Schizosaccharomyces pombe: Methods and Protocols

239 Pages·2018·6.946 MB·English

by  Teresa L. Singleton (eds.)

Methods in
Molecular Biology 1721
Teresa L. Singleton Editor
Schizo-
saccharomyces
pombe
Methods and Protocols
M M B
ethods in olecular iology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springernature.com/series/7651
Schizosaccharomyces pombe
Methods and Protocols
Edited by
Teresa L. Singleton
Department of Biological Sciences, Winston-Salem State University, Winston-Salem, NC, USA
Editor
Teresa L. Singleton
Department of Biological Sciences
Winston-Salem State University
Winston-Salem, NC, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-7545-7 ISBN 978-1-4939-7546-4 (eBook)
https://doi.org/10.1007/978-1-4939-7546-4
Library of Congress Control Number: 2017963311
© Springer Science+Business Media, LLC 2018
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.
The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Printed on acid-free paper
This Humana Press imprint is published by Springer Nature
The registered company is Springer Science+Business Media, LLC
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Dedication
I dedicate my first book editing experience to God, my lovely daughter Shakyrah, and all
undergraduate student researchers.
v
Preface
The unicellular organism has facilitated our understanding of cellular mechanisms in higher
eukaryotes for years. The prokaryote, Escherichia coli, has served as a model organism in the
genetic engineering laboratory due to the inexpensive means to grow it and its fast genera-
tion time. E. coli serves as an excellent host for the production of proteins, bacteriophage
infection, and the use of conjugation to study antibiotic resistance.
The rise of more genetic diseases in higher eukaryotes has forced the research commu-
nity to adopt yet another model organism. Schizosaccharomyces pombe (fission yeast), where
“pombe” means “beer” in Swahili, has become an essential laboratory model organism.
Complete DNA sequencing of S. pombe’s genome in 2002 began to attract many research
groups. S. pombe has become the new “workhorse” in the biomedical research laboratory
because of the many protein homologs to higher eukaryotic organisms (humans), inexpen-
sive conditions and ease of growing, and haploid genome. S. pombe’s debut as a model
organism for understanding the cell cycle in humans has catapulted it into various areas of
research study. Many areas include centromeres, heterochromatin silencing components,
the RNA interference (RNAi) machinery, microtubules in mitosis and interphase, cell cycle
checkpoints, chromosome cohesion and segregation, DNA repair pathways, mitotic and
meiotic DNA recombination, mating-type switching, and sporulation, just to name a few!
The central dogma (DNA > RNA > protein) is essential to understanding how genes
work in organisms. This methods and protocols series highlights each phase of the central
dogma. Chapter 1 is a technical section dedicated to the undergraduate and graduate stu-
dent researcher. I have dedicated almost 20 years of my career toward teaching undergradu-
ate student researchers the essential skills to perform well in the research lab. The ability to
understand and correctly prepare solutions and reagents adds to the success of experiments
and respect in a lab. It is my hope that this chapter will help strengthen the research skills
and create better “lab hands” for our future student researchers.
Chapters 2–5, 6–7, 8–11, and 12–18 discuss protocols targeted at working with DNA,
RNA, protein, and S. pome as a “whole cell,” respectively.
Biotechnology is an area of science that affects the everyday life of every living organ-
ism. By definition, biotechnology is a science that deals with the use of an organism or the
by-products from an organism to develop technologies and products that will better the
lives of living organisms on this planet. Chapters 19 and 20 highlight the many facets of S.
pombe, including functioning as an agent in “winemaking.”
Each protocol in this book highlights working with S. pombe in the research commu-
nity, but the protocols can address any molecular biological experiment. I have included a
glossary that defines terms strategically chosen from each chapter.
I am very grateful to all authors for their commitment and contribution toward this
project and highlighting S. pombe.
vii
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Preparation of Solutions and Reagents .................................. 1
Teresa L. Singleton
2 Analysis of Fission Yeast Single DNA Molecules on the Megabase Scale
Using DNA Combing .............................................. 9
Atanas Kaykov and Paul Nurse
3 Chromatin Immunoprecipitation-Polymerase Chain Reaction (ChIP-PCR)
Detects Methylation, Acetylation, and Ubiquitylation in S. pombe .............. 25
Valerie Migeot and Damien Hermand
4 Primer Design and Inverse PCR on Yeast Display Antibody Selection Outputs .... 35
Fortunato Ferrara, Andrew R.M. Bradbury, and Sara D’Angelo
5 Molecular Cloning and Characterization of Small Viral Genome in Fission Yeast... 47
Ge Li and Richard Y. Zhao
6 Total RNA Isolation and Quantification of Specific RNAs in Fission Yeast........ 63
Robert Roth, Hiten D. Madhani, and Jennifer F. Garcia
7 Analysis of Reverse Transcribed mRNA Using PCR and Polyacrylamide
Gel Electrophoresis................................................. 73
Pranjal Biswas, Uddalak Majumdar, and Sanjay Ghosh
8 The No-Nonsens SDS-PAGE......................................... 89
Lars Backman and Karina Persson
9 Crystallization of Recombinant α-Actinin and Related Proteins................ 95
Karina Persson and Lars Backman
10 Estimation of GFP-Nucleoporin Amount Based on Fluorescence Microscopy..... 105
Haruhiko Asakawa, Yasushi Hiraoka, and Tokuko Haraguchi
11 Antibody Pull-Down Experiments in Fission Yeast ......................... 117
Qianhua Dong and Fei Li
12 Preparation of Cell Lysates of Fission Yeast for Immunoprecipitation ........... 125
Akihisa Matsuyama, Atsuko Shirai, and Minoru Yoshida
13 Wide-band Electrical Impedance Spectroscopy (EIS) Measures S. pombe
Cell Growth in vivo ................................................ 135
Zhen Zhu, Olivier Frey, and Andreas Hierlemann
14 In Situ Chromatin-Binding Assay Using Epifluorescent
Microscopy in S. pombe.............................................. 155
Jinpu Yang and Fei Li
15 High-Frequency Lithium Acetate Transformation
of Schizosaccharomyces pombe.......................................... 167
Sudhir Kumar Rai, Angela Atwood-Moore, and Henry L. Levin
ix
x Contents
16 Tetrad Dissection in Fission Yeast ...................................... 179
Wilber Escorcia and Susan L. Forsburg
17 Random Spore Analysis in Fission Yeast ................................. 189
Wilber Escorcia and Susan L. Forsburg
18 Duplication and Transformation of the Schizosaccharomyces
pombe Collection of Deletion Strains.................................... 197
Sudhir Kumar Rai, Angela Atwood-Moore, and Henry L. Levin
19 Schizosaccharomyces pombe Biotechnological Applications in Winemaking......... 217
Ángel Benito, Fernando Calderón, and Santiago Benito
20 Schizosaccharomyces pombe Isolation Protocol ............................. 227
Ángel Benito, Fernando Calderón, and Santiago Benito
Glossary.................................................................. 235
Index............................................................... 243
Contributors
HaruHiko asakawa • Graduate School of Frontier Biosciences, Osaka University, Suita,
Japan
angela atwood-Moore • Section on Eukaryotic Transposable Elements, Division
of Molecular and Cellular Biology, The Eunice Kennedy Shriver National Institute
of Child Health and Human Development, National Institutes of Health, Bethesda,
MD, USA
lars BackMan • Department of Chemistry, Umeå University, Umeå, Sweden
Ángel Benito • Department of Chemistry and Food Technology, Polytechnic University
of Madrid, Madrid, Spain
santiago Benito • Department of Chemistry and Food Technology, Polytechnic University
of Madrid, Madrid, Spain
Pranjal Biswas • Department of Genetics, Cell Biology and Anatomy, University
of Nebraska Medical Center, Omaha, NE, USA; Department of Biochemistry,
University of Calcutta, Kolkata, West Bengal, India
andrew r.M. BradBury • Specifica Inc., Santa Fe, NM, USA
Fernando calderón • Department of Chemistry and Food Technology,
Polytechnic University of Madrid, Madrid, Spain
sara d’angelo • Specifica Inc., Santa Fe, NM, USA
QianHua dong • Department of Biology, New York University, New York, NY, USA
wilBer escorcia • Molecular and Computational Biology Section, University of Southern
California, Los Angeles, CA, USA
Fortunato Ferrara • Specifica Inc., Santa Fe, NM, USA
susan l. ForsBurg • Molecular and Computational Biology Section, University
of Southern California, Los Angeles, CA, USA
olivier Frey • Department of Biosystems Science and Engineering, ETH Zurich, Basel,
Switzerland
jenniFer F. garcia • Department of Molecular Biology, Colorado College, Colorado
Springs, CO, USA
sanjay gHosH • Department of Biochemistry, University of Calcutta, Kolkata, West
Bengal, India
tokuko HaragucHi • Graduate School of Frontier Biosciences, Osaka University, Suita,
Japan; Advanced ICT Research Institute Kobe, National Institute of Information and
Communications Technology, Kobe, Japan
daMien HerMand • URPHYM-GEMO, The University of Namur, Namur, Belgium
andreas HierleMann • Department of Biosystems Science and Engineering, ETH Zurich,
Basel, Switzerland
yasusHi Hiraoka • Graduate School of Frontier Biosciences, Osaka University, Suita,
Japan; Advanced ICT Research Institute Kobe, National Institute of Information
and Communications Technology, Kobe, Japan
atanas kaykov • Avangard Genetics, Plovdiv, Bulgaria; The Rockefeller University,
New York, NY, USA
xi
xii Contributors
Henry l. levin • Section on Eukaryotic Transposable Elements, Division of Molecular
and Cellular Biology, The Eunice Kennedy Shriver National Institute of Child Health
and Human Development, National Institutes of Health, Bethesda, MD, USA
Fei li • Department of Biology, New York University, New York, NY, USA
ge li • University of Maryland School of Medicine, Baltimore, MD, USA
Hiten d. MadHani • Deptartment of Biochemistry and Biophysics, University of
California, San Francisco, CA, USA
uddalak MajuMdar • Center for Cardiovascular Research, Nationwide Children’s
Hospital, Columbus, OH, USA; Department of Biochemistry, University of Calcutta,
Kolkata, West Bengal, India
akiHisa MatsuyaMa • Chemical Genomics Research Group, RIKEN Center for
Sustainable Resource Science, Saitama, Japan; Chemical Genetics Laboratory, RIKEN,
Saitama, Japan
valerie Migeot • URPHYM-GEMO, The University of Namur, Namur, Belgium
Paul nurse • The Rockefeller University, New York, NY, USA; The Francis Crick
Institute, London, UK
karina Persson • Department of Chemistry, Umeå University, Umeå, Sweden
sudHir kuMar rai • Section on Eukaryotic Transposable Elements, Division of Molecular
and Cellular Biology, The Eunice Kennedy Shriver National Institute of Child Health
and Human Development, National Institutes of Health, Bethesda, MD, USA;
Division of Hematology/Oncology, Department of Medicine, University of Florida,
Gainesville, FL, USA
roBert rotH • Department of Molecular Biology, Colorado College, Colorado Springs,
CO, USA
atsuko sHirai • Chemical Genetics Laboratory, RIKEN, Saitama, Japan;
Cellular Memory Laboratory, RIKEN, Saitama, Japan
teresa l. singleton • Department of Biological Science, Winston-Salem State University,
Winston-Salem, NC, USA
jinPu yang • Department of Biology, New York University, New York, NY, USA
Minoru yosHida • Chemical Genomics Research Group, RIKEN Center for Sustainable
Resource Science, Saitama, Japan; Chemical Genetics Laboratory, RIKEN, Saitama,
Japan; Department of Biotechnology, The University of Tokyo, Tokyo, Japan
ricHard y. ZHao • University of Maryland School of Medicine, Baltimore, MD, USA
ZHen ZHu • Southeast University, Key Laboratory of MEMS of Ministry of Education,
Nanjing, China