Yeast Genetics: Methods and Protocols

382 Pages·2014·8.701 MB·English

by  Jeffrey S. Smith, Daniel J. Burke (eds.)

Methods in
Molecular Biology 1205
Jeff rey S. Smith
Daniel J. Burke Editors
Yeast Genetics
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
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Yeast Genetics
Methods and Protocols
Edited by
Jeffrey S. Smith
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine,
Charlottesville, VA, USA
Daniel J. Burke
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine,
Charlottesville, VA, USA
Editors
Jeffrey S. Smith Daniel J. Burke
Department of Biochemistry Department of Biochemistry
and Molecular Genetics and Molecular Genetics
University of Virginia School of Medicine University of Virginia School of Medicine
Charlottesville, VA, USA Charlottesville, V A , USA
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ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-1362-6 ISBN 978-1-4939-1363-3 (eBook)
DOI 10.1007/978-1-4939-1363-3
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Prefa ce
Almost anyone working in a laboratory that utilizes S accharomyces cerevisiae for their
research projects has no doubt heard the phrase, “The awesome power of yeast genetics.”
This statement has long been a source of pride within the yeast research community. It
refers to the simple nature of a single-cell eukaryotic organism, the relative ease of manipu-
lating its genome, and the ability to interchangeably exist in both haploid and diploid
states. Genes can be deleted, mutated, engineered, and tagged at will. Tetrad dissection of
diploids is the workhorse of classical yeast genetics and remains a critical technique, comple-
menting the widespread use of synthetic genetic array (SGA) technologies that can generate
various mutant combinations in high throughput. Protocols for all of these methods are
included in this book.
Saccharomyces cerevisiae has played a major role in the elucidation of multiple conserved
cellular processes including MAP kinase signaling, splicing, transcription, and many others.
With the advent of RNAi, the ability to reduce gene expression in higher eukaryotes has
allowed some model organisms to “catch up” with the yeast system in terms of high-
throughput analysis, and CRISPR technology is sure to push this even further in mamma-
lian cells. However, the simplicity of the yeast system still makes it a highly attractive, and
truly genetic powerhouse. This is especially true given the wide range of genome-wide
resources that are readily available for analysis. Such collections simply do not exist for most
other model organisms, at least not to such a degree. The general idea of this protocols
book is to provide a balanced blend of classic and more modern genetic methods relevant
to a wide range of research areas. It should be widely used as a reference in yeast labs.
Charlottesville, VA, USA J effrey S . S mith
Daniel J . Burke
v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
1 Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method . . . . . . . . . 1
R. Daniel Gietz
2 T etrad, Random Spore, and Molecular Analysis of Meiotic Segregation
and Recombination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Michael Lichten
3 P CR Mutagenesis and Gap Repair in Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 9
Mark Weir and Jill B . K eeney
4 P CR-Mediated Epitope Tagging of Genes in Yeast . . . . . . . . . . . . . . . . . . . . . 3 7
Radhika Mathur and P eter K aiser
5 M anipulating the Yeast Genome: Deletion, Mutation,
and Tagging by PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Jennifer M . Gardner and Sue L . J aspersen
6 P reparation of Yeast Cells for Live-Cell Imaging and Indirect
Immunofluorescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Lucy F. Pemberton
7 S ingle Yeast Cell Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1
Heimo W olinski and Sepp D . K ohlwein
8 M icrofluidic Platforms for Generating Dynamic Environmental
Perturbations to Study the Responses of Single Yeast Cells . . . . . . . . . . . . . . . 111
Anjali B isaria, P ascal H ersen, and M egan N. McClean
9 Using Two-Hybrid Interactions to Identify
Separation-of-F unction Mutations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Brian H aarer and David C . A mberg
10 Synthetic Genetic Array Analysis for Global Mapping
of Genetic Networks in Yeast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Elena Kuzmin, S ara S harifpoor, A nastasia Baryshnikova,
Michael C ostanzo, C had L . Myers, Brenda J. Andrews,
and Charles B oone
11 C hemical Genetic and Chemogenomic Analysis in Yeast . . . . . . . . . . . . . . . . . 1 69
Namal V. C . C oorey, L iam D . P . Sampson, Jacqueline M. Barber,
and David S. Bellows
12 P henomic Assessment of Genetic Buffering by Kinetic Analysis
of Cell Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 87
John Rodgers, J ingyu Guo, and J ohn L. H artman IV
vii
viii Contents
13 Detection of Short-Range Chromatin Interactions
by Chromosome Conformation Capture (3C) in Yeast . . . . . . . . . . . . . . . . . . 209
Badri Nath Singh and Michael H ampsey
14 C hromosome Conformation Capture (3C) of Tandem Arrays in Yeast. . . . . . . 2 19
Maria D. Mayán and Luis A ragón
15 G lobal Analysis of Transcription Factor-Binding Sites in Yeast
Using ChIP-Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 31
Philippe L efrançois, J ennifer E . G. G allagher, and M ichael Snyder
16 H igh-Density Tiling Microarray Analysis of the Full Transcriptional
Activity of Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Lior D avid, S andra C lauder-Münster, and L ars M. Steinmetz
17 Analysis of Silencing in Saccharomyces cerevisiae. . . . . . . . . . . . . . . . . . . . . . . . 275
Andrew M iller and Ann L. K irchmaier
1 8 A User’s Guide to the Ribosomal DNA in Saccharomyces cerevisiae . . . . . . . . . 3 03
Joseph M. Johnson, Jeffrey S. S mith, and D avid A. Schneider
19 Two-Dimensional Agarose Gel Electrophoresis for Analysis
of DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 29
Sandra K. Villwock and Oscar M . Aparicio
20 R eplicative Life Span Analysis in Budding Yeast. . . . . . . . . . . . . . . . . . . . . . . . 3 41
George L. Sutphin, J oe R . D elaney, and Matt K aeberlein
21 M etabolomic and Lipidomic Analyses of Chronologically Aging Yeast. . . . . . . 3 59
Vincent R. Richard, S imon D . B ourque, and Vladimir I . Titorenko
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 75
Contributors
DAVID C. AMBERG • Department of Biochemistry and Molecular Biology,
SUNY Upstate Medical University , S yracuse, N Y , U SA
BRENDA J. ANDREWS • The Department of Molecular Genetics, University of Toronto ,
Toronto , O N , C anada ; D onnelly Center for Cellular and Biomedical Research , T oronto ,
ON, C anada ; B anting and Best Department of Medical Research , T oronto , O N, Canada
OSCAR M. APARICIO • Molecular and Computational Biology Program , U niversity of
Southern California , Los Angeles, C A , USA
LUIS ARAGÓN • Cell Cycle Group, MRC Clinical Sciences Centre , I mperial College ,
London, U K
JACQUELINE M. B ARBER • School of Biological Sciences, V ictoria University of Wellington ,
Wellington, N ew Zealand
ANASTASIA B ARYSHNIKOVA • The Department of Molecular Genetics, U niversity of Toronto ,
Toronto , O N , C anada ; Donnelly Center for Cellular and Biomedical Research ,
Toronto , O N , C anada
DAVID S. B ELLOWS • School of Biological Sciences, V ictoria University of Wellington ,
Wellington, N ew Zealand
ANJALI BISARIA • Lewis-Sigler Institute for Integrative Genomics, P rinceton University ,
Princeton, N J , USA
CHARLES BOONE • The Department of Molecular Genetics, U niversity of Toronto , T oronto ,
ON, C anada ; D onnelly Center for Cellular and Biomedical Research , T oronto , O N,
Canada ; B anting and Best Department of Medical Research , T oronto , O N , C anada
SIMON D . B OURQUE • Department of Biology, Concordia University , M ontreal, Q C , C anada
DANIEL J . BURKE • Department of Biochemistry and Molecular Genetics,
University of Virginia School of Medicine , Charlottesville, V A , USA
SANDRA CLAUDER-MÜNSTER • Genome Biology Unit, E MBL , H eidelberg, G ermany
NAMAL V. C. C OOREY • School of Biological Sciences, Victoria University of Wellington ,
Wellington, N ew Zealand
MICHAEL COSTANZO • Donnelly Centre for Cellular and Biomolecular Research ,
Toronto , O N, C anada
LIOR DAVID • Department of Animal Sciences, R.H. Smith Faculty of Agriculture,
Food and Environment , T he Hebrew University of Jerusalem , Rehovot, Israel
JOE R . DELANEY • Department of Pathology, University of Washington , Seattle , WA, USA ;
The Molecular and Cellular Biology Program , University of Washington , Seattle , WA , USA
JENNIFER E. G. GALLAGHER • Department of Biology, West Virginia University ,
Morgantown, W V , U SA
JENNIFER M. GARDNER • Stowers Institute for Medical Research , Kansas City, M O, U SA
R. D ANIEL GIETZ • Department of Biochemistry and Medical Genetics,
University of Manitoba , Winnipeg, MB , Canada
JINGYU G UO • Department of Genetics, U niversity of Alabama at Birmingham ,
Birmingham, A L , U SA
BRIAN H AARER • Department of Biochemistry and Molecular Biology,
SUNY Upstate Medical University , S yracuse, N Y , U SA
ix