Molecular Biotechnology

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

• ISBN13: 9781555812249
• ISBN10: 1555812244
• Edition: 3rd
• Format: Hardcover
• Copyright: 2003-03-01
• Publisher: Amer Society for Microbiology

Summary

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

Author Biography

Bernard R. Glick, University of Waterloo, Waterloo, Ontario, Canada Jack J. Pasternak, University of Waterloo, Waterloo, Ontario, Canada

Table of Contents

Preface	xix
Preface to the First Edition	xxi
I Fundamentals of Molecular Biotechnology	1	(224)
The Molecular Biotechnology Revolution	3	(11)
Recombinant DNA Technology	3	(2)
Emergence of Molecular Biotechnology	5	(2)
Commercialization of Molecular Biotechnology	7	(3)
Concerns and Consequences	10	(4)
Summary	12	(1)
References	13	(1)
Review Questions	13	(1)
Molecular Biotechnology Biological Systems	14	(9)
Prokaryotic and Eukaryotic Organisms	14	(1)
Escherichia coli	15	(2)
Saccharomyces cerevisiae	17	(1)
Secretion Pathways in Prokaryotic and Eukaryotic Organisms	18	(3)
Eukaryotic Cells in Culture	21	(2)
Summary	22	(1)
References	22	(1)
Review Questions	22	(1)
DNA, RNA, and Protein Synthesis	23	(24)
Structure of DNA	23	(4)
DNA Replication	27	(1)
Decoding Genetic Information: RNA and Protein	28	(6)
Translation	34	(4)
Regulation of mRNA Transcription in Bacteria	38	(4)
Regulation of mRNA Transcription in Eukaryotes	42	(5)
Summary	45	(1)
References	46	(1)
Review Questions	46	(1)
Recombinant DNA Technology	47	(44)
Restriction Endonucleases	49	(8)
Plasmid Cloning Vectors	57	(7)
Plasmid Cloning Vector pBR322	58	(2)
Transformation and Selection	60	(1)
Other Plasmid Cloning Vectors	61	(3)
Creating and Screening a Library	64	(11)
Making a Gene Library	64	(3)
Screening by DNA Hybridization	67	(6)
Screening by Immunological Assay	73	(1)
Screening by Protein Activity	73	(2)
Cloning DNA Sequences That Encode Eukaryotic Proteins	75	(5)
Vectors for Cloning Large Pieces of DNA	80	(6)
Bacteriophage λ Vectors	80	(3)
Cosmids	83	(3)
High-Capacity Bacterial Vector Systems	86	(1)
Genetic Transformation of Prokaryotes	86	(5)
Transferring DNA into E. coli	86	(1)
Electroporation	86	(1)
Conjugation	87	(2)
Summary	89	(1)
References	89	(1)
Review Questions	90	(1)
Chemical Synthesis, Sequencing, and Amplification of DNA	91	(30)
Chemical Synthesis of DNA	91	(10)
The Phosphoramidite Method	92	(4)
Uses of Synthesized Oligonucleotides	96	(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