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: [email protected])
Meena Srivastava(Email: [email protected])
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