Saccharomyces

BIOTECHNOLOGY HANDBOOKS
Series Editors: Tony Atkinson and Roger F. Sherwood
PHLS Centre for Applied Microbiology and Research
Division of Biotechnology
Salisbury, Wiltshire, England

Volume 1 PENICILLIUM ANDACREMONIUM

Edited by John F. Peberdy

Volume 2 BA GILL US
Edited by Colin R. Harwood

Volume 3 CLOSTRIDIA
Edited by Nigel P. Minton and David]. Clarke

Volume 4 SACCHAROMYCES
Edited by Michael F. Tuite and Stephen G. Oliver

A Continuation Order Plan is available for this series. A continuation order will bring
delivery of each new volume immediately upon publication. Volumes are billed only upon
actual shipment. For further information please contact the publisher.
Saccharo"!)Jces

Edited by
Michael F. Tuite
The University of Kent, Canterbury
Kent, England

and
Stephen G. Oliver
Manchester Biotechnology Centre, UMIST
Manchester, England

Springer Science+Business Media, LLC

 Ltbrary of Congraee Catalogtng-tn-Publtcatton Data

Saccharo•yces 1 ed1ted by M1chae1 F. Tu1te and Stephen G. 011var.

p. c1. -- <B1otechnology handbooks ; v. 4l
Includas b1b11ograph1ca1 raferancas and Index.
ISBN 978-1-4899-2643-2 ISBN 978-1-4899-2641-8 (eBook)
DOI 10.1007/978-1-4899-2641-8
1. Saccharo•yces--B1otechnology. 2. Saccharoaycas. I. Tu1te,
M1chu1 F. II. 011ver, s. D. <Staphen George>, 1949-
III. Ser tes.
TP248.27.Y43S22 1991
6B0'.62--dc20 90-19442
CIP

ISBN 978-1-4899-2643-2

© 1991 Springer Science+Business Media New York

Originally published by Plenum. Press, New York in 1991
Softcover reprint of the hardcover 1st edition 1991
AII rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted
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Contributors

J. R. Dickinson • School of Pure and Applied Biology, University of

Wales College of Cardiff, Cardiff CF1 3TL, Wales
M. J.Dobson • Department of Botany, University of Nottingham,
Nottingham NG7 2RD, England
A. J. Kingsman • Department of Biochemistry, University of Oxford,
Oxford OX1 3QU, England
S. M. Kingsman • Department of Biochemistry, University of Ox-
ford, Oxford OX1 3QU, England
C. Kreutzfeldt • Institut fur Pharmakologie und Toxikologie, Phil-
ipps-Universitat Marburg, Lahnberge, 3550 Marburg, Federal Re-
public of Germany
T. M. Matthews • Department of Chemical Engineering, University
of Manchester, Institute for Science and Technology, Manchester
M60 1QD, England
E. J. Mellor • Department of Biochemistry, University of Oxford,
Oxford OX 1 3QU, England
Stephen G. Oliver • Manchester Biotechnology Centre, University of
Manchester, Institute for Science and Technology, Manchester M60
1QD, England
Michael F. Tuite • Biological Laboratory, University of Kent, Canter-
bury, Kent CT2 7NJ, England
C. Webb • Department of Chemical Engineering, University of Man-
chester, Institute for Science and Technology, Manchester M60
1QD, England
R. B. Wickner • National Institutes of Health, Bethesda, Maryland
20892
W. Witt • Institut fur Pharmakologie und Toxikologie, Philipps-Uni-
versitat Marburg, Lahnberge, 3550 Marburg, Federal Republic of
Germany

v
Preface

In this volume we aim to present an easy-to-read account of the genus

Saccharomyces that we hope will be of value to all students and researchers
wishing to exploit this important genus, be it for academic or commer-
cial purposes. Individual chapters have been commissioned to cover
specific aspects of the biology of Saccharomyces species: growth, genetics,
and metabolism, with the emphasis on methodology. Basic principles are
discussed without an over-detailed, step-by-step breakdown of specific
techniques, and lengthy discussions of standard molecular, biological,
and biochemical techniques (e.g., polyacrylamide gel electrophoresis,
protein purification, DNA sequencing) have been avoided. We hope the
volume will provide a quick reference to the current status of a wide
range of Saccharomyces-specific methodologies without focusing ex-
clusively on recent developments in molecular techniques which can be
found in the ever increasing numbers of "cloning manuals." By necessity,
much of what is described in this volume concentrates on one particular
species of Saccharomyces, namely Saccharomyces cerevisiae. This is not just a
reflection of the authors' interests, but indicates the extent to which this
simple eukaryote has been studied by biologists from all walks of life, for
all sorts of reasons. If this volume can provide a broader knowledge base
to the experienced yeast researcher, or ease the path of someone just
starting work with Saccharomyces, then we will have achieved our aim.
We are grateful to all the authors and to Ken Derham at Plenum
Press for their patience during the preparation of this volume.

Michael F. Tuite
University of Kent, Canterbury
Stephen G. Oliver
University of Manchester

vii
Contents

Chapter 1

Introduction 1

Michael F. Tuite and Stephen G. Oliver

Chapter 2

Structural Biochemistry 5

C. Kreutzfeldt and W. Witt

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2. Basic Macromolecules in Saccharomyces . . . . . . . . . . . . . . . . . . . 5
2.1. Proteins and Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2. Polysaccharides, Structural Mannoprotein, and
Polyphosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.3. Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.4. Ribonucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3. Cell Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1. Cell Envelope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2. Vacuoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3. Endoplasmic Reticulum and Secretory and Endocytic
Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.4. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.1. Mating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4.2. Meiosis and Sporulation............. .............. 34
4.3. Vegetative Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
5. Cell Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
5.1. Isolation of Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
5.2. Identification of Yeast Cell Subfractions . . . . . . . . . . . . 43
5.3. Autolytic Enzymes in Yeast Cells . . . . . . . . . . . . . . . . . . . 43
6. Methods of Synchronization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
ix
x CONTENTS

6.1. Imposition of a Cell Cycle Block . . . . . . . . . . . . . . . . . . . 44

6.2. Centrifugational Methods . . . . . . . . . . . . . . . . . . . . . . . . . 44
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Chapter 3

Metabolism and Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

J. R. Dickinson

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2. Outline of Carbohydrate Metabolism . . . . . . . . . . . . . . . . . . . . 60
2.1. Catabolite Repression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.2. Catabolite Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
2.3. Effect of Oxygen .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 62
2.4. The Pasteur Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2.5. Catabolite Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.6. Molecular Genetics of Carbon Metabolism . . . . . . . . . . 66
3. Outline of Nitrogen Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.1. Amino Acid Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.2. Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.3. Nucleotide Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.4. Molecular Genetics of Nitrogen Metabolism . . . . . . . . . 74
4. Transport of Substrates into the Cell . . . . . . . . . . . . . . . . . . . . 74
5. Regulation of Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5.1. Genetic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5.2. Physiological and Biochemical Studies . . . . . . . . . . . . . . 83
5.3. Further Aspects of Regulation . . . . . . . . . . . . . . . . . . . . . 87
6. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Chapter 4

Methods in Classical Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

R. B. Wickner

1. Introduction 101
2. Life Cycle of Saccharomyces Cerevisiae .................... . 101
3. Tetrad Analysis ............ , .......................... . 105
4. Aneuploidy .......................................... . 112
5. Mutant Induction and Isolation ........................ . 115
 CONTENTS xi

5.1. Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

5.2. Mutant Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
6. Genetic Mapping Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
6.1. Meiotic Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
6.2. Mitotic Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
6.3. Aneuploid Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
6.4. The spoll Mapping Method . . . . . . . . . . . . . . . . . . . . . . . 120
6.5. Chromosome-Loss Methods . . . . . . . . . . . . . . . . . . . . . . . 121
6.6. 2-~J.m DNA Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
6.7. Overall Mapping Strategies . . . . . . . . . . . . . . . . . . . . . . . . 122
6.8. The Genetic Map............ ................... .. 122
7. Non-Mendelian Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.1. The Mitochondrial Genome . . . . . . . . . . . . . . . . . . . . . . . 123
7.2. The Killer Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
7.3. 2-j..l.m DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
8. Appendix I: Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
9. Appendix II: Sample Tetrad Data . . . . . . . . . . . . . . . . . . . . . . . 140
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Chapter 5

Recombinant DNA Techniques 149

A. J. Kingsman, E. J. Mellor, M. J. Dobson, and S.M. Kingsman

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
2. Yeast Transformation .................. ............ :. . . 150
3. Plasmid Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
3.1. ARS-Based Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
3.2. 2-~J.m-Based Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
3.3. Yeast Gene Isolation Using Plasmid Vectors . . . . . . . . . 152
3.4. Site-Directed Mutagenesis in Yeast Using Single-
Stranded Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
4. Minichromosome Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
5. YAC Cloning Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
6. Integrative Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
6.1. Targeted Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
6.2. Allelic Rescue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
6.3. Gene Disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
6.4. Refinements of Transplacement . . . . . . . . . . . . . . . . . . . 163
7. Selection Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
xii CONTENTS

Chapter 6

Expression of Heterologous Genes . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Michael F. Tuite

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
2. Introduction of Heterologous DNA into Yeast . . . . . . . . . . . . 171
2.1. Basic Cloning Technology . . . . . . . . . . . . . . . . . . . . . . . . . 171
2.2. Basic Vector Design............................... 171
3. Transcription of Heterologous Genes . . . . . . . . . . . . . . . . . . . . 173
3.1. The Problem of Introns . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
3.2. Encoded Pre-Pro Sequences . . . . . . . . . . . . . . . . . . . . . . 174
3.3. Transcriptional Promoters . . . . . . . . . . . . . . . . . . . . . . . . . 176
3.4. Termination of Transcription . . . . . . . . . . . . . . . . . . . . . . 179
3.5. Regulated Promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
4. Translation of Heterologous mRNAs . . . . . . . . . . . . . . . . . . . . 182
4.1. Codon Bias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
4.2. 5' Untranslated mRNA Leader . . . . . . . . . . . . . . . . . . . . 184
4.3. AUG Context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
5. Posttranslational Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . 186
5.1. Yeast Secretion Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
5.2. Use of Heterologous Signal Sequences . . . . . . . . . . . . . 187
5.3. Homologous Signal Sequences . . . . . . . . . . . . . . . . . . . . . 189
5.4. Posttranslational Modification and Secretion . . . . . . . . 191
5.5. Other Posttranslational Modification Events . . . . . . . . . 192
6. Maximizing Product Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
6.1. Stability of Recombinant Plasmids in Yeast . . . . . . . . . . 195
6.2. Host Strains for Heterologous Gene Expression . . . . . 200
7. Summary and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Chapter 7

"Classical" Yeast Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

Stephen G. Oliver

1. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
2. Baking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
3. Beer Brewing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
4. Sake Brewing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
5. Wine Making . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
5.1. White Wine Production . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
 CONTENTS xiii

5.2. Red Wine Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

6. Strain Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
7. Ethanol Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
8. Flocculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
9. Polysaccharide Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
10. Rare Mating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
11. Protoplast Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
12. Recombinant DNA Technology . . . . . . . . . . . . . . . . . . . . . . . . . 240
12.1. Cloning and Expression in Yeast of Genes
Encoding Amylolytic Enzymes . . . . . . . . . . . . . . . . . . . . 240
12.2. Cloning and Expression of Endoglucanase
Genes in Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
12.3. Cloning and Expression of Complete
Metabolic Pathways: The Way Ahead . . . . . . . . . . . . . . 243
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

Chapter 8

Culture Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

T. M. Matthews and C. Webb

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
2. Nutritional Requirements of Saccharomyces . . . . . . . . . . . . . . . . 250
2.1. Carbon .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 250
2.2. Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
2.3. Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
2.4. Sulfur . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
2.5. Trace Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
2.6. Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
3. Process Variables that Influence the Growth
of Saccharomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
3.1. Hydrogen Ion Concentration . . . . . . . . . . . . . . . . . . . . . . 254
3.2. Temperature and Ethanol Inhibition Effects . . . . . . . . 255
3.3. Dissolved Oxygen and Substrate Inhibition Effects . . . 256
3.4. Carbon Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
4. The Theory and Practice of Yeast Culture Systems . . . . . . . . 258
4.1. Batch Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
4.2. Continuous Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
4.3. Practical Continuous Culture Systems . . . . . . . . . . . . . . 266
5. Monitoring the Growth of Saccharomyces . . . . . . . . . . . . . . . . . . 271
6. Cell Separation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
7. Immobilized Cell Systems . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . 272
xiv CONTENTS

7.1. Passive Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

7.2. Active Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
7.3. Fermenters for Immobilized Saccharomyces Cells . . . . . 275
8. Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
8.1. Pressed Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
8.2. Dried Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
8.3. Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
8.4. Alcoholic Beverages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
8.5. Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

Chapter 9

Biochemical Techniques 283

Michael F. Tuite and Stephen G. Oliver

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
2. Cell Disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
2.1. Whole Cell Disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
2.2. Protoplast Lysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
3. Radioactive Labeling of Macromolecules . . . . . . . . . . . . . . . . . 286
3.1. RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
3.2. DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
3.3. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
4. RNA Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
4.1. Differential Extraction Techniques . . . . . . . . . . . . . . . . . 290
4.2. Messenger RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
4.3. Transfer RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
4.4. Ribosomal RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
4.5. Double-Stranded RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
5. DNA Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
5.1. Chromosomal DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
5.2. Mitochondrial DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
5.3. Plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
6. In vitro Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
6.1. In vitro Transcription Systems . . . . . . . . . . . . . . . . . . . . . 298
6.2. In vitro Translation Systems . . . . . . . . . . . . . . . . . . . . . . . 300
6.3. In vitro DNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
6.4. Two-Dimensional Gel Electrophoresis in the Analysis
of DNA Replication ............................... 306
7. Pulsed Field Gel Electrophoresis of Yeast Chromosomes . . . 307
71. Theoretical Background . .. . . .. .. .. .. .. .. .. . . . . . . . 308
 CONTENTS xv

7.2. Pulsed Field Gradient Gel Electrophoresis . . . . . . . . . . 309

7.3. Orthogonal-Field-Alteration Gel Electrophoresis..... 310
7.4. Contour-Clamped Homogeneous Electric Field . . . . . . 311
7.5. Vertical Pulsed-Field Gradient Gel Electrophoresis
and Constant Electric Field with Rotating Gel
Platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
7.6. Field Inversion Gel Electrophoresis . . . . . . . . . . . . . . . . 312
7. 7. General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
7.8. Sample Preparations .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 313
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

Index ................................................... 321