Molecular Biotechnology

, by Glick, Bernard R.; Pasternak, Jack J.

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ISBN: 9781555812249 | 1555812244
Cover: Hardcover
Copyright: 3/1/2003

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Completely revised, updated, and expanded, the third edition of the best-selling Molecular Biotechnology: Principles and Applications of Recombinant DNA covers the underlying scientific principles and deals extensively with the many industrial, agricultural, pharmaceutical, and biomedical applications of recombinant DNA technology. The latest edition offers greatly expanded coverage of directed mutagenesis and protein engineering, therapeutic agents, and genetic engineering of plants. Updated chapters reflect recent developments in biotechnology and the societal issues related to it, such as cloning, gene therapy, and patenting and releasing genetically engineered organisms. Over 500 figures, including 200 that are new in this edition, illustrate all key concepts. "Milestones" summarize important research papers in the history of biotechnology and their effects on the field. As with previous editions, the authors clearly explain all concepts and techniques to provide m

Preface

xix

Preface to the First Edition

xxi

I Fundamentals of Molecular Biotechnology

(224)

The Molecular Biotechnology Revolution

(11)

Recombinant DNA Technology

(2)

Emergence of Molecular Biotechnology

(2)

Commercialization of Molecular Biotechnology

(3)

Concerns and Consequences

(4)

Summary

(1)

References

(1)

Review Questions

(1)

Molecular Biotechnology Biological Systems

(9)

Prokaryotic and Eukaryotic Organisms

(1)

Escherichia coli

(2)

Saccharomyces cerevisiae

(1)

Secretion Pathways in Prokaryotic and Eukaryotic Organisms

(3)

Eukaryotic Cells in Culture

(2)

Summary

(1)

References

(1)

Review Questions

(1)

DNA, RNA, and Protein Synthesis

(24)

Structure of DNA

(4)

DNA Replication

(1)

Decoding Genetic Information: RNA and Protein

(6)

Translation

(4)

Regulation of mRNA Transcription in Bacteria

(4)

Regulation of mRNA Transcription in Eukaryotes

(5)

Summary

(1)

References

(1)

Review Questions

(1)

Recombinant DNA Technology

(44)

Restriction Endonucleases

(8)

Plasmid Cloning Vectors

(7)

Plasmid Cloning Vector pBR322

(2)

Transformation and Selection

(1)

Other Plasmid Cloning Vectors

(3)

Creating and Screening a Library

(11)

Making a Gene Library

(3)

Screening by DNA Hybridization

(6)

Screening by Immunological Assay

(1)

Screening by Protein Activity

(2)

Cloning DNA Sequences That Encode Eukaryotic Proteins

(5)

Vectors for Cloning Large Pieces of DNA

(6)

Bacteriophage λ Vectors

(3)

Cosmids

(3)

High-Capacity Bacterial Vector Systems

(1)

Genetic Transformation of Prokaryotes

(5)

Transferring DNA into E. coli

(1)

Electroporation

(1)

Conjugation

(2)

Summary

(1)

References

(1)

Review Questions

(1)

Chemical Synthesis, Sequencing, and Amplification of DNA

(30)

Chemical Synthesis of DNA

(10)

The Phosphoramidite Method

(4)

Uses of Synthesized Oligonucleotides

(5)

DNA Sequencing Techniques

101

(9)

Dideoxynucleotide Procedure for Sequencing DNA

102

(4)

Automated DNA Sequencing

106

(1)

Using Bacteriophage M13 as a DNA Sequencing Vector

107

(3)

Primer Walking

110

(1)

PCR

110

(11)

Gene Synthesis by PCR

115

(1)

Cycle Sequencing

115

(3)

Summary

118

(1)

References

119

(1)

Review Questions

120

(1)

Manipulation of Gene Expression in Prokaryotes

121

(42)

Gene Expression from Strong and Regulatable Promoters

122

(8)

Regulatable Promoters

122

(4)

Increasing Protein Production

126

(1)

Large-Scale Systems

126

(2)

Expression in Other Microorganisms

128

(2)

Fusion Proteins

130

(6)

Cleavage of Fusion Proteins

131

(1)

Uses of Fusion Proteins

131

(4)

Surface Display

135

(1)

Unidirectional Tandem Gene Arrays

136

(2)

Translation Expression Vectors

138

(4)

Increasing Protein Stability

142

(1)

Protein Folding

142

(3)

Overcoming Oxygen Limitation

145

(1)

Use of Protease-Deficient Host Strains

145

(1)

Bacterial Hemoglobin

145

(1)

DNA Integration into the Host Chromosome

146

(5)

Removing Selectable Marker Genes

149

(2)

Increasing Secretion

151

(4)

L-Form Bacteria

153

(2)

Metabolic Load

155

(8)

Summary

159

(1)

References

159

(2)

Review Questions

161

(2)

Heterologous Protein Production in Eukaryotic Cells

163

(27)

Saccharomyces cerevisiae Expression Systems

165

(6)

S. cerevisiae Vectors

166

(3)

Intracellular Production of Heterologous Proteins in S. cerevisiae

169

(2)

Secretion of Heterologous Proteins by S. cerevisiae

171

(1)

Pichia pastoris and Other Yeast Expression Systems

171

(3)

Baculovirus--Insect Cell Expression System

174

(7)

Baculovirus Expression Vector System

175

(1)

Increasing the Yield of Recombinant Baculovirus

175

(2)

Construction of an E. coli--Insect Cell Baculovirus Shuttle Vector

177

(3)

Mammalian Glycosylation and Processing of Precursor Proteins in Insect Cells

180

(1)

Mammalian Cell Expression Systems

181

(9)

Selectable Marker Systems for Mammalian Expression Vectors

185

(2)

Summary

187

(1)

References

187

(2)

Review Questions

189

(1)

Directed Mutagenesis and Protein Engineering

190

(35)

Directed Mutagenesis Procedures

191

(14)

Oligonucleotide-Directed Mutagenesis with M13 DNA

192

(2)

Oligonucleotide-Directed Mutagenesis with Plasmid DNA

194

(1)

PCR-Amplified Oligonucleotide-Directed Mutagenesis

195

(3)

Random Mutagenesis with Degenerate Oligonucleotide Primers

198

(3)

Random Mutagenesis with Nucleotide Analogues

201

(1)

Error-Prone PCR

201

(2)

DNA Shuffling

203

(1)

Mutant Proteins with Unusual Amino Acids

204

(1)

Protein Engineering

205

(20)

Adding Disulfide Bonds

205

(4)

Changing Asparagine to Other Amino Acids

209

(1)

Reducing the Number of Free Sulfhydryl Residues

209

(2)

Increasing Enzymatic Activity

211

(1)

Modifying Metal Cofactor Requirements

212

(2)

Decreasing Protease Sensitivity

214

(1)

Modifying Protein Specificity

215

(3)

Increasing Enzyme Stability and Specificity

218

(1)

Altering Multiple Properties

219

(2)

Summary

221

(1)

References

222

(1)

Review Questions

223

(2)

II Molecular Biotechnology of Microbial Systems

225

(286)

Molecular Diagnostics

227

(29)

Immunological Diagnostic Procedures

228

(2)

Enzyme-Linked Immunosorbent Assay

229

(1)

Monoclonal Antibodies

230

(4)

Formation and Selection of Hybrid Cells

231

(1)

Identification of Specific Antibody-Producing Hybrid Cell Lines

232

(2)

DNA Diagnostic Systems

234

(11)

Hybridization Probes

235

(1)

Diagnosis of Malaria

236

(1)

Detection of Trypanosoma cruzi

236

(1)

Nonradioactive Hybridization Procedures

237

(3)

Molecular Beacons

240

(1)

DNA Fingerprinting

241

(1)

Random Amplified Polymorphic DNA

242

(2)

Bacterial Biosensors

244

(1)

Molecular Diagnosis of Genetic Disease

245

(11)

Screening for Cystic Fibrosis

246

(1)

Sickle-Cell Anemia

246

(2)

The PCR/OLA Procedure

248

(2)

Padlock Probes

250

(1)

Genotyping with Fluorescence-Labeled PCR Primers

250

(3)

Summary

253

(1)

References

253

(1)

Review Questions

254

(2)

Therapeutic Agents

256

(53)

Pharmaceuticals

257

(6)

Isolation of Interferon cDNAs

257

(1)

Human Interferons

258

(1)

Human Growth Hormone

259

(1)

Tumor Necrosis Factor Alpha

260

(1)

Optimizing Gene Expression

261

(1)

Therapeutics Produced and Delivered by Intestinal Bacteria

262

(1)

Enzymes

263

(4)

DNase I

263

(2)

Alginate Lyase

265

(1)

Phenylalanine Ammonia Lyase

266

(1)

α1-Antitrypsin

267

(1)

Monoclonal Antibodies as Therapeutic Agents

267

(10)

Structure and Function of Antibodies

269

(1)

Preventing Rejection of Transplanted Organs

270

(1)

Treating Brain Tumors

270

(1)

Chemically Linked Monoclonal Antibodies

271

(1)

Human Monoclonal Antibodies

272

(2)

Hybrid Human-Mouse Monoclonal Antibodies

274

(3)

Production of Antibodies in E. coli

277

(6)

Phage Combinatorial Libraries

277

(3)

Shuffling CDR Sequences

280

(2)

Single-Chain Antibodies

282

(1)

Nucleic Acids as Therapeutic Agents

283

(12)

Antisense RNA

284

(1)

Antisense Oligonucleotides

284

(4)

Ribozymes

288

(3)

Chimeric RNA--DNA Molecules

291

(1)

Interfering RNAs

292

(2)

Antibody Genes

294

(1)

Treating Genetic Disorders

295

(8)

Human Gene Therapy

298

(5)

Prodrug Activation Therapy

303

(6)

Summary

304

(1)

References

305

(2)

Review Questions

307

(2)

Vaccines

309

(31)

Subunit Vaccines

312

(10)

Herpes Simplex Virus

312

(1)

Foot-and-Mouth Disease

313

(2)

Peptide Vaccines

315

(2)

Genetic Immunization: DNA Vaccines

317

(5)

Attenuated Vaccines

322

(5)

Cholera

322

(2)

Salmonella Species

324

(1)

Leishmania Species

325

(1)

Herpes Simplex Virus

326

(1)

Vector Vaccines

327

(13)

Vaccines Directed against Viruses

327

(6)

Vaccines Directed against Bacteria

333

(2)

Bacteria as Antigen Delivery Systems

335

(1)

Summary

336

(1)

References

337

(1)

Review Questions

338

(2)

Synthesis of Commercial Products by Recombinant Microorganisms

340

(38)

Restriction Endonucleases

340

(4)

Small Biological Molecules

344

(11)

Synthesis of L-Ascorbic Acid

345

(4)

Microbial Synthesis of Indigo

349

(1)

Synthesis of Amino Acids

350

(4)

Removal of Lipids

354

(1)

Antibiotics

355

(12)

Cloning Antibiotic Biosynthesis Genes

356

(4)

Synthesis of Novel Antibiotics

360

(1)

Engineering Polyketide Antibiotics

361

(4)

Improving Antibiotic Production

365

(2)

Peptide Antibiotics

367

(1)

Biopolymers

367

(11)

Engineering Xanthomonas campestris for Xanthan Gum Production

367

(2)

Isolation of Melanin Biosynthesis Genes

369

(1)

Synthesis of an Animal Adhesive Biopolymer in Microbial Cells

370

(2)

Microbial Synthesis of Rubber

372

(1)

Microbial Production of Polyhydroxyalkanoates

372

(3)

Summary

375

(1)

References

375

(2)

Review Questions

377

(1)

Bioremediation and Biomass Utilization

378

(38)

Microbial Degradation of Xenobiotics

378

(4)

Genetic Engineering of Biodegradative Pathways

382

(12)

Manipulation by Transfer of Plasmids

384

(1)

Manipulation by Gene Alteration

385

(9)

Utilization of Starch and Sugars

394

(11)

Commercial Production of Fructose and Alcohol

395

(1)

Improving Alcohol Production

396

(2)

Improving Fructose Production

398

(2)

Zymomonas mobilis

400

(4)

Silage Fermentation

404

(1)

Utilization of Cellulose

405

(11)

Components of Lignocellulose

406

(2)

Isolation of Prokaryotic Cellulase Genes

408

(2)

Isolation of Eukaryotic Cellulase Genes

410

(2)

Manipulation of Cellulase Genes

412

(1)

Summary

413

(1)

References

414

(1)

Review Questions

415

(1)

Plant Growth-Promoting Bacteria

416

(39)

Nitrogen Fixation

417

(10)

Nitrogenase

419

(1)

Components of Nitrogenase

419

(2)

Genetic Engineering of the Nitrogenase Gene Cluster

421

(4)

Glycogen Synthase Mutants

425

(2)

Engineering Oxygen Levels

427

(1)

Hydrogenase

427

(3)

Hydrogen Metabolism

428

(1)

Genetic Engineering of Hydrogenase Genes

428

(2)

Nodulation

430

(5)

Competition among Nodulating Organisms

430

(1)

Genetic Engineering of Nodulation Genes

431

(4)

Growth Promotion by Free-Living Bacteria

435

(5)

Decreasing Plant Stress

438

(2)

Phytoremediation

440

(2)

Biocontrol of Pathogens

442

(13)

Siderophores

443

(2)

Antibiotics

445

(2)

Enzymes

447

(1)

Ice Nucleation and Antifreeze Proteins

447

(2)

Ethylene

449

(2)

Root Colonization

451

(1)

Summary

451

(1)

References

452

(1)

Review Questions

453

(2)

Microbial Insecticides

455

(26)

Insecticidal Toxin of B. thuringiensis

456

(17)

Mode of Action and Use

456

(4)

Toxin Gene Isolation

460

(2)

Engineering of B. thuringiensis Toxin Genes

462

(8)

Preventing the Development of Resistance

470

(2)

Genetic Engineering for Improved Biocontrol

472

(1)

Baculoviruses as Biocontrol Agents

473

(8)

Mode of Action

473

(2)

Genetic Engineering for Improved Biocontrol

475

(2)

Summary

477

(1)

References

478

(2)

Review Questions

480

(1)

Large-Scale Production of Proteins from Recombinant Microorganisms

481

(30)

Principles of Microbial Growth

483

(5)

Batch Fermentation

483

(2)

Fed-Batch Fermentation

485

(1)

Continuous Fermentation

486

(2)

Maximizing the Efficiency of the Fermentation Process

488

(2)

High-Density Cell Cultures

489

(1)

Bioreactors

490

(4)

Typical Large-Scale Fermentation Systems

494

(5)

Two-Stage Fermentation in Tandem Airlift Reactors

495

(1)

Two-Stage Fermentation in a Single Stirred-Tank Reactor

496

(1)

Batch versus Fed-Batch Fermentation

497

(2)

Harvesting Microbial Cells

499

(3)

Disrupting Microbial Cells

502

(2)

Downstream Processing

504

(2)

Protein Solubilization

506

(1)

Large-Scale Production of Plasmid DNA

506

(5)

Summary

507

(1)

References

507

(2)

Review Questions

509

(2)

III Eukaryotic Systems

511

(160)

Genetic Engineering of Plants: Methodology

513

(25)

Plant Transformation with the Ti Plasmid of Agrobacterium tumefaciens

514

(3)

Ti Plasmid-Derived Vector Systems

517

(3)

Physical Methods of Transferring Genes to Plants

520

(2)

Microprojectile Bombardment

522

(1)

Use of Reporter Genes in Transformed Plant Cells

523

(2)

Manipulation of Gene Expression in Plants

525

(7)

Isolation and Use of Different Promoters

526

(1)

Targeted Alterations in Plant DNA

527

(1)

Targeted Alterations in Plant RNA

528

(1)

Targeting Foreign DNA to the Chloroplast Genome

529

(3)

Secretion of Foreign Proteins

532

(1)

Production of Marker-Free Transgenic Plants

532

(6)

Removing Marker Genes from Nuclear DNA

533

(1)

Removing Marker Genes from Chloroplast DNA

534

(1)

Summary

535

(1)

References

535

(2)

Review Questions

537

(1)

Genetic Engineering of Plants: Applications

538

(56)

Development of Insect-, Pathogen-, and Herbicide-Resistant Plants

538

(22)

Insect-Resistant Plants

538

(9)

Virus-Resistant Plants

547

(7)

Herbicide-Resistant Plants

554

(2)

Fungus- and Bacterium-Resistant Plants

556

(4)

Development of Stress- and Senescence-Tolerant Plants

560

(6)

Oxidative Stress

561

(1)

Salt Stress

562

(2)

Fruit Ripening and Flower Wilting

564

(2)

Genetic Manipulation of Flower Pigmentation

566

(3)

Modification of Plant Nutritional Content

569

(8)

Amino Acids

569

(2)

Lipids

571

(2)

Vitamins

573

(2)

Iron

575

(2)

Modification of Food Plant Taste and Appearance

577

(5)

Preventing Discoloration

577

(1)

Sweetness

578

(1)

Starch

579

(3)

Plants as Bioreactors

582

(3)

Antibodies

583

(1)

Polymers

583

(1)

Foreign Proteins in Plants

584

(1)

Edible Vaccines

585

(1)

Plant Yield

586

(8)

Increasing Iron Content

587

(1)

Altering Lignin Content

587

(1)

Increasing Oxygen Content

588

(1)

Summary

589

(1)

References

590

(3)

Review Questions

593

(1)

Transgenic Animals

594

(39)

Transgenic Mice: Methodology

596

(13)

Retroviral Vector Method

596

(1)

DNA Microinjection Method

596

(3)

Engineered Embryonic Stem Cell Method

599

(4)

Genetic Modification with the Cre-loxP Recombination System

603

(4)

Transgenesis with High-Capacity Vectors

607

(2)

Transgenic Mice: Applications

609

(7)

Transgenic Models for Alzheimer Disease

610

(1)

Using Transgenic Mice as Test Systems

611

(2)

Conditional Regulation of Gene Expression

613

(2)

Conditional Control of Cell Death

615

(1)

Cloning Livestock by Nuclear Transfer

616

(2)

Transgenic Cattle, Sheep, Goats, and Pigs

618

(5)

Transgenic Birds

623

(3)

Transgenic Fish

626

(7)

Summary

629

(1)

References

630

(2)

Review Questions

632

(1)

Human Molecular Genetics

633

(38)

Modes of Human Inheritance

634

(2)

Genetic Linkage and Gene Mapping

636

(3)

Detection and Estimation of Genetic Linkage in Humans

639

(4)

Genetic Mapping of Human Chromosomes

643

(7)

Genetic Polymorphism

643

(1)

Restriction Fragment Length Polymorphism

644

(3)

Short Tandem Repeat Polymorphism

647

(3)

Comprehensive Human Linkage Maps

650

(2)

Mapping of a Genetic Disease Locus to a Chromosome Location

650

(2)

Whole-Genome BAC Map

652

(3)

Radiation Hybrid Mapping

653

(2)

Human Genome Sequence

655

(2)

Detection of Mutations in Human Genes

657

(6)

Single-Strand Conformation Analysis

657

(2)

Denaturing Gradient Gel Electrophoresis

659

(1)

Heteroduplex Analysis

660

(1)

Chemical Mismatch Cleavage

661

(2)

Direct DNA Sequencing

663

(1)

Determining Gene Function

663

(8)

Summary

668

(1)

References

669

(1)

Review Questions

670

(1)

IV Regulating and Patenting Molecular Biotechnology

671

(36)

Regulating the Use of Biotechnology

673

(24)

Regulating Recombinant DNA Technology

674

(2)

Regulating Food and Food Ingredients

676

(4)

Chymosin

676

(1)

Tryptophan

677

(1)

Bovine Somatotropin

678

(2)

Deliberate Release of GMOs

680

(5)

Ice-Minus P. syringae

680

(2)

Open-Field Tests of Other GMOs

682

(3)

Controversy about GMOs

685

(3)

Human Gene Therapy

688

(9)

Development of a Policy for Somatic Cell Gene Therapy

688

(3)

Accumulation of Defective Genes in Future Generations

691

(1)

Human Germ Line Gene Therapy

692

(1)

Cloning Humans?

692

(1)

Summary

693

(1)

References

694

(1)

Review Questions

695

(2)

Patenting Biotechnology Inventions

697

(10)

Patenting

698

(2)

Patenting in Different Countries

700

(1)

Patenting DNA Sequences

701

(1)

Patenting Multicellular Organisms

702

(1)

Patenting and Fundamental Research

703

(4)

Summary

704

(1)

References

705

(1)

Review Questions

705

(2)

Glossary

707

(26)

Index

733

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Molecular Biotechnology

Summary

Author Biography

Table of Contents

Supplemental Materials