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Fission Yeast: A Laboratory Manual


Subject Area(s):  YeastCell BiologyMicrobiology

Edited by Iain Hagan, Cancer Research UK Manchester Institute, University of Manchester; Antony M. Carr, University of Sussex; Agnes Grallert, Cancer Research UK Manchester Institute, University of Manchester; Paul Nurse, The Francis Crick Institute

Download a Free Excerpt from Fission Yeast:

Preface
Intro: Introduction to Fission Yeast as a Model System
Chapter 2: Growth and the Environment of Schizosaccharomyces pombe
Protocol: Mating, Spore Dissection, and Selection of Diploid Cells in Schizosaccharomyces japonicus
Index


© 2016 • 490 pages, illustrated (32 color, 17 B&W), index
Paperback • $90 72.00 (click here to price in UK Pounds)
ISBN  978-1-621820-82-6
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Description

Fission yeast are unicellular, rod-shaped fungi that divide by medial fission. Studies using fission yeast were instrumental in identifying fundamental mechanisms that govern cell division, differentiation, and epigenetics, to name but a few. Their rapid growth rate, genetic malleability, and similarities to more complex eukaryotes continue to make them excellent subjects for many biochemical, molecular, and cell biological studies.

This laboratory manual provides an authoritative collection of core experimental procedures that underpin modern fission yeast research. The contributors describe basic methods for culturing and genetically manipulating fission yeast, synchronization strategies for probing the cell cycle, technologies for assessing proteins, metabolites, and cell wall constituents, imaging methods to visualize subcellular structures and dynamics, and protocols for investigating chromatin and nucleic acid metabolism. Modifications to techniques commonly used in related species (e.g., budding yeast) are noted, as are useful resources for fission yeast researchers, including various databases and repositories.

The well-studied fission yeast Schizosaccharomyces pombe is the focus throughout, but the emerging model S. japonicus—a larger, dimorphic species with several desirable characteristics—is also covered. This manual is an important reference for existing fission yeast laboratories and will serve as an essential start-up guide for those working with fission yeast for the first time.

Contents

Preface
CHAPTER 1 INTRODUCTION
Introduction to Fission Yeast as a Model System
Jacqueline Hayles and Paul Nurse
CHAPTER 2 INTRODUCTION
Growth and the Environment of Schizosaccharomyces pombe
Janni Petersen and Paul Russell
CHAPTER 3 INTRODUCTION
Genetic Analysis of Schizosaccharomyces pombe
Karl Ekwall and Geneviève Thon
PROTOCOLS
1 Setting Up Schizosaccharomyces pombe Crosses/Matings
Karl Ekwall and Geneviève Thon
2 Selecting Schizosaccharomyces pombe Diploids
Karl Ekwall and Geneviève Thon
3 Spore Analysis and Tetrad Dissection of Schizosaccharomyces pombe
Karl Ekwall and Geneviève Thon
4 Mating-Type Determination in Schizosaccharomyces pombe
Karl Ekwall and Geneviève Thon
5 Ethyl Methanesulfonate Mutagenesis in Schizosaccharomyces pombe
Karl Ekwall and Geneviève Thon
CHAPTER 4 INTRODUCTION
Molecular Genetic Tools and Techniques in Fission Yeast
Johanne M. Murray, Adam T. Watson, and Antony M. Carr
PROTOCOLS
1 Transformation of Schizosaccharomyces pombe: Electroporation Procedure
Johanne M. Murray, Adam T. Watson, and Antony M. Carr
2 Transformation of Schizosaccharomyces pombe: Protoplast Procedure
Johanne M. Murray, Adam T. Watson, and Antony M. Carr
3 Identifying Products of Recombinase-Mediated Cassette Exchange (RMCE) in Schizosaccharomyces pombe
Johanne M. Murray, Adam T. Watson, and Antony M. Carr
4 Transformation of Schizosaccharomyces pombe: Lithium Acetate/ Dimethyl Sulfoxide Procedure
Johanne M. Murray, Adam T. Watson, and Antony M. Carr
5 Extraction of Chromosomal DNA from Schizosaccharomyces pombe
Johanne M. Murray, Adam T. Watson, and Antony M. Carr
6 Colony Polymerase Chain Reaction with Schizosaccharomyces pombe
Johanne M. Murray, Adam T. Watson, and Antony M. Carr
CHAPTER 5 INTRODUCTION
Analysis of RNA Metabolism in Fission Yeast
Jo Ann Wise and Olaf Nielsen
PROTOCOLS
1 Preparation of Total RNA from Fission Yeast
Jürg Bähler and Jo Ann Wise
2 Schizosaccharomyces pombe Polysome Profile Analysis and RNA Purification
Dieter A. Wolf, Jürg Bähler, and Jo Ann Wise
3 4-Thiouridine Labeling to Analyze mRNA Turnover in Schizosaccharomyces pombe
Juan Mata and Jo Ann Wise
CHAPTER 6 INTRODUCTION
Elementary Protein Analysis in Schizosaccharomyces pombe
Agnes Grallert and Iain M. Hagan
PROTOCOLS
1 Preparation of Protein Extracts from Schizosaccharomyces pombe Using Trichloroacetic Acid Precipitation
Agnes Grallert and Iain M. Hagan
2 Small-Scale Immunoprecipitation from Fission Yeast Cell Extracts
Agnes Grallert and Iain M. Hagan
3 Large-Scale Immunoprecipitation from Fission Yeast Cell Extracts
Agnes Grallert and Iain M. Hagan
4 Large-Scale Purification of Small Ubiquitin-Like Modifier (SUMO)-Modified Proteins from Schizosaccharomyces pombe
Minghua Nie and Michael N. Boddy
5 Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe
Nicola Zilio and Michael N. Boddy
CHAPTER 7 INTRODUCTION
Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Technology in Fission Yeast
Boris Maček, Alejandro Carpy, André Koch, Claudia C. Bicho, 
Weronika E. Borek, Silke Hauf, and Kenneth E. Sawin
PROTOCOLS
1 Construction, Growth, and Harvesting of Fission Yeast Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Strains
André Koch, Claudia C. Bicho, Weronika E. Borek, Alejandro Carpy, Boris Maček, Silke Hauf, and Kenneth E. Sawin
2 Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-Based Quantitative Proteomics and Phosphoproteomics in Fission Yeast
Alejandro Carpy, André Koch, Claudia C. Bicho, Weronika E. Borek, Silke Hauf, Kenneth E. Sawin, and Boris Maček
CHAPTER 8 INTRODUCTION
Electron Microscopy of Fission Yeast
J. Richard McIntosh, Mary K. Morphew, and Thomas H. Giddings, Jr.
PROTOCOLS
1 Preparing Fission Yeast for Electron Microscopy
Thomas H. Giddings, Jr., Mary K. Morphew, and J. Richard McIntosh
2 Immunolocalization of Proteins in Fission Yeast by Electron Microscopy
Mary K. Morphew, Thomas H. Giddings, Jr., and J. Richard McIntosh
3 Cryoelectron Microscopy of Fission Yeast
Mary K. Morphew, Thomas H. Giddings, Jr., and J. Richard McIntosh
CHAPTER 9 INTRODUCTION
Fixed-Cell Imaging of Schizosaccharomyces pombe
Iain M. Hagan and Steven Bagley
PROTOCOLS
1 Immunofluorescence Microscopy of Schizosaccharomyces pombe Using Chemical Fixation
lain M. Hagan
2 Chromatin and Cell Wall Staining of Schizosaccharomyces pombe
lain M. Hagan
3 Staining Fission Yeast Filamentous Actin with Fluorescent Phalloidin Conjugates
lain M. Hagan
CHAPTER 10 INTRODUCTION
Live Cell Imaging in Fission Yeast
Daniel P. Mulvihill
PROTOCOLS
1 Microscopic Observation of Living Cells Stained with Fluorescent Probes
Haruhiko Asakawa, Da-Qiao Ding, Tokuko Haraguchi, and 
Yasushi Hiraoka
2 Visualization of a Specific Genome Locus by the lacO/Lacl-GFP System
Da-Qiao Ding and Yasushi Hiraoka
3 Live Cell Imaging of the Schizosaccharomyces pombe Sexual Life Cycle
Laura Merlini, Aleksandar Vjestica, Omaya Dudin, Felipe Bendezú, and Sophie G. Martin
CHAPTER 11 INTRODUCTION
Analysis of Schizosaccharomyces pombe Meiosis
Akira Yamashita, Takeshi Sakuno, Yoshinori Watanabe, and 
Masayuki Yamamoto
PROTOCOLS
1 Live Imaging of Chromosome Segregation during Meiosis in the Fission Yeast Schizosaccharomyces pombe
Akira Yamashita, Takeshi Sakuno, Yoshinori Watanabe, and Masayuki Yamamoto
2 Synchronous Induction of Meiosis in the Fission Yeast Schizosaccharomyces pombe
Akira Yamashita, Takeshi Sakuno, Yoshinori Watanabe, and 
Masayuki Yamamoto
3 A Simple Method to Induce Meiosis and Sporulation Semisynchronously in the Fission Yeast Schizosaccharomyces pombe
Akira Yamashita, Takeshi Sakuno, Yoshinori Watanabe, and 
Masayuki Yamamoto
CHAPTER 12 INTRODUCTION
Analysis of the Schizosaccharomyces pombe Cell Cycle
lain M. Hagan, Agnes Grallert, and Viesturs Simanis
PROTOCOLS
1 Cell Cycle Synchronization of Schizosaccharomyces pombe by Centrifugal Elutriation of Small Cells
lain M. Hagan, Agnes Grallert, and Viesturs Simanis
2 Cell Cycle Synchronization of Schizosaccharomyces pombe by Lactose Gradient Centrifugation to Isolate Small Cells
lain M. Hagan, Agnes Grallert, and Viesturs Simanis
3 Synchronizing Progression of Schizosaccharomyces pombe Cells from
Prophase through Mitosis and into S Phase with nda3-KM311 Arrest Release
lain M. Hagan, Agnes Grallert, and Viesturs Simanis
4 Synchronizing Progression of Schizosaccharomyces pombe Cells from G2 through Repeated Rounds of Mitosis and S Phase with cdc25-22 Arrest Release
lain M. Hagan, Agnes Grallert, and Viesturs Simanis
5 Synchronization of S Phase in Schizosaccharomyces pombe Cells by Transient Exposure to M-Factor Pheromone
Olaf Nielsen
6 Analyzing Schizosaccharomyces pombe DNA Content by Flow Cytometry
Eric Boye, Silje Anda, Christiane Rothe, Trond Stokke, and Beata Grallert
CHAPTER 13 INTRODUCTION
Analysis of DNA Metabolism in Fission Yeast
Francisco Antequera and Timothy C. Humphrey
PROTOCOLS
1 Determination of the Frequency of Minichromosome Loss to Assess Chromosomal Stability in Fission Yeast
Osami Niwa
2 Two-Dimensional Gel Electrophoresis of DNA Replication Intermediates in Schizosaccharomyces pombe
Mar Sánchez and Francisco Antequera
3 Analysis of DNA Replication in Fission Yeast by Combing
Divya R. lyer, Shankar Das, and Nick Rhind
4 Using Pulsed-Field Gel Electrophoresis to Analyze Schizosaccharomyces pombe Chromosomes and Chromosomal Elements
Chen-Chun Pai, Carol Walker, and Timothy C. Humphrey
5 DNA Double-Strand Break Repair Assay
Chen-Chun Pai, Elizabeth Blaikley, and Timothy C. Humphrey
CHAPTER 14 INTRODUCTION
Analysis of Heterochromatin in Schizosaccharomyces pombe
Hugh P. Cam and Simon Whitehall
PROTOCOLS
1 Reporter Gene Silencing Assays in Fission Yeast
Hugh P. Cam and Simon Whitehall
2 Analysis of Schizosaccharomyces pombe Heterochromatin-Associated Short Interfering RNAs
Ke Zhang
3 Micrococcal Nuclease Digestion of Schizosaccharomyces pombe Chromatin
Hugh P. Cam and Simon Whitehall
4 Chromatin Immunoprecipitation (ChIP) in Schizosaccharomyces pombe
Hugh P. Cam and Simon Whitehall
CHAPTER 15 INTRODUCTION
Fission Yeast Cell Wall Analysis
Pilar Pérez and Juan C. Ribas
PROTOCOL
1 Radioactive Labeling and Fractionation of Fission Yeast Walls
Pilar Pérez and Juan C. Ribas
CHAPTER 16 INTRODUCTION
High-Throughput Quantitative Genetic Interaction Mapping in the Fission Yeast Schizosaccharomyces pombe
Assen Roguev, Colm J. Ryan, Edgar Hartsuiker, and Nevan J. Krogan
PROTOCOLS
1 Transformation of Schizosaccharomyces pombe in a 96-Well Format
Assen Roguev, Jiewei Xu, and Nevan J. Krogan
2 DNA Preparation from Schizosaccharomyces pombe
Assen Roguev, Jiewei Xu, and Nevan J. Krogan
3 Genetic Interaction Screens in Schizosaccharomyces pombe Using the Pombe Epistasis Mapper (PEM) System and a Manual Colony Replicator
Isabelle Colson and Edgar Hartsuiker
4 Genetic Interaction Mapping in Schizosaccharomyces pombe Using the Pombe Epistasis Mapper (PEM) System and a ROTOR HDA Colony Replicating Robot in a 1536 Array Format
Assen Roguev, Jiewei Xu, and Nevan J. Krogan
5 Genetic Interaction Score (S-Score) Calculation, Clustering, and Visualization of Genetic Interaction Profiles for Yeast
Assen Roguev, Colm J. Ryan, Jiewei Xu, Isabelle Colson, Edgar Hartsuiker, and Nevan J. Krogan
CHAPTER 17 INTRODUCTION
Metabolomic Analysis of Schizosaccharomyces pombe: Sample Preparation, Detection, and Data Interpretation
Tomáš Pluskal and Mitsuhiro Yanagida
PROTOCOLS
1 Preparation of Intracellular Metabolite Extracts from Liquid Schizosaccharomyces pombe Cultures
TomᚠPluskal, Takahiro Nakamura, and Mitsuhiro Yanagida
2 Measurement of Metabolome Samples Using Liquid Chromatography-Mass Spectrometry, Data Acquisition, and Processing
Tomas Pluskal and Mitsuhiro Yanagida
CHAPTER 18 INTRODUCTION
Schizosaccharomyces japonicus: A Distinct Dimorphic Yeast among the Fission Yeast
Keita Aoki, Kanji Furuya, and Hironori Niki
PROTOCOLS
1 Mating, Spore Dissection, and Selection of Diploid Cells in Schizosaccharomyces japonicus
Kanji Furuya and Hironori Niki
2 Transformation of Schizosaccharomyces japonicus
Keita Aoki and Hironori Niki
3 Induction of Hyphal Growth in Schizosaccharomyces japonicus
Hironori Niki
APPENDIX General Safety and Hazardous Material Information
INDEX