Biotechnology A Problem Approach PDF

Biotechnology
A problem approach
Third edition
Pranav Kumar
Former faculty,
Department of Biotechnology
Jamia Millia Islamia,
New Delhi, India
Usha Mina
Scientist,
Division of Environmental Sciences
Indian Agricultural Research Institute (IARI),
New Delhi, India
Pathfinder Publication
New Delhi, India
Pranav Kumar
Former faculty,
Department of Biotechnology
Jamia Millia Islamia,
New Delhi, India
Usha Mina
Scientist,
Division of Environmental Sciences
Indian Agricultural Research Institute (IARI),
New Delhi, India
Biotechnology A problem approach, Third edition

ISBN: 978-93-80473-00-0 (paperback)
Copyright © 2014 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
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private use, without written permission from the publisher.
Publisher : Pathfinder Publication

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Preface
The present century has been considered as one that belongs to biotechnology. This
branch of science has been viewed as something vital for life with numerous scientific
applications in several fields of human endeavours. The branch of science is significant for
mankind that many of the big discoveries of the second half of the last century and early
this century would not have been possible in the absence of our accomplishments in this
discipline. Biotechnology – A problem approach, covers fundamentals and techniques.
This book provides a balanced introduction to all major areas of the subject. The chap-
ters such as Biomolecules and catalysis, Bioenergetics and metabolism, Cell structure
and functions, Immunology, Bioinformatics and Bioprocess engineering were selected
in a sharply focused manner without overwhelming or excessive detail. 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 knowl-
edge.
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, Abhai Kumar, Rizwan Ansari, Lekha Nath, Harleen Kaur and Mr. 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.
Pranav Kumar
Usha Mina
iii
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Contents
Chapter 1
Biomolecules and Catalysis
1.1 Amino acids and Proteins 1
1.1.1 Optical properties 2
1.1.2 Absolute configuration 4
1.1.3 Standard and non-standard amino acids 5
1.1.4 Titration of amino acids 8
1.1.5 Peptide and polypeptide 11
1.1.6 Peptide bond 12
1.1.7 Protein structure 14
1.1.8 Denaturation of proteins 18
1.1.9 Solubilities of proteins 19
1.1.10 Simple and conjugated proteins 20
1.2 Fibrous and globular proteins 20
1.2.1 Collagen 21
1.2.2 Elastin 22
1.2.3 Keratins 23
1.2.4 Myoglobin 23
1.2.5 Hemoglobin 25
1.2.6 Models for the behavior of allosteric proteins 29
1.3 Protein folding 31
1.3.1 Molecular chaperones 32
1.3.2 Amyloid 33
1.4 Protein sequencing and assays 34
1.5 Nucleic acids 42
1.5.1 Nucleotides 42
1.5.2 Chargaff’s rules 46
1.6 Structure of dsDNA 47
1.6.1 B-DNA 47
1.6.2 Z-DNA 49
1.6.3 Triplex DNA 49
1.6.4 G-quadruplex 50
1.6.5 Stability of the double helical structure of DNA 51
1.6.6 Thermal denaturation 51
1.6.7 Quantification of nucleic acids 53
1.6.8 Supercoiled forms of DNA 53
1.6.9 DNA: A genetic material 56
v
1.7 RNA 58
1.7.1 Alkali-catalyzed cleavage of RNA 60
1.7.2 RNA world hypothesis 61
1.7.3 RNA as genetic material 61
1.8 Carbohydrates 63
1.8.1 Monosaccharide 63
1.8.2 Epimers 64
1.8.3 Cyclic forms 65
1.8.4 Derivatives of monosaccharide 67
1.8.5 Disaccharides and glycosidic bond 68
1.8.6 Polysaccharides 70
1.8.7 Glycoproteins 72
1.8.8 Reducing and non-reducing sugar 73
1.9 Lipids 73
1.9.1 Fatty acids 74
1.9.2 Triacylglycerol and Wax 75
1.9.3 Phospholipids 76
1.9.4 Glycolipids 78
1.9.5 Steroid 79
1.9.6 Eicosanoid 79
1.9.7 Plasma lipoproteins 81
1.10 Vitamins 82
1.10.1 Water-soluble vitamins 82
1.10.2 Fat-soluble vitamins 86
1.11 Enzymes 89
1.11.1 Naming and classification of enzyme 90
1.11.2 What enzyme does? 91
1.11.3 How enzymes operate? 92
1.11.4 Enzyme kinetics 94
1.11.5 Enzyme inhibition 102
1.11.6 Regulatory enzymes 105
1.11.7 Isozymes 106
1.11.8 Zymogen 107
1.11.9 Ribozyme 108
1.11.10 Examples of enzymatic reactions 108
Chapter 2
Bioenergetics and Metabolism
2.1 Bioenergetics 117
2.2 Metabolism 122
vi
2.3 Respiration 123
2.3.1 Aerobic respiration 123
2.3.2 Glycolysis 124
2.3.3 Pyruvate oxidation 129
2.3.4 Krebs cycle 131
2.3.5 Anaplerotic reaction 134
2.3.6 Oxidative phosphorylation 135
2.3.7 Inhibitors of electron transport 139
2.3.8 Electrochemical proton gradient 140
2.3.9 Chemiosmotic theory 141
2.3.10 ATP synthase 142
2.3.11 Uncoupling agents and ionophores 144
2.3.12 ATP-ADP exchange across the inner mitochondrial membrane 144
2.3.13 Shuttle systems 145
2.3.14 P/O ratio 147
2.3.15 Fermentation 148
2.3.16 Pasteur effect 150
2.3.17 Warburg effect 150
2.3.18 Respiratory quotient 151
2.4 Glyoxylate cycle 151
2.5 Pentose phosphate pathway 152
2.6 Entner-Doudoroff pathway 154
2.7 Photosynthesis 154
2.7.1 Photosynthetic pigment 155
2.7.2 Absorption and action spectra 158
2.7.3 Fate of light energy absorbed by photosynthetic pigments 160
2.7.4 Concept of photosynthetic unit 161
2.7.5 Hill reaction 162
2.7.6 Oxygenic and anoxygenic photosynthesis 162
2.7.7 Concept of pigment system 163
2.7.8 Stages of photosynthesis 165
2.7.9 Light reactions 165
2.7.10 Prokaryotic photosynthesis 171
2.7.11 Non-chlorophyll based photosynthesis 173
2.7.12 Dark reaction: Calvin cycle 174
2.7.13 Starch and sucrose synthesis 177
2.8 Photorespiration 178
2.8.1 C4 cycle 179
2.8.2 CAM pathway 180
2.9 Carbohydrate metabolism 182
2.9.1 Gluconeogenesis 182
vii
2.9.2 Glycogen metabolism 187
2.10 Lipid metabolism 192
2.10.1 Synthesis and storage of triacylglycerols 192
2.10.2 Biosynthesis of fatty acid 194
2.10.3 Fatty acid oxidation 198
2.10.4 Biosynthesis of cholesterol 205
2.10.5 Steroid hormones and Bile acids 206
2.11 Amino acid metabolism 208
2.11.1 Amino acid synthesis 208
2.11.2 Biological nitrogen fixation 211
2.11.3 Amino acid catabolism 214
2.11.4 Molecules derived from amino acids 220
2.12 Nucleotide metabolism 221
2.12.1 Nucleotide synthesis 221
2.12.2 Nucleotide degradation 228
Chapter 3
Cell Structure and Functions
3.1 What is a Cell? 234
3.2 Structure of eukaryotic cells 235
3.2.1 Plasma membrane 235
3.2.2 ABO blood group 243
3.2.3 Transport across plasma membrane 245
3.3 Membrane potential 252
3.4 Transport of macromolecules across plasma membrane 262
3.4.1 Endocytosis 262
3.4.2 Fate of receptor 266
3.4.3 Exocytosis 267
3.5 Ribosome 268
3.5.1 Protein targeting and translocation 269
3.6 Endoplasmic reticulum 270
3.6.1 Endomembrane system 275
3.6.2 Transport of proteins across the ER membrane 275
3.6.3 Transport of proteins from ER to cis Golgi 280
3.7 Golgi complex 281
3.7.1 Transport of proteins through cisternae 283
3.7.2 Transport of proteins from the TGN to lysosomes 284
3.8 Vesicle fusion 285
3.9 Lysosome 286
3.10 Vacuoles 288
viii
3.11 Mitochondria 288
3.12 Plastids 291
3.13 Peroxisome 292
3.14 Cytoskeleton 293
3.14.1 Microtubules 293
3.14.2 Kinesins and Dyneins 296
3.14.3 Cilia and Flagella 296
3.14.4 Centriole 299
3.14.5 Actin filament 299
3.14.6 Myosin 301
3.14.7 Muscle contraction 302
3.14.8 Intermediate filaments 306
3.15 Cell junctions 307
3.16 Cell adhesion molecules 310
3.17 Extracellular matrix of animals 311
3.18 Plant cell wall 312
3.19 Nucleus 314
3.20 Cell signaling 317
3.20.1 Signal molecules 318
3.20.2 Receptors 319
3.20.3 GPCR and G-proteins 321
3.20.4 Ion channel-linked receptors 330
3.20.5 Enzyme-linked receptors 330
3.20.6 Nitric oxide 336
3.20.7 Two-component signaling systems 337
3.20.8 Chemotaxis in bacteria 338
3.20.9 Quorum sensing 339
3.20.10 Scatchard plot 340
3.21 Cell Cycle 342
3.21.1 Role of Rb protein in cell cycle regulation 346
3.21.2 Role of p53 protein in cell cycle regulation 347
3.21.3 Replicative senescence 348
3.22 Mechanics of cell division 348
3.22.1 Mitosis 348
3.22.2 Meiosis 355
3.22.3 Nondisjunction and aneuploidy 361
3.23 Apoptosis 362
3.24 Cancer 365
3.25 Stem cells 372
ix
Chapter 4
Prokaryotes and Viruses
4.1 General features of Prokaryotes 377
4.2 Phylogenetic overview 378
4.3 Structure of bacterial cell 378
4.4 Bacterial genome : Bacterial chromosome and plasmid 389
4.5 Bacterial nutrition 393
4.5.1 Culture media 395
4.5.2 Bacterial growth 395
4.6 Horizontal gene transfer and genetic recombination 399
4.6.1 Transformation 400
4.6.2 Transduction 402
4.6.3 Conjugation 406
4.7 Bacterial taxonomy 411
4.8 General features of important bacterial groups 413
4.9 Archaebacteria 415
4.10 Bacterial toxins 416
4.11 Control of microbial growth 418
4.12 Virus 422
4.12.1 Bacteriophage (Bacterial virus) 423
4.12.2 Life cycle of bacteriophage 424
4.12.3 Plaque assay 427
4.12.4 Genetic analysis of phage 430
4.12.5 Animal Viruses 433
4.12.6 Plant viruses 443
4.13 Prions and Viroid 444
4.13.1 Bacterial and viral disease 445
Chapter 5
Immunology
5.1 Innate immunity 448
5.2 Adaptive immunity 450
5.3 Cells of the immune system 452
5.3.1 Lymphoid progenitor 453
5.3.2 Myeloid progenitor 455
5.4 Organs involved in the adaptive immune response 456
5.4.1 Primary lymphoid organs 456
5.4.2 Secondary lymphoid organs/tissues 457
5.5 Antigens 458
5.6 Major-histocompatibility complex 462
x
5.6.1 MHC molecules and antigen presentation 464
5.6.2 Antigen processing and presentation 465
5.6.3 Laboratory mice 467
5.7 Immunoglobulins : Structure and function 468
5.7.1 Basic structure of antibody molecule 468
5.7.2 Different classes of immunoglobulin 470
5.7.3 Action of antibody 473
5.7.4 Antigenic determinants on immunoglobulins 473
5.8 B-cell maturation and activation 475
5.9 Kinetics of the antibody response 481
5.10 Monoclonal antibodies and Hybridoma technology 482
5.10.1 Engineered monoclonal antibodies 483
5.11 Organization and expression of Ig genes 485
5.12 Generation of antibody diversity 491
5.13 T-cells and CMI 494
5.13.1 Superantigens 504
5.14 Cytokines 505
5.15 The complement system 509
5.16 Hypersensitivity 512
5.17 Autoimmunity 515
5.18 Transplantation 515
5.19 Immunodeficiency diseases 516
5.20 Failures of host defense mechanisms 516
5.21 Vaccines 518
Chapter 6
Genetics
Classical genetics
6.1 Mendel’s principles 525
6.1.1 Mendel’s laws of inheritance 527
6.1.2 Incomplete dominance and codominance 531
6.1.3 Multiple alleles 532
6.1.4 Lethal alleles 534
6.1.5 Penetrance and expressivity 534
6.1.6 Probability 534
6.2 Chromosomal basis of inheritance 537
6.3 Gene interaction 539
6.3.1 Dominant epistasis 540
6.3.2 Recessive epistasis 541
6.3.3 Duplicate recessive epistasis 542
xi
6.3.4 Duplicate dominant interaction 542
6.3.5 Dominant and recessive interaction 543
6.3.6 Pleiotropy 544
6.3.7 Genetic dissection to investigate gene action 544
6.4 Genetic linkage and gene mapping 546
6.4.1 Genetic mapping 550
6.4.2 Gene mapping from two point cross 551
6.4.3 Gene mapping from three point cross 552
6.4.4 Interference and coincidence 554
6.5 Tetrad analysis 555
6.5.1 Analysis of ordered tetrad 557
6.5.2 Analysis of unordered tetrad 558
6.6 Sex chromosomes and sex determination 560
6.6.1 Sex chromosome 560
6.6.2 Chromosomal basis of sex determination 561
6.6.3 Sex determination in humans 561
6.6.4 Genic balance theory of sex determination in Drosophila 562
6.6.5 Sex determination in plants 563
6.6.6 Non-chromosomal basis of sex determination 563
6.6.7 Mosaicism 564
6.6.8 Sex-linked traits and sex-linked inheritance 564
6.6.9 Sex-limited traits 566
6.6.10 Sex-influenced traits 566
6.6.11 Pedigree analysis 566
6.7 Quantitative inheritance 570
6.7.1 Quantitative trait locus analysis 574
6.7.2 Heritability 574
6.8 Extranuclear inheritance and maternal effect 575
6.8.1 Maternal effect 577
6.9 Cytogenetics 578
6.9.1 Human karyotype 579
6.9.2 Chromosome banding 579
6.9.3 Ploidy 581
6.9.4 Chromosome aberrations 582
6.9.5 Position effect 585
6.10 Population genetics 586
6.10.1 Calculation of allelic frequencies 586
6.10.2 Hardy-Weinberg Law 587
Molecular genetics
6.11 Genome 594
6.11.1 Genome complexity 595
xii
6.11.2 Transposable elements 598
6.11.3 Gene 604
6.11.4 Introns 605
6.11.5 Acquisition of new genes 607
6.11.6 Fate of duplicated genes 607
6.11.7 Gene families 608
6.11.8 Human nuclear genome 610
6.11.9 Organelle genome 611
6.11.10 Yeast S. cerevisiae genome 612
6.11.11 E. coli genome 612
6.12 Eukaryotic chromatin and chromosome 612
6.12.1 Packaging of DNA into chromosomes 614
6.12.2 Histone modification 618
6.12.3 Heterochromatin and euchromatin 619
6.12.4 Polytene chromosomes 623
6.12.5 Lampbrush chromosomes 623
6.12.6 B-chromosomes 624
6.13 DNA replication 624
6.13.1 Semiconservative replication 624
6.13.2 Replicon and origin of replication 626
6.13.3 DNA replication in E. coli 628
6.13.4 Telomere replication 638
6.13.5 Rolling circle replication 639
6.13.6 Replication of mitochondrial DNA 640
6.14 Recombination 640
6.14.1 Homologous recombination 641
6.14.2 Site-specific recombination 646
6.15 DNA repair 648
6.15.1 Direct repair 648
6.15.2 Excision repair 648
6.15.3 Mismatch repair 650
6.15.4 Recombinational repair 651
6.15.5 Repair of double strand DNA break 653
6.15.6 SOS response 654
6.16 Transcription 654
6.16.1 Transcription unit 656
6.16.2 Prokaryotic transcription 656
6.16.3 Eukaryotic transcription 662
6.16.4 Role of activator and co-activator 667
6.16.5 Long-range regulatory elements 668
6.16.6 DNA binding motifs 670
xiii
6.17 RNA processing 672
6.17.1 Processing of eukaryotic pre-mRNA 673
6.17.2 Processing of pre-rRNA 681
6.17.3 Processing of pre-tRNA 684
6.18 mRNA degradation 685
6.19 Regulation of gene transcription 686
6.19.1 Operon model 686
6.19.2 Tryptophan operon system 693
6.19.3 Riboswitches 697
6.20 Bacteriophage lambda : a transcriptional switch 698
6.21 Regulation of transcription in eukaryotes 701
6.21.1 Influence of chromatin structure on transcription 701
6.21.2 DNA methylation and gene regulation 703
6.21.3 Post-transcriptional gene regulation 705
6.22 RNA interference 706
6.23 Genetic code 709
6.24 Protein synthesis 714
6.24.1 Incorporation of selenocysteine 725
6.24.2 Cap snatching 725
6.24.3 Translational frameshifting 725
6.24.4 Antibiotics and toxins 726
6.24.5 Post-translational modification of polypeptides 727
6.24.6 Ubiquitin mediated protein degradation 730
6.25 Mutation 732
6.25.1 Mutagen 737
6.25.2 Types of mutation 740
6.25.3 Fluctuation test 744
6.25.4 Replica plating experiment 745
6.25.5 Ames test 746
6.25.6 Complementation test 746
6.26 Developmental genetics 748
6.26.1 Genetic control of embryonic development in Drosophila 748
6.26.2 Genetic control of vulva development in C. elegans 754
6.26.3 Genetic control of flower development in Arabidopsis 755
Chapter 7
Recombinant DNA technology
7.1 DNA cloning 762
7.2 Enzymes for DNA manipulation 764
7.2.1 Template-dependent DNA polymerase 764
xiv
7.2.2 Nucleases 764
7.2.3 End-modification enzymes 768
7.2.4 Ligases 770
7.2.5 Linkers and adaptors 770
7.3 DNA and RNA purification 771
7.4 Vectors 773
7.4.1 Vectors for E. coli 774
7.4.2 Cloning vectors for yeast, S. cerevisiae 780
7.4.3 Vectors for plants 781
7.4.4 Vectors for animals 784
7.5 Introduction of DNA into the host cells 784
7.5.1 In bacterial cells 784
7.5.2 In plant cells 784
7.5.3 In animal cells 785
7.6 Selection of transformed bacterial cells 787
7.7 Recombinant screening 789
7.8 Expression vector 789
7.8.1 Reporter gene 790
7.8.2 Expression system 791
7.8.3 Fusion protein 792
7.9 DNA library 793
7.10 Polymerase chain reaction 796
7.11 DNA sequencing 800
7.12 Genome mapping 803
7.12.1 Genetic marker 804
7.12.2 Types of DNA markers 804
7.12.3 Physical mapping 808
7.12.4 Radiation hybrids 810
7.13 DNA profiling 811
7.14 Genetic manipulation of animal cells 812
7.14.1 Transgenesis and transgenic animals 812
7.14.2 Gene knockout 814
7.14.3 Formation and selection of recombinant ES cells 815
7.15 Nuclear transfer technology and animal cloning 816
7.16 Gene therapy 817
7.17 Transgenic plants 822
7.17.1 General procedure used to make a transgenic plant 822
7.17.2 Antisense technology 826
7.17.3 Molecular farming 827
7.18 Plant tissue culture 828
7.18.1 Cellular totipotency 828
xv
7.18.2 Tissue culture media 829
7.18.3 Types of cultures 830
7.18.4 Somaclonal and gametoclonal variation 835
7.18.5 Somatic hybridization and cybridization 835
7.18.6 Applications of cell and tissue culture 836
7.19 Animal cell culture 839
7.19.1 Primary and secondary cultures 839
7.19.2 Cell line 839
7.19.3 Culture media 840
7.19.4 Growth pattern 841
7.19.5 Application of animal cell culture 841
Chapter 8
Bioprocess engineering
8.1 Concept of material and energy balance 847
8.1.1 Material balance 847
8.1.2 Energy balance 853
8.2 Microbial growth kinetics 855
8.3 Fermentation 862
8.3.1 Fermentation processes 862
8.3.2 Fermentation media 863
8.4 Bioreactor 864
8.4.1 Agitation and aeration 864
8.4.2 Types of bioreactor 865
8.4.3 Mass balances for bioreactor 869
8.4.4 Ideal batch reactor 870
8.5 Basic operation and process control 875
8.6 Sterilization 877
8.7 Genetic instability 880
8.8 Mass and Heat transfer 881
8.8.1 Mass transfer 881
8.8.2 Heat transfer 886
8.9 Rheology of fermentation fluids 889
8.10 Enzyme immobilization 890
8.11 Scale up 894
8.12 Downstream processing 895
8.13 Industrial production of chemicals 900
8.14 Wastewater treatment 903
8.15 Bioremediation 905
xvi
Chapter 9
Bioinformatics
9.1 Introduction 912
9.2 Biological databases 912
9.3 Sequence formats 915
9.4 Biosequence analysis 917
9.5 Sequence alignment 918
9.6 Molecular phylogenetics 923
9.7 Protein structure prediction 927
9.8 Bioinformatics resources on the web 930
9.9 Genomics and proteomics 931
9.9.1 Genomics 931
9.9.2 Proteomics 932
Answers of self test 937
Index
xvii
Chapter 01
A biomolecule is an organic molecule that is produced by a living organism. Biomolecules act as building blocks of
life and perform important functions in living organisms. More than 25 naturally occurring chemical elements are
found in biomolecules. Most of the elements have relatively low atomic numbers. Biomolecules consist primarily of
carbon, hydrogen, nitrogen, oxygen, phosphorus and sulfur. The four most abundant elements in living organisms,
in terms of the percentage of the total number of atoms, are hydrogen, oxygen, nitrogen, and carbon, which
together make up over 99% of the mass of most cells.
Nearly all of the biomolecules in a cell are carbon compounds, which account for more than one-half of the dry
weight of the cells. Covalent bonding between carbon and other elements permit formation of a large number of
compounds. Most biomolecules can be regarded as derivatives of hydrocarbons. The hydrogen atoms may be
replaced by a variety of functional groups to yield different families of organic compounds. Typical families of
organic compounds are the alcohols, which have one or more hydroxyl groups; amines, which have amino groups;
aldehydes and ketones, which have carbonyl groups; and carboxylic acids, which have carboxyl groups. Many
biomolecules are polyfunctional, containing two or more different kinds of functional groups. Functional groups
determine chemical properties of biomolecules.
Sugars, fatty acids, amino acids and nucleotides constitute the four major families of biomolecules in cells. Many of
the biomolecules found within cells are macromolecules and mostly are polymers (composed of small, covalently
linked monomeric subunits). These macromolecules are proteins, carbohydrates, lipids and nucleic acids.
Small biomolecules Macromolecules
Sugars Polysaccharide
Fatty acids Fats/Lipids
Amino acids Proteins
Nucleotide Nucleic acid
Nucleic acids and proteins are informational macromolecules. Proteins are polymers of amino acids and constitute
the largest fraction (besides water) of cells. The nucleic acids, DNA and RNA, are polymers of nucleotides. They
store, transmit and translate genetic information. The polysaccharides, polymers of simple sugars, have two major
functions. They serve as energy-yielding fuel stores and as extracellular structural elements.
1.1 Amino acids and Proteins

Amino acids are compounds containing carbon, hydrogen, oxygen and nitrogen. They serve as monomers (building
blocks) of proteins and composed of an amino group, a carboxyl group, a hydrogen atom, and a distinctive side
chain, all bonded to a carbon atom, the α-carbon.
In an α-amino acid, the amino and carboxylate groups are attached to the same carbon atom, which is called the
α-carbon. The various α-amino acids differ with respect to the side chain (R group) attached to their α-carbon. The
general structure of an amino acid is:
1
—
COO
+ a
H3N C H
R (side chain)
Figure 1.1 General structure of an amino acid.
This structure is common to all except one of the α-amino acids (proline is the exception). The R group or side chain
attached to the α-carbon is different in each amino acid. In the simplest case, the R group is a hydrogen atom and
amino acid is glycine.
—
COO e d g b a
+ 6 5 4 3 2 1
+ —
H3N C H NH3 CH2 CH2 CH2 CH2 CH COO
+
H NH3
Figure 1.2 Structure of glycine and lysine.
In α-amino acids both the amino group and the carboxyl group are attached to the same carbon atom. However,
many naturally occurring amino acids not found in protein, have structures that differ from the α-amino acids. In
these compounds the amino group is attached to a carbon atom other than the α-carbon atom and they are called
β, γ, δ, or ε amino acids depending upon the location of the C-atom to which amino group is attached.
Amino acids can act as acids and bases

When an amino acid is dissolved in water, it exists in solution as the dipolar ion or zwitterion. A zwitterion can act as
either an acid (proton donor) or a base (proton acceptor). Hence, an amino acid is an amphoteric molecule. At
high concentrations of hydrogen ions (low pH), the carboxyl group accepts a proton and becomes uncharged, so
that the overall charge on the molecule is positive. Similarly at low concentrations of hydrogen ion (high pH), the
amino group loses its proton and becomes uncharged; thus the overall charge on the molecule is negative.
R O R O R O
+ +
— —
H3N C C OH H3N C C O H2N C C O
H H H
Low pH (pH < pI) Intermediate pH High pH (pH > pI)

(pH = pI)
Figure 1.3 The acid-base behavior of an amino acid in solution. At low pH, the positively charged species
predominates. As the pH increases, the electrically neutral zwitterion becomes predominant. At higher pH, the
negatively charged species predominates.
1.1.1 Optical properties

All amino acids except glycine are optically active i.e. they rotate the plane of plane polarized light. Optically active
molecules contain chiral carbon. A tetrahedral carbon atom with four different constituents are said to be chiral. All
amino acids except glycine have chiral carbon and hence they are optically active.
2
Problem
A solution of L-leucine (3.0 g/50 ml of 6 N HCl) had an observed rotation of +1.81° in a 20 cm polarimeter tube.
Calculate the specific rotation of L-leucine in 6 N HCl.
Solution
Å
[a]DT =
l ´C
+1.81 3g
[α]DT = where, l = 20 cm = 2 dm and C = = 0.06 g / ml
2 × 0.06 50 ml
[α] = +15.1°.
1.1.2 Absolute configuration

An amino acid with a chiral carbon can exist in two configurations that are non-superimposable mirror images of
each other. These two configurations are called enantiomers. An enantiomer is identified by its absolute configuration.
For example, glyceraldehyde has two absolute configurations. When the hydroxyl group attached to the chiral
carbon is on the left in a Fischer projection, the configuration is L; when the hydroxyl group is on the right, the
configuration is D.
CHO CHO
HO C H H C OH
CH2OH CH2OH
L-Glyceraldehyde D-Glyceraldehyde
In the above figure, prefixes D- and L- refer to absolute configuration of glyceraldehyde. Similarly, absolute
configuration of amino acids are specified by the D- and L- system. The designation of D or L to an amino acid refers
to its absolute configuration relative to the structure of D- or L-glyceraldehyde, respectively.
— —
COO COO
+ +
H3N C H H C NH3
CH3 CH3
L-Alanine D-Alanine
All amino acids except glycine exist in these two different enantiomeric forms. However, all the amino acids
ribosomically incorporated into proteins exhibit L-configuration. Therefore, they are all L-α-amino acids. The basis
for preference for L-amino acids is not known. D-form of amino acids are not found in proteins, although they exist
in nature. D-form of amino acids are found in some peptide antibiotics and peptidoglycan cell wall of eubacteria.
A second absolute configuration notation using the symbols R (from rectus, Latin for right) and S (from sinister,
Latin for left) can also be used. In this approach, the substituents on an asymmetric carbon (a chiral carbon with
four different substituents) are prioritized by decreasing the atomic number. Atoms of higher atomic number
bonded to a chiral centre are ranked above those of lower atomic number. For example, the oxygen atom of a —OH
group takes precedence over the carbon atom of the —CH3 group that is bonded to the same chiral carbon atom.
If any of the first substituent atoms are of the same element, the priority of these groups is established from the
4
atomic number of the second, third etc, atoms outward from the chiral carbon atom. Hence, a CH2OH group takes
precedence over a CH3 group. The prioritized groups are assigned the letters W, X, Y and Z with the order of priority
rating is W > X > Y > Z. Configuration is assigned by looking down the bond to the lowest priority substituent, Z. If
the order of the group W → X → Y is clockwise, then the configuration of the chiral centre is designated R. If the
order of W → X → Y is counterclockwise, the chiral centre is designated S.
Y
CH2OH CH2OH CH3
Y Y
C C C
— +
OHC OH HO CHO OOC NH3
X W W X X H w
H H
Z Z Z
D-Glyceraldehyde L-Glyceraldehyde D-Alanine

R-Glyceraldehyde S-Glyceraldehyde R-Alanine
The absolute configuration of the amino acids at the α-carbon is typically described by D-L system rather than the
more modern R-S system. According to the R-S system, all the L-amino acids from proteins are S-amino acids, with
the exception of L-cysteine, which is R-cysteine.
1.1.3 Standard and non-standard amino acids

More than 300 amino acids are present in cells; however, only 22 amino acids participate in protein synthesis
ribosomically. Such amino acids are called standard or proteinogenic amino acids. Some amino acids are more
abundant in proteins than other amino acids. Four amino acids - leucine, serine, lysine, and glutamic acid- are the
most abundant amino acid residues in a typical protein. Tryptophan and methionine are rare amino acids in a
protein. Standard L-α-amino acids are specified by simple three letter codons. α-amino acids can be classified on
the properties of their side chain (or R group), in particular, their polarity, or tendency to interact with water at
physiological pH (near pH 7). The polarity of the side chain varies widely, from nonpolar and hydrophobic to highly
polar and hydrophilic.
1. Amino acids with nonpolar side chain

Among standard amino acids, nine amino acids contain nonpolar side chain or R group. These are glycine,
alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine and tryptophan. Proline differs from
other members in having its side chain bonded to both the nitrogen and the α-carbon atoms. Phenylalanine and
tryptophan have aromatic side chains. The side chain of phenylalanine contains a phenyl ring whereas tryptophan
has an indole ring.
2. Amino acids with uncharged polar side chain

Six amino acids contain uncharged polar side chain – serine, threonine, cysteine, asparagine, glutamine and
tyrosine. Three amino acids, serine, threonine and tyrosine contain hydroxyl groups attached to a side chain.
Cysteine is structurally similar to serine but contains a sulfhydryl, or thiol group (–SH) in place of the hydroxyl
group.
3. Amino acids with charged polar side chain

Positively charged R group : Lysine and arginine have side chains that contain positively charged groups at
neutral pH or physiological pH. Lysine has an amino group whereas arginine contains a guanidinium group.
Histidine contains an imidazole group, an aromatic ring. The imidazole group can be uncharged or positively
charged near neutral pH, depending on its local environment.
Negatively charged R group : Amino acids aspartate and glutamate contain acidic side chains that contain
negatively charged groups at physiological pH.
5
Amino acids with charged polar side chain
Nonstandard amino acids

More than three hundred amino acids have been found in cells. Twenty-two amino acids are ribosomically incorporated
into proteins and are called proteinogenic or standard amino acids. Apart from the 22 standard amino acids, all
other amino acids are not ribosomically incorporated into proteins are called non-standard. In addition to the
standard amino acids, some proteins may contain non-standard amino acid residues formed by post-translational
modification of standard amino acid residues already incorporated into a polypeptide. These modifications are
often essential for the function or regulation of a protein. Examples of some of these amino acids are 4-Hydroxyproline
(derivative of proline), 5-Hydroxylysine (derivative of lysine), desmosine (derivative of lysine), N-acetylserine,
N-formylmethionine and γ-carboxyglutamate (found in the blood clotting protein prothrombin).
Besides their role in proteins, amino acids and their derivatives have many other biologically important functions.
Many nonstandard amino acids are not found in proteins. These amino acids often occur as intermediates in the
metabolic pathways for standard amino acids. For example, ornithine and citrulline are key intermediates in the
biosynthesis of arginine and in the urea cycle. Similarly, azaserine, a nonstandard amino acid, acts as an antibiotic.
It was originally thought that all unconventional amino acids were made by modifying one of the standard amino
acids after it was incorporated into protein, a process called a post-translational modification. But amino acids like
selenocysteine, pyrrolysine are inserted into proteins by the translational machinery. Selenocysteine is introduced
during protein synthesis rather than created through a postsynthetic modification. It contains selenium rather than
sulphur of its structural analog, cysteine. Since selenocysteine is incorporated into polypeptides during translation,
it is referred to as 21st amino acid. However, it is specified by a triplet codon, UGA (a stop codon). Selenocysteine
has its own tRNA containing the anticodon UCA and it is formed by modifying a serine that has been attached to the
selenocysteine tRNA. Enzymes like glutathione peroxidase and formate dehydrogenase contain selenocysteine in
their catalytic center. Pyrrolysine is similar to lysine and is present in some bacterial proteins. It is coded by UAG
codon.
— —
COO COO
+ +
H3N C H H3N C H
CH2 CH2
SH SeH
Cysteine Selenocysteine
7
1.1.4 Titration of amino acids

Because amino acids contain ionizable groups, the predominant ionic form of these molecules in solution depends
on the pH. Titration of an amino acid illustrates the effect of pH on amino acid structure. Consider alanine, a simple
amino acid, which has two titrable groups (α-amino and α-carboxyl group). During titration with a strong base such
as NaOH, alanine loses two protons in a stepwise fashion. In a strongly acidic solution, alanine is present mainly in
the form in which the carboxyl group is uncharged. Under this condition the molecule’s net charge is +1, since the
ammonium group is protonated. However, an increase in the pH results in the deprotonation of α-carboxyl group. At
this point, alanine has no net charge and is electrically neutral. The pH at which this occurs is called the isoelectric
point (pI).
Net charge: +1 0 –1
CH3 O CH3 O CH3 O

+ pk1 + pk2
— —
H3N C C OH H3N C C O H2N C C O
H H H
Low pH (pH < pI) Intermediate pH High pH (pH > pI)

(pH = pI)
Because there is no net charge at the isoelectric point, amino acids are electrophoretically non-mobile and least
soluble at this pH. Further increase in pH i.e. lowering of the H+ concentration results in the deprotonation of the
charged amino group and an uncharged amino group forms. So at high pH, the net charge on the molecule is –1,
since the ammonium group is deprotonated and a net negative charge develops due to the presence of the
carboxylate group.
13
Alanine
pK2 = 9.7
7
pH pI = 6
pK1 = 2.34
0 0.5 1 1.5 2
—
OH (equivalents)
Figure 1.6 Titration curve of alanine (monoamino and monocarboxylic acid). A plot of the dependence of
the pH on the amount of OH– added is called a titration curve.
The isoelectric point for alanine may be calculated as follows:
pK1 + pK2
pI =
2
8
Table 1.2 pKa values for the ionizing groups of the standard amino acids
Amino acid pK1 (—COOH) pK2 (—NH3+) pKR
Glycine (Gly, G) 2.34 9.6
Alanine (Ala, A) 2.34 9.69
Valine (Val, V) 2.32 9.62
Leucine (Leu, L) 2.36 9.6
Isoleucine (Ile, I) 2.36 9.6
Serine (Ser, S) 2.21 9.15
Threonine (Thr, T) 2.11 9.43
Methionine (Met, M) 2.28 9.21
Phenylalanine (Phe, F) 1.83 9.13
Tryptophan (Trp, W) 2.83 9.39
Asparagine (Asn, N) 2.02 8.8
Glutamine (Gln, Q) 2.17 9.13
Proline (Pro, P) 1.99 10.6
Cysteine (Cys, C) 1.71 10.78 8.33
Histidine (His, H) 1.82 9.17 6.04
Aspartic acid (Asp, D) 2.09 9.82 3.86
Glutamic acid (Glu, E) 2.19 9.67 4.25
Tyrosine (Tyr, Y) 2.2 9.11 10.46
Lysine (Lys, K) 2.18 8.95 10.54
Arginine (Arg, R) 2.17 9.04 12.48
Note: Seven of the 20 amino acids have ionizable side chains. These 7 amino acids are able to donate or accept protons.
Absorption of UV radiation by aromatic amino acids

Aromatic amino acids such as tryptophan, tyrosine and phenylalanine absorb ultraviolet (UV) light. The aromatic
side chains of these amino acids are responsible for UV absorption. Tryptophan and tyrosine absorb maximum near
a wavelength of 280 nm. However phenylalanine absorbs maximum at 257.4 nm. Absorbance at 280 nm is used for
detection and quantification of purified proteins. The absorbance of each protein depends on the number and
positions of its aromatic amino acid residues.
8000
6000 Trp
Extinction coefficient
—1 —1
(M cm ) 4000
2000 Tyr
0
220 240 260 280 300 320
Wavelength (nm)
Figure 1.9 Absorption of UV-light. Maximum radiation absorption for both tryptophan and tyrosine occur
near a wavelength of 280 nm and absorbance of tryptophan is as much as four times that of tyrosine.
10
1.1.6 Peptide bond

Peptides and polypeptides are linear and unbranched polymers composed of amino acids linked together by peptide
bonds. Peptide bonds are amide linkages formed between α-amino group of one amino acid and the α-carboxyl
group of another. This reaction is a dehydration reaction, that is, a water molecule is removed and the linked amino
acids are referred to as amino acid residues. Peptide bond formation is an endergonic process, with ΔG ~ +21kJ/mol.
H O H O
H2N C C OH H N C C OH
R1 H R2
H2O
H O H O
H2N C C N C C OH
R1 H R2
Figure 1.11 The formation of a peptide bond (also called an amide bond) between the α-carboxyl group of
one amino acid to the α-amino group of another amino acid is accompanied by the loss of a water molecule.
The peptide C—N bond has a partial double bond character that keeps the entire six-atom peptide group in a rigid
planar configuration. Consequently, the peptide bond length is only 1.33 Å, shorter than the usual C—N bond length
of 1.45 Å. The peptide bond appears to have approximately 40 percent double-bonded character. As a result, the
rotation of this bond is restricted.
Od
—
O O
C Ca C d
+
Ca C + Ca
Ca N Ca N Ca N
H H H
Figure 1.12 The peptide bond has some double-bond character due to resonance. The carbonyl oxygen
has a partial negative charge and the amide nitrogen a partial positive charge, setting up a small electric
dipole. Virtually all peptide bonds in proteins occur in trans configuration.
The angle of rotation around the peptide bond, ω, usually has the value ω = 180° (trans) and occasionally ω = 0°
(cis). The trans form is favoured by a ratio of approximately 1000:1 over the cis form because in the cis form the
Cα atom and the side chains of neighbouring residues are in close proximity.
O O
C Ca C H
N N
Ca Ca
H Ca
trans cis
However, the rotation is permitted about the N–Cα and the Cα–C bonds. Rotation about bonds are described as
torsion or dihedral or conformational angle. By convention, the bond angles resulting from rotations at Cα are
labeled φ (phi) for the N–Cα bond and ψ (psi) for the Cα–C bond.
12
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isoelectric precipitation. Salting out is dependent on the hydrophobic nature of the surface of the protein. Hydrophobic
groups predominate in the interior of the protein, but some are located at the surface. Water is forced into contact
with these groups. When salts are added to the system, water solvates the salt ions and as salt concentration
increases water is removed from around the protein, eventually exposing the hydrophobic groups. Hydrophobic
groups on one protein molecule can interact with those of another, resulting in aggregation and thus precipitation.
Effect of solvent
Organic solvents such as acetone, ethanol, decrease the dielectric constant of the aqueous solution, which in effect
allows two proteins to come close together through electrostatic force of attraction. These solvents due to their low
dielectric constants lower the solvating power of aqueous solutions.
1.1.10 Simple and conjugated proteins

On the basis of composition, proteins are classified as simple or conjugated. Simple proteins, such as serum
albumin, contain only amino acids. In contrast, conjugated protein consists of a simple protein combined with a
non-protein component. The non-protein component is called a prosthetic group. A conjugated protein without its
prosthetic group is called an apoprotein. Apoprotein combined with its prosthetic group is referred to as a holoprotein.
Conjugated proteins are further classified according to the nature of their prosthetic groups. For example,
glycoproteins contain a carbohydrate component, lipoproteins contain lipid molecules, and metalloproteins contain
metal ions. Similarly, phosphoproteins contain phosphate groups and hemoproteins possess heme groups.
Table 1.5 Examples of few conjugated proteins

Class
Glycoproteins Fibronectin
Cadherin
Lipoproteins Chylomicron
High Density Lipoprotein (HDL)
Metalloproteins Ferritin (Iron)
Alcohol dehydrogenase (Zinc)
Cytochrome oxidase (Copper and iron)
Nitrogenase (Molybdenum and iron)
Hemoprotein Hemoglobin (Transport of oxygen in blood)
Myoglobin (Storage of oxygen in muscle)
Cytochrome C (Involvement in electron transport chain)
Cytochrome P450 (Hydroxylation of xenobiotics)
Catalase (Degradation of hydrogen peroxide)
1.2 Fibrous and globular proteins

Proteins are also classified into two categories – fibrous and globular proteins, on the basis of shape and solubility.
Fibrous proteins are long, rod-shaped molecules that are insoluble in water and physically tough. Fibrous proteins,
such as keratins have structural and protective functions. These proteins usually consist largely of a single type of
secondary structure. Globular proteins are compact spherical molecules that are usually water-soluble. These
proteins often contain several types of secondary structure. In globular proteins, the nonpolar residues Val, Leu,
Ile, Met and Phe largely occur in the interior of a protein, out of contact with the aqueous solvent. The charged
polar residues Arg, His, Lys, Asp and Glu are largely located on the surface of the protein in the contact with the
aqueous solvent. Uncharged polar residues Ser, Thr, Asn, Gln and Tyr are usually present on the protein surface.
20
Table 1.6 The major functions of proteins and their examples

Function Class of protein Example
Structure Fibers Collagen, Keratin, Fibrin
Metabolism Enzymes Lysosomes, Proteases, Polymerase, Kinases
Membrane transport Channels/Carriers Proton pump, Anion channels
Cell recognition Cell surface antigens MHC proteins, ABO blood group
Osmotic regulation Albumin Serum albumin
Regulation of gene action Repressors lac repressor
Regulation of body functions Hormones Insulin, Vasopressin, Oxytocin
Transport throughout body Globins Hemoglobin, Myoglobin, Cytochromes
Storage Ion-binding Ferritin, Casein, Calmodulin
Contraction Muscle Actin, Myosin
Defense Ig, Toxins Antibodies, Snake venom
1.2.1 Collagen
Collagen is the major structural protein in the extracellular matrix. It is the most abundant protein in vertebrates.
Collagens are a large family of proteins containing at least 19 different members. A typical collagen molecule is
long, inelastic, stiff, triple stranded helical structure. The fundamental unit of collagen is tropocollagen (length
~300nm). Tropocollagen consists of 3-coiled polypeptides called α-chains. The α-chains are left-handed polypeptide
helices and have 3.3 amino acid residues per turn. Three α-chains wind around one another in a characteristic
right-handed triple helix. Vertebrates have about 25 different kinds of α-chains, each coded by different genes and
has its own unique amino acid sequence. These different types of α-chains combine in various ways to form at least
19 different types of collagen molecules. Types I, II, and III represent 90% of collagens.
The amino acid sequence in α-chain is generally a repeating tripeptide unit, Gly–X–Y, where X is often proline and
Y is often 3- or 4-hydroxyproline or 5-hydroxylysine. Glycine constitutes approximately one-third of the amino acid
residues. Proline and hydroxyproline confer rigidity on the collagen molecule. The hydroxylation is carried out
during post-translational modifications of α-chains by two enzymes: prolyl hydroxylase and lysyl hydroxylase.
Ascorbate (vitamin C) acts as cofactors for these enzymes and hence is essential for hydroxylation of proline and
lysine residues. Hydroxylation results in formation of interchain H-bonds. It also allows the glycosylation of
hydroxylysine residues. Deficiency of ascorbic acid causes scurvy, a disease that affects the structure of collagen.
It occurs due to impaired synthesis of collagen as a result of deficiencies of prolyl and lysyl hydroxylases.
The α-chains are synthesized on membrane-bound ribosomes and enter into the lumen of the endoplasmic reticulum
(ER). In the lumen of the ER selected proline and lysine residues are hydroxylated to form hydroxyproline and
hydroxylysine, respectively, and some of the hydroxylysine residues are glycosylated. Each α-chain then combines
with two others to form a hydrogen-bonded, triple-stranded helical molecule known as tropocollagen (or collagen
molecule). Tropocollagen is secreted into the extracellular space. After secretion the tropocollagens assemble in
the extracellular space to form collagen fibrils.
Cross-linking of collagen
Covalent cross-links are formed both within a tropocollagen molecule and between different molecules. Intramolecular
cross-links form through the action of lysyl oxidase, a copper-dependent enzyme that oxidatively deaminates the
ε-amino groups of lysine residues, yielding reactive aldehydes of allysine residues. Such aldehydes of two side
chains then link covalently in a spontaneous nonenzymatic aldol condensation. Histidine may also be involved in
certain cross-links.
21
H H O
N C C
(CH2)2
H N N H
CH2
H C CH2 H2C CH2 CH2 C H
O C C O
+
N
CH2
(CH2)3
N C C
H H O
Figure 1.20 Intramolecular desmosine cross-links in elastin.
1.2.3 Keratins
Keratins are fibrous proteins present in eukaryotes. They form a large family, with about 30 members being
distinguished. Keratins have been classified as either α-keratins or β-keratins.
Proteins α-keratin β-keratin
Characteristics Tough, insoluble Soft, flexible
Conformation Helical Extended chain
Basic unit Protofibril Antiparallel β-pleated sheet
α-keratins are intermediate filament proteins present only in many metazoans, including vertebrates. In vertebrates,
α-keratins constitute almost the entire dry weight of hair, wool, feathers, nails, claws, scales, horns, hooves, and
much of the outer layer of skin. The α-keratin polypeptide chain which forms polymerized α-keratin structure, is a
right-handed α-helix and rich in hydrophobic amino acid residues Ala, Val, Leu, Ile, Met and Phe. Every α-keratin
polypeptide chain dimerizes to form heterodimer. The heterodimer is made up of type I (acidic) and the type II
(neutral/basic) α-keratin polypeptide chains. The two chains in heterodimer have a parallel arrangement. Two
heterodimers join in an antiparallel manner to form the fundamental tetrameric subunit (a protofilament). Two
protofilaments constitute a protofibril. Four protofibrils constitute a microfibril, which associates with other microfibrils
to form a macrofibril.
1.2.4 Myoglobin
Myoglobin (Mb), a globular protein, contains a single polypeptide chain of 153 amino acid residues (molecular
weight 17,800), and a single heme group. The inside of myoglobin consists almost exclusively of nonpolar residues,
whereas the outside contains both polar and nonpolar residues. About 75% of the polypeptide chain is α-helical.
There are eight helical segments. These eight helical segments are commonly labeled A–H, starting from the NH2-
terminal end. The interhelical regions are designated as AB, BC, CD,..., GH, respectively. The iron atom of the
heme is directly bonded to a nitrogen atom of a histidine side chain of globin.
Heme
Globin of Mb binds a single heme group by forming a co-ordinate bond. The heterocyclic ring system of heme is a
porphyrin derivative. The porphyrin in heme is known as protoporphyrin IX. It is made up of 4-pyrrole ring and
4-pyrroles are linked by methylene bridges to form a tetrapyrrole ring. The Fe atom is present either in Fe2+ or Fe3+
oxidation state in the center of the porphyrin ring.
23
the native structure and lacks the proper packing interactions in the interior of the protein. The interior side chains
remain mobile, more closely resembling a liquid than the solid-like interior of the native state.
Fast Slow
Molten globule Folded
Unfolded
Figure 1.32 The molten globule state is an intermediate state in the folding pathway when a
polypeptide chain converts from an unfolded to a folded native state.
1.3.1 Molecular chaperones

Not all proteins fold spontaneously after or during synthesis in the cell. Folding of many proteins requires molecular
chaperones. Molecular chaperones are a class of proteins which bind to incompletely folded or assembled proteins
in order to assist their folding or prevent them from aggregating. Chaperones function mainly by preventing
formation of incorrect structures rather than by promoting formation of correct structures. Chaperones may also
be required to assist the formation of oligomeric structures and for the transport of proteins through membranes.
Molecular chaperones were first identified in bacteria E. coli but are present in both prokaryotes and eukaryotes
(ubiquitous). Several molecular chaperones are included among the heat-shock proteins (hence their designation
as Hsp), because they are synthesized in increased amounts after a brief exposure of cells to an elevated temperature
(for example, 42°C for cells that normally live at 37°C).
Eukaryotic cells have at least two major families of molecular chaperones known as the Hsp60 and Hsp70
families. Hsps have been classified by molecular mass, for example: Hsp70 for the 70 kDa hsp. The members of
these two chaperone families function differently. The members of Hsp70 (Hsp70, Hsc70, Hsp40 and GrpE) act early
in the life of many proteins, binding to a string of about seven hydrophobic amino acids before the protein leaves
the ribosome. The Hsp70 polypeptide chain is divided into two functional regions, one that binds and hydrolyses ATP
and a second that binds hydrophobic segments of unfolded polypeptide chains. The polypeptide binding domain is
an antiparallel C-terminal region. Hsp70 is induced by stress (e.g. heat shock) whereas Hsc70 is constitutively
expressed in cells. Cytosolic Hsp70s prevent misfolding and maintain the polypeptide chain in unfolded condition.
Cytosolic Hsp70s are also necessary for normal translocation of protein from cytosol into either ER or mitochondria.
The Hsp70 family is found in bacteria, eukaryotic cytosol, in the endoplasmic reticulum, and in chloroplasts and
mitochondria.
In contrast, the Hsp60 family of molecular chaperones (sometime also called chaperonins) forms a large barrel-
shaped structure that acts later in a protein’s life, after it has been fully synthesized. Chaperonins bind unfolded,
partly folded and incorrectly folded protein molecules but not protein in their native state. This type of chaperone
forms an isolation chamber into which misfolded proteins are fed, preventing their aggregation and providing them
a favorable environment to refold. The typical structure is a ring of many subunits, forming a cylinder. Hsp60 itself
(known as GroEL in E. coli) forms a structure consisting of 14 subunits that are arranged in two heptameric rings
stacked on top of each other in an inverted orientation. This structure associates with a ring shaped heptamer
formed of subunits of Hsp10 (GroES in E. coli), also described as co-chaperonin.
32
1.4 Protein sequencing and assays

Determination of amino acid compositions
Peptide bonds of proteins are hydrolyzed by either strong acid or strong base. In acid hydrolysis, the peptide can
be hydrolyzed into its constituent amino acids by heating it in 6 M HCl at 110°C for 24 hours. Base hydrolysis of
polypeptides is carried out in 2 to 4 M NaOH at 100°C for 4 to 8 hours. A mixture of amino acids in hydrolysates can
be separated by ion exchange chromatography or by reversed phase HPLC. The identity of the amino acid is
revealed by its elution volume and quantified by reaction with ninhydrin.
N-terminal analysis
Reagent 1-fluoro-2,4-dinitrobenzene (FDNB) and Dansyl chloride are used for determination of N-terminal amino
acid residue. FDNB reacts in alkaline solution (pH 9.5) with the free amino group of the N-terminal amino acid
residue of a peptide to form a characteristic yellow dinitrophenyl (DNP) derivative. It can be released from the
peptide by either acid or enzymic hydrolysis of the peptide bond and subsequently identified. Sanger first used this
reaction to determine the primary structure of the polypeptide hormone insulin. This reagent is often referred to as
Sanger’s reagent.
NO2 NO2
R R
O2N F + NH2 C COOH O2N N C COOH + HF
H H H
FDNB Yellow-coloured derivative
Figure 1.34 FDNB reacts with free amino group to produce dinitrophenyl (DNP) derivative of amino acid.
Similarly, Dansyl chloride reacts with a free amino group of the N-terminal amino acid residue of a peptide in alkaline
solution to form strongly fluorescent derivatives of free amino acids and N-terminal amino acid residue of peptides.
Edman degradation
Edman degradation method for determining the sequence of peptides and proteins from their N-terminus was
developed by Pehr Edman. This chemical method uses phenylisothiocyanate (also termed Edman reagent) for
sequential removal of amino acid residues from the N-terminus of a polypeptide chain.
Polypeptide
R
A1 R
A2 R
A3 R
A4 R
A5
First round
Labeling
R
A1 R
A2 R
A3 R
A4 R
A5
Release
Figure 1.35
R
A1 R
A2 R
A3 R
A4 R
A5
Edman degradation sequentially
Labeling removes one residue at a time from

Second round
the amino end of a peptide. The labeled

R
A2 R
A3 R
A4 R
A5 amino-terminal residue (R1) can be released
without hydrolyzing the rest of the peptide
Release
bonds. Hence, the amino-terminal residue
of the shortened peptide (R2—R3—R4—R5)
R
A2 R
A3 R
A4 R
A5
can be determined in the second round.
34
trypsin, chymotrypsin, elastase, thermolysin and pepsin. Various other chemicals also cleave polypeptide chains at
specific locations. The most widely used is cyanogen bromide (CNBr), which cleaves peptide bond at C-terminal of
Met residues. Similarly hydroxylamine cleaves the polypeptide chain at Asn-Gly sequences.
Table 1.8 Specificities of proteolytic enzymes.
Rn–1 O Rn O
NH CH C NH CH C
Agents Site of Cleavage

Trypsin Carboxyl side of Lys or Arg, Rn ≠ Pro
Chymotrypsin Carboxyl side of aromatic amino acid residues, Rn ≠ Pro
Pepsin Amino side of aromatic amino acids like Tyr, Phe and Trp, Rn–1 ≠ Pro
Elastase Carboxyl side of Ala, Gly and Ser, Rn ≠ Pro
Carboxypeptidases and aminopeptidases are exopeptidases that remove terminal amino acid residues from C and
N-termini of polypeptides, respectively. Carboxypeptidase A cleaves the C-terminal peptide bond of all amino acid
residues except Pro, Lys and Arg. Carboxypeptidase B is effective only when Arg or Lys are the C-terminal residues.
Carboxypeptidase C acts on any C-terminal residue. Aminopeptidases catalyze the cleavage of amino acids from
the amino terminus of the protein. Aminopeptidase M catalyzes the cleavage of all free N-terminal residues.
Cleavage of disulfide bonds
If protein is made up of two or more polypeptide chains and held together by noncovalent bonds then denaturing
agents, such as urea or guanidine hydrochloride, are used to dissociate the chains from one another. But polypeptide
chains linked by disulfide bonds can be separated by two common methods. These methods are used to break
disulfide bonds and also to prevent their reformation.
Oxidation of disulfide bonds with performic acid produces two cysteic acid residues. Because these cysteic acid side
chains are ionized SO3– groups, electrostatic repulsion prevents S-S recombination. The second method involves
the reduction by β-mercaptoethanol or dithiothreitol (Cleland’s reagent) to form cysteine residues. This reaction is
followed by further modification of the reactive –SH groups to prevent reformation of the disulfide bond. Acetylation
by iodoacetate serves this purpose which modifies the –SH group.
Protein assays
To determine the amount of protein in an unknown sample is termed as protein assays. The simplest and most
direct assay method for proteins in solution is to measure the absorbance at 280 nm (UV range). Amino acids
containing aromatic side chains (i.e. tyrosine, tryptophan and phenylalanine) exhibit strong UV-light absorption.
Consequently, proteins absorb UV-light in proportion to their aromatic amino acid content and total concentration.
Several colorimetric, reagent-based protein assay techniques have also been developed. Protein is added to the
reagent, producing a color change in proportion to the amount added. Protein concentration is determined by
reference to a standard curve consisting of known concentrations of a purified reference protein. Some most
commonly used colorimetric, reagent-based methods are:
Biuret method : Biuret method is based on the direct complex formation between the peptide bonds of the
protein and Cu2+ ion. This method is not highly sensitive since the complex does not have a
high extinction coefficient.
Folin method : The Folin assay (also called Lowry method) is dependent on the presence of aromatic amino
acids in the protein. First, a cupric/peptide bond complex is formed and then this is enhanced
by a phosphomolybodate complex with the aromatic amino acids.
Bradford method : Bradford method is based on a blue dye (Coomassie Brilliant Blue) that binds to free amino
groups in the side chains of amino acids, especially Lys. This assay is as sensitive as the Folin
assay.
36
1.5 Nucleic acids

Nucleic acid was first discovered by Friedrich Miescher from the nuclei of the pus cells (Leukocytes) from discarded
surgical bandages and called it nuclein. Nuclein was later shown to be a mixture of a basic protein and a phosphorus-
containing organic acid, now called nucleic acid. There are two types of nucleic acids (polynucleotides): ribonucleic
acid (RNA) and deoxyribonucleic acid (DNA).
1.5.1 Nucleotides
The monomeric units of nucleic acids are called nucleotides. Nucleic acids therefore are also called polynucleotides.
Nucleotides are phosphate esters of nucleosides and made up of three components:
1. A base that has a nitrogen atom (nitrogenous base)
2. A five carbon sugar
3. An ion of phosphoric acid
Nitrogenous bases
Nitrogenous bases are heterocyclic, planar and relatively water insoluble aromatic molecules. There are two general
types of nitrogenous bases in both DNA and RNA, pyrimidines and purines.
H H
7
C6 5 N C4 5
1N C 3N CH
8
2 CH 2
HC C HC CH
4 N9 6
N H N
3 1
Purine Pyrimidine
Purines
Two different nitrogenous bases with a purine ring (composed of carbon and nitrogen) are found in DNA. The two
common purine bases found in DNA and RNA are adenine (6-aminopurine) and guanine (6-oxy-2-aminopurine).
Adenine has an amino group (–NH2) on the C6 position of the ring (carbon at position 6 of the ring). Guanine has an
amino group at the C2 position and a carbonyl group at the C6 position.
Pyrimidines
The two major pyrimidine bases found in DNA are thymine (5-methyl-2,4-dioxypyrimidine) and cytosine (2-oxy-4-
aminopyrimidine) and in RNA they are uracil (2,4-dioxypyrimidine) and cytosine. Thymine contains a methyl group
at the C5 position with carbonyl groups at the C4 and C2 positions. Cytosine contains a hydrogen atom at the C5
position and an amino group at C4. Uracil is similar to thymine but lacks the methyl group at the C5 position. Uracil
is not usually found in DNA. It is a component of RNA.
NH2 O NH2 O O
C C C C C
N N
N C HN C N CH HN CH HN C CH3
CH CH
HC C C C C CH C CH C CH
N H2N N O N O N O N
N H N H H H H
Adenine Guanine Cytosine Uracil Thymine
Sugars
Naturally occurring nucleic acids have two types of pentose sugars: Ribose and deoxyribose sugar. All known
sugars in nucleic acids have the D-stereoisomeric configuration.
42
The base is free to rotate around the glycosidic bond. Due to rotation of the glycosidic bond, two different conformations
are possible. The two standard conformations of the base around the glycosidic bond are syn and anti. Pyrimidines
tend to adopt the anti conformation almost exclusively, because of steric interference between O2 and C5' in the
syn-conformation, whereas purines are able to assume both forms (syn as well as anti).
Nucleotides
The nucleotides are phosphoric acid esters of nucleosides, with phosphate at position C5’. The nucleotide can have
one, two, or three phosphate groups designated as α, β and γ for the first, second and third, respectively.
O 9
5’
—
O P O CH2
O
—
O H H
H
HO OH
Figure 1.40 Structure of nucleotide.
Nucleotides are found primarily as the monomeric units comprising the major nucleic acids of the cell, RNA and DNA.
However, they also are required for numerous other important functions within the cell. These functions include:
• Formation of energy currency like ATP, GTP.
• Act as a precursor for several important coenzymes such as NAD+, NADP+, FAD and coenzyme A.
• Serving as a precursor for secondary messengers like cyclic AMP (cAMP), cGMP.
ATP
ATP is the chemical link between catabolism and anabolism. It is the energy currency of the living cells. It acts as
a donor of high energy phosphate. ATP consists of an adenosine moiety to which three phosphoryl groups (—PO32–)
are sequentially linked via a phosphoester bond followed by two phosphoanhydride bonds, referred to as a high
energy bond. The active form of ATP is usually a complex of ATP with Mg2+ or Mn2+.
NH2
Phosphoester
bonds
N N
— — —
O O O
N N
—
g b a
O P O P O P O CH2 O
O O O H H
H H
Phosphoanhydride OH OH
bonds
Adenosine
Figure 1.41 Structure of ATP indicating phosphoester and phosphoanhydride bonds.
44
1.6.2 Z-DNA
Left-handed Z-DNA has been mostly found in alternating purine-pyrimidine sequences (CG)n and (TG)n.
Z-DNA is thinner (18 Å) than B-DNA (20 Å), the bases are shifted to the periphery of the helix, and there is only one
deep, narrow groove equivalent to the minor groove in B-DNA. In contrast to B-DNA where a repeating unit is a 1
base pair, in Z-DNA the repeating unit is a 2 base pair. The backbone follows a zigzag path as opposed to a smooth
path in B-DNA. The sugar and glycosidic bond conformations alternate; C2’ endo in anti dC and C3’ endo in syn dG.
Electrostatic interactions play a crucial role in the Z-DNA formation. Therefore, Z-DNA is stabilized by high salt
concentrations or polyvalent cations that shield interphosphate repulsion better than monovalent cations.
Z-DNA can form in regions of alternating purine-pyrimidine sequence; (GC)n sequences form Z-DNA most easily.
(GT)n sequences also form Z-DNA but they require a greater stabilization energy. (AT)n sequences generally does
not form Z-DNA since it easily forms cruciforms.
Table 1.10 Comparisons of different forms of DNA
Geometry attribute A-form B-form Z-form

Helix sense Right-handed Right-handed Left-handed
Repeating unit 1 bp 1 bp 2 bp
Rotation/bp (Twist angle) 33.6° 34.3° 60°/2
Mean bp/turn 10.7 10.4 12
Base pair tilt 20° –6° 7°
Rise/bp along axis 2.3Å 3.32Å 3.8Å
Pitch/turn of helix 24.6Å 33.2Å 45.6Å
Mean propeller twist +18° +16° 0°
Glycosidic bond conformation Anti Anti Anti for C, Syn for G
Sugar pucker C3'-endo C2'-endo C:C2'-endo, G:C3'-endo
Diameter 23Å 20Å 18Å
Major groove Narrow and deep Wide and deep Flat
Minor groove Wide and shallow Narrow and deep Narrow and deep
1.6.3 Triplex DNA

In certain circumstances (e.g. low pH), a DNA sequence containing a long segment consisting of a polypurine
strand, hydrogen bonded to a polypyrimidine strand and form a triple helix. The triple helix will be written as
(dT).(dA).(dT) with the third strand in italics. Triple-stranded DNA is formed by laying a third strand into the major
groove of DNA. A third strand makes a hydrogen bond to another surface of the duplex. The third strand pairs in
a Hoogsteen base-pairing scheme. The central strand of the triplex must be purine rich. Thus, triple-stranded
DNA requires a homopurine: homopyrimidine region of DNA. If the third strand is purine rich, it forms reverse
Hoogsteen hydrogen bonds in an antiparallel orientation with the purine strand of the Watson-Crick helix. If the
third strand is pyrimidine rich, it forms Hoogsteen bonds in a parallel orientation with the Watson-Crick-paired
purine strand.
Triple helix can be intermolecular or intramolecular. In the intermolecular Pu.Pu.Py triple helix, the poly-purine
third strand is organized antiparallel with respect to the purine strand of the original Watson-Crick duplex. In the
intermolecular Py.Pu.Py triplex, the polypyrimidine third strand is organized parallel with respect to the purine
strand and the phosphate backbone is positioned.
49
3' 3'
3' 3' 5' 3'
G G
G G
G G G G
G G G G
G G G G
G G G G
G G G G
G G G G
T T G G
T T
T G G
T T
T T T
T T
G G T T
G G T T
G G G G
G G G G
G G G G
G G G G
G G G G
G G G G
T T G G
T T G G
T T
T T
5' 5'
5' 5' 5' 3'
Parallel Antiparallel
Figure 1.47 G-Quadruplex DNA. Quadruplex structures may be parallel or antiparallel.
1.6.5 Stability of the double helical structure of DNA

First : Internal and external hydrogen bonds stabilize the double helix. The two strands of DNA are held together by
H-bonds that form between the complementary purines and pyrimidines, two H-bonds in an A:T pair and three
H-bonds in a G:C pair, while the polar atoms in the sugar-phosphate backbone form external H-bonds with surrounding
water molecules.
Second : The core of the helix consists of the base pairs, which, in addition to being H-bonded, stack together
through stacking interactions. These interactions include hydrophobic interactions and van der Waals interactions
between base pairs that contribute significantly to the overall stability. Base stacking helps to minimize contact of
the bases with water.
Third : The negatively charged phosphate groups are all situated on the exterior surface of the helix in such a way
that they have minimal effect on one another and are free to interact electrostatically with cations in solution such
as Mg2+.
1.6.6 Thermal denaturation

When duplex DNA molecules are subjected to specific conditions of pH, temperature or ionic strength that disrupt
the hydrogen bonds and stacking interactions, the strands are no longer held together. That is, the double helix is
denatured and the strands separate as individual random coils. If temperature is the denaturing agent, the double
helix is said to have melted. DNA denaturation is a co-operative process. Denaturation process is accompanied by
a change in the DNA’s physical properties. Denaturation increases the relative absorbance of the DNA solution at
260 nm (as much as 40%). This increase in the absorbance is known as hyperchromic shift. The increased
absorbance is due to the fact that the aromatic bases in DNA interact via their π-electron clouds when stacked
together in the double helix. Because the absorbance of the bases at 260 nm is a consequence of π-electron
51
1.8 Carbohydrates
Carbohydrates are polyhydroxy aldehydes or polyhydroxy ketones, or compounds that can be hydrolyzed to them.
In the majority of carbohydrates, H and O are present in the same ratio as in water, hence also called as hydrates
of carbon. Carbohydrates are the most abundant biomolecules on Earth. Carbohydrates are classified into following
classes depending upon whether these undergo hydrolysis and if so on the number of products form:
Monosaccharides are simple carbohydrates that cannot be hydrolyzed further into polyhydroxy aldehyde or ketone
unit.
Oligosaccharides are polymers made up of two to ten monosaccharide units joined together by glycosidic linkages.
Oligosaccharides can be classified as di-, tri-, tetra- depending upon the number of monosaccharides present.
Amongst these the most abundant are the disaccharides, with two monosaccharide units.
Polysaccharides are polymers with hundreds or thousands of monosaccharide units. Polysaccharides are not sweet
in taste hence they are also called non-sugars.
1.8.1 Monosaccharide
Monosaccharides consist of a single polyhydroxy aldehyde or ketone unit. Monosaccharides are the simple sugars,
which cannot be hydrolyzed further into simpler forms and they have a general formula CnH2nOn. Monosaccharides
are colourless, crystalline solids that are freely soluble in water but insoluble in nonpolar solvents. The most
abundant monosaccharide in nature is the D-glucose. Monosaccharides can be further sub classified on the basis of:
The number of the carbon atoms present

Monosaccharides can be named by a system that is based on the number of carbons with the suffix-ose added.
Monosaccharides with four, five, six and seven carbon atoms are called tetroses, pentoses, hexoses and heptoses,
respectively.
System for numbering the carbons : The carbons are numbered sequentially with the aldehyde or ketone group
being on the carbon with the lowest possible number.
1 CHO 6 CHO
H C OH H C OH
2 5
HO C H HO C H
3 4
H C OH H C OH
4 3
H C OH H C OH
5 2
6 CH2OH 1 CH2OH
Correct Incorrect
Presence of aldehyde or ketone groups
Aldoses are monosaccharides with an aldehyde group.

Ketoses are monosaccharides containing a ketone group.
The monosaccharide glucose is an aldohexose; that is, it is a six-carbon monosaccharide (-hexose) containing an
aldehyde group (aldo-). Similarly fructose is a ketohexose; that is, it is a six-carbon monosaccharide (-hexose) and
containing a ketone group (keto-).
Trioses are simplest monosaccharides. There are two trioses– dihydroxyacetone and glyceraldehyde.
Dihydroxyacetone is called a ketose because it contains a keto group, whereas glyceraldehyde is called an aldose
because it contains an aldehyde group.
63
Leukotrienes are hydroxy fatty acid derivatives of arachidonic acid and do not contain a ring structure. Leukotrienes
are distinguished by containing a conjugated triene double-bond arrangement. They are involved in chemotaxis,
inflammation, and allergic reactions.
H
O
COOH
H
CH3
Figure 1.73 Structure of leukotriene A.
Table 1.17 Biological effects of eicosanoids

Type Major functions
Prostaglandins Mediation of inflammatory response
Regulation of nerve transmission
Inhibition of gastric secretion
Sensitization to pain
Stimulation of smooth muscle contraction
Thromboxanes Platelet aggregation
Aorta constriction
Prostacyclins Thromboxane antagonists
Leukotrienes Bronchoconstriction
Leukotaxis
1.9.7 Plasma lipoproteins

Triacylglycerols, phospholipids, cholesterol and cholesterol esters are transported in human plasma in association
with proteins as lipoproteins. Blood plasma contains a number of soluble lipoproteins, which are classified, according
to their densities, into four major types. These lipid-protein complexes function as a lipid transport system because
isolated lipids are insoluble in blood. There are four basic types of lipoproteins in human blood : chylomicrons, very
low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). A lipoprotein
contains a core of neutral lipids, which includes triacylglyerols and cholesterol esters. This core is coated with a
monolayer of phospholipids in which proteins (called apolipoprotein) and cholesterol are embedded.
Table 1.18 Some properties of major classes of human plasma lipoproteins
Lipoprotein Density Protein Phospho- Free Cholesterol Triacyl- Apolipo-

(g/mL) lipids cholesterol esters glycerols protein
Chylomicrons <1.006 1.5–2.5 7–9 1–3 3–5 85 A-I, C-I, B-48
VLDL 0.95–1.006 5–10 15–20 5–10 10–15 50 B-100, C-I, C-II
LDL 1.006–1.063 20–25 15–20 7–10 35–40 7–10 B-100
HDL 1.063–1.210 50–55 20–25 3–4 15 3–4 A-I, A-II, C-I
81
1.10 Vitamins
Vitamins are organic compounds required by the body in trace amounts to perform specific cellular functions. They
can be classified according to their solubility and their functions in metabolism. The requirement for any given
vitamin depends on the organisms. Not all vitamins are required by all organisms. Vitamins are not synthesized by
humans, and therefore must be supplied by the diet. Vitamins may be water soluble or fat soluble. Nine vitamins
(thiamines, riboflavin, niacin, biotin, pantothenic acid, folic acid, cobalamin, pyridoxine, and ascorbic acid) are
classified as water soluble, whereas four vitamins (vitamins A, D, E and K) are termed fat-soluble. Except for
vitamin C, the water soluble vitamins are all precursors of coenzymes.
1.10.1 Water-soluble vitamins

Thiamine (vitamin B1)
Thiamine pyrophosphate (TPP) is the biologically active form of the vitamin, formed by the transfer of a pyrophosphate
group from ATP to thiamine. Thiamine is composed of a substituted thiazole ring joined to a substituted pyrimidine
by a methylene bridge.
Thiazolium Aminopyrimidine
Reactive H NH2
H NH2 carbon +
S N N
+
S N N AMP
ATP
N CH3
CH3
N CH3
CH3 O
TPP synthetase
O —
O P O
H
Thiamine O
—
O P O
—
O
Thiamine pyrophosphate (TPP)
Figure 1.74 Structure of thiamine and thiamine pyrophosphate.
TPP serves as a coenzyme in the oxidative decarboxylation of α-keto acid, and in the formation or degradation of
α-ketols (hydroxy ketones) by transketolase.
Pyruvate
decarboxylase
Pyruvate (a-keto acid) Acetaldehyde + CO2
Transketolase
Xylulose-5-Phosphate + Ribose-5-Phosphate Glyceraldehyde–3–Phosphate + Sedoheptulose–7-Phosphate
Beri-Beri is a severe thiamine-deficiency syndrome found in areas where polished rice is the major component of the
diet.
Riboflavin (vitamin B2)
Riboflavin is a constituent of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). FMN is synthesized
after the addition of phosphate in riboflavin and FAD formed by the transfer of an AMP moiety from ATP to FMN. FMN
and FAD are each capable of reversibly accepting two hydrogen atoms, forming FMNH2 or FADH2. The oxidized form
of the isoalloxazine structure absorbs light around 450 nm. The colour is lost, when the ring is reduced.
82
C
2'
HN 1' NH
3'
H C C H
4 3
5 2 H
H2C 1
C
—
S CH2 CH2 CH2 CH2 COO
Valerate side chain
Figure 1.77 Structure of Biotin.
Most biotin-dependent carboxylations use bicarbonate as the carboxylating agent and transfer the carboxyl
group to a substrate carbanion. Examples of some important biotin-dependent carboxylations are given below:
Pyruvate
— carboxylase
ATP + HCO 3 + Pyruvate Oxaloacetate + ADP
Acetyl-CoA
— carboxylase
ATP + HCO3 + Acetyl-CoA Malonyl-CoA + ADP
Propionyl-CoA
— carboxylase
ATP + HCO3 + Propionyl-CoA Methylmalonyl-CoA + ADP
Biotin deficiency does not occur naturally because the vitamin is widely distributed in foods. Raw egg white contains
a glycoprotein, avidin, which tightly binds biotin and prevents its absorption from the intestine. The avidin homolog
streptavidin, which is secreted by the Streptomyces avidinii, also has high affinity for biotin.
Pantothenic acid
Pantothenic acid is a component of coenzyme A, which is responsible for the transfer of acyl groups. Coenzyme
A contains a thiol group that carries acyl compounds as activated thiol esters. Pantothenic acid is also a constituent
of acyl carrier protein (ACP). Coenzyme A performs two main functions:
• Activation of acyl groups for transfer by nucleophilic attack.
• Activation of the α-hydrogen of the acyl group.
O O CH3 OH O O
NH2 —
O P O P O C C C C N C C C N C C SH
N O O— H H
CH3
N N 5' H2C Pantothenic acid b-Mercaptoethylamine

O
1'
H H
H
OP OH
Adenosine
Figure 1.78 Structure of Coenzyme A (CoA-SH).
84
1.11.5 Enzyme inhibition

Inhibition of enzyme activity may be irreversible or reversible. Irreversible inhibitors usually bound covalently to
the enzyme and destroy the functional group in the active site. Most of irreversible inhibitors are toxic substances.
The antibiotic penicillin acts as an irreversible inhibitor of the enzyme glycopeptide transpeptidase (also known as
glycoprotein peptidase). Penicillin exerts its effects by covalently reacting with an essential serine residue in the
active site of glycopeptide transpeptidase, an enzyme that acts to cross-link the peptidoglycan chains during the
synthesis of bacterial cell walls. Once the cell wall synthesis is blocked, the bacterial cells undergo osmotic lysis and
bacterial growth is halted.
Table 1.25 Examples of irreversible enzyme inhibitors

Name Mode of action
Cyan ide Reacts with enzyme metal ions (i.e. Fe, Zn, Cu); respiratory chain
enzymes are primary targets.
Diisopropyl phosphofluoridate (DIPF) Inhibits enzymes with serine at active site e.g. acetylcholinesterase.
Sarin (nerve gas) Like DIPF
Physostigmine Like DIPF
Parathion (insecticide) Like DIPF, but especially inhibitory to insect acetylcholinesterase.
In reversible inhibition, the inhibitor can dissociate from the enzyme. Reversible inhibitors involve the non-covalent
binding with enzymes. Three common types of reversible inhibition are competitive, uncompetitive and noncompetitive
inhibition.
Competitive inhibition
The structure of a competitive inhibitor closely resembles that of the enzyme’s normal substrate. Because of its
structure, a competitive inhibitor binds reversibly to the enzyme’s active site. The inhibitor forms an enzyme-
inhibitor complex (EI) that is equivalent to the ES complex. The effect of a competitive inhibitor on activity can be
reversed by increasing the concentration of substrate. At high [S], all the active sites are filled with substrate, and
reaction velocity reaches the value observed without an inhibitor.
E + S ES E + P
+
I [E] [I]
KI 
[EI]
KI
EI
In the presence of a competitive inhibitor, the Michaelis-Menten equation becomes
Vmax [S] [I]

V  Where,   1 
 Km  [S] KI
In a double-reciprocal form the equation will be
1   Km  1 1
  
V  Vmax  [S] Vmax
In competitive inhibition, Vmax stays same and Km increases, but the inhibitor does not affect the turnover number
of the enzyme. Clinical treatment of methanol poisoning is a classical example of the exploitation of competitive
inhibitory mechanism. In the case of methanol poisoning, methanol in the body is converted to harmful formaldehyde
by alcohol dehydrogenase. A high dose of ethanol is used to alleviate the effect of methanol because ethanol
competitively binds with the active site of alcohol dehydrogenase.
102
Chapter 02
2.1 Bioenergetics
Bioenergetics is the quantitative study of the energy transductions that occur in living cells and of the nature and
functions of the chemical processes underlying these transductions.
Thermodynamic principles
The First law of thermodynamics states that the energy is neither created nor destroyed, although it can be
transformed from one form to another i.e. the total energy of a system, including surroundings, remains constant.
Mathematically, it can be expressed as:

ΔU = Δq – Δw
ΔU is the change in internal energy,
Δq is the heat exchanged from the surroundings,
Δw is the work done by the system.
If Δq is positive, heat has been transferred to the system, giving an increase in internal energy. When Δq is
negative, heat has been transferred to the surroundings, giving a decrease in internal energy. When Δw is positive,
work has been done by the system, giving a decrease in internal energy. When Δw is negative, work has been done
by the surroundings, giving an increase in internal energy.
The Second law of thermodynamics states that the total entropy of a system must increase if a process is to occur
spontaneously. Mathematically, it can be expressed as:
Dq
DS ³ where, ΔS is the change in entropy of the system
T
Entropy is unavailable form of energy and it is very difficult to determine it, so a new thermodynamic term called
free energy is defined.
Free energy
Free energy or Gibb’s free energy indicates the portion of the total energy of a system that is available for useful
work (also known as chemical potential). The change in free energy is denoted as ΔG.
Under constant temperature and pressure, the relationship between free energy change (ΔG) of a reacting system
and the change in entropy (ΔS) is expressed by following equation:
ΔG = ΔH – TΔS
Where, ΔH is the change in enthalpy and T is absolute temperature. ΔH is the measure of change in heat content of
reactants and products. The change in the free energy, ΔG, can be used to predict the direction of a reaction at
constant temperature and pressure.
117
If G is negative, the reaction proceeds spontaneously with the loss of free energy (exergonic),
G is positive, the reaction proceeds only when free energy can be gained (endergonic),
G is 0, the system is at equilibrium; both forward and reverse reactions occur at equal rates,
G of the reaction A  B depends on the concentration of reactant and product. At constant temperature and
pressure, the following relation can be derived:
[B]
G = G0 + RT ln
[A]
Where, G0 is the standard free energy change;
R is the gas constant;
T is the absolute temperature;
[A] and [B] are the actual concentrations of reactant and product.
Standard free energy change

The actual change in free energy (G) during a reaction is influenced by temperature, pressure and the initial
concentrations of reactants and products, and usually differs from standard free energy change, G0.
The chemical reaction has a characteristic standard free energy change and it is constant for a given reaction. It
can be calculated from the equilibrium constant of the reaction under standard conditions i.e., at a solute concentration
of 1.0M, at temperature of 25°C and at 1.0 atm pressure. The free energy change which corresponds to this
standard state is known as standard free energy change, G0.
Relationship between G0 and Keq
In a reaction A  B, a point of equilibrium is reached at which no further net chemical change takes place–that is,
when A is being converted to B, B is also being converted to A, as fast as A into B. In this state, the ratio of [B] to
[A] is constant, regardless of the actual concentrations of the two compounds:
[B]eq
K eq =
[A]eq
where Keq is the equilibrium constant, and [A]eq and [B]eq are the concentrations of A and B at equilibrium. The
concentration of reactants and products at equilibrium define the equilibrium constant, Keq. The equilibrium constant
Keq depends on the nature of reactants and products, the temperature and the pressure. Under standard physical
conditions (25°C and 1 atm pressure, for biological systems), the Keq is always the same for a given reaction,
whether or not a catalyst is present.
If the reaction A B is allowed to go to equilibrium at constant temperature and pressure, then at equilibrium
the overall free energy change (G) is zero. Therefore,
[B]eq
G0 = –RT ln
[A]eq
So, G0 = –RT ln Keq

This equation allows some simple predictions:
Keq G 0 Reaction
> 1.0 Negative proceeds forward
1.0 Zero is at equilibrium
< 1.0 Positive proceeds in reverse
As we know, the ionic composition of an acid or base varies with pH. So, the standard free energy calculated
according to the biochemistry convention is valid only at pH=7. Hence, under biochemistry convention, G0 is
symbolized by G0’ and likewise, the biochemical equilibrium constant is represented by K’eq.
SoG0’ = –RT ln K’eq
118
2.2 Metabolism
Metabolism (derives from the Greek word for change) is a series of interconnected chemical reactions occurring
within a cell; the chemical compounds involved in this process are known as metabolites. It consists of hundreds of
enzymatic reactions organized into discrete pathways. These pathways proceed in a stepwise manner, transforming
substrates into end products through many specific chemical intermediates. Each step of metabolic pathways is
catalyzed by a specific enzyme.
Reaction 1 Reaction 2 Reaction 3

A B C D
Enzyme 1 Enzyme 2 Enzyme 3
Starting Product
molecule
Metabolic pathways can be linear (such as glycolysis), cyclic (such as the citric acid cycle) or spiral (such as the
biosynthesis of fatty acids). Metabolism serves two fundamentally different purposes: generation of energy to drive
vital functions and the synthesis of biological molecules. To achieve these, metabolic pathways fall into two catego-
ries: anabolic and catabolic pathways. Anabolic pathways are involved in the synthesis of compounds and ender-
gonic in nature. Catabolic pathways are involved in the oxidative breakdown of larger complex molecules and
usually exergonic in nature. The basic strategy of catabolic metabolism is to form ATP and reducing power for
biosyntheses. Some pathways can be either anabolic or catabolic, depending on the energy conditions in the cell.
They are referred to as amphibolic pathways. Amphibolic pathways occur at the ‘crossroads’ of metabolism, acting
as links between the anabolic and catabolic pathways, e.g. the citric acid cycle.
Characteristics of metabolic pathways are:

1. They are irreversible.
2. Each one has a first committed step.
3. Those in eukaryotic cells occur in specific cellular locations.
4. They are regulated. Regulation occurs in following different ways:
I. Availability of substrate; the rate of reaction depends on substrate concentration.
II. Allosteric regulation of enzymes by a metabolic intermediate or coenzyme.
III. By extracellular signal such as growth factors and hormones that act from outside the cell in multicellular
organisms; changes the cellular concentration of an enzyme by altering the rate of its synthesis or degradation.
A number of central metabolic pathways are common to most cells and organisms. These pathways, which serve
for synthesis, degradation, interconversion of important metabolites, and energy conservation, are referred to as
the intermediary metabolism.
Metabolic pathways involve several enzyme-catalyzed reactions. Most of the reactions in living cells fall into one of
five general categories: oxidation-reductions; reactions that make or break carbon–carbon bonds; group transfers;
internal rearrangements, isomerizations and eliminations; and free radical reactions.
Feedback inhibition and feedback repression

In feedback inhibition (or end product inhibition), the end product of a biosynthetic pathway inhibits the activity of
the first enzyme that is unique to the pathway, thus controlling production of the end product. The first enzyme in
the pathway is an allosteric enzyme. Its allosteric site will bind to the end product of the pathway which alters its
active site so that it cannot mediate the enzymatic reaction.
The feedback inhibition is different from feedback repression. An inhibitory feedback system in which the end
product produced in a metabolic pathway acts as a co-repressor and represses the synthesis of an enzyme that
is required at an earlier stage of the pathway is called feedback repression.
122
2.3 Respiration
Living cells require an input of free energy. Energy is required for the maintenance of highly organized structures,
synthesis of cellular components, movement, generation of electrical currents and for many other processes. Cells
acquire free energy from the oxidation of organic compounds that are rich in potential energy.
Respiration is an oxidative process, in which free energy released from organic compounds is used in the formation
of ATP. The compounds that are oxidized during the process of respiration are known as respiratory substrates,
which may be carbohydrates, fats, proteins or organic acids. Carbohydrates are most commonly used as respiratory
substrates.
During oxidation within a cell, all the energy contained in respiratory substrates is not released free in a single step.
Free energy is released in multiple steps in a controlled manner and used to synthesise ATP, which is broken down
whenever (and wherever) energy is needed. Hence, ATP acts as the energy currency of the cell.
During cellular respiration, respiratory substrates such as glucose may undergo complete or incomplete oxidation.
The complete oxidation of substrates occurs in the presence of oxygen, which releases CO2, water and a large
amount of energy present in the substrate. A complete oxidation of respiratory substrates in the presence of
oxygen is termed as aerobic respiration.
Although carbohydrates, fats and proteins can all be oxidized as fuel, but here processes have been described by
taking glucose as a respiratory substrate. Oxidation of glucose is an exergonic process. An exergonic reaction
proceeds with a net release of free energy. When one mole of glucose (180 g) is completely oxidized into CO2 and
water, approximately 2870 kJ or 686 kcal energy is liberated. Part of this energy is used for synthesis of ATP. For
each molecule of glucose degraded to carbon dioxide and water by respiration, the cell makes up to about 30 or 32
ATP molecules, each with 7.3 kcal/mol of free energy.
C6H12O6 + 6O2 6CO2 + 6H2O + Energy (ATP + Heat)
The incomplete oxidation of respiratory substrates occurs under anaerobic conditions i.e. in the absence of oxygen.
As the substrate is never totally oxidized, the energy generated through this type of respiration is lesser than that
during aerobic respiration.
2.3.1 Aerobic respiration

Enzyme catalyzed reactions during aerobic respiration can be grouped into three major processes: glycolysis, citric
acid cycle and oxidative phosphorylation. Glycolysis takes place in the cytosol of cells in all living organisms. The
citric acid cycle takes place within the mitochondrial matrix of eukaryotic cells and in the cytosol of prokaryotic
cells. The oxidative phosphorylation takes place in the inner mitochondrial membrane. However, in prokaryotes,
oxidative phosphorylation takes place in the plasma membrane.
Table 2.3 Intracellular location of major processes of aerobic respiration

In eukaryotes,
Glycolysis – Cytosol
Citric acid cycle – Mitochondrial matrix
Oxidative phosphorylation – Inner mitochondrial membrane
In prokaryotes,
Glycolysis – Cytosol
Citric acid cycle – Cytosol
Oxidative phosphorylation – Plasma membrane
123
2.3.2 Glycolysis
Glycolysis (from the Greek glykys, meaning sweet, and lysis, meaning splitting) also known as Embden-Meyerhof
pathway, is an oxidative process in which one mole of glucose is partially oxidized into the two moles of pyruvate
in a series of enzyme-catalyzed reactions. Glycolysis occurs in the cytosol of all cells. It is a unique pathway that
occurs in both aerobic as well as anaerobic conditions and does not involve molecular oxygen.
6 CH2OH
5 O
Glucose (G) 4
OH
1
3 2
HO OH
2+ ATP
Hexokinase, Mg OH
1
DG° (kJ/mol) = –16.7 ADP CH2OP
O
Glucose-6-phosphate (G6P) OH
HO OH
Preparatory phase (Energy investment phase)
Phosphoglucoisomerase OH
2
DG° (kJ/mol) = +1.7
POH2C O CH2OH
Fructose-6-phosphate (F6P) HO
OH
2+ ATP
Phosphofructokinase, Mg OH
3
DG° (kJ/mol) = –14.2 ADP

POH2C O CH2OP
Fructose-1,6-bisphosphate (FBP)
HO
OH
2+
Aldolase, Zn OH
4
DG° (kJ/mol) = +23.9
OH
Glyceraldehyde-3-phosphate (G3P) POH2C CH CHO
Triose phosphate isomerase

5
DG° (kJ/mol) = +7.6
O
Dihydroxyacetone phosphate HOH2C C CH2OP
Step 1 : (Phosphorylation) Glucose is phosphorylated by ATP to form a glucose 6-phosphate. The negative
charge of the phosphate prevents the passage of the glucose 6-phosphate through the plasma membrane, trapping
glucose inside the cell. This irreversible reaction is catalyzed by hexokinase. Hexokinase is present in all cells of all
organisms. Hexokinase requires divalent metal ions such as Mg2+ or Mn2+ for activity. Hepatocytes and β-cells of
the pancreas also contain a form of hexokinase called glucokinase (hexokinase D). Hexokinase and glucokinase
are isozymes. Glucokinase is present in liver and beta-cells of the pancreas and has a high Km and Vmax as
compared to hexokinase.
Step 2 : (Isomerization) A readily reversible rearrangement of the chemical structure (isomerization) moves the
carbonyl oxygen from carbon 1 to carbon 2, forming a ketose from an aldose sugar. Thus, the isomerization of
glucose 6-phosphate to fructose 6-phosphate is a conversion of an aldose into a ketose.
124
CoA CH3 C S CoA
O 3
H3C C
SH
S
SH
HS
O E2
CH3 C COO— TPP FAD
NADH
Pyruvate
2 4
1 E1 E3 5
+
NAD
CO2 CH3 FADH2
S
CH OH S
TPP
Hydroxyethyl-TPP
Figure 2.6 Structure of pyruvate dehydrogenase and its catalytic activities. Catalytic activities occur in
four steps:
Step 1 : Decarboxylation of pyruvate occurs with formation of hydroxy ethyl – TPP.
Step 2 : Transfer of the two carbon unit to lipoic acid.
Step 3 : Formation of acetyl-CoA.
Step 4 : Lipoic acid is re-oxidized.
2.3.4 Krebs cycle

Krebs cycle (also known as the citric acid cycle or tricarboxylic acid cycle) was discovered by H. A. Kreb, a German
born British Biochemist, who received the Nobel prize in 1953. This cycle occurs in the matrix of mitochondria
(cytosol in prokaryotes). The whole cycle is explained in the following figure. The net result of Krebs cycle is that for
each acetyl group entering the cycle as acetyl-CoA, two molecules of CO2 are produced.
Step 1 : The Krebs cycle begins with the condensation of an oxaloacetate (four carbon unit), and the acetyl group
of acetyl-CoA (two-carbon unit). Oxaloacetate reacts with acetyl-CoA and H2O to yield citrate and coenzyme A.
This reaction, which is an aldol condensation followed by a hydrolysis, is catalyzed by citrate synthase.
Step 2a and 2b : An isomerization reaction, in which water is first removed and then added back, moves the
hydroxyl group from one carbon atom to its neighbour. The enzyme catalyzing this step, aconitase (nonheme iron
protein), is the target site for the toxic compound fluoroacetate (used as a pesticide). Fluoroacetate blocks the
citric acid cycle by its metabolic conversion of fluorocitrate, which is a potent inhibitor of aconitase.
Step 3 : Isocitrate is oxidized and decarboxylated to α-ketoglutarate. In the first of four oxidation steps in the
cycle, the carbon carrying the hydroxyl group is converted to a carbonyl group. The immediate product is unstable,
losing CO2 while still bound to the enzyme. The oxidative decarboxylation of isocitrate is catalyzed by isocitrate
dehydrogenase.
Step 4 : A second oxidative decarboxylation reaction results in the formation of succinyl-CoA from α-ketoglutarate.
α-ketoglutarate dehydrogenase catalyzes this oxidative step and produces NADH, CO2, and a high-energy thioester
bond to coenzyme A.
131
integrity requires a plentiful supply of reduced glutathione (GSH), a Cys-containing tripeptide (γ-glutamyl-
cysteinylglycine). A major function of GSH in the erythrocyte is to eliminate H2O2 and organic hydroperoxides.
H2O2, a toxic product of various oxidative processes, reacts with double bonds in the fatty acid residues of the
erythrocyte cell membrane to form organic hydroperoxides. These, in turn, result in premature cell lysis. Peroxides
are eliminated through the action of glutathione peroxidase, yielding glutathione disulfide (GSSG). So, G6PD deficiency
results in hemolytic anemia caused by the inability to detoxify oxidizing agents.
Pentose NADPH G–S–S–G 2H2O

Phosphate
Pathway Glutathione Glutathione
Reductase Peroxidase
+
2H NADP 2G–SH H2O2
Figure 2.30 Role of the pentose phosphate pathway in the reduction of oxidized glutathione.
2.6 Entner-Doudoroff pathway

Entner-Doudoroff pathway is an alternative pathway that catabolizes glucose to pyruvate using a set of enzymes
different from those used in either glycolysis or the pentose phosphate pathway. This pathway, first reported by
Michael Doudoroff and Nathan Entner, occurs only in prokaryotes, mostly in gram-negative bacteria such as
Pseudomonas aeruginosa, Azotobacter, Rhizobium.
In this pathway, glucose phosphate is oxidized to 2-keto-3-deoxy-6-phosphogluconic acid (KDPG) which is cleaved
by 2-keto-3-deoxyglucose-phosphate aldolase to pyruvate and glyceraldehyde-3-phosphate. The latter is oxidized
to pyruvate by glycolytic pathway where in two ATPs are produced by substrate level phosphorylations. This
process yields one ATP as well as one NADH and one NADPH for every glucose molecule.
— —
COO COO O O
—
H C OH C O CH3 C C O
6 CH2OH CH2OP
5 O ATP ADP O NADP NADPH HO C H H2O H C H Pyruvate
4 1
OH OH H C OH H C OH
3 2
HO OH HO OH
OH
OH OH H C OH H C OH
POH2C CH CHO
CH2O P CH2O P
Glucose Glucose-6-phosphate
6-Phosphogluconate Glyceraldehyde-3-
2-Keto-3-deoxy-
phosphate
6-phosphogluconate
+
NAD
2 ADP
NADH
2 ATP
Pyruvate
Figure 2.31 Entner-Doudoroff pathway.
2.7 Photosynthesis
Photosynthesis is a physiochemical process by which photosynthetic organisms convert light energy into chemical
energy in the form of reducing power (as NADPH) and ATP, and use these chemicals to drive carbon dioxide
fixation.
154
molecules act together as one photosynthetic unit in which only one member of the group- the reaction center
chlorophyll- actually transfers electrons to an electron acceptor. The majority of the chlorophyll molecules serve as
an antenna complex, collecting light and transferring the energy to the reaction center, where the photochemical
reaction takes place.
Chl
Light
—
Chl Chl e Acceptor
Chl Chl Chl Reaction center
Chl Chl Donor

e—
Chl
Antenna Chl molecules
Figure 2.40 Simplified representation of the photosynthetic unit consisting of the light-harvesting antenna
chlorophyll molecules and the reaction center, small arrows in the chlorophyll antenna represent transfer of
excitation energy.
2.7.5 Hill reaction

In 1937, Robert Hill found that in the presence of light, isolated chloroplast from green leaves reduce a variety of
compounds. Hill’s isolated chloroplast did not evolve O2 when illuminated, but did so when the suitable electron
acceptor (oxidants) like potassium ferrioxalate or potassium ferricyanide was added to the illuminated suspension.
This phenomenon is known as Hill reaction. It is a light-driven transfer of electrons from water to non-physiological
oxidants (Hill reagent). One of the non-physiological oxidants used by Hill was the dye 2,6-dichlorophenolindophenol
(DCPIP), now called a Hill reagent, which in its oxidized form is blue and in its reduced form is colourless. Later S.
Ochoa showed that NADP+ is the biological electron acceptor in the chloroplast. Light reduces NADP+ which in turn
serves as the reducing agent for carbon fixation in the Calvin cycle.
2H2O + 2NADP+ 2NADPH + 2H+ + O2
2.7.6 Oxygenic and anoxygenic photosynthesis

In anoxygenic photosynthesis, light energy is captured and converted into ATP, without the production of oxygen.
Water is, therefore, not used as an electron donor. In oxygenic photosynthesis, light energy is captured and
converted into ATP, with the production of oxygen. Here, synthesis of oxygen occurs due to photooxidation or
photolysis of water.
Before 1930, reserchers considered carbon dioxide as source of oxygen in oxygenic photosynthetic organisms.
This idea was challenged in the 1930s by C. B. van Niel of Stanford University. According to him, the O2 produced
by plants is derived from H2O and not from CO2. van Niel found that photosynthetic bacteria Chromatium vinosum
assimilates CO2 in light without evolving O2. Such bacteria use H2S, instead of water as an electron donor and forms
sulphur instead of O2.
CO2 + 2H2S (CH2O) + H2O + 2S
The chemical similarity between H2S and H2O led van Niel to propose the general photosynthetic reaction:
CO2 + 2H2A (CH2O) + 2A + H2O
where H2A is H2O in green plants and cyanobacteria and H2S in photosynthetic sulfur bacteria. Thus, he hypothesized
that in oxygenic photosynthetic organisms water acts as a source of oxygen.
162
The validity of van Niel’s hypothesis was established in the year 1941 by Ruben and Kamen. They directly demonstrated
18
by isotopic study using O labeled water and CO2 on green alga chlorella that the source of oxygen formed in
photosynthesis is water. In the following equations, bold letter denotes labeled atom of oxygen (18O).
Experiment 1: CO2 + 2H2O ⎯→ [CH2O] + H2O + O2
Experiment 2: CO2 + 2H2O ⎯→ [CH2O] + H2O + O2
2.7.7 Concept of pigment system

In 1943, Robert Emerson and Charlton Lewis examined the action spectrum in the visible region for oxygen
evolution in the green algae Chlorella pyrenoidosa. They found that the quantum yield remained fairly constant
upto 680 nm, beyond which it declined sharply. This drop in quantum yield in the far–red region of the spectrum was
called the red drop phenomenon. This suggests that light with a wavelength greater than 680 nm is much less
efficient than light of shorter wavelengths. Quantum yield is the number of oxygen molecules produced per photon
absorbed. The reciprocal of quantum yield is quantum requirement i.e. number of photons needed for each oxygen
molecule produced.
In the another experiment, Emerson and his colleagues set up two beams of light – one in the red region (wavelengths
less than 680 nm) and the other in the far red region (wavelengths greater than 680 nm). Emerson found that when
the two beams were applied simultaneously, the rate of photosynthesis was 2–3 times greater than the sum of the
rates obtained with each beam separately. This phenomenon is known as a Emerson enhancement effect. The
enhancement effect suggests that photosynthesis involves two photosystems – one driven by light of long wavelength
(greater than 680 nm) and other driven by light of short wavelength (less than or equal to 680 nm).
Far-red and
Relative rate of photosynthesis
red light on Off
Far-red Red light

light on Off on Off
Time
Figure 2.41 Emerson enhancement effect. The rate of photosynthesis when red and far-red light are given
together is greater than the sum of the rates when they are given apart. The enhancement effect provided
essential evidence in favor of the concept that photosynthesis is carried out by two different photosystems
working in series but with slightly different wavelength optima.
Pigment systems
In all natural photosynthetic systems, pigment molecules are bound to proteins forming pigment–protein complexes
called pigment system (or photosystem). The pigment systems have two components: Photochemical reaction
center and antenna complex.
Photochemical reaction center carries out photochemical reaction. In all oxygenic photosynthetic organisms, the
reaction center contains the special pair of chlorophyll a molecules associated with specific proteins that participate
in photochemical reactions.
163
2.7.8 Stages of photosynthesis

Photosynthesis is a two-stage process: one stage is dependent on the light and another independent of it.
The light reactions, a light-dependent reactions which occur in the grana of chloroplast, and require the direct
energy of light to make NADPH and ATP that are used in the dark reaction. A process of formation of ATP from ADP
and inorganic phosphate by utilizing light energy is called photophosphorylation.
The dark reaction, a light-independent reaction, occurs in the stroma of the chloroplasts when the products of the
light reaction, ATP and NADPH, are used to make glyceraldehyde 3-phosphate (a triose phosphate) from reduction
of carbon dioxide.
Chloroplast
Light
Stroma
CO2
Granum
ATP Calvin
Light
+ cycle
reaction
NADPH
H2O O2
Triose phosphate
Glucose
Figure 2.44 The chemical reactions in which water is oxidized to oxygen, NADP is reduced, and ATP is formed
are known as the thylakoid reactions because almost all the reactions up to NADP reduction take place within
the thylakoids. The carbon fixation and reduction reactions are called the stroma reactions because the
carbon reduction reactions take place in the aqueous region of the chloroplast, the stroma.
Table 2.11 Differences between light and dark reactions in plants

Light reaction Dark reaction
Light-dependent phase Light-independent phase
Occurs in the grana of chloroplast Occurs in the stroma of the chloroplasts
Photochemical reaction occurs Chemical reaction occurs
Formation of ATP and NADPH occurs Utilization of ATP and NADPH occurs
Oxidation of H2O occurs Reduction of CO2 occurs
2.7.9 Light reactions

Light reaction (photochemical reaction) in the photosystem starts electron flow. In oxygenic photosynthetic organisms,
flow of electron is of two types: non-cyclic as well as cyclic.
Noncyclic electron flow
It is a light-induced electron transport from water to NADP+ and a concomitant evolution of oxygen. It involves a
collaboration of two photosystems: PSII and PSI. Electrons move from water through PSII to PSI and then to
NADP+. Electron transport leads to generation of a proton-motive force and synthesis of ATP. Formation of ATP due
to light-induced non-cyclic electron flow is called non-cyclic photophosphorylation.
The diagram in figure 2.45 often called the Z scheme because of its overall form, outlines the pathway of electron
flow between the two photosystems and the energy relationship in the light reactions.
165
2.8 Photorespiration
Otto Warburg made an observation that O2 inhibits photosynthesis in C3 plants. This phenomenon, originally known
as the Warburg effect, was later recognized as the light-dependent release of CO2 due to oxygenase activity of
RuBisCo. RuBisCo is a bifunctional enzyme. It catalyzes both the carboxylation and the oxygenation of RuBP. At low
CO2 concentration, RuBisCo performs oxygenase activity. Oxygenation of RuBP leads to the production of one
molecule of 3-phosphoglycerate and one of 2-phosphoglycolate.
CH2OP
C O CH2OP
O2 H2O CH2OP
H C OH + H C OH
—
Rubisco COO
H C OH COOH
CH2OP 2-phosphoglycolate 3-phosphoglycerate
Ribulose-1,5-bisphosphate
2-Phosphoglycolate produced by RuBisCo when it oxygenates RuBP cannot be utilized within the Calvin cycle. The
pathway by which 2-Phosphoglycolate is further metabolized is described as glycolate pathway (C2 cycle or
oxidative photosynthetic carbon cycle).
The pathway involves three subcellular compartments, the chloroplasts, peroxisomes and mitochondria. The key
features of the pathway are the conversion of two-carbon molecule, 2-phosphoglycolate to glycine and decarboxylation
of two molecules of glycine to serine, CO2 and NH3. The three-carbon molecule, serine, is then converted into
3-phosphoglycerate, which re-enters the Calvin cycle. Release of CO2 in this process decreases the photosynthetic
output and limits the plant biomass production.
O2 O2 H2O2
2-Phospho- Glycolate Glycolate Glyoxylate Gly 2 Glycine

RuBP
glycolate +
NAD
Pi
Calvin
cycle NADH CO2
Hydroxy- +
3-Phospho- Glycerate pyruvate Ser Serine NH3
Glycerate
glycerate
+
ADP ATP NAD NADH
Chloroplast Peroxisome Mitochondrion

Figure 2.56 Photorespiration. Operation of the C2 cycle involves the cooperative interaction among three
organelles: chloroplasts, mitochondria and peroxisomes. Glycolate transported from the chloroplast into
the peroxisome are converted to glycine, which in turn is exported to the mitochondrion and transformed to
serine with the concurrent release of carbon dioxide. Serine is transported to the peroxisome and transformed
to glycerate. The latter flows to the chloroplast where it is phosphorylated to 3-phosphoglycerate and
incorporated into the Calvin cycle.
Effect of temperature
Apart from the ambient concentrations of O2 and CO2, the factor influencing the enzyme’s oxygenase activity most
is the temperature. High temperatures promote oxygenation, and hence the photorespiration, because the solubility
of CO2 in water declines more rapidly than that of O2 as the temperature is increased. Second the specificity factor
of RuBisCo also decreases with increasing temperature.
178
In fact, the C4 cycle concentrates CO2 in the bundle sheath cells, keeping the CO2 concentration in the bundle
sheath cells high enough for RuBisCo to bind carbon dioxide rather than oxygen. In this way, C4 photosynthesis
minimizes photorespiration and enhances carbohydrate production.
Chloroplast Chloroplast
+
NADPH NADP
Oxaloacetate
(OAA) OAA Malate Malate
+
NADP
+
NADP
– PEP NADPH
CO2 HCO3 carboxylase Malic
enzyme CO2 Calvin
cycle
Phosphoenol- PEP Pyruvate Pyruvate
pyruvate (PEP)
AMP ATP
Cytosol Cytosol
Mesophyll cell Bundle-sheath cell
Figure 2.58 There are three distinct biochemical subtypes of C4 cycle. These are classified on the basis of
the enzyme which is employed to decarboxylate C4 acids in the bundle sheath. These are NADP+–malic
enzyme type, NAD+–malic enzyme type and PEP carboxykinase type. In the figure, reactions of the NADP+–
malic enzyme type C4 cycle is described. CO2 is transported from mesophyll cells into the bundle sheath
cells by coupling it to phosphoenolpyruvate, forming oxaloacetate. Oxaloacetate is then reduced to malate,
which is passed to bundle sheath cells and decarboxylated. The pyruvate product is returned to the mesophyll
cells, where it is phosphorylated to regenerate phosphoenolpyruvate.
Leaf anatomy of C4 plants

C4 plants are unique in possessing two types of photosynthetic cell. A layer of cells surrounding the vascular
bundle, the bundle-sheath, is a common structural feature, but only in C4 plants it contains chloroplasts. The
bundle-sheath is thick-walled, sometimes suberized and there is no direct access from the intercellular spaces of
the mesophyll. The appearance of a wreath of cells surrounding the vasculature gives rise to the term Kranz
(German: wreath) anatomy. The distance between bundle-sheath cells are normally only two or three mesophyll
cells, so that no mesophyll cell is more than one cell away from a bundle-sheath cell. Mesophyll cells are also
connected to the bundle-sheath cells by large numbers of plasmodesmata.
2.8.2 CAM pathway

Crassulacean Acid Metabolism (CAM) is a photosynthetic adaptation in succulent plants. Succulent plants, also
known as fat plants, are xerophytic plants adapted to arid climates or soil conditions. Succulent plants store water
in their leaves and stems. The storage of water often gives succulent plants a swollen or fleshy appearance than
other plants, a characteristic known as succulence. The best-known succulents are cacti. These plants open their
stomata during the night and close them during the day. Closing stomata during the day helps succulent plants
conserve water, but it also prevents CO2 from entering the leaves. During the night, when their stomata are open,
these plants take up CO2. Assimilation of CO2 occurs into malic acid at night which is stored in the vacuole. This
mode of carbon fixation is called crassulacean acid metabolism, or CAM, after the plant family Crassulaceae, the
succulents in which the process was first discovered.
During the day time, when the light reactions can supply ATP and NADPH for the Calvin cycle, CO2 is released from
the malate for fixation through Calvin cycle. This cycle differs from the C4 cycle. In C4 plants, formation of the
180
Glycogen storage diseases
Glycogen storage diseases are caused by a genetic deficiency of one or another of the enzymes of glycogen
metabolism. Many diseases have been characterized that result from an inherited deficiency of the enzyme.
These defects are listed in the table.
Table 2.17 Glycogen storage diseases

Name Enzyme deficiency
Von Gierke’s disease Liver glucose-6-phosphatase
Pompe’s disease Lysosomal α1 → 4 and α1 → 6 glucosidase (acid maltase)
Hers’ disease Liver phosphorylase
Tarui’s disease Muscle and erythrocyte phosphofructokinase 1
McArdle’s disease Muscle glycogen phosphorylase
Andersen’s disease Amylo (1,4 → 1,6) transglycosylase (Branching enzyme)
2.10 Lipid metabolism

2.10.1 Synthesis and storage of triacylglycerols
All animals and plants have the ability to synthesize triacylglycerol (TAG). In animals, many cell types and organs
have the ability to synthesise triacylglycerols, but the liver and intestines are most active. Within all cell types, even
those of the brain, triacylglycerols are stored as cytoplasmic lipid droplets (also termed fat globules, oil bodies, lipid
particles, adiposomes, etc.) enclosed by a monolayer of phospholipids and hydrophobic proteins, such as the
perilipins in adipose tissue or oleosins in seeds. Two main biosynthetic pathways are known, the sn-glycerol-3-
phosphate pathway, which predominates in liver and adipose tissue, and a monoacylglycerol pathway in the intestines.
The most important route to triacylglycerol biosynthesis is the sn-glycerol-3-phosphate or Kennedy pathway.
O O O
CH2 OH CH2 O C R1 CH2 O C R1 CH2 O C R1
O O
1 2 3
CH OH CH OH CH O C R2 CH O C R2
Fatty Fatty
acyl-CoA acyl-CoA Pi
CH2 OP CH2 OP CH2 OP CH2 OH
Glycerol-3-phosphate Lysophosphatidic acid Phosphatidic acid Diacylglycerol
Fatty 4
acyl-CoA
Enzymes
1 Glycerol-3-phosphate acyltransferase O
2 Acylglycerophosphate acyltransferase CH2 O C R1
3 Phosphatidic acid phosphohydrolase O
4 Diacylglycerol acyltransferase CH O C R2
O
CH2 O C R3
Triacylglycerol
Figure 2.68 Triacylglycerol biosynthetic pathway.
192
Glucose
Glucose 6-phosphate
Ribose 5-phosphate
Histidine
Erythrose 3-Phosphoglycerate Serine

4-phosphate
Glycine
Cysteine
Phosphoenolpyruvate
Tryptophan Pyruvate Alanine

Phenylalanine Valine
Tyrosine Citrate Leucine

Isoleucine
Aspartate Oxaloacetate -Ketoglutarate Glutamate
Asparagine Glutamine
Methionine Proline
Threonine Arginine
Lysine
Figure 2.85 Overview of amino acid biosynthesis. The carbon skeleton precursors are derived from
three sources—glycolysis, TCA cycle and pentose phosphate pathway.
2.11.2 Biological nitrogen fixation

Nitrogen is present in many forms in the biosphere. 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 includes lightning, photochemical reactions and biological
nitrogen fixation. Approximately 90% of nitrogen fixation is biological nitrogen fixation, in which prokaryotic
organisms fix molecular nitrogen into ammonium ions. 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
211
Chapter 03
3.1 What is a Cell?

The basic structural and functional unit of cellular organisms is the cell. It is an aqueous compartment bound by cell
membrane, which is capable of independent existence and performing the essential functions of life. All organisms,
more complex than viruses, consist of cells. Viruses are noncellular organisms because they lack cell or cell-like
structure. In the year 1665, Robert Hooke first discovered cells in a piece of cork and also coined the word cell. The
word cell is derived from the Latin word cellula, which means small compartment. Hooke published his findings in
his famous work, Micrographia. Actually, Hooke only observed cell walls because cork cells are dead and without
cytoplasmic contents. Anton van Leeuwenhoek was the first person who observed living cells under a microscope
and named them animalcules, meaning little animals.
On the basis of the internal architecture, all cells can be subdivided into two major classes, prokaryotic cells and
eukaryotic cells. Cells that have unit membrane bound nuclei are called eukaryotic, whereas cells that lack a
membrane bound nucleus are prokaryotic. Eukaryotic cells have a much more complex intracellular organization
with internal membranes as compared to prokaryotic cells. Besides the nucleus, the eukaryotic cells have other
membrane bound organelles (little organs) like the endoplasmic reticulum, Golgi complex, lysosomes, mitochondria,
microbodies and vacuoles. The region of the cell lying between the plasma membrane and the nucleus is the
cytoplasm, comprising the cytosol (or cytoplasmic matrix) and the organelles. The prokaryotic cells lack such unit
membrane bound organelles.
Cell theory
In 1839, Schleiden, a German botanist, and Schwann, a British zoologist, led to the development of the cell theory
or cell doctrine. According to this theory all living things are made up of cells and cell is the basic structural and
functional unit of life. In 1855, Rudolf Virchow proposed an important extension of cell theory that all living cells
arise from pre-existing cells (omnis cellula e cellula). The cell theory holds true for all cellular organisms. Non-
cellular organisms such as virus do not obey cell theory. Over the time, the theory has continued to evolve. The
modern cell theory includes the following components:
• All known living things are made up of one or more cells.
• The cell is the structural and functional unit of life.
• All cells arise from pre-existing cells by division.
• Energy flow occurs within cells.
• Cells contain hereditary information (DNA) which is passed from cell to cell.
• All cells have basically the same chemical composition.
Evolution of the cell

The earliest cells probably arose about 3.5 billion years ago in the rich mixture of organic compounds, the primordial
soup, of prebiotic times; they were almost certainly chemoheterotrophs. Primitive heterotrophs gradually acquired
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the capability to derive energy from certain compounds in their environment and to use that energy to synthesize
more and more of their own precursor molecules, thereby becoming less dependent on outside sources of these
molecules-less extremely heterotrophic. A very significant evolutionary event was the development of photosynthetic
ability to fix CO2 into more complex organic compounds. The original electron (hydrogen) donor for these
photosynthetic organisms was probably H2S, yielding elemental sulfur as the byproduct, but at some point, cells
developed the enzymatic capacity to use H2O as the electron donor in photosynthetic reactions, producing O2. The
cyanobacteria are the modern descendants of these early photosynthetic O2 producers.
One important landmark along this evolutionary road occurred when there was a transition from small cells with
relatively simple internal structures - the so-called prokaryotic cells, which include various types of bacteria - to a
flourishing of larger and radically more complex eukaryotic cells such as are found in higher animals and plants.
The fossil record shows that earliest eukaryotic cells evolved about 1.5 billion years ago. Details of the evolutionary
path from prokaryotes to eukaryotes cannot be deduced from the fossil record alone, but morphological and
biochemical comparison of modern organisms has suggested a reasonable sequence of events consistent with the
fossil evidence.
Three major changes must have occurred as prokaryotes gave rise to eukaryotes. First, as cells acquired more
DNA, mechanisms evolved to fold it compactly into discrete complexes with specific proteins and to divide it equally
between daughter cells at cell division. These DNA-protein complexes called chromosomes become especially
compact at the time of cell division. Second, as cells became larger and intracellular membrane organelles developed.
Eukaryotic cells have a nucleus which contains most of the cell’s DNA, enclosed by a double layer of membrane.
The DNA is, thereby, kept in a compartment separate from the rest of the contents of the cell, the cytoplasm, where
most of the cell’s metabolic reactions occur.
Finally, primitive eukaryotic cells, which were incapable of photosynthesis or of aerobic metabolism, pooled their
assets with those of aerobic bacteria or photosynthetic bacteria to form symbiotic associations that became
permanent. Some aerobic bacteria evolved into the mitochondria of modern eukaryotes, and some photosynthetic
cyanobacteria became the chloroplasts of modern plant cells.
3.2 Structure of eukaryotic cells

3.2.1 Plasma membrane
Plasma membrane is a dynamic, fluid structure and forms the external boundary of cells. It acts as a selectively
permeable membrane and regulates the molecular traffic across the boundary. The plasma membrane exhibits
selective permeability; that is, it allows some solutes to cross it more easily than others. Different models were
proposed to explain the structure and composition of plasma membranes. In 1972, Jonathan Singer and Garth
Nicolson proposed fluid-mosaic model, which is now the most accepted model. In this model, membranes are viewed
as quasi-fluid structures in which proteins are inserted into lipid bilayers. It describes both the mosaic arrangement
of proteins embedded throughout the lipid bilayer as well as the fluid movement of lipids and proteins alike.
Peripheral protein
Phospholipid
bilayer
Integral
protein Peripheral
protein
Figure 3.1 Fluid mosaic model for membrane structure. The fatty acyl chains in the lipid bilayer form a
fluid, hydrophobic region. Integral proteins float in this lipid bilayer. Both proteins and lipids are free to move
laterally in the plane of the bilayer, but movement of either from one face of the bilayer to the other is restricted.
235
Glycolipids
Glycolipids contain carbohydrate (either monosaccharide or oligosaccharide) covalently attached to the lipid. These
can derive from either glycerol or sphingosine. The simplest glycolipid, called a cerebroside, contains a single sugar
residue, either glucose or galactose. Gangliosides are more complex glycolipids, containing a branched chain of as
many as seven sugar residues. The glycolipids are found exclusively in the outer leaflet of the plasma membrane,
with their carbohydrate portions exposed on the cell surface.
Sterols
The basic structure of sterol is a steroid nucleus, consisting of four fused rings, three with six carbons and one with
five. It is planar, and relatively a rigid structure. Cholesterol is the major sterol present in the plasma membrane of
animal cells. The plasma membrane of plant cells lacks cholesterol, but they contain other sterols like stigmasterol,
sitosterol. With rare exceptions like Mycoplasma, bacterial plasma membrane also lacks cholesterol.
Table 3.1 Major lipid components of plasma membranes

Source PC PE + PS SM Cholesterol
Plasma membrane (human RBC) 21 29 21 26
Plasma membrane (E. coli) 0 85 0 0
Myelin membrane (human neurons) 16 37 13 34
Composition in mol %
PC – phosphatidylcholine; PE – phosphatidylethanolamine; PS – phosphatidylserine; SM – sphingomyelin.
Lipids are not randomly mixed in each leaflet of a bilayer. Certain lipids in the plasma membrane, particularly
cholesterol and sphingolipids, are organized into aggregates called lipid rafts. Lipid rafts are membrane
microdomains that are enriched with cholesterol and glycosphingolipids. These microdomains also contain specific
proteins. In mammalian cells, lipid rafts termed caveolae are marked by the presence of caveolin proteins. The
rafts in cells appear to be heterogeneous both in terms of their protein and lipid content, and can be localized to
different regions of the cell. Lipid rafts have been implicated in processes as diverse as signal transduction,
endocytosis and cholesterol trafficking.
When amphipathic lipids are mixed with water, three types of lipid aggregates can form. In the case of fatty acid
salt, which contains only one fatty acid chain, the molecules form a small and spherical micellar structure (diameter
usually <20nm) in which the hydrophobic fatty acid chains are hidden inside the micelle. A second type of lipid
aggregate in water is the bilayer, in which two lipid monolayers combine to form a two-dimensional sheet. In the
third type of lipid aggregate, lipid bilayer forms a hollow sphere called a liposome. Liposomes are closed, self
sealing, solvent filled vesicles that are bound by only a single bilayer.
Asymmetry of lipid bilayer

The phospholipids in plasma membranes are asymmetrically distributed across the bilayer; the amine-containing
phospholipids are enriched on the cytoplasmic surface of the plasma membrane, while the choline-containing and
sphingolipids are enriched on the outer surface. This asymmetry is usually not absolute, except for glycolipids. In
the human erythrocyte, for example, the phospholipids, such as sphingomyelin and phosphatidylcholine are mostly
found in the extracytoplasmic leaflet, whereas phosphatidylserine and phosphatidylethanolamine are preferentially
located on the cytoplasmic face.
The maintenance of transbilayer lipid asymmetry is essential for normal membrane function. Once lipid asymmetry
has been established, it is maintained by a combination of slow transbilayer diffusion, protein-lipid interactions and
protein-mediated transport.
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3.3 Membrane potential

Electrical character of ion transport may be electroneutral i.e. electrically silent either by symport of the oppositely
charged ions or antiport of similarly charged ions or electrogenic i.e. result in charge separation across the
membrane. Electrogenic transport affects and can be affected by the membrane potential. For example, the Na+–
K+ pump imports 2K+ and simultaneously exports 3Na+; that is, it moves 1 positive charge out of the cell. Its
electrogenic operation directly contributes to the negative inside membrane potential, which is evidenced by the
fact that stopping the pump using an alkaloid inhibitor, ouabain, causes an immediate and slight depolarization of
the cell membrane.
All cells have an electrical potential difference, or membrane potential, across their plasma membrane. Electrical
potential across cell membranes is a function of the electrolyte concentrations in the intracellular and extracellular
solutions and of the selective permeabilities of the ions. Active transport of ions by ATP-driven ion pumps, generate
and maintain ionic gradients. In addition to ion pumps, which transport ions against concentration gradients, plasma
membrane contains channel protein that allows ions to move through it at different rates down their concentration
gradient. Ion concentration gradients and selective movements of ions create a difference in electric potential or
voltage across the plasma membrane. This is called membrane potential.
How membrane potentials arise?

To help explain how an electric potential across the plasma membrane can arise, we first consider a set of simplified
experimental systems in which a membrane, which is only permeable for K+ separates a 1 M KCl solution on the left
from a 1 M KCl solution on the right. Because the concentrations of K+ across the membrane are equal, there is no
net flow of ions across the membrane and thus no electric potential is generated. If the concentration of K+ ions
across the membrane is different as shown in the figure, then K+ ions tend to move down their concentration
gradient from the left side to the right, leaving an excess of negative Cl— ions compared with K+ ions on the left side
and generate an excess of positive K+ ions compared with Cl— ions on the right side. The resulting separation of
charge across the membrane constitutes an electric potential, or voltage, with the left side of the membrane having
excess negative charge with respect to the right. However, continued left-to-right movement of the K+ ions eventually
is inhibited by the mutual repulsion between the excess positive charges accumulated on the right side of the
membrane and by the attraction of K+ ions to the excess negative charges built up on the left side. The system soon
reaches an equilibrium point at which the two opposing factors that determine the movement of K+ ions—the
membrane electric potential and the ion concentration gradient—balance each other out. At equilibrium, no net
movement of K+ ions occurs across the membrane.
+ +
(a) No net flow of K (b) Net flow of K
— +
— +
— +
— +
— +
+
K
— +
— +
— +
1 M KCl 1 M KCl 1 M KCl — + 0.1 M KCl
+ +
Membrane only permeable to K Membrane only permeable to K
No membrane potential Membrane potential established
Figure 3.16 Two compartments are separated by a membrane permeable only to K+ ions. (a) Because the
concentrations in the two compartments are equal, there is no net flow of ions across the membrane and no
electrical potential. (b) A difference in concentration causes K+ ions to move from the left compartment to
the right one. At equilibrium, an electrical potential is established across the membrane due to an accumulation
of negative charges on the left side and positive charges on the right.
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3.4 Transport of macromolecules across plasma membrane

The plasma membrane is a dynamic structure that functions to segregate the chemically distinct intracellular milieu
(the cytoplasm) from the extracellular environment by regulating and coordinating the entry and exit of small and
large molecules. Essential small molecules, such as amino acids, sugars and ions, can traverse the plasma membrane
through the action of integral membrane protein pumps or channels. Macromolecules must be carried into the cell
in membrane bound vesicles derived by the invagination and pinching-off of pieces of the plasma membrane in a
process termed endocytosis.
3.4.1 Endocytosis
The term endocytosis was coined by Christian de duve in the year 1963. Endocytosis is a process whereby eukaryotic
cells internalize material from their surrounding environment. Internalization is achieved by the formation of
membrane-bound vesicles at the cell surface that arise by progressive invagination of the plasma membrane,
followed by pinching off and release of free vesicles into the cytoplasm.
Classically, endocytosis has been divided into phagocytosis (cellular eating) and pinocytosis (cellular drinking).
Phagocytosis or cell eating (first reported by Metchnikoff) describes the internalization of large particles following
particle binding to specific plasma membrane receptors and by the formation of large endocytic vesicles (generally
>250 nm in diameter) called phagosomes. Phagocytosis occurs in specialized mammalian cells (macrophage,
monocytes, neutrophils). It is an active and highly regulated process involving specific cell-surface receptors and
signalling cascades mediated by Rho-family GTPases.
Pinocytosis or cell drinking (also termed as fluid-phase endocytosis) involves the ingestion of fluid and solutes via
small vesicles (<150 nm in diameter). Uptake of material dissolved in extracellular fluid during pinocytosis occurs
both selectively as well as non-selectively. Selective and efficient uptake occurs when solutes are captured by
specific high-affinity receptors (receptor mediated endocytosis). In receptor-mediated endocytosis, a specific receptor
on the cell surface binds tightly to the extracellular macromolecule (the ligand) that it recognizes. The plasma
membrane region containing the receptor-ligand complex then undergoes endocytosis, becoming a transport vesicle.
Receptor ligand complexes are selectively incorporated into the intracellular transport vesicles. Pinocytosis occurs
in all cells by at least four basic mechanisms: macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated
endocytosis and clathrin- and caveolae independent endocytosis.
Phagocytosis Macropinocytosis
(>1µm)
Clathrin-mediated Caveolae-mediated Clathrin- and

endocytosis endocytosis caveolae-independent
(~120 nm) (~80 nm) endocytosis (~90 nm)
Figure 3.22 The endocytic pathways differ with regard to the size of the endocytic vesicle, the nature
of the cargo (ligands, receptors and lipids) and the mechanism of vesicle formation.
Macropinocytosis
Macropinocytosis involves the membrane ruffling that is induced in many cell types upon stimulation by growth
factors or other signals. Like phagocytosis, the signalling cascades that induce macropinocytosis involve Rho-
family GTPases, which trigger the actin-driven formation of membrane protrusions. However, unlike phagocytosis,
262
plasma membrane at the opposite side. An example of transcytosis is the movement of maternal antibodies across
the intestinal epithelial cells of the newborn rat. A newborn rat obtains antibodies from its mother’s milk by transporting
them across the epithelium of its gut. The lumen of the gut is acidic, and, at this low pH, the antibodies in the milk
bind to specific receptors on the apical (absorptive) surface of the gut epithelial cells. The receptor-antibody
complexes are internalized via clathrin coated vesicles and are delivered to early endosomes. The complexes
remain intact and are retrieved in transport vesicles that bud from the early endosome and subsequently fuse with
the basolateral domain of the plasma membrane. On exposure to the neutral pH of the extracellular fluid that
bathes the basolateral surface of the cells, the antibodies dissociate from their receptors and eventually enter the
newborn’s bloodstream.
3.4.3 Exocytosis
Transport vesicles destined for the plasma membrane undergo fusion with the plasma membrane and release the
contents outside the cell in the process called exocytosis. It may be a constitutive secretory pathway (carried out
by all cells) or regulated secretory pathway (carried out by specialized cells). Examples of proteins released by
such constitutive (or continuous) secretion include collagen by fibroblasts, serum proteins by hepatocytes, and
antibodies by activated B-lymphocytes.
Vesicle containing
soluble proteins for
constitutive secretion
Constitutive
secretory
pathway
Trans-Golgi
network Extracellular space
Regulated
secretory
pathway
Secretory
Golgi complex
vesicle containing
secretory proteins
Plasma
membrane
Figure 3.27 Constitutive and regulated secretory pathways. The two pathways diverge in the trans Golgi
network. The constitutive secretory pathway operates in all cells. Many soluble proteins are continually
secreted from the cell by this pathway. This pathway also supplies the plasma membrane with newly
synthesized lipids and proteins. Specialized secretory cells also have a regulated secretory pathway, by
which selected proteins in the trans Golgi network are diverted into secretory vesicles, where the proteins
are concentrated and stored until an extracellular signal stimulates their secretion. The regulated secretion
of small molecules, such as histamine and neurotransmitters, occurs by a similar pathway.
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The regulated secretory pathway is found mainly in cells specialized for secreting products rapidly on demand such
as hormones, neurotransmitters, or digestive enzymes. In this secretory pathway, secretory vesicles form from
the trans Golgi network, and they release their contents to the cell exterior by exocytosis in response to specific
signals. The secreted product can be either a small molecule (such as histamine) or a protein (such as a hormone
or digestive enzyme). Proteins destined for secretion (called secretory proteins) are packaged into appropriate
secretory vesicles in the trans Golgi network. The signal that directs secretory proteins into such vesicles is not known.
3.5 Ribosome
The ribosomes are large ribonucleoproteins consisting of RNAs and proteins, ubiquitous in all cells, that translate
genetic information stored in the messenger RNA into polypeptides. The ribosome is approximately globular structure,
its average diameter ranging from 2.5 nm (Escherichia coli) to 2.8 nm (mammalian cells). The functional ribosomes
consist of two subunits of unequal size, known as the large and small subunits. Ribosomes consist of rRNA and
r-proteins. The r-proteins are termed as L or S depending on whether the protein is from the large or small subunit.
Table 3.5 Ribosome structure and chemical composition

Property Prokaryote Eukaryote
Overall size 70S 80S
Small subunit 30S 40S
Number of proteins ~21 ~30
RNA size (number of bases) 16S (1500) 18S (2300)
Large subunit 50S 60S
Number of proteins ~34 ~50
RNA size (number of bases) 23S (2900) 28S (4200)
5S (120) 5.8S (160)
5S (120)
‘S’ stands for the sedimentation coefficient. It is the ratio of a velocity to the centrifugal acceleration. The sedimentation
coefficient has units of second. A sedimentation coefficient of 1 × 10–13 second is defined as one Svedberg, S.
rDNA organization
In prokaryotes such as Escherichia coli, there are three ribosomal RNAs (16S, 23S and 5S), which are organized as
a single transcription unit. In all eukaryotes studied so far, the organization of the ribosomal RNA genes is recognizably
similar to that of prokaryotes, but with major differences; the size of the small subunit RNA has increased from 16S
to 18S, and that of the large subunit from 23S and 28S; a new small 5.8S rRNA has become interspersed between
the 18S and the 28S rRNA, and the 5S rRNA has become separated from the other rRNAs in a different transcription
unit. The former transcription unit is generally referred to as the rRNA gene or the ribosomal DNA (rDNA). 5S genes
are transcribed by a different RNA polymerase from rRNA genes (RNA polymerase III rather than RNA polymerase I).
There are generally more copies of the 5S genes than of the rRNA genes. The human genome contains about 100
copies of rRNA genes per haploid set. Many other species, including most plants, have several thousand copies. The
rRNA gene is transcribed to give a precursor the 45S pre-rRNA, which is processed in a series of post-transcriptional
modifications to the mature rRNA species.
Table 3.6 Different types of ribosomes and their rRNAs

Ribosome source Sedimentation coefficient rRNA (large subunit/small subunit)
Bacterial 70S 5S, 23S/16S
Chloroplast 70S 5S, 23S/16S
Mitochondria (human) 55S 16S/12S
Archaebacteria 70S 5S,23S/16S
Eukaryotes (cytosol) 80S 5S, 5.8S, 28S/18S
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Unfolded protein Folded protein
NH2
COOH Signal peptide

H2N
COOH H2N Signal patch

H2N
Figure 3.28 Signal peptide and signal patch.
Protein translocation describes the movement of a protein across a membrane. Within the cell, translocation of
proteins from cytosol to specific organelle or organelle to cytosol and from one organelle to another occur in three
different ways:
1. Gated transport : The protein translocation between the cytosol and nucleus occurs through the nuclear pore
complexes. This process is called gated transport because the nuclear pore complexes function as selective
gates that can actively transport specific macromolecules.
2. Transmembrane transport : In transmembrane transport, membrane-bound protein translocators directly

transport specific proteins across a membrane from the cytosol into a organelle. The transport of selected
proteins from the cytosol into the ER lumen or into mitochondria is an example of transmembrane transport.
3. Vesicular transport : In vesicular transport, proteins move from one organelle to another through transport
vesicles. The transfer of proteins from the endoplasmic reticulum to the Golgi apparatus, for example, occurs
in this way.
Protein translocation may occur co-translationally or post-translationally. Proteins synthesized by membrane bound
ribosomes are translocated co-translationally. All proteins synthesized by membrane free ribosomes are translocated
post-translationally.
3.6 Endoplasmic reticulum

Endoplasmic reticulum (ER) is the largest single membrane bound intracellular compartment. It is an extensive
network of closed and flattened membrane-bound structure. The enclosed compartment is called the ER lumen. ER
membranes are physiologically active, interact with the cytoskeleton and contain differentiated domains specialized
for distinct functions.
ER membranes are differentiated into rough and smooth regions (RER and SER, respectively), depending on
whether ribosomes are associated with their cytoplasmic surfaces. Regions of ER that lack bound ribosomes are
called SER (sometime also called transitional ER). The membranes and luminal spaces of the ER are normally
continuous throughout the cell and that RER and SER form an interconnected membrane system. When cells are
disrupted by homogenization, the ER breaks into fragments and reseals into small vesicles called microsomes.
Microsomes derived from RER are studded with ribosomes on the outer surface and are called rough microsomes.
Microsomes lacking attached ribosomes are called smooth microsome.
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Table 3.8 Coated vesicles found within eukaryotic cells

Coated vesicle Coat proteins Transport
Clathrin Clathrin, AP1 Golgi complex to endosome
Clathrin Clathrin, AP2 Plasma membrane to endosome
COPI COPI Golgi complex to the ER or intra Golgi complex
COPII COPII ER to Golgi complex
The coat proteins surrounding transport vesicles that move from the late endosome to lysosomes and to the
plasma membrane have not yet been identified.
ER-resident proteins often are retrieved from the Cis-Golgi

As we have mentioned in the previous section that proteins entering into the lumen of the ER are of two types-
resident proteins and export proteins. How, then, are resident proteins retained in the ER lumen to carry out their
work?
The answer lies in a specific C-terminal sequence present in resident ER proteins. Most ER-resident proteins have
a Lys-Asp-Glu-Leu (KDEL in the one-letter code) sequence at their C-terminus. Several experiments demonstrated
that the KDEL sequence which acts as sorting signal, is both necessary and sufficient for retention in the ER. If this
ER retention signal is removed from BiP, for example, the protein is secreted from the cell; and if the signal is
transferred to a protein that is normally secreted, the protein is now retained in the ER. The KDEL sorting signal is
recognized and bound by the KDEL receptor found on the ER and the cis-Golgi. The KDEL receptor acts mainly to
retrieve proteins with the KDEL sorting signal that have escaped to the cis-Golgi network and returns them to the
ER. The finding that most KDEL receptors are localized to the membranes of small transport vesicles shuttling
between the ER and the cis-Golgi also supports this concept. The KDEL receptor acts mainly to retrieve soluble
proteins containing the KDEL sorting signal. The retention of transmembrane proteins in the ER is carried out by
short C-terminal sequences that contain two lysine residues (KKXX sequences).
The affinity of the KDEL receptor for proteins with KDEL sorting signal changes in different compartments. How can
the affinity of the KDEL receptor change depending on the compartment in which it resides? The answer may be
related to the differences in pH. In the low-pH environment of cis-Golgi and transport vesicles, the KDEL receptor
has greater binding affinity with the KDEL sorting signal whereas in the neutral-pH environment of the ER, the ER
proteins dissociate from the KDEL receptor due to lesser affinity.
Clearly, the transport of newly synthesized proteins from the RER to the Golgi cisternae is a highly selective and
regulated process. The selective entry of proteins into membrane-bound transport vesicles is an important feature
of protein targeting as we will encounter them several times in our study of the subsequent stages in the maturation
of secretory and membrane proteins.
3.7 Golgi complex

The Golgi complex was first discovered in 1897 by Italian physician Camillo Golgi. The Golgi complex, also termed
as Golgi body or Golgi apparatus, is a single membrane bound organelle and part of endomembrane system. It
consists of five to eight flattened membrane-bound sacs called the cisternae. Each stack of cisternae is termed as
Golgi stack (or dictyosome). The cisternae in Golgi stack vary in number, shape and organization in different cell
types. The typical diagrammatic representation of three major cisternae (cis, medial and trans) as shown in the
figure 3.42 is actually a simplification. In some unicellular flagellates, however, as many as 60 cisternae may
combine to make up the Golgi stack. The number of Golgi complexes in a cell varies according to its function. A
mammalian cell typically contains 40 to 100 stacks. In mammalian cells, multiple Golgi stacks are linked together
at their edges.
Each Golgi stack has two distinct faces: a cis face (or entry face or forming face) and a trans face (or maturing
face). Both cis and trans faces are closely associated with special compartments: the cis Golgi network (CGN)
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After the v-SNAREs and t-SNAREs have mediated the fusion of a vesicle on a target membrane, the NSF (NEM
Sensitive Factor) binds to the SNARE complex via adaptor proteins, SNAPs (Soluble NSF Attachment Proteins)
proteins. NSF, a hexamer of identical subunits, and SNAPs are not necessary for actual membrane fusion but rather
are required for regeneration of free SNARE proteins. NSF is a soluble ATPase and hydrolyzes ATP to dissociate the
SNAREs apart.
3.9 Lysosome
Lysosomes are membrane-enclosed compartments filled with hydrolytic enzymes that are used for the controlled
intracellular digestion of macromolecules. They contain about 40 types of hydrolytic enzymes, including proteases,
nucleases, glycosidases, lipases, phospholipases, phosphatases and sulfatases. All are acid hydrolases because for
optimal activity they require an acid environment and the lysosome provides this by maintaining a pH of about 5.0
in its interior. A H+ pump in the lysosomal membrane uses the energy of ATP hydrolysis to pump H+ into the
lysosome, thereby maintaining the acidic pH of lumen. Lysosomes greatly vary in size and shape. There are two
types of lysosomes: Primary lysosomes (do not contain particle or membrane for digestion) and Secondary
lysosomes (contain particles or membranes in the process of being digested).
Lysosome
ATP
+
pH ~5 H
ADP
Figure 3.46 The interior of lysosomes has a pH of about 5.0. To create the low pH environment, transport
proteins located in the lysosomal membrane pump hydrogen ions into the lysosome using energy supplied
from ATP. All the lysosomal enzymes work most efficiently at acidic pH and collectively are termed acid
hydrolases.
Lysosomes are responsible for the degradation of large particles taken up by phagocytosis and for the gradual
digestion of the cell’s own components by autophagy. On this basis lysosome can be divided into:
Heterophagic vacuoles (or heterolysosomes or phagolysosomes): They are formed by the fusion of primary lysosome
with cytoplasmic vacuoles containing extracellular substances brought into the cell by an endocytic process.
Autophagic vacuoles (or autolysosomes): Autophagic vacuoles contain particles isolated from the cells own cytoplasm
including mitochondria, microbodies etc.
Autophagy: A process of self-digestion

During autophagy, sequestration begins with the formation of a phagophore. Phagophores form de novo in the
cytoplasm from a cup-shaped membrane that expands into a double-membrane bound autophagosome surrounding
a portion of the cytoplasm. The autophagosome may fuse with an endosome. The product of the endosome-
autophagosome fusion is called an amphisome. The completed autophagosome or amphisome fuses with a lysosome,
which supplies acid hydrolases. The enzymes in the resulting compartment, an autolysosome, break down the
inner membrane from the autophagosome and degrade the cargo. The resulting macromolecules are released and
recycled in the cytosol.
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3.10 Vacuoles
Most plants and fungal cells contain one or several very large, fluid-filled vesicles called vacuoles. They are
surrounded by single membrane called tonoplast and related to the lysosomes of animal cells, containing a variety
of hydrolytic enzymes, but their functions are remarkably diverse. Like a lysosome, the lumen of a vacuole has an
acidic pH, which is maintained by similar transport proteins in the vacuolar membrane. The plant vacuole contains
water and dissolved inorganic ions, organic acids, sugars, enzymes and a variety of secondary metabolites. Solute
accumulation causes osmotic water uptake by the vacuole, which is required for plant cell enlargement. This water
uptake generates the turgor pressure.
The vacuole is different from contractile vacuole. A contractile vacuole is an organelle involved in osmoregulation.
It pumps excess water out of the cell. It is found predominantly in protists (such as Paramecium, Amoeba) and in
unicellular algae (Chlamydomonas). It was previously known as pulsatile or pulsating vacuole.
3.11 Mitochondria
Mitochondria (term coined by C. Benda) are energy-converting organelles, which are present in virtually all eukaryotic
cells. They are the sites of aerobic respiration. They produce cellular energy in the form of ATP, hence they are
called ‘power houses’ of the cell. Mitochondria are membrane-bound mobile as well as plastic organelle. Each
mitochondrion is a double membrane-bound structure with outer and inner membranes. The outer membrane is
fairly smooth. But the inner membrane is highly convoluted; forming folds called cristae. The inner membrane is
also very impermeable to many solutes due to very high content of a phospholipid called cardiolipin. The cristae
greatly increase the inner membrane’s surface area. The two faces of this membrane are referred to as the matrix
side (N-side) and the cytosolic side (P-side). Inner membrane contains enzyme complex called ATP synthase (or
F0-F1 ATPase or oxysome) that makes ATP. The outer membrane protects the organelle, and contains specialized
transport proteins such as porin which allows free passage for various molecules into the intermitochondrial space
(the space between the inner and outer membranes) of the mitochondria. Mitochondrial porins, or voltage-dependent
anion-selective channels (VDAC) allow the passage of small molecules across the mitochondrial outer membrane.
Inter-mitochondrial space
Inner membrane
Matrix Outer membrane
ATP synthase (F0-F1 ATPase)
Figure 3.48 A mitochondrion has double-membraned organization and contains: the outer mitochondrial
membrane, the intermembrane space (the space between the outer and inner membranes), the inner
mitochondrial membrane, and the matrix (space within the inner membrane).
The matrix (large internal space) contains several identical copies of the dsDNA (as genetic material), mitochondrial
ribosomes (ranging from 55S-75S), tRNAs and various proteins. Mitochondrial dsDNA is mostly circular. The size of
mitochondrial DNA also varies greatly among different species.
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3.14.8 Intermediate filaments

Intermediate filaments are rope-like cytoplasmic filaments of about 10-nm diameter. These filaments are found in
many metazoans, including vertebrates, nematodes and molluscs but not in plants and fungi. Unlike the actin and
tubulin proteins, the intermediate filament proteins are chemically heterogenous and show species-specific variations
in molecular weight. The principal functions of intermediate filaments are structural to reinforce cells and to organize
cells into tissues. Unlike microfilaments and microtubules, intermediate filaments do not participate in cell motility.
All intermediate filaments share a common structural organization. The individual polypeptide of intermediate
filament is an elongated molecule consisting of a non-α-helical N-terminal head domain, a central α-helical rod
domain and a non-α-helical C-terminal tail domain. The central rod domain consists of long tandem repeats of a
distinctive seven amino acid sequence called the heptad repeat. Polypeptide chain forms a parallel coiled coil
dimeric structure with another. Two dimers then line up side by side to form an antiparallel tetramer of four
polypeptide chains. Tetramer, the soluble subunit of intermediate filament, further organizes to form higher level
organization. Tetramer is analogous to the αβ-tubulin heterodimer or G-actin. Unlike the actin or tubulin subunits,
the intermediate filament subunits do not contain a binding site for a nucleoside triphosphate. The antiparallel
arrangement of dimers implies that the tetramer, and hence the intermediate filament that it forms, is a non-
polarized structure.
Monomer
(Single polypeptide chain)
Coiled-coil dimer
(Hetero or homodimer,
Parallel coiled coil structure)
Tetramer
(Staggered arrangement of dimer,
Lacks structural polarity)
Intermediate filament
(10 nm diameter)
Figure 3.62 A model of intermediate filament construction.
Intermediate filament proteins are classified into four major types based on their sequences and tissue distribution:
nuclear, vimentin-like, epithelial and axonal.
Types Component polypeptides Features
Nuclear Lamins A, B and C Most ubiquitous group of intermediate filaments and found
exclusively in the nucleus. Lamins form a network structure that
lines the inside surface of the inner nuclear membrane termed
nuclear lamina.
Vimentin-like Vimentin Most widely distributed of all intermediate filament proteins is
vimentin, which is typically expressed in leukocytes, blood vessel
endothelial cells, some epithelial cells, and mesenchymal cells
such as fibroblasts.
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Desmin Desmin expressed in skeletal, cardiac and smooth muscles.

Epithelial Type I keratins (acidic) The largest group of intermediate filament proteins.
Type II keratins (basic/neutral) Keratins are obligatory heterodimers containing equimolar
amounts of type I plus type II keratin polypeptide chains.
Axonal Neurofilament Forms primary cytoskeletal component in mature nerve cells.
(NF-L, NF-M and NF-H) In mammals, three different neurofilament proteins have been
recognized: NF-L, NF-M and NF-H, for low, middle, and high
molecular weight, respectively. All three are usually found in each
neurofilament.
3.15 Cell junctions

Many cells in tissues are linked to one another and to the extracellular matrix at specialized contact sites called cell
junctions. The cell junctions are critical to the development and functions of multicellular organisms. Cell junctions
can be classified into three functional groups: occluding junctions, anchoring junctions and communicating junctions.
1. Occluding junctions
Occluding junctions seal cells together in an epithelium in a way that prevents even small molecules from leaking
from one side of the sheet to the other (i.e. forms permeability barrier across epithelial cell sheets). These junctions
are of two types– tight junction and septate junction.
Tight junctions (or zonula occludens) are cell-cell occluding junctions mediated by two major transmembrane
proteins-claudins and occludin. Claudins and occludins associate with intracellular peripheral membrane proteins
called ZO proteins. Tight junctions make the closest contact between adjacent cells and prevent the free passage
of molecules (including ions) across an epithelial sheet in the spaces between cells. They also maintain the polarity
of epithelial cells by preventing the diffusion of molecules between the apical and the basolateral regions of the
plasma membrane. Septate junctions are the main occluding junctions in invertebrates.
Lumen
Tight
junction
Cell 1 Cell 2 Cell 3 Cell 4
Figure 3.63 Tight junctions allow cell sheets to serve as barriers to solute diffusion. Schematic drawing
showing how a small extracellular molecule present on one side of an epithelial cell sheet cannot traverse
the tight junctions that seal adjacent cells together.
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3.19 Nucleus
The nucleus is the controlling center of eukaryotic cell. It contains most of the genetic materials of cell. Most
eukaryotic cells have one nucleus (uninucleate) each, but some have many nuclei (multinucleate) and certain cells,
such as mature red blood cells, do not have it. Paramecium (unicellular ciliate protozoa) have two nuclei - a
macronucleus and a micronucleus. Genes in the macronucleus control the everyday functions of the cell, such as
feeding, waste removal, and maintenance of water balance. Micronucleus controls the sexual reproduction.
Nuclei differ in size depending on the cell type. Most nuclei are spherical, but multilobed nuclei are also common,
such as those found in polymorphonuclear leukocytes or mammalian epididymal cells. A nucleus has four components:
Nuclear envelope, nucleolus, nucleoplasm and chromosomes.
Nuclear envelope
The nuclear envelope consists of two concentric membranes called the inner and outer nuclear membranes. The
outer nuclear membrane is continuous with the endoplasmic reticulum, so the space between the inner and outer
nuclear membranes, the perinuclear space, is directly connected with the lumen of the endoplasmic reticulum. In
addition, the outer nuclear membrane is functionally similar to the membranes of the endoplasmic reticulum and
has ribosomes bound to its cytoplasmic surface. In contrast, the inner nuclear membrane carries unique proteins
that are specific to the nucleus.
A network of intermediate filaments present on the nuclear side of the inner membrane is known as nuclear lamina.
The nuclear lamina is made up of lamin proteins. The nuclear lamina provides the mechanical support to the
nucleus. The critical function of the nuclear membrane is to act as a barrier that separates the contents of the
nucleus (nucleoplasm) from the cytoplasm. The nuclear matrix or the nucleoplasm contains nucleolus and chromatin.
The nuclear envelope contains nuclear pores for transport of macromolecules between the cytoplasm and nucleus.
Each nuclear pore is formed from an elaborate structure termed the nuclear pore complex. Each nuclear pore
complex is a cylindrical structure comprised of eight spokes surrounding a central channel. The inner and outer
membranes fuse at the nuclear pore complexes. Nuclear pore complexes are made up of some 50 to 100 different
proteins. The proteins that make up the nuclear pore complex are known as nucleoporins. The nucleus of a
typical mammalian cell contains about 3000 to 4000 pores.
Cytoplasmic
filament
Cytoplasm
Central
Spoke channel
complex Cytoplasmic ring
Luminal Nuclear
ring envelope
Nuclear ring
Nucleus
Nuclear
basket
Distal ring
Figure 3.69 The nuclear pore complex is cylindrical and displays an octagonal symmetry. At the center of
the pore is a spoke assembly of 8 annular units anchored to the membrane by luminal ring. Attached by
column subunits are two rings, one facing the nucleus and the other the cytoplasm. The nucleoplasmic side
of the nuclear pore complex is associated with fibrils. On the nucleoplasmic side, a nuclear basket is attached.
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interacts with iron bound to the active site of the enzyme guanylyl cyclase. This increases enzymatic activity,
resulting in the synthesis of the second messenger cyclic GMP, which induces muscle cell relaxation and blood
vessel dilation.
Effect of Viagra
Concentration of cGMP decreases because a specific phosphodiesterase convert cGMP to the inactive 5’-GMP.
Sildenafil (Viagra) causes cGMP levels to remain high by inhibiting the activity of phosphodiesterase.
Acetylcholine
GPCR
PLC IP3
2+
Ca /calmodulin complex
Activates
NO synthase
Activation of
Synthesis
Guanylyl cyclase PKG Vasodilation
of cGMP
(NO receptor) Activates
Figure 3.86 Action of nitric oxide.
3.20.7 Two-component signaling systems

Two-component signaling system is the most common form of signaling pathway that responds to extracellular
events in bacteria and plants. The canonical two-component system in bacteria consists of a sensor that is an
autophosphorylating histidine kinase and a response regulator, which transfers the phosphate from sensor kinase
to a conserved aspartate within itself.
The sensor histidine kinase is located in the membrane. It can be activated by binding a ligand that is in the
extracellular medium. Activation causes the kinase to autophosphorylate. The reaction transfers the phosphate
from ATP on to a histidine residue in the kinase domain. The sensor interacts with an effector protein i.e. response
regulator. The response regulator has two domains - conserved receiver domain and effector domain. The receiver
domain catalyzes transfer of the phosphate group from the histidine on the sensor to an aspartic acid residue in its
own domain. This activates the effector domain. The usual end target of a two-component pathway is the regulation
of gene transcription.
H D
Sensor histidine kinase Response regulator
Figure 3.87 Two component system.
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concentration of an attractant or repellent is only transient, even if the higher level of ligand is maintained, as the
bacteria desensitize, or adapt, to the increased stimulus. The adaptation is mediated by the covalent methylation. A
methyltransferase, CheR, catalyzes methylation of the MCP. [In other species of bacteria such as B. subtilis, attractants
may stimulate and repellents inhibit CheA activity].
Repellent CheA Attractant
CheY CheYP
CheZ
Flagellum Counterclockwise rotation Clockwise rotation

Sensor Degree of methylation increases Degree of methylation decreases
Behavior Run Tumbling
Chemotaxis proteins
1. CheA, a cytoplasmic sensor kinase.
2. CheW, an adaptor protein linking the sensor protein with CheA.
3. CheY, the response regulator controlling the flageller motor.
4. CheZ, an Asp–specific protein phosphatase for signal termination.
5. CheR, a methyltransferase catalyzing methylation of the MCP.
3.20.9 Quorum sensing

The term quorum sensing describes a bacterial communication phenomenon that allows bacteria to communicate
using secreted signal molecules to assess their population density. This process enables a population of bacteria to
collectively regulate gene expression and, therefore, behaviour. In quorum sensing, bacteria assess their population
density by detecting the concentration of a particular signal molecule termed autoinducer, which is correlated with
cell density.
Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density. Bacteria
that use quorum sensing constantly produce and secrete certain signaling molecules (called autoinducers). These
bacteria also have a receptor for the autoinducer. When the inducer binds to the receptor, it activates the transcription
of a set of genes, including those responsible for the synthesis of the autoinducer itself. The concentration of the
autoinducer in the surrounding medium depends on cell-population density. As the bacterial population grows, the
concentration of the autoinducer in the surroundings increases, causing more autoinducer molecules to be synthesized.
The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene
expression. Both gram-positive and gram-negative bacteria use quorum sensing communication circuits to regulate
a diverse array of physiological activities. These processes include symbiosis, virulence, competence, conjugation,
antibiotic production, motility and sporulation. In general, Gram-negative bacteria use N-Acyl-L-Homoserine Lactones
(AHLs) as autoinducers, and Gram-positive bacteria use processed oligo-peptides to communicate.
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The proteasomal degradation of p53 results from its polyubiquitination by a ubiquitin ligase called Mdm2. In the
case of DNA damage, DNA dependent protein kinase ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-
related, Rad3 is a DNA dependent protein kinase of yeast) are activated. ATM primarily is a sensor of DNA defects
caused by ionizing radiation, while ATR is more specialized for UV induced DNA damage and inhibitors of DNA
replication. In the case of DNA damage, the rapid degradation of p53 is inhibited by ATM, which phosphorylates p53
at a site that interferes with binding to Mdm2. Hence, in response to DNA damage, p53 levels increase and arrest
the cell at the G1-phase of the cell cycle. If all of the repairs have been made to the DNA, the cell divides normally
and completes the cycle. However, if the cell still contains mutated or duplicated DNA sequences, it dies by a
suicidal apoptotic mechanism to prevent its proliferation. In those cells that have mutated or lost p53 genes, the
arrest at G1 does not occur, and the cells that have mutated genomes proliferate and become cancerous.
3.21.3 Replicative senescence

Normal eukaryotic cells have only a limited capacity for cell division. The process that limits the cell division has
been termed replicative senescence. It appears to be a fundamental feature of somatic cells, with the exception of
most tumor cells and possibly certain stem cells. In the early 1960s, Leonard Hayflick observed that human cells
placed in tissue culture stop dividing after a limited number of cell divisions. The number of mitosis a cell is capable
of undergoing in tissue culture before it stops dividing is described as the Hayflick limit. Telomere shortening is
considered as the main causal mechanism of replicative senescence. When telomeres become critically short, the
nucleus signals the cell to cease proliferation, provoking cell senescence or cell death. The telomeres are short
tandemely repetitive DNA sequences that cap the ends of eukaryotic chromosomes. It serves a dual role in
protecting the chromosome ends and in intracellular signaling for regulating cell proliferation. A complex of six
telomere-associated proteins has been identified – the telosome or shelterin complex - that is crucial for both the
maintenance of telomere structure and its signaling functions. The length of telomeric DNA is maintained by the
enzyme telomerase. It is a reverse transcriptase that maintains the length of telomeres by overcoming ‘end
replication problem’. Most human somatic cells are telomerase negative and thereby experience progressive telomere
shortening, at each cell division, due to the end replication problem. As human cells proliferate in culture, their
telomeres get progressively shorter and shorten down to a point when they elicit a DNA-damage response. It leads
to an irreversible growth arrest, a phenomenon called cellular senescence. Telomerase therefore constitutes a
telomere maintenance mechanism conferring infinite replicative potential. However, telomerase is normally expressed
in stem cells, tumor cells and germ-line cells, but is nearly absent in most somatic cells. Induction of telomerase
synthesis bypasses normal cellular senesce in cancer cells and endows them with unlimited replicative potential.
A number of human tumors and cell immortalized in culture maintain their telomeres by a telomerase independent
mechanism termed Alternative Lengthening of Telomeres (ALT). The available data indicate that ALT involves
homologous recombination-mediated DNA replication.
3.22 Mechanics of cell division

In eukaryotes, two types of cell divisions partition the genetic material into progeny, or daughter cells. In one
process called mitosis, a parent cell divides into two daughter cells and each receives an exact copy of the
chromosomes (genetic material) in the parent cell. Since the number of chromosomes in the parent and progeny
cells is the same, it is also called as equational division. In the other partitioning process, the genetic material must
precisely halve so that fertilization will restore the diploid complement. This cellular process is termed meiosis.
3.22.1 Mitosis
Mitosis is the process that partitions newly replicated chromosomes equally into two daughter cells. The term
mitosis (derived from the Greek word, meaning thread) was introduced by Walther Flemming in 1882. During
mitosis, one round of DNA replication is followed by a single round of chromosome segregation and generate two
genetically identical daughter cells.
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pro-apoptotic. Mammalian Bcl2 family of proteins regulate the intrinsic pathway of apoptosis mainly by controlling
the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol.
The anti-apoptotic Bcl2 proteins include Bcl2 itself and Bcl-XL. Bcl2 was the first protein shown to cause an inhibition
of apoptosis. It is the mammalian homologue of the CED-9 in C. elegans. The pro-apoptotic Bcl2 proteins consist of
two subfamilies - the BH123 proteins and the BH3-only proteins. The main BH123 proteins are Bax and Bak, which
are structurally similar to Bcl2. Important members of the BH3-only proteins are Bid, Bim, Bik, Bad and Bmf.
When an apoptotic stimulus triggers the intrinsic pathway, the pro-apoptotic BH123 proteins become activated and
induces the release of cytochrome c and other intermembrane proteins by an unknown mechanism. In the absence
of an apoptotic stimulus, anti-apoptotic Bcl2 proteins bind to and inhibit the BH123 proteins on the mitochondrial outer
membrane and in the cytosol. In the presence of an apoptotic stimulus, BH3-only proteins are activated and bind to
the anti-apoptotic Bcl2 proteins so that they can no longer inhibit the BH123 proteins. Some activated BH3-only
proteins may stimulate mitochondrial protein release more directly by binding to and activcting the BH123 proteins.
3.24 Cancer
A normal cell undergoes regulated division, differentiation and apoptosis (programmed cell death). When normal
cells have lost the usual control over their division, differentiation and apoptosis they become tumor cells. So, a
tumor is the result of an abnormal proliferation of cells without differentiation and apoptosis. Tumor or neoplasm
(any abnormal proliferation of cells) may be of two types: Benign tumor and Malignant tumor.
Benign and malignant tumor
In benign tumor, neoplastic cells remain clustered together in a single mass and cannot spread to other sites. It
contains cells that closely resemble normal cells and that may function like normal cells.
Neoplastic cells that don’t remain localized and encapsulated and becomes progressively invasive and malignant
are described as malignant tumors. They invade surrounding normal tissues (called invasiveness) and spread
throughout the body through circulatory or lymphatic systems (called metastasis). The term cancer refers specifically
to malignant tumors.
Table 3.20 Comparison of benign and malignant tumours

Characteristics Benign Malignant
Differentiation Well differentiated Lack differentiation
Rate of growth Slow Rapid
Invasiveness Absent Present
Metastasis Absent Present
Both benign and malignant tumors are classified according to the type of cell from which they arise. Most cancers
fall into three main groups– Carcinomas (tumors that arise from endodermal or ectodermal tissues), Sarcomas
(malignancies of mesodermal connective tissues) and Leukemia/Lymphomas (from blood forming tissues and from
cells of the immune system).
Most cancers originate from single abnormal cell i.e. monoclonal origin. Cancers are probably initiated by changes
in the cell’s DNA sequence (genetic changes) or change in pattern of gene expression without a change in DNA
sequences (epigenetic changes).
Most cancers are initiated by genetic changes and majority of them are caused by changes in somatic cells and
therefore are not transmitted to the next generation. About 1% of all cancers is due to genetic changes in germinal
cells and is therefore inherited. About 80% of these inherited cancers are dominant in nature.
The transition of a normal cell into a tumor cell is referred to as transformation. The transition from a normal to a
transformed state is a multisteps process involving genetic/epigenetic changes and selection of cells with the
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3.25 Stem cells

Stem cells are unspecialized (undifferentiated) cells that have the ability to differentiate into other cells and self-
regenerate. These cells divide to produce one daughter cell that remains a stem cell and one that divides and
differentiates. Because the division of stem cells produces new stem cells as well as differentiated daughter cells,
stem cells are self renewing populations of cells that can serve as a source for the production of differentiated cells
throughout life. Typically, stem cells generate an intermediate cell type or types before they achieve their fully
differentiated state.
The intermediate cell is called a precursor or progenitor cell. The ability to differentiate is the potential to develop
into other cell types. Depending on the ability to differentiate into other cell types, stem cells can be classified as
totipotent, pluripotent and multipotent stem cells. Totipotent stem cells are cells that can give rise to a fully functional
organism as well as to every cell type of the body. Pluripotent stem cells can differentiate into nearly all cell types.
Multipotent stem cells can differentiate into a limited number of closely related families of cells.
There are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of
blastocysts, and adult stem cells, which are found in various tissues. Embryonic stem cells can become all cell types
of the body because they are pluripotent. An adult stem cell (also termed as somatic stem cell) is an undifferentiated
cell found among differentiated cells in a tissue or organ, can renew itself and differentiate to yield the major
specialized cell types of the tissue or organ. The primary roles of adult stem cells in a living organism are to
maintain and repair the tissue in which they are found. Unlike embryonic stem cells, which are defined by their
origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is unknown. Most adult
stem cells are multipotent. The bone marrow contains two kinds of stem cells. One population, called hematopoietic
stem cells, forms all the types of blood cells in the body. A second population called bone marrow stromal cells
generates bone, cartilage, fat and fibrous connective tissue. The adult brain also contains stem cells that are able
to generate the brain’s three major cell types—astrocytes and oligodendrocytes, which are non-neuronal cells and
neurons or nerve cells.
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Chapter 04
4.1 General features of Prokaryotes

Prokaryotes (pro means before and karyon means kernel or nucleus) consist of eubacteria and the archaea (also
termed as archaebacteria or archaeobacteria). The term eubacteria refer specifically to bacteria. The informal
name bacteria are occasionally used loosely in the literature to refer to all the prokaryotes, and care should be
taken to interpret its meaning in any particular context. Prokaryotes can be distinguished from eukaryotes in terms
of their cell structure and molecular make-up. Prokaryotic cells have a simpler internal structure than eukaryotic cells.
Although many structures are common to both cell types, some are unique to prokaryotes. Most prokaryotes lack
extensive, complex, internal membrane systems. The major distinguishing characteristics of prokaryotes and
eukaryotes are as follows:
Features of prokaryotic organisms

True membrane bound nucleus – Absent
DNA complexed with histone – Absent
Number of chromosomes – One (mostly)
Mitosis and meiosis – Absent
Genetic recombination – Partial (unidirectional transfer of DNA)
Sterol in plasma membrane – Absent (Except Mycoplasma)
Ribosome – 70S
Unit membrane bound organelles – Absent
Cell wall – Present in most of prokaryotic cells. In eubacteria, it is made up of peptidoglycan.
Features of eukaryotic organisms

True membrane bound nucleus – Present
DNA complexed with histone – Present
Number of chromosomes – More than one
Mitosis and meiosis – Present
Genetic recombination – By crossing over during meiosis
Sterol in the plasma membrane – Present
Ribosome – 80S (in cytosol) and 70S (in organelles)
Unit membrane bound organelles – Present
Cell wall – Made up of cellulose in plant and chitin in fungi. Absent in animal cells.
Prokaryotic cells show similarities with eukaryotic organelles like mitochondria and chloroplast. The endosymbiotic
theory (Margulis, 1993) proposes that the mitochondria and chloroplasts of eukaryotic cells originated as symbiotic
prokaryotic cells. The presence of circular, covalently closed DNA and 70S ribosomes in mitochondria and chloroplast
support this theory.
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Table 4.1 Similarities between prokaryotic cells and eukaryotic organelles

Prokaryotic cells Eukaryotic organelles
Nature of DNA ds circular ds circular
Histone protein Absent Absent
Ribosome type 70S 70S
Growth Binary fission Binary fission
4.2 Phylogenetic overview

Historically, prokaryotes were classified on the basis of their phenotypic characteristics. Prokaryotic taxonomy
therefore involved measuring a large number of characteristics, including morphology and biochemical characteristics
(e.g. ability to grow on different substrates, cell wall structure, antibiotic sensitivities, and many others). This
contrasts with the classification of eukaryotic organisms, for which phylogenetic (evolution-based) classification
was possible through the availability of fossil evidence.
A major revolution occurred with the realization that evolutionary relationships could be deduced on the basis of
differences in gene sequence. The most important gene for prokaryote phylogeny is the 16S ribosomal RNA (rRNA)
gene, which is present in all cells. The gene is approximately 1500 bp in length and possesses signature sequences.
These sequences are conserved and found in the organisms of one taxonomic group but not in other groups.
Bacteria Archaea Eukaryotes
Green
filamentous Entamoebae Slime Animals
Spirochetes bacteria molds
Gram Methanosarcina Fungi
positive
Methanobacterium Halophiles
Proteobacteria Plants
Methanococcus
Cyanobacteria Ciliates
T. Celer
Planctomyces Thermoproteus Flagellates
Pyrodicticum
Cytophaga Trichomonads
Microsporidia
Thermotoga
Diplomonads
Aquifex
Phylogenetic tree of life
Figure 4.1 A phylogenetic tree of living things, based on RNA data (proposed by Carl Woese), showing
the separation of bacteria, archaea, and eukaryotes from a common ancestor.
Based on ribosomal RNA signature sequences, Carl Woese proposed a radical reorganization of the five kingdoms
into three domains. In his classification system, Woese placed all four eukaryotic kingdoms (protista, fungi, plantae,
animalia) into a single domain called Eukarya, also known as the eukaryotes. He then split the former kingdom of
Monera into the Eubacteria and the Archaea domains. Unlike Whittaker’s five kingdom system, Woese’s three
domain system organizes biodiversity by evolutionary relationships.
4.3 Structure of bacterial cell

Bacteria (eubacteria) are microscopic, relatively simple, prokaryotic organisms whose cells lack a nucleus. Prokaryotes
can be distinguished from eukaryotes in terms of their cell structure and molecular make-up. Prokaryotic cells are
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Bacterial staining protocols can be divided into three basic types – simple, differential, and specialized. Simple
stains react uniformly with all cell types and only distinguish the organisms from their surroundings. Differential
stains do not stain all types of cells with the same colour. It discriminates different cell types depending upon the
chemical or physical composition of the cells. The differential stains most frequently used for bacteria are the Gram
stain and the acid-fast stain. Specialized stains detect specific structures of cells such as flagella and endospores.
These stains are used to color and isolate specific parts of organisms.
Gram staining
Gram staining (or Gram’s method) is a differential staining method for differentiating bacterial species into two
groups based on the physical properties of their cell walls. The method is named after the inventor, the Danish
scientist Hans Christian Gram, who developed the technique in 1884.
The gram staining procedure involves four basic steps:
1. The bacteria are first stained with the basic dye crystal violet. Crystal violet (it is referred to as a primary stain)
imparts purple colour to all cells.
2. The bacteria are then treated with Gram’s iodine solution. This allows the stain to be retained better by forming
an insoluble crystal violet-iodine complex. Iodine is used as a mordant. A mordant is used to increase the
affinity of a stain for a biological specimen.
3. Gram’s decolorizer, a mixture of ethyl alcohol and acetone, is then added. A decolorizer or decolouring agent
removes the stain from the specimen. This is the differential step. After this step some bacteria retain the
purple colour while some other loose purple colour. Bacteria that retain colour are classified as gram-positive
and bacteria that lose the colour after decolorization are classified as gram negative.
4. Because gram-negative bacteria are colourless after the treatment with decolorizer, they are no longer visible.
Thus, the counterstain safranin (also a basic dye) is applied. Since the gram-positive bacteria are already
stained purple, they are not affected by the counterstain. Gram-negative bacteria, that are now colourless,
become directly stained by the safranin. Thus, gram-positive appear purple, while gram-negative appear red
or pink.
Gram positive
Gram negative
Application of Application of Alcohol wash Application of

crystal violet iodine (mordant) (decolorization) safranin
(counterstain)
Figure 4.2 The Gram-staining procedure. In the first step of the Gram-staining procedure, the smear is
stained with the basic dye crystal violet, the primary stain. It is followed by treatment with an iodine solution
functioning as a mordant. The decolorization with ethanol or acetone removes crystal violet from gram-
negative cells but not from gram-positive cells. The gram-negative cells then turn pink to red when
counterstained with safranin.
Acid-fast staining
The acid-fast stain is a differential stain used to identify acid-fast organisms such as members of the genus
Mycobacterium. The acid-fast staining procedure involves heating of bacteria with a mixture of basic fuchsin and
phenol (also known as Ziehl-Neelsen stain). The presence of phenol and heat treatment helps the stain to penetrate
the cell wall. Once basic fuchsin has penetrated the cell wall, acid-fast cells are not easily decolorized by an acid-
alcohol treatment and hence remain red. It occurs due to the presence of large amounts of mycolic acid, a
branched chain hydroxy fatty acid. Non-acid-fast bacteria are decolorized by acid-alcohol. Because non-acid-fast
bacteria are colourless after the treatment with decolorizer, they are no longer visible. Finally, the counterstain
methylene blue is applied. Methylene blue colours non-acid-fast bacteria blue.
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Partitioning
Partitioning is an active process which assures that after cell division each daughter cell gets a copy of plasmid. For
plasmids present in high copy numbers (50 to 100 copies per cell), random diffusion may be enough to get at least
one copy of the plasmid to each daughter cell. However, random segregation of low-copy-number plasmids (only 1
to 2 copies per cell) would most likely mean that, following cell division, one of the daughter cells would not receive
a plasmid. The plasmid would eventually be diluted from the population. Consequently, regulated partitioning
mechanisms are essential for these plasmids. The mechanism used for partitioning differs depending on the plasmid.
Partitioning, especially of low-copy-number plasmids, is regulated by par genes present on plasmids. The par
systems consist of a cis-acting centromere-like site, often called parS and two genes termed parA and parB, which
encode trans-acting proteins, Par A and B.
Functions encoded by plasmids
Depending on their size, plasmids can encode a few or hundreds of different proteins. However, plasmids rarely
encode gene products that are essential for growth, such as RNA polymerase, ribosomal subunits, or enzymes of
the tricarboxylic acid cycle. Instead, plasmid genes usually give bacteria a selective advantage under only some
conditions. Gene products encoded by plasmids include enzymes for the utilization of unusual carbon sources such
as toluene, resistance to substances such as heavy metals and antibiotics, synthesis of antibiotics, and synthesis of
toxins and proteins that allow the successful infection of higher organisms. A plasmid that confers no identified
functions or phenotypic properties is termed as cryptic plasmid.
Table 4.8 List of some plasmid-coded traits

Trait Organisms in which trait is found
Antibiotic resistance E. coli, Salmonella sp., Staphylococcus sp.
Pilus synthesis E. coli, Pseudomonas sp.
Tumor formation in plants Agrobacterium tumefaciens
Nitrogen fixation (in plants) Rhizobium sp.
Oil degradation Pseudomonas sp.
Gas vacuole production Halobacterium sp.
Insect toxin synthesis Bacillus thuringiensis
Plant hormone synthesis Pseudomonas sp.
Antibiotic synthesis Streptomyces sp.
Increased virulence Yersinia enterocolitica
Plasmids in eukaryotic organisms
Plasmids are not limited to prokaryotes only. Plasmids are also found in eukaryotic organisms like yeast. One yeast
plasmid is called the 2μ circle. The 2μ circle is a 6.3 kb circular, extrachromosomal element found in the nucleus of
most Saccharomyces cerevisiae strains. It is stably maintained at about 50 to 100 copies per haploid genome of the
yeast cell. Like the nuclear chromosomes, the 2μ circle is coated with nucleosomes and replication is initiated by
host replication enzymes once per cell cycle.
4.5 Bacterial nutrition

Nutrients are substances used in biosynthesis and energy production and therefore are required for bacterial
growth. All bacteria require several macro- and micronutrients. Macronutrients (C, O, H, N, S, P, K, Ca, Mg and Fe)
are needed in relatively large quantities; micronutrients (e.g. Mn, Zn, Co, Mo, Ni and Cu) are used in very small
amounts. In addition to the need for carbon, hydrogen and oxygen, all organisms require sources of energy and
electrons for growth to take place. On the basis of sources of carbon, energy and hydrogen/electrons bacteria can
be categorized into following types:
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Actinomycetes
Actinomycetes are aerobic, gram-positive, mold-like bacteria that form branched, septate hyphae and asexual
spores. The thin walled asexual spores are conidiospores or conidia (located at the tip of hyphae) and sporangiospores
(located in a sporangium). Classification of actinomycetes is primarily based on the properties like conidia arrangement,
the presence or absence of the sporangium, cell wall type. Most actinomycetes are nonmotile. When motility is
present, it is confined to flagellated spores only.
Streptomyces is the largest genus of actinomycetes. Members of the genus are strict aerobes, mostly non pathogenic
saprophytes and bear the chains of nonmotile conidia. The natural habitat of most streptomycetes is the soil. In fact,
the odor of moist soil is largely due to the production of volatile substances such as geosmin from streptomycetes.
Streptomycetes are best known for their synthesis of a vast array of antibiotics like amphotericin B, chloramphenicol,
erythromycin, neomycin, nystatin, streptomycin and tetracycline.
Spirochetes
Spirochetes are a group of gram-negative, chemoheterotrophic bacteria. They are slender, long organisms with a
flexible, helical shape. Spirochetes lack external rotating flagella. They exhibit creeping or crawling movements.
Their unique pattern of motility is due to an unusual morphological structure called the axial filament. The central
protoplasmic cylinder, which contains cytoplasm and the nucleoid, is bounded by a plasma membrane and gram-
negative type cell wall. Two or more than a hundred prokaryotic flagella, called axial filaments or periplasmic
flagella, extend from both ends of the cylinder often overlap. The whole complex of periplasmic flagella lies inside
a flexible outer membrane. The outer membrane contains lipid, protein, and carbohydrate. Treponema pallidum
(causes syphilis) and Borrelia burgdorferi (responsible for Lyme disease) are examples of spirochetes.
Outer Protoplasmic
Cell Wall membrane cylinder
Flagella
Periplasmic space
Figure 4.30 The most peculiar feature of spirochetes may be the location of their flagella. Flagella present
in the periplasmic space between the plasma membrane and outer membranes. Spirochete Borrelia burgdorferi
has 7-11 flagella attached near each end of the ‘protoplasmic’ or cell cylinder, with each flagellum extending
through the periplasm towards the center of the spirochete.
Mycoplasmas
Mycoplasmas are the smallest and simplest self-reproducing gram negative bacteria. Mycoplasmas lack cell walls
and thus placed in a separate class Mollicutes (mollis, soft; cutis, skin). Formerly, Mycoplasmas were called
pleuropneumonia-like organisms (PPLO) because it was first isolated from cattle suffering from pleuropneumonia.
The trivial term mollicutes is frequently used as a general term to describe any member of the class, replacing in
this respect the older term mycoplasmas. Mycoplasmas are pleomorphic (vary in shape) and mostly non-motile.
General metabolic nature is chemoorganoheterotrophic and require cholesterol for growth. They can be saprophytes
or parasites and usually facultative anaerobes. Characteristically, mycoplasmas growing on solid media produce
fried egg colonies with a central dense region surrounded by a lighter peripheral zone.
Cyanobacteria
Cyanobacteria are gram negative bacteria. They are oxygenic photosynthetic and obligate photolitho-autotrophs.
Photosynthetic pigments present in cyanobacteria are chlorophyll a, carotenoids and phycobilins (phycocyanin and
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Features of cell wall and plasma membrane lipid

The cell walls of archaebacteria are distinctive from those of eubacteria. Archaebacterial cell walls are composed of
different polysaccharides, glycoproteins or proteins, with no peptidoglycan. Many archaebacteria have cell walls
made of the polysaccharide pseudomurein (a modified peptidoglycan lacking D-amino acids and containing N-
acetyltalosaminuronic acid instead of N-acetylmuramic acid; found in methanogenic bacteria). All archaebacteria
are resistant to lysozyme and beta-lactam antibiotics such as penicillin.
The nature of archaebacterial plasma membrane lipids differs from both eubacteria and eukaryotes. Archaebacterial
membrane lipids contain branched chain hydrocarbons attached to glycerol by ether links. Sometimes two glycerols
are linked to form a long tetraether. Usually the diether chains are 20 carbons in size and the tetraether chains are
40 carbons. Eubacterial and eukaryotic lipids have glycerol connected to fatty acids by ester bonds.
Phytanyl (20C)
CH3
H2C — O — C
CH3
HC — O — C
H2C — O — P
Glycerol diether
Biphytanyl
OHCH2
H2C — O — C C — O — CH
HC — O — C C — O — CH2
H2C — O — P
Diglycerol tetraether
Major groups of archaebacteria

There are three major known groups of archaebacteria: methanogens, extreme halophiles, and thermophiles.
Methanogens
Methanogens are methane producing obligate anaerobes. They comprise the largest group of archaebacteria.
Extreme halophiles
Extreme halophiles are aerobic chemoorganoheterotrophs. They thrive in very high salt concentrations. The best-
studied member of the family is Halobacterium salinarium. H. salinarium can carry out photosynthesis without
chlorophyll or bacteriochlorophyll by using bacteriorhodopsin.
Thermophiles
Thermophiles are heat-loving archaebacteria found near hydrothermal vents and hot springs. The optimum growth
temperature is between 70–110°C. They are gram-negative and usually strict anaerobes.
4.10 Bacterial toxins

A toxin (Latin toxicum, poison) is a specific substance, often a metabolic product of the organism that damages the
host. Toxins can even induce disease in the absence of the organism that produced them. Diseases that result from
the entrance of a specific toxin into the body of a host is called intoxication. The term toxemia refers to the condition
caused by toxins that have entered the blood of the host. Toxins produced by organisms can be divided into two
main categories: exotoxins and endotoxins.
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4.12 Virus
Viruses are simple, noncellular entities consisting of one or more molecules of either DNA or RNA enclosed in a coat
of protein. They can reproduce only within living cells and are obligate intracellular parasites. Viruses are smaller
than prokaryotic cells ranging in size from 0.02 to 0.3 μm (smallpox virus is largest virus about 200 nm in diameter
and polio virus is the smallest virus about 28 nm in diameter). A fully assembled infectious virus is called a virion.
The main function of the virion is to deliver its DNA or RNA genome into the host cell so that the genome can be
expressed (transcribed and translated) by the host cell. Each viral species has a very limited host range; i.e. it can
reproduce in only a small group of closely related species.
Viral structure
The structure of virions are very diverse, varying widely in size, shape and chemical composition. All viruses have
a nucleocapsid composed of nucleic acid surrounded by a protein capsid.
A protein coat, the capsid, which functions as a shell to protect the viral genome from nucleases and which during
infection attaches the virion to specific receptors exposed on the prospective host cell. Capsids are formed as
single or double protein shells and consist of only one or a few structural protein species. The proteins used to build
the capsid are called capsomeres. The nucleic acid together with the genome forms the nucleocapsid. Some viruses
have a membranous envelope that lies outside the nucleocapsid. Those virions having an envelope are called
enveloped viruses; whereas those lacking an envelope are called naked viruses. In enveloped viruses, the
nucleocapsid is surrounded by a lipid bilayer and glycoprotein derived from the modified host cell membrane.
Enveloped viruses often exhibit a fringe of glycoprotein spikes, also called peplomers. In viruses that acquire their
envelope by budding through the plasma membrane or another intracellular cell membrane, the lipid composition
of the viral envelope closely reflects that of the particular host membrane.
Viral genomes are smaller in size. The largest known viral genome, that of bacteriophage G, is 670 kbs. The
genome of a virus may consist of DNA or RNA, which may be single stranded (ss) or double stranded (ds), linear or
circular. The genomic RNA strand of single-stranded RNA viruses is called sense (positive sense, plus sense) in
orientation if it can serve as mRNA, and antisense (negative sense, minus sense) if a complementary strand
synthesized by a viral RNA transcriptase serves as mRNA.
RNA genomes of certain viruses may be segmented in nature. The segmented genomes are those which are
divided into two or more physically separate molecules of nucleic acid, all of which are then packaged into a single
viral particle. The segmented genome is different from multipartite genome. Multipartite genomes are also segmented,
but each genome segment is packaged into a separate virus particle. These discrete particles are structurally
similar and may contain the same component proteins, but often differ in size depending on the length of the
genome segment packaged. Multipartite viruses are only found in plants.
Table 4.18 Types of viral nucleic acids
Nucleic acid type Nucleic acid structure
DNA
Single stranded Linear, single-stranded DNA
Circular, single-stranded DNA
Double stranded Linear, double-stranded DNA
Linear double-strand DNA with single chain breaks
Circular, double-strand DNA
RNA
Single stranded Linear, single-stranded, positive-strand RNA
Linear, single-stranded, negative-strand RNA
Linear, single-stranded, segmented RNA
Double stranded Linear, double-stranded, segmented RNA
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Protease inhibitors work by blocking the activity of the HIV protease and thus interfere with virion assembly.
Examples include indinavir (Crixivan), ritonavir (Norvir), nelfinavir (Viracept), and saquinavir (Invirase).
However, the most successful treatment approach is to use drug combinations. An effective combination is a
cocktail of AZT, lamivudine, and a protease inhibitor such as ritonavir.
Hepatitis virus
Hepatitis is a liver inflammation commonly caused by an infectious agent. Hepatitis sometimes results in destruction
of functional liver anatomy and cells, a condition known as cirrhosis. Some forms of hepatitis may lead to liver
cancer. Although many viruses and a few bacteria can cause hepatitis, a restricted group of viruses is often
associated with liver disease termed hepatitis viruses. Hepatitis viruses are diverse, and none of these viruses are
genetically related, but all infect cells in the liver, causing hepatitis.
Characteristics of hepatitis viruses

Features Incubation period
Hepatitis A ssRNA; No envelope 2–6 week
Hepatitis B dsDNA; enveloped 4–26 week
Hepatitis C ssRNA; enveloped 2–22 week
Hepatitis D ssRNA; enveloped 6–26 week
Hepatitis E ssRNA; No envelope 2–6 week
The genome of hepatitis B virus (hepadnavirus) is among the smallest known of any viruses, 3-4 kb. Like retroviruses,
hepatitis B virus uses reverse transcriptase during replication cycle. However, unlike retroviruses the DNA genome
of hepatitis B virus is replicated through an RNA intermediate, the opposite of what occurs in retroviruses. Hepatitis
D virus, classified as a hepatitis delta virus, is considered to be a subviral satellite because it can propagate only in
the presence of the hepatitis B virus. Transmission of hepatitis D virus can occur either via simultaneous infection
with hepatitis B virus (coinfection) or via infection of an individual previously infected with hepatitis B virus
(superinfection). The hepatitis D virus genome consists of a single stranded, negative sense, circular RNA.
4.12.6 Plant viruses

Plant viruses exist in rod and polyhedral shape. Most plant viruses have genomes consisting of a single RNA strand
of plus (+) sense type. The best-known plant virus is the rod-shaped tobacco mosaic virus (TMV). Relatively few
plant viruses have DNA genomes. There are only two classes of DNA containing plant viruses. The cauliflower
mosaic virus belongs to the first class, which contains a double-stranded DNA genome in a polyhedral capsule. The
second class of DNA containing plant viruses are the geminiviruses (gemini = twins), characterized by a connected
pair of capsids, each containing a circular, single-stranded DNA molecule of about 2500 nucleotides.
Tobacco Mosaic Virus (TMV) causes leaf mottling and discoloration in tobacco and many other plants. It was the
first virus to be discovered (by Dmitri Iwanowasky) and first virus to be crystallized (by W. Stanley). TMV is a rod
shaped virus with ~2130 capsomeres arranged in a hollow right handed helix. It contains a single genetic RNA
(ss, plus sense) of ~6400 nucleotides.
RNA
Figure 4.51
Tobacco mosaic virus has a rod-like
appearance. Its capsid is made of
Capsid ~2130 capsomeres. One molecule
of genomic ssRNA, 6400 nucleotides
long, present in the centre of the capsid.
The capsomere self-assembles into the
rod like helical structure (16.3 capsomeres
per helical turn) around the RNA.
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4.13 Prions and Viroid

Prions are proteinaceous infectious agents that are responsible for neurodegenerative diseases in animals including
human. Prions are devoid of nucleic acid. The word prion, coined in 1982 by Stanley B. Prusiner, is derived from the
words protein and infection. Prion proteins are designated as PrP. The endogenous, normal form is denoted PrPC
(for Cellular) while the disease-causing, misfolded form is denoted PrPSc (for Scrapie, after one of the diseases first
linked to prions).
The normal cellular form, PrPC, is converted into PrPSc through a process whereby a portion of its α-helical and coil
structure is refolded into a β-sheet. This structural transition is accompanied by profound changes in the
physicochemical properties of the PrP. PrPC is sensitive to proteases whereas PrPSC is protease resistant. High
content of β-sheet in PrPSc results in the formation of amyloid fibrillous structure that is absent from the PrPC form.
Both proteins are glycosylated and linked to the membrane by a GPI-linkage. The PrPSc form can perpetuate itself
by causing the newly synthesized PrP protein to take up the PrPSc form instead of the PrPC form.
Prions are novel transmissible pathogens causing a group of neuro-degenerative diseases that can be perpetuated
by inoculating animal with tissue extracts from infected one. Collectively, prion diseases are described as spongiform
encephalopathies. No prion diseases of plants are known. In 1997, American scientist Stanley B. Prusiner won
the Nobel Prize for this pioneering work with these diseases and with the prion proteins. Kuru was the first naturally
occurring spongiform encephalopathy of humans shown to be caused by prions. It was first described by Gajdusek
and Zigas in 1957. Kuru is characterized by cerebellar ataxia and a shivering-like tremor that produces complete
motor incoordination.
Table 4.23 Prion disease of human/animals

Disease Organism
Creutzfeldt-Jakob Human
Kuru Human
Bovine spongiform encephalopathy Cow
(Also known as Mad cow disease)
Scrapie Sheep
Chronic wasting disease Mule deer
Viroid and virusoid

Viroid is an infectious agent of plants that is a single-stranded, covalently closed circular RNA (about 250 to 400
nucleotides long) not associated with any protein. Viroid RNA does not code for any proteins. Viroids (discovered
and named by Otto Diener) have so far been shown to infect plants only. A few well-studied viroids include coconut
cadang-cadang viroid and Potato Spindle-Tuber Viroid (PSTV). No viroid diseases of animals are known, and the
precise mechanisms by which viroids cause plant diseases remain unclear. Although the viroid encodes no protein
enzymes, the viroid RNA itself acts as a ribozyme.
Table 4.24 Comparison of viruses and viroids

Features Virus Viroid
Nucleic acid DNA or RNA (ss or ds) RNA (ss)
Protein Present Absent
Capsid Present Absent
Host Bacteria, animal and plants Plants
Virusoid are satellite nucleic acids. Satellite nucleic acids may be single stranded RNA, single-stranded DNA, or
double-stranded RNA. Most of the characterized satellites are associated with plant viruses, and most are single-
stranded RNA. Satellite nucleic acids are always functionally dependent on specific helper viruses and are encapsidated
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Chapter 05
Immunology
Immunology is the science that is concerned with immune response to foreign challenges. Immunity (derived
from Latin term immunis, meaning exempt), is the ability of an organism to resist infections by pathogens or state
of protection against foreign organisms or substances. The array of cells, tissues and organs which carry out this
activity constitute the immune system. Immunity is typically divided into two categories—innate and adaptive immunity.
5.1 Innate immunity

Innate (native/natural) immunity is present since birth and consists of many factors that are relatively nonspecific—
that is, it operates against almost any foreign molecules and pathogens. It provides the first line of defense against
pathogens. It is not specific to any one pathogen but rather acts against all foreign molecules and pathogens. It also
does not rely on previous exposure to a pathogen and response is functional since birth and has no memory.
Elements of innate immunity
Physical barriers
Physical barriers are the first line of defense against microorganisms. It includes skin and mucous membrane. Most
organisms and foreign substances cannot penetrate intact skin but can enter the body if the skin is damaged.
Secondly, the acidic pH of sweat and sebaceous secretions and the presence of various fatty acids and hydrolytic
enzymes like lysozyme inhibit the growth of most microorganisms. Similarly, respiratory and gastrointestinal tracts
are lined by mucous membranes. Mucous membranes entrap foreign microorganisms. The respiratory tract is also
covered by cilia, which are hair like projections of the epithelial-cell membranes. The synchronous movement of
the cilia propels mucus-entrapped microorganisms out of these tracts. Similarly, the conjunctiva is a specialized,
mucus-secreting epithelial membrane that lines the interior surface of each eyelid. It is kept moist by the continuous
flushing action of tears (lacrimal fluid) from the lacrimal glands. Tears contain lysozyme, lactoferrin, IgA and thus
provide chemical as well as physical protection.
Microorganisms do occasionally breach the epithelial barricades. It is then up to the innate and adaptive immune
systems to recognize and destroy them, without harming the host. In case of innate immune response several
antimicrobial chemicals and phagocytic cells provide protection against pathogens.
Chemical mediator
A variety of chemicals mediate protection against microbes during the period before adaptive immunity develops.
The molecules of the innate immune system include complement proteins, cytokines, pattern recognition molecules,
acute-phase proteins, cationic peptides, enzyme like lysozyme and many others.
Complement proteins
The complement proteins are soluble proteins/glycoproteins that are mainly synthesized by liver and circulate in
the blood and extracellular fluid. They were originally identified by their ability to amplify and complement the
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Immunology
neutrophils, macrophages, monocytes and dendritic cells. In vertebrates, macrophages reside in tissues throughout
the body. Macrophages are long lived cells, which patrol the tissues of the body. The second major type of phagocytic
cells in vertebrates, the neutrophils, are short lived cells which are abundant in blood but are not present in normal
healthy tissues. Phagocytosis is the ingestion of invading foreign particles, such as bacteria by individual cell.
Phagocytosis may be enhanced by a variety of factors collectively referred to as opsonins (Greek word meaning
‘prepared food for’), which consist of antibodies and various serum components of complement. The process by
which particulate antigens are rendered more susceptible to phagocytosis is called opsonization. After ingestion,
the foreign particle is entrapped in a phagocytic vacuole (phagosome), which fuses with lysosomes forming the
phagolysosome. The antimicrobial and cytotoxic substances present within the lysosome destroy the phagocytosed
microorganisms in the following ways:
Oxygen dependent killing mechanisms

During phagocytosis, a metabolic process known as the respiratory burst occurs in activated phagocytes. It results
in a transient increase in oxygen consumption by cell. Activated phagocytes generate a number of toxic products
such as reactive oxygen intermediates (such as hydroxyl radicals, hypochlorite anion, superoxide anions, hydrogen
peroxide) and reactive nitrogen intermediates (like NO, NO2, HNO2•) which have potent antimicrobial activity.
Oxygen independent killing mechanisms

Activated macrophages also synthesize lysozyme, defensins (cysteine rich cationic peptides containing 29–35
amino acid residues) and various hydrolytic enzymes/cytotoxic peptides whose degradative activities do not require
oxygen.
Inflammatory barriers
Inflammation is an important nonspecific defense reaction to cell injury. The hallmark signs of inflammation are
pain, redness (erythema), swelling (edema) and heat. Each of these is the result of specific changes in the local
blood vessels. Erythema is caused by increased vascular diameter, which leads to increased blood flow, thereby
causing heat and redness in the area. The blood vessels become permeable to fluid and proteins, leading to local
swelling and an accumulation of blood proteins that aid in defense. At the same time, the endothelial cells lining the
local blood vessels are stimulated to express cell adhesion proteins that facilitate the attachment and extravasion
(movement of blood cells through the vessel wall into the surrounding tissue) of white blood cells, including neutrophils,
lymphocytes, and monocytes.
The inflammatory response is mediated by a variety of signaling molecules. Activated macrophages produce
chemoattractants (known as chemokines). Some of these attract neutrophils, which are the first cells recruited in
large numbers to the site of the new infection. Others later attract monocytes and dendritic cells. The dendritic cells
pick up antigens from the invading pathogens and carry them to nearby lymph nodes, where they present the
antigens to lymphocytes to marshal the forces of the adaptive immune system. Two principal mediators of the
inflammatory response are histamine (released by a variety of cells in response to tissue injury) and kinins
(present in blood plasma in an inactive form). Both cause vasodilation and increased permeability of capillaries.
Kinins are also very potent nerve stimulators and are the molecules most responsible for pain associated with
inflammation.
5.2 Adaptive immunity

Adaptive immunity, also known as specific or acquired immunity, is capable of recognizing and selectively eliminating
specific foreign antigens. It does not come into play until there is an antigenic challenge to the organism. Adaptive
immunity displays four characteristic features:
1. Antigenic specificity : It is the ability to discriminate among different epitopes/antigens.
2. Immunologic memory : It is the ability to recall previous contact with a foreign molecule and respond to it in a
learned manner-that is, with a more rapid and larger response.
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Immunology
Innate immunity
Physical barriers Cells Chemical barriers
Skin, pH, lipids,

Pattern recognition
mucous membranes enzymes, etc.
molecules
PMN’s, monocytes,
macrophages,
eosinophils,
NK cells
Cytokines Cytokines
Antibodies Cytokines
B-cells T-cells
Antigen-Specific receptors
Acquired immunity
Figure 5.2 The interrelationship between innate and acquired immunity.

Adapted and modified from Immunology A short Course, R. Coico et. al, Wiley-Liss Publication.
The encounter between macrophages and microbes can generate ‘danger’ signals that stimulate and direct adaptive
responses. It may increase the ability of macrophages to display antigen for recognition by antigen specific T-cells.
Macrophage stimulated by encounters with microbes also secrete immunoregulatory molecules, called cytokines.
These cytokines guide adaptive immune response. Vice-versa, adaptive immune system also produces signals and
components which stimulate and increase the efficacy of innate response. The cells of adaptive system (e.g.
T-cells) secrete cytokines and increase the ability of macrophage to kill the ingested microbes. By binding to the
pathogens, antibodies mark it as a target for attack by complement.
5.3 Cells of the immune system

The immune system is a defensive system in a host consisting of widely distributed cells, tissues and organs that
recognize foreign substances and microorganisms and acts to neutralize or destroy them. The cells responsible for
both nonspecific and specific immunity are the leukocytes or white blood cells. All leukocytes arise from a type of
cell called the hematopoietic stem cell. A hematopoietic stem cell is multipotent cell. During hematopoiesis,
hematopoietic stem cell differentiates along one of two pathways, giving rise to either a common lymphoid
progenitor cell or a common myeloid progenitor cell. Common lymphoid progenitor cells give rise to B-cells,
T-cells and natural killer cells and some dendritic cells. The common myeloid progenitor cells give rise to red blood
cells (erythrocytes), white blood cells (neutrophils, eosinophils, basophils, monocytes, mast cells, dendritic cells)
and platelets.
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Immunology
Hematopoietic stem cells

(Present in bone marrow)
Myeloid progenitor Lymphoid progenitor

Mature into
Monocyte Macrophage
Basophil
Eosinophil Dendritic cell Natural killer cell
Neutrophil
Mast cell
Platelets B-cell T-cell
RBC
Dendritic cell
5.3.1 Lymphoid progenitor

Lymphocytes (responsible for adaptive immune response) are mononuclear leukocytes which constitute 20 to 40%
of total white blood cells (or leukocytes). They occur in large numbers in the blood and lymph and in lymphoid
organs such as the thymus, lymph nodes, spleen and appendix. Up to 99% of lymphocytic cells are found in lymph.
Lymphocytes are of three main types:
1. B-lymphocytes or B-cells
2. T-lymphocytes or T-cells
3. Natural killer (NK) cells
B-lymphocytes
The B-lymphocyte matures in the bone marrow in many mammalian species, including humans (in birds it is Bursa
of Fabricius) and expresses membrane-bound antibody. After interacting with antigen, it differentiates into antibody-
secreting plasma cells and memory cells. They are the only cell type capable of producing antibody molecules and
therefore the central cellular component of humoral immune responses. B-cells also serve as Antigen Presenting
Cells (APCs).
Properties of B-cells
Origin — Bone marrow
Maturation — Bone marrow (Bursa of Fabricius in bird)
Expression of Ag receptor — Bone marrow
Differentiation — In lymphoid tissue
Surface immunoglobulin — Present
Immunity — Humoral
Distribution — Spleen, Lymph nodes, Bone marrow and other lymphoid tissue
Secretory product — Antibodies and cytokines
Complement receptors — Present
T-lymphocytes
T-lymphocytes arise in the bone marrow. Unlike B-cells, which mature within the bone marrow, T-cells migrate to
the thymus gland to mature. During its maturation within the thymus, the T-cell comes to express a unique antigen-
binding molecule, called the T-cell receptor, on the membrane. T-cells do not make antibodies but perform various
effector functions when APC bring antigens into the secondary lymphoid organ. T-cells help in eliminating APCs,
cancer cells, virus-infected cells or grafts which have altered self-cells.
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Immunology
Thymus
Thymus is the site where T-cells mature. Progenitor cells from the bone marrow migrate into the thymus gland,
where they differentiate into T-cells. It is a flat, bilobed organ situated above the heart. Each lobe is surrounded by
a capsule and is divided into lobules, which are separated from each other by strands of connective tissue called
trabeculae. Each lobule is organized into two compartments: the outer compartment, or cortex, and the inner
compartment, or medulla. T-lymphocytes mature in the cortex and migrate to the medulla, where they encounter
macrophages and dendritic cells. Here, they undergo thymic selection, which results in the development of mature,
functional T-cells, which then leave to enter the peripheral blood circulation, through which they are transported to
the secondary lymphoid organs. It is in these secondary lymphoid organs where the T-cells encounter and respond
to foreign antigens.
5.4.2 Secondary lymphoid organs/tissues

Mature B and T-lymphocytes migrate from bone marrow and thymus, respectively, through the bloodstream to the
secondary (peripheral) lymphoid organs. These secondary (peripheral) lymphoid organs are those organs in which
antigen-driven proliferation and differentiation take place.
The major secondary lymphoid organs are the spleen, the lymph nodes and mucosa associated lymphoid
tissue (MALT). Spleen and lymph nodes are the highly organized secondary lymphoid organs. The secondary
lymphoid organs have two major functions: They are highly efficient in trapping and concentrating foreign substances,
and they are the main sites of production of antibodies and the induction of antigen-specific T- lymphocytes.
Spleen
The spleen is the largest of the secondary lymphoid organs. It is highly efficient in trapping and concentrating
foreign substances carried in the blood. It is the major organ in the body in which antibodies are synthesized and
from which they are released into the circulation.
The interior of the spleen is a compartmentalized structure. The compartments are of two types – Red pulp and
white pulp. Red pulp is the site where old and defective RBCs are destroyed and removed, whereas white pulp forms
PALS (Periarteriolar lymphoid sheath) which are rich in T-cells. The marginal zone, located peripheral to the PALS, is
rich in lymphocyte and macrophage. Approximately 50% of spleen cells are B-lymphocytes; 30-40% are T-lymphocytes.
Lymph nodes
Lymph nodes are small encapsulated bean shaped structures (normally <1cm in diameter) found in various regions
throughout the body. The lymph nodes are composed of a medulla and a cortex, which is surrounded by a capsule
of connective tissue. They are packed with lymphocytes, macrophages, and dendritic cells. The cortical region
contains primary lymphoid follicles. After antigenic stimulation, these structures enlarge to form secondary lymphoid
follicles with germinal centers containing dense populations of lymphocytes (mostly B-cells). The deep cortical area
or paracortical region contains T-cells and dendritic cells. Antigens are brought into these areas by dendritic cells,
which present antigen fragments to T-cells. The medullary area of the lymph node contains antibody-secreting
plasma cells that have traveled from the cortex to the medulla via lymphatic vessels.
Lymph nodes are highly efficient in trapping antigen that enters through the afferent lymphatic vessels. In the node,
the antigen interacts with macrophages, T-cells, and B-cells, and that interaction brings about in immune response,
manifested by the generation of antibodies and antigen-specific T-cells.
Mucosa associated lymphoid tissue

The majority of secondary lymphoid tissue in the human body is located within the lining of respiratory, digestive
and genitourinary tracts. These are collectively called as Mucosa Associated Lymphoid Tissue (MALT). There are
several types of MALT. Two major MALT includes Bronchial Associated Lymphoid Tissue (BALT) and Gut-Associated
Lymphoid Tissue (GALT). GALT includes the tonsils, adenoids, and specialized regions in the small intestine called
Peyer’s patches.
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5.5 Antigens
Adaptive immune responses arise as a result of exposure to foreign compounds. The compound that evokes the
response is referred to as antigen, a term initially coined due to the ability of these compounds to cause antibody
responses to be generated. An antigen is any agent capable of binding specifically to T-cell receptor (TCR) or an
antibody molecule (membrane bound or soluble). The ability of a compound to bind with an antibody or a TCR is
referred to as antigenicity. There is a functional distinction between the term antigen and immunogen. An
immunogen is any agent capable of inducing an immune response and is therefore immunogenic. The distinction
between the terms is necessary because there are many compounds that are incapable of inducing an immune
response, yet they are capable of binding with components of the immune system that have been induced specifically
against them. Thus all immunogens are antigens, but not all antigens are immunogens.
Requirements for immunogenicity
A substance must possess the following characteristics to be immunogenic:
1. Foreignness
The most important feature of an immunogen is that an effective immunogen must be foreign with respect to
the host. The adaptive immune system recognizes and eliminates only foreign (nonself) antigens. Self antigens
are not recognized and thus individuals are tolerant to their own self molecules, even though these same
molecules have the capacity to act as immunogens in other individuals of the same species.
2. Size
The second requirement for being immunogenic is that the compound must have a certain minimal molecular
weight. There is a relationship between the size of immunogen and its immunogenicity. In general, small
compounds with a molecular weight <1000 Da (e.g. penicillin, aspirin) are not immunogenic; those of molecular
weight between 1000 and 6000 Da (e.g. insulin, adrenocorticotropic hormone) may or may not be immunogenic;
and those of molecular weight >6000 Da (e.g. albumin, tetanus toxin) are generally immunogenic. The most
active immunogens tend to have a molecular mass of 100,000 Da or more. In short relatively small substances
have decreased immunogenicity, whereas large substances have increased immunogenicity.
3. Chemical complexity
The third characteristic necessary for a compound to be immunogenic is a certain degree of chemical complexity.
For example, homopolymers of amino acids or sugars are seldom good immunogens regardless of their size.
Similarly, a homopolymer of poly-γ-D-glutamic acid (the capsular material of Bacillus anthracis) with a molecular
weight of 50,000 Da is not immunogenic. The absence of immunogenicity is because these compounds, although
of high molecular weight, are not sufficiently chemically complex.
Virtually all proteins are immunogenic. Furthermore, the greater the degree of complexity of the protein, the
more vigorous will be the immune response to that protein. Carbohydrates are immunogenic only if they have
a complex polysaccharide structure or part of complex molecules such as glycoproteins. Nucleic acids and
lipids are poor immunogens by themselves, but they become immunogenic when they are conjugated to
protein carriers.
4. Dosage and route of administration

The insufficient dose of immunogen may not stimulate an immune response either because the amount
administered fails to activate enough lymphocytes or because such a dose renders the responding cells
unresponsive. Besides the need to administer a threshold amount of immunogen to induce an immune response,
the number of doses administered also affects the outcome of the immune response generated.
The route of administration also affects the outcome of the immunization because this determines which organs
and cell populations will be involved in the response. Immunogens can be administered through a number of
common routes: Intravenous (into a vein); intradermal (into the skin); subcutaneous (beneath the skin);
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Immunology
Exogenous antigen
4 Plasma membrane
CLIP
3 5
CLIP
1. Class II MHC binds invariant chain blocking

Golgi apparatus
2
binding of endogenous antigen
2. MHC complex is routed through Golgi to endocytic
Invariant chain pathway compartments
3. Invariant chain is degraded, leaving CLIP fragment
ER
4. Exogenous antigen is taken up, degraded
1 Class II MHC fragments routed to endocytic pathway
compartments
5. Class II-MHC peptide is transported to PM
Figure 5.11 The processing of an exogenous protein antigen for presentation to a helper T-cell.
5.6.3 Laboratory mice

Mice are the most commonly used mammalian research model. They are common experimental animals in biology,
primarily because they are mammals, are relatively easy to maintain and handle, reproduce quickly, and share a
high degree of homology with humans. Laboratory mice include several inbred, outbred, knockout and transgenic
mice strains. Many laboratory strains are inbred. An inbred strain is one that is produced using at least 20 consecutive
generations of sister and brother or parent and offspring matings. The mating of two genetically related parents is
called inbreeding. Inbreeding results in increased homozygosity. In contrast to inbred mice, outbred mice are
usually heterozygous at many loci.
If mice are inbred (that is, have identical alleles at all loci), each H-2 locus will be homozygous because the
maternal and paternal haplotypes are identical, and all offspring therefore express identical haplotypes. Inbred
mouse strains are syngeneic or identical at all genetic loci. Two strains are considered congenic if they are genetically
identical except at a single genetic locus.
Some inbred mouse strains have been designated as prototype strains and the MHC haplotype expressed by these
strains is designated by an arbitrary italic superscript (e.g. H-2a, H-2b). If another inbred strain has the same set of
alleles as the prototype strain, its MHC haplotype is the same as the prototype strain.
Table 5.5 H-2 haplotypes of some mouse strains

H-2 alleles
Prototype strain Other strains with the same haplotype Haplotype K IA IE S D
CBA AKR, C3H, C57BR k k k k k k
DBA/2 BALB/c, SEA, YBR d d d d d d
C57BL/10 (B10) C57BL/6, C57L b b b b b b
A A/He, A/Sn a k k k d d
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5.7 Immunoglobulins : Structure and function

Antibodies, the antigen-binding glycoproteins are synthesized exclusively by B-cells and in billions of forms, each
with a different amino acid sequence and a different antigen binding site. Collectively called immunoglobulins (Ig),
they are among the most abundant protein components in the blood, constituting about 20% of the total protein
components in the blood plasma. Antibodies (Ab) are present on the B-cell membrane and also are secreted by
plasma cells.
5.7.1 Basic structure of antibody molecule

The simplest antibodies are Y-shaped molecules with two identical antigen-binding sites, one at the tip of each arm
of the Y. Because of their two antigen-binding sites, they are described as bivalent.
Antibody has a common structure of 4 polypeptide chains. It is a heterodimer and consists of two identical light (L)
chains (each containing about 220 amino acids residues, about 25000 MW) and two identical heavy (H) chains
(each usually containing about 440 amino acids residues, about 50000 MW). Each light chain is bound to heavy
chain by disulfide bridges and other non-covalent linkages. Thus, antibody is a dimer of H—L chain.
All species studied have the two major classes of light chains: κ and λ. Any one individual of a species produces both
types of light chain. However, in any one immunoglobulin molecule, the light chains are always either both κ or both
λ, never one of each. While there are two types of light chains, the immunoglobulins of virtually all species have
been shown to consist of five different types of heavy chains- α, γ, δ, ε and μ. These five different types of heavy
chains are called isotypes. The heavy-chains of a given antibody molecule determine the class of that antibody:
IgM (μ), IgG (γ), IgA (α), IgD (δ) or IgE (ε). Each class can have either κ or λ light chains. Any individual of a species
makes all heavy chains, but in any one antibody molecule, both heavy chains are identical. Thus an antibody molecule
of the IgG class could have the structure κ2γ2 with two identical κ light chains and two identical γ heavy chains.
Alternatively, it could have the structure λ2γ2 with two identical λ light chains and two identical γ heavy chains.
Immunoglobulin heavy chain isotypes

Isotype Heavy chain
IgM μ
IgD δ
IgG γ
IgA α
IgE ε
Minor differences in the amino-acid sequences of the α and the γ heavy chains led to further classification of the
heavy chains into subclasses. In humans, there are two subclasses of α heavy chains (α1 and α2) and four subclasses
of γ heavy chains (γ1, γ2, γ3 and γ4).
Both light and heavy chains have a variable sequence at their N-terminal ends but a constant sequence at their
C-terminal ends. Light chains have a constant region (CL) about 110 amino acids long and a variable region
(VL) of the same size. The variable region (VH) of the heavy chains (at their N-terminus) is also about 110 amino
acids long, but the heavy-chain constant region (CH) is about three to four times longer (330 or 440 amino acids),
depending on the class. It is the N-terminal ends of the light and heavy chains that come together to form the
antigen-binding site.
The diversity in the variable regions of both light and heavy chains is for the most part restricted to three small
hypervariable regions (each ~10 amino acid residues long) in each chain called complementarity determining
regions (CDR); the remaining parts of the variable region, known as framework regions, are relatively constant.
Proceeding from either the VL or VH amino terminus, these regions are called CDR1, CDR2 and CDR3. The CDR3 is
the most variable of the CDRs.
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Clonal selection theory

The clonal selection theory is a central paradigm of adaptive immunity. The most remarkable feature of the
adaptive immune system is that it can respond to millions of different foreign antigens in a highly specific way.
B-cells, for example, make antibodies that react specifically with the antigen that induced their production. The
clonal selection theory (formulated by Sir Macfarlane Burnet) explains how the adaptive immune system can
respond to millions of different antigens in a highly specific way.
According to this theory, an animal first randomly generates a vast diversity of lymphocytes, and then those
lymphocytes that can react against the foreign antigens that the animal actually encounters are specifically selected
for proliferation. As each lymphocyte develops in a central lymphoid organ, it becomes committed to react with a
particular antigen before ever being exposed to the antigen. It expresses this commitment in the form of cell-
surface receptor proteins that specifically fit the antigen.
Precursor cell
B1 B2 B3 Different
non-activated B-cells
Ag
Antigen binding to
specific B-cell
B2
Proliferation and
differentiation
B2 B2 B2 B2
Figure 5.23 The clonal selection theory of B-cells leading to antibody production.
Adapted from Molecular Biology of the cell, Albert et al., Garland Science.
When a lymphocyte encounters its antigen in a peripheral lymphoid organ, the binding of the antigen to the
receptors activates the lymphocyte, causing it both to proliferate and to differentiate into an effector cell. An
antigen therefore selectively stimulates those cells that express complementary antigen-specific receptors and are
thus already committed to respond to it. This arrangement is what makes adaptive immune responses antigen-
specific. According to the clonal selection theory, then, the immune system functions on the ready-made principle
rather than the made-to-order one.
The term clonal in clonal selection theory derives from the postulate that the adaptive immune system is composed
of millions of different families, or clones, of lymphocytes, each consisting of T or B-cells descended from a
common ancestor. Each ancestral cell was already committed to make one particular antigen-specific receptor
protein, and so all cells in a clone have the same antigen specificity.
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5.9 Kinetics of the antibody response

Humoral immunity is mediated by serum antibodies which are the proteins secreted by the B-cells. B-cells are
initially activated to secrete antibodies after the binding of antigens to specific membrane immunoglobulin molecules
(B-cells receptors), which are expressed by these cells. Once bound, the B-cell receives signals to begin making
the secreted form of this immunoglobulin, a process that initiates the full-blown antibody response whose purpose
is to eliminate the antigen from the host. Antibodies are a heterogeneous mixture of serum globulins, all of which
share the ability to bind individually to specific antigens.
Primary and secondary responses
The first exposure of an individual to an immunogen is referred to as the primary immunization, which generates a
primary response. The primary antibody response may be divided into several phases, as follows:
1. Lag or latent phase: It is the immediate stage following antigenic stimulus during which no antibody is detectable
in circulation. The length of this period is generally one to two weeks.
2. Log or exponential phase: In this phase there is a steady rise in the titer of antibody and the concentration of
antibody in the serum increases exponentially.
3. Plateau or steady state: During this phase there is an equilibrium between antibody synthesis and degradation.
4. Declining phase: The concentration of antibody in serum declines rapidly.
A second exposure to the same immunogen results in a secondary response. This second exposure may occur
after the response to the first immune event has leveled off or has totally subsided. The secondary response is also
called the memory or anamnestic response and the B-and T-lymphocytes that participate in the memory response
are termed memory cells.
The primary response is slow and short lived with a long lag phase and low titer of antibodies that do not persist for
long. However the secondary response is prompt, powerful and prolonged, with a short or negligible lag phase and
a much higher level of Ab that lasts for long periods.
Antibody concentration in serum
Primary response Secondary response
IgG
De
IgG
cli
ne
IgM
ph
IgM
as
e
Latent period
10 days 15 days 5 days 10 days
First exposure to antigen Second exposure to antigen
Figure 5.24 Antibody production and kinetics.
In the primary response, the first class of antibody detected is generally IgM, then IgG, or another antibody class.
There is a marked change in the type and quality of antibody produced in the secondary response. There is a shift
in class response, known as class switching, with IgG antibodies appearing at higher concentrations and with
greater persistence than IgM, which may be greatly reduced or disappear altogether. This may be also accompanied
by the appearance of IgA and IgE. The IgG, IgE, and IgA molecules are collectively referred to as secondary
classes of antibodies because they are thought to be produced only after antigen stimulation and because they
dominate secondary antibody responses.
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Immunology
Antigen
Spleen cells
(contain B-cells that produce
Spleen antibodies against antigen)
Unfused spleen cells
Unfused myeloma cells
Cultured mouse Myeloma cells Fused hybrid cells

myeloma cells
(cancerous B-cells)
Cells transferred to a medium in which

only hybridoma cells can grow
Hybridoma cells grow, others die
Clone of
hybridoma cells
Hybridoma cells that produce a

desired antibody are cultured
Desired monoclonal
antibodies
Figure 5.25 Production of monoclonal antibodies.
In the procedure, myeloma cells are engineered to be deficient in enzyme HGPRT. After fusion of lymphocytes with
HGPRT-negative myeloma cells, aminopterin-containing medium, supplemented with hypoxanthine and thymidine
to ensure and adequate supply of substrates for the salvage pathway (HAT medium) is added, which kills myeloma
cells but allows hybridomas to survive as they inherit HGPRT from the lymphocyte parent. Unfused lymphocytes die
after a short period of culture, which results in a pure preparation of hybridomas.
5.10.1 Engineered monoclonal antibodies

Immunotoxins
Immunotoxins are protein-based drugs contain two functional domains, one allowing them to bind specific target
cells (target-specific binding domain), and one that kills the cells following internalization (cytotoxic domain). An
immunotoxin is prepared by replacing the target-specific binding domain of toxin with a monoclonal antibody that
is specific for a particular antigen. The toxins used may be bacterial toxins such as diphtheria toxins or plant toxins
like ricin, abrin, etc. Toxins used to prepare immunotoxins include ricin, Shigella toxin and diphtheria toxin, all of
which inhibit protein synthesis.
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Immunology
Immunological tolerance of B-cells are also mediated by the process of clonal anergy or inactivation. When a
mature B-cell escapes tolerance in primary lymphoid organ and bind self antigens in the peripheral lymphoid organ,
self-reactive B-cell may either die by apoptosis or be functionally inactivated and cannot amplify the immune response.
5.13 T-cells and CMI

Ig molecules secreted from B-cells, play a critical role in interacting with antigens when they are present outside
the cells; for example, when viruses are encountered in blood plasma or at mucosal surfaces. Once an antigen gets
into a cell however, antibodies do not generally have access to it, and so antibodies are ineffective in dealing with
antigens inside cells. T-cells deal with pathogens – such as viruses, bacteria, and parasites – that resides inside the
cells of the host. T-cells responses differ from B-cell responses in at least two crucial ways.
First, T-cells are activated by foreign antigen to proliferate and differentiate into effector cells only when the antigen
is displayed on the surface of antigen-presenting cells/target cells in peripheral lymphoid organs.
The second difference is that, once activated, effector T-cells act only at short range, either within a secondary
lymphoid organ or after they have migrated into a site of infection. They interact directly with another cell in the
body, which they either kill or signal in some way.
T-cell receptor
T-cells, like B-cells, express antigen specific receptors. The T-cell receptor (TCR) is a heterodimer and composed of
two transmembrane glycoprotein chains, α and β. The extracellular portion of each chain consists of two domains,
resembling immunoglobulin variable (V) and constant (C) domains, respectively. Both chains are glycosylated and
connected with each other with the help of interchain disulfide bond. The transmembrane helices of both chains are
unusual in containing positively charged (basic) amino acid residues within the hydrophobic transmembrane segment.
The α-chains carry two such residues; the β-chains have one.
a-chain b-chain
or or
d-chain g-chain
N N
S S
Va S S Vb
S S
Ca S S Cb
S S
+
+ +
C C
Figure 5.38 The predominant form of the antigen-binding chains of TCR.
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Immunology
Class I Inhibitory
MHC receptor
–
No killing
+
Ligand
Activating
Normal cell receptor NK cell
Killing
+
Ligand
Activating
Altered self cell receptor NK cell
Figure 5.46 An activating receptor on NK cells interacts with its ligand on normal and altered self cells,
inducing an activation signal that results in killing. However, interaction of inhibitory NK-cell receptors with
class I MHC molecules delivers an inhibition signal that counteracts the activation signal. Expression of class I
molecules on normal cells thus prevents their destruction by NK cells. Because class I expression is often
decreased on altered self cells (virus infected cells and tumor cells), the killing signal predominates, leading
to their destruction.
5.13.1 Superantigens
Superantigens are viral or bacterial proteins that bind simultaneously to the variable domain of β of a T-cell
receptor (TCR) and to the α-chain of a class II MHC molecule (i.e. outside the peptide-binding groove). Because of
their unique binding ability, superantigens can activate large numbers of T-cells irrespective of their antigenic
specificity. Superantigens can be exogenous and endogenous. Exogenous superantigens are soluble proteins secreted
by bacteria whereas endogenous superantigens are cell-membrane proteins encoded by certain viruses that infect
mammalian cells.
b Ag
a
MHC TCR
a b
Superantigen
APC TH cell
Figure 5.47 Superantigen-mediated cross-linkage of T-cell receptor (TCR) and class II MHC molecules.
Superantigen binds to class II MHC molecule and a part of the Vβ chain of the T-cell receptor that is outside
the normal antigen-binding site and this binding is sufficient to trigger T-cell activation. A superantigen binds
to all TCRs bearing a particular V sequence regardless of their antigen specificity.
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5.14 Cytokines
Cytokines are low-molecular-mass (generally less than 30 kDa) soluble proteins/glycoproteins, non-immunoglobulin
in nature, secreted by a variety of cell types and act nonenzymatically through specific receptors to regulate host
cell function. They do not include the peptide and steroid hormones of the endocrine system. Cytokines play major
roles in the development of cellular and humoral immune responses, induction of the inflammatory response,
regulation of hematopoiesis, control of cellular proliferation and differentiation.
Cytokines can affect the same cell responsible for their production (an autocrine function) or nearby cells (a
paracrine function), or they can be distributed by the circulatory system to distant target cells (an endocrine
function). They are highly potent hormone-like substances, active even at femto molar concentration. However,
they differ from endocrine hormones as being not produced by glands but by widely distributed cells. Cytokines
produce biological actions only when they bind to specific, high-affinity receptors on the surface of target cells. The
biological activities of cytokines exhibit pleiotropy (a given cytokines that has different biological effect on different
target cells), redundancy (two or more cytokines that mediates similar functions), synergy (combined effect of two
cytokines on cellular activity is greater than the additive effect of the individual cytokines) and antagonism (effect
of one cytokines inhibit the effect of another cytokines).
Target cell Effect

PLEIOTROPY
B cell Activation, proliferation, differentiation
Activated TH cells IL-4 Thymocyte Proliferation
Mast cell Proliferation
REDUNDANCY
IL-2
Activated TH cells IL-4 B cell Proliferation
IL-5
SYNERGY
IL-4
Activated TH cells + B cell Induces class switch to IgE
IL-5
ANTAGONISM
Activated TH cells IL-4 B cell Blocks class switch of IgE induced by IL-4
IFN-g
Figure 5.48 Cytokine attributes of pleiotropy, redundancy, synergy (synergism), antagonism.
Cytokines differ from hormones and growth factors. All three are secretory proteins that elicit their biological
effects at very low concentrations by binding to receptors on target cells. Growth factors tend to be produced
constitutively, whereas cytokines and hormones are secreted in response to discrete stimuli. Unlike hormones,
which generally act long range in an endocrine fashion, most cytokines act over a short distance in an autocrine or
paracrine fashion. In addition, most hormones are produced by specialized glands and tend to have a unique action
on one or a few types of target cell. In contrast, cytokines are often produced by, and bind to, a variety of cells.
There are over 100 different cytokines. The generic name of cytokines includes all proteins with a small molecular
weight, released by cells of the immune system, especially by monocytes and T-lymphocytes. But they are also
secreted by many cells in addition to those of the immune system, such as endothelial cells and fibroblasts. They
used to have different names depending either on their origin, such as lymphokines (produced by lymphocytes),
monokines (substances produced by monocytes or macrophages) or on their activity: chemokines, interleukins,
interferons.
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Immunology
Biologically active functions mediated by complement products

Activation of complement results in the production of several biologically active molecules which contribute to
killing of cell, opsonization, chemotaxis, anaphylaxis and inflammation.
The most important action of complement is to facilitate the uptake and destruction of pathogens by phagocytes.
This occurs by the specific recognition of bound complement components by complement receptors on phagocytes.
These complement receptors bind pathogens opsonized with complement components: opsonization of pathogens
is a major function of C3b and its proteolytic derivatives. C4b also acts as an opsonin but has a relatively minor
role. There are several different complement receptors (CRs). CR1 and CR3 are especially important in inducing
phagocytosis of bacteria with complement components on their surface.
The small complement fragments C3a, C4a and C5a act on specific receptors to produce local inflammatory
responses. When produced in large amounts they induce a shocklike syndrome similar to that seen in a systemic
allergic reaction involving IgE antibodies. Such a reaction is termed anaphylactic shock and these small fragments
of complement are therefore often referred to as anaphylotoxins. Of the three, C5a is the most stable and has the
highest specific biological activity.
Biologically active functions Complement component

Cell lysis C5b–9 (membrane-attack complex)
Inflammatory response C3a, C4a, and C5a (anaphylatoxins)
Chemotaxis of leukocytes C3a, C5a
Opsonization of particulate antigens C3b, C4b
Viral neutralization C3b, C5b–9 (membrane-attack complex)
Solubilization and immune clearance C3b
5.16 Hypersensitivity
Hypersensitivity is an exaggerated immune response that results in tissue damage and is manifested in the individual
on a second or subsequent contact with an antigen.
Hypersensitivity has been traditionally classified into immediate and delayed types based on the time required for
a sensitized host to develop clinical reactions on re-exposure to the antigen. Later, Gell and Coombs proposed a
classification scheme which defined four types of hypersensitivity reactions.
Type I Hypersensitivity
Type I hypersensitivity (also known as allergic reaction) is induced by antigens referred to as allergens. The term
allergen refers specifically to nonparasitic antigens capable of stimulating type I hypersensitive responses. Type I
hypersensitive reactions are IgE-mediated humoral antibody responses. These IgE-mediated reactions are stimulated
by the binding of IgE (via its Fc region) to high-affinity IgE-specific Fc receptors expressed on mast cells and
basophils. When cross linked by antigens, the IgE antibodies trigger the mast cells and basophils to release primary
mediators, vasoactive amines, stored in the granules (degranulation). The most significant primary mediators are
histamine, proteases, eosinophil chemotactic factor, neutrophil chemotactic factor, and heparin.
These mediators cause all the normal consequences of an acute inflammatory reaction - increased vascular
permeability, smooth muscle contraction, granulocyte chemotaxis and extravasation etc. Mast cell activation via Fc
also leads to the production of two other types of mediators. These secondary mediators, unlike the stored granule
contents, must be synthesized de novo and comprise arachidonic acid derivatives (prostaglandins and leukotrienes),
platelet-activating factor, bradykinins, and various cytokines.
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Immunology
Allergen Allergen
FC receptor
IgE for IgE
Subsequent Degranulation
exposures
Mast cell to allergen
binding
fragment
Sensitized Release of allergic
Mast cell mediators
Figure 5.53 Ag induces crosslinking of IgE bound to mast cells and basophils with release of
vasoactive mediators.
Type I hypersensitivity can be anaphylaxis or atopy. Anaphylaxis is a very rapid, life-threatening, severe whole
body allergic reaction. It is caused by re-exposure to a previously encountered antigen. Atopy (atopic allergy) is a
hereditary tendency to develop allergic reaction to substances such as pollen, food, insect venom etc.
Type II Hypersensitivity
Type II hypersensitivity is generally called a cytolytic or cytotoxic reaction because it results in the destruction of
host cells, either by lysis or toxic mediators. Type II Hypersensitivity is caused by antibodies binding to cells or
tissue antigens. The antibodies are of the IgM or IgG classes and cause cell destruction by Fc dependent mechanisms
either directly or by recruiting complement via the classical pathway. Classical examples of type II hypersensitivity
reactions are the response exhibited by a person who receives a transfusion with blood from a donor with a
different blood group and erythroblastosis fetalis.
Two different antibody-mediated mechanisms are involved in these cytotoxic reactions. In complement-mediated
hypersensitivity reactions, the antibodies react with a cell membrane component, leading to complement fixation.
This activates the complement cascade and leads either to lysis of the cell or opsonization. Blood cells are most
commonly affected by this mechanism.
+ Complement
activation
Target cell
Figure 5.54 Antibody subclasses activate the complement system, creating pores in the membrane
of a foreign cell.
Antibody-dependent cell mediated cytotoxicity (ADCC) used Fc receptors expressed on many cell types (e.g.
natural killer cells, macrophages, neutrophils, eosinophils) as a means of bringing these cells into contact with
antibody-coated target cells. Lysis of these target cells requires contact but does not involve phagocytosis or
complement fixation. Instead, ADCC lysis of target cells is analogous to that of cytotoxic T cells and involves the
release of cytoplasmic granules containing perforin and granzymes that activate events leading to apoptosis.
ADCC reactions involve IgG and IgG Fc receptors.
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Immunology
5.17 Autoimmunity
The body is normally able to distinguish its own self-antigens from foreign nonself antigens and does not mount an
immunologic attack against itself. This phenomenon is called immune tolerance. Autoimmunity is a condition in
which structural or functional damage is produced by the action of immunologically competent cells or Ab against
self antigen. Autoimmunity literally means protection against self, but actually it implies injury to self, and therefore
sometimes the term is also under criticism.
Autoimmune disease results from the activation of self-reactive T and B-cells that, following stimulation by
genetic or environmental triggers, cause actual tissue damage. Four factors influence the development of autoimmune
disease. These factors are genetic, viral, hormonal and psycho-neuro-immunological (the influence of stress and
neurochemicals). All four of these factors can affect gene expression, which directly or indirectly interferes with
important immunoregulatory actions. Based on the site of involvement and nature of lesions autoimmune diseases
may be classified as hemocytolytic, localized (or organ specific), systemic (or non-specific) and transitory diseases.
Important examples of autoimmune diseases in human and their respective autoantigen are given below in the table.
Table 5.14 Some autoimmune diseases in humans

Disease Autoantigen
Autoimmune hemolytic anemia Rh blood group
Graves disease Thyroid-stimulating hormone receptor
Multiple sclerosis Myelin basic protein
Myasthenia gravis Acetylcholine receptor
Rheumatoid arthritis Unknown synovial joint antigen
Systemic lupus erythematosus DNA, histones, snRNP
Type 1 diabetes mellitus Pancreatic beta cell antigen
5.18 Transplantation
The immune system has evolved as a way of discriminating between self and non-self. This discriminating power of
the immune system between self and non-self is undesirable in the case of tissue transplant from one individual to
another for therapeutic purposes. Indeed, result of transplants culminates in the phenomenon of graft rejection.
Before the discussion about the immunological mechanisms associated with graft rejection, it is important to
understand the various gradations in relationship from donor to recipient.
Isograft : Graft between genetically identical individuals (syngeneic). In humans, an isograft (or syngraft) can
be performed between monozygotic twins.
Allograft : Transplants between genetically different individuals within a species.
Xenograft : A graft between individuals from different species.
Autograft : A graft or transplant from one body part to another on the same individual.
Transplanting tissue that is not immunologically privileged generates the possibility that the recipient’s cells will
recognize the donor’s tissue as foreign. This triggers the recipient’s immune mechanisms, which may destroy the
donor tissue. Such a response is called a graft rejection reaction. Some transplanted tissues do not stimulate an
immune response. For example, a transplanted cornea is rarely rejected because lymphocytes do not circulate into
the anterior chamber of the eye. This site is considered an immunologically privileged site. Another example of a
privileged tissue is the heart valve.
A tissue rejection reaction can occur by two different mechanisms. First, foreign class II MHC molecules on transplanted
tissue, or the graft is recognized by host T-helper cells, which aid cytotoxic T-cells in graft destruction. Cytotoxic
T-cells then recognize the graft through the foreign class I MHC molecules. This response is much like the activation
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Chapter 06
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
6.1 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
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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 colour Violet White
Seed coat colour Grey White
4. Pod shape Inflated Constricted
5. Pod colour Green Yellow
6. Cotyledon colour Yellow Green
7. Seed form Round Wrinkled
Flower colour is positively correlated with seed coat colours. 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 inher-
ited character. We may also define alleles as genes occupying corresponding positions on homologous chromo-
somes 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:
1. Homozygous, indicating they possess two identical alleles for a trait.
a. Homozygous dominant genotypes possess two dominant alleles for a trait (T T ).
b. Homozygous recessive genotypes possess two recessive alleles for a trait (tt).
2. Heterozygous genotypes possess one of each allele for a particular trait (Tt).
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Genetics
A B a b A B A b
a b A A B A b
c. and
B d. or
a b A B a b a B
A B a b a b a B
50% 50% 100% 100%
Solution
Choice ‘a’ represents the correct chromosomal arrangement in meiotic metaphase I. In this situation, as a result of
independent assortment – 50% meiotic product will be AB + ab and 50% will be Ab + aB.
6.3 Gene interaction

According to Mendel, genes are functioning independently of each other i.e. each of seven traits considered was
controlled by a single gene. But many traits of an organism are determined by the complex contribution of many
different genes. When two or more different genes (non-allelic) influence the outcome of single trait, this is known
as a gene interaction.
The first case of two different genes interacting to affect a single trait was discovered by William Bateson and
Reginald Punnett in 1906. They discovered an unexpected gene interaction when they studied crosses involving the
sweet pea, Lathyrus odoratus. When they crossed true breeding purple flowered plant to a true breeding white
flowered plant, the F1 generation was all purple flowered plants and the F2 generation (produced by self fertilization
of the F1 generation) contained purple and white flowered plants in a 3 : 1 ratio. But when they crossed two
different varieties of white flowered plants then all F1 generation plants had purple flowers. When these purple
flower plants were allowed to self fertilized, the F2 generation contained purple and white flowers in a ratio of
9 purple : 7 white. How can this unexpected result be explained? This surprising result was explained by Bateson
and Punnett by considering the involvement of two different (non-allelic) genes; because the F2 9 : 7 ratio is a
variation of the 9 : 3 : 3 : 1 ratio. Let us consider the formation of the purple pigment in which products of two
different genes are involved.
Genotype Genotype
(CC or Cc) (PP or Pp)
Enzyme A Enzyme B
Colourless precursor Colourless intermediate Purple pigment
(Anthocyanin)
C (purple colour producing) allele is dominant to c (white)

P (purple colour producing) allele is dominant to p (white)
In the above pathway, a colourless precursor molecule must be acted on by two different enzymes to produce the
purple pigment. Gene C encodes a functional enzyme A, which converts the colourless precursor into a colourless
intermediate and finally gene P encodes enzyme B, which gives purple colour by converting colourless intermediate.
If any of these two genes will be in homozygous recessive condition (cc or pp) then purple colour will not appear.
Thus the genotype cc can hide or mask the phenotype expression of genotype PP or Pp.
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P generation White flowered plant × White flowered plant

(CCpp) (ccPP)
F1 generation All purple

(CcPp)
The F1 hybrid plants are allowed to self fertilize

CcPp × CcPp
CP Cp cP cp
CP CCPP CCPp CcPP CcPp

Purple Purple Purple Purple
Cp CCPp CCpp CcPp Ccpp

Purple White Purple White
F2 generation
cP CcPP CcPp ccPP ccPp
Purple Purple White White
cp CcPp Ccpp ccPp ccpp

Purple White White White
Figure 6.8 9 : 7 phenotypic ratio in F2 generation.
The purple colour appears only when dominant alleles of both genes are present. When one or both genes have
only recessive alleles, the colour will be white.
Epistasis
The term epistasis (Greek for standing upon) describes a type of gene interaction when one gene masks or
modifies the expression of another gene at distinct locus. Any gene that masks the expression of another non-
allelic gene is epistatic to that gene. The gene suppressed is hypostatic. In the pathway discussed for formation of
purple colour, when either is homozygous recessive (cc or pp) that gene is epistatic to the other.
Epistasis is different from dominance. Epistasis is the interaction between different genes (non-alleles). Dominance
is the interaction between different alleles of the same gene i.e. intraallelic.
Table 6.3 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.
6.3.1 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
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Haploid cell Haploid cell
Diploid zygote
Zygotic meiosis
Tetrad of cells contained

within an ascus
Mitosis
Figure 6.15
Sexual reproduction in ascomycetes.
For simplicity, this diagram shows
each haploid cell as having only one
chromosome per haploid set. However,
fungal species actually contain several
chromosomes per haploid set.
Ordered or unordered tetrad/octad

The arrangement of spores within an ascus varies from species to species. In some cases, the ascus provides
enough space for the tetrads or octads of spores to randomly mix together. This is known as an unordered tetrad
or octad. These occur in fungal species such as S. cerevisiae. By comparison, other species of fungi produce a very
tight ascus that prevents spores from randomly moving around. This can create a linear tetrad or octad found in
N.crassa.
Saccharomyces cerevisiae
Figure 6.16 Different arrangements of fungal spores.
A key feature of linear tetrads or octads is that the position and order of spores within the ascus reflects their
relationship to each other as they were produced by meiosis and mitosis. This idea is schematically shown in figure 6.17.
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After the original diploid cell has undergone chromosome replication, the first meiotic division produces two cells
that are arranged next to each other within the sac. The second meiotic division then produces four cells that are
also arranged in a straight row. Due to the tight enclosure of the sac around the cells, each pair of daughter cells is
forced to lie next to each other in a linear fashion. Likewise, when each of these four cells divides by mitosis, each
of the daughter cells is located next to each other.
A A
A A A
A Meiosis II Mitosis
a a a
a
Meiosis I a a
Figure 6.17 Formation of a linear octad in N. crassa.
6.5.1 Analysis of ordered tetrad

Linear tetrad analysis can be used to map the distance between a gene and the centromere. This approach has
been extensively exploited in N. crassa. In N. crassa, the products of meiosis are contained in an ordered array of
spores. Each mature ascus contains eight ascospores in four pairs, each pair representing one of the products of
meiosis. The ordered arrangement of meiotic product makes it possible to map each gene with respect to its
centromere; i.e. to determine the recombination frequency between a gene and its centromere. Two cases are
possible depending on whether or not there is a crossover between the locus and its centromere.
First case
In the absence of crossing over between a gene and its centromere, the alleles of the gene (for example A and a)
must separate in the first meiotic division, this separation is called First Division Segregation (FDS). Octad
contains a linear arrangement of four haploid cells carrying the A allele, which are adjacent to four haploid cells that
contain an allele i.e. 4:4 arrangement of spores within the ascus (figure 6.18).
A
A
A 4
A
A
A
A Mitosis
A A
a a a
a
a a a 4
a
a
Segregation of homologous
chromosome during meiosis I a
Segregation of sister
chromatids during meiosis II
Figure 6.18 First Division Segregation (FDS) : No crossing over produces a 4 : 4 arrangement.
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6.6.7 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.
6.6.8 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 for X-linked genes in males. Since only one allele is present, a single copy of a recessive allele can determine
the phenotype, a phenomenon called pseudodominance. This is the same way that one copy of a dominant
autosomal allele would determine the phenotype of a normal diploid organism; hence the term pseudodominance.
The genes on the differential regions of the sex chromosomes show patterns of inheritance related to sex. The
inheritance patterns of genes on the autosomes produce male and female progeny in the same phenotypic proportions,
as typified by Mendel’s data (for example, both sexes might show a 3:1 ratio). However, crosses following the
inheritance of genes on the sex chromosomes often show male and female progeny with different phenotypic
ratios. T.H.Morgan demonstrated the X-linked pattern of inheritance in Drosophila in 1910, when a white eyed male
appeared in a culture of wild type (red-eyed) flies.
Let’s look at an example from Drosophila. When white-eyed males are crossed with red-eyed females, all the
F1 progeny have red eyes, showing that the allele for white is recessive. Crossing the red-eyed F1 males and
females produces a 3:1 F2 ratio of red-eyed to the white-eyed flies, but all the white-eyed flies are males.
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Female (Dextral) Male (Sinistral) Male (Dextral) Female (Sinistral)
Parent
DD × dd DD × dd
F1 generation
Dd Dd
(Dextral) (Sinistral)
F2 generation
DD Dd Dd dd DD Dd Dd dd
(Dextral) (Dextral) (Dextral) (Dextral) (Dextral) (Dextral) (Dextral) (Dextral)
F3 generation
Dextral Dextral Dextral Sinistral Dextral Dextral Dextral Sinistral
Figure 6.33 Inheritance of the direction of shell coiling in the snail Lymnaea. Sinistral coiling is determined
by recessive allele d and dextral coiling by dominant allele D. The F2 and F3 generations are obtained by
self-fertilization.
The next observation is that the phenotype of the F1 generation is always that of the female parent. One hypothesis
would suggest that the genotype of the female controls the genotype of its offspring. Can these results be confirmed
in the subsequent generations? If the genotypes we assigned to the parents are correct, then the genotype of F1
individuals from each cross are Dd (from DD×dd and dd×DD). If the female genotype does control the phenotype
of its offspring, then we would predict that all the F2 snails would have right coils. This is the exact result that is
seen. But what would the genotypes of the F2 snails be? If we intermate snails with the genotype Dd, the genotypic
ratio should be 3 D_ to 1 dd. These genotypes would not be expressed as a phenotype until the F3 generation.
These are the results that were obtained. A general conclusion from all traits that express a maternal effect is that
the normal Mendelian ratios are expressed one generation than expected. Cytological analysis of developing eggs
has provided the explanation of above mentioned result: the genotype of the mother determines the orientation of
the mitotic spindle during the second cleavage (mitotic) division in the zygote, and this, in turn, controls the
direction of shell coiling of the offspring.
6.9 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.
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Molecular genetics
6.11 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 6.43 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.
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6.11.10 Yeast S. cerevisiae genome

The yeast genome consists of 16 linear chromosomes, each containing a centromeric region required for chromosome
segregation. The nucleotide sequence of the entire S. cerevisiae genome has been determined and found to contain
12,068 kb of DNA. Sequence analysis has identified 5885 potential protein coding genes and another 45S RNA
coding genes (rRNA, snRNA and tRNA genes). Almost 70% of the yeast genome is devoted to protein coding
sequences. Interestingly, unlike most other eukaryotic genes, only about 4% of the about 6000 yeast genes have
introns, and even then, most of these genes contain only a single intron within the coding sequence.
6.11.11 E. coli genome

E. coli genome comprises single main chromosome and plasmids. The main chromosome is made up of circular
dsDNA with a homogeneous distribution of genes. Computer analysis of the E. coli DNA sequence identified 4288
actual and proposed gene-coding sequences. It was found that approximately 88% of the genome encodes proteins
or RNAs, ~11% appears to be utilized for gene regulatory functions, and <1% consists of repetitive DNA sequences.
The average distance between E. coli genes is only 120 bp.
6.12 Eukaryotic chromatin and chromosome

A chromatin is an organized structure of DNA and protein that is found in the nucleus of eukaryotic cells. It contains
a single dsDNA in coiled and condensed form. Chromatin and chromosomes are basically the same thing. The
difference is that chromatin is less condensed, extended DNA while chromosomes are highly condensed DNA. The
word chromosome comes from the Greek word chroma, color and soma, body due to their property of being very
strongly stained by particular dyes. The extent of chromatin condensation varies during the life cycle of the cell. In
non-dividing as well as interphase stages of cell, most of the chromatin remain relatively decondensed. The light-
staining, less condensed portions of chromatin is termed euchromatin. The darkly stained and highly condensed
regions of chromatin is termed as heterochromatin. In interphase nuclei, chromatin appears to be attached to a
nuclear matrix, a proteinaceous structure. DNA sequence attached to nuclear matrix are called MAR (matrix
attachment regions). MAR are usually ~70% A·T-rich, but lack any consensus sequences. A chromatin DNA molecule
contains three specific nucleotide sequences: Centromere, Telomere and Origin of replication.
Centromere
The centromere is a constricted region of a eukaryotic chromatin/chromosome where the kinetochore is assembled
and sister chromatids are held together. Although this constriction is termed as centromere, it is usually not located
exactly in the center of the chromosome and, in some cases, is located almost at the chromosome’s end. The
regions on either side of the centromere are referred to as the chromosome’s arms. Kinetochore associated with
the centromere is a complex of proteins where spindle fibers attach to the chromosome during mitosis/meiosis and
help in the proper segregation of sister chromatids or homologous chromosomes. The centromere has no defined
DNA sequence. It typically consists of large arrays of tandemly repeated DNA sequences. In humans, the centromeric
sequences are made up of 171 bp repeating unit and are called alphoid DNA. In the yeast, Saccharomyces cerevisiae,
the centromeric sequence (CEN) is about 110 bp long and it consists of three types of sequence element:
• CDE-I - 9 bp sequence;
• CDE-II - >90% A·T-rich sequence of 80–90 bp;
• CDE-III - 11 bp highly conserved sequence.
TC A C ATG AT TG ATTTC C G A A
A G TG TA C TA A C TA A A G G C TT
{
{
{
CDE-I CDE-II CDE-III

80–90 bp, > 90% (A+T)
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Chromosomes can be classified into following types based on the position of the centromere:
Metacentric: If the centromere is located exactly in the middle of the chromosome, the two arms of the chromosome
are nearly equal (median centromere). The chromosome appears V-shaped during anaphasic movement.
Submetacentric: If the centromere is situated some distance away from the middle (submedian centromere), one
arm of the chromosome will be shorter than the other, such a chromosome will appear L-shaped during anaphasic
movement.
Acrocentric: If the centromere is situated near the end of the chromosome, one arm will be extremely short and
other very long (subterminal centromere). These chromosomes appear rod shaped during anaphase.
Telocentric: If the centromere is truly terminal, i.e. situated at the tip of the chromosome, the chromosome is said
to be telocentric (terminal centromere).
In chromosome that is not metacentric, p represents the short arm of chromosome and q represents the long arm
of chromosome. Most eukaryotic chromosomes are monocentric, having a single centromere, but some are
holocentric (holokinetic or polycentric) and have diffused centromere. Every point along the length of the
chromosome exhibits centromeric activity. The nematode C. elegans has holocentric chromosome. In holocentric
chromosome, spindle fibers attach along the entire length of chromosome.
During interphase stage of cell cycle, chromatin replicates, resulting in the formation of two copies of each chromatin.
As the cell enters M-phase, chromatin condensation leads to the formation of metaphase chromosomes consisting
of two identical sister chromatids. These sister chromatids are held together at the centromere, which is seen as a
constricted chromosomal region. A cohesin protein play a role in linking together sister chromatids immediately
after replication and keeping them together at centromere.
Telomeres
Telomeres are specialized structures which cap the ends of eukaryotic chromosomes. They have several likely
functions – maintaining the structural integrity of a chromosome (if a telomere is lost, the resulting chromosome
end is unstable) and ensuring complete replication of the extreme ends of chromosomes. Eukaryotic telomeres
consist of a long array of short and tandemly repeated sequences. There may be 100–1000 repeats, depending on
the organism. One unusual property of the telomeric sequence is the presence of the G-rich single strand 3’
overhang, measuring between 50 to 300 nucleotides. The G-rich sequence is generated because there is a limited
degradation of the C-rich complementary strand. Unlike centromeres, the sequence of telomeres has been highly
conserved in evolution – there is considerable similarity in the simple sequence repeat, for example T T GGGG
(Paramecium), TAGGG (Trypanosoma), TTTAGGG (Arabidopsis) and T TAGGG (Homo sapiens). Two sequence-
specific DNA binding proteins – telomeric repeat binding factor 1 (TRF1) and telomeric repeat binding factor 2
(TRF2) bind directly with telomeric sequences, which in turn interact with a larger number of proteins.
Single-stranded 3’ overhang
{
5’ 3’
3’ 5’
5’
3’
t-loop
Figure 6.59 DNA at the telomeres consists of G-rich tandem sequences. The G-strand overhangs are
important for telomeric protection by formation of a duplex loop. Telomeric duplex DNA forms a loop (t-loop),
thus avoiding the sticky end problem. The loop formation is mediated by the TRF2, which bind to telomere
repeats and the loop is anchored by the insertion of the G-strand overhang into a proximal segment of duplex
telomeric DNA.
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Maternal chromosome
Paternal chromosome
Chromomere
Enlarged section of
a chromosome
Chromatin loop
Chromatin
loop
Chromatid
Chromatid
Chromomere
Figure 6.71 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.
6.12.6 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.
6.13 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.
6.13.1 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
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6.13.2 Replicon and origin of replication

DNA replication does not start at random locations but at particular sites, called the origins of DNA replication. A unit
of DNA in which replication starts from an origin and proceeds bidirectionally or unidirectionally to terminus site is
called a replicon, a unit of DNA replication. Replicon can be linear or circular. Prokaryotic replicons are usually
circular.
In bacterial cells, the circular chromosome contains a unique origin and DNA replication proceeds bidirectionally
from the origin to the terminus. Therefore, the whole bacterial genome (~4.6 Mbp for Escherichia coli) is a single
replicon (monorepliconic). On the other hand, eukaryotic cells contain multiple replication origins on single
chromosome and hence many replicons (multirepliconic). Individual replicons in eukaryotic genomes are relatively
small and generally 40 to 100 kb in size.
~ 245 bp
13 mer 13 mer 13 mer 9 mer 9 mer 9 mer 9 mer 9 mer
GATCTATTTATTT TTATCCACA
13 bp sequence (called 13 mer) 9 bp sequence (called 9 mer)
Binding sites for DnaA protein
Figure 6.73 E. coli origin of replication, oriC. oriC contains repetitive 9-bp and A.T rich 13-bp sequences,
referred to as 9-mers and 13-mers, respectively. Multiple copies of DnaA protein bind to the 9-mer and then
‘melt’ the 13-mer segments.
The origin of replication is a cis acting sequence. In E. coli single origin of the replication present in the chromosome
is referred to as oriC. It spans approximately 245 bp of DNA. It contains two short repeat motifs, one of nine
nucleotides and the other of 13 nucleotides. The nine-nucleotide repeat, five copies of which are dispersed throughout
oriC, is the binding site for a protein called DnaA. The result of DnaA binding is that the double helix opens up
(‘melts’) within the tandem array of three AT-rich, 13-nucleotide repeats located at one end of the oriC sequence.
9 mers ATP+DnaA
Ori C
Open
Initial complex
complex
13 mers
Supercoiled
template
Figure 6.74 Initiation at oriC occurs after DnaA protein binds the five 9 mers. The 13 mer region is then
denatured, and this open complex serves as a replication start site. Adapted and redrawn from D. Bramhill
and A. Kornberg. Cell, 1988,54; 915-918.
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Genetics
6.13.6 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 6.86 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 is separate origins for L
and H strand.
6.14 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:
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6.22 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 6.155 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
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Genetics
Problem
If poly-G is used as a messenger RNA in an incorporation experiment, glycine is incorporated into a polypeptide. If
poly-C is used, proline is incorporated. If both poly-G and poly-C are used, no amino acids are incorporated into
protein. Why?
Solution
We are mixing two RNA strands that are complementary; these strands will form a double-stranded RNA molecule.
Since we observed the incorporation of no amino acids, the ribosome must not be able to read a double-stranded
molecule.
Wobble hypothesis
It was first proposed that a specific tRNA anticodon would exist for every codon. If that were the case, at least 61
different tRNAs, possibly with an additional 3 for the chain-terminating codons, would be present. In 1966, Francis
Crick devised the wobble concept to explain these observations. It states that the base at the 5’ end of the
anticodon also shows non-standard base pairing with any of several bases located at the 3’ end of a codon. So, first
base of anticodon and third base of codon is the wobble position. For example, U at the wobble position can pair with
either adenine or guanine, while I can pair with U, C or A. However, the wobble rules do not permit any single tRNA
molecule to recognize four different codons. Three codons can be recognized only when inosine occupies the first
(5’) position of the anticodon.
tRNA anticodon loop
3’ 5’
A G G
mRNA 5’ 3’
U C C
or
U
tRNA anticodon loop
3’ 5’
GU U
mRNA 5’ 3’
C A A
or
G
tRNA anticodon loop
3’ A G I 5’
mRNA 5’ 3’
U C A
or
C
or
U
Figure 6.158 Wobble pairing between the anticodon on the tRNA and the codon in the mRNA.
Table 6.34 Pairing combinations with the Wobble concept

Base in anticodon Base in codon
A U
G U or C
C G
U A or G
I A, U, or C
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Genetics
6.24 Protein synthesis

The fundamental process of protein synthesis is the formation of a peptide bond between the carboxyl group of one
amino acid or at the end of a growing polypeptide chain and a free amino group on an amino acid. Proteins are
made up from a set of 20 amino acids called standard amino acids. Each of the 20 amino acids is specified by specific
codon/s. One additional amino acid - selenocysteine present in some polypeptide is directed by a modified reading
of the genetic code (5’UGA3’). Polypeptide synthesis proceeds from N-terminus to C-terminus and ribosome read
mRNA in the 5’ to 3’ direction. Three kinds of RNA molecules perform different but cooperative functions in protein
synthesis:
mRNA
mRNA carries the genetic information copied from DNA in the form of a series of three-base code words (codons),
each of which specifies a particular amino acid.
Comparison of the structures of prokaryotic and eukaryotic mRNA

Eukaryotic mRNAs are mostly monocistronic; having an average size of 1500 to 2000 nucleotides. It has a 5' cap,
which is recognized by the small ribosomal subunit. Protein synthesis, therefore, begins at an initiation codon near
the 5' end of the mRNA. Upstream of the initiation codon contains a non-translatable sequences called 5' UTR
(5’-untranslated region) or leader sequence. Similarly non translatable sequences at the 3’ end after stop codon is
termed as 3’ UTR (3’-untranslated region) or trailer sequence, which varies in length and sequence.
In prokaryotes, most of the mRNAs are polycistronic. In contrast to eukaryotic mRNAs, the 5' end has no cap-like
structure, and there are multiple ribosome-binding sites (called Shine-Dalgarno sequences) within the polycistronic
mRNA chain, each resulting in the synthesis of a different protein. Just like prokaryotic mRNA, eukaryotic mRNA
also contains 5’ UTR and 3’ UTR.
Prokaryotic mRNA
5' UTR Intercistronic region 3' UTR

{
5' P P P AUG
Initiation
codon
UAA
Stop
codon
AUG
Initiation
codon
{
UAA
Stop
codon
3'
Protein A Protein B
Eukaryotic mRNA
5' UTR 3' UTR Poly-A tail

{
{
{
5' G P P P AUG UAA AAAAAAn 3'
5' cap Initiation Stop
codon codon
Protein A
Figure 6.159 All mRNAs (monocistronic and polycistronic) contain two types of region – the coding region
(which starts with initiation codon and ends with a stop codon) and untranslated region (5'- and 3'-UTR). A
polycistronic mRNA also contains intercistronic regions. They vary greatly in size: they may be as long as 30
nucleotides.
An mRNA can be translated in three different reading frames, depending on where the decoding process begins.
However, only one of the three possible reading frames in an mRNA encodes the required protein. Any sequence of
bases (in DNA or RNA) that could, at least theoretically, encode a polypeptide, is known as an open reading frame,
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Genetics
Ubiquitination or ubiquitylation of substrates

The most well-established means of targeting proteins to proteasomes is by their modification with chains of
ubiquitin. Ubiquitin, a highly conserved eukaryotic protein of 76 amino acid residues, is usually attached to
substrates by an isopeptide bond between a substrate’s lysine residue and the C-terminal glycine of ubiquitin. The
process of ubiquitylation occurs in following steps:
Activation of ubiquitin: Ubiquitin is activated by an E1; ubiquitin-activating enzyme. E1 becomes covalently linked to
free ubiquitin through the free C-terminal residue of ubiquitin, in an energy-dependent manner.
Transfer of ubiquitin from E1 to E2: The activated ubiquitin is subsequently transferred to a cysteine residue
present on an E2; ubiquitin-conjugating enzyme.
Ligation of ubiquitin to target protein: Finally, E3; ubiquitin ligases (~500 in humans) transfer the activated ubiquitin
from E2 to a Lys amino acid residue of its target protein, forming an isopeptide bond. A ubiquitinated protein is
proteolytically degraded to short peptides in an ATP-dependent process mediated by proteasome.
E1 —SH
E2 —SH
AMP
Target
ATP
protein
O O
Target
O C—Ubiquitin S—C—Ubiquitin S—C—Ubiquitin protein
Ubiquitin
E1 —SH E3
OH E1 E2
+
E2—SH
Figure 6.176 The reactions involved in the attachment of ubiquitin to a protein. In the first part of the process,
ubiquitin's terminal carboxyl group is joined, via a thioester linkage, to E1 in a reaction driven by ATP hydrolysis.
The activated ubiquitin is subsequently transferred to a sulfhydryl group of E2 and then in a reaction catalyzed
by E3, to the amino group of a lysine residue on a target protein.
6.24.7 N-end rule

The half-lives of proteins in a living cell range from a few seconds to many days. The metabolic stability of proteins
is guided by the presence of degradation signals (or degrons). The essential component of one degradation signal
is a destabilizing N-terminal residue of a protein. This signal is called the N-degron. A set of N-degrons containing
different destabilizing residues yields a rule, termed the N-end rule which relates the in vivo half-life of a protein
based on its N-terminal residue and its post-translational modification. The N-end rule operates in all organisms
examined, including the bacterium E. coli, the yeast S. cerevisiae and mammals. In eukaryotes, the N-degron
comprises at least two determinants: a destabilizing N-terminal residue and an internal lysine (or lysines). The Lys
residue is the site of ubiquitin ligation.
N-end rule pathway

Proteins with N-degron are degraded via the N-end rule pathway. In eukaryotes, the N-end rule pathway is a part
of the ubiquitin system. This pathway is present in both the cytosol and the nucleus. In this pathway, proteins
bearing a destabilizing amino acid residue at their N-terminus are degraded by the ubiquitin proteasome system (in
bacteria by the ATP-dependent protease ClpAP, a functional counterpart of the eukaryotic 26S proteasome).
In bacteria, destabilizing residues are divided into two groups- primary and secondary destabilizing residues. In
contrast to bacteria, destabilizing residues in eukaryotes can be classified into three hierarchical levels - primary,
secondary and tertiary. The primary destabilizing residues fall into two categories – type 1 (basic N-terminal
residues Arg, Lys and His) and type 2 (bulky hydrophobic N-terminal residues Ile, Leu, Phe, Tyr and Trp). In
general, tertiary destabilizing residues (Asn, Gln and Cys) are first modified to generate a secondary destabilizing
residue (Asp, Glu and oxidized Cys). Finally, the modified N-terminal amino acid is arginylated to create a substrate
bearing a type 1 primary destabilizing residue (Arg). All primary destabilizing residues are recognized by an N-recognins
(also called E3).
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Genetics
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.
6.25.3 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.
E. coli : Wild type
Normal receptor
Lysis
T1
Mutant type
Mutant receptor
T1 cannot bind
Figure 6.184 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.
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Genetics
6.25.5 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, auxotropic 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.
6.25.6 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.
If the two mutations affect the same gene and are alleles of one another. Complementation does not occur. Because
the two mutations affect the same gene, the F1 is homozygous for the two mutant alleles. No normal product of the
gene is produced.
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Chapter 07
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.
7.1 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.
762
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.
7.2 Enzymes for DNA manipulation

The enzymes used in the recombinant DNA technology fall into four broad categories:
7.2.1 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.
7.2.2 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. Nuclease 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.
764
7.7 Recombinant screening

A selective medium enables transformants to be distinguished from non-transformants. The next problem is to
determine which of the transformed colonies comprise cells that contain recombinant DNA molecules, and which
contain self-ligated vector molecules. With most cloning vectors insertion of a DNA fragment into the plasmid destroys
the integrity of one of the genes present on the molecule. Recombinants can, therefore, be identified because the
characteristic coded by the inactivated gene is no longer displayed by the host cells (called insertional inactivation).
Most commonly recombinant selection is carried out by insertional inactivation of antibiotic resistance gene. In
this case the insertion of new DNA fragments (insert) occurs at the site within the gene that confers resistance
towards a particular antibiotic.
R
R amp
amp New DNA inserted
BamHI in BamHI site
R
tet
pBR322 pBR322
Origin of
replication Origin of
replication
R R S
Normal vector (amp tetR) Recombinant (amp tet )
Insertional inactivation does not always involve antibiotic resistance genes. For example in pUC8, gene LacZ’,
which codes for part of enzyme β-galactosidase is used for insertional inactivation. Recombinant pUC8 involves
insertional inactivation of the lac Z’ gene, can be identified because of their inability to synthesize β-galactosidase.
β-galactosidase, coded by lacZ gene, causes the breakdown of lactose to glucose plus galactose. lacZ’, a modified
lacZ gene, codes for the α peptide portion of β-galactosidase.
7.8 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. Such vectors are equipped with transcriptional regulatory sequences
and sequences that control the RNA processing and protein synthesis.
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. A ribosome binding site upstream from the start codon is also
present in many of the expression vectors. This site is required for the efficient initiation of translation in bacteria.
Promoter is the most critical component of an expression vector since it controls the very first stage of gene
expression and 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.
789
Automated sequencing
The standard chain termination sequencing methodology employs radioactive labels, and the banding pattern in the
polyacrylamide gel is visualized by autoradiography. Fluorescent primers are the basis of automated sequencing.
The fluorolabel is attached to the ddNTPs, with a different fluorolabel used for each one. Chains terminated with A
are therefore labeled with one fluorophore, chains terminated with C are labeled with a second fluorophore, and so
on. Now it is possible to carry out the four sequencing reactions - for A, C, G and T - in a single tube and to load all
four families of molecules into just one lane of the polyacrylamide gel, because the fluorescent detector can
discriminate between the different labels and hence determine if each band represents an A, C, G or T. The
sequence can be read directly as the bands pass in front of the detector and either printed out in a form readable
by eye or sent straight to a computer for storage.
Genome sequencing
The first genome to be completely sequenced was the genome of bacteriophage φX174. Although sequencing can
be performed directly on genomic DNA, this is generally impractical on a large scale. Hence genomes have to be
split into fragments of a suitable size such that they can be maintained within a vector. Genomic DNA fragments are
therefore cloned into a vector and each fragment is subsequently sequenced. The problem then is how to reconstruct
the original genome sequence based on the small fragments that are cloned into individual vectors. Two different
approaches have been developed for sequence assembly.
• The clone contig approach : The simplest way to generate overlapping DNA sequence is to isolate and sequence
one clone, from a library, then identify (by hybridization) a second clone, whose insert overlaps with the first.
The second clone is then sequenced and the information used to identify a third clone, whose insert overlaps
with the second clone, and so on. This is used to build up large continuous DNA sequences (contigs) from small
fragments cloned into vectors. This method is, however, laborious. A single clone has been isolated and sequenced
before the next overlapping clone can be sought. Additionally, repetitive sequences within the genome can give
rise to incorrect contig assignment.
• The shotgun approach : The fragments of the genome, which have been randomly generated, are cloned into
a vector and each insert is sequenced. The sequence is then examined for overlaps (sequences that occur in
more than one clone) and the genome is reconstructed by assembling the overlapping sequences together.
This approach was first used to sequence the genome of the bacterium Haemophilus influenzae. The main
advantage of the shotgun approach is that no prior knowledge of the sequence of the genome is required. The
approach is, however, limited by the ability to identify overlapping sequences. Every sequence obtained must
be compared with every other sequence in order to identify the overlaps.
Table 7.6 Genome sequencing of some model organisms

Genome sequenced Year Genome size Comment
Bacteriophage φX174 1977 5.38 kb First genome sequenced
Plasmid pBR322 1979 4.3 kb First plasmid sequenced
Bacteriophage λ 1982 48.5 kb
Yeast chromosome III 1992 315 kb First chromosome sequenced
Haemophilus influenzae 1995 1.8 Mb First genome of a cellular organism to be sequenced
Saccharomyces cerevisiae 1996 12 Mb First eukaryotic genome to be sequenced
Ceanorhabditis elegans 1998 97 Mb First genome of multicellular organism to be sequenced
Homo sapiens 2000 3000 Mb First mammalian genome to be sequenced
Arabidopsis thaliana 2000 125 Mb First plant genome to be sequenced
802
7.12.1 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.
There are three major types of genetic markers:
1. Morphological (also classical or visible) markers which are based on phenotypic traits or characters;
2. Biochemical markers, which are based on gene products; and
3. DNA (or molecular) markers, which reveal sites of variation in DNA.
Morphological markers are usually visually characterized phenotypic characters such as flower colour, seed shape,
growth habits or pigmentation. Biochemical markers are differences in gene products that are detected by
electrophoresis and specific staining. The major disadvantages of morphological and biochemical markers are that
they may be limited in number and are influenced by environmental factors or the developmental stages. A
molecular or DNA marker is defined as a particular segment of DNA that is representative of the differences at the
genome level. Molecular 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 molecular marker should have the following criteria:
1. be polymorphic and evenly distributed throughout the genome,
2. provide adequate resolution of genetic differences,
3. have linkage to distinct phenotypes.
7.12.2 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 tech-
niques (such as SSLP, SNP).
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 7.23 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.
804
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 7.33 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.
7.16 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 immunodeficiency
817
Secondly, the triggering of sense strand (mRNA) cleavage by incorporating ribozyme catalytic centres into antisense
RNA. A number of ribozymes have been characterized, including the most studied form called the hammerhead
ribozyme (first isolated from viroid RNA).
Thirdly, RNA interference induced by small interfering RNA molecules. This naturally occurring phenomenon, a
potent sequence specific mechanism for post-transcriptional gene silencing, was first described for the nematode
worm Caenorhabditis elegans.
DNA
mRNA
Antisense
siRNA
oligonucleotide
Ribozyme
RISC
RNase H
No protein No protein No protein

synthesis synthesis synthesis
Figure 7.40 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.
7.17.3 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.
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Table 7.10 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
7.18 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 is referred to as plant tissue
culture.
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.
7.18.1 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
cells’ functions. Isolated cells from differentiated tissues are generally non-dividing and quiescent; to express
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 dedifferentiation, 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 this 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 express totipotency. This phenom-
enon of totipotency is an amazing developmental plasticity that sets plant cells apart from most of their animal
counterparts. In animals the differentiation is irreversible.
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7.19 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.
7.19.1 Primary and secondary 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.
Lymphocytes are anchorage independent cells commonly grown in culture.
A secondary culture is prepared by subculturing a primary culture. Subculture (or passage) refers to the transfer of
cells from one culture vessel to another. In most cases, cells in primary cultures can be removed from the culture
dish and made to proliferate to form a large number of secondary cultures.
7.19.2 Cell line

When a primary cell culture is subcultured, it becomes a cell line. 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 passages. Normal
mammalian cells generally have a finite life span in culture; that is, after a number of divisions characteristic of the
species and cell type, the cells stop dividing. These cell lines exhibit the property of contact inhibition, density
limitation and anchorage dependence.
A cell line that has the potential to be subcultured indefinitely is termed infinite (immortal or continuous) cell line.
Tumor cells or normal cells that have undergone transformation induced by chemical carcinogens or viruses can be
propagated indefinitely in tissue culture; thus, have unlimited number of possible subcultures.
Infinite cell lines are also known as transformed cell lines due to altered growth properties of immortalized cells.
Transformed cells do not necessarily mean cancer or tumor cells. Transformed cell lines do not exhibit the property
of contact inhibition, density-dependent inhibition of proliferation and anchorage dependence. They have a reduced
requirement for serum or growth factors for optimal growth. A transformed cell line often has an abnormal chromosome
number (aneuploid) and overproduces different proteins. Cancer cells are naturally immortal. Thus all cancerous
cell lines are transformed, although it is not clear whether all transformed cell lines are cancerous.
The first cell line—the mouse fibroblast L cell—was derived from cultured mouse subcutaneous connective tissue
by exposing the cultured cells to a chemical carcinogen. Another important cell line, the HeLa cell, was derived
from a 31-year-old black woman named Henrietta Lacks, who died of cervical cancer in 1951. Since these early
cell lines, hundreds of cell lines have been established.
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defined quantities of purified growth factors, lipoproteins and other proteins usually provided by the serum or
extract supplement. Since the components (and concentrations thereof) in such culture media are precisely known,
these media are generally referred to as defined culture media and often as serum-free media (SFM). A number of
SFM formulations are commercially available, such as those designed to support the culture of endothelial cells,
monocytes/macrophages, fibroblasts, neurons, lymphocytes, chondrocytes or hepatocytes.
Some extremely simple SFM, which consist essentially of vitamins, amino acids, organic and inorganic salts and
buffers have been used for cell culture. Such media (often called basal media), however, are usually, seriously
deficient in the nutritional content required by most animal cells. Accordingly, most SFM incorporate additional
components into the basal media to make the media more nutritionally complex, while maintaining the serum-free
and low protein content of the media. Examples of such components include serum albumin from bovine (BSA) or
human (HSA), animal-derived lipids such as human excyte, sterols, etc., and certain growth factors or hormones
derived from natural (animal) or recombinant sources.
7.19.4 Growth pattern

Animal cells growth in culture have a characteristic growth pattern similar to bacteria. The cell growth is typically
divided into three phases: Lag phase, Log phase and Plateau phase.
Lag phase
The lag phase is a period of zero growth when cells are first inoculated into the growth medium. The length of this
phase depends on the type of cells and their metabolic state at inoculation. It is a period of adaptation during which
the cell replaces elements of the glycocalyx lost during trypsinization, attaches to the substrate and spreads out.
Log phase
The exponential growth phase is a period of continuous cell doubling. Animal cells normally exhibit a doubling time
of between 15 and 25 hours. The length of the log phase depends on the seeding density, the growth rate of the
cells and the density at which cell proliferation is inhibited by density.
Plateau (or stationary) phase

The stationary phase is a period after growth when there is no change in the culture cell density. The phase occurs
when the nutrients have been depleted or inhibitory metabolites have accumulated in the culture. All the available
growth surface is occupied and all the cells are in contact with surrounding cells. Further growth of cells can be
obtained by subculturing the cells in fresh medium.
7.19.5 Application of animal cell culture

Cell culture has become one of the major tools used in cell and molecular biology. Some of the important areas
where cell culture is currently playing a major role are briefly described below:
Model systems
Cell cultures provide a good model system for studying 1) basic cell biology and biochemistry, 2) the interactions
between disease-causing agents and cells, 3) the effects of drugs on cells, 4) the process and triggers for ageing
and 5) nutritional studies.
Toxicity testing
Cultured cells are widely used alone or in conjunction with animal tests to study the effects of new drugs, cosmetics
and chemicals on survival and growth in a wide variety of cell types. Especially important are liver- and kidney-
derived cell cultures.
Cancer research
Since both normal cells and cancer cells can be grown in culture, the basic differences between them can be closely
studied. In addition, it is possible, by the use of chemicals, viruses and radiation, to convert normal cultured cells
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Chapter 08
Bioprocess engineering is a specialization of chemical engineering that deals with the design and development of
equipment and processes for the manufacturing of products such as food, pharmaceuticals and polymers from
biological materials. It uses the capabilities of organisms in industrial, medical, environmental or agricultural processes
in order to produce useful biological materials.
Application of bioprocess engineering includes:

• Design and operation of fermentation systems,
• Development of food processing systems,
• Application and testing of product separation technologies,
• Design of instrumentation to monitor and
• Control biological processes and many more.
Bioprocess engineers work at the frontiers of biological and engineering sciences to Bring engineering to Life
through the conversion of biological materials into other forms needed by mankind. One of the main tasks of a
bioprocess engineer is control and maintenance of a biological processes such as the production of beverages,
pharmaceuticals, antibiotics, enzymes, biochemicals, enzyme-catalyzed reactions, food processing and biological
waste treatment. These processes require a well-designed growth environment to obtain the maximum yield of the
product and consequently these conditions need to be carefully controlled. Environmental design comprises the
determination of the environment of the process, while fermentation engineering provides the means for meeting
those requirements.
8.1 Concept of material and energy balance

8.1.1 Material balance
Material balances (mass balances) are based on the law of conservation of mass. The law of ’conservation of mass’
states that mass cannot be created or destroyed. In performing the material balance we apply thermodynamic
terms – system and process. A system is defined as that part of the universe that is under consideration. All space
outside the system is known as the surroundings. A system is separated from the surrounding by a system
boundary, which may be real or imaginary. If the boundary doesn’t allow mass to pass from system to surroundings
and vice versa, the system is considered as a closed system with constant mass. If the system boundary allows the
mass to pass from system to surroundings and vice versa, then it is an open system. A process causes changes in
the system or surroundings. In bioprocess, the process can be batch, fed batch and continuous processes. A batch
process operates in a closed system. All materials are added to the system at the start of the process, the system is
then closed and products removed only when the process is complete. A fed-batch process allows input of material to
the system but not output and a continuous process allows matter to flow in and out of the system.
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A cell or a bioreactor is defined as a system, and mass balance is performed on the system. Doing a mass balance
is similar in principle to accounting. In accounting, accountants do balances of what happens to a company’s
money. In the process of mass balance, the first step is to look at the three basic categories: mass in, mass out
and mass stored. The mass can be total mass, the mass of a particular molecular or atomic species, or biomass.
Total mass balance for the system can be written in a general way:
Mass in (input) = Mass stored (accumulation) + Mass out (output)
System
Products out
Raw materials in Stored materials
Waste products
Mass in Mass stored Mass out
Figure 8.1 Mass balance.
Bioprocess engineers do a mass balance to account for what happens to each of the chemicals that is used in a
chemical process. For example, in a plant that is producing sugar, if the total quantity of sugar going into the plant
is not equalled by the total of the purified sugar and the sugar in the waste liquors, then there is something wrong.
Sugar is either being burned (chemically changed) or accumulating in the plant or else it is going unnoticed down
the drain somewhere. In this case the mass balance is;
Raw materials = Products + Waste products + Stored products + Losses
Mass balances can be based on total mass, mass of dry solids or mass of particular components, for example
protein. If a mass balance is written using the total mass in each process stream, then it is called total balance. A
separate mass balance can be written for a particular chemical component in the total mass. This is called component
balance. Thus, for a component mass balance the simplest expression is:
Input – Output + Formation – Disappearance = Accumulation
Problem
In a filtration device, the input concentration of the cell is 5g/litre which is pumped in at 100 litre/hr. The desired
output concentration is 50 g/litre. The system runs continuously so there is no accumulation. Calculate the rate of
removal of the permeate supernatant.
Solution
In this case, the system runs continuously and there is no reaction, hence
Input = Output
Input cells = 5 gram/litre × 100 litre/hr = 500 gram/hr
If ‘Y’ litres is the output of concentrated cells then,
Output cells = 50 gram/litre × Y litre/hr = 50 Y gram/hr
So, 500 = 50 Y
Y = 10 litre/hr
Hence, supernatant volume = 100 – 10 = 90 litre. Thus, supernatant has to be removed at a rate of 90 litre/hr.
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Concept of degree of reduction

The degree of reduction, γ, is defined as the number of equivalents of available electrons in the quantity of material
containing 1 g atom (1 mole) carbon. Therefore, for substrate CwHxOyNz, the number of available electrons is
(4w + x – 2y – 3z). The degree of reduction for the substrate, γs, is therefore (4w + x – 2y – 3z)/w.
The degree of reduction relative to CO2, H2O and NH3 is zero.
Yield coefficient
The yield coefficient is the ratio of the amount produced to the amount consumed for any product/reactant pair. It is
a ratio having no unit. A yield coefficient is often used to describe the conversion efficiency. Virtually any pair of output
and input can be combined to give a yield coefficient. For example, the yield coefficient of biomass, YX/S, is the
biomass of cells formed per unit of substrate consumed for biosynthesis. Yield coefficient can be related to ATP
consumption. The ATP yield coefficient, YX/ATP, represents the amount of biomass synthesized per mole of ATP consumed.
Cell biomass and product formation can be described quantitatively by yield coefficients. Let's consider the overall
stoichiometric equation for growth and production:
sS + nN + oO2 ⎯→ X + pP + wH2O + eCO2
where S, carbon source; N, nitrogen source; X, biomass; P, product and s, n, o, p, w, e are stoichiometric coeffi-
cients. The theoretical yield coefficients can be determined from the above stoichiometry with a known chemical
formula for S, N, X and P. The cell biomass yield coefficient and the product yield coefficient are
YX/S = MX / sMS and YP/S = pMP / sMS
respectively, where Mx, Mp and Ms are the molecular weights of cell biomass, product and carbon source.
8.1.2 Energy balance

Energy balances are used to quantify the energy used or produced by a system. In bioprocessing, energy accounting
system can be set up to determine the amount of steam or cooling water required to maintain optimum process
temperatures. The principle underlying all energy-balance calculations is the law of conservation of energy, which
states that the energy can be neither created nor destroyed. Although this law does not apply to nuclear reaction,
conservation of energy remains a valid principle for bioprocesses.
The law of conservation of energy can be represented as:

(Energy in through system boundaries) – (Energy out through system boundaries) = (Energy accumulated within
the system).
Energy in products
System
Energy in Stored energy Energy in waste
Energy in through Energy accumulated Energy losses

system boundaries within the system to surrounding
Energy out through

system boundaries
Figure 8.2 Energy balance.
Energy takes many forms, such as heat, kinetic energy, chemical energy and potential energy but because of
interconversions it is not always easy to isolate separate constituents of energy balances. However, under some
circumstances certain aspects predominate. In many heat balances in which other forms of energy are insignificant;
in some chemical situations mechanical energy is insignificant.
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Phase change
When a substance changes from one phase of matter to another, we say that it has undergone a change of phase.
These changes of phase always occur with a change of heat. Heat either comes into the material during a change
of phase or heat comes out of the material during this change. However, the heat content of the material changes,
the temperature does not. The amount of energy released or absorbed during a phase change is called latent heat.
The latent heat for a different mass of the substance can be calculated using the equation:
Q = ML
where, Q = is the amount of energy released or absorbed during the change of phase of the substance
M = is the mass of the substance and

L = is the specific latent heat per gram for a particular substance ; substituted as Lf to represent the
specific latent heat of fusion, Lv as the specific latent heat of vaporization.
Enthalpy change due to mixing
When compounds are mixed or dissolved, the bonds between molecules in the solvent and solute are broken and
reformed so a net absorption or release of energy takes place due to which internal energy and enthaply of mixture
change. The enthaply change during mixing of non-ideal two compounds A and B is given by
ΔHmixing = Hmixture – (HA + HB)
For an ideal solution or ideal mixture, ΔHmixing = 0
Enthalpy change due to reaction
Bioprocessing involves enzyme catalyzed reactions. During the reaction, relatively large changes in internal energy
and enthalpy occur. Enthalpy of a reaction is the amount of heat released or absorbed during the reaction and equal
to the difference in enthalpy of reactants and products. In case of an exothermic reaction, the enthalpy of a
reaction is negative. On the other hand, enthalpy of a reaction is positive for an endothermic reaction.
ΔHreaction = Hproduct – Hreactant
8.2 Microbial growth kinetics

Microbial growth is a result of both cell division and change in cell size. Microorganisms can grow under a variety of
physical, chemical and nutritional conditions. In a suitable nutrient medium, organisms extract nutrients from the
medium and convert them into biological compounds. Part of these nutrients are used for energy production and
part are used for biosynthesis and product formation. Thus microbial growth is an orderly increase in the quantity
of cellular constituents (i.e. cell mass) and number. It depends on the ability of the cell to form new protoplasm from
nutrients available in the environment. Microbial growth is a good example of an autocatalytic reaction.
Microbial batch growth
When a microbe (such as bacterial cell) is inoculated into a flask containing fresh culture medium and incubated, it
enters into a rapid growth phase during which the microbe divides and increases its population in the flask medium.
Since the microbes are not transferred to a new medium or no fresh nutrients are added to the medium, the
increasing population of microbial cells, after sometime, enters into a stationary-phase with the exhaustion of the
required nutrients and the accumulation of inhibitory end products in the medium. Eventually, the stationary phase
of microbial population culminates into death-phase when the viable microbial cells begin to die. A batch culture can
be considered to be a closed system.
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Consequently, the residual substrate concentration in the reactor is controlled by the dilution. Any alteration to this
dilution rate results in a change in the growth rate of the cells that will be dependent on substrate availability at the
new dilution rate. Thus, growth is controlled by the availability of a rate-limiting nutrient. This system, where the
concentration of the rate-limiting nutrient entering the system is fixed, is often described as a chemostat as
opposed to operation as a turbidostat, where nutrients in the medium are not limiting. In turbidostat, turbidity of
the culture is monitored and maintained at a constant value by regulating the dilution rate, i.e. cell concentration is
held constant.
The concentration of biomass or microbial metabolites in a continuous fermenter under steady-state conditions can
be related to the yield coefficient as described in the batch fermentation section. Inserting the equation for residual
substrate into the biomass or a metabolic product yield coefficient equation gives, in this case, for steady-state
biomass (x),
 DKs 
x  YX / S  SR  
 µmax  D 
where SR is the substrate concentration of inflowing medium or
x  YX / S (SR  Sr )
Therefore, the biomass concentration under steady-state conditions is controlled by the substrate concentration of
inflowing medium and the operating dilution rate. Under non-inhibitory conditions, where there is no substrate or
product inhibition, the higher the feed concentration the greater the biomass concentration and residual substrate
concentration remains constant. However, the higher the dilution rate, the faster the cells grow, which results in a
simultaneous increase in the residual substrate concentration and a consequent reduction in the steady-state
biomass concentration. As D approaches µmax, the biomass concentration becomes even lower, yet the cells grow
faster and there is a concurrent increase in the residual substrate concentration.
8.3 Fermentation
Fermentation (derived from the Latin verb fervere, to boil) is the production of carbon dioxide by the anaerobic
catabolism of the sugars. The term fermentation has been used in a strict biochemical sense which mean an
energy-generation process in which organic compounds act as both electron donors and terminal electron acceptors.
However, industrial microbiologists have extended the term fermentation to describe any process for the production
of the product by the mass culture of a microorganism.
8.3.1 Fermentation processes

On the commercial scale, there are five major groups of fermentation processes:
1. Produce microbial cells (or biomass) as a product.
Bakers’ yeast, used in the baking industry, is an example of a produced cell mass. Others include single-cell
proteins for food sources.
2. Produce microbial enzymes.
3. Produce microbial metabolites.
4. Produce recombinant products.
5. Processes that modify a compound that is added to the fermentation process are referred to as biotransformations.
Biotransformations occur using the inherent enzymatic capability of most cells. Cells of all types can be employed
to biocatalyze a transformation of certain compounds via dehydration, oxidation, hydroxylation, amination or
isomerization.
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For increased documentation and reproducibility in the fermentation industry, there is a trend towards application of
defined media with a carbon and energy source (glucose, sucrose or starch), an inorganic nitrogen source (ammonia
or urea), a mixture of minerals and perhaps a few vitamins.
8.4 Bioreactor
A bioreactor (in biochemical engineering, we also use terms like biochemical reactor, biological reactor, fermenter
or microbial reactor, which are all synonymous) is a vessel in which the growth and metabolism of cells take place.
What makes the bioreactors different from the chemical reactors is the presence of the living organisms. Bioreactors
are commonly cylindrical, ranging in size from some liter to cubic meters and are often made of stainless steel. The
process in bioreactor can either be aerobic or anaerobic. The term bioreactor is often used synonymously with
fermenter; however, in the strict sense, a fermenter is a system in which anaerobic process is carried out.
Bioreactor design
Bioreactor design is a relatively complex engineering task. The goal of an effective bioreactor is to control, contain
and positively influence the biological reaction. Suitable bioreactor design criteria include:
• Microbiological and biochemical characteristics of the cell systems (microbial, mammalian, plant cell).
• Hydrodynamic characteristics of the bioreactor.
• Mass and heat characteristics of the bioreactor.
• Kinetics of cell growth and product formation.
• Genetic stability characteristics of the cell system.
• Sterilization and maintenance of sterility.
• Agitation (for mixing of cells and medium) and aeration (aerobic fermenters; for O2 supply).
• Process monitoring and control (regulation of factors like temperature, pH, pressure, aeration, nutrient).
• Implication of bioreactor design on downstream product separation.
• Capital and operating costs of the bioreactor.
• Potential for bioreactor scale-up.
In addition to controlling these, the bioreactor must be designed to both promote formation of the optimal morphology
of the organism and eliminate or reduce contaminations by unwanted organisms or mutation of the organisms.
There are a wide variety of bioreaction systems, and any attempt to categorize them by their various attributes will
naturally result in some overlap of system characteristics.
8.4.1 Agitation and aeration

Agitation
Mixing is one of the most important operations in bioprocessing. Within a fermenter, there is a need to mix three
different phases:
• Liquid phase, which contains dissolved nutrients and metabolites.
• Gaseous phase, which is predominantly oxygen and CO2.
• Solid phase, which is made up of the cells and any solid substance that may be present.
Purpose of mixing
• Air bubble dispersion;
• Mass transfer from air bubbles (i.e. oxygen supply) to the liquid and then to the cells;
• Supply of the nutrient components to cells (more precisely, cell agglomerates);
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During bioprocessing, it may reduce the overall rate of synthesis of plasmid-encoded products in bioreactor. Plasmid
instability occurs as a result of DNA mutation or defective plasmid segregation. For segregational stability, the total
number of plasmids present in the culture must double once per generation, and the plasmid copies must be
equally distributed between mother and daughter cells.
A simple model has been developed for batch culture to describe changes in the fraction of plasmid-bearing cells as
a function of time. The important parameters in this model are the probability of plasmid loss per generation of
cells, and the difference in the growth rates of plasmid-bearing and plasmid-free cells. If x+ is the concentration of
plasmid-carrying cells and x– is the concentration of plasmid-free cells, the rates at which the two cell populations
grow are:
rX+ = (1 – p)μ+ x+ and
r X - = p μ+ x + + μ– x –
where, rX+ is the rate of growth of the plasmid-bearing population,

rX- is the rate of growth of the plasmid-free population,
p is the probability of plasmid loss per cell division (p ≤ 1),
+
μ is the specific growth rate of plasmid-carrying cells, and
–
μ is the specific growth rate of plasmid free cells.
This model is based on the following assumptions:

1. Exponential growth of the host cells
2. All plasmid-containing cells are identical in growth rate and probability of plasmid loss:
3. All plasmid-containing cells have the same copy number.
8.8 Mass and Heat transfer

8.8.1 Mass transfer
Mass is transferred from one location to another under the influence of a concentration gradient in the system. Mass
transfer takes place by two basic processes: diffusion and convection. In bioreactions, the transport of nutrients to
the cell surface and the removal of metabolites from the cell surface to the bulk of the medium are rate processes
with time constants. The driving forces for mass transfer are concentration, temperature or pressure gradients.
An example of mass transfer is the supply of oxygen in fermenters for aerobic culture. The concentration of oxygen
at the surface of air bubbles is high compared with the rest of the fluid; this concentration gradient promotes
oxygen transfer from the bubbles into the medium.
Diffusion
Diffusion is the movement of component molecules in a mixture under the influence of a concentration difference
in the system. In single-phase systems, the rate of mass transfer due to molecular diffusion is given by Fick’s law
of diffusion, which states that mass flux is proportional to the concentration gradient.
dCA
JA = −DAB
dx
where, JA = the mass flux of component A,

CA = the concentration of component A,
X = distance,
dCA
= the concentration gradient, or change in concentration of A with distance,
dx
DAB = the binary diffusion coefficient or diffusivity of component A in a mixture of A and B.
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Due to the typical fragility of the engineered microorganisms, large-scale fermentation vessels must be designed
with the ability to:
• Remove the heat buildup that results from metabolic processes;
• Manage agitation and mixing with minimal shear damage;
• Effectively control the highly variable liquid flow rates and turndowns that are associated with batch fermentation;
• Execute safeguards and sterilization techniques to guard against potential contamination.
8.12 Downstream processing

Bioprocessing treats raw materials and generates useful products. A problem common to all biological processes,
whether based on fermentation or cell culture technology, is the need to recover the product. Fermentation broths
are complex, aqueous mixtures of cells, comprising the soluble extracellular, intracellular products and any
unconverted substrates. The fermentation broth has to be processed and passed through several stages of
separation and purification.
In the case of protein production especially human therapeutic proteins, these products must be recovered in a
highly purified form, with the molecule in its proper 3-D configuration. The need for extremes in purity, retention
of molecular configuration and efficiency in recovery are major challenges. Downstream processing refers to the
recovery and purification of biosynthetic products. The problem of recovery depends very much on the type of
cell and how the bioreactor is designed and operated. The selection of appropriate purification step depends on
the nature of the end product, its concentration, the side product present, the stability of the biological materials
and necessary degree of purification.
Upstream processes
Production fermenter
Stock culture Seed fermenter
Media formulation Media sterilization
Oxygen
pH control
Antifoam
Cooling/heating
Downstream processes
Culture fluid
Cell separation Biomass
Cell free supernatant
Product isolation (concentration)
Product purification
Figure 8.14 An outline of upstream and downstream processing operations.
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Lactic acid Lactobacillus delbrueckii (bacteria)

Propionic acid Propionibacterium (bacteria)
Malic acid Leuconostoc brevis (bacteria)
Penicillins Penicillium chrysogenum (fungi)
Cephalosporins Acremonium chrysogenum (fungi)
Bacitracin Bacillus licheniformis (bacteria)
Gramicidin Bacillus brevis (bacteria)
B 12 (Cyanocobalamin) Pseudomonas denitrificans (bacteria)
β-Carotene (Provitamin A) Blakeslea trispora (fungi)
Ascorbic acid (vitamin C) Acetobacter suboxydans (bacteria)
Alginate Azotobacter vinelandii (bacteria)
Cellulose Acetobacter xylinum (bacteria)
Dextran Leuconostoc mesenteroides (bacteria)
Pullulan Aureobasidium pullulans (fungi)
Xanthan Xanthomonas campestris (bacteria)
8.14 Wastewater treatment

Waste materials generated in a society can be classified into three major categories: industrial wastes, domestic
wastes and agricultural wastes. Each of these waste materials has its own characteristics, and thus treatment
methods vary. Three major waste treatment methods are the following:
1. Physical treatment
Physical treatment includes screening, flocculation, sedimentation and filtration, which are usually used for the
removal of insoluble materials.
2. Chemical treatment
Chemical treatment includes chemical oxidations and chemical precipitation.
3. Biological treatment
Biological treatment includes the aerobic and anaerobic treatment of wastewater by a mixed culture of
microorganisms.
Quantification of biodegradable material in wastewater

The biodegradable materials of a waste-water sample can be expressed in two ways: biological oxygen demand
(BOD) and chemical oxygen demand (COD). The BOD test estimates the amount of oxygen required by aerobic
microorganisms to oxidize biodegradable materials in the wastewater- over a fixed period of time (normally 5
days), at constant temperature (20°C) in the dark. A wastewater sample is saturated with oxygen and seeded with
an inoculum containing a diverse range of microbes. Its oxygen concentration is measured before and after a 5
days incubation period and the results are expressed as milligrams of oxygen per litre of waste.
COD determines the amount of oxygen required to chemically oxidize any oxidizable organic material present in a
waste water. Organic compounds are oxidized by a strong chemical oxidant, and using the reaction stoichiometry,
the organic content is calculated. This test involves the addition of a known volume of sample to a mixture of
oxygen-rich potassium dichromate and concentrated sulphuric acid. Almost all organic compounds present in waste
water are oxidized by strong chemical oxidants. Therefore, the COD content of a waste-water sample usually
exceeds the measured BOD (COD > BOD). The BOD:COD ratios for sewage are normally between 0.2:1 and 0.5:1.
903
Oxidation ponds
Oxidation ponds are large, shallow ponds, typically 1-2 m deep. It acts as a shallow waste-treatment reactor where
raw or partially treated sewage is decomposed by microorganisms. The conditions are similar to eutrophic lake.
The ponds can be designed to maintain aerobic conditions. Oxidation ponds are also used to augment secondary
treatment, in which case they are often called polishing ponds.
Advanced wastewater treatment

Advanced wastewater treatments are designed for the purpose of removing nitrogen and phosphorus. Nitrogen
containing organic compounds are first oxidized biologically to ammonium ions which is further oxidized to nitrite
and nitrate by genera nitrosomonas and nitrobacter, respectively. The second phase is anaerobic denitrification
which releases nitrogen gas. A number of bacteria can act as denitrifiers such as Pseudomonas, Alcaligenes, Arthrobacter.
Phosphorus in wastewater exists in many forms but all of it ends up as orthophosphate. Removing phosphate is
most often accomplished by adding a coagulant, usually alum or lime. Phosphate removal from wastewater by
biological means involves assimilation or storage.
8.15 Bioremediation
Bioremediation is a biological process whereby organic wastes are biologically degraded under controlled conditions.
This process involves the use of living organisms, primarily microorganisms, to degrade the environmental
contaminants. In this process, contaminant compounds are transformed by living organisms through reactions that
take place as a part of their metabolic processes. For bioremediation to be effective, microorganisms must
enzymatically attack the contaminants and convert them to harmless products. Hence, it is effective only where
environmental conditions permit microbial growth and activity. Thus, its application involves the manipulation of
environmental parameters to allow microbial growth and degradation to proceed at a faster rate. The control and
optimization of bioremediation processes is a complex phenomenon. Various factors influencing this process include:
the existence of a microbial population capable of degrading the pollutants; the availability of contaminants to the
microbial population; and the environment factors (type of soil, temperature, pH, the presence of oxygen or other
electron acceptors, and nutrients).
Bioremediation strategies
Bioremediation strategies can be in-situ or ex-situ. In-situ bioremediation involves treating the contaminated material
at the site while ex-situ bioremediation involves the removal of the contaminated material to be treated elsewhere.
In-situ bioremediation techniques are generally the most desirable options due to lower cost and less disturbance
since they provide the treatment at a site avoiding excavation and transport of contaminants. Ex-situ bioremediation
requires transport of the contaminated water or excavation of contaminated soil prior to remediation treatments.
In-situ and ex-situ bioremediation strategies involve different technologies such as bioventing, biosparging, bioreactor,
composting, landfarming, bioaugmentation and biostimulation.
Bioventing is an in-situ bioremediation technology that uses microorganisms to biodegrade organic constituents
adsorbed on soils in the unsaturated zone (extends from the top of the ground surface to the water table). Bioventing
enhances the activity of indigenous bacteria and stimulates the natural in-situ biodegradation of contaminated
materials in soil by inducing air or oxygen flow into the unsaturated zone and, if necessary, by adding nutrients.
Biosparging is also an in-situ bioremediation technology that uses indigenous microorganisms to biodegrade or-
ganic constituents in the saturated zone. In biosparging, air (or oxygen) and nutrients (if needed) are injected into
the saturated zone to increase the biological activity of the indigenous microorganisms.
Biostimulation involves the modification of the environment to stimulate the existing bacteria capable of bioremediation.
This can be done by the addition of various forms of rate limiting nutrients and electron acceptors, such as phosphorus,
nitrogen, oxygen or carbon (e.g. in the form of molasses).
905
Chapter 09
Bioinformatics
9.1 Introduction
Bioinformatics is a discipline at the intersection of biology, computer science, information technology and mathematics.
It aims at integrating and analyzing a wealth of biological data with the aim of identifying and assigning a function
to each. It is applied, for example, in the construction of genetic and physical maps of genomes, gene discovery,
the inference of the molecular function and three-dimensional structure of their products, the interpretation of the
effect of gene variations on the phenotype, the reconstruction of interaction and signal transduction pathways and
the simulation of biological systems.
9.2 Biological databases

Bioinformatics is about exploring biological information. This information is kept safely in databases. A database
consists of an organized collection of persistent data that provides a standardized way for locating, adding, and
changing data. Biological data are available in the form of sequences and structures of proteins and nucleic acids.
The biological information of nucleic acids is available as sequences while the data of proteins is available as
sequences and structures. Sequences are represented in a single dimension whereas the structure contains the
three dimensional data of sequences.
The first database was created after the insulin protein sequence was made available in 1956. Insulin (consists of
51 residues) is the first protein to be sequenced. Later, three dimensional structure of proteins were studied and the
well known Protein Data Bank was developed as the first protein structure database.
Database classification
Biological databases can be classified into sequence and structure databases or primary and secondary databases.
Primary and secondary databases are classified on the basis of source of data.
Primary databases
Databases consisting of data derived experimentally such as nucleotide sequences and three dimensional structures
are known as primary databases. Examples of these include GenBank, EMBL and DDBJ for nucleotide sequences
and the Protein Data Bank (PDB) for 3D-protein structures.
Secondary databases
A secondary database derives from the analysis or treatment of the primary database. A secondary sequence
database contains information like the conserved sequence, signature sequence and active site residues of the
protein families arrived by multiple sequence alignment of a set of related proteins.
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Bioinformatics
• ALN: a database of protein sequence alignments.

• RESID: a database of covalent protein structure modifications.
In 2002 PIR, along with its international partners, EBI (European Bioinformatics Institute) and SIB (Swiss Institute
of Bioinformatics), were awarded a grant from NIH to create UniProt, a single worldwide database of protein
sequence and function, by unifying the PIR-PSD, Swiss-Prot, and TrEMBL databases. The UniProt database has
larger coverage than any one of the three databases while at the same time maintaining the original SWISS-PROT
feature of low redundancy, cross-references and a high quality of annotation.
Protein Data Bank (PDB)

The PDB archive is the single worldwide repository of information about the 3D structures of large biological
molecules, including proteins and nucleic acids. Understanding the shape of a molecule helps to understand how it
works. The PDB was established in 1971 at Brookhaven National Laboratory and originally contained 7 structures.
In 1998, the Research Collaboratory for Structural Bioinformatics (RCSB) became responsible for the management
of the PDB. As of Aug 2012, around 83000 structures are deposited so far in PDB. In 2003, the wwPDB was formed
to maintain a single PDB archive of macromolecular structural data that is freely and publicly available to the global
community. It consists of organizations that act as deposition, data processing and distribution centers for PDB
data.
Structural Classification of Proteins (SCOP)

The SCOP database provides a detailed and comprehensive description of the relationships of known protein
structures. PDB contains many protein entries. These proteins have structural similarities with other proteins and,
in many cases, share a common evolutionary origin. To facilitate access to this information, the Structural
Classification of Proteins (SCOP) database was constructed. The classification of proteins in SCOP has been constructed
by visual inspection and comparison of structures. The unit of classification is usually the protein domain. The
classification of the proteins in SCOP is on hierarchical levels are as follows: Family, Superfamily, Common fold and
Class. There are now a number of other databases which classify protein structures, such as CATH, FSSP, Entrez
and DDBASE, however, the distinction between evolutionary relationships and those that arise from the physics and
chemistry of proteins is a feature that is so far unique to SCOP. Because functional similarity is implied by an
evolutionary relationship but not necessarily by a physical relationship, we believe that this classification level is of
considerable value, for example as a way of reliably linking very distant sequence families.
Class, Architecture, Topology and Homology (CATH)

The CATH classification of protein domain structures was established in 1993 as a hierarchical clustering of protein
domain structures into evolutionary families and structural groupings, depending on sequence and structure similarity.
There are four major levels, corresponding to protein class, architecture, topology or fold and homologous family.
CATH consists of both phylogenetic and phenetic descriptors for protein domain relationships.
Molecular Modeling Database (MMDB)

The three-dimensional structures of biomolecules provide a wealth of information on their biological function and
evolutionary relationships. The MMDB, as part of the Entrez system, facilitates access to structure data by connecting
them with associated literature, protein and nucleic acid sequences, chemicals, biomolecular interactions, and
more. It is possible, for example, to find 3D structures for homologs of a protein of interest by following the ‘Related
Structures’ link in an Entrez Protein sequence record.
Genome databases
Genome sequences form entries in the standard nucleic acid sequence databases. Many species like Arabidopsis
thaliana, C. elegans, Rice etc., have special databases that bring together the genome sequence and its annotation
with other data related to the species.
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Bioinformatics
• Microbial genome database

http://www.ncbi.nlm.nih.gov:80/PMGGifs/Genomes/micr.html
• TIGR: The comprehensive Microbial Resource
http://www.tigr.org/tigr-scripts/CMR2:CMRHomepage.spl
• Arabidopsis thaliana genome displayer
http://www.kazusa.or.jp/kaos
• Caenorhabditis elegans (worm) database
http://www.wormbase.org/
• EBI genomes
http://www.ebi.ac.uk/genomes/
Superspecialized databases
Many individuals or groups select, annotate, and recombine data focused on particular topics, and include links
affording streamlined access to information about subjects of interest. The protein kinase resource is a specialized
compilation that includes sequences, structures and functional information, laboratory procedures, list of interested
scientists, tools for analysis, a bulletin board and links. The HIV protease database store structures of HIV1
proteinases, HIV2 proteinases and SIV proteinases, and their complexes and provides tools for their analysis and
other links.
9.3 Sequence formats

The protein and nucleic acids sequences can be stored in computer files. Once in the computer, the sequences can
be analyzed by a variety of methods. Most sequence analysis programs require that the information in a sequence
file be stored in a particular format. Format refers to the arrangement of data within a document file that typically
permits the document/data to be read or written by certain application. In other words, it is an organization of data
in a particular order. Some of the commonly used sequence formats are discussed below:
GenBank sequence entry

It has the following features:
• LOCUS: Short name for this sequence (Maximum of 32 characters).
• DEFINITION: Definition of sequence (Maximum of 80 characters).
• ACCESSION: accession number of the entry.
• VERSION: Version of the entry.
• DBSOURCE: Shows the source, the date of creation and last modification of the database entry.
• KEYWORDS: Keywords for the entry.
• AUTHORS: Authors of the work.
• TITLE: Title of the publication.
• JOURNAL: Journal reference for the entry.
• MEDLINE: Medline ID.
• COMMENT: Lines of comments.
• SOURCE ORGANISM: The organism from which the sequence was derived.
• ORGANISM: Full name of organism (Maximum of 80 characters).
• AUTHORS: Authors of this sequence (Maximum of 80 characters).
• ACCESSION: ID Number for this sequence (Maximum of 80 characters).
• FEATURES: Features of the sequence.
• ORIGIN: Beginning of sequence data.
• // End of sequence
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Bioinformatics
FASTA sequence format
The FASTA sequence format includes three parts shown in the figure below:
• A comment line identified by a “>” character in the first column followed by the name and origin of the
sequence;
• The sequence is standard one-letter symbol and
• An optional “*” which indicates the end of the sequence and which may or may not be present.
>MCHU - Calmodulin - Human, rabbit, bovine, rat, and chicken

ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTID
FPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREA
DIDGDGQVNYEEFVQMMTAK*
Figure 9.3 FASTA sequence entry format.
NBRF/PIR sequence format
The NBRF (National Biomedical Research Foundation) format has the following features. The first line includes an
initial “>” character followed by a two-letter code such as P for complete sequence or F for fragment, followed by
a 1 or 2 to indicate type of sequence, then a semicolon, then a four- to six-character unique name for the entry.
There is also an essential second line with the full name of the sequence, a hyphen, then the species of origin. The
sequence terminates with an asterisk.
>P1;CRAB_ANAPL
ALPHA CRYSTALLIN B CHAIN (ALPHA(B)-CRYSTALLIN).
MDITIHNPLIRRPLFSWLAPSRIFDQIFGEHLQESELLPASPSLSPFLMRSPIFRMPSWL
ETGLSEMRLEKDKFSVNLDVKHFSPEELKVKVLGDMVEIHGKHEERQDEHGFIAREFN
RKYRIPADVDPLTITSSLSLDGVLTVSAPRKQSDVPERSIPITREEKPAIAGAQRK*
Figure 9.4 NBRF/PIR sequence entry format.
9.4 Biosequence analysis

The determination of the linear sequence of amino acids in proteins and the nucleotides in DNA and RNA leads to
the requisite for compiling and analyzing sequence data. Sequence analysis is the process of investigating the
information content of linear raw nucleic acid and protein sequence data.
Amino acid sequence analysis

Apart from maintaining the large database, mining useful information from these sets of primary and secondary
databases is very important. Linear chains of amino acids, in proteins, the product of gene translation, are normally
found in cells folded into functionally active structures. It is established that the primary sequence of the protein,
that is, its amino acid sequence, determines the ultimate conformation of the protein and therefore its biological
function. However, the flexibility of long-chain polypeptides can generate an almost infinite number of shapes, and
the computational task of predicting correct structures is beyond the reach of current knowledge. Predicting the
shape of a protein from its linear amino acid sequence is one of the important goals of computational biology.
A lot of efficient algorithms have been developed for data mining and knowledge discovery. These are computation
intensive and need fast and parallel computing facilities for handling multiple queries simultaneously. It is these
search tools that integrate the user and the databases. One of the widely used search program is BLAST (Basic
Local Alignment Search Tool).
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Bioinformatics
Nucleic acid sequence analysis

Nucleic acid sequence analysis includes assembling partially overlapping fragments, analyzing sequences, comparing
sequences and detecting functional (RNA coding) regions. The bulk of genomic DNA does not code for proteins, and
the protein-coding regions of human genes are not collinear but arranged with exons interspersed with introns. Therefore,
an important question for computational biology is how to detect protein-coding regions within genomic DNA.
Current DNA sequencing technologies are not capable of generating a complete sequence of long nucleic acid
molecules in a single sequencing run and so it is necessary to utilize computational methods to assemble contiguous
sequences from individual short-sequence determinations. If a large DNA molecule is randomly broken into smaller
pieces for the actual sequence determinations then a contiguous linear sequence can be reconstructed by aligning
the overlapping portions from different random fragments.
A common question arising when new genes are cloned and sequenced is whether the sequence is already known
or does not occur in current databases. Answering this question requires comparing the newly obtained sequence
to every sequence in the database.
9.5 Sequence alignment

Sequence alignment refers to the procedure of comparing two or more sequences of nucleic acid or protein by
looking for a series of individual characters or character patterns that are in the same order in the sequences. It is
used to identify regions of similarity that may be a consequence of functional, structural or evolutionary relationships
between the sequences.
Global alignments and local alignments
Computational approaches to sequence alignment generally fall into two categories: global alignments and local
alignments.
Global alignment is an attempt to match as many characters as possible, from end to end, in a set of two or more
sequences. It attempts to align every residue in every sequence. Sequences that are quite similar and approximately
of the same length are suitable candidates for global alignment. A general global alignment technique is the
Needleman-Wunsch algorithm, which is based on dynamic programming.
Local alignment searches for regions of local similarity need not include the entire length of the sequences. Local
alignments are more useful for dissimilar sequences that are suspected to contain regions of similarity or similar
sequence motifs within their larger sequence context. The Smith-Waterman algorithm is a general local alignment
algorithm, also based on dynamic programming. With sufficiently similar sequences, there is no difference between
local and global alignments.
Pairwise and multiple sequence alignments
Pairwise sequence alignment
Pairwise alignment is used between two query sequences at a time. It is used to identify regions of similarity that
may indicate functional, structural and/or evolutionary relationships between two biological sequences (protein or
nucleic acid). It involves matching of homologous positions in two sequences. Positions with no homologous pair
are matched with a space ‘–’ and a group of consecutive spaces is a gap.
The three primary methods of producing pairwise alignments are dot-matrix methods, dynamic programming, and
word methods. Although each method has its individual strengths and weaknesses, all three pairwise methods have
difficulty with highly repetitive sequences.
918
Bioinformatics
The E-value is a parameter that describes the number of hits one can ‘expect’ to see by chance when searching a
database of a particular size. It decreases exponentially with the score (S) that is assigned to a match between two
sequences. Essentially, the E-value describes the random background noise that exists for matches between
sequences. For example, an E-value of 1 assigned to a hit can be interpreted as in a database of the current size,
one might expect to see one match with a similar score simply by chance. This means that the lower the E-value,
or the closer it is to ‘0’, the higher is the ‘significance’ of the match. However, it is important to note that searches
with short sequences can be virtually identical and have relatively high E-value. This is because the calculation of
the E-value also takes into account the length of the query sequence. This is because shorter sequences have a
high probability of occurring in the database purely by chance.
BLAST family
There are a number of different versions of the BLAST program for comparing either nucleic acid or protein
sequence with nucleic acid or protein sequence databases. These programes are:
• BLASTP compares an amino acid query sequence against a protein sequence database.
• BLASTN compares a nucleotide query sequence against a nucleotide sequence database.
• BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence
database.
• TBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated
in all reading frames.
• TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations
of a nucleotide sequence database.
PSI-BLAST (Position-Specific-Iterated BLAST)
PSI-BLAST uses a method that involves a series of repeated steps or iterations. First, a database search of a
protein sequence database is performed using a query sequence. Second, the results of the search are presented
and can be assessed visually to see whether any database sequences that are significantly related to the query
sequence are present. Third, if such is the case, the mouse is clicked on a decision box to go through another
iteration of the search. The high-scoring sequence matches found in the first step are aligned, and from the
alignment, a type of scoring matrix that indicates the variations at each aligned position is produced. The database
is then again searched with this scoring matrix.
PHI-BLAST (Pattern-Hit Initiated BLAST)
This program functions much like PSI-BLAST except that the query sequence is first searched for a complex pattern
provided by the investigator. The subsequent search for similarity in the protein sequence database is then focused
on regions containing the pattern. PSI-BLAST like other programs are – sSEARCH, MAXHOM.
FASTA
FASTA is a software program for rapid alignment of pairs of protein and DNA sequences. FASTA is pronounced ‘fast
A’, where A stands for All, because it works with any alphabet, an extension of ‘FAST-P’ (protein) and ‘FAST-N’
(nucleotide) alignment. It is a heuristic approximation to the Smith-Waterman algorithm. It is a two step algorithm.
The first step is a search for highly similar segments in the two sequences. In this search a word with a specific word
size is used to find regions in a two-dimensional table similar to the Smith-Waterman algorithm. These regions are
a diagonal or a few closely spaced diagonals in the table which have a high number of identical word matches
between the sequences. The second step is a Smith-Waterman alignment centered on the diagonals that correspond
to the alignment of the highly similar sequence segments.
Version of FASTA
1. FASTA compares a query protein sequence to a protein sequence library to find similar sequences. FASTA also
compares a DNA sequence to a DNA sequence library.
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Bioinformatics
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.
Phylogenetic trees
In phylogenetic studies, the most convenient way of visually presenting evolutionary relationships among a group
of organisms is through illustrations called phylogenetic trees. Phylogenetic tree is represented by lines and nodes.
Nodes can be internal or external (terminal). The different sequences of DNA/proteins compared are located at
external nodes but connected via branches to interior nodes which represent ancestral forms for two or more
sequences. The terminal nodes at the tips of trees represent operational taxonomic units (OTUs). Branch defines
the relationship between the taxa in terms of descent and ancestry. The lengths of the branches indicate the degree
of difference between the sequence represented by the nodes. The branch lengths are proportional to the predicted
evolutionary time between organisms or sequences. The branching pattern of the tree is termed a topology.
A phylogenetic tree may be rooted or unrooted. A rooted tree infers the existence of a common ancestor and
indicates the direction on the evolutionary process. A rooted tree in which every node has two descendants is called
a binary tree. An unrooted tree does not infer a common ancestor and shows only the evolutionary relationships
between the organisms.
Gene trees versus species trees
A gene tree is a model of how a gene evolves through duplication, loss, and nucleotide substitution. It is constructed
from comparisons between the sequences of orthologous genes. A species tree depicts the pattern of branching of
species lineages via the process of speciation. When reproductive communities are split by speciation, the gene
copies within these communities likewise are split into separate bundles of descent.
An internal node in a gene tree indicates the divergence of an ancestral gene into two genes with different DNA
sequences, usually resulting from a mutation of one sort or another. An internal node in a species tree represents
what is called a speciation event, whereby the population of the ancestral species splits into two groups that are no
longer able to interbreed. These two events, mutation and speciation, do not always occur at the same time.
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Bioinformatics
which reproduce the original data as closely as possible. An example of the distance method for a dataset of 4
nucleic-acid sequences is given below. The diagram below summarizes the calculation of pairwise distances between
the gene sequences for four hypothetical species.
Proportional distance (p)

DNA site
Species 1 2 3 4 5 6 7 8 9 10
1 A T A T A C G T A T
2 A T G T A C G T A T
3 G T A – A C G T G C
4 G C G T A T G C A C
Differences
p =
Sites
1 2 3 4
1 – 0.1 0.4 0.6
2 – 0.5 0.5
3 – 0.6
4 –
The coefficients provide a simple summary of how similar (or different) each sequence is from the other. Sequence
1 and 2 are more alike to each other than either is to 3. In this example, we calculated the distances across the
length of the whole sequence (10 bases); distances can be calculated for different sections of a sequence to see if
some parts are more conserved than others.
Maximum likelihood approach

This method uses probability calculations to find a tree that best accounts for the variation in a set of sequences. All
possible trees are considered, Hence, the method is only feasible for a small number of sequences. For each tree,
the number of sequence changes or mutations that may have occurred to the given sequence variation is considered.
Because the rate of appearance of new mutations is very small, the more mutations needed to fit a tree to the data,
the less likely the tree.
The maximum likelihood method presents an additional opportunity to evaluate trees with variations in mutation
rates in different lineages, and to use explicit evolutionary models such as the Jukes-Cantor and Kimura models.
The method can be used to explore relationships among more diverse sequences and conditions that are not well
handled by maximum parsimony methods.
9.7 Protein structure prediction

Genome sequencing projects are producing linear amino acid sequences, but full understanding of the biological
role of these proteins will require knowledge of their structure and function. One of the major goals of bioinformatics
is to understand the relationship between amino acid sequence and the three dimensional structure in proteins. If
these relationships are known then the structure of a protein could be reliably predicted from the amino acid
sequence. Although experimental structure determination methods are providing high-resolution structure information
about a subset of the proteins, computational structure prediction methods will provide valuable information for the
large fraction of sequences whose structures will not be determined experimentally.
Methods for prediction of protein structure from amino acid sequence include:
• Attempts to predict secondary structure without attempting to assemble these regions in three dimensions.
• Homology modeling prediction of the three-dimensional structure of a protein from the known structures of one
or more related proteins.
927

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Biotechnology

Biotechnology A problem approach, Third edition

Copyright © 2014 by Pathfinder Publication, all rights reserved.

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1.7.1 Alkali-catalyzed cleavage of RNA 60

1.7.2 RNA world hypothesis 61

1.7.3 RNA as genetic material 61

1.8.3 Cyclic forms 65

1.8.4 Derivatives of monosaccharide 67

1.8.5 Disaccharides and glycosidic bond 68

1.8.8 Reducing and non-reducing sugar 73

1.9.1 Fatty acids 74

1.9.2 Triacylglycerol and Wax 75

1.9.7 Plasma lipoproteins 81

1.10.1 Water-soluble vitamins 82

1.10.2 Fat-soluble vitamins 86

1.11.1 Naming and classification of enzyme 90

1.11.2 What enzyme does? 91

1.11.3 How enzymes operate? 92

1.11.4 Enzyme kinetics 94

1.11.5 Enzyme inhibition 102

1.11.6 Regulatory enzymes 105

1.11.7 Isozymes 106

1.11.8 Zymogen 107

1.11.9 Ribozyme 108

1.11.10 Examples of enzymatic reactions 108

2.2 Metabolism 122

2.3.1 Aerobic respiration 123

2.3.2 Glycolysis 124

2.3.3 Pyruvate oxidation 129

2.3.4 Krebs cycle 131

2.3.5 Anaplerotic reaction 134

2.3.6 Oxidative phosphorylation 135

2.3.7 Inhibitors of electron transport 139

2.3.8 Electrochemical proton gradient 140

2.3.9 Chemiosmotic theory 141

2.3.10 ATP synthase 142

2.3.11 Uncoupling agents and ionophores 144

2.3.12 ATP-ADP exchange across the inner mitochondrial membrane 144

2.3.13 Shuttle systems 145

2.3.14 P/O ratio 147

2.3.15 Fermentation 148

2.3.16 Pasteur effect 150

2.3.17 Warburg effect 150

2.3.18 Respiratory quotient 151

2.4 Glyoxylate cycle 151

2.5 Pentose phosphate pathway 152

2.6 Entner-Doudoroff pathway 154

2.7 Photosynthesis 154

2.7.1 Photosynthetic pigment 155

2.7.2 Absorption and action spectra 158

2.7.3 Fate of light energy absorbed by photosynthetic pigments 160

2.7.4 Concept of photosynthetic unit 161

2.7.5 Hill reaction 162

2.7.6 Oxygenic and anoxygenic photosynthesis 162

2.7.7 Concept of pigment system 163

2.7.8 Stages of photosynthesis 165

2.7.9 Light reactions 165