Molecular Biology and Genetic Engineering of Yeasts

406 Pages·1992·42.579 MB·\406

by  Henri Heslot (Author)

Molecular Biology
and
Genetic Engineering
of
Yeasts
Authors
Henri Heslot, D.Sc.
Emeritus Professor
Department of Genetics
Institut National Agronomique
Paris-Grignon, France
Claude Gaillardin, Ph.D.
Professor
Department of Molecular and Cellular Genetics
Institut National Agronomique
Paris-Grignon, France
Boca Raton London New York
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Library of Congress Cataloging-in-Publication Data
Heslot, H.
Molecular biology and genetic engineering of yeasts/authors,
Henri Heslot, Claude Gaillardin.
p. cm.
Includes bibliographical references and index.
ISBN 0-8493-5645-8
1. Yeast fungi—Biotechnology. 2. Genetic engineering.
3. Molecular biology. I. Gaillardin, Claude. II. Title.
TP248.27.Y43H47 1991
660’.65—dc20 91-33676
A Library of Congress record exists under LC control number: 91033676
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THE AUTHORS
Henri Heslot, D.Sc., is Emeritus Professor of Genetics at the Institut National Agron-
omique Paris-Grignon (France). He is Vice-Chairman of the French Society of Microbiology
and member of the "Comite des Applications de I' Academie des Sciences".
He is an author of over 100 articles on yeast genetics and molecular biology and has
presented his research findings at numerous national and international meetings.
Claude Gaillardin, Ph.D., is Professor of Molecular and Cellular Genetics at the Institut
National Agronomique Paris-Grignon (France), Director of the CNRS-INRA associated
Laboratory of Genetics of Industrial Microorganisms.
After entering Ecole Normale Superieure in 1966, he received his degrees from the
Universite de Paris and completed a Ph.D. in 1976 on yeast genetics at the Institut National
Agronomique. He entered the INRA in 1976 and after a postdoctoral stay at MIT (Cambridge,
U.S.) held successive appointments at INRA as Research Fellow, Research Director, and
currently Professor at the Institut National Agronomique.
His major interests are on the molecular genetics of nonconventional yeasts with par-
ticular emphasis on their biotechnological applications. In addition to his research activities,
he is active as a teacher and as a member of several scientific evaluation or advisory
committees.
TABLE OF CONTENTS
Chapter 1
Yeast Transformation .................................................................. .
I. Yeast Transformation ............................................................ .
A. Procedures for Yeast Transformation ...................................... 1
B. Fate of Transforming DNA ................................................ 2
C. Marker Genes ............................................................. 4
II. Gene Manipulation with Integrative Vectors ...................................... 4
A. Basic Features of Integrative Transformation .............................. 4
B. Gene Replacement Strategies .............................................. 7
III. Replication Origins (ARS) and YRp Vectors ...................................... 8
A. Chromosome Structure and Replication ................................... 9
B. Autonomously Replicating Sequences (ARS) ............................. 12
C. Stability of ARS Plasmids ................................................ 14
D. ARS Structure ............................................................ 15
IV. Centromeres and YCp Vectors ................................................... 17
A. Background .............................................................. 17
B. Isolation of Yeast Centromeres ........................................... 17
C. Structure of Saccharomyces cerevisiae Centromeres ...................... 18
D. CEN Plasmids (YCp) in Saccharomyces cerevisiae ....................... 20
V. Telomeres and YA C vectors ..................................................... 22
A. Telomeres and 3' End Replication in Ciliates ............................ 22
B. Isolation and Structure of Yeast Telomeres ............................... 23
C. Telomere Elaboration in Yeast ........................................... 25
D. Yeast Artificial Chromosomes (YAC) .................................... 25
References ............................................................................... 28
Chapter 2A
Yeast Plasmids: The 2-JJ.m Circle Plasmid of Saccharomyces cerevisiae ............. 41
I. Introduction ...................................................................... 41
II. Stability of the 2-1..1-m Circle in Yeast Populations ............................... 41
III. Recombination ................................................................... 41
IV. Control of 2-j.Lm Copy Number. ................................................. 46
A. Partition System .......................................................... 47
B. Amplification System .................................................... 48
V. Regulation of 2-j.Lm Copy Number .............................................. 49
VI. A Model for Plasmid Copy Control. ............................................. 50
VII. 2-j.Lm-Like Plasmids in Other Yeast Species ..................................... 51
VIII. Vectors Derived from the 2-1..1-m Circle Plasmid .................................. 51
References ............................................................................... 52
Chapter 2B
Yeast Plasmids: Linear and Circular Plasmids from Kluyveromyces. ................ 57
I. Linear Plasmids .................................................................. 57
A. Killer Character .......................................................... 57
B. Genetic Control of Killer and Immunity Characters ...................... 58
C. Sequence of Linear Plasmids ............................................. 59
D. Lack of Expression of k1 Plasmid Genes inside the Nucleus ............. 61
E. Replication of k1 and k2 ................................................. 61
F. Structure of Deleted Plasmids ............................................ 62
G. Transmission of k 1 and k2 to Heterologous Yeasts ....................... 64
H. Incompatibility of k Plasmids with Mitochondrial DNA in
Saccharomyces cerevisiae ................................................ 64
I. Autonomously Replicating Sequence Elements ........................... 64
J. Vector Construction ...................................................... 65
K. Other Linear Plasmids .................................................... 66
II. Circular Plasmids ................................................................ 66
References ............................................................................... 67
Chapter 3
Transposable Elements in Yeast ....................................................... 71
I. Introduction ...................................................................... 71
II. Structure and Variability of Ty .................................................. 71
III. Transcription - Influence of Mating Type ...................................... 73
IV. Proteins Encoded by Ty ......................................................... 73
V. Effects of Ty on Expression of Adjacent Genes- the ROAM Effect ........... 74
VI. Gene Conversions Associated with Ty ........................................... 75
VII. DNA Rearrangements Induced by Ty ............................................ 75
VIII. Mobilization of Ty ............................................................... 77
IX. Transposition and VLPs ......................................................... 77
X. Relationship of Ty to Retroviruses ............................................... 80
XI. Cell-Free Ty Transportation ...................................................... 81
XII. Mutagenic Effects of Transposition .............................................. 83
XIII. Tagging with Ty and Gene Amplification ........................................ 83
XIV. Production of Fusion Proteins and Potential Vaccines ........................... 85
XV. Other Transposable Elements in Yeast ........................................... 86
References ............................................................................... 87
Chapter 4
Transcriptional Control of Gene Expression .......................................... 91
I. Introduction ...................................................................... 91
A. Overview of a Yeast Polymerase II Promoter ............................ 91
II. Upstream Regulatory Regions ................................................... 92
A. Upstream Activating Sequences .......................................... 93
1. General Properties ................................................ 93
2. Isocytochrome c Genes UAS ..................................... 95
a. UASl ..................................................... 96
b. UAS2 ..................................................... 98
3. Other specific UASs .............................................. 98
a. Repeated UAS ............................................. 98
b. GCN4 UAS ............................................... 99
c. Other Palindromic UASs .................................. 99
d. Complex UASs ........................................... 101
e. PGK UAS ................................................ 101
f. P and Q Boxes ........................................... 101
4. Ubiquitous UASs ................................................ 102
a. RPG Box ................................................. 103
b. ABF1 Box ................................................ 103
c. Perspectives .............................................. 104
B. Downstream Activating Sequences? ..................................... 106
1. Evidence for Possible Downstream Activation ................... 106
2. DAS in Ty ....................................................... 106
3. Other Possible DASs in PYK and PGK .......................... 107
C. Negative Regulatory Sites ............................................... 107
1. Bacterial Operators in Yeast ..................................... 107
2. Mata2 and Mata l/Mata2 Operators .............................. 108
a. a2 Operator .............................................. 109
b. a1/a2 Operator ........................................... 110
c. Overlapping Operators and UAS Sites? .................. 111
3. GAL Operators .................................................. 111
4. Silencers ......................................................... 114
D. Constitutive Expression, Poly (dNdT) Tracts, and Chromatin
Structure ................................................................. 116
III. Basal Promoter Sequences ...................................................... 118
A. Initiator Element ........................................................ 118
B. Selector Elements ....................................................... 120
IV. Regulatory Proteins ............................................................. 125
A. Structure of Regulatory Proteins ......................................... 125
1. Mata2 Repressor and Homeodomain ............................ i25
2. HAP1, GAL4, and Zinc Fingers ................................. 129
3. GCN4 and Leucine Zippers ...................................... 133
4. Other Leucine Zippers ........................................... 136
a. yAP1 ..................................................... 136
b. HSF ...................................................... 137
5. Complex Activators and HAP 2/3/4 Trimers ..................... 138
B. Interactions of Regulatory Proteins ...................................... 140
1. Signal Transmission to the Regulatory Proteins .................. 140
a. Binding of a Small Molecule Modifies DNA-
Binding ................................................... 140
b. Covalent Modifications Modify Activity of the
Bound Factor ............................................. 141
c. Binding of Another Protein Blocks and the
Activation Domain ....................................... 141
d. Synthesis of the Activator Is Controlled .................. 141
e. Combinatorial Complexes Binding Specificity ............ 141
2. Signal Transmission from the Regulatory Proteins ............... 141
a. Which Targets? ........................................... 142
b. Control at a Distance ..................................... 145
c. Mechanisms of Activation ................................ 146
References .............................................................................. 148
Chapter 5
Regulation of the Galactose Genes of Saccharomyces cerevisiae ..................... 161
I. Introduction ..................................................................... 161
II. Organization and Transcription of GAL Genes ................................. 161
III. Upstream Activating Sequences ................................................. 161
IV. Properties of the GAL4 Protein ................................................. 162
V. Properties of the GAL80 Protein ................................................ 166
VI. Role of the GAL3 Protein ...................................................... 167
VII. Interactions between GAL4 Protein and RNA Polymerase II ................... 168
VIII. Glucose Repression ............................................................. 168
IX. UAS and GAL4 Protein Activate Gene Expression in Heterologous
0
Cells ............................................................................ 169
References .............................................................................. 170
Chapter 6A
Protein Localization and Processing in Yeast: Nuclear Targeting .................. 175
I. Introduction ..................................................................... 175
II. Nuclear Targeting Sequences ................................................... 175
A. MATu2 Protein ......................................................... 175
B. GAL4 Protein ........................................................... 175
C. Histones ................................................................. 176
D. L3 Ribosomal Protein ................................................... 177
III. Search for Nuclear Protein Receptors ........................................... 177
IV. Nuclear Targeting in Other Systems ............................................ 179
V. In Vitro Studies ................................................................. 179
References .............................................................................. 180
Chapter 6B
Protein Localization and Processing in Yeast: Vacuolar Protein Targeting ........ 183
I. Introduction ..................................................................... 183
II. Lysosome Targeting in Mammalian Cells ....................................... 183
III. Transit of Vacuolar Proteins through the Early Part of the Secretory
Pathway ......................................................................... 183
IV. Gene Dosage-Dependent Secretion of Vacuolar Proteins ........................ 186
V. Role of the PEP4 Gene Product. ................................................ 187
VI. Processing of Proteinase B ...................................................... 188
VII. Identification of Vacuolar Targeting Sequences ................................. 189
VIII. Mutants Affected in Vacuolar Protein Targeting ................................ 190
IX. Characteristics of Mutants Defective in Vacuolar Protein Localization .......... 191
References .............................................................................. 192
Chapter 6C
Protein Localization and Processing in Yeast: Targeting Proteins
into Mitochondria ..................................................................... 195
I. Structure and Functions of Mitochondria ........................................ 195
II. Gene Fusion Approach .......................................................... 195
A. Strategy ................................................................. 195
B. Matrix Proteins .......................................................... 19 5
C. Inner Membrane Proteins ................................................ 196
D. Outer Membrane Proteins ............................................... 197
III. Physiological and Biochemical Studies ......................................... 197
A. In Vitro Experiments .................................................... 197
B. Outer-Inner Membrane Contact Sites: Means of Blocking
Translocation ............................................................ 198
C. Unfolding of Precursors ................................................. 199
D. Mitochondrial Receptors: General Insertion Protein ..................... 201
E. Blocking of Import Sites with Specific Fusion Proteins:
Isolation of an Import Protein ........................................... 202
F. Import of Other Artificial Precursors .................................... 204
G. Structure and Function of Presequence .................................. 205
H. Matrix Processing Protease .............................................. 207
I. Chaperonins: Role of HSP60 ............................................ 208
IV. Evolutionary Significance ....................................................... 208
V. Relocation of Mitochondrial Genes to the Nucleus .............................. 210
A. Subunit 8 of mit ATPase ................................................ 210
B. Intron-Encoded bl4 Maturase ............................................ 210
References .............................................................................. 211
Chapter 6D
Protein Localization and Processing in Yeast: Peroxisomes ......................... 215
I. Introduction ..................................................................... 215
II. Role of Peroxisomes in Alkane Catabolism ..................................... 215
III. Role of Peroxisomes in Methanol-Utilizing Yeasts .............................. 217
IV. Peroxisomes in Saccharomyces cerevisiae ...................................... 218
V. Molecular Cloning of Genes Coding for Peroxisomal Proteins .................. 219
A. Methanol Oxidase Gene ................................................. 219
B. Dihydroxyacetone Synthase Gene ....................................... 220
C. Acyl-CoA Oxidase Genes ............................................... 220
D. Catalase Gene ........................................................... 221
E. POX18 Gene ............................................................ 221
F. Gene Coding for the Peroxisomal Trifunctional Enzyme
Hydratase-Dehydrogenase-Epimerase of Candida tropicalis ............. 221
VI. In Vitro Systems ................................................................ 221
VII. Heterologous Expression of Genes Coding for Peroxisomal Proteins ........... 222
VIII. Overproduction of Methanol Oxidase in H ansenula polymorpha ................ 224
IX. The Firefly Luciferase System .................................................. 224
X. Use in Biotechnology ........................................................... 228
XI. Peroxisomes Biogenesis and Evolutionary Origin ............................... 228
References .............................................................................. 229
Chapter 6E
Protein Localization and Processing in Yeast: Targeting of Integral Membrane
Proteins ................................................................................ 233
I. Introduction ..................................................................... 233
II. Topology of Integral Membrane Proteins ....................................... 233
III. Structure Predictions ............................................................ 233
IV. Transmembrane Integration Signals ............................................. 234
A. Stop-Transfer Signals .................................................... 236
B. Uncleaved Signal Peptide ............................................... 236
C. Start-Stop Signals ....................................................... 236
D. Polytopic Membrane Proteins ........................................... 236
V. Tools for Molecular Genetic Analysis of Membrane Protein Topology
in Bacteria ...................................................................... 23 7
VI. Yeast Integral Membrane Proteins .............................................. 238
A. Transit through the Secretory Pathway .................................. 238
B. Specific Examples ....................................................... 239
VII. Prospects ........................................................................ 241
References .............................................................................. 242
Chapter 7
Protein Secretion ...................................................................... 245
I. Introduction ..................................................................... 245
II. Brief Overview of the Secretory Pathway ....................................... 245
III. Thermosensitive sec Mutants ................................................... 245
IV. Early Steps of the Secretory Pathway in Mammalian Cells and in
Yeasts ........................................................................... 247
A. Signal Recognition Particle and Its Receptor ............................ 247
B. Isolation of Mutants Blocked at a Very Early Stage of Import
into the Endoplasmic Reticulum ......................................... 251
C. Role of 70-kDa Stress Proteins in Protein Translocation through
the Endoplasmic Reticulum ............................................. 252
V. The Signal Peptide and Its Processing .......................................... 253
VI. Glycosylation of Secretory Proteins ............................................. 254
VII. Role of SEC Genes in the Assembly and Fusion of a Vesicle
Intermediate in Endoplasmic Reticulum to Golgi Transport ..................... 256
VIII. Retention of Soluble Proteins in the ER ........................................ 258
IX. Mutations Affecting the Golgi Apparatus and Secretory Vesicles ............... 260
X. Role of GTP-Binding Proteins in Membrane Targeting and Secretion .......... 261
A. Membrane Targeting .................................................... 261
B. Secretion ................................................................ 262
XI. In Vitro Translocation Systems ................................................. 265
A. Mammalian System ..................................................... 265
B. Yeast Systems ........................................................... 267
XII. Endocytosis ..................................................................... 270
XIII. Conclusion ...................................................................... 272
References .............................................................................. 272
Chapter 8A
Saccharomyces cerevisiae Mating Pheromones ....................................... 279
I. Biological Basis ................................................................. 279
II. a-Factor Mating Pheromone .................................................... 279
A. Cloning and Sequencing of the MFal Gene ............................. 279
B. Glycosylation and Processing of Prepro-a-Factor. ....................... 279
C. Gene Fusions to MFa 1 .................................................. 281
III. a-Factor Mating Pheromone ..................................................... 282
References .............................................................................. 284
Chapter 8B
The Killer Toxin of Saccharomyces cerevisiae ........................................ 287
I. Introduction ..................................................................... 287
II. Cloning and Sequencing of the Preprotoxins dsRNA Gene ..................... 287
III. Processing of Preprotoxin ....................................................... 287
IV. Functional Domains of the Preprotoxin ......................................... 288
V. Mechanisms of Killing .......................................................... 289
VI. Mechanisms of Immunity ....................................................... 289
References .............................................................................. 290
Chapter 8C
Saccharomyces cerevisiae Invertase ................................................... 293
I. Invertase Gene Family .......................................................... 293
II. SUC2 Encodes Two Forms of Invertase ........................................ 293
III. Structure of Invertase - Effect of Glycosylation ............................... 293
IV. Secretion of Octameric Invertase ................................................ 294
V. Failure of Secretion of SUC2-LacZ Hybrid Proteins ............................ 295