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

Cover......Page 1
Methods in Enzymology, Volume 470......Page 2
Series Editors......Page 3
ISBN 978-0-12-375172-0......Page 4
Contributors......Page 5
Preface......Page 13
Contents of Previous Volumes......Page 14
Analysis of Gene Function Using DNA Microarrays......Page 42
Single-mutant analysis......Page 43
Double-mutant analysis......Page 45
Experimental design......Page 47
Total RNA isolation and purification......Page 48
Purification of poly-A RNA......Page 49
Reverse transcription and dye labeling......Page 50
Hybridization......Page 51
Microarray washing......Page 52
Gridding and normalization......Page 53
References......Page 55
An Introduction to Microarray Data Analysis and Visualization......Page 57
Introduction......Page 58
Protocols......Page 144
Commonly used terms in microarray data analysis......Page 60
Background on MS instrumentation for ``shotgun´´ proteomics......Page 61
Protocol-Membrane Preparation and Solubilization
......Page 627
Measuring labeling efficiency......Page 62
Experimental Design......Page 406
Image Analysis......Page 64
What is a digital image?......Page 65
The pαF retrotranslocation assay
......Page 691
Extraction protocol for cells on filters (from vacuum filtration of liquid culture, or from filter cultures)
......Page 66
Software tools......Page 67
[R] and Bioconductor......Page 68
Calculating ratio values......Page 70
Global mean or median normalization......Page 71
Adjusting for spatial or print-tip bias......Page 73
Array quality......Page 74
Preprocessing Affymetrix arrays......Page 75
How hierarchical clustering works......Page 76
Software......Page 77
Partitioning and network-based approaches......Page 78
Significance analysis of microarrays......Page 79
Gene Ontology term mapping......Page 80
Graphing array data on genome tracks......Page 81
Data persistence and integrity......Page 82
Web-based access......Page 83
Public data repositories and MIAME compliance......Page 84
References......Page 87
Genome-Wide Approaches to Monitor Pre-mRNA Splicing......Page 89
Introduction......Page 90
Microarray Design......Page 91
Life Cycle......Page 94
C. albicans Total RNA Purification
......Page 760
RNA isolation......Page 95
Significance of Ultradian Cycles......Page 875
Protocol for RNA isolation......Page 97
cDNA synthesis......Page 98
Protocol for cDNA synthesis......Page 100
Imaging samples using fluorescent microscope......Page 102
Chromatin immunoprecipitation protocol......Page 765
Microarray hybridization......Page 104
Quantifying gene expression......Page 173
Double mutant array image acquisition and processing......Page 190
Materials for microarray washing......Page 105
Copy-number variation profiles......Page 380
Sampling regimen......Page 525
Microarray Data Analysis......Page 107
Transformation of DNA into fission yeast......Page 108
Extracting biological meaning......Page 109
Future Methodologies......Page 110
Acknowledgments......Page 360
References......Page 112
ChIP-Seq: Using High-Throughput DNA Sequencing for Genome-Wide Identification of Transcription Factor Binding Site......Page 114
Introduction......Page 115
Generation and Measurement of Double Mutant Strains......Page 118
Transformation buffers......Page 122
Illumina sequencing DNA library generation......Page 123
Barcode design and adapter annealing......Page 128
Illumina sequencing......Page 130
Sup35p-based prion assay......Page 482
Genome Analysis Pipeline......Page 131
Visualization in Genome Browser......Page 132
Basic selection on liquid versus solid media
......Page 549
Data analyses......Page 224
Track length......Page 138
References......Page 139
Genome-Wide Mapping of Nucleosomes in Yeast......Page 142
Introduction......Page 663
Other media supplements-Phloxin B......Page 149
Labeling of Mononucleosomal DNA for Tiling Microarray Analysis......Page 150
Protocol......Page 151
Generation of Nucleosomal DNA Libraries for Deep Sequencing......Page 152
References......Page 621
Genome-Wide Translational Profiling by Ribosome Footprinting......Page 156
Introduction......Page 157
Ribosome Footprint Generation and Purification......Page 158
Production and verification of Y2H datasets......Page 159
Nuclease digestion and monosome purification......Page 160
Footprint fragment purification......Page 161
Turbidostats......Page 517
Identifying orthogroups......Page 165
Polyadenylation......Page 166
Circularization......Page 167
PCR amplification......Page 168
Data Analysis......Page 170
Protocols......Page 171
Reference databases......Page 172
Sterol analysis by GC-MS......Page 279
Solutions......Page 175
Molecular Analysis......Page 176
Nucleic acid gel extraction......Page 177
References......Page 638
References......Page 554
Synthetic Genetic Array (SGA) Analysis in Saccharomyces cerevisiae and Schizosaccharomyces pombe......Page 180
Introduction......Page 181
SGA query strain construction......Page 183
Nonessential query strain: Gene deletion marker switch method......Page 184
Examination of P-bodies and stress granule in mid-log glucose-deprived cells
......Page 350
Manual pin tools (for low throughput: 1-2 genome-wide screens/month)......Page 185
BioMatrix robot (for ultrahigh-throughput SGA analysis)......Page 186
Constructing a 1536-density DMA......Page 187
MATa/α diploid selection and sporulation......Page 188
Schematic of the 13x myc tagging procedure
......Page 764
Freeze-substitution......Page 636
Antibody purification and specificity......Page 580
Interpretation and analysis of genetic interactions......Page 194
S. pombe SGA......Page 200
Monitoring disease progression......Page 834
SGA media and stock solutions......Page 203
RNA preparation from fission yeast
......Page 206
Protocol 8: Assessing equal representation of AD-Y clones in pools......Page 330
Preprocessing and normalization......Page 255
Chemical genomics......Page 210
References......Page 211
Making Temperature-Sensitive Mutants......Page 215
Introduction......Page 216
General Considerations......Page 604
General description of the diploid shuffle-plasmid method......Page 217
Other media supplements-Adenine and low-Ade media......Page 219
The in vitro degradation assay for pαF
......Page 370
Conditions to observe increases or decreases in P-bodies and stress granules
......Page 220
Freeze-substitution......Page 629
Breaking candidate orthogroups......Page 223
Probes used for the hybridization shown in Figs.26.1 and 26.3
......Page 573
Transforming yeast cells......Page 225
Screening for Ts alleles......Page 226
Confirming Ts mutants......Page 227
A general description......Page 228
Materials......Page 230
Triple quadrupole mass spectrometers......Page 439
Strains......Page 231
Cloning the PCR products......Page 232
Yeast transformation......Page 233
Screening for Ts alleles......Page 234
Allele transfer-out......Page 235
Perspectives......Page 236
References......Page 237
Quantitative Genetic Interaction Mapping Using the E-MAP Approach......Page 239
Introduction......Page 240
Selection of Mutations for Genetic Analysis......Page 243
Random mutagenesis by PCR......Page 248
Plates nomenclature......Page 250
Preparation of hybridization and wash buffers......Page 251
Recombination control......Page 253
Fission yeast plasmids......Page 791
Copy-number volatility......Page 501
Computing genetic interaction scores......Page 257
Identifying genes acting in the same pathway using patterns of interactions......Page 258
Using individual interactions to predict enzyme-substrate relationships......Page 259
Using individual interactions to predict opposing enzyme relationships......Page 261
Dissecting multiple roles of a single gene by detailed comparison of interaction patterns......Page 262
References......Page 263
Exploring Gene Function and Drug Action Using Chemogenomic Dosage Assays......Page 266
Introduction......Page 267
Promoter selection......Page 536
Yeast deletion strain pool construction......Page 270
MSP pool construction......Page 271
Freeze-substitution......Page 606
Additional tips for optimal yeast fluorescence microscopy of P-bodies and stress granules
......Page 650
Cameras......Page 273
Deletion profiling (HIP/HOP)......Page 276
Growth in liquid media......Page 317
Reconstitution of cytosol- and ATP-dependent degradation
......Page 689
Population size......Page 277
Hybridization......Page 278
Outlier masking (for HIP and HOP)......Page 280
Reagents......Page 379
Intranasal infection......Page 281
Special notes......Page 282
Confirmation of microarray data......Page 283
Experimental Considerations......Page 285
Perspectives......Page 286
Acknowledgments......Page 287
Yeast Expression Proteomics by High-Resolution Mass Spectrometry......Page 289
Introduction......Page 290
DHFR PCA Survival-Selection for Large-Scale Analysis of PPIs......Page 368
Quantitative proteomics......Page 297
Mounting and Storage of Slides......Page 596
Perspective and outlook......Page 300
Performing genetic crosses......Page 783
Yeast strains for SILAC proteomics experiments......Page 302
Media for SILAC labeling......Page 303
Single stage (nontargeted) profiling......Page 408
Biological evaluation of binary interactome maps......Page 304
Peptide IEF (Optional)......Page 305
Protein extraction for SDS-PAGE......Page 306
SOC medium......Page 307
Acknowledgments......Page 308
References......Page 471
High-Quality Binary Interactome Mapping......Page 311
Introduction......Page 312
High-Quality Binary Interactome Mapping......Page 313
Genomic Potential for Sex......Page 860
Cell Fixation, Preparation, and Storage......Page 668
Validation of Y2H datasets to produce reliable binary interactome maps......Page 318
Quality control I: Experimental assessment of dataset precision......Page 320
Minimizing autofluorescence......Page 321
Concluding Remarks......Page 553
Protocol 3: Bacterial transformation......Page 324
Protocol 4: Bacterial culture PCR......Page 325
Protocol 5: Yeast transformation......Page 326
Protocol 6: Identification of autoactivating DB-X hybrid proteins......Page 328
Efficient screening by AD pooling......Page 329
Screening and phenotyping......Page 331
Protocol 11: Y2H primary screening......Page 333
Protocol 12: Phenotyping......Page 334
Protocol 13: Verification of candidate Y2H interaction pairs......Page 335
YEPD agar plates......Page 337
Preparation of cells for bioluminescence assay......Page 393
Synthetic complete (Sc) media......Page 338
Validation Using Orthogonal Binary Interaction Assays......Page 339
Protocol 14: Yellow fluorescent protein complementation assay (YFP-PCA)......Page 340
Protocol 15: Well-nucleic acid programmable protein array (wNAPPA)......Page 341
Acknowledgments......Page 342
References......Page 343
Quantitative Analysis of Protein Phosphorylation on a System-Wide Scale by Mass Spectrometry-Based Proteomics......Page 346
Introduction......Page 347
Generation of peptide samples......Page 348
Calculation of phenotypic diversity
......Page 518
Phosphopeptide isolation......Page 351
Phosphopeptide isolation using a TiO2 resin......Page 352
Ure2p-based prion assay......Page 740
Phosphopeptide isolation using phosphoramidate chemistry......Page 353
Isolation of yeast microsomes containing integral membrane ERAD substrates
......Page 693
Quantification......Page 358
Simulated orthogroups......Page 491
References......Page 361
A Toolkit of Protein-Fragment Complementation Assays for Studying and Dissecting Large-Scale and Dynamic Protein-Protein Interactions in Living Cells
......Page 364
Introduction......Page 365
Techniques for Basic Culture......Page 816
Filter culture......Page 371
Materials......Page 569
Mounting......Page 375
A Life and Death Selection PCA Based on the Prodrug-Converting Cytosine Deaminase for Dissection of PPIs......Page 377
Lactose gradient centrifugation......Page 789
Death selection screen......Page 381
Survival selection screen......Page 382
Additional information......Page 383
Visualizing the Localization of PPIs with GFP Family Fluorescent Protein PCAs......Page 385
Equipment......Page 387
Cotransformation of competent yeast......Page 388
Timeline......Page 389
Studying Dynamics of PPIs with Luciferase Reporter PCAs......Page 390
Reagents......Page 391
Cotransformation of competent yeast......Page 392
References......Page 395
Yeast Lipid Analysis and Quantification by Mass Spectrometry......Page 398
Introduction......Page 399
Methods......Page 402
Chemostats......Page 404
Quantification of total sequence diversity and library maintenance
......Page 542
Sample Preparation......Page 615
Protocol for biolistic transformation
......Page 823
Quantitative analysis by multiple-reaction monitoring......Page 414
Acknowledgments......Page 418
Mass Spectrometry-Based Metabolomics of Yeast......Page 421
LC-MS Basics......Page 423
Determining the position of the nucleus......Page 562
Strains......Page 426
Choice of fluorophore and appropriate filter sets......Page 460
Short-Period Cycles......Page 874
Protocol for harvesting yeast by vacuum filtration......Page 428
Preparation of antibleach solution and enzymes (``GLOX solution´´)......Page 466
Metabolite Extraction......Page 430
Chemical Derivatization of Metabolites......Page 433
LC-MS for Mixture Analysis......Page 434
G1/G0 arrest of fission yeast by nitrogen starvation
......Page 435
Electrospray Ionization......Page 438
High-resolution mass analyzers......Page 441
Targeted Data Analysis......Page 443
Adducts......Page 446
Isotopic variants......Page 448
Future Outlook......Page 449
Soluble protein extract......Page 798
References......Page 450
Introduction......Page 455
RNA FISH Protocol......Page 458
Preparation of Different Conformations of In Vitro Sup-NM Amyloid
......Page 459
Purification of probes using HPLC......Page 461
Fusion polymerase chain reaction......Page 463
Hybridizing probes to target mRNA......Page 464
Image processing: Detecting diffraction-limited mRNA spot......Page 468
Example: STL1 mRNA Detection in Response to NaCl Shock......Page 470
Reconstructing Gene Histories in Ascomycota Fungi......Page 473
Introduction......Page 474
Chemostat setup......Page 477
Scoring gene similarity......Page 479
Growing and concentrating yeast......Page 480
Interpreting alterations in P-body/stress granule size and number
......Page 654
Evaluating Orthogroup Quality......Page 485
Fungal orthogroup robustness......Page 487
Comparison to curated resource......Page 489
Defining orthogroup categories......Page 492
Singletons and ORF predictions......Page 494
ECVPs within orthogroup classes......Page 497
ECVPs within interaction networks......Page 498
Estimating functional divergence between paralogous genes......Page 504
Estimating divergence based on degree of conserved interactions......Page 505
Discussion and General Applicability......Page 506
References......Page 508
Experimental Evolution in Yeast: A Practical Guide
......Page 512
Experiment Rationale......Page 513
C. albicans DNA Transformation......Page 758
Serial dilution......Page 515
Experiment duration......Page 520
Strains and markers......Page 521
Media......Page 522
Growth rate......Page 523
Good sterile practices......Page 524
Fitness......Page 526
Chemostat preparation......Page 527
Weekly sampling......Page 528
Conclusions......Page 529
References......Page 530
Enhancing Stress Resistance and Production Phenotypes Through Transcriptome Engineering
......Page 533
Introduction......Page 534
Transcription Factor Selection......Page 535
Acknowledgments......Page 724
Evaluation of mutant libraries......Page 545
Selecting for Phenotypes of Interest
......Page 548
Validation......Page 552
Visualizing Yeast Chromosomes and Nuclear Architecture
......Page 557
Introduction......Page 558
Tagging chromatin in vivo with lac and tet operator arrays
......Page 559
Basic Molecular Biology Techniques......Page 564
ATP regenerating system
......Page 565
Testing mating type and sporulation by iodine staining
......Page 784
Spinning-disk confocal microscopy
......Page 568
Quantification of locus mobility......Page 574
Mean-squared displacement analysis
......Page 576
IF and FISH on Fixed Samples......Page 579
Choice of fluorophores......Page 581
Protocol......Page 582
References......Page 588
Quantitative Localization of Chromosomal Loci by Immunofluorescence
......Page 590
Yeast Strain Construction......Page 591
Immunofluorescence......Page 593
Fixing Cells......Page 594
Spheroplasting......Page 595
Long-Period Cycles......Page 873
Microscopy and Analysis......Page 597
References......Page 600
Spinning-Disk Confocal Microscopy of Yeast
......Page 602
Introduction......Page 603
Choice of objective......Page 607
Other hardware considerations......Page 614
Correlative GFP-Immunoelectron Microscopy in Yeast
......Page 624
Introduction......Page 625
Recent Advances in High-Pressure Freezing and Freeze-Substitution
......Page 626
High-pressure freezing......Page 628
Conditions for amplification of 5prime and 3prime flanks and antibiotic resistance cassette
......Page 631
Sectioning......Page 632
Immunolabeling......Page 633
Semiautomated quantification of P-body size and number
......Page 634
Filtration and HPF......Page 635
Embedding......Page 637
Analyzing P-Bodies and Stress Granules in Saccharomyces cerevisiae
......Page 640
Introduction......Page 641
Markers of P-bodies and stress granules
......Page 643
Preparation of samples......Page 647
Quantification of P-Body Size and Number
......Page 656
Colony PCR......Page 826
Acknowledgments......Page 658
Analyzing mRNA Expression Using Single mRNA Resolution Fluorescent In Situ Hybridization
......Page 662
Probe Design......Page 664
Materials......Page 666
Protocol......Page 667
Materials......Page 669
Hybridization......Page 671
Protocol......Page 673
Image Acquisition......Page 674
Image Analysis......Page 675
Summary and Perspectives......Page 678
References......Page 679
The Use of In Vitro Assays to Measure Endoplasmic Reticulum-Associated Degradation
......Page 681
Introduction......Page 682
In Vitro ERAD Assays Using a Soluble Substrate, pαF
......Page 685
Microsome preparation......Page 686
The ubiquitination reaction
......Page 694
Analysis of integral membrane protein retrotranslocation
......Page 696
References......Page 697
A Protein Transformation Protocol for Introducing Yeast Prion Particles into Yeast
......Page 700
Introduction......Page 701
Purification of Sup-NM
......Page 702
Dominant drug selection markers......Page 817
Preparation of In Vivo Prions from Yeast......Page 704
Protein Transformation......Page 705
Determination of Prion Conversion Efficiency and Prion Strain Phenotypes
......Page 707
References......Page 711
Overexpression and Purification of Integral Membrane Proteins in Yeast
......Page 713
Introduction......Page 714
Protocol-Molecular Biology......Page 715
Protocol-Cell Growth......Page 717
Protocol-Protein Purification......Page 719
Protocol-Protein Characterization......Page 723
References......Page 773
Biochemical, Cell Biological, and Genetic Assays to Analyze Amyloid and Prion Aggregation in Yeast
......Page 726
Introduction......Page 727
Fluorescence microscopy and staining of amyloid-like aggregates
......Page 729
Sedimentation assay......Page 733
Filter retardation assay using yeast protein lysates
......Page 735
Assays for prion behavior......Page 736
Tetrad dissection......Page 742
Methods to analyze prion inheritance
......Page 743
Transformation of prion particles
......Page 744
References......Page 748
Genetics and Molecular Biology in Candida albicans
......Page 752
Homozygous Gene Disruption in C. albicans
......Page 753
Homozygous gene disruption by fusion PCR
......Page 755
C-Terminal Epitope Tagging in C. albicans
......Page 761
Transformation......Page 762
Transformation......Page 763
Chromatin immunoprecipitation buffers......Page 768
Strand displacement amplification of ChIP samples
......Page 769
Dye coupling......Page 771
ChIP-chip hybridization protocol (adapted from the Agilent oligo aCGH/chip-on-chip hybridization kit)
......Page 772
Molecular Genetics of Schizosaccharomyces pombe
......Page 774
Introduction......Page 775
Biology, Growth, and Maintenance of Fission Yeast
......Page 776
Genetics and Physiology......Page 782
Random spore analysis......Page 785
Bulk spore germination......Page 786
Isolating diploids......Page 787
Cell synchrony in fission yeast cultures......Page 788
Block and release using temperature sensitive alleles
......Page 790
Integrations in fission yeast......Page 793
Electroporation of fission yeast......Page 794
Rapid lithium acetate transformation......Page 795
Colony PCR......Page 796
Whole cell protein extract with trichloroacetic acid (TCA)
......Page 799
Flow cytometry (whole cells)......Page 800
Concluding Remarks......Page 842
Flow cytometry (nuclear "ghosts")
......Page 801
DNA and septum staining in fixed cells......Page 802
Fission yeast whole-cell immunofluorescence......Page 803
Appendix: Schizosaccharomyces pombe Online Resources
......Page 806
References......Page 808
Applying Genetics and Molecular Biology to the Study of the Human Pathogen Cryptococcus neoformans
......Page 811
Introduction......Page 812
Serotypes, Strains, and Sequences......Page 813
Fusion PCR for targeted gene deletion......Page 819
Conditions for fusion PCR
......Page 821
Genomic DNA extraction......Page 828
RNA extraction......Page 829
Murine model of infection......Page 831
Considerations of murine strain and age......Page 832
Signature-tagged mutagenesis screening......Page 835
STM qPCR conditions
......Page 837
Calculating STM score......Page 838
Assay for killing of C. neoformans by macrophages
......Page 839
Melanization......Page 840
Melanization test protocol
......Page 841
References......Page 843
The Fungal Genome Initiative and Lessons Learned from Genome Sequencing
......Page 846
Introduction: Yeast Genomes and Beyond
......Page 847
Computational Prediction of Genes and Noncoding Elements
......Page 854
Mechanisms of Genome Evolution......Page 856
Gene Family Conservation and Evolution......Page 861
Impact of Next-Generation Sequencing......Page 862
Future Directions......Page 864
Acknowledgments......Page 865
References......Page 866
Introduction......Page 869
Induction of Ultradian Cycles of Oxygen Consumption Using a Chemostat
......Page 870
Comments......Page 872
References......Page 877
Author Index......Page 879
Subject Index......Page 891