Genetic Techniques for Biological Research Corinne A. Michels Copyright q 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)
Genetic Techniques for Biological Research A case study approach
For Harold and for our F1 generation Catherine and Bill
Genetic Techniques for Biological Research A case study approach CORINNE A. MICHELS Department of Biology, Queen$ College of the City University of New York, New York, USA
@
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Library of Congress Cataloging-in-Publication Data Genetic techniques for biological research : a case study approach / [edited by] Corinne A. Michels. p. cm. Includes bibliographical references and index. ISBN 0-471-89919-4 (alk. paper) ISBN 0-471-89921-6 (pbk.) 1. Molecular genetics-Methodology-Case studies. 2. Saccharomyces cerevisiae. I. Michels, Corrinne C. Anthony, 1943~
QH440.4 .G464 2001 2001055948
British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 0-471-89921-6 Typeset in 10/12pt Times by Mayhew Typesetting, Rhayader, Powys Printed and bound in Great Britain by TJ International Ltd, Padstow This book is printed on acid-free paper responsibly manufactured from sustainable forestry, in which at least two trees are planted for each one used for paper production.
Contents lntroduction
ix
Section I Saccharomyces cevevisiae as aGeneticResearchOrganism
1
1
3 3 3
2
Saccharomyces cevevisiae as a GeneticModelOrganism Overview Culture Conditions The Mitotic Life Cycle Mating Type, Mating, and the Sexual Life Cycle Saccharomyces Genome and Nomenclature Genome Sequence Genetic Nomenclature Phenotype Nomenclature Strain Nomenclature Protein Nomenclature Genetic Crosses and Linkage Analysis Single Gene Cross Two Gene Cross Classes of Saccharomyces Cloning Plasmid Vectors YIP Plasmid YRp Plasmid YEp Plasmid YCp Plasmid YAC Plasmid Libraries Gene DisruptionlDeletion in Saccharomyces (One-Step Gene Replacement) Gap Repair Reporter and Other Types of Fusion Gene Expression Vectors References and Further Reading
16 17 18 20 20
Techniquesin Cell andMolecular Biology Cell Fractionation Preparation of the Cell Extract Differential-Velocity Centrifugation Equilibrium Density Gradient Centrifugation Microscropy Techniques Fluorescence Microscropy, Immunofluorescence, and GFP Confocal Scanning Microscropy Nomarski Interference Microscropy Electron Microscropy
23 23 23 24 24 26 26 30 30 31
5
5 7 7 7 8 8 8 8 9 10 12 13 14 15 15 15 16
vi
3
CONTENTS
Flow Cytometry Protein Extraction and Purification Western Analysis Epitope-Tagging and Immunodetection of Epitope-Tagged Proteins Hemagglutinin (HA) Epitope FLAG Epitope Myc Epitope Immunoprecipitation and Related Methods Immunoprecipitation Metal Chelate Affinity Purification GST-Tagged and MalB-Tagged Proteins References and Further Reading
32 32 35 37 39 39 39 39 39 40 41 41
Saccharomyces Cell Structure Cell Shape and Growth Patterns Cell Wall, Cell Surface Morphology, and Morphological Variation Cell Wall Composition and Synthesis Bud Scars, Birth Scars, and Budding Patterns Schmoo Formation and Mating Bud Site Selection and Polarized Cell Growth Spore Formation Nucleus Nuclear Envelope Spindle Pole Body Cytoskeleton Actin Cytoskeleton Microtubule Cytoskeleton Microtubule Morphology in Cell Division and Mating Plasma Membrane, Endoplasmic Reticulum, Golgi Complex, Vacuole, and Membrane Trafficking Endoplasmic Reticulum Golgi Complex Vacuole Membrane Trafficking Mitochondrion Peroxisome References and Further Reading
43 43 44 45 45 47 47 51 51 52 52 52 53 55 55 57 57 59 59 60 61 61 62
Section I1 Techniques of Genetic Analysis
65
4
MutantHunts-To
67
5
ComplementationAnalysis: How Many GenesareInvolved? References
73 77
6
Epistasis Analysis Overview Epistasis Analysis of a Substrate-Dependent Pathway Epistasis Analysis of a Switch Regulatory Pathway
79 79 81 82
Select or to Screen (Perhaps EvenbyBruteForce)
CONTENTS
vii
Epistasis Group References and Further Reading
84 84
Gene Isolation and Analysis of Multiple Mutant Alleles Preparation of the Library Cloning by Complementation Positional Cloning Cloning by Sequence Homology Analysis of Multiple Mutant Alleles Reference
85 85 86 87 89 89 90
Suppression Analysis Overview Intragenic Suppression Intergenic Suppression By-Pass Suppression Allele-Specific Suppression Suppression by Epistasis Overexpression Suppression By-Pass Suppression by Overexpression Allele-Specific Suppression by Overexpression Overexpression Suppression by Epistasis References and Further Reading
91 91 91 92 93 94 95 96 97 97 97 97
Enhancement and Synthetic Phenotypes Overview Mechanisms of Enhancement Synthetic Enhancement Conditional Lethal Mutations for the Isolation of Enhancer Mutations Genetic Interaction Further Reading
99 99 99 100 101 102 102
10 Two-Hybrid Analysis Two-Hybrid Analysis One-Hybrid and Three-Hybrid Analysis References and Further Reading
103 103 105 106
11 Advanced Concepts in Molecular Genetic Analysis Reverse Genetics Cold-Sensitive Conditional Mutations Dominant Negative Mutations Charged-Cluster to Alanine Scanning Mutagenesis References and Further Reading
107 107 109 109 111 111
12 Genomic Analysis Databases Biochemical Genomic Analysis DNA Microarray Analysis Genome-Wide Two-Hybrid Screens
113 114 115 115 116
...
CONTENTS
v111
Genome-Wide Generation of Null Mutations Gene Disruption Strains Transposon Mutagenesis References and Further Reading Section 111 Case Studies fromthe Saccharomyces GeneticLiterature Case Study I
Glucose Sensing and Signaling Mechanisms in Sacchavomyces
117 117 117 118 121 123
Case Study I1 Secretion, Exocytosis, and Vesicle Trafficking in Saccharomyces
143
Case Study 111 The Cell Division Cycle of Saccharomyces
173
Case Study IV Mating-type Pheromone Response Pathway of Saccharomyces
205
Index
235
Introduction Molecular genetics is a tool used by today’s biologist interested in understandingnot simply describing-the underlying mechanisms of processes observed in cellular and developmental biology. It is a fusion of the biochemical and genetic approaches to problem solving developed over the past decades and the resulting synergy of these approaches has produced an extremely powerful tool for the investigation of living systems. The biochemical approach has beenvery productive in identifying the major macromolecular components of cells and the pathways of metabolism. Nevertheless, usedexclusively, it is not an adequate tool for elucidating the details of the regulation of these pathways and their physiological coordination. The biochemist’s tools, although powerful, are limited. The biochemist identifies and characterizes a component of interest (such asa protein) by purifying it or by monitoring its presence based on an assay of the reaction or cellular process it catalyzes. It is hoped that investigations of characteristics such as subcellular localization, structure, and identification of interacting proteins will provide clues to its cellular function. But, if these studies are uninformative, if the component is present at a verylowlevel or is unstable, oran assay method cannot be developed, the biochemical approach will fall short. The genetic approach does not have these limitations but does have others. No information regarding the number, function, location, or structure of the gene functions involved is required. One only needs to be able to observe the process of interest (the wild-type phenotype) and identify individuals exhibiting alterations or aberrations in this process (the mutant phenotype). The genetic approach assumes that few, if any, cellular processes occur spontaneously in vivo, and that there is a gene(s) encoding a protein(s) or RNA(s) that is responsible for catalyzing the process and allowing it to occur at a rate that is adequate for sustaining growth and development. The geneticist isolates mutant individuals exhibiting alterations in the process, uses genetic analysis to identify the full battery of genes encoding the products involvedin regulating the process of interest, and explores the genetic interactions among these genes. To carry these studies further, the geneticist needs to isolate and functionally characterize the gene products and this requires the tools of biochemical analysis. Moreover, major limitations for the geneticist come from the availability of specificgenetic techniques for the particular organism under study. Thus, through the skilled use of the techniques of genetic analysis and biochemical methods, molecular genetic analysis allows the researcher to identify all the genes controlling a process, isolate the protein(s) or RNA(s) involved, and reveal their molecular mechanism of action. Numerous reference books, review articles, and journal articles are available to the laboratory researcher to learn the theory and practice of the vast array of biochemical methods available. Only a very few review articles on some methods of genetic analysis havebeen published. Thus,
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INTRODUCTION
learning the tools of the trade for geneticists has been largely a hands-on experience and only those fortunate enough to be trained in genetic model systemslike Escherichia coli, bacteriophage, Saccharomyces, Drosophila, and more recently Caenorhabditis elegans and Arabidopsis thaliana completely integrate these methods into their research. The genetic approach is straightforward but not easy. One needs to be a creative and shrewd observer with a critical, clear-thinking mind. The geneticist’s tools include mutant selections/screens, complementation analysis, fine structure mutation analysis, suppressor and enhancer analysis, and more recentlygene cloning, sequence analysis, and genomics. This book outlines the tools of molecular genetic analysis and presents examples of their use through case studies. The goal is to provide the novice geneticist with the skill to use these tools forhidher own research. The case studies use Saccharomyces because the tools of molecular genetic analysis available for Saccharomyces are the most straightforward and highly developed of all of the eukaryotic research organisms. As similar tools develop for genetic analysis of other systems, particularly the mammalian systems, the ability to carry out sophisticated genetic analysis to the level seen in Saccharomyces will also develop. Nevertheless, the theoretical basis of the methods will remain the same. To quote David Botstein (1993), a renowned geneticist who has contributed greatly to the theoretical development of molecular genetics, ‘The many different organisms upon which we practice genetics present diverse difficulties and opportunities in execution, but underneath the fundamentals remain always the same.’ The methods of molecular genetic analysis learned using Saccharomyces are directly applicable to other organisms. Section I of this book describes Saccharomyces cerevisiae asa geneticmodel organism. The genome, life cycle, sexual cycle,basic genetic methods, plasmids, and tools for molecular genetic manipulation are described. An overview of important standard techniques in cell and molecular biology is presented along with Saccharomyces cell structure. This summary is presented largely to facilitate reading of the research literature articles included in the case studies. Section I1 presents the various methods and tools of molecular genetic analysis and takes a theoretical approach. Specific protocols for procedures are not presented. These are available from the literature and differ from organism to organism. The methods described in Section I1 are intended to be general in nature and adaptable to any organism. Section I11 consists of the Saccharomyces case studies. With each case study one is expected to read, interpret,and critique a series of original research articles by responding to a series of homework questions based on each article. These articles were published over the past several decades and illustrate, step by step, the molecular genetic analysis of important cellular processes in the budding yeast S. cerevisiue. Along the way, the reader will develop an appreciation for the molecular genetic method of analysis and the synergy between the genetic, biochemical, and cytological approaches to problem-solving in biological systems. More important, the critical thinking skills illustrated by the case studies presented here should translate quite readily to the reader’s own research projects and scientific decisionmaking. The following fable, ‘A Tale of Two Retired Scientists and Some Rope’, by William T. Sullivan (1993), describes in anecdotal fashion the differences between
INTRODUCTION
xi
the biochemical approach and the genetic approach to problem-solving. The real take-home message of this story,and also of this book, is that while both the biochemical and genetic approaches are very valuable, the synthesis of the two, that is the molecular genetic approach, is far more powerful than either method used exclusively. ‘The Salvation of DougA Tale of Two Retired Scientists and Some Rope’, by William T. Sullivan On a hill overlooking an automobile factory, lived Doug, a retired biochemist, and a retired geneticist (nobody knew his name). Every morning, over a cup of coffee, and every afternoon, over a beer, they would discuss and argue over many issues and philosophical points.During their morning conversations, they would watch the employees entering the factory below to begin their workday. Some would be dressed in work clothes carrying a lunch pail, others, dressed in suits, would be carrying briefcases. Every afternoon, as they waited for the head on their beers to settle, they would see fully built automobiles being driven out of the other side of the factory. Having spent a life in pursuit of higher learning, both were wholly unfamiliar with how cars worked. They decided that they would like to learn about the functioning of cars and having different scientific backgrounds they each tooka very different approach. Doug immediately obtained 100 cars (he is a rich man, typical of most biochemists) and ground them up. He found that cars consist of the following: 10% glass, 25% plastic, 60% steel, and 5% other materials that he could not easily identify. He felt satisfied that he had learned of the types and proportions of material that made up each car. His next task was to mix these fractions to see if he could reproduce some aspects of the automobile’s function. As you can imagine, this proved daunting. Doug put in long hard hours between his morning coffee and afternoon beer. The geneticist, not being inclined toward hard work (as is true for most geneticists) pursued a less strenuous (and less expensive) approach. One day, before his morning coffee, he hiked down the hill, selected a worker at random, and tied his hands. After coffee, while the biochemist zipped up his blue jump suit, adjusted his welder’s goggles, and lit his blowtorch to begin another day of grinding, the geneticist puttered around the house, made himself another pot of coffee, and browsed through the latest issue of Genetics. That afternoon, while the automobiles were rolling off the assembly line, Doug, wet with the sweat of his day’s exertions, took a sip of beer and as soon as he caught his breath began discussing his progress. ‘I have been focusing my efforts on a component I consistently find in the plastic fraction. It looks like this (he draws the shape of a steering wheel on the edge of a napkin). Presently I have been mixing it with the glass fraction to seeif it has any activity. I am hoping that with the right mixture I may get motion, although I have not hadany success so far. I believe with a biggerblow torch, perhaps even a flame thrower, I will get better results.’ The geneticist was only half listening because his attention was drawn to the cars rolling off the assembly line. He noticed that they were missing the front and rear windows, but notthe side windows. As soon as the biochemist finished speaking (geneticists are very polite conversationalists), the geneticist proclaimed, ‘I have learned two facts today.The worker whose hands I tied thismorning is responsible for installing car windows and the installation of the front and back windows.’ The following day the geneticist tied the hands of another worker. That afternoon he noticed that the cars were being produced without the plastic devices the biochemist was working on (steering wheels). In addition, he noticed that as the cars were being
xii
INTRODUCTION driven off to the parking lot, none of them make the first turn in the road and they begin piling up on the lawn. That evening, to Doug’s dismay, the geneticist concluded that steering wheels were responsible for turning thecarand, in addition, that hehad identified the worker responsible for installing the steering wheels. Emboldened by his successes, the next morning the geneticist tied the hands of an individual dressed in a suit and carrying a briefcase in one hand and a laser pointer in the other (he was a vice president). That evening the geneticist, and Doug (although he would not openly admit it), anxiously awaited to see the effect on the cars. They speculated that the effect might be so great as to prevent the production of the cars entirely. To their surprise, however, that afternoon the cars rolled off the assembly line with no discernible effect. The two scientists conversed late into the evening about the implications of this result. The geneticist, always having had a dislike for men in suits, concluded that the vice-president sat around drinking coffee all day (much like geneticists) and had no role in the production of the automobiles. Doug, however, held the view that there was more than one vice president so that if one was unable to perform, others could take over his duties. The next morning Doug watched as the geneticist, in an attempt toresolve this issue, headed off towards the factory carrying a large rope to tie the hands of all the men in suits. Doug, aftera slight hesitation, abandoned his goggles and blowtorch, and stumbled down the hill to join him. (Reproduced by permission of the Genetics Society of America.)
REFERENCES AND FURTHER READING Botstein, D. (1993) From phage to yeast. In The Early Days of Yeast Genetics, M.N. Hall & Linder, eds. Cold Spring Harbor Laboratory Press, New York. Botstein, D. & G.R.Fink (1998) Yeast: an experimental organism for modern biology. Science 240: 1439-1443. Botstein, D., S.A. Chervitz, & J.M. Cherry (1997) Yeast as a model organism. Science 277: 1259-1260. Hall, M.N. & P. Linder, editors (1993) The Early Days of Yeast Genetics. Cold Spring Harbor Laboratory Press, New York. Lander, E.S. & R.A. Weinberg (2000) Genomics: journey to the center of biology. Science 287: 1777-1982. Sullivan, W.T. (1993) The salvation of Doug. GENErations 1: 3.
Genetic Techniques for Biological Research Corinne A. Michels Copyright q 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)
Index Numbers in italics indicate figures. Entries for other than general or specific topics refer to Saccharomyces cerevisiue. 2p circle 15 a-factor 50, 207 acid phosphatase 145 actin cytoskeleton 51,53-5 ADE 4 ADHl promoter 20 Aeyuorea victoriu 28 affinity purification 40- 1 agglutinins 207 alanine-scanning mutagenesis 111 allele-specific enhancement 100, 101 allele-specific suppression 94-5, 97 alleles cold-sensitive 70, 109 dominant/recessive 69 mutant 69 mutation analysis 89-90 nomenclature 7 temperature-sensitive 70 wild-type 69 see also genes a-factor 49, 50, 148, 207, 218 apicalgrowth 51 artificial chromosome vectors 15, 86 ascospores 6, 51 autonomously replicating sequences (ARS) 14 autophagy 60 auxotropes 4 axial budding pattern 44, 45, 47, 49 bacterial artificial chromosomes (BACs) 86 bacteriophage vectors (y and P1) 86 bacteriophages complementation analysis of T4 r l l 73 epistasis analysis of P22 morphogenesis 79 103, 104,116 baitfusions BEMl 230 Benzer, S. 73 BFP (blue fluorescent protein) 30 biochemical genomic analysis 115 BioKnowledge Library 114 BiP 152 bipolarbuddingpattern 47, 48, 49
birth scars 44, 45 blue fluorescent protein (BFP) 30 bud scars 44, 45 budding 5, 6 actin cytoskeleton in 54 axial 44, 45, 47, 49 bipolar 47, 48, 49 microtubulemorphology during 55-6 site selection 47, 49, 50 unipolar 47, 48, 49 by-pass suppression 93-4,97 cables, actin 53, 54 calcofluor white 27 canavanine 208 case studies 121 cell division cycle173-203 glucose sensing and signaling mechanisms 123-41 mating-type pheromone response pathway 205-33 secretion, exocytosis and vesicle trafficking 143-71 CAT 19 Cdc7-Dbf4protein kinase 197,198 Cdc28 proteinkinase 110, 185,188,192, 218 cDNA libraries 86 cellcycle arrest by mating pheromones 218 cell division see meiosis; mitosis cell fractionation 23 differential-velocity centrifugation 24 equilibrium density gradient centrifugation 24-6 extract preparation 23 cell structure cell shape 43, 44 cell walls 44-5, 46 composition 45 surface morphology 44, 45, 47, 48 synthesis 45 cytoskeleton 52 actin 51, 53-5 microtubule 55-7 growth patterns 43, 44
236 cell structure (cont.) organelles endoplasmic reticulum see endoplasmic reticulum Golgi complex 43, 45, 58, 59 mitochondria 28, 61, 62 nucleus 5 1-2 peroxisomes 61 vacuoles 31, 59-60 schmoos 47, 49, 50 vesicles 59-61, 159-60 centromere sequences (CENs) 15, 27 change of function mutations 69 charged-cluster to alanine scanning mutagenesis 11 1 checkpoints 200 chitin 45, 46 chromosome walking 88 cistrons see genes clathrin-coated vesicles60-1 CLB 218 CLN 218 cloning see gene cloning coat proteins (vesicle) see COPI; COPII coatomer 61 coimmunoprecipitation(CO-IP) 40 cold-sensitive mutations 70, 109 comparative genomic hybridization 116 complementationanalysis 73-7 complementation, cloning by 86-7 complementation groups see genes conditional mutations 70, 101 lethal 71, 101-2 confocalscanningmicroscopy 30 congenic strains 68 constitutive mutants 80 contigs 88 COPI 61 COPII 61, 160 copper-regulated promoters 20 corticalpatches(actin) 53, 54, 55 cosmids 86 culture of Saccharomyces 3-4 cvt (cytoplasm-to-vacuole targeting) pathway 60 CYCI promoter 20 cyclin-dependent protein kinase see Cdc28 protein kinase cyclins 110, 192, 218 cytochrome c l 154 cytoduction 152 cytokinesis 5 cytokinesis tags 47 cytoplasm-to-vacuole targeting (cvt) pathway 60 cytoplasmicmicrotubules 55
INDEX DAPI (4,6-diamidino-2-phenylindole) 27, 28 databases 114 defined media 3-4 denaturing conditions 35 densitygradients 24 4, 6-diamidino-2-phenylindole (DAPI) 27, 28 differential interference contrast (DIC) microscopy 30- 1 differential velocity centrifugation 24 diploid cells bud site selection 47, 48, 49 M A TaIMA Tu 2 l 0- l 1 meiosis 6 mitosis 5, 6 disruptionconstructs 16- 17 cDNA libraries 86 DNA microarrays 1 15- 16 DNA synthesis, initiation of 196, 197, 198 DNA-protein interactions, one-hybrid analysis 105-6 dominantmutations 69 dominant negative mutations 109- 10 doublemutant phenotypes 12 doublemutants 70, 81, 82 Drosophila eye color 82 dyneins 55 electron microscopy (EM) 3 1-2 endocytosis 33, 57, 60 endoplasmic reticulum (ER) 45, 57-9, 147-8,156, 159 endosomes 59, 60 enhancement mechanisms 99-100, 101 overview 99 synthetic 100, 101 enrichment for desired mutants 68-9 enzymes mitochondrial 61 see also specific enzymes epistasis analysis overview 79-81 substrate-dependentpathways 80, 81-2 switch regulatorypathways 80-1,82-4 epistasis groups 84 epistasis, suppression by95-6,97 epitopetagging 37-9 equilibrium density gradient centrifugation 24-6 ER see endoplasmic reticulum Escherichia coli plasmids 12 Escherichia colilyeast shuttlevectors 12 essential genes 70- 1, 108
INDEX ethidiumbromide 152 exocytosis see secretory pathway expression vectors 20 FACS (fluorescence activated cell sorter) analysis 32, 34 a-factor 49, 50, 148, 207, 218 a-factor 50, 207 Farlp 36, 229 Fields, S. 103, 116 fine structure mapping of recessive mutant alleles 18 FISH (fluorescence in situ hybridization) 27 FLAG epitope 39 flow cytometry 32, 34 fluorescein 26 fluorescence activated cell sorter (FACS) analysis 32, 34 fluorescence in situ hybridization (FISH) 27 fluorescence microscopy 26 confocal scanning microscopy 30 FISH 27 green fluorescent protein (GFP) 28-30 immunofluorescence 26 fluorescent dyes 26 5-fluor0 orotic acid (5FOA) 160 forward mutations 70 fosmids 86 French press 23 functional analysis of the genome see genomic analysis functionally-related genes, identification see enhancement; suppression FUSl 219 fusion genes 18-20 bait and prey 103, 104, 116 epitope 38 G F P 28-30 GST 41 MalB 41 reporter 18-19, 103, 104, 105 GAL1 and GAL10 promoters20 /?-galactosidase 19 gap repair 17-18 gene cloning by complementation 86-7 by sequence homology 89 libraryconstruction 85-6 positional 87-8 see also vectors gene disruption 16-17, 117
237 gene isolation 85 see also gene cloning genes essential 70-1, 108 function determination by mutation analysis 89-90 functionally-related see enhancement; suppression fusion see fusion genes heterologous expression in Saccharomyces 113 involved in mating 50, 207 linked 10, 209 marker 12, 16, 69, 108 see also specific genes genetic crosses 9 single gene 9-10 two gene 10-12 genetic distance 88 genetic interaction 102 genetic markers see marker genes genetic nomenclature7 genomic analysis 113-14 biochemical screening method1 15 databases 114 DNA microarrays 1 1 5- 16 genome-wide generation of null mutations 117-18 genome-wide two-hybrid screens 116 genomic libraries 85-6 G F P (green fluorescent protein) 19, 28-30 P-glucan 45, 46 glucose 4 glucose repression resistant (Grrl) protein 135-6 glutathione S-transferase (GST) fusion proteins 41, 115 glycosyl phosphatidylinositol (GPI) 45 Golgi complex 43, 45, 58, 59 GPDl promoter 20 green fluorescent protein (GFP) 19, 28-30 growth media 3-4 Grr 1 (glucose repression resistant) protein 135-6 GST (glutathione S-transferase) fusion proteins 41, 115 haploid cells bud site selection 44, 45, 47, 49 see also mating; mating types hemagglutinin (HA) epitope 39 heterozygosity, loss of210 His-tag 40 HIS3 12, 105 H M L a 222-3
238 HMRa 223 H 0 5, 68 HXTIIHXT2 132 hybridomas 37 61 hydrogenperoxide immuno-gold localization 3 1-2, 33 immunofluorescence 26 immunoprecipitation 39-40 intergenicsuppression92 functionsuppression 93 allele-specific 94-5, 97 by-pass 93-4,97 epistatic 95-6, 97 overexpression 96-7 information suppression 92-3 intrageniccomplementation 76-7 intragenicsuppression9 1-2 intranuclear microtubules 55 invasive growth 43 invertase 45, 145, 148 isogenic strains 67, 68 isotropicgrowth 51 karyokinesis 5, 6 kinesins 55 knock-outstrains 1 17
lacZ 19 lethal mutations 71 LEU2 12 85 libraryconstruction cDNA 86 genomic 85-6 prey 104 linked genes 10, 209 logarithmicphase3 loss of function mutations 69, 109, 110 luciferase gene 19 MalB-taggedproteins 41 mannoproteins(mannans) 45, 46 maps distancecalculations 76 fine structure 18 order-of-function 79 marker genes 12, 16, 69, 108 M A T locus 5 , 207,222 MATalMATa diploids 210-1 1 mating 47, 50, 207 pheromones a-factor see a-factor
INDEX a-factor 50, 207 cell cycle arrest 2 18 in mating projection formation 51 receptors 2 12 response pathway, interactions of components 225-6 quantitative assays 152 roles of microtubules 50, 57 mating types 5, 6, 49, 207 Mcm (mini-chromosome maintenance) complex 197-8 MCS (multiplecloning sites) 38 meiosis 6, 51 see also tetrad analysis membrane trafficking 43, 57,60-1 see also secretory pathway metalchelate affinity purification 40-1 MFcvl 148 microarray analysis 1 15-16 microfilaments 52 microscopy techniques 26 electron microscopy 3 1-2 fluorescence microscopy see fluorescence microscopy Nomarski interference microscopy 30-1 microtubules 55 in budding 55-6 in mating 50, 57 Mig2 protein 140 mini-chromosome maintenance (Mcm) complex 197-8 MIPS (Munich Information Center for Protein Sequences) 114 mitochondria 28, 61, 62 mitochondrial DNA(mtDNA) 152 mitosis 5, 6 roles of microtubules 55-6 see also budding monoclonalantibodies 37 motor proteins 55, 57 mtDNA (mitochondrial DNA) 152 multicopy suppression 87, 96-7 multiple cloning sites (MCS) 38 Munich Information Center for Protein Sequences (MIPS) 114 mutagenesis 67 mutanthunts 67-71 mutantslmutations change of function 69 cold-sensitive 70, 109 conditional see conditional mutations constitutive 80 dominant 69 dominant negative 109-10 double 70, 81, 82 forward 70
INDEX lethal 71 loss of function 69,109, 1 10 null 70,117-18 recessive 69 reverse 70 suppressor 91 temperature-sensitive 70 mutation analysis 89-90 Myc epitope 39 nickel (Ni2+)ions 40 Nomarski interference microscopy 30- l nomenclature (Saccharomyces) 7-8 nonallelic noncomplementation 77 nondenaturing conditions 35 nonparental ditype (NPD)tetrads 11 nuclear envelope 52, 58 nuclear fusion 50 nuclei 5 1-2 null mutations 70, 117-18 one-hybrid analysis 105-6 one-step gene replacement 16-17 open reading frame (ORF) nomenclature 7 order-of-functionmaps 79 organelles see cell structure: organelles origins of replication (ORI) 12, 13,14, 15 overexpression (multicopy)suppression 87, 96-7 P1-derived artificial chromosomes (PACs) 86 parentalditype (PD) tetrads 11 parental strains 69, 74 PCR see polymerase chain reaction (PCR) peptideepitopes 38-9 peptone 4 periplasmic space 44, 45, 145 peroxisomes 61 phalloidin 27, 28 phenotype 67 determination, mutant alleles 108 nomenclature 8 pheromones see mating: pheromones plasma membrane 43, 57, 58 plasmid shuffle 108 plasmid vectors cosmids 86 Escherichiacoli 12 fosmids 86 G F P O R F 28
239 Saccharomyces libraries 16 transformation 12-1 3 YAC 15 YCp 15 YEp 15 YIP 13-14 YRp 14-15 pleiotropy 10, 81 polarized growth 47-51,230 polyclonal antibodies 37 polymerase chain reaction (PCR) construction of disruptionfragments 16, 17 epitope tagging 38 identification of functional homologues 89 positionalcloning 87-8 prepro-cu-factor 148 prey fusions 103,104, I 16 promoters in expression vectors 20, 96 protein A 31,39, 40 protein databases 114 protein G 39-40 protein-DNA interactions, one-hybrid analysis 105-6 protein-protein interactions alanine-scanning mutagenesis 11 1 two-hybrid analysis 103-5,116 protein-RNA interactions, three-hybrid analysis 106 protein(s) bud site selection 50 cellwall 44, 45 conserved 89 cytokinesis tag 47 epitope tagging 37, 38-9 function, study methods alanine-scanning mutagenesis 111 dominant negative mutations 109-10 reverse genetics 107-8 motor 55, 57 nomenclature 8 overexpression 34-5 periplasmic 44, 45, 145 polarity-establishment 50-1 purification 32, 34-5 affinity methods 40-1 immunoprecipitation 39-40 SNARE complexes 61 spindle pole body 53 structure-function analysis 90, 181-2 vesicle coat 60-1 Western analysis 35-7,37-8 see also secretory pathway;speciJicproteins prototropes 4
240
INDEX
pseudohyphalgrowth 43, 44 unipolarbudding 47, 48, 49 quantitative matingassays
152
RAD52 epistasis group 84 RAD53 200-1 recessive mutations 69 recombination 10, 11, 13, 14, 88 regulatory factors in switch regulatory pathways 80-1 replica plating method 68 reporter genes 18-19, 103, 104, 105 restrictionendonucleases 18, 85, 86 restriction fragment length polymorphisms (RFLPs) 88 reverse genetics 107-8 reverse mutations (reversions) 70 rhodamine 26 rhoclrho- strains 152 ribosomes 57 rich media 4 RNA-protein interactions, three-hybrid analysis 106 Rsrlp 50 Saccharomyces cerevisiae 3 cell structure see cell structure cultureconditions 3-4 genome 7 life cycle 5-6 nomenclature 7-8 Saccharomyces Genome Database (SGD) 7. 114 saturated cultures3 scanningelectronmicroscopy (SEM) 32 schmooformation 47, 49, 50 screening formutants 68 SEC13 160 Sec63 protein 156, 159 secretion 144-5 assay of secreted proteins 145 secretory pathway 57 ER see endoplasmic reticulum Golgi complex see Golgi complex organization of theorganelles 58 overview 60 substrate dependence 150 vacuole 59-60 vesicles 59-61, 159-60 selectable marker genes 12, 16, 69, 108 selection of mutant phenotypes 68 SEM (scanningelectron microscopy) 32
Sepharosebeads 39, 40 septins 52 sequence homology,cloning by 89 sexual lifecycle 5-6 SGD (Saccharomyces Genome Database) 7, 114 short tandemrepeats(STRs) 88 single gene crosses 9-10 SM (synthetic minimal media) 4 SNARE complexes 61 SnD protein 130 spheroplasts 145 spindle pole bodies (SPBs) 52, 53, 55, 57 spindles 55,56 spores6, 51 Staphylococcus aureus protein A 3 1, 39, 40 stationary phase3 Ste2 protein 33 STE2 and STE3 212 step density gradients 24 strains congenic 68 isogenic 67, 68 knock-out1 17 nomenclature 8 parental 69, 74 revertant 70 wild-type 69 STRs (shorttandemrepeats) 88 substrate-dependent pathways 80, 8 1-2 SUC2 148 suppression 70, 91 intergenic see intergenic suppression intragenic9 1-2 switch regulatory pathways 80- 1,82-4, 95-6. 97 syntheticenhancement 100,101 synthetic lethality 101-2 synthetic minimal media (SM) 4 targeted integration of YIP plasmids 14, 87 TEF2 promoter 20 temperature-sensitive mutants 70 tetrad analysis 8-9 combined with complementation analysis 74, 75, 76 single gene crosses 10 two gene crosses 10-12 tetratype (TT) tetrads 11 three-hybridanalysis 106 Tn3 117 transcription activators 103 transformation of Saccharomyces 12- 13 transposon mutagenesis 117-18 TRPl 12
INDEX
24 1
TT (tetratype) tetrads 11 TUB genes 55
Western analysis 35-7,37-8 wild-type strains 69
tubulins 52, 55 two gene crosses 10-12 two-hybrid analysis 103-5,116
X-gal 19
ultracentrifugation 24-6 unipolarbudding 47, 48, 49 LIRA3 12, 13, 160 uracil 13 vacuoles 31, 59-60 vectors artificial chromosome 15, 86 bacteriophage 86 for epitopefusions 38 expression 20 plasmid see plasmid vectors vesicles 59-61, 159-60
YCp plasmids 15 yeast artificial chromosomes (YACs) 15 yeast form of Saccharomyces 43, 44, 48, 49 YeastProteome Database(YPD) 114 YEp plasmids 15 YEP (YP) medium 4 YEPD (YPD) medium4 Yer028 protein 140 YIP plasmids 13-14 YPD (YeastProteome Database) 114 YRp plasmids 14-15
