Guide to Yeast Genetics: Functional Genomics, Proteomics, and Other Systems Analysis (eBook)

Front Cover 1
Methods in Enzymology 4
Copyright Page 5
Contents 6
Contributors 16
Preface 24
Volumes in Series 26
Section 1: Functional Genomics 54
Chapter 1: Analysis of Gene Function Using DNA Microarrays 56
1. Introduction and Experimental Design 57
1.1. Single-mutant analysis 57
1.2. Double-mutant analysis 59
2. Methods 61
2.1. Experimental design 61
2.2. Cell growth 62
2.3. Total RNA isolation and purification 62
2.4. Purification of poly-A RNA 63
2.5. Reverse transcription and dye labeling 64
2.6. Hybridization 65
2.7. Microarray washing 66
2.8. Array scanning 67
2.9. Gridding and normalization 67
References 69
Chapter 2: An Introduction to Microarray Data Analysis and Visualization 72
1. Introduction 73
1.1. Overview 73
1.2. Commonly used terms in microarray data analysis 75
1.3. A simple case study 76
2. Experimental Design: Single-Sample Versus Competitive Hybridization 76
2.1. Single-sample hybridization 77
2.2. Competitive hybridization 77
2.3. Choosing the best approach 78
3. Image Analysis 79
3.1. What is a digital image? 80
3.2. Data files 80
3.3. Software tools 81
4. Preprocessing 82
4.1. Software tools 82
4.2. Calculating ratio values 85
4.3. Normalizing ratio values 86
4.4. Quality assessment, filtering, and handling replicates 89
4.5. Preprocessing Affymetrix arrays 90
5. Visualizing Data Using Cluster Analysis 91
5.1. Hierarchical clustering 91
5.2. Partitioning and network-based approaches 93
6. Assessing the Statistical Evidence for Differential Expression 94
6.1. Significance analysis of microarrays 94
6.2. Limma 95
7. Exploring Gene Sets 95
7.1. Gene Ontology term mapping 95
7.2. Motif searching 96
7.3. Network visualization 96
7.4. Graphing array data on genome tracks 96
8. Managing Data 97
8.1. Data persistence and integrity 97
8.2. Public data repositories and MIAME compliance 99
Acknowledgments 102
References 102
Chapter 3: Genome-Wide Approaches to Monitor Pre-mRNA Splicing 104
1. Introduction 105
2. Microarray Design 106
3. Sample Preparation 109
3.1. Cell collection 109
3.2. RNA isolation 110
3.3. cDNA synthesis 113
3.4. Fluorescent labeling of cDNA 117
3.5. Microarray hybridization 119
3.6. Microarray washing 120
4. Microarray Data Collection 121
5. Microarray Data Analysis 122
5.1. Data normalization 123
5.2. Replication 123
5.3. Splicing specific data 124
5.4. Extracting biological meaning 124
6. Future Methodologies 125
Acknowledgments 126
References 127
Chapter 4: ChIP-Seq: Using High-Throughput DNA Sequencing for Genome-Wide Identification of Transcription Factor Binding Site 130
1. Introduction 131
2. Protocols 134
2.1. Chromatin immunoprecipitation 134
2.2. Input DNA preparation 138
2.3. Illumina sequencing DNA library generation 139
2.4. Barcode design and adapter annealing 144
2.5. Illumina sequencing 146
3. Sequencing Data Management 146
4. Genome Analysis Pipeline 147
5. Examining Data Quality and Parsing Barcoded Data 148
6. Visualization in Genome Browser 148
6.1. Low-level analysis 149
6.2. High-level analysis 154
6.3. Troubleshooting 154
7. Conclusion and Future Directions 155
Acknowledgments 155
References 155
Chapter 5: Genome-Wide Mapping of Nucleosomes in Yeast 158
1. Introduction 158
2. Isolation of Mononucleosomal DNA 160
3. Variation in Titration Level Used for Nucleosome Purification 165
4. Labeling of Mononucleosomal DNA for Tiling Microarray Analysis 166
4.1. Protocol 167
5. Generation of Nucleosomal DNA Libraries for Deep Sequencing 168
References 170
Chapter 6: Genome-Wide Translational Profiling by Ribosome Footprinting 172
1. Introduction 173
2. Ribosome Footprint Generation and Purification 174
2.1. Extract preparation 175
2.2. Nuclease digestion and monosome purification 176
2.3. Footprint fragment purification 177
2.4. Fragmented mRNA preparation 179
3. Sequencing Library Preparation 181
3.1. Polyadenylation 182
3.2. Reverse transcription 183
3.3. Circularization 183
3.4. PCR amplification 184
4. Data Analysis 186
4.1. Mapping polyadenylated sequences 187
4.2. Reference databases 188
4.3. Selecting high-quality alignments 189
4.4. Quantifying gene expression 189
5. Solutions and Common Procedures 191
5.1. Solutions 191
5.2. Oligonucleotides 192
5.3. Nucleic acid precipitation 192
5.4. Nucleic acid gel extraction 193
Acknowledgments 193
References 193
Section 2: Systematic Genetic Analysis 196
Chapter 7: Synthetic Genetic Array (SGA) Analysis in Saccharomyces cerevisiae and Schizosaccharomyces pombe 198
1. Introduction 199
2. Methodology 201
2.1. SGA query strain construction 201
2.2. Pin tool sterilization procedures 203
2.3. Constructing a 1536-density DMA 205
2.4. SGA procedure 206
2.5. Double mutant array image acquisition and processing 208
2.6. Quantitative scoring of genetic interactions using colony size-based fitness measurements 210
2.7. Interpretation and analysis of genetic interactions 212
2.8. S. pombe SGA 218
3. Media and Stock Solutions 221
3.1. SGA media and stock solutions 221
3.2. SpSGA media and stock solutions 224
4. Applications of SGA Methodology 224
4.1. Integrating SGA and high-content screening 224
4.2. Essential gene and higher order genetic interactions 227
4.3. Combining SGA and gene overexpression libraries 227
4.4. Applying SGA as a method for high-resolution genetic mapping (SGAM) 228
4.5. Chemical genomics 228
References 229
Chapter 8: Making Temperature-Sensitive Mutants 234
1. Introduction 235
2. Diploid Shuffle-Plasmid Method 236
2.1. General description of the diploid shuffle—plasmid method 236
2.2. Materials 238
2.3. Methods 239
3. Diploid Shuffle-Chromosome Method 247
3.1. A general description 247
3.2. Materials 249
3.3. Methods 251
4. Perspectives 255
References 256
Chapter 9: Quantitative Genetic Interaction Mapping Using the E-MAP Approach 258
1. Introduction 259
2. Selection of Mutations for Genetic Analysis 262
3. Generation and Measurement of Double Mutant Strains 264
3.1. Basic SGA protocol 267
3.2. Basic PEM protocol 272
3.3. Digital photography 274
4. Data Processing and Computation of Scores 274
4.1. Preprocessing and normalization 274
4.2. Computing expected colony sizes 276
4.3. Computing genetic interaction scores 276
4.4. Quality control 277
5. Extraction of Biological Hypotheses 277
5.1. Identifying genes acting in the same pathway using patterns of interactions 277
5.2. Using individual interactions to predict enzyme–substrate relationships 278
5.3. Using individual interactions to predict opposing enzyme relationships 280
5.4. Dissecting multiple roles of a single gene by detailed comparison of interaction patterns 281
6. Perspective 282
References 282
Chapter 10: Exploring Gene Function and Drug Action Using Chemogenomic Dosage Assays 286
1. Introduction 287
2. Methodology 290
2.1. Yeast deletion strain pool and MSP pool construction 290
2.2. Determining the drug dosage 291
2.3. Experimental pool growth for deletion and overexpression collections 293
2.4. Purification and amplification of barcodes and ORFs 296
2.5. Hybridization 298
2.6. Analysis of results 300
2.7. Confirmation of microarray data 303
3. Experimental Considerations 305
4. Perspectives 306
Acknowledgments 307
References 307
Section 3: Proteomics 310
Chapter 11: Yeast Expression Proteomics by High-Resolution Mass Spectrometry 312
1. Introduction 313
2. Background, Methods, and Applications 314
2.1. The challenge 314
2.2. Background on MS instrumentation for ‘‘shotgun’’ proteomics 316
2.3. Quantitative proteomics 320
2.4. Computational proteomics and data ana 323
2.5. Perspective and outlook 323
3. Protocols 325
3.1. Yeast strains for SILAC proteomics experiments 325
3.2. Media for SILAC labeling 326
3.3. Growing yeast cultures for SILAC labeling 327
3.4. Extract Preparation for SILAC experiments 327
3.5. In-solution digest of proteins for MS 327
3.6. Test of label incorporation 327
3.7. Peptide IEF (Optional) 328
3.8. MS analysis 329
3.9. Identification and quantitation of peptides and proteins 330
Acknowledgments 331
References 331
Chapter 12: High-Quality Binary Interactome Mapping 334
1. Introduction 335
2. High-Quality Binary Interactome Mapping 336
2.1. Production and verification of Y2H datasets 338
2.2. Validation of Y2H datasets to produce reliable binary interactome maps 341
2.3. Biological evaluation of binary interactome maps 344
3. High-Throughput Y2H Pipeline 345
3.1. Assembly of DB-X and AD-Y expression plasmids 345
3.2. Yeast transformation 349
3.3. Autoactivator removal and AD-Y pooling 351
3.4. Screening and phenotyping 354
3.5. Verification 358
3.6. Media and plates 360
4. Validation Using Orthogonal Binary Interaction Assays 362
5. Conclusion 365
Acknowledgments 365
References 366
Chapter 13: Quantitative Analysis of Protein Phosphorylation on a System-Wide Scale by Mass Spectrometry-Based Proteomics 370
1. Introduction 371
2. Protocols 372
2.1. Generation of peptide samples 372
2.2. Phosphopeptide isolation 375
2.3. Mass spectrometric analyses of the phosphopeptide isolates 379
2.4. Data analyses 382
Acknowledgments 384
References 385
Chapter 14: A Toolkit of Protein-Fragment Complementation Assays for Studying and Dissecting Large-Scale and Dynamic Protein-Protein Interactions in Living Cells 388
1. Introduction 389
2. General Considerations in Using PCA 390
3. DHFR PCA Survival-Selection for Large-Scale Analysis of PPIs 392
3.1. Materials 394
3.2. Procedure 394
4. A Life and Death Selection PCA Based on the Prodrug-Converting Cytosine Deaminase for Dissection of PPIs 401
4.1. Preparation for a two-step OyCD PCA screen 403
4.2. Materials 403
4.3. Procedure 405
5. Visualizing the Localization of PPIs with GFP Family Fluorescent Protein PCAs 409
5.1. Materials 411
5.2. Procedure 412
6. Studying Dynamics of PPIs with Luciferase Reporter PCAs 414
6.1. Materials 415
6.2. Procedure 416
References 419
Chapter 15: Yeast Lipid Analysis and Quantification by Mass Spectrometry 422
1. Introduction 423
2. Methods 426
2.1. Sample preparation 426
2.2. Normalization for starting amount of material 428
2.3. Lipid extraction (for glycerophospholipids and sphingolipids) 428
2.4. Sterol isolation 429
2.5. Lipid analysis 430
Acknowledgments 442
References 442
Chapter 16: Mass Spectrometry-Based Metabolomics of Yeast 446
1. Introduction 447
2. LC-MS Basics 448
3. Experimental Design 448
4. Strains 451
5. Culture Conditions 451
5.1. Batch liquid culture and chemostats 451
5.2. Protocol for harvesting yeast by vacuum filtration 453
5.3. Protocol for harvesting yeast by centrifugation after methanol quenching 453
5.4. Filter culture 454
5.5. Protocol for growth of yeast on filters atop agarose support 455
6. Metabolite Extraction 455
6.1. Extraction protocol for cells on filters (from vacuum filtration of liquid culture, or from filter cultures) 457
6.2. Extraction protocol for cells after methanol quenching 457
7. Chemical Derivatization of Metabolites 458
7.1. Amino acid derivatization 459
7.2. Thiol and disulfide derivatiz 459
8. LC-MS for Mixture Analysis 459
9. Liquid Chromatography 460
10. Electrospray Ionization 463
11. Mass Spectrometry 464
11.1. Triple quadrupole mass spectrometers 464
11.2. High-resolution mass analyzers 466
11.3. Hybrid instruments 468
12. Targeted Data Analysis 468
13. Untargeted Data Analysis 471
13.1. Adducts 471
13.2. Isotopic variants 473
13.3. In-source fragmentation 474
13.4. Unknown identification 474
14. Future Outlook 474
References 475
Section 4: Systems Analysis 480
Chapter 17: Imaging Single mRNA Molecules in Yeast 482
1. Introduction 482
2. RNA FISH Protocol 485
2.1. Designing oligonucleotides 485
2.2. Coupling fluorophores to oligonucleotides 486
2.3. Purification of probes using HPLC 488
2.4. Fixing S. cerevisiae 490
2.5. Hybridizing probes to target mRNA 491
2.6. Image processing: Detecting diffraction-limited mRNA spot 495
3. Example: STL1 mRNA Detection in Response to NaCl Shock 497
4. Conclusions 498
Acknowledgments 498
References 498
Chapter 18: Reconstructing Gene Histories in Ascomycota Fungi 500
1. Introduction 501
2. Synergy 504
2.1. Overview 504
2.2. Defining orthogroups 504
2.3. Scoring gene similarity 506
2.4. Gene similarity graph 507
2.5. Identifying orthogroups 507
3. Evaluating Orthogroup Quality 512
3.1. Fungal orthogroup robustness 514
3.2. Comparison to curated resource 516
3.3. Simulated orthogroups 518
4.Biological Analysis of Gene Histories 519
4.1. Defining orthogroup categories 519
4.2. Singletons and ORF predictions 521
4.3. Gene sets and orthogroup projections 521
4.4. Copy-number variation profiles 524
5. Analysis of Paralogous Genes 531
5.1. Estimating functional divergence between paralogous genes 531
5.2. Estimating divergence based on degree of conserved interactions 532
6. Discussion and General Applicability 533
References 535
Chapter 19: Experimental Evolution in Yeast: A Practical Guide 540
1. Introduction 541
2. Experiment Rationale 541
3. Experimental Evolution Approaches 543
3.1. Serial dilution 543
3.2. Chemostats 544
3.3. Turbidostats 545
3.4. More specialized systems 546
3.5. Miniaturization 546
4. Experimental Design 546
4.1. Growth conditions 546
4.2. Population size 547
4.3. Experiment duration 548
5. Practical Considerations 549
5.1. Strains and markers 549
5.2. Media 550
5.3. Growth rate 551
5.4. Good sterile practices 552
5.5. Good strain hygiene 552
5.6. Record-keeping 553
6. Analysis Techniques 553
6.1. Sampling regimen 553
6.2. Population genetics 554
6.3. Fitness 554
7. Example Protocol 555
7.1. Medium formulation 555
7.2. Chemostat preparation 555
7.3. Chemostat assembly 556
7.4. Inoculation 556
7.5. Daily sampling 556
7.6. Weekly sampling 556
7.7. Analysis 557
8. Conclusions 557
Acknowledgments 558
References 558
Chapter 20: Enhancing Stress Resistance and Production Phenotypes Through Transcriptome Engineering 562
1. Introduction 563
2. Transcription Factor Selection 564
3. Plasmid Library Construction 565
3.1. Promoter selection 565
3.2. Random mutagenesis by PCR 568
3.3. Quantification of total sequence diversity and library maintenance 571
4. Assessment of Phenotypic Diversity 572
4.1. Determination of yeast transformation efficiency 573
4.2. Evaluation of mutant libraries 574
4.3. Calculation of phenotypic diversity 575
5. Selecting for Phenotypes of Interest 577
5.1. Creation and maintenance of yeast library 578
5.2. Basic selection on liquid versus solid media 578
5.3. Alternative selection strategies and postselection screening 579
6. Validation 581
7. Concluding Remarks 582
References 583
Section 5: Advances in Cytology/Biochemistry 586
Chapter 21: Visualizing Yeast Chromosomes and Nuclear Architecture 588
1. Introduction 589
2. Strain Constructions and Image Acquisition for Nuclear Architecture Analysis in Living Cells 590
2.1. Tagging chromatin in vivo with lac and tet operator arrays 590
2.2. Determining the position of the nucleus 593
2.3. Immobilizing cells for microscopy 593
2.4. Controlling temperature 595
2.5. Image acquisition set-ups 595
3. Data Analysis and Quantitative Measurements 600
3.1. Accurate determination of the 3D position of a tagged locus 600
3.2. Colocalization of a DNA locus with a subnuclear structure 604
3.3. Quantification of locus mobility 605
4. IF and FISH on Fixed Samples 610
4.1. Yeast strains and media 611
4.2. Antibody purification and specificity 611
4.3. Choice of fluorop 612
4.4. Protocol 613
4.5. Special notes 618
References 619
Chapter 22: Quantitative Localization of Chromosomal Loci by Immunofluorescence 622
1. Yeast Strain Construction 623
2. Immunofluorescence 625
3. Fixing Cells 626
4. Spheroplasting 627
5. Preparing Slides 627
6. Antibody Incubations 628
7. Mounting and Storage of Slides 628
8. Microscopy and Analysis 629
Acknowledgments 631
References 632
Chapter 23: Spinning-Disk Confocal Microscopy of Yeast 634
1. Introduction 635
2. Building a Spinning-Disk Confocal Microscope 636
2.1. Microscope base 638
2.2. Scanhead 638
2.3. Lasers and filters 638
2.4. Choice of objective 639
2.5. Cameras 643
2.6. Other hardware considerations 646
2.7. Software 647
2.8. System integration 647
3. Sample Preparation 647
3.1. Fluorescent tagging and choice of fluorescent protein 647
3.2. Minimizing autofluores 650
3.3. Mounting 651
Acknowledgments 653
References 653
Chapter 24: Correlative GFP-Immunoelectron Microscopy in Yeast 656
1. Introduction 657
2. Recent Advances in High-Pressure Freezing and Freeze-Substitution 658
2.1. High-pressure freezing 658
2.2. Freeze-substitution 658
3. How to Prepare Yeast by HPF/FS 659
3.1. Growing and concentrating yeast 660
3.2. High-pressure freezing 660
3.3. Freeze-substitution 661
3.4. Embedding 663
3.5. Sectioning 664
4. Immunolabeling 665
5. Conclusions 666
6. Protocols 667
6.1. Filtration and HPF 667
6.2. Freeze-substitution 668
6.3. Embedding 669
6.4. Anti-GFP immunolabeling 670
Acknowledgments 670
References 670
Chapter 25: Analyzing P-Bodies and Stress Granules in Saccharomyces cerevisiae 672
1. Introduction 673
2. Determining If a Specific Protein can Accumulate in P-Bodies or Stress Granules 675
2.1. Markers of P-bodies and stress granules 675
2.2. Preparation of samples 679
3. Monitoring Messenger RNA in P-Bodies 683
4. Determining If a Mutation/Perturbation Affects P-Body or Stress Granule Size and Number 684
4.1. Conditions to observe increases or decreases in P-bodies and stress granules 684
4.2. Interpreting alterations in P-body/stress granule size and number 686
5. Quantification of P-Body Size and Number 688
5.1. Semiautomated quantification of P-body size and number 689
5.2. Manual quantification of stress granule size and number 690
Acknowledgments 690
References 690
Chapter 26: Analyzing mRNA Expression Using Single mRNA Resolution Fluorescent In Situ Hybridization 694
1. Introduction 695
2. Probe Design 696
3. Probe Labeling 698
3.1. Materials 698
3.2. Protocol 699
3.3. Measuring labeling efficiency 699
4. Cell Fixation, Preparation, and Storage 700
4.1. Materials 701
4.2. Protocol 701
5. Hybridization 703
5.1. Materials 703
5.2. Probes used for the hybridization shown in Figs. 26.1 and 26.3 704
5.3. Protocol 705
6. Image Acquisition 706
6.1. Microscope (example) 707
7. Image Analysis 707
8. Summary and Perspectives 710
Acknowledgments 711
References 711
Chapter 27: The Use of In Vitro Assays to Measure Endoplasmic Reticulum-Associated Degradation 714
1. Introduction 715
2. In Vitro ERAD Assays Using a Soluble Substrate, p?F 718
2.1. Materials 719
2.2. The in vitro degradation assay for p?F 722
2.3. The p?F retrotranslocation assay 724
3. In Vitro Assays for Integral Membrane Proteins that are ERAD Substrates 726
3.1. In vitro ubiquitination assay 726
3.2. Analysis of integral membrane protein retrotranslocation 729
References 730
Chapter 28: A Protein Transformation Protocol for Introducing Yeast Prion Particles into Yeast 734
1. Introduction 735
2. Purification of Bacterially Expressed Sup-NM 736
2.1. Purification of Sup-NM 736
3. Preparation of Different Conformations of In Vitro Sup-NM Amyloid 737
3.1. Melting temperature analysis of Sup-NM amyloid 738
4. Preparation of In Vivo Prions from Yeast 738
5. Preparation of Lyticase 739
6. Protein Transformation 739
7. Determination of Prion Conversion Efficiency and Prion Strain Phenotypes 741
Acknowledgments 745
References 745
Chapter 29: Overexpression and Purification of Integral Membrane Proteins in Yeast 748
1. Introduction 749
2. General Considerations 749
3. Protocol-Molecular Biology 750
4. Protocol-Cell Growth 752
5. Protocol-Membrane Preparation and Solubilization 752
6. Protocol-Protein Purification 754
7. Protocol-Protein Characterization 758
8. Conclusion 759
Acknowledgments 759
References 759
Chapter 30: Biochemical, Cell Biological, and Genetic Assays to Analyze Amyloid and Prion Aggregation in Yeast 762
1. Introduction 763
2. Methods 765
2.1. Detecting protein aggregation in yeast cells 765
2.2. Assays for prion behavior 772
3. Concluding Remarks 784
References 784
Section 6: Other Fungi 788
Chapter 31: Genetics and Molecular Biology in Candida albicans 790
1. Homozygous Gene Disruption in C. albicans 791
1.1. Homozygous gene disruption by fusion PCR 793
2. C. albicans DNA Transformation 796
2.1. Transformation buffers 797
3. C. albicans Total RNA Purification 798
4. C-Terminal Epitope Tagging in C. albicans 799
4.1. Primer design 800
4.2. PCR conditions 800
4.3. Transformation 800
4.4. Integration confirmation 801
4.5. SAT1 marker excision 801
4.6. Tag sequence confirmation 801
4.7. Schematic of the 13×myc tagging procedure 802
5. C. albicans Chromatin Immunoprecipitation 803
5.1. Chromatin immunoprecipitation protocol 803
5.2. Chromatin immunoprecipitation buffers 806
5.3. Strand displacement amplification of ChIP samples 807
5.4. Strand displacement amplification solutions 809
5.5. Dye coupling 809
5.6. ChIP-chip hybridization protocol (adapted from the Agilent oligo aCGH/chip-on-chip hybridization kit) 810
References 811
Chapter 32: Molecular Genetics of Schizosaccharomyces pombe 812
1. Introduction 813
2. Biology, Growth, and Maintenance of Fission Yeast 814
2.1. Other media supplements—Phloxin B 814
2.2. Other media supplements—Adenine and low-Ade media 819
2.3. Storage of fission yeast 819
2.4. Growth in liquid media 819
3. Genetics and Physiology 820
3.1. Performing genetic crosses 821
3.2. Testing mating type and sporulation by iodine staining 822
3.3. Random spore analysis 823
3.4. Tetrad dissection 823
3.5. Bulk spore germination 824
3.6. Isolating diploids 825
3.7. Cell synchrony in fission yeast cultures 826
4. Molecular Analysis 829
4.1. Fission yeast plasmids 829
4.2. Integrations in fission yeast 831
4.3. Isolation and analysis of novel mutations 832
4.4. Transformation of DNA into fission yeast 832
4.5. Harvesting DNA, RNA, and protein from fission yeast 834
5. Cell Biology 838
5.1. Preparation and analysis of cell populations 838
5.2. Flow cytometry (whole cells) 838
5.3. Flow cytometry (nuclear ‘‘ghosts’’) 839
5.4. DNA and septum staining in fixed cells 840
5.5. Fission yeast whole-cell immunofluorescence 841
6. Conclusion 844
Appendix: Schizosaccharomyces pombe Online Resources 844
References 846
Chapter 33: Applying Genetics and Molecular Biology to the Study of the Human Pathogen Cryptococcus neoformans 850
1. Introduction 851
2. Serotypes, Strains, and Sequences 852
3. Life Cycle 853
4. Techniques for Basic Culture 855
4.1. Dominant drug selection markers 856
5. Basic Molecular Biology Techniques 857
5.1. Fusion polymerase chain reaction 857
5.2. Transformation 861
5.3. Colony PCR 865
5.4. Genomic DNA extraction 867
5.5. RNA extraction 868
5.6. Protein extraction for SDS–PAGE 870
6. Methods for Assaying Pathogenesis 870
6.1. Murine model of infection 870
6.2. Tissue culture 878
6.3. Assays for characterized virulence factors 879
7. Concluding Remarks 881
Acknowledgments 882
References 882
Chapter 34: The Fungal Genome Initiative and Lessons Learned from Genome Sequencing 886
1. Introduction: Yeast Genomes and Beyond 887
2. Computational Prediction of Genes and Noncoding Elements 894
3. Mechanisms of Genome Evolution 896
4. Genomic Potential for Sex 900
5. Gene Family Conservation and Evolution 901
6. Impact of Next-Generation Sequencing 902
7. Future Directions 904
Acknowledgments 905
References 906
Chapter 35: Ultradian Metabolic Cycles in Yeast 910
1. Introduction 910
2. Induction of Ultradian Cycles of Oxygen Consumption Using a Chemostat 911
2.1. Chemostat setup 912
2.2. Comments 913
3. Long-Period Cycles 914
4. Short-Period Cycles 915
5. Significance of Ultradian Cycles 916
Acknowledgments 918
References 918
Author Index 920
Subject Index 932
Color Plates 946