Karps Cell Biology / Janet Iwasa and Wallace F Marshall

1 Introduction to Cell Biology 11.1 The Discovery of Cells 2Microscopy 2Cell Theory 31.2 Basic Properties of Cells 3Cells are Highly Complex and Organized 3Cells Possess a Genetic Program and the Means to Use It 5Cells Are Capable of Producing More of Themselves 5Cells Acquire and Utilize Energy 5Cells Carry Out a Variety of Chemical Reactions 6Cells Engage in Mechanical Activities 6Cells are Able to Respond to Stimuli 6Cells Are Capable of Self-Regulation 6Cells Evolve 71.3 Two Fundamentally Different Classes of Cells 81.4 Types of Prokaryotic Cells 14Domain Archaea and Domain Bacteria 14Prokaryotic Diversity 141.5 Types of Eukaryotic Cells 15Cell Differentiation 16Model Organisms 161.6 The Sizes of Cells and Their Components 181.7 Viruses 19Viroids 22THE HUMAN PERSPECTIVE 23The Prospect of Cell Replacement Therapy 23EXPERIMENTAL PATHWAYS 27The Origin of Eukaryotic Cells 272 The Structure and Functions of Biological Molecules 332.1 Covalent Bonds 34Polar and Nonpolar Molecules 35Ionization 362.2 Noncovalent Bonds 36Ionic Bonds: Attractions between Charged Atoms 36Hydrogen Bonds 36Hydrophobic Interactions and van der Waals Forces 37The Life-Supporting Properties of Water 382.3 Acids, Bases, and Buffers 392.4 The Nature of Biological Molecules 40Functional Groups 41A Classification of Biological Molecules by Function 412.5 Carbohydrates 42The Structure of Simple Sugars 43Stereoisomerism 43Linking Sugars Together 44Polysaccharides 452.6 Lipids 47Fats 47Steroids 48Phospholipids 482.7 Building Blocks of Proteins 49The Structures of Amino Acids 50The Properties of the Side Chains 512.8 Primary and Secondary Structures of Proteins 54Primary Structure 54Secondary Structure 552.9 Tertiary Structure of Proteins 56Myoglobin: The First Globular Protein Whose Tertiary Structure Was Determined 57Tertiary Structure May Reveal Unexpected Similarities between Proteins 58Protein Domains 58Dynamic Changes within Proteins 592.10 Quaternary Structure of Proteins 60The Structure of Hemoglobin 60Protein-Protein Interactions 612.11 Protein Folding 61Dynamics of Protein Folding 62The Role of Molecular Chaperones 632.12 Proteomics and Interactomics 64Proteomics 64Interactomics 662.13 Protein Engineering 67Production of Novel Proteins 67Structure-Based Drug Design 682.14 Protein Adaptation and Evolution 692.15 Nucleic Acids 712.16 The Formation of Complex Macromolecular Structures 72The Assembly of Tobacco Mosaic Virus Particles 73The Assembly of Ribosomal Subunits 73THE HUMAN PERSPECTIVE 73I. Do Free Radicals Cause Aging? 73II. Protein Misfolding Can Have Deadly Consequences 74EXPERIMENTAL PATHWAYS 79Chaperones-Helping Proteins Reach Their Proper Folded State 793 Bioenergetics, Enzymes, and Metabolism 873.1 Bioenergetics 88The First Law of Thermodynamics 88The Second Law of Thermodynamics 893.2 Free Energy 90Free?Energy Changes in Chemical Reactions 91Free?Energy Changes in Metabolic Reactions 923.3 Coupling Endergonic and Exergonic Reactions 943.4 Equilibrium versus Steady?State Metabolism 943.5 Enzymes as Biological Catalysts 95The Properties of Enzymes 96Overcoming the Activation Energy Barrier 96The Active Site 983.6 Mechanisms of Enzyme Catalysis 99Substrate Orientation 100Changing Substrate Reactivity 100Inducing Strain in the Substrate 1003.7 Enzyme Kinetics 103The Michaelis?Menten Model of Enzyme Kinetics 103Enzyme Inhibitors 1053.8 Metabolism 106Oxidation and Reduction: A Matter of Electrons 107The Capture and Utilization of Energy 1083.9 Glycolysis and ATP Production 108ATP Production in Glycolysis 109Anaerobic Oxidation of Pyruvate: The Process of Fermentation 1113.10 Reducing Power 1123.11 Metabolic Regulation 113Altering Enzyme Activity by Covalent Modification 113Altering Enzyme Activity by Allosteric Modulation 1133.12 Separating Catabolic and Anabolic Pathways 114THE HUMAN PERSPECTIVE 115I. The Growing Problem of Antibiotic Resistance 115II. Caloric Restriction and Longevity 1184 Genes, Chromosomes, and Genomes 1234.1 The Concept of a Gene as a Unit of Inheritance 1244.2 The Discovery of Chromosomes 1254.3 Chromosomes: The Physical Carriers of the Genes 126The Chromosome as a Linkage Group 1274.4 Genetic Analysis in Drosophila 127Crossing Over and Recombination 128Mutagenesis and Giant Chromosomes 1294.5 The Structure of DNA 129The Watson?Crick Proposal 132The Importance of the Watson?Crick Proposal 1324.6 DNA Supercoiling 1344.7 The Structure of the Genome 136DNA Denaturation 137DNA Renaturation 1374.8 The Stability of the Genome 141Whole?Genome Duplication (Polyploidization) 141Duplication and Modification of DNA Sequences 141Evolution of Globin Genes 1424.9 "Jumping Genes" and the Dynamic Nature of the Genome 143Transposons 144The Role of Mobile Genetic Elements in Genome Evolution 1444.10 Sequencing Genomes: The Footprints of Biological Evolution 1464.11 Comparative Genomics: "If It's Conserved, It Must Be Important" 1484.12 The Genetic Basis of "Being Human" 1484.13 Genetic Variation within the Human Species Population 150DNA Sequence Variation 150Structural Variation 151Copy Number Variation 152THE HUMAN PERSPECTIVE 152I. Diseases That Result from Expansion of Trinucleotide Repeats 152II. Application of Genomic Analyses to Medicine 154EXPERIMENTAL PATHWAYS 157The Chemical Nature of the Gene 1575 The Path to Gene Expression 1655.1 The Relationship between Genes, Proteins, and RNAs 166Evidence That DNA Is the Genetic Material 166An Overview of the Flow of Information through the Cell 1675.2 The Role of RNA Polymerases in Transcription 1695.3 An Overview of Transcription in Both Prokaryotic and Eukaryotic Cells 171Transcription in Bacteria 171Transcription and RNA Processing in Eukaryotic Cells 1725.4 Synthesis and Processing of Eukaryotic Ribosomal and Transfer RNAs 174Synthesis and Processing of the rRNA Precursor 174The Role of snoRNAs in the Processing of Pre?rRNA 176Synthesis and Processing of the 5S rRNA 176Transfer RNAs 1775.5 Synthesis and Structure of Eukaryotic Messenger RNAs 178The Formation of Heterogeneous Nuclear RNA (hnRNA) 178The Machinery for mRNA Transcription 178The Structure of mRNAs 1815.6 Split Genes: An Unexpected Finding 1815.7 The Processing of Eukaryotic Messenger RNAs 1845' Caps and 3' Poly(A) Tails 185RNA Splicing: Removal of Introns from a Pre?RNA 1865.8 Evolutionary Implications of Split Genes and RNA Splicing 1895.9 Creating New Ribozymes in the Laboratory 1915.10 Small Regulatory RNAs and RNA Silencing Pathway 1915.11 Small RNAs: miRNAs and piRNAs 193miRNAs: A Class of Small RNAs that Regulate Gene Expression 193piRNAs: A Class of Small RNAs that Function in Germ Cells 1945.12 CRISPR and other Noncoding RNAs 195CRISPR: Noncoding RNA in Bacteria 195Other Noncoding RNAs 1955.13 Encoding Genetic Information 196The Properties of the Genetic Code 196Identifying the Codons 1975.14 Decoding the Codons: The Role of Transfer RNAs 198The Structure of tRNAs 198tRNA Charging 2005.15 Translating Genetic Information: Initiation 201Initiation of Translation in Prokaryotes 202Initiation of Translation in Eukaryotes 203The Role of the Ribosome 2035.16 Translating Genetic Information: Elongation and Termination 205Elongation Step 1: Aminoacyl?tRNA Selection 205Elongation Step 2: Peptide Bond Formation 205Elongation Step 3: Translocation 205Elongation Step 4: Releasing the Deacylated tRNA 206Termination 2075.17 mRNA Surveillance and Quality Control 2085.18 Polyribosomes 209THE HUMAN PERSPECTIVE 210Clinical Applications of RNA Interference 210EXPERIMENTAL PATHWAYS 212The Role of RNA as a Catalyst 2126 Controlling Gene Expression 2206.1 Control of Gene Expression in Bacteria 221Organization of Bacterial Genomes 221The Bacterial Operon 221Riboswitches 2246.2 Control of Gene Expression in Eukaryotes: Structure and Function of the Cell Nucleus 225The Nuclear Pore Complex and Its Role in Nucleocytoplasmic Trafficking 227RNA Transport 2306.3 Chromosomes and Chromatin 230Nucleosomes: The Lowest Level of Chromosome Organization 230Higher Levels of Chromatin Structure 2326.4 Heterochromatin and Euchromatin 234X Chromosome Inactivation 234The Histone Code and Formation of Heterochromatin 2356.5 The Structure of a Mitotic Chromosome 238Telomeres 240Centromeres 2436.6 Epigenetics: There's More to Inheritance than DNA 2436.7 The Nucleus as an Organized Organelle 2446.8 An Overview of Gene Regulation in Eukaryotes 2476.9 Transcriptional Control 248DNA Microarrays 249RNA Sequencing 2516.10 The Role of Transcription Factors in Regulating Gene Expression 252The Role of Transcription Factors in Determining a Cell's Phenotype 2526.11 The Structure of Transcription Factors 253Transcription Factor Motifs 2536.12 DNA Sites Involved in Regulating Transcription 2566.13 The Glucocorticoid Receptor: An Example of Transcriptional Activation 2586.14 Transcriptional Activation: The Role of Enhancers, Promoters, and Coactivators 259Coactivators That Interact with the Basal Transcription Machinery 260Coactivators That Alter Chromatin Structure 2606.15 Transcriptional Activation from Paused Polymerases 2636.16 Transcriptional Repression 264DNA Methylation 264Genomic Imprinting 265Long Noncoding RNAs (lncRNAs) as Transcriptional Repressors 2666.17 RNA Processing Control 2676.18 Translational Control 269Initiation of Translation 269Cytoplasmic Localization of mRNAs 270The Control of mRNA Stability 2716.19 The Role of MicroRNAs in Translational Control 2736.20 Posttranslational Control: Determining Protein Stability 274THE HUMAN PERSPECTIVE 275Chromosomal Aberrations and Human Disorders 2757 DNA Replication and Repair 2827.1 DNA Replication 2837.2 DNA Replication in Bacterial Cells 286Replication Forks and Bidirectional Replication 287Unwinding the Duplex and Separating the Strands 287The Properties of DNA Polymerases 288Semidiscontinuous Replication 2897.3 The Machinery Operating at the Replication Fork 2917.4 The Structure and Functions of DNA Polymerases 293Exonuclease Activities of DNA Polymerases 293Ensuring High Fidelity during DNA Replication 2947.5 Replication in Viruses 2967.6 DNA Replication in Eukaryotic Cells 296Initiation of Replication in Eukaryotic Cells 297Restricting Replication to Once Per Cell Cycle 297The Eukaryotic Replication Fork 298Replication and Nuclear Structure 3007.7 Chromatin Structure and Replication 3007.8 DNA Repair 302Nucleotide Excision Repair 302Base Excision Repair 303Mismatch Repair 304Double?Strand Breakage Repair 3047.9 Between Replication and Repair 305THE HUMAN PERSPECTIVE 306Consequences of DNA Repair Deficiencies 3068 Cellular Membrane 3118.1 Introduction to the Plasma Membrane 312An Overview of Membrane Functions 312A Brief History of Studies on Plasma Membrane Structure 3138.2 The Chemical Composition of Membranes 315Membrane Lipids 316The Nature and Importance of the Lipid Bilayer 317The Asymmetry of Membrane Lipids 3198.3 Membrane Carbohydrates 3198.4 The Structure and Functions of Membrane Proteins 320Integral Membrane Proteins 321Peripheral Membrane Proteins 322Lipid?Anchored Membrane Proteins 3228.5 Studying the Structure and Properties of Integral Membrane Proteins 323Identifying Transmembrane Domains 324Experimental Approaches to Identifying Conformational Changes within an Integral Membrane Protein 3258.6 Membrane Lipids and Membrane Fluidity 327The Importance of Membrane Fluidity 328Maintaining Membrane Fluidity 328Lipid Rafts 3298.7 The Dynamic Nature of the Plasma Membrane 329The Diffusion of Membrane Proteins after Cell Fusion 330Restrictions on Protein and Lipid Mobility 3318.8 The Red Blood Cell: An Example of Plasma Membrane Structure 334Integral Proteins of the Erythrocyte Membrane 334The Erythrocyte Membrane Skeleton 3368.9 The Movement of Substances across Cell Membranes 336The Energetics of Solute Movement 336Formation of an Electrochemical Gradient 3378.10 Diffusion through the Lipid Bilayer 338Diffusion of Substances through Membranes 338The Diffusion of Water through Membranes 3388.11 The Diffusion of Ions through Membranes 3408.12 Facilitated Diffusion 3458.13 Active Transport 346Primary Active Transport: Coupling Transport to ATP Hydrolysis 346Other Primary Ion Transport Systems 347Using Light Energy to Actively Transport Ions 348Secondary Active Transport (or Cotransport): Coupling Transport to Existing Ion Gradients 3488.14 Membrane Potentials 350The Resting Potential 350The Action Potential 3528.15 Propagation of Action Potentials as an Impulse 3538.16 Neurotransmission: Jumping the Synaptic Cleft 354Actions of Drugs on Synapses 356Synaptic Plasticity 357THE HUMAN PERSPECTIVE 357Defects in Ion Channels and Transporters as a Cause of Inherited Disease 357EXPERIMENTAL PATHWAYS 359The Acetylcholine Receptor 3599 Mitochondrion and Aerobic Respiration 3689.1 Mitochondrial Structure and Function 369Mitochondrial Membranes 370The Mitochondrial Matrix 3729.2 Oxidative Metabolism in the Mitochondrion 372The Tricarboxylic Acid (TCA) Cycle 373The Importance of Reduced Coenzymes in the Formation of ATP 3759.3 The Role of Mitochondria in the Formation of ATP 377Oxidation-Reduction Potentials 377Electron Transport 379Types of Electron Carriers 3799.4 Electron?Transport Complexes 381Complex I (NADH dehydrogenase) 383Complex II (succinate dehydrogenase) 384Complex III (cytochrome bc1) 384Complex IV (cytochrome c oxidase) 3849.5 Translocation of Protons and the Establishment of a Proton? Motive Force 3859.6 The Machinery for ATP Formation 386The Structure of ATP Synthase 3879.7 The Binding Change Mechanism of ATP Formation 388Components of the Binding Change Hypothesis 388Evidence to Support the Binding Change Mechanism and Rotary Catalysis 3899.8 Using the Proton Gradient 391The Role of the Fo Portion of ATP Synthase in ATP Synthesis 391Other Roles for the Proton?Motive Force in Addition to ATP Synthesis 3929.9 Peroxisomes 392THE HUMAN PERSPECTIVE 394I. The Role of Anaerobic and Aerobic Metabolism in Exercise 394II. Diseases that Result from Abnormal Mitochondrial or Peroxisomal Function 39510 Chloroplast and Photosynthesis 40110.1 The Origin of Photosynthesis 40210.2 Chloroplast Structure and Function 40310.3 An Overview of Photosynthetic Metabolism 40410.4 The Absorption of Light 405Photosynthetic Pigments 40610.5 Photosynthetic Units and Reaction Centers 407Oxygen Formation: Coordinating the Action of Two Different Photosynthetic Systems 40810.6 The Operations of Photosystem II and Photosystem I 409PSII Operations: Obtaining Electrons by Splitting Water 409PSI Operations: The Production of NADPH 41210.7 An Overview of Photosynthetic Electron Transport 413Killing Weeds by Inhibiting Electron Transport 41410.8 Photophosphorylation 415Noncyclic Versus Cyclic Photophosphorylation 41510.9 Carbon Dioxide Fixation and the Carbohydrate Synthesis 415Carbohydrate Synthesis in C3 Plants 416Redox Control 416Photorespiration 417Peroxisomes and Photorespiration 41810.10 Carbohydrate Synthesis in C4 and CAM Plants 420THE HUMAN PERSPECTIVE 421Global Warming and Carbon Sequestration 42111 The Extracellular Matrix and Cell Interactions 42611.1 Overview of Extracellular Interactions 42711.2 The Extracellular Space 428The Extracellular Matrix 42811.3 Components of the Extracellular Matrix 430Collagen 430Proteoglycans 432Fibronectin 433Laminin 43311.4 Dynamic Properties of the Extracellular Matrix 43511.5 Interactions of Cells with Extracellular Materials 436Integrins 43611.6 Anchoring Cells to Their Substratum 438Focal Adhesions 438Hemidesmosomes 44011.7 Interactions of Cells with Other Cells 441Selectins 441The Immunoglobulin Superfamily 442Cadherins 44311.8 Adherens Junctions and Desmosomes: Anchoring Cells to Other Cells 44511.9 The Role of Cell?Adhesion Receptors in Transmembrane Signaling 44711.10 Tight Junctions: Sealing the Extracellular Space 44711.11 Gap Junctions and Plasmodesmata: Mediating Intercellular Communication 449Gap Junctions 449Plasmodesmata 45111.12 Cell Walls 453THE HUMAN PERSPECTIVE 455The Role of Cell Adhesion in Inflammation and Metastasis 455EXPERIMENTAL PATHWAYS 457The Role of Gap Junctions in Intercellular Communication 45712 Cellular Organelles and Membrane Trafficking 46312.1 An Overview of the Endomembrane System 46412.2 A Few Approaches to the Study of Endomembranes 466Insights Gained from Autoradiography 466Insights Gained from the Use of the Green Fluorescent Protein 467Insights Gained from the Analysis of Subcellular Fractions 468Insights Gained from the Use of Cell?Free Systems 469Insights Gained from the Study of Mutant Phenotypes 47012.3 The Endoplasmic Reticulum 472The Smooth Endoplasmic Reticulum 473The Rough Endoplasmic Reticulum 47312.4 Functions of the Rough Endoplasmic Reticulum 473Synthesis of Proteins on Membrane?Bound versus Free Ribosomes 473Synthesis of Secretory, Lysosomal, or Plant Vacuolar Proteins 475Processing of Newly Synthesized Proteins in the Endoplasmic Reticulum 476Synthesis of Integral Membrane Proteins on ER?Bound Ribosomes 47612.5 Membrane Biosynthesis in the Endoplasmic Reticulum 47712.6 Glycosylation in the Rough Endoplasmic Reticulum 47912.7 Mechanisms That Ensure the Destruction of Misfolded Proteins 48112.8 ER to Golgi Vesicular Transport 48212.9 The Golgi Complex 482Glycosylation in the Golgi Complex 484The Movement of Materials through the Golgi Complex 48512.10 Types of Vesicle Transport and Their Functions 487COPII?Coated Vesicles: Transporting Cargo from the ER to the Golgi Complex 488COPI?Coated Vesicles: Transporting Escaped Proteins Back to the ER 48912.11 Beyond the Golgi Complex: Sorting Proteins at the TGN 491Sorting and Transport of Lysosomal Enzymes 491Sorting and Transport of Nonlysosomal Proteins 49312.12 Targeting Vesicles to a Particular Compartment 49312.13 Exocytosis 49612.14 Lysosomes 49612.15 Plant Cell Vacuoles 49812.16 Endocytosis 498Receptor?Mediated Endocytosis and the Role of Coated Pits 499The Role of Phosphoinositides in the Regulation of Coated Vesicles 50112.17 The Endocytic Pathway 50212.18 Phagocytosis 50512.19 Posttranslational Uptake of Proteins by Peroxisomes, Mitochondria, and Chloroplasts 505Uptake of Proteins into Peroxisomes 506Uptake of Proteins into Mitochondria 506Uptake of Proteins into Chloroplasts 507THE HUMAN PERSPECTIVE 508Disorders Resulting from Defects in Lysosomal Function 508EXPERIMENTAL PATHWAYS 510Receptor?Mediated Endocytosis 51013 The Cytoskeleton 51713.1 Overview of the Major Functions of the Cytoskeleton 51813.2 Microtubules: Structure and Function 520Structure and Composition of Microtubules 520Microtubule?Associated Proteins 521Microtubules as Structural Supports and Organizers 521Microtubules as Agents of Intracellular Motility 52213.3 Motor Proteins: Kinesins and Dyneins 524Motor Proteins Traverse the Microtubular Cytoskeleton 524Kinesins 524Cytoplasmic Dynein 52613.4 Microtubule?Organizing Centers (MTOCs) 527Centrosomes 528Basal Bodies and Other MTOCs 530Microtubule Nucleation 53013.5 Microtubule Dynamics 530The Dynamic Properties of Microtubules 530The Underlying Basis of Microtubule Dynamics 53213.6 Cilia and Flagella: Structure and Function 534Structure of Cilia and Flagella 535Growth by Intraflagellar Transport 537The Mechanism of Ciliary and Flagellar Locomotion 53913.7 Intermediate Filaments 541Intermediate Filament Assembly and Disassembly 541Types and Functions of Intermediate Filaments 54313.8 Microfilaments 544Microfilament Structure 544Microfilament Assembly and Disassembly 54513.9 Myosin: The Molecular Motor of Actin Filaments 547Conventional (Type II) Myosins 547Unconventional Myosins 54813.10 Muscle Contractility 552Organization of Sarcomeres 552The Sliding Filament Model of Muscle Contraction 55313.11 Nonmuscle Motility 557Actin-Binding Proteins 55813.12 Cellular Motility 56013.13 Actin?Dependent Processes During Development 564Axonal Outgrowth 56413.14 The Bacterial Cytoskeleton 567THE HUMAN PERSPECTIVE 568The Role of Cilia in Development and Disease 568EXPERIMENTAL PATHWAYS 569I. The Step Size of Kinesin 569II. Studying Actin?Based Motility without Cells 57114 Cell Division 57814.1 The Cell Cycle 579Phases of the Cell Cycle 579Cell Cycles in Vivo 58014.2 Regulation of the Cell Cycle 58114.3 Control of the Cell Cycle: The Role of Protein Kinases 582Cyclin Binding 583Cdk Phosphorylation/Dephosphorylation 583Cdk Inhibitors 584Controlled Proteolysis 584Subcellular Localization 58414.4 Control of the Cell Cycle: Checkpoints, Cdk Inhibitors, and Cellular Responses 58614.5 M Phase: Mitosis and Cytokinesis 58814.6 Prophase 588Formation of the Mitotic Chromosome 588Centromeres and Kinetochores 590Formation of the Mitotic Spindle 591The Dissolution of the Nuclear Envelope and Partitioning of Cytoplasmic Organelles 59414.7 Prometaphase 59414.8 Metaphase 59614.9 Anaphase 598The Role of Proteolysis in Progression through Mitosis 598The Events of Anaphase 600Forces Required for Chromosome Movements at Anaphase 601The Spindle Assembly Checkpoint 60214.10 Telophase and Cytokinesis 603Motor Proteins Required for Mitotic Movements 603Cytokinesis 603Cytokinesis in Plant Cells: Formation of the Cell Plate 60714.11 Meiosis 60814.12 The Stages of Meiosis 61014.13 Genetic Recombination during Meiosis 613THE HUMAN PERSPECTIVE 615Meiotic Nondisjunction and Its Consequences 615EXPERIMENTAL PATHWAYS 616The Discovery and Characterization of MPF 61615 Cell Signaling Pathways 62415.1 The Basic Elements of Cell Signaling Systems 62515.2 A Survey of Extracellular Messengers and their Receptors 62815.3 Signal Transduction by G Protein?Coupled Receptors 629Receptors 629G Proteins 630Termination of the Response 631Bacterial Toxins 63215.4 Second Messengers 632The Discovery of Cyclic AMP 633Phosphatidylinositol?Derived Second Messengers 633Phospholipase C 63515.5 The Specificity of G Protein?Coupled Responses 63615.6 Regulation of Blood Glucose Levels 636Glucose Mobilization: An Example of a Response Induced by cAMP 637Signal Amplification 638Other Aspects of cAMP Signal Transduction Pathways 63815.7 The Role of GPCRs in Sensory Perception 64015.8 Protein?Tyrosine Phosphorylation as a Mechanism for Signal Transduction 641Receptor Dimerization 641Protein Kinase Activation 643Phosphotyrosine?Dependent Protein-Protein Interactions 643Activation of Downstream Signaling Pathways 643Ending the Response 64515.9 The Ras?MAP Kinase Pathway 645Accessory Proteins 645Adapting the MAP Kinase to Transmit Different Types of Information 64715.10 Signaling by the Insulin Receptor 648The Insulin Receptor Is a Protein?Tyrosine Kinase 648Insulin Receptor Substrates 1 and 2 649Glucose Transport 650Diabetes Mellitus 65015.11 Signaling Pathways in Plants 65115.12 The Role of Calcium as an Intracellular Messenger 651IP3 and Voltage?Gated Ca2+ Channels 651Visualizing Cytoplasmic Ca2+ Concentration in Living Cells 651Ca2+?Binding Proteins 654Regulating Calcium Concentrations in Plant Cells 65415.13 Convergence, Divergence, and Cross?Talk among Different Signaling Pathways 65515.14 The Role of NO as an Intercellular Messenger 657NO as an Activator of Guanylyl Cyclase 658Inhibiting Phosphodiesterase 65815.15 Apoptosis (Programmed Cell Death) 659The Extrinsic Pathway of Apoptosis 660The Intrinsic Pathway of Apoptosis 661Necroptosis 662Signaling Cell Survival 663THE HUMAN PERSPECTIVE 663Disorders Associated with G Protein?Coupled Receptors 663EXPERIMENTAL PATHWAYS 665The Discovery and Characterization of GTP?Binding Proteins 66516 Cancer 67316.1 Basic Properties of a Cancer Cell 67416.2 The Causes of Cancer 67716.3 The Genetics of Cancer 67816.4 An Overview of Tumor?Suppressor Genes and Oncogenes 68016.5 Tumor?Suppressor Genes: The RB Gene 68116.6 Tumor?Suppressor Genes: The TP53 Gene 684The Role of p53: Guardian of the Genome 684The Role of p53 in Promoting Senescence 68616.7 Other Tumor?Suppressor Genes 68716.8 Oncogenes 688Oncogenes That Encode Growth Factors or Their Receptors 688Oncogenes That Encode Cytoplasmic Protein Kinases 689Oncogenes That Encode Transcription Factors 689Oncogenes That Encode Proteins That Affect the Epigenetic State of Chromatin 689Oncogenes That Encode Metabolic Enzymes 690Oncogenes That Encode Products That Affect Apoptosis 69016.9 The Mutator Phenotype: Mutant Genes Involved in DNA Repair 69116.10 MicroRNAs: A New Player in the Genetics of Cancer 69116.11 The Cancer Genome 69116.12 Gene?Expression Analysis 69416.13 Strategies for Combating Cancer 69616.14 Immunotherapy 69616.15 Inhibiting the Activity of Cancer?Promoting Proteins 69816.16 The Concept of a Cancer Stem Cell 70116.17 Inhibiting the Formation of New Blood Vessels (Angiogenesis) 701EXPERIMENTAL PATHWAYS 702The Discovery of Oncogenes 70217 Immunity 70917.1 An Overview of the Immune Response 710Innate Immune Responses 711Adaptive Immune Responses 71317.2 The Clonal Selection Theory as It Applies to B Cells 71417.3 Vaccination 71517.4 T Lymphocytes: Activation and Mechanism of Action 71717.5 The Modular Structure of Antibodies 72017.6 DNA Rearrangements That Produce Genes Encoding B? and T?Cell Antigen Receptors 72317.7 Membrane?Bound Antigen Receptor Complexes 72517.8 The Major Histocompatibility Complex 72617.9 Distinguishing Self from Nonself 73017.10 Lymphocytes Are Activated by Cell?Surface Signals 731Activation of Helper T Cells by Professional APCs 731Activation of B Cells by TH Cells 73217.11 Signal Transduction Pathways in Lymphocyte Activation 732THE HUMAN PERSPECTIVE 733Autoimmune Diseases 733EXPERIMENTAL PATHWAYS 736The Role of the Major Histocompatibility Complex in Antigen Presentation 73618 Techniques in Cell and Molecular Biology 74218.1 The Light Microscope 743Resolution 744Visibility 74518.2 Bright?Field and Phase?Contrast Microscopy 745Bright?Field Light Microscopy 745Phase?Contrast Microscopy 74618.3 Fluorescence Microscopy (and Related Fluorescence?Based Techniques) 746Laser Scanning Confocal Microscopy 749Super?Resolution Fluorescence Microscopy 750Light Sheet Fluorescence Microscopy 75118.4 Transmission Electron Microscopy 75218.5 Specimen Preparation for Electron Microscopy 753Cryofixation and the Use of Frozen Specimens 754Negative Staining 755Shadow Casting 755Freeze?Fracture Replication and Freeze Etching 75618.6 Scanning Electron Microscopy 75718.7 Atomic Force Microscopy 75818.8 The Use of Radioisotopes 75918.9 Cell Culture 76018.10 The Fractionation of a Cell's Contents by Differential Centrifugation 76218.11 Purification and Characterization of Proteins by Liquid Column Chromatography 762Ion?Exchange Chromatography 763Gel Filtration Chromatography 763Affinity Chromatography 76418.12 Determining Protein-Protein Interactions 76418.13 Characterization of Proteins byPolyacrylamide Gel Electrophoresis 766SDS-PAGE 767Two?Dimensional Gel Electrophoresis 76718.14 Characterization of Proteins by Spectrometry 76718.15 Characterization of Proteins by Mass Spectrometry 76718.16 Determining the Structure of Proteins and Multisubunit Complexes 76818.17 Fractionation of Nucleic Acids 770Separation of DNAs by Gel Electrophoresis 770Separation of Nucleic Acids by Ultracentrifugation 77118.18 Nucleic Acid Hybridization 77318.19 Chemical Synthesis of DNA 77418.20 Recombinant DNA Technology 774Restriction Endonucleases 774Formation of Recombinant DNAs 775DNA Cloning 77618.21 Enzymatic Amplification of DNA by PCR 778Process of PCR 778Applications of PCR 77818.22 DNA Sequencing 78018.23 DNA Libraries 782Genomic Libraries 782cDNA Libraries 78318.24 DNA Transfer into Eukaryotic Cells and Mammalian Embryos 783Transgenic Animals 785Transgenic Plants 78518.25 Gene Editing and Silencing 786In Vitro Mutagenesis 786Knockout Mice 787RNA Interference 788Genome Editing Using Engineered Nucleases 78918.26 The Use of Antibodies 789Glossary G-1Additional Reading A-1Index I-1