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Essentials of Molecular Genetics

Book · January 2015

DOI: 10.13140/2.1.3513.3769

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Gurbachan Miglani
Punjab Agricultural University
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Foreword

Advances in molecular biology and cutting edge science of genomics are leading to breakthroughs in
life sciences, particularly in agricultural and medical sciences. Genetic control of biological
characteristics of an organism involves complex interactions of DNA, RNA and proteins at molecular
and cellular levels. Molecular genetics provides the basic knowledge in understanding the
complexity of various biological processes both in prokaryotes and eukaryotes. Genes produce
structural and functional molecules that are so essential in controlling the synthesis of different bio-
molecules and for modifying metabolic pathways and gene expression, leading to phenotypes. Thus,
it is important to understand the complexities of structural and functional components of genetic
material. In addition to the complexities of gene expression, epigenetic variation also plays an
important role in development and evolution.

There is a gap between the knowledge that is required and what is actually being taught in
molecular genetics courses at bachelor’s, master’s and doctoral levels. Thus, there is urgent need to
upgrade the teaching level of molecular genetics courses in various subjects, e.g., basic, agricultural,
medical and veterinary sciences. Good textbooks on molecular genetics are therefore essential for
this purpose.

I am happy to note that Dr. Gurbachan Singh Miglani has produced a textbook entitled,
“Essentials of Molecular Genetics.” This book will fill the above-mentioned need for upgrading the
level of teaching of molecular biology. Dr. Miglani who is teaching Genetics and Molecular Biology
at the Punjab Agricultural University has 35 years teaching experience. He is eminently qualified to
prepare this text book. He has also published nine books earlier, including Dictionary of Plant
Genetics and Molecular Biology (1998), Advanced Genetics (2002), Genetic Material (2013) and
Gene Regulation (2013).

The book under review has 27 chapters. It is very well written and covers contemporary
subjects such as nature, structure and organization of genetic material, packaging and replication of
nucleic acids, genetic recombination, gene structure, gene organization, gene function, transcription,
RNA processing, genetic code, translation and molecular mechanisms of regulation of gene
expression in viruses, bacteria and eukaryotes, role of DNA and histone modifications and non-
coding RNAs and gene silencing in bacteria, viruses and eukaryotes. Author has given due emphasis
to molecular techniques and application of new breakthroughs in molecular genetics in
understanding basic biology of complex living systems.

At the end of each chapter, a few problems have been included to test the comprehension level of
the readers. There is an extensive glossary of 58 pages on more than 720 different terms related to
VIII Foreword

molecular genetic techniques. The book is written in simple and easy to read language. Illustrations
are clear and easy to understand. Subject index is comprehensive. The book is co-published by
Narosa Publishing House, Pvt. limited, New Delhi and Alpha Science International, Oxford, U.K. It
will be available to Indian as well as to international students. It will also be an excellent source of
references for teachers and researchers alike.

I would like to congratulate Dr. Miglani for his labor of love in producing this valuable text.

July 9, 2014 Gurdev S. Khush,

Adjunct Professor
University of California, Davis
Preface

The present era belongs to molecular genetics and biotechnology. Therefore, to become proficient in
teaching, research, and applications of the subject matter, the underlying concepts, phenomena,
hypotheses, theories, laws and principles must be thoroughly understood at the molecular level. The
blueprint or genetic material responsible for the physical appearance of an organism resides inside the
cell. This genetic material stores all the information for development and survival of the individual and
transmission of biological properties from one generation to the next. The first step for studying
molecular biology/genetics/biotechnology is to gain an understanding of the nature, structure,
molecular forms, location, organization, analysis, sequencing, synthesis, packaging, recombination,
damage, repair, and protection of genetic material in viruses, prokaryotes and eukaryotes. Genetic
material performs its function by organizing itself in the form of gene, the unit of heredity. Knowing
above-mentioned aspects of genetic material sets the stage for knowing about gene. In order to
understand how a gene expresses itself to perform its function, its various aspects, viz., gene structure,
gene organization, genome evolution, gene function, transcription, RNA processing, genetic code,
translation, and fate of finished proteins, used ribosomes and messenger RNAs need to be understood.
Finely regulated mechanisms exist in the cell to control expression of gene at every level of its
expression. Every gene requires a large number of proteins to help it perform its assigned function.
DNA and protein modifications influence gene expression. Without proper coordination of gene
regulatory mechanisms, which “switch on” or “switch off” particular genes, whose products are
required by the cell in a spacio-temporal manner, development and evolution cannot be thought of in
the present era of molecular biology. Thus gene regulation is the molecular basis of development and
evolution.
Essentials of Molecular Genetics has been written with the objective of providing concise but
complete knowledge on the above-mentioned aspects of the chemical basis of life, genetic material
(deoxyribonucleic acid and ribonucleic acid), and expression and regulation of gene in viruses, bacteria
and eukaryotes. This book also briefly deals with the role of epigenetic modifications in gene
regulation. This book is primarily designed as a text book for undergraduate students studying
molecular genetics in any discipline of life sciences, agricultural sciences, medicine, and
biotechnology in all the conventional, medical, and agricultural universities. However, postgraduate
students, teachers, research workers, and biotechnology professionals working with molecular
biotechnology companies/colleges/institutes/schools across the world can very conveniently use it as a
reference book.
x Essentials of Molecular Genetics

This book provides a brief historical background in introductory paragraph(s) of every chapter.
Recent progress on the topic is discussed. Work of 70 Nobel laureates finds a special mention. Various
hypotheses, principles, concepts, phenomena of molecular genetics have been dealt with in a simple
and lucid language. The text is supported by a number of important original and recent references,
tables, figures and flow diagrams where necessary. Special attention has been paid to precise
definitions of various terms in the subject in light of the present day knowledge. For this, there is a
separate section on glossary of important terms used in the book. Important literature relevant to the
subject matter has been cited in the text and all such references have been listed at the end of every
chapter. A few thought-provoking problems are also included in every chapter. Where there are six or
more authors, only first three have been mentioned, followed by et al. Throughout efforts have been
made to briefly pin-point applications/implications of different discoveries in the area of molecular
genetics and biotechnology. Extensive cross-referencing has been done.
Essentials of Molecular Genetics should be extremely useful to those who are preparing for
national and international level competitive examinations, entrance tests, and interviews for
jobs/fellowships. I trust the book will provide enjoyable reading experience. Strenuous efforts have
been made to include all important information relevant at undergraduate level; however, if something
important is missing, the readers are urged to inform the author so that deficiencies can be corrected in
a subsequent printing/edition. Style of presentation and selection of examples are purely my choice. I
am responsible for any omissions and commissions. Readers’ feedback will be appreciated.
Class-room has been my lab for my teaching experiments. Students have always been a guiding
force to me in an indirect way in deciding order of different chapters in the book and organization of
the contents in a chapter. Thus contribution of my present and past students in my books has been
tremendous. During the writing of this book I have consulted a large number of original research
papers, review articles, books, monographs, and web sites. My head bows with respect before all these
great authors for their landmark work. I thank various copyright holders to grant me permission for use
of their published work in this book.
I am grateful to Dr. Kuldeep Singh, Director, School of Agricultural Biotechnology, Punjab
Agricultural University, Ludhiana, India for his moral support and providing me facilities for this
work. Dr. S.S. Gosal, Director of Research, Punjab Agricultural University, Ludhiana, India, who is an
experienced author, made very useful suggestions in the preparation of this book. Stewardship
provided by Dr. Manjit Singh Kang, former Vice-Chancellor, Punjab Agricultural University,
Ludhiana, India, who has edited and authored several books in the area of genetics, is gratefully
acknowledged. I enjoyed full support of Dr. Baldev Singh Dhillon, Vice-Chancellor, Punjab
Agricultural University, Ludhiana, India in the writing of this book.
Dr. Darshan S. Brar, former Head, Plant Breeding, Genetics and Biotechnology Division, Inter-
national Rice Research Institute, Los Baños, The Philippines, presently Honorary Adjunct Professor,
School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India advised me to
include some thought-provoking questions/problems at the end of every chapter to test comprehension
of the reader; for this and many other useful pieces of advice, I am thankful to Dr. Brar.
I am indebted to Dr. Gurdev S. Khush, former Principal Plant Breeder and Head, Division of Plant
Breeding Genetics and Biochemistry, International Rice Research Institute, Los Baños, The
Preface xi
Philippines, and presently Adjunct Professor, University of California, Davis, USA for showering his
blessings on me by writing Foreword of this book.
I must make mention of my immediate family, relatives and friends without whose involvement
and support, completion of this book would not have been possible. No one can do any creative work,
such as writing of a book, without the cooperation of his/her spouse. My wife Harjit showed utmost
patience and cooperation during the preparation of this book. My son Jemmy and daughter-in-law
Parvi made sure that I was kept away from tedious household duties like depositing bills and making
purchases from the market. My daughter Simmi and son-in-law Shally frequently motivated me by
checking up on the progress of the book. My grandchildren, Prabhasis, Harmehar and Bhavneet were
more of an encouragement rather than a disturbance to me. Because of his experience in writing
manuals and books, I often consulted my dear nephew, Dr. Sandeep Singh, Assistant Entomologist,
Punjab Agricultural University, Ludhiana, India, who also assisted me in compiling the subject index.
Mr. Gagandeep Singh typed the manuscript and drew figures meticulously. Mr. Gurdeep Singh of
ALPS Educational Services, Ludhiana formatted the book for camera-ready printouts. I like the way
the book Essentials of Molecular Genetics has come up with respect to format and design. For this, I
whole heartedly thank Mr. N.K. Mehra, Managing Director, Narosa Publishing House Pvt. Ltd., New
Delhi.

March 4 , 2014 Gurbachan S. Miglani

Residence: Visiting Professor

147-D, BRS Nagar School of Agricultural Biotechnology
Ludhiana – 141 012 Punjab Agricultural University
India Ludhiana 141 004, Punjab, India

Email: Professor of Genetics (Retired)

[email protected] Department of Plant Breeding, Genetics and Biotechnology
[email protected] Former Adjunct Professor
[email protected] School of Agricultural Biotechnology
Punjab Agricultural University
Ludhiana 141 004, Punjab, India
Contents
Foreword by Dr. Gurdev S. Khush vii
Preface ix
1. Introduction 1.1
Birth of Molecular Genetics 1.1
Mendelism Down to Molecular Level 1.2
Sperm RNAs are delivered to Oocytes at Fertilization 1.3
Nature, Structure and Organization of the Genetic Material 1.3
Gene Structure, Function and Expression 1.3
Gene Regulation 1.4
Applying Knowledge about Genetic Material and Genes 1.4
References 1.4
Problems
2. Nature of the Genetic Material 2.1
Characteristics of Genetic Material 2.1
Discovery of DNA as Genetic Material 2.2
Discovery of RNA as Genetic Material 2.5
Viral Genomes 2.7
Latent Viruses 2.9
References 2.10
Problems 2.10
3. Structure of the Genetic Material 3.1
Structure of DNA 3.1
Alternate Structural Forms of DNA 3.6
Structure of RNA 3.13
Fundamental Properties of Genetic Material 3.15
References 3.17
Problems 3.18
4. Extranuclear Genomes 4.1
Chloroplast Genomes 4.1
Mitochondrial Genomes 4.3
Kinetoplast DNA 4.7
Centriole DNA 4.9
Cyanelle DNA 4.9
Promiscuous DNA 4.10
Coupled Expression of Nuclear and Organelle Genomes 4.11
References 4.11
Problems 4.14
xiv Contents

5. Organization of the Genetic Material 5.1

DNA Kinetics 5.1
Organization of Genetic Material 5.8
Prokaryotic Genome 5.10
Eukaryotic Genome 5.10
Repeated DNA Sequences and Diseases 5.19
References 5.20
Problems 5.20
6. Packaging of Nucleic Acids 6.1
DNA Packaging in Viruses/Bacteriophages 6.1
DNA Packaging in Bacteria 6.3
DNA Packaging in Nucleus of Eukaryotic Cells 6.6
DNA Packaging in Eukaryotic Organelles 6.19
References 6.19
Problems 6.22
7. Replication of Nucleic Acids 7.1
Different Modes of DNA Replication 7.1
DNA Replication in Prokaryotes 7.1
Steps of DNA Replication 7.6
Plasmid Replication 7.12
Replication in Viruses 7.12
DNA Replication in Eukaryotic Nuclear Chromosomes 7.12
Replication of Mitochondrial DNA 7.24
Replication of Chloroplast DNA 7.25
Models of DNA Replication 7.26
Duplication of RNA 7.28
Polymerase Chain Reaction 7.29
References 7.30
Problems 7.32

8. Genetic Recombination 8.1

DNA Recombination in Bacteriophages 8.1
DNA Recombination in Bacteria 8.1
DNA Recombination in Eukaryotes 8.4
Types of Recombination 8.8
Models of Genetic Recombination 8.10
Crossing Over Event 8.17
Gene Conversion 8.18
Genetics and Enzymology of Recombination 8.20
Mitochondrial DNA Recombination 8.23
Chloroplast DNA Recombination 8.23
Intragenic Recombination 8.23
RNA Recombination 8.25
References 8.28
Problems 8.30
Contents xv

9. Mutation 9.1
Classification of Mutations 9.1
Detection of Mutations 9.17
Molecular Basis of Mutation 9.27
Site-directed Mutagenesis 9.41
In Vitro Mutagenesis 9.42
Mutagenesis in Organelle Genomes 9.42
Usefulness of Studies on Mutations 9.44
Environmental Mutagenesis 9.46
References 9.47
Problems 9.49
10. DNA Transposition 10.1
General Characteristics of Transposons 10.1
Bacterial Transposable Elements 10.3
Transposable DNA Phages 10.4
Eukaryotic Transposable Elements 10.5
Transposon Mutagenesis 10.18
Illegitimate Recombination 10.18
Mechanism of Transposition 10.20
Role of Transposons 10.23
Uses of Transposons 10.24
Limitations of Transposons 10.25
Mobile Elements and Genome Evolution 10.25
References 10.25
Problems 10.27
11. DNA and Non-DNA Repair Mechanisms 11.1
DNA Damage Checkpoints and Response 11.1
DNA Repair Mechanisms in Prokaryotes 11.1
DNA Repair Mechanisms in Eukaryotes 11.20
Diseases due to DNA Repair Defects 11.34
References 11.37
Problems 11.40
12. Gene Structure 12.1
Gene Concept 12.1
Types of Genes 12.6
Defining Gene? 12.18
Introns 12.19
Why are the Eukaryotic Genes Split? 12.21
Overlapping Genes 12.23
Pseudogenes 12.24
Colinearity 12.24
References 12.28
Problems 12.30

13. Defense Genes 13.1

Immunoglobulin Genes 13.1
Human Leukocyte Antigen Complex 13.8
xvi Contents

Oncogenes 13.10
Proto-oncogenes 13.18
Epigenetic Alterations and Neoplasia 13.20
How Immune System Counteracts Oncogenes? 13.22
Antioncogenes 13.22
Antisense Molecules as Anticancer Drugs 13.23
References 13.24
Problems 13.26

14. Gene Organization 14.1

Gene Families 14.1
Changes in Genome Size 14.10
Changes in DNA Sequences 14.12
Nucleotide Polymorphism 14.13
References 14.14
Problems 14.14
15. Gene Function 15.1
Relationship Between Gene and Enzyme 15.1
Genetic Control of Biochemical Reactions 15.1
One Gene-One Reaction Hypothesis 15.3
Relationship Between Genotype and Phenotype 15.3
One Gene-One Enzyme Hypothesis 15.5
One Gene-One Polypeptide Hypothesis 15.8
One Gene-One Chromomere Hypothesis 15.9
One Gene-One Antigen Hypothesis 15.10
One Cistron-One Polypeptide Hypothesis 15.10
One Gene-One Ribosome-One Protein Hypothesis 15.10
One Gene-One mRNA-One Protein Hypothesis 15.10
One Gene-One Primary Cellular Function Hypothesis 15.11
Gene Discoveries 15.11
Two Genes-One Polypeptide Hypothesis 15.12
Knowing Gene Function by Knocking off Genes 15.13
One Gene-Many Proteins Hypothesis 15.13
One Enzyme-Two Functions Concept 15.14
Recent Thoughts on Gene Function 15.14
References 15.14
Problems 15.16
16. Transcription 16.1
Central Dogma and its Modification 16.1
Transcription in Bacteria 16.4
Transcription in Viruses 16.10
Reverse Transcription 16.12
Transcription of Eukaryotic Genes 16.13
Chloroplast Transcription 16.26
Mitochondrial Transcription 16.26
References 16.26
Problems 16.28
Contents xvii

17. RNA Processing and RNA Editing 17.1

Pre-Messenger RNA Processing 17.1
Pre-Transfer (Soluble) RNA Processing 17.6
Pre-Ribosomal RNA Processing 17.12
Self-Splicing of rRNA In Tetrahymena 17.16
Alternative RNA Splicing 17.18
RNA Editing 17.21
References 17.27
Problems 17.29
18. Genetic Code 18.1
Size of Genetic Code 18.1
Glossary of Terms on Genetic Code 18.2
Properties of Genetic Code 18.2
Deciphering the Genetic Code 18.7
Genetic Code at Work 18.9
Genetic Code Specificity 18.9
Expansion of Genetic Alphabet 18.10
Recoding 18.10
The Second Code 18.12
References 18.13
Problems 18.14
19. Protein Biosynthesis 19.1
Translation in Prokaryotes 19.1
Ribosome – Site of Protein Synthesis 19.4
Translation in Eukaryotic Cytoplasm 19.16
Protein Synthesis in Chloroplasts and Mitochondria 19.20
Role of MicroRNAs in Protein Synthesis 19.23
Hybrid Arrested Translation 19.24
Hybrid Released Translation 19.24
Protein Engineering 19.24
References 19.25
Problems 19.27

20. Fate of Nascent Proteins, Ribosomes, and Messenger RNAs 20.1

Protein Modification 20.1
Protein Secretion in Prokaryotes 20.2
Protein Translocation in Prokaryotes 20.5
Co-Translational Targeting (Secretory Pathways) in Eukaryotes 20.7
Post-Translational Targeting in Eukaryotes 20.9
Protein Folding 20.12
Molecular Chaperone 20.13
Protein Degradation 20.14
Fate of Ribosomes After Translation 20.16
Messenger RNA Decay 20.16
References 20.19
Problems 20.20
xviii Contents

21. Gene Expression Analysis 21.1

Glossary Related to Gene Expression Analysis 21.1
Global Gene Expression Profiling 21.2
Expression System 21.2
Gene Expression Networks 21.3
mRNA Quantification 21.3
Protein Quantification 21.6
Applications of Gene Expression Analysis 21.8
Limitations of Gene Expression Analysis 21.10
Systems Biology Approach in Gene Expression Analysis 21.10
References 21.13
Problems 21.14

22. Gene Regulation in Bacteria 22.1

Gene Regulation at DNA Level 22.1
Gene Regulation at Transcriptional Level 22.2
Antisense RNA in Prokaryotic Gene Regulation 22.18
Alternative Sigma Factors 22.20
Post-Transcriptional Control 22.20
Control at Translational Level 22.22
Post-Translational Control 22.22
Alarmones 22.24
References 22.25
Problems 22.26
23. Gene Regulation in Viruses 23.1
SpoI Bacteriophage 23.3
Lambda Bacteriophage 23.4
T4 Bacteriophage 23.14
T7 bacteriophage 23.15
Human Immunodeficiency Virus 23.15
References 23.16
Problems 23.16
24. Gene Regulation in Eukaryotes 24.1
Gene Control Levels in Eukaryotic Cells 24.2
Transcriptional Initiation in Mammalian Genes 24.12
Molecular Zippers in Gene Regulation 24.15
Gene Regulation by Non-Histone Proteins 24.16
Artificial Gene Repressors 24.17
Specificity in Eukaryotic Gene Transcription 24.18
Metal-Regulated Transcription in Eukaryotes 24.20
Regulation of Transposable Elements 24.21
Control of Cell Cycle 24.21
Cohesion Complex Regulates Gene Expression 24.23
Gene Regulation by Hormonal Action 24.24
References 24.28
Problems 24.29
Contents xix

25. Epigenetics 25.1

Epigenetic Patterns 25.1
Bacterial Methylase Systems 25.2
Characteristics of DNA Methylation 25.3
Host Restriction and Modification 25.5
Cytosine Methylation in Vertebrates 25.7
DNA Methylation and Gene Expression 25.8
DNA Methylation in Gene Regulation and Differentiation 25.10
Mutations and Epimutations 25.10
DNA Methylation in Genome Stability and Gene Silencing 25.11
Epigenetic Reprogramming 25.11
Chromosome Imprinting 25.13
Dosage Compensation 25.16
Epigenetic Variations 25.17
Paternal X chromosome Inactivation 25.18
DNA Methylation in Invertebrates 25.18
DNA Methylation in Plants 25.18
Paramutation 25.19
Defense Against Spread of Transposable Elements 25.19
Cyclic Changes in DNA Methylation 25.20
RNA Methylation 25.20
Histone Modification 25.21
General Functions of Histone Modification 25.25
Consequences of Histone Modifications 25.26
References 25.27
Problems 25.30
26. Noncoding RNAs and Gene Silencing 26.1
Noncoding RNAs 26.1
Antisense Noncoding RNAs 26.2
Gene Silencing 26.3
Transcriptional Gene Silencing 26.4
Phenomenon Associated with TGS 26.7
Post-Transcriptional Gene Silencing 26.12
Methylation of Non-Coding RNAs 26.21
Translational Gene Silencing 26.21
Exploitation of Gene Silencing 26.22
References 26.23
Problems 26.26
27. Molecular Techniques and Tools 27.1
Separation of DNA/RNA 27.1
In situ Hybridization 27.3
Squash Dot Hybridization 27.5
Southern Blotting 27.5
Northern Blotting 27.6
Western Blotting 27.7
Eastern Blotting 27.8
Dot Blots and Slot blots 27.8
xx Contents

Electrophoresis 27.9
Colony or Plaque Hybridization 27.10
Chromosome Walking 27.11
Chromosome Jumping/Hopping 27.12
Chromosome Landing 27.12
Nick Translation 27.13
RNA Sequencing 27.13
DNA Sequencing 27.17
RNA Synthesis 27.22
DNA Synthesis 27.23
Gene Synthesis Machines 27.29
DNA Fingerprinting 27.31
DNA Markers 27.33
Microarrays 27.39
Restriction Endonucleases 27.41
Recombinant DNA Technology 27.43
Quantitative Trait Loci Mapping 27.45
References 27.47
Problems 27.52
Glossary G1
Subject Index S1
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