YEAST MOLECULAR BIOLOGY
A SHORT COMPENDIUM
ON BASIC FEATURES
AND NOVEL ASPECTS
Horst Feldmann
Adolf-Butenandt-Institute
University of Munich
2005
Table of Contents
| 1 Introduction - Yeast as a Versatile Model System |
| 1.1 Yeast History |
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1.2 Yeast - an Experimental System for Molecular Biology |
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1.3 Experimental Approaches in Yeast Molecular Biology |
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2.1 General Cellular Characteristics of Yeast |
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2.2 Cytological Methods |
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2.3 Yeast Cell Organelles and Compartments |
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2.3.1 Cell Envelope |
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2.3.2 Cell Wall |
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2.3.3 Cytosol and Cytoskeleton |
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2.3.4 Nucleus and Extrachromosomal Elements |
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2.3.5 Secretory System and Vacuoles |
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2.3.6 Peroxisomes |
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2.3.7 Mitochondria |
| 3 Yeast Metabolism |
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3.1 Sugar Catabolism in Yeast |
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3.1.1 Principal Pathways |
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3.1.2 Regulation of Biochemical Pathways |
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3.1.3 Respiration versus Fermentation |
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3.1.4 Other sugars - Galactose |
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3.1.5 Metabolism of Non-Hexose Carbon Sources |
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3.2 Gluconeogenesis and Carbohydrate Biosynthesis |
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3.3 Fatty Acid and Lipid Metabolism |
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3.4 Nitrogen Metabolism |
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3.4.1 Catabolic Pathways |
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3.4.2 Amino Acid Biosynthesis Pathways |
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3.4.3 Protein Biosynthesis |
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3.5 Phosphate Metabolism |
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3.6 Sulphur Metabolism |
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3.7 Transition Metals |
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4.1 Isolation of Particular Cell Types and Components |
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4.2 Yeast Vectors |
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4.3 Yeast Expression Vectors |
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4.3.1 Regulated Promoters |
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4.3.2 Secretion of Heterologous Proteins from Yeast |
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4.3.3 Post-translational Processing and Modification of Heterologous Proteins in Yeast |
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4.3.4 GFP Fusion Proteins |
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4.4 Yeast Cosmid Vectors |
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4.5 Yeast Artificial Chromosomes (YACs) |
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4.6 The Yeast Two-Hybrid System |
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4.7 The Yeast One-Hybrid (Matchmaker) System |
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5.1 The Yeast Genome Sequencing Project |
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5.1.1 Cloning and Mapping Procedures |
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5.1.2 Sequencing Strategies, Sequence Assembly and Quality Control |
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5.1.3 Sequence Analysis |
| 5.2 The Yeast Genome: Life with 6000 Genes |
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5.2.1 The Proteome: Open Reading Frames and Gene Function |
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5.2.2 Overlapping ORFs, Pseudogenes and Introns |
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5.2.3 Putative Membrane Proteins, Mitochondrial Proteins |
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5.2.4 Other Genetic Entities |
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5.2.4.1 Yeast Ribosomal Genes |
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5.2.4.2 Yeast tRNA Genes and Codon Selection |
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5.2.4.2.1 Number and Genomic Distribution of Yeast tRNA Genes |
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5.2.4.2.2 tRNA Families, tRNA Gene Redundancy, and tRNA Gene Variants |
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5.2.4.2.3 Introns and Processing of Precursor tRNAs |
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5.2.4.2.4 tRNA Multiplicity and Codon Capacity in Yeast |
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5.2.4.2.5 Correlation of tRNA Abundance to Gene Copy Number |
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5.2.4.2.6 tRNA Gene Redundancy and Codon Selection in Yeast |
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5.2.4.2.7 Genomic Organisation of Yeast tRNA Genes and Evolutionary Aspects |
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5.2.4.3 Yeast Ty Elements |
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5.2.5 Genome Architecture and Gene Organisation |
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5.2.5.1 Gene Density and Gene Arrangement of Protein-encoding Genes |
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5.2.5.2 Base Composition and Gene Density |
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5.2.5.3 Functional Elements of Yeast Chromosomes |
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5.2.5.3.1 Centromeres |
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5.2.5.3.2 Origins of Replication |
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5.2.5.3.3 Telomeres |
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5.2.5.4 Complex and Simple Repeats |
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5.2.5.5 Comparison of Genetic and Physical Maps |
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5.2.5.6 Genome Organisation and Evolutionary Aspects |
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5.2.5.6.1 Genetic Redundancy in Yeast |
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5.2.5.6.1.1 Duplicated Genes in Subtelomeric Regions |
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5.2.5.6.1.2 Duplicated Genes Internal to Chromosomes |
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5.2.5.6.1.3 Duplicated Genes in Clusters |
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5.2.5.6.1.4 Cluster Homology Regions (CHRs) |
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5.2.5.6.1.5 Redundancy and Gene Organization |
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5.2.5.6.2 Sequence Variation Among Yeast Strains |
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5.2.6 Gene Families |
| 5.2.7 Comparison of Yeast with Other Genomes |
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5.2.7.1 The Human-Yeast Connection |
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5.2.7.2 Other Model Organisms |
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6.1 Genomics and Gene Function |
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6.2 Analysis of Gene Functions in International Programmes |
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6.2.1 The EUROFAN Programme |
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6.2.2 The TRANSATLANTIC Programme |
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6.2.3 Other Programmes |
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6.3 Genome-wide Microarrays |
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6.4 Yeast Proteomics |
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6.4.1 2D-Gels in Proteomics |
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6.4.2 Systematic Two-Hybrid Analysis |
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6.5 Further Recent Developments |
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6.6 Tools and Databases |
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7 Yeast Gene Families for Multiple Functions |
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7.1.1 General Features |
| 7.1.2 Cellular Activities of Representative AAA+ Proteins |
| 7.1.2.1 ATP-dependent Proteases |
| 7.1.2.1.1 Proteasomal ATPases |
| 7.1.2.1.2 AAA Metallo-Proteases |
| 7.1.2.1.3 Other ATP-dependent Proteolytic Complexes |
| 7.1.2.2 Membrane Fusion Proteins |
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7.1.2.3 Biogenesis and Transport |
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7.1.2.4 DNA Replication Proteins |
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7.1.3 Disorders |
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7.1.4 Structure and Mechanisms of AAA ATPases |
| 7.1.4.1 Structures |
| 7.1.4.2 Mechanisms |
| 7.2 Ubiquitin and Related Proteins |
| 7.2.1 Ubiquitin |
| 7.2.1.1 Programmed Proteolysis By The Proteasome |
| 7.2.1.2 Programmed Proteolysis By The Vacuolar System |
| 7.2.2 Ubiquitin-Like Molecules (UBLs) |
| 7.2.2.1 SUMO |
| 7.2.2.2 Rub1/Nedd8 |
| 7.2.3 Ubiquitin-Domain Proteins (UDPs) |
| 7.2.3.1 Rad23 |
| 7.3 Programmed Proteolysis in Yeast |
| 7.3.1 General Features |
| 7.3.2 General Features of the Ubiquitin-Proteasome System |
| 7.3.3 26 S Proteasomes |
| 7.3.3.1 Occurrence and Structures |
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7.3.3.2 The Yeast 26S Proteasome as a Model System |
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7.3.3.3 Regulated Expression of the RPT Genes |
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7.3.3.4 Regulatory Networks Involving Rpn4 |
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7.3.4 Controlled Degradation of Cellular Proteins |
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7.3.4.1 Programmed Proteolysis Coupled to Retrograde Protein Transport (ERAD) |
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7.3.4.2 Controlled Degradation of Regulatory Proteins |
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7.3.4.2.1 Cell Cycle Control Factors |
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7.3.4.2.2 Transcriptional Regulators |
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7.3.4.2.3 Developmental Factors |
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7.3.4.2.4 Membrane Receptors and Hormonal Control |
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7.3.5 Immunoproteasomes |
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7.3.6 Selective Inhibitors of Proteasome Activity |
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7.3.7 Cellular Distribution of Proteasomes and Regulation of Proteasome Activity |
| 7.3.8 Proteasome-like Particles |
| 7.3.9 Conclusions |
| 7.4 Small ATP-and GTP-binding Proteins (Non-AAA+ Proteins) |
| 7.5 Proteinkinases and Proteinphosphatases |
| 7.6 DExD-Proteins and Helicases |
| 7.7 Aminoacyl tRNA Synthases |
| 8 Transport in Yeast |
| 8.1 Internal Transport and Import/Export of Proteins |
| 8.2 Membrane Transporters in Yeast |
| 8.2.1 Channels and ATPases |
| 8.2.2 Transition Metal Transport |
| 8.2.2.1 Iron |
| 8.2.2.2 Copper |
| 8.2.2.3 Zinc |
| 8.2.2.4 Manganese |
| 8.2.3 Phosphate Transport |
| 8.2.4 Nutrient Transporters |
| 8.2.5 ABC Transporters |
| 8.3 Nuclear Transport |
| 8.4 Transport in Peroxisome Biosynthesis |
| 8.5 Mitochondrial Transport |
| 8.5.1 Transport of Substrates |
| 8.5.2 Electron Transport Chain |
| 8.5.3 Proton Transport – ATP Synthesis |
| 8.5.4 Import of Proteins into Mitochondria |
| 9 Chromatin |
| 9.1 Chromatin Structure |
| 9.2 Chromatin Remodelling |
| 9.2.1 Chromatin Modifying Activities |
| 9.2.2 Chromatin Remodelling Complexes |
| 9.3 Chromatin During Replication, Transcription, and Repair |
| 9.3.1 Replication |
| 9.3.2 Transcription |
| 9.3.3 Repair |
| Appendix: Structure of Nucleosomes |
| 10 Yeast Growth and Cell Cycle |
| 10.1 Vegetative Reproduction in Yeast |
| 10.1.1 Budding |
| 10.1.2 Spindle Pole Body |
| 10.1.3 Septins |
| 10.2 The Yeast Cell Cycle |
| 10.2.1 General |
| 10.2.2 DNA Replication |
| 10.2.3 Spindle Dynamics |
| 10.2.4 Sister Chromatid Cohesion and Separation |
| 10.2.5 Spindle Checkpoint |
| 10.3 Sexual Reproduction |
| 10.3.1 Mating Types and Mating |
| 10.3.2 Meiosis and Sporulation |
| 11 Yeast Retrotransposons |
| 11.1 Classes of Transposable Elements |
| 11.2 Ty Elements and Their Genomes |
| 11.3 Genomic Organisation and Transposition Activity of the Ty Elements |
| 11.4 Ty Expression |
| 11.5 Codon Usage in Ty Open Reading Frames |
| 11.6 Retrotranscription |
| 11.7 Interactions Between Retroelements and Host |
| 12 Gene Expression in Yeast |
| 12.1 Transcription and Chromatin |
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12.1.1 Basal Transcription Machineries |
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12.1.2 RNA Polymerase I |
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12.1.3 RNA Polymerase II |
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12.1.3.1 The Basal Pol II Transcription Machinery in Yeast |
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12.1.3.2 Conservation of Transcription Machineries |
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12.1.3.3 Chromatin Structure in Polymerase II Genes |
| 12.1.3.3.1 Remodeling Machines |
| 12.1.3.3.2 Histone Modifiers |
| 12.1.3.4 Gene-specific Factors, DNA-binding Proteins, and Promoters in Pol II Transcription |
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12.1.3.4.1 Yeast Transcription Factors |
| 12.1.3.4.2 Regulation of PolII Gene Transcription |
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12.1.4 RNA Polymerase III |
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12.1.4.1 tRNA Gene Expression |
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12.1.4.2 5S RNA transcription |
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12.2 Processing of RNA Precursors and Splicing |
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12.2.1 Processing of rRNA Precursors |
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12.2.2 Processing and Splicing of tRNA Precursors |
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12.2.3 Processing of mRNA Precursors |
| 12.2.3.1 Capping |
| 12.2.3.2 3’end Processing and Polyadenylation |
| 12.2.3.3 Splicing of mRNA |
| 12.3 Posttranscriptional Control of Gene Expression |
| 12.3.1 Generation of Functional mRNA |
| 12.3.2 Yeast Translation Apparatus |
| 12.3.3.1 Initiation |
| 12.3.3.2 Elongation and Termination |
| 12.3.3.3 Mitochondrial Translation |
| 12.3.3 Pathways of Translational Initiation |
| 12.3.4 Mechanisms of Translational Regulation |
| 12.3.4.1 Modulation of Factor Activities |
| 12.3.4.2 Gene Specific Regulation via Trans-acting Factors |
| 12.3.5 Control and Pathways of mRNA Decay |
| 13 Signalling and Regulatory Circuits in Yeast |
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13.1 MAP Kinase Pathways in Yeast |
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13.1.1 Mating Response and Mating Type Switch |
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13.1.2 Filamentation/Invasion Pathway |
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13.1.3 Control of Cell Integrity |
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13.1.4 High Osmolarity Growth (HOG) Pathway |
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13.1.5 Spore Wall Assembly Pathway |
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13.2 General Control by Gene Repression |
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13.3 Gene Regulation by Nutrients |
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13.3.1 General Control of Cell Growth by Nutrients |
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13.3.1.1 The TOR System |
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13.3.1.2 Activation and Repression by Rap1 |
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13.3.2 Regulation of Glucose Metabolism |
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13.3.3 Regulation of Galactose Metabolism |
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13.3.4 General Amino Acid Control |
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13.3.5 Regulation of Arginine Metabolism |
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13.4 Stress Responses in Yeast |
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13.4.1 Temperature Stress and Heat-shock Proteins |
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13.4.2 Oxidative and Chemical Stresses |
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13.4.2.1 AP-1 Transcription Factors in Yeast |
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13.4.2.1.1 Yap1p, the Major Regulon in Oxidative Stress Response |
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13.4.2.1.2 Yap2p/Cad1p in Response to Toxic Compounds |
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13.4.2.1.4 Yap4p and Yap6p in the Response to Osmotic Stress |
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13.4.2.1.6 Yap8p/Acr1p and its Role upon Arsenic Conditions |
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13.4.2.1.5 Remaining Yap Members: Yap3p, Yap5p, and Yap7p |
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13.4.2.2 The STRE-dependent System |
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13.4.2.3 Pleiotropic Drug Resistance |
| 14 Hemiascomycetous Yeasts |
| 14.1 The GÉNOLEVURES Project |
| 14.2 Lessons from Comparative Analyses among Hemiascmycetes |
| 14.2.1 Common and Species Specific Functions |
| 14.2.2 Evolutionary Aspects |
| 15 Aspects of Fungi Related to Yeast |
| 15.1 Diseases Caused by Fungi |
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15.1.1 Superficial Mycoses |
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15.1.2 Subcutaneous Mycoses |
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15.1.3 Systemic Mycoses |
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15.2 Examples and Potential Therapy |
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15.2.1 Pseudohyphal Growth in Yeasts |
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15.2.2 Candida albicans |
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15.3 Other Pathogenic Fungi |
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15.3.1 Histoplasma capsulatum |
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15.3.2 Cryptococcus performans |
| 15.3.3 Blastomyces dermatitidis |
| 15.3.4 Coccidioides |