A TEXT BOOK OF

MOLECULAR

CYTOGENETICS

A TEXT BOOK OF

MOLECULAR

CYTOGENETICS

Ashok KumarAshok KumarAshok KumarAshok KumarAshok KumarAssistant Professor

Dept. of Biotecnology Himachal Institute of Life Science,Paonta Sahib – 173025 (Himachal Pradesh) India

AndAndAndAndAnd

Meena SrivastavaMeena SrivastavaMeena SrivastavaMeena SrivastavaMeena SrivastavaAssociate Professor

Dept. of Zoology, Maharana Pratap Government (PG) CollegeHardoi – 241001 (Uttar Pradesh) India

NARENDRA PUBLISHING HOUSEDELHI-110006 (INDIA)

Copyright © 2012, Narendra Publishing House, Delhi (India)

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First Edition 2012

ISBN: 978-93-80428-79-6

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This Book is Dedicated toThis Book is Dedicated toThis Book is Dedicated toThis Book is Dedicated toThis Book is Dedicated toMy Divine MotherMy Divine MotherMy Divine MotherMy Divine MotherMy Divine Mother“Smt. Kamla Devi”“Smt. Kamla Devi”“Smt. Kamla Devi”“Smt. Kamla Devi”“Smt. Kamla Devi”

(Mummi)(Mummi)(Mummi)(Mummi)(Mummi)

Contents

Preface xxi

Acknowledgements xxiii

1. MOLECULAR BIOLOGY ........................................................................ 1

Introduction ..................................................................................................... 1

Molecular Biology ........................................................................................... 5

Chromosomes and Inherited Traits ................................................................ 7

Discovery of Structure of DNA .................................................................... 8

Complementary Nucleotides ........................................................................... 8

Central Dogma ............................................................................................... 8

History of RNA Tertiary Structure ................................................................ 9

Crystal Structure of tRNA ............................................................................. 9

Hammerhead Ribozyme ............................................................................... 10

Protein ............................................................................................................11

Biotechnology ................................................................................................ 13

Development of Molecular Biology and Cytogenetics ................................ 16

Components of Molecular Biology ............................................................... 20

Restriction Enzymes ..................................................................................... 21

Restriction Endonucleases ..................................................................... 21

Type II Restriction Endonucleases ........................................................ 22

Isoschizomers ......................................................................................... 23

Restriction Mapping ............................................................................... 23

Host Cells ............................................................................................... 24

Nucleic Acid Hybridization ........................................................................... 24

Polymerase Chain Reaction ......................................................................... 25

Nucleic Acid Sequencing .............................................................................. 26

Nucleic Acids ................................................................................................ 27

Types of Nucleic Acids ................................................................................ 28

Ribonucleic Acid ........................................................................................... 28

Deoxyribonucleic Acid (DNA) ..................................................................... 29

Nucleic Acid Components ............................................................................ 29

viii

Nucleobases .................................................................................................. 29

Nucleosides ................................................................................................... 29

Nucleotides and Deoxynucleotides ............................................................... 30

History of Nucleic Acid ................................................................................ 30

Experimental Proof of the Genetic Function of DNA ................................ 31

Structure of DNA......................................................................................... 34

Properties of DNA ....................................................................................... 35

Bases ...................................................................................................... 36

Base Pairing ........................................................................................... 39

Grooves ................................................................................................... 40

Sense and Antisense .............................................................................. 41

DNA Super Coiling ................................................................................ 41

Role of Supercoiling ............................................................................... 42

Occurrence of DNA super coiling ........................................................ 43

Alternate DNA Structures ........................................................................... 46

Quadruplex Structures .................................................................................. 47

B-DNA ......................................................................................................... 48

Branched DNA ............................................................................................. 49

A-DNA ......................................................................................................... 49

Z-DNA .......................................................................................................... 50

Biological Significance of Z DNA ......................................................... 51

Chemical Modifications of DNA ................................................................. 52

Base Modifications ................................................................................. 52

Biological Functions ................................................................................ 52

Genes and Genomes ..................................................................................... 52

Interactions with Proteins ............................................................................. 53

DNA-binding Proteins .................................................................................. 53

Watson and Crick Model of DNA ............................................................... 54

Features of the Watson-Crick base pair ............................................... 55

Features of the Watson-Crick model of B-DNA ................................. 56

Real Structure of B-DNA ............................................................................ 56

History of DNA Research ........................................................................... 57

ix

Important Contributors to the Field of DNA ............................................... 57

Ribonucleic Acid ........................................................................................... 61

Discovery of RNA ................................................................................. 63

Type of RNA ......................................................................................... 64

Ribosomal RNA (rRNA) ....................................................................... 64

Prokaryotes vs. Eukaryotes ................................................................... 64

Prokaryotes ............................................................................................. 65

Eukaryotes .............................................................................................. 65

Importance of rRNA ............................................................................. 65

Messenger RNA (mRNA) .................................................................... 66

Synthesis, Processing and Function ....................................................... 66

Transcription ........................................................................................... 66

Eukaryotic pre-mRNA Processing ........................................................ 66

5' cap addition ........................................................................................ 67

Splicing .................................................................................................... 67

Editing ..................................................................................................... 67

Polyadenylation ....................................................................................... 67

Transport ................................................................................................. 68

Translation ............................................................................................... 68

Structure of mRNA ...................................................................................... 68

5' cap ...................................................................................................... 68

Coding regions ........................................................................................ 68

Untranslated regions .............................................................................. 69

Poly (A) tail ............................................................................................ 70

Monocistronic Versus Polycistronic mRNA .......................................... 70

mRNA Circularization ............................................................................ 70

Degradation ............................................................................................ 70

Prokaryotic mRNA Degredation ........................................................... 70

Eukaryotic mRNA Turnover .................................................................. 71

AU-rich Element Decay ........................................................................ 71

Nonsense Mediated Decay.................................................................... 71

Small Interfering RNA (siRNA) .................................................................. 72

x

Micro RNA (miRNA) .................................................................................. 72

Small Nucleolar RNA (snRNA) .................................................................. 72

XIST RNA .................................................................................................... 73

Transfer RNA (tRNA) ................................................................................. 73

Secondary Structure of tRNA ............................................................... 73

3D Structure of tRNA ........................................................................... 75

Anticodon ................................................................................................ 76

Aminoacylation ....................................................................................... 76

Binding to Ribosome .............................................................................. 77

tRNA Genes ........................................................................................... 77

Formation and Processing ...................................................................... 77

Cellular Functions ................................................................................... 79

Gene Activation ...................................................................................... 79

Detecting and Manipulating miRNA Signaling ............................................ 80

miRNA and Disease ..................................................................................... 80

Similarities and Differences between DNA and RNA ............................... 81

Selfish DNA .................................................................................................. 82

Satellite DNA ............................................................................................... 82

Minisatellite DNA .................................................................................. 82

Microsatellites DNA .............................................................................. 83

Central Dogma of Molecular Biology .......................................................... 84

2. REPLICATION .......................................................................................... 88

DNA Replication-Some Facts ...................................................................... 88

DNA Polymerase ......................................................................................... 93

Helicases ....................................................................................................... 94

Structural Feature of Helicases ............................................................. 94

Superfamilies of Helicase ...................................................................... 94

Function of Helicase .............................................................................. 95

Origin of Replication ..................................................................................... 95

Types of Origin of Replication ............................................................... 96

Prokaryotic Origin of Replication .......................................................... 96

Eukaryotic Origin of Replication ............................................................ 97

Replication fork ............................................................................................. 98

xi

Leading strand synthesis ........................................................................ 98

Lagging strand synthesis ........................................................................ 98

Prokaryotic DNA Replication ..................................................................... 100

Initiation ................................................................................................ 100

Elongation ............................................................................................. 101

Termination ........................................................................................... 101

Eukaryotic DNA replication ....................................................................... 102

Regulation of Replication ..................................................................... 103

Termination of Replication ................................................................... 104

Rolling circle Replication ............................................................................ 105

Replication of Plasmids ........................................................................ 105

Control of Plasmid Replication and Copy-number .............................. 106

3. TRANSCRIPTION ..................................................................................108

Introduction ................................................................................................. 108

RNA Polymerase I ..................................................................................... 108

Initiation ................................................................................................ 109

Elongation ............................................................................................. 109

Termination ............................................................................................ 110

RNA Polymerase II .....................................................................................110

Stages of Transcription .........................................................................110

Initiation ................................................................................................. 111

Initiation Regulation ............................................................................... 112

C-terminal Domain ................................................................................ 113

5’Capping ...............................................................................................113

Spliceosome ........................................................................................... 113

RNA polymerase III ....................................................................................113

Initiation ........................................................................................................ 114

Class I ....................................................................................................114

Class II ..................................................................................................114

Class III .................................................................................................114

Termination ............................................................................................ 115

Operon Concepts .........................................................................................115

xii

1. Lac Operon .....................................................................................115

Lactose analogues ..........................................................................118

Allolactose .......................................................................................119

b-galactosidase Activity .................................................................. 119

Mechanism of Induction .................................................................119

Application of lac Operon ............................................................. 120

2. Trp Operon ..................................................................................... 120

Repression ...................................................................................... 121

Attenuation ..................................................................................... 121

Eukaryotic Gene Regulation ................................................................ 123

Enzyme Regulation ............................................................................... 124

a. Genetic Control .............................................................................. 124

b. Controlling the enzyme’s activity (feedback inhibition) ................ 126

Post Transcriptional Modification ......................................................... 127

1. 5' caping ......................................................................................... 127

Capping Process ............................................................................ 127

5' Capping Targeting ...................................................................... 128

2. Polyadenylation .............................................................................. 129

Nuclear polyadenylation ................................................................ 129

Mechanism of Nuclear Poly ademylation ..................................... 130

a. Factor (Proteins involded) ....................................................... 130

b. Downstream Effects ............................................................... 131

c. Deadenylation .......................................................................... 131

3. Alternative Polyadenylation ........................................................... 131

Cytoplasmic Polyadenylation ......................................................... 132

Tagging for Degradation in Eukaryotes ........................................ 132

4. RNA splicing .................................................................................. 132

i) Splicing Mechanism ................................................................ 133

Beta-globin gene ..................................................................... 134

ii) Sex Lethal Gene ..................................................................... 134

5. RNA Editing ................................................................................... 136

Importance of RNA Editing .......................................................... 136

xiii

4. TRANSLATION.......................................................................................138Introduction ................................................................................................. 138

Genetic Code .............................................................................................. 138

History of Genetic Code ...................................................................... 139

Transfer of Information via Genetic Code .......................................... 139

Salient Features of Genetic Code ........................................................ 142

Theories on the Origin of the Genetic Code ....................................... 145

Wobble Base Pair and Wobble Hypothesis ............................................... 146

Aminoacyl-tRNA ........................................................................................ 148

Translation in Prokaryotes .......................................................................... 150

Translation in Eukaryotes ........................................................................... 152

Posttranslational Modification ..................................................................... 153

Molecular chaperones and HSP .......................................................... 165

Molecular Chaperone ........................................................................... 165

Location and Functions ........................................................................ 165

Bacterial and Archeal Chaperones ...................................................... 166

Chaperonins .......................................................................................... 166

Categories of Chaperonins ................................................................... 167

Mechanism of Action ................................................................................. 167

Heat Shock Proteins (HSP) ....................................................................... 168

Classification ........................................................................................ 168

Function of Heat Shock Protein .......................................................... 169

Applications .......................................................................................... 170

Protein Processing ............................................................................... 171

Proteasomes ......................................................................................... 172

Structure and Organization .................................................................. 172

20S Core Particle ................................................................................. 173

19S Regulatory Particle ....................................................................... 174

Regulation of the 20S by 19S .............................................................. 174

11S regulatory particle ......................................................................... 174

Assembly .............................................................................................. 175

Ubiquitylation and Targeting ....................................................................... 175

Unfolding and Translocation ................................................................ 176

Proteolysis ............................................................................................. 177

Ubiquitin-independent Degradation ...................................................... 177

xiv

Cell Cycle Control ...................................................................................... 178

Regulation of Plant Growth ........................................................................ 178

Apoptosis ................................................................................................. 179

Response to Cellular Stress ........................................................................ 179

Proteasome Inhibitors ................................................................................. 180

5. DNA DAMAGE AND REPAIR ............................................................181

DNA Repair ................................................................................................ 181

DNA Damage ............................................................................................. 181

Types of Damage................................................................................. 182

Nuclear Versus Mitochondrial DNA Damage .................................... 183

Senescence and Apoptosis ......................................................................... 183

DNA Damage and Mutation ...................................................................... 184

Agents that Damage DNA ........................................................................ 184

DNA Repair Mechanisms .......................................................................... 185

Direct Reversal ........................................................................................... 186

Single Strand Damage ................................................................................ 186

1. DNA Mismatch Repair ........................................................................ 186

i) Mismatch Repair Proteins ............................................................. 188

a) MutS Homologs ...................................................................... 188

b) MutL Homologs ...................................................................... 189

c) MutH ....................................................................................... 189

d) b-sliding clamp ......................................................................... 189

ii) Defects in Mismatch Repair ......................................................... 190

2. Base Excision Repair ........................................................................... 190

i) Lesions Processed by BER .......................................................... 191

ii) Proteins for Base Excision Repair ................................................ 192

3. Nucleotide Excision Repair (NER) ...................................................... 193

Double-strand Breaks .......................................................................... 194

Translesion Synthesis ........................................................................... 195

DNA Damage Checkpoints ................................................................. 195

SOS Response ...................................................................................... 196

Eukaryotic Transcriptional Responses to DNA Damage ................... 197

DNA Repair and Evolution .................................................................. 197

Xeroderma Pigmentosum ..................................................................... 198

xv

i) Types of XP ................................................................................... 199

ii) Symptoms of XP ........................................................................... 199

iii) Treatment ....................................................................................... 200

Mutation ......................................................................................... 200

Mutagens ........................................................................................ 201

6. CYTOGENETICS ....................................................................................202

Organization of DNA in chromosome ....................................................... 202

i) Introduction ........................................................................................... 202

ii) Chromosome Structure ........................................................................ 203

a) Chromatin ....................................................................................... 204

b) Packaging of DNA in Chromatin .................................................. 204

Interphase Chromatin ................................................................................. 205

Metaphase Chromatin and Division ........................................................... 205

Chromatin and Watson/Crick base Pairing ................................................ 206

Histone ........................................................................................................ 206

Nucleosome and Beads-on-a-string ........................................................... 207

Chromatin Fibre .......................................................................................... 209

DNA Superhelix .......................................................................................... 210

Spatial Organization of Chromatin ............................................................. 210

Nucleoid ....................................................................................................... 211

Circular Bacterial Chromosome .................................................................. 211

Structure of Eukaryotic Chromosome ........................................................ 212

Centromere ................................................................................................. 212

Centromere Positions .................................................................................. 212

Telomere ...................................................................................................... 213

Structure and Functions of Telomere ......................................................... 214

Number of Chromosomes .......................................................................... 215

i) Eukaryotes ............................................................................................ 215

ii) Prokaryotes ........................................................................................... 216

Heterochromatin ......................................................................................... 216

i) Constitutive Heterochromatin ............................................................... 217

ii) Facultative Heterochromatin ................................................................ 217

iii) Yeast Heterochromatin ........................................................................ 218

xvi

Gene Family ................................................................................................ 218

Interrupted Gene ......................................................................................... 219

Euchromatin ................................................................................................ 219

Function ....................................................................................................... 220

C-value Paradox ......................................................................................... 220

C-value Paradox History ............................................................................ 220

Polytene Chromosome ................................................................................ 222

Chromosome Puffs ..................................................................................... 223

Lampbrush Chromosomes .......................................................................... 224

Linkage ....................................................................................................... 224

Frequency of Recombination ............................................................... 225

Importance of Linkage ......................................................................... 225

Sex Linkage ................................................................................................ 225

Genes on the Sex Chromosomes ......................................................... 226

Genetic and Hereditary Disorders ....................................................... 227

Types of Genetic Disorders ........................................................................ 227

Genetic Linkage .......................................................................................... 227

Linkage Mapping ........................................................................................ 228

Linkage Map ............................................................................................... 229

LOD Score Method .................................................................................... 230

Recombination Frequency .......................................................................... 230

Back cross .................................................................................................. 232

Test Cross ................................................................................................... 233

Two Point Test Cross ................................................................................. 234

Chi square Analysis for Linkage ................................................................ 237

Map Distance .............................................................................................. 238

Hardy-Weinberg Equilibrium ...................................................................... 239

Random Mating .......................................................................................... 239

7. SEX DETERMINATION .......................................................................244

Indroduction ................................................................................................. 244

Sex Determination in Mammal ................................................................... 244

Sex Determination Mechanisms .......................................................... 245

Primary Sex Determination .................................................................. 245

Secondary Sex Determination ............................................................. 246

xvii

Testis Determining Factor (TDS) ............................................................... 246

Function of SRY ......................................................................................... 247

Role of SOX9 ............................................................................................. 248

Female Development .................................................................................. 248

Gonadal Sex ................................................................................................ 249

Fetal Production of Gonadal Hormones .................................................... 249

External Genitalia ........................................................................................ 249

Chromosomal Sex Determination (CSD) ............................................ 250

Environmental Sex Determination (ESD) ........................................... 250

Sex Determination System in Insects ........................................................ 251

Sex chromosomes in Insects ...................................................................... 254

ZW Sex Chromosomes .............................................................................. 255

Haplodiploidy ............................................................................................... 255

Non genetic Sex determination Systems ................................................... 256

Sex Determination Mechanisms in Insects ................................................ 256

Comparison of Sex Determination System ................................................ 257

Dosage Compensation ................................................................................ 259

Barr Bodies ................................................................................................. 262

8. KARYOTYPING AND CHROMOSOMAL DISORDERS ..............264

Human Chromosomes ................................................................................ 264

Gene Complexity ........................................................................................ 266

Single Nucleotide Polymorphisms ............................................................... 267

Chromosomes in Eukaryotes ...................................................................... 267

Chromosomes in Prokaryotes ..................................................................... 267

Karyotype .................................................................................................... 268

History of Karyotype Studies .............................................................. 270

Observations on karyotypes ................................................................. 270

Human Karyotype ................................................................................ 271

Diversity and Evolution of Karyotypes ............................................... 272

i) X inactivation ................................................................................. 272

ii) Number of Chromosomes in a Set ............................................... 272

iii) Fundamental Number .................................................................... 273

iv) Ploidy .............................................................................................. 273

xviii

v) Aneuploidy ..................................................................................... 274

vi) Chromosomal Polymorphism ......................................................... 274

Karyotyping ................................................................................................. 274

i) Classic Karyotype Cytogenetics ................................................... 274

ii) Spectral karyotype (SKY Technique) ........................................... 275

iii) Digital Karyotyping ........................................................................ 275

Chromosome Abnormalities ........................................................................ 276

Chromosomal Banding ................................................................................ 277

Types of Banding ........................................................................................ 278

Staining Protocols ....................................................................................... 279

i) Protocol-1: Giemsa Staining ................................................................. 279

ii) Protocol-2: Leishman’s Stain ............................................................... 280

iii) Protocol-3: G-Banding .......................................................................... 281

iv) Protocol-4: C-Banding .......................................................................... 282

v) Protocol-5: Giemsa Reverse Banding (RHG) ..................................... 284

vi) Protocol-6: R Banding by Fluorescence usingvii) Protocol-7: T-Banding .......................................................................... 286

Chromosomal Aberrations .................................................................... 287

Genetic and Hereditary Disorders ....................................................... 288

Types of Genetic Disorders ................................................................. 288

1. Single Gene Disorders ................................................................... 290

2. Multifactorial and Polygenic Disorders ......................................... 300

3. Disorders with Variable Modes of Transmission .......................... 301

4. Cytogenetic Disorders ................................................................... 301

9. HUMAN GENOME PROJECT AND GENE LIBRARY ................302

Human Genome Project ............................................................................. 302

Indroduction .......................................................................................... 302

Complation of Human Genome project ............................................... 304

Findings ................................................................................................. 306

Ethical, Legal and Social Issues .......................................................... 306

Concerns of the Sequencing Project ................................................... 307

Public Versus Private Approaches ...................................................... 308

Advantages of Human Genome Project ............................................. 309

Benefits of Human Genome Project ................................................... 309

xix

Genotype ..................................................................................................... 310

Gene Library ................................................................................................311

Principle of Gene Cloning ..................................................................... 311

Basic Steps ........................................................................................... 312

Genomic Library .................................................................................. 315

i) Constructing Genomic Libraries using Plasmids ................................. 316

Types of Vectors .................................................................................. 316

Steps: .................................................................................................... 316

ii) Constructing Genomic Libraries using Bacteriophage Vectors .......... 318

iii) Constructing Genomic Libraries with Yeast ArtificialChromosomes ....................................................................................... 320

Applications of Genomic Libraries ...................................................... 320

cDNA Library ............................................................................................. 321

Construction of Library ........................................................................ 322

Uses of cDNA Library ........................................................................ 325

10. CANCER BIOLOGY..............................................................................326

Introduction ................................................................................................. 326

Historical Perspective ................................................................................. 327

Classification of Cancer ....................................................................... 330

Signs and Symptoms ............................................................................ 331

Causes of Cancer ....................................................................................... 331

Mechanism of Cancer ................................................................................ 335

Epigenetics ............................................................................................ 336

Oncogenes ............................................................................................ 337

Proto-oncogene .................................................................................... 338

Activation .............................................................................................. 338

Conversion of Proto-oncogenes ................................................................. 338

Tumor Suppressor Genes ..................................................................... 339

Protein 53 ............................................................................................. 340

Structure of p53 ................................................................................... 341

Significance of p53 ............................................................................... 342

Regulation of p53 Activity ................................................................... 342

Role in Disease .................................................................................... 343

Biological Properties of Cancer Cells ........................................................ 344

xx

Comparison to Non-biological Organisms .................................................. 345

Prevention of Cancer ................................................................................. 345

Diagnosis .............................................................................................. 348

Treatment .............................................................................................. 349

Control of Symptom ............................................................................. 351

11. MOLECULAR EVOLUTION ..............................................................354

Beginning of Life ........................................................................................ 354

RNA World ................................................................................................. 356

DNA World ................................................................................................. 357

Phylogenetic Analysis ................................................................................. 358

Evolution of Eukaryotic Cells ..................................................................... 358

GLOSSARY ...............................................................................................361

SUBJECT INDEX ...................................................................................376

LIST OF BOX

1. Molecule ................................................................................................. 16

2. Nucleosides, Phosphate and Nucleotides .............................................. 34

3. X-ray crystallography and Watson and Crick ....................................... 60

4. Ubiquitin ................................................................................................ 160

5. Genetic Sex .......................................................................................... 249

6. History of Sex Determination in Insects ............................................. 251

7. Sex Determination in Amphibian ......................................................... 258

8. Genetic Characters Due to Chromosomal Abnormalitiy ..................... 289

9. Important Terms of Cancer Biology .................................................... 329

Preface

The present book entitled A Text Book of “Molecular Cytogenetics” aimsto cover a wide area of molecular biology and cytogenetic biology in aform especially suitable for graduate (PG) and undergraduate students

(UG). We have deliberately kept the book to a manageable size so that neitherthe cost and content nor the weight is too overwhelming for the student. Thisbook will widely help to students, teachers, academicians to aid the knowledgeabout the course and beyond the course related to the subject matter as givenin the box. Present book contains 11 chapters. We begin by describing the historyof molecular and cytogenetic biology. Further, we turn to the nucleic acid, typesand its structure. The chapter 2 describes replication of DNA and enzymology,chapter 3 dscribes the transcription and modification and translation of protein isdiscussed in chapter 4. The next chapter 5 has DNA damage and repairmechanisms. The cytogenetics describes in the chapter 6 and sex determinationand differentiation is discussed in chapter 7. The chapter 8 has karyotyping andchromosomal disorder, chapter 9 has human genome project and gene library.The chapter 10 has cancer biology, in which the role of proto-oncogenes andoncogenes, its conversion, cancer treatment, drugs and therapy is discussed.Finally, the chapter 11 includes the molecular evolution. Boxed material throughoutthe book is divided into examples to illustrate the topics covered in the main text,explanations of the medical relevance of the material and in depth sections thatextend the coverage beyond the content of the main text.

Ashok Kumar(Email: asokumr@gmail.com)

Meena Srivastava(Email: meena.zoology@gmail.com)

Acknowledgements

By the grace of almighty, we have reached this opportunity to thank allthose who have been instrumental in bringing this book to completion.

It’s our immense pleasure to thank Dr. Ramanand Prasad, Director HigherEducation, Uttar Pradesh (India) for motivation to write this manuscript. Weexpress our sincere gratitude to Dr. D.K. Singh, Principal, M.P. Govt. (PG)

College, Hardoi (UP) India, Prof. N. Singh Dept. of Biotechnology and Zoology,Prof. S.C. Tiwari and Prof. J.P. Mehta, Dept. of Microbiology and BotanyHNBGU (A Central University), Srinagar-Garhwal (Uttarakhand), India for theircritical suggestions and inspirations. Our special thanks to Prof. B.S. Bisht, Dept.of Zoology, HNBGU (A Central University), SRT Campus Badshahithaul Tehri(Uttarakhand) India, Dr. D. K. Srivastava, Principal, Govt PG College, Rajajipuram,Lucknow (India), Dr. I.D. Singh, Ex Head, Dept. of Botany, M. P. Govt (PG)College Hardoi (UP) India, Dr. V.D. Joshi, Principal, Govt PG College Purola,Uttarkashi (Uttarakhand) India, Dr. Arun Bhatt, Dr. Harish Chandra and Dr.Manoj Bhatt, Dept. of Biotechnology, G.B. Pant Engineering College Pauri-Garhwal,Dr. Arun Kumar, Director Research, Dolphin Institute of Biomedical and NaturalSciences, Dr. A.K. Singh, Scientist E, Wadia Institute of Himalayan Geology,Dehradun, Dr. S.P. Goyal, Scientist F and Head, Forensic Science Laboratory,Wild Life Institute of India, Dehradun, Er. P. Dwivedi Dept. of Biothchnology,Amity Institute Noida, India, Dr. Saswat Katiyar Dept. of Biochemistry, CSJMUKanpur (UP) India, Dr. Sandip Kumar and Dr. Sandip Tripathi, NIMS UniversityJaipur India, Dr. Ashish Thapliyal, Scientist State Biotech Program Utterakhand,Prof. U.K. Atheya, Dr. N. Rai Dept. of Biotech, GEU Dehradun, Dr. AftabAhmad, Director Beehive College Dehradun, Dr. G. Sunil Babu, Dept. of Env.Biothech BRA University Lucknow (U.P.), Dr. M.S. Khan, Dept. of MicrobiologyAMU. Aligarh, Dr. A. K. Chopra, Dr. P.C. Joshi, Dept. of Zoology, GurukulKangari University Haridwar, Dr. R. Kumar, Scientist, IARI Delhi, Dr. SanjayGupta Dept. of Biotech, SBS College Dehradun, for discussions and valuablecomments which have helped to improve the matter.

xxiv

We thank our colleagues Drs. Kiran Yadav, Archana Rajan, Anju Agarwal,Sangeeta Awasthi, V.L. Saxena, V.K. Dhatwalia, Pankaj Dhiman, Dinesh Kumarand Navneet Kumar and for their scientific discussions and valuable company tokeep cheerful and focused. We sincerely appreciate the help and support renderedby Mrs. Kanchan Prabha Rathoure, Er. Jitendra Kumar and Mr. Atul KumarSingh. Our sincere thanks to all other friends and colleagues whose names wehave not mentioned here for want of space.

Thank you all forever!!!

Authors