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,
X
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