
present in seminiferous tubule divide by mitosis. Some of these cells enter meiosis I to become primary spermatocytes;
they then complete meiosis I to become secondary spermatocytes. The secondary spermatocytes then complete
meiosis II to become spermatids, which differentiate into spermatozoa (sperms) and are released into the lumen
of the tubule. Within the seminiferous tubules, interspersed with spermatogonia, there are large cells that extend
4.10 Embryonic development

Embryogenesis is a developmental process that usually begins once the egg has been fertilized. It involves
multiplication of cells by mitosis and their subsequent growth, movement, and differentiation into all the tissues
and organs of a living baby.
4.10.1 Fertilization
Fertilization is a process whereby two gametes fuse together to form a zygote. The male gamete, the sperm cell, is
a small cell with a greatly reduced cytoplasm and a haploid nucleus. Sperm usually consist of two morphologically
and functionally distinct regions enclosed by a single plasma membrane: the tail and the head. The head contains a
nucleus and an acrosomal vesicle. The DNA in the nucleus is highly condensed and transcriptionally inactive because
the normal histones are replaced by a special class of packaging proteins known as protamines. The acrosomal
vesicle (or acrosome) is a specialized secretory vesicle present in most animal sperm. It is derived from the Golgi
apparatus and contains hydrolytic enzymes needed to digest extracellular coats surrounding the egg. It can be
considered as a modified secretory vesicle.
The motile tail of a sperm is a long flagellum. The active bending of the flagellum is caused by the sliding of
adjacent microtubule doublets past one another, driven by dynein motor proteins, which use the energy from ATP
hydrolysis to slide the microtubules. The ATP is generated by a large number of highly specialized mitochondria
that are concentrated in the anterior part of the sperm tail (called the midpiece). The neck (region between head
and midpiece) contains the two centrioles (proximal and distal). Axonemal structure of flagella grows out of distal
centriole.
Acrosomal
vesicle Midpiece
Plasma membrane
Nucleus
Mitochondria
Head Tail
Centrioles
Figure 4.87 A longitudinal section of human sperm.
The female gamete (egg) of most animals is giant single cell. It contains all the materials needed for initial
development of the embryo. There are three main ways to provide nutrition to the developing embryo: 1. supply
the embryo with yolk; 2. form a larval feeding stage between the embryo and the adult; or 3. create a placenta
between the mother and the embryo.
A placental mammalian egg is not as large as the egg of a frog or bird. Because the embryo can start to grow
early by taking up nutrients from the mother via the placenta. The egg cytoplasm usually contains nutritional
reserves in the form of yolk, which is rich in lipids, proteins and polysaccharides and is often contained within
discrete structures called yolk granules. In some species, a membrane encloses each yolk granule. Yolk is usually
synthesized outside the ovary and imported into the oocyte. In birds, amphibians and insects, yolk proteins
are made by liver cells (or their equivalents), which secrete these proteins into the blood. Within the ovaries,
oocytes use receptor-mediated endocytosis to take up the yolk proteins from the extracellular fluid. Yolk is often
concentrated toward one pole of the egg, called the vegetal pole; the yolk concentration decreases significantly
toward the opposite pole, the animal pole. The animal pole is also the site where the polar bodies of oogenesis
bud from the cell. In eggs that develop into large animals outside the mother’s body, yolk can account for more
than 95% of the volume of the cell. In mammals, whose embryos are largely nourished by their mothers via
the placenta, there is little, if any, yolk. The egg coat is another peculiarity of eggs. It is a specialized form of
Chapter 05
Ecology
5.1 What is Ecology?

Ecology is the study of relationships between living organisms and their environments, the interaction of organisms
with each other and the pattern and cause of the abundance and distribution of organisms in nature. Thus, ecology
is the science that attempts to answer questions about how the nature works. The term ecology was coined by
German biologist Ernst Haeckel combining two Greek words, oikos (meaning ‘house’ or ‘dwelling place’) and logos
(meaning the study of) to denote such relationship between the organisms and their environment.
Atmosphere
Organisms Lithosphere
Hydrosphere
Figure 5.1 Organisms interact with physical environment comprised of atmosphere, hydrosphere and lithosphere.
Level of organization
Ecological patterns and processes vary as a function of the scale at which they operate. The scales may be biological
and spatial.
The biological scale includes individual organism, population and community. The basic level of the ecological
organization starts with the individual (a single plant, insect or bird). The next level of organization is the population.
Populations are a collection of individuals of the same species within an area or region. The next, more complex,
level of organization is the community. Communities are made up of populations of different species within some
defined geographical area.
The spatial scale in ecology includes ecosystem, biome and biosphere.
An ecosystem is the interacting system made up of all the living and non-living components in a physically defined
space. A biome is a distinct ecological community of plants and animals living together in a particular climate. It
is characterized by distinctive vegetation distributed over wide geographical area and defined largely by regional
climatic conditions. A biome is the largest scale at which ecologists classify vegetation. In a strict sense, the
biosphere represents all the living organisms of the Earth. But in ecology, the biosphere is a functional concept
which emphasizes the interrelationship between all living organisms and their environment on a planetary scale.
It is an ultimate ecosystem.
562 Ecology
Based on the level of organization, ecology is classified into autecology and synecology. Autecology is the study
of interaction between organisms and their environments at the level of an individual, a population or an entire
species. Synecology is the study of a biotic community. It is also called community ecology. It is the synecology
which describes the biotic community as a whole, especially the links between organisms.
5.2 Environment
Organisms and their environments are dynamic and interdependent. The term ‘environment’ etymologically means
surroundings. Thus, the environment includes everything (biotic as well as abiotic) that surrounds an organism.
Any factor, abiotic or biotic, that influences living organisms is called environmental factor (or ecological factor or
ecofactor). Abiotic factors include ambient temperature, amount of sunlight and pH of the water and soil in which
an organism lives. Biotic factors include the availability of prey, competitors, predators and parasites.
Soil
Soil is the uppermost weathered layer of the earth’s crust. It is a mixture of weathered mineral rock particles,
organic matter (i.e. both living and dead), water and air. Soil is a biologically active matrix and home for plant
roots, seeds, animals, bacteria, fungi, algae and viruses. The study of soil is called pedology.
Soil composition
Soils are composed of mineral particles, organic matters, air and water. Soil mineral particles include sand
(0.05-2.0 mm), silt (0.002-0.05 mm) and clay (<0.002 mm). The relative proportions of sand, silt, and clay in a
soil are referred as soil texture. Soils are also composed of organic matter which include living biomass, detritus
and humus. Humus is an amorphous and a colloidal mixture of complex organic substances. It is made up of humic
and non-humic substances. Non-humic substances include carbohydrates, proteins, lignins, lipids, organic acids
etc. Humic substances are stable end products derived from the decomposition of plant and animal residues. It
comprises about 80 to 90% of the soil organic matter and characterized by dark colored amorphous substance. The
three fractions of humic substances are fulvic acid, humic acid and humin. Humin is the most insoluble fraction.
Soil air is the mixture of gases that are present in soil pores that are not filled with water. Oxygen and carbon
dioxide are important constituents, and their concentration in the soil affects many processes (e.g. nitrification and
denitrification). Soil water can contribute up to 30% of soil volume, and is essential for the activity and physiological
functioning of organisms in the soil.
Soil profile
The mineral and organic components of soil are differentiated into horizons or strata of variable depth. Each horizon
differs in morphology, physical structure, and chemical and biological characteristics. These horizons are evident
when a vertical cut is made through the soil, revealing the soil profile. The widely accepted structure of the soil
profile is as follows:
O Organic litter of loose leaves and debris.

A1 Rich in humus and dark in color.
A2 Zone of maximum leaching of minerals; readily available minerals to plant roots present in this layer.
B Little organic material and chemical composition is largely that of the underlying rock; also referred to as
the zone of accumulation since minerals from above and below tend to concentrate here.
C Parent rock, which is weakly weathered.
D Unweathered bedrock.
The soil profile and the relative thickness of the horizons are generally characteristic for different climatic regions and
different topographical situations. For example, in grassland soil humification is rapid, but mineralization is slow. In
forest soil litter and root decay slowly. Hence humus layer is narrow, but mineralization is rapid so B horizon is broad.
Ecology 563
Soil water
Within the soil system, the storage of water is influenced by several forces. According to the nature of interaction
between soil particles and water molecules, soil water may be classified into the following types:
Hygroscopic water Water present as a thin film around soil particles and remains firmly attached is called as
the hygroscopic water. It is not utilized by plants hence it is unavailable to plants.
Capillary water Water present in thin and narrow capillaries formed by soil particles and widely utilized
by plants. The water present in soil, which can be utilized by plants is called chresard
(available water).
Gravitational water Water percolate deep into the soil due to the gravitational force of the Earth that constitutes
ground water. It is not available to plants.
Chemically bound water Water present in the form of hydrated oxides of iron, aluminium, silicon, etc. is described
as chemically bound water and is not available to plants.
Air and Atmosphere

Atmosphere is the gaseous mass or envelope surrounding a celestial body, especially the one surrounding the Earth
and retained by the celestial body’s gravitational field. The atmosphere is densest at the surface of the Earth because
gravity pulls the molecules of gas toward the center of the Earth. The atmosphere is divided into four general layers:
Troposphere More than 80 percent of the mass of the atmosphere and virtually all of the water vapour,
clouds, and precipitation occur in the troposphere. At the poles, the troposphere may extend
up to 5–6 km, while at the equator it is about 18 km. In the troposphere, temperature
typically decreases with altitude at 5–7°C per km.
Stratosphere This layer extends up to 50 km and a thin layer of ozone is present at the height of 15 to
30 km. The troposphere and stratosphere together account for about 99.9 percent of the
mass of the atmosphere.
Mesosphere This layer starts at 50 km above Earth’s surface and goes up to 85 km. As we move up in
the mesosphere, the temperature gets colder. The top of the mesosphere is the coldest
part of Earth’s atmosphere (the temperature is around –90°C).
Thermosphere It extends from about 90 km to between 500 and 1000 km above the Earth’s surface.
Within the thermosphere temperature rises continually and go beyond 1000°C. Within the
thermosphere, there is a relatively dense band of charged particles called the ionosphere.
Thermosphere
100
80
Altitude (km)
Mesosphere
60
40
Stratosphere
20
Troposphere
0 100 200 300 400 500
Temperature (K)
Figure 5.2 Temperature profile of the atmosphere.

Ecology 571
5.7.3 Productivity
The rate of biomass production per unit area is called productivity. The primary productivity of a community is
the rate at which biomass (biomass is a measure of the mass of the living organic material in a specific area or
ecosystem) produced per unit area by plants, the producers. The term ‘primary’ indicates that we are concerned
with the first trophic level in the system. It can be expressed either in units of energy (e.g. Jm–2 day–1) or dry
organic matter (e.g. kg ha–1 year–1) or carbon (e.g. g Cm–2 year–1). The total fixation of energy by photosynthesis
is referred to as gross primary productivity (GPP). The proportion which remains after respiration losses in the
plant is termed net primary productivity (NPP). Thus, the difference between GPP and respiration is known as net
primary productivity. NPP represents the actual rate of production of new biomass that is available for consumption
by heterotrophic organisms (bacteria, fungi and animals). An ecosystem’s NPP should not be confused with the
standing crop (a measure of total biomass of photosynthetic autotrophs present at a given time). NPP is the amount
of new biomass added in a given period of time. The rate of production of new biomass by heterotrophs is called
secondary productivity. The rate of storage of organic matter not used by heterotrophs during the period under
consideration is termed as net community productivity.
Secondary productivity by herbivores is invariably less than that of the plants on which they feed. Where has the
missing energy gone? First, not all of the plant biomass produced is consumed alive by herbivores. Second, not all
the plant biomass that is eaten by herbivores (or herbivores biomass eaten by carnivores) is assimilated and made
available for incorporation into consumer’s biomass. Some is lost in the form of feces. Third, not all the energy that
has been assimilated is actually converted into biomass. A proportion is lost as respiratory heat. This occurs both
because no energy conversion process is ever 100% efficient and also because animals do work which requires
energy, again released as heat.
Patterns in primary productivity: The net primary production of the planet is estimated to be about 105 petagrams
of carbon per year (1 Petagram, Pg=1015 g). Of this, 56.4 Pg C year–1 is produced in terrestrial ecosystems and
48.3 Pg C year–1 in aquatic ecosystems.
Open ocean 65.0 125 24.4

Continental shelf 5.2 360 5.6
Extreme desert, rock, sand, ice 4.7 3 0.04
Desert and semidesert scrub 3.5 90 0.9
Tropical rain forest 3.3 2200 22
Savanna 2.9 900 7.9
Cultivated land 2.7 600 9.1
Boreal forest (taiga) 2.4 800 9.6
Temperate grassland 1.8 600 5.4
Woodland and shrubland 1.7 700 3.5
Tundra 1.6 140 0.6
Tropical seasonal forest 1.5 1600 7.1
Temperate deciduous forest 1.3 1200 4.9
Temperate evergreen forest 1.0 1300 3.8
Swamp and marsh 0.4 2000 2.3
Lake and stream 0.4 250 0.3
Estuary 0.3 1500 1.2
Algal beds and reefs 0.1 2500 0.9
Upwelling zones 0.1 500 0.1
0 1 2 3 4 5 6 0 500 1000 1500 2000 2500 0 5 10 15 20 25
(a) (b) (c)

2
Terrestrial ecosystem Percentage of Earth’s Average NPP (g/m /yr) Percentage of Earth’s NPP
Aquatic ecosystem surface area
Figure 5.6 Net Primary Productivity (NPP) of different ecosystems. Different ecosystems vary considerably in their
NPP as well as in their contribution to the total production on the Earth. Tropical rain forests and algal beds and coral
reefs are among the most productive ecosystems. Although estuaries and coral reefs have very high NPP, their total
contribution to global production is relatively small because these ecosystems are not very large. Similarly, the open
ocean contributes maximum in terms of Earth’s total NPP than any other ecosystem, because of its very large size
but average NPP is small.
572 Ecology
Thus, although oceans cover about two-thirds of the Earth’s surface, they account for less than half of its production.
Terrestrial primary production varies considerably across the surface of the Earth and among different ecosystem
types. Terrestrial primary production, both NPP and GPP, vary from north to south (or latitudinally) due to gradients
in plant community composition, growing season length, precipitation, temperature and solar radiation. NPP generally
declines from tropical regions to the poles because of temperature and light limitations. Tropical forests tend to
be much more productive than other terrestrial ecosystems, with temperate forests, tropical savannah, croplands
and boreal forests. Desert and Tundra biomes, limited by precipitation and temperature respectively, contain the
least productive ecosystems.
Measurement of GPP and NPP in an aquatic ecosystem
The general procedure for measurement of GPP and NPP in aquatic system is very simple. One takes a series of
small glass bottles with stoppers and half of them are wrapped with some material such as tinfoil so that no light
penetrates. These are called the light and dark bottles, respectively. The bottles are filled with water taken from a
particular place (e.g. ponds); this water contains aquatic organisms (plants and animals). The oxygen concentration
in each bottle is measured, the bottles are sealed and then suspended for few hours (suppose 1 hour) at the same
depth from which the water was originally taken. After 1 hour, the oxygen concentration is again measured in each
bottle. In the light bottle there is photosynthesis (or GPP) as well as respiration (R). The difference between these
two processes is NPP = GPP – R. In the dark bottle, there is no photosynthesis and only respiration.
Let us assume,
Initial oxygen concentration = 8 mg O2 /L
Oxygen concentration in light bottle after 1 hour = 10 mg O2 /L
Oxygen concentration in dark bottle after 1 hour = 5 mg O2 /L
The oxygen increased in the light bottle compared to the initial is due to photosynthesis, and the oxygen decreased
in the dark bottle due to respiration. With this information we can calculate the respiration, NPP, and GPP for our
system:
(Light – Initial) = (10 – 8) = 2 mg/L/hr = NPP
(Initial – Dark) = (8 – 5) = 3 mg/L/hr = Respiration
(Light – Dark) = (10 – 5) = 5 mg/L/hr = GPP
5.7.4 Energy flow

The Earth is an open system for energy. Energy flow is the key function in the ecosystem. The operation of an
ecosystem, is consistent with the laws of thermodynamics that deal with the relationships between energy and
matter in a system. The behaviour of the energy in the ecosystem is based on two basic laws of thermodynamics.
The first law of thermodynamics which states that energy cannot be created or destroyed but only transformed.
The first law is also called the law of conservation of energy.
The Sun is the ultimate source of energy for almost all ecosystems present on the Earth. Photosynthetic organisms
convert solar energy to chemical energy, but the total amount of energy does not change. The total amount of
energy stored in organic molecules synthesized by the process of photosynthesis plus the amounts dissipated as
heat must equal the total solar energy intercepted by the photosynthetic organism. In other words, energy may
change form (e.g. from radiant to chemical) but not amount.
As the energy moves through an ecosystem, it does change form. During energy transformation, some amount of
energy is converted to a form that’s unusable (unavailable to do work). In most cases, this unusable energy takes
the form of heat. So, every time an energy transformation happens, some amount of useful energy will move from
the useful to the useless category. Energy in the form of heat that does not do work goes to increase the randomness
(disorder) of the universe. The degree of randomness or disorder in a system is called its entropy. The second
law of thermodynamics states that every energy transformation that takes place will increase the entropy of the
universe. In other words, energy transformations cannot be 100% efficient; some energy is always lost as heat.
Ecology 573
In ecosystems, energy flows unidirectionally. The conversion of solar energy to chemical energy by the process of
photosynthesis is the starting point of energy flow within ecosystems. The fraction of incoming solar radiant energy
that the producers capture is small. Only about 1–5 percent energy of incident solar radiation, or 2–10 percent of
PAR (Photosynthetically Active Radiation) is actually captured by the photosynthetic process. The solar energy not
used for photosynthesis is immediately converted to heat.
The producers carry out respiration simultaneously in which they break down some of the organic compounds in
their bodies to release chemical energy. A portion of this chemical energy is used to make ATP, which in turn uses
to power various metabolic processes. Ultimately, the chemical energy released by respiration is converted to heat.
If organisms convert some chemical energy to heat, the conversion is one-way; they cannot use heat as a source
of energy. The chemical energy from producer passes from one heterotroph trophic level to the next. Only the
energy captured in net productivity of producers can be used by other trophic levels. As chemical energy move from
one trophic level to the next, a great deal of the energy is diverted all along the way. It means that, the amount
of chemical energy available to secondary consumers (i.e. primary carnivores) is far less than that available to
primary consumers (i.e. herbivores) and the amount available to tertiary consumers (i.e. secondary carnivores) is
far less than that available to secondary consumers.
Why does the amount of energy decrease as energy is passed from one trophic level to the next? Consider the use
of energy by the herbivore (i.e. primary consumers) as an example. After an herbivore ingests some plant biomass
(i.e. food), it produces faeces. The chemical energy in the faecal matters is not passed along to the primary carnivore.
The chemical energy of the food that is assimilated by the herbivore is used for a number of functions. Part of the
assimilated energy is liberated by cellular respiration to be used for tissue repair, body movements, and other such
functions. The energy used in these ways turns to heat and is not passed along to the primary carnivore. Some of
the ingested chemical energy is converted into the biomass of the herbivore and can serve as food for a primary
carnivore. However, some herbivore individuals die rather than being eaten by carnivores. At the end, much of the
chemical energy preset in herbivores do not transfer to carnivores. Ecologists figure as a rule of thumb that the
amount of energy available to a trophic level over time is about 10% of that available to the preceding level over
the same period of time. Essentially all of the chemical-bond energy captured by photosynthesis in an ecosystem
eventually becomes heat as the chemical energy is used by various trophic levels.
Sun
Solar energy
Producers
D R
D Primary consumers R
Herbivores
Heat Decomposers Heat
D R
Secondary consumers
Primary carnivores
D R
Tertiary consumers
R = Respiration | D = Death Secondary carnivores
Figure 5.7 The flow of energy through an ecosystem. The energy that enters the ecosystem as solar energy (radiant
energy) and is than passed along as chemical energy to successive trophic levels. At each step energy is diverted,
meaning that the chemical energy available to each trophic level is less than that available to the preceding trophic
level. Death at each level transfers energy to decomposers. Energy lost as heat at each level is returned to the external
environment.
574 Ecology
5.7.5 Energy flow model

A simplified representation of energy flow through ecosystem has been made in figure 5.8. The model attempts to
recognize the various inputs and fates of energy. Two aspects with respect to energy flow in the ecosystem need
careful consideration. First, the energy flows unidirectionally, i.e. from producers through herbivores to carnivores;
it cannot be transferred in the reverse direction. Second, the amount of energy flow decreases with successive
trophic levels.
Import of organic matter
DC
AL
PS GP RS NP HB CR TC
Storage
Export
PS=Photosynthesis CR=Carnivores
Community AL=Absorbed light DC=Decomposers
respiration
RS=Respiration GP=Gross production
TC=Top carnivores NP=Net production
HB=Herbivores
Figure 5.8 Energy flow diagram of a generalized ecosystem.
5.7.6 Transfer efficiencies

In ecosystems, living organisms are linked together by feeding relationships. Producers (or autotrophs) have the
ability to fix carbon through photosynthesis via chlorophylls in their leaves. Herbivores are the primary consumers
of organic molecules fixed by the producers. Carnivores are secondary consumers, living on the organic molecules
of the herbivores. There may be several levels of carnivores in any one ecosystem; in such cases the ultimate level
will be occupied by the top carnivore. The final groups of organisms in an ecosystem are decomposers, bacteria
and fungi which can break down the complex organic chemicals of dead materials and waste products.
The proportions of net primary production that flow along each of the possible energy pathways depend on transfer
efficiencies in the way energy is used and passed from one step to the next. A knowledge of the values of just three
categories of transfer efficiency is all that is required to predict the pattern of energy flow. These are consumption
efficiency (CE), assimilation efficiency (AE) and production efficiency (PE).
Consumption efficiency is the percentage of total productivity available at one trophic level (Pn–1) that is actually
consumed (ingested) by a trophic compartment one level up (In).
In
Consumption efficiency (CE) = ´ 100
Pn-1
In the case of secondary consumers, it is the percentage of herbivore productivity eaten by carnivores. Consumption
efficiencies of herbivores are very low, reflecting either the difficulty of utilizing plant material or the low herbivore
densities.
592 Ecology
Temperate deciduous forest biome
Location Eastern part of the United States and Canada, most of Europe and parts of China and Japan.
Precipitation 75–150 cm annually.
Temperature –30°C to 30°C, yearly average is 10°C, hot summers, cold winters
Taiga biome
Location 50 and 60° North latitudes.
Precipitation 40–100 cm annually.
Temperature –40°C to 20°C.
Climate
Climate is the long-term pattern of weather in a locality, region or even over the entire globe. It is the statistics
of weather, usually over a 30-year interval. It is measured by assessing the patterns of variation in temperature,
humidity, atmospheric pressure, wind, precipitation and other meteorological variables in a given region over long
periods of time.
The terms ‘climate’ and ‘weather’ have different meanings. Weather is the short-term properties (such as tempera-
ture, pressure, moisture) of atmospheric conditions for a specific place and time. Weather differs both spatially and
temporally. Two of the most important factors determining an area’s climate are air temperature and precipitation.
The climate of a region will determine which plants will grow there and which animals will inhabit it.
Climatic zone
Climatic zone represents any of the geographical zones (regions on the surface of the Earth loosely divided according
to latitude or longitude) loosely divided according to the prevailing climate and latitude. On the basis of variation in
mean temperature along latitude, the main climatic zones are:
1. Tropical (0°–20° latitude)
2. Subtropical (20°–40° latitude)
3. Temperate (40°–60° latitude)
4. Arctic and Antarctic (60°–80° latitude).
The mean temperature declines as we move from tropical to arctic region. A similar climatic zonation occurs with
increasing altitude in the mountains. A mountain located in a tropical regions will successively have tropical, subtrop-
ical, temperate and alpine zones with increasing altitude. Within each temperature-based climatic zone, the annual
precipitation (rainfall and /or snowfall) varies considerably.
5.9 Population ecology

A population is a collection of individuals of the same species that live together in a region. Population ecology is
the study of populations (especially population abundance) and how they change over time. It studies the spatial
and temporal patterns in the abundance and distribution of organisms and of the mechanisms that produce those
patterns.
5.9.1 Population characteristics

A population has several characteristics or attributes which is a function of the whole group and not of the individual.
Different populations can be compared by measuring these attributes. These attributes are population density,
natality, mortality, growth forms, distributions, etc. The study of the group characteristics or parameters of the
human population, their changes over time and prediction of future changes is known as demography.
Ecology 593
Population density
The size of the population is represented by its fundamental property called density. Density is expressed as the
total number of individuals per unit area or volume at a given time. Two types of densities are described – crude
density (it is the density per unit total space) and specific (ecological) density (it is the density per unit of habitat
space i.e. available area or volume that can actually be colonized by the population).
Determining population size: Usually population size is estimated by counting all the individuals from a smaller
sample area, then extrapolated over a larger area. Another common method is mark-recapture technique. Using
this method, a small random sample of the population is captured, marked, then released to disperse within the
general population. The marked individuals mix freely with unmarked individuals and within a short time are
randomly mixed within the population. The population is resampled and the numbers of marked and unmarked
individuals are recorded. We then assume that the ratio of marked to unmarked individuals in the second sample
taken is the same as the ratio of marked to unmarked individuals in the first sample. We can use a simple formula
for estimating total population size (N):
Total individuals marked in first sample × Size of second sample

N=
Number of marked individuals recaptures in second sample
Natality
Natality refers to the birth of individuals in a population. The natality rate (or birth rate) is expressed as the number
of individuals produced per female per unit time. To describe ‘rate’, we must specify time interval (such as one year
or one month) and a base population. Two bases are commonly used – Per capita or per individual (it is equivalent
to the number of births per individual per unit of time) or Per 1000 individuals.
Natality may be maximum natality or ecological natality. Maximum (sometimes called absolute or physiological)
natality is the theoretical maximum number of individuals produced under ideal environmental conditions (i.e. no
ecological limiting factors) and is a constant for a given population. Ecological or realized natality refers to number
of individuals produced under an actual or specific environmental condition. It is not a constant for a population
but may vary with the size and age composition of the population and the physical environmental conditions.
In ecology, fecundity and fertility is not same. The ‘fecundity’ can be described as the maximum reproductive output
potential of an individual over its lifetime under ideal environmental conditions. The term ‘fertility’ differs from
fecundity in that it describes the actual reproductive performance of an individual under prevailing environmental
conditions and it is a generalization of the terms ‘birth rate’ and ‘natality rate’.
Mortality
Mortality refers to the death of individuals in a population. Like natality rate, mortality rate (death rate) may be
expressed as the number of individuals dying in a given period. Mortality may be minimum mortality or ecological
mortality. A theoretical minimum mortality, a constant for a population, represents the loss under ideal or non-
limiting conditions. Ecological or realized mortality is the loss of individuals, under a given environmental condition.
Like ecological natality, it is not a constant but varies with population and environmental conditions.
Information of the death and survivor of a population with respect to age is represented in the form of table known
as life table. It is simply an age-specific account of mortality.
What is really vital for the population is not which members die, but which member survives? Consequently specific
mortality rate of a population is expressed by survivorship curve. Survivorship curves plot the number of surviving
individuals to a particular age. Survivorship curves are of three general types:
A highly convex curve (type I) is characteristic of the species in which the population mortality rate is low until near
the end of the life span. Many species of large animals such as deer, mountain sheep and man, show such curves.
A highly concave curve (type III) is characteristic of those species where the mortality rate is high during the young
stages. Oysters or shell fish show this type of curve. In oysters mortality is extremely high during free swimming larval
stages, but once an individual is well established on a favourable substrate, life expectancy improves considerably.
Chapter 06
Evolution
6.1 Origin of Life

Abiogenesis
Abiogenesis is the generation of life from non-living matter. Abiogenesis is now more precisely known as spontaneous
generation. This theory states that complex living organisms are generated from decaying organic substances e.g.
organism like mice spontaneously appears in stored grain or maggots spontaneously appear in meat.
Francesco Redi, an Italian physician, was the first who disproved the Theory of Spontaneous Generation by
performing a controlled experiment. In 1668, Redi performed an experiment to check whether maggots really
came from decaying meat. He did this by placing meat in a number of jars and covering half of them with fine
gauze while leaving the others uncovered. Maggots developed only on the meat in the uncovered jars. From this,
Redi concluded that the maggots did not come from the meat, but from tiny eggs that flies had laid on the meat.
Since flies could not land on the meat in the covered jars, they could not lay eggs on that meat, and no maggots
formed. Therefore, decaying meat could not produce maggots.
Later, Lazzaro Spallanzani (an Italian naturalist) performed a similar experiment with broth. He put broth into two
glass flasks and sterilized them by boiling the flask containing broth. One of the flasks was left open to the air. The
other flask was sealed up to keep out any organisms that might be floating in the air. Microorganisms developed
only in the uncovered flask. From this, Spallanzani concluded that the microorganisms did not come from the broth,
but were in the air that entered the flask.
Cool Wait
Broth heated Flask left open Growth
Open
Wait flask
Broth heated Flask sealed No growth Growth
Figure 6.1 Sterilized broth in open flask developed microbes whereas in sealed flask microbes not developed.
Unfortunately, many scientists were not convinced by his experiment. Louis Pasteur, a French chemist, finally
disproved the Theory of Spontaneous Generation in the mid 1800’s. He performed the same type of experiment
as Spallanzani. Louis Pasteur, however, allowed air to enter into the flask of sterile broth.
658 Evolution
He performed experiments with two flasks – one with a straight neck and other with S-shaped neck. Flask with a
straight neck allowed both air and microorganisms to enter whereas the other flask with S-shaped neck allowed only
air to enter but not microorganisms. The broth in the straight neck flask became contaminated with microorganisms
but the broth in the flask with an S-shaped neck did not become contaminated. Therefore, Louis Pasteur showed
that even though air could get in the flask, the broth did not produce microorganisms.
Wait
Boil No growth
Wait
Boil Break stem Microbial growth
Figure 6.2 No microbial growth occurs in flask with an S-shaped neck whereas microbes growth occurs in flask with
broken stem.
Scientists finally were convinced that living things, no matter how small, do not come from nonliving things. The
present theory of where living things come from is called biogenesis. This theory states that living things come
only from other living things. For example, mice come only from mice, and microorganisms such as bacteria can
only come from other bacteria. Since spontaneous generation was now proved incorrect, many scientists began to
wonder how life started on the Earth. Oparin and Haldane attempted to answer this question. They proposed that
life had arisen from simpler molecules on the lifeless earth under much different atmospheric conditions than exist
today. However, instead of life arising suddenly, as previous spontaneous generation theories proposed, Oparin
and Haldane believed that it occurred over a very long period of time.
Chemical evolution
Our current understanding of conditions on prebiotic Earth and the idea of a gradual chemical evolution toward life
were first proposed independently by A.I. Oparin and J.B.S. Haldane. The Oparin-Haldane model suggested that
under the strong reducing conditions theorized to have been present in the atmosphere of the early Earth (between
4.0–3.5 billion years ago), inorganic molecules would spontaneously form organic molecules (simple sugars and
amino acids). Oparin argued that a primeval soup (or Primordial soup) of organic molecules could be created in
an oxygenless atmosphere through the action of sunlight. These would combine in evermore complex ways until
they formed coacervate droplets. These droplets would grow by fusion with other droplets, and reproduce through
fission into daughter droplets.
Around the same time in the year 1929, J.B.S. Haldane published a paper in which he proposed that the Earth's
prebiotic oceans would have formed a hot dilute soup in which organic compounds could have formed.
In 1953, Stanley Miller, along with his graduate advisor Harold Urey, tested this hypothesis by constructing an
apparatus that simulated the Oparin-Haldane early earth.
Miller-Urey experiment
The Miller-Urey experiment, a classic experiment fundamentally established that the Earth’s primitive atmosphere
was capable of producing the building blocks of life from inorganic materials. It established that the conditions that
existed in the Earth’s primitive atmosphere were sufficient to produce organic molecules like amino acids.
Evolution 669
Problem
Whether the limbs modified into wings of bats and the wings of birds is an example of evolutionary analogy or homology?
What about whale fins compared to fish fins?
Solution
Bat and bird wings have the same function and the same origin (they are modified limbs) so they are analogous and ho-
mologous organs. Whale fins are a modification of the posterior limbs while fish fins although having the same function,
do not come from modified limbs; so they are analogous but not homologous structures.
6.4 Natural selection

Selection is a composite of all the forces that cause differential survival and differential reproduction among genetic
variants. When the selective agencies are primarily those of human choice, the process is called artificial selection.
When the selective agencies are not those of human choice, it is called natural selection. Natural selection is the
nonrandom process by which biological traits become more or less common in a population as a function of the
differential reproduction of their bearers or differences in the rate of survival. Natural selection can act on any
heritable phenotypic trait and operate among any entities that reproduce, show inheritance of their characteristics
from one generation to the next, and vary in fitness.
Natural selection is the machine that drives evolution. It also explains adaptation. Some evidence of natural selection
has been seen in nature, but not to an extent that would change a species in any meaningful way.
Evidences of natural selection

Industrial melanism
Industrial melanism is a phenomenon used to describe the evolutionary process in which initially light colored
organism’s population become dark as a result of natural selection. Industrial melanism affected over 70 species
of moths in England. It has been best studied in the peppered moth, Biston betularia. Prior to 1800, the typical
moth species had a light color pattern. Dark colored or melanic moths were rare. A population that contains more
than one recognizable form is polymorphic (the condition is called polymorphism). During the industrial revolution,
soot and other industrial wastes darkened tree trunks and killed off lichens. Because light colored peppered moths
rely on camouflage to avoid predation, this sudden change in their environment made them highly vulnerable to
predators. In course of time, the light-colored moth became rare and the dark colored moth became abundant.
The cause of this change was thought to be selective predation by birds, which favoured camouflage coloration in
the moth. By 1886, dark colored (melanic) were far more common — illustrating rapid evolutionary change.
Evolution of drug resistant HIV
The evolution of drug resistance in HIV also provides an example of natural selection. Researchers have developed
numerous drugs to combat this pathogen. Drug 3TC (lamivudine) is one of them. It is a nucleoside inhibitor and
acts as a molecular analog of cytidine, C. When reverse transcriptase places a 3TC molecule, instead of a C, in a
replicating DNA chain, chain elongation is stopped and the reproduction of HIV is also stopped. This drug-susceptible
reverse transcriptase binds both 3TC and C. The drug 3TC resistant varieties of HIV carry slightly different versions
of reverse transcriptase that are able to discriminate between the drug and the normal C. It binds only C, and not
3TC. The viruses that carry these genes have no advantage in the absence of 3TC; in fact, they replicate slowly
than those that carry the most common form. But once 3TC is added to their environment it becomes a powerful
selecting force, favouring reproduction of resistant individuals. Here drug does not create resistant HIV; it selected
resistant HIV that was already present in the population. The increase in the frequency of drug-resistant HIV is
almost certainly driven by natural selection.
670 Evolution
Modes of natural selection

If an individual has some form of a trait that gives him a better chance of survival in a given environment, evidently
his offspring inherit the trait. This is known as differential survival. Differential survival would refer to the survival
of individuals in an environment to which they are well-adapted. The adaptations are genetically controlled and
heritable. Differential survival leads to differential reproduction of alleles responsible for the survival of the next
generation. Natural selection means differential reproduction among members of the same gene pool. Natural
selection can alter the frequency of heritable traits in three ways. These three modes of natural selection are:
1. Directional selection
2. Stabilizing selection
3. Disruptive selection
Under directional selection, individuals at one end of the frequency distribution do especially well, and so the
frequency distribution of the trait in the subsequent generation is shifted from where it was in the parental
generation. In nature, directional selection can occur when one of the phenotypic extremes becomes selected for
or against, usually as a result of changes in the environment. The industrial melanism provides an example of
directional selection.
Features of directional selection

• Occurs due to the change in the environment in a particular direction.
• Favours the phenotype which is non-average or extreme.
• Alters the mean value of the trait in the population in one direction.
• Favours accumulation of those mutations that increases fitness in the changing environment.
• Eliminate normal or average individuals.
Under stabilizing selection, extreme varieties from both ends of the frequency distribution are eliminated. This type
of selection tends to favour intermediate types. At the genetic level, stabilizing selection acts to keep a population
well adapted to its environment. In this situation, individuals closer to the average for a given trait will have higher
fitness. A real-life example is that of birth weight of human babies.
Features of stabilizing selection

• Operates in the constant or unchanging environment.
• Keeps a population genetically constant.
• Favours the average or normal phenotypes.
• Introduces homozygosity in the population.
• Checks accumulation of mutation.
Under disruptive selection, both extremes are favoured at the expense of intermediate varieties. It can be viewed
as the opposite of stabilizing selection because the intermediate types are selected against. A clear example is
provided by the different color pattern of the butterfly Papilio dardanus.
Features of disruptive selection

• Previously homologous populations breakup into several different adaptive forms.
• Extreme values have highest fitness and intermediate or mean values are relatively disadvantageous.
• Occurs when a population previously adapted to a non-homologous environment is subjected to divergent
selection pressure in different parts of its distributional area.
680 Evolution
æ 1 ö
H' = ç1 - ÷H
è 2Nø
This equation tells us that in one generation, random genetic drift causes the heterozygosity to decline by a factor
of 1/2N. Over many generations, the heterozygosity will eventually be reduced to 0, at which point all genetic
variability in the population will be lost. At this point the population will possess only one allele of the gene, and
either p = 1 and q = 0, or p = 0 and q = 1. Thus, through random changes in allele frequencies, drift steadily
erodes the genetic variability of a population, ultimately leading to the fixation and loss of alleles.
Problem
If the frequency of a homozygous dominant genotype in a randomly mating population is 0.09, what is the frequency of
the dominant allele? What is the combined frequency of all the other alleles of this gene?
Solution
p2 = 0.09, and so p = (0.09)1/2 = 0.30. All other alleles have a combined frequency of 1 – 0.30 = 0.70.
Problem
A particular recessive disorder is present in one in ten thousand individuals. If the population is in Hardy-Weinberg equi-
librium, what are the frequencies of the two alleles?
Solution
If the population is in equilibrium, there should be p2 of AA + 2pq of Aa + q2 of aa individuals.

Since, 1/10,000 shows the recessive trait, this is q2.
Therefore, q = 1 / 10,000 = 0.0001 = 0.01.
Since, p + q = 1, then p = 1 – 0.01 = 0.99.
6.6.3 Inbreeding
Inbreeding is a mating between individuals that are closely related through common ancestry. The extent of inbreeding
occurring in a population is measured by inbreeding coefficient. The inbreeding coefficient (expressed as F) is
the probability that two alleles of a given gene in an individual are identical by descent. Such a genotype would be
homozygous and considered autozygous since the alleles were inherited from a common ancestor (homozygosity
by descent). Hence, inbreeding coefficient is also defined as the probability of autozygosity. When two alleles are
not identical by descent, we call the genotype allozygous (allo- means other). Note that allozygous can be either
homozygous or heterozygous.
First generation Aa Aa
Second generation aA aa AA Aa
Third generation aa aA AA
Autozygous
Allozygous
Figure 6.11 A diagram showing how both allozygous and autozygous individuals can be generated within the same
family. Two unrelated heterozygotes in first generation produced four offspring, all with different genotypes (second
generation). Inbreeding occurs among the siblings of second generation resulting in two allozygous and one autozygous
individual in third generation. The allozygous individuals include both a heterozygote and a homozygote.
Evolution 693
Punctuated equilibria and phyletic gradualism

According to the theory of punctuated equilibrium (evolution by jerks), evolution proceeds relatively rapidly during
speciation: between speciation events, the population remains relatively constant in a condition called stasis.
Stasis is broken by rare events of large net change, characterized by rapid events of branching speciation called
cladogenesis. It considers that most sexually reproducing species will show little change for most of their geological
history. When phenotypic evolution occurs, it is localized in rare events of branching speciation and occurs relatively
quickly compared to the species full and stable duration on earth.
Theory of punctuated equilibrium is commonly contrasted against the hypothesis of phyletic gradualism, which states
that most evolution occurs uniformly and by the steady and gradual transformation of whole lineages (anagenesis).
According to phyletic gradualism:
• Evolution occurs at a constant rate.
• New species arise by the gradual transformation of ancestral species.
Species descended from A new species changes

a common ancestor gradually most as it buds from a
diverge more and more in parent species and then
their morphology as they changes little for the
acquire unique adaptations. rest of its existence.
Time
Phyletic gradualism Punctuated equilibrium
Figure 6.15 Phyletic gradualism and punctuated equilibrium.
6.11 Molecular phylogeny

Phylogenetics is the science of estimating and analyzing evolutionary relatedness among various group of organisms.
Molecular phylogenetics is the use of the structure of molecules to gain information on an organism’s evolutionary
relationships. Evolution, at the molecular level, is observable as nucleotide changes in the nucleic acids and amino
acid changes in proteins. Nucleic acids (DNA and RNA) and proteins are ‘information molecules’ in that they retain
information of an organism’s evolutionary history. The approach is to compare nucleic acid or protein sequences
from different organisms using computer programmes and estimate the evolutionary relationships based on the
degree of homology between the sequences. Nucleic acids and proteins are linear molecules made of smaller units
called nucleotides and amino acids, respectively. The nucleotide differences within a gene or amino acid differences
within a protein reflect the evolutionary distance between two organisms. In other words, closely related organisms
will exhibit fewer sequence differences than distantly related organisms. Thus, the field of molecular phylogenetics
can be defined as the study of evolutionary relationships of genes or proteins by analyzing mutations at various
positions in their sequences and developing hypotheses about the evolutionary relatedness of the biomolecules.
Based on the sequence similarity of the molecules, evolutionary relationships between the organisms can often be
inferred. Phylogenetic studies construct the tree like pattern that describes the evolutionary relationship between
the organisms being studied.
Selection of molecular markers: To construct a molecular phylogenetic tree, it is necessary to compare nucleic
acid sequence (or protein sequence). The decision to use nucleotide or protein sequences depends on the properties
of the sequences and the purposes of the study. A nucleotide sequence yields more phylogenetic information than
protein sequences, the nucleotide sequences of a pair of homologous genes having a higher information content
than the amino acid sequences of the corresponding proteins, because mutations that result in synonymous changes
alter the DNA sequence, but do not affect the amino acid sequence.
694 Evolution
Gly Ala Ile Leu Asp Arg
{
{
{
{
{
{
GGAGCCATATTAGATAGA
GGAGCAATTTTAGATAGA
{
{
{
{
{
{
Gly Ala Ile Leu Asp Arg
Figure 6.16 DNA yields more phylogenetic information than protein.
Hence, for studying very closely related organisms, nucleotide sequences, which evolve more rapidly than proteins,
can be used. If the phylogenetic relationships to be delineated are at the deepest level, such as between bacteria
and eukaryotes, using conserved protein sequences makes more sense than using nucleotide sequences because
protein sequences are relatively more conserved as a result of the degeneracy of the genetic code. Protein sequences
can remain the same while the corresponding DNA sequences have more room for variation.
Nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) have been most commonly used for phylogenetic
studies. The DNA sequence chosen for phylogenetic analysis must display variability in the organisms being studied.
If there is no variability then there is no phylogenetic information. For example, for evolutionary analysis of different
individuals within a population, mtDNA are often used. mtDNA is known to evolve much faster than the nuclear
genome. Consequently, mtDNA have been used mostly to examine phylogenetic relationships in relatively lower
categorical levels such as in families, genera, species or populations. Although mtDNA has evolved faster than nuclear
genome, 12S rDNA, however, is highly conserved. For studying the evolution of more widely divergent groups of
organisms, one may choose either slowly evolving nucleotide sequences, such as nuclear rDNA or protein sequences.
Molecular clock
Rates of molecular evolution and amounts of genetic variation can be measured. It can be estimated from the amino
acid sequence of a protein, or nucleotide sequence of a region of DNA, in two or more species. The molecular clock
is a technique in molecular evolution to relate the time that the two species diverged to the number of molecular
differences measured between the species’ DNA sequences or proteins. It is sometimes called a gene clock or
evolutionary clock. The concept of molecular clock is based on hypothesis that DNA and protein sequences evolve
at a rate that is relatively constant over time and among different organism. This constancy is used to estimate the
length of time that various organisms have been diverging from one another by measuring the degree of difference
between two sequences. The molecular clock hypothesis was originally proposed by researchers Emile Zuckerkandl
and Linus Pauling on the basis of empirical observations, but it soon received theoretical backing when biologist
Motoo Kimura developed the neutral theory of molecular evolution in 1968.
Neutral theory of molecular evolution

The neutral theory of molecular evolution (also, simply the neutral theory of evolution) is an influential theory
introduced by Motoo Kimura. Kimura suggested that a large fraction of new mutations do not have an effect on
evolutionary fitness, so natural selection would neither favour nor disfavour them. Eventually, each of these neutral
mutations would either spread throughout a population and become fixed in all of its members, or they would
be lost entirely in a stochastic process called genetic drift. Kimura then showed that the rate at which neutral
mutations become fixed in a population (known as the substitution rate) is equivalent to the rate of appearance of
new mutations in each member of the population (the mutation rate). Provided that the mutation rate is consistent
across species, the substitution rate would remain constant throughout the tree of life.
Kimura subsequently summarized his theory as follows:

‘This neutral theory claims that the overwhelming majority of evolutionary changes at the molecular level are not
caused by selection acting on advantageous mutants, but by random fixation of selectively neutral or very nearly
neutral mutants through the cumulative effect of sampling drift (due to finite population number) under continued
input of new mutations.’

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Life Sciences Fundamentals and Practice - II

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Life Sciences

Fundamentals and Practice II

Pranav Kumar | Usha Mina

Life Sciences : Fundamentals and Practice

ISBN: 978-81-906427-7-4 (paperback)

Copyright © 2017 by Pathfinder Publication, all rights reserved.

This book contains information obtained from authentic and highly

Publisher : Pathfinder Publication

1.1.1 Mendel’s laws of inheritance 3

Law of independent assortment 5

1.1.2 Incomplete dominance and codominance 7

1.1.3 Multiple alleles 8

1.1.4 Lethal alleles 10

1.1.5 Penetrance and expressivity 11

1.2 Chromosomal basis of inheritance 14

1.3 Gene interaction 15

1.3.1 Dominant epistasis 17

1.3.2 Recessive epistasis 18

1.3.3 Duplicate recessive epistasis 19

1.3.4 Duplicate dominant interaction 19

1.3.5 Dominant and recessive interaction 19

1.3.6 Genetic dissection to investigate gene action 21

1.4 Genetic linkage and gene mapping 22

1.4.1 Genetic mapping 26

1.4.2 Gene mapping from two point cross 28

1.4.3 Gene mapping from three point cross 29

1.4.4 Interference and coincidence 31

1.5 Tetrad analysis 32

1.5.1 Analysis of ordered tetrad 34

1.5.2 Analysis of unordered tetrad 35

1.6 Sex chromosomes and sex determination 36

1.6.1 Sex chromosome 36

1.6.2 Sex determination in animals 37

Sex determination in humans 39

Genic balance theory of sex determination in Drosophila 40

1.6.3 Sex determination in plants 41

1.6.5 Sex-linked traits and sex-linked inheritance 41

1.6.6 Sex-limited traits 43

1.6.7 Sex-influenced traits 43

1.6.8 Pedigree analysis 43

1.7 Quantitative inheritance 47

1.7.1 Quantitative trait locus analysis 51

1.8 Extranuclear inheritance and maternal effect 52

1.8.1 Maternal effect 55

1.9.1 Human karyotype 57

1.9.2 Chromosome banding 58

1.9.3 Variation in chromosome number 59

1.9.4 Chromosome aberrations 63

1.9.5 Position effect 68

1.10.1 Genome complexity 70

1.10.2 Transposable elements 73

1.10.5 Acquisition of new genes 84

1.10.6 Fate of duplicated genes 85

1.10.7 Gene families 86

1.10.8 Human nuclear genome 87

1.10.9 Organelle genome 88

1.10.10 Yeast S. cerevisiae genome 89

1.10.11 E. coli genome 89

1.11 Eukaryotic chromatin and chromosome 90

1.11.1 Packaging of DNA into chromosomes 92

1.11.2 Histone modification 95

1.11.3 Heterochromatin and euchromatin 97

1.11.4 Polytene chromosomes 100

1.11.5 Lampbrush chromosomes 101

1.11.6 B-chromosomes 102

1.12 DNA replication 102

1.12.1 Semiconservative replication 102