Thông tin tài liệu
GENETIC MANIPULATION
OF DNA AND PROTEIN –
EXAMPLES FROM
CURRENT RESEARCH
Edited by David Figurski
Genetic Manipulation of DNA and Protein – Examples from Current Research
Edited by David Figurski
Contributors
Deepak Bastia, S. Zzaman, Bidyut K. Mohanty, J. Esclapez, M. Camacho, C. Pire, M.J. Bonete,
David H. Figurski, Daniel H. Fine, Brenda A. Perez-Cheeks, Valerie W. Grosso, Karin E. Kram,
Jianyuan Hua, Ke Xu
,
Jamila Hedhli, Jürgen Ludwig, Holger Rabe, Anja Höffle-Maas, Marek
Samochocki, Alfred Maelicke, Titus Kaletta, Luis Eduardo S. Netto, Marcos Antonio Oliveira,
Toni Petan, Petra Prijatelj Žnidaršič, Jože Pungerčar, Ewa Sajnaga, Ryszard Szyszka, Konrad
Kubiński, Jane E. Carland, Amelia R. Edington, Amanda J. Scopelliti, Renae M. Ryan, Robert J.
Vandenberg, José Manuel Pérez-Donoso, Claudio C. Vásquez, Kevin Hadi, Oznur Tastan,
Alagarsamy Srinivasan, Velpandi Ayyavoo, Ahmed Chraibi, Stéphane Renauld, M. Tang, K.J.
Wierenga, K. Lai, Christelle Bonod-Bidaud, Florence Ruggiero, Silvio Alejandro López-Pazos,
Jairo Cerón, Juanita Yazmin Damián-Almazo, Gloria Saab-Rincón, Stathis Frillingos, Roman G.
Gerlach, Kathrin Blank, Thorsten Wille, Nathan A. Sieracki, Yulia A. Komarova, Shona A.
Mookerjee, Elaine A. Sia, Joy Sturtevant, James W. Wilson, Clayton P. Santiago, Jacquelyn
Serfecz, Laura N. Quick
Published by InTech
Janeza Trdine 9, 51000 Rijeka, Croatia
Copyright © 2013 InTech
All chapters are Open Access distributed under the Creative Commons Attribution 3.0 license,
which allows users to download, copy and build upon published articles even for commercial
purposes, as long as the author and publisher are properly credited, which ensures maximum
dissemination and a wider impact of our publications. After this work has been published by
InTech, authors have the right to republish it, in whole or part, in any publication of which they
are the author, and to make other personal use of the work. Any republication, referencing or
personal use of the work must explicitly identify the original source.
Notice
Statements and opinions expressed in the chapters are these of the individual contributors and
not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy
of information contained in the published chapters. The publisher assumes no responsibility for
any damage or injury to persons or property arising out of the use of any materials,
instructions, methods or ideas contained in the book.
Publishing Process Manager Ivana Zec
Typesetting InTech Prepress, Novi Sad
Cover InTech Design Team
First published January, 2013
Printed in Croatia
A free online edition of this book is available at www.intechopen.com
Additional hard copies can be obtained from orders@intechopen.com
Genetic Manipulation of DNA and Protein – Examples from Current Research,
Edited by David Figurski
p. cm.
ISBN 978-953-51-0994-5
Contents
Preface IX
Section 1 Molecular Genetics in Basic Research 1
Chapter 1 Site-Directed Mutagenesis and Yeast Reverse
2-Hybrid-Guided Selections to Investigate
the Mechanism of Replication Termination 3
Deepak Bastia, S. Zzaman and Bidyut K. Mohanty
Chapter 2 Biochemical Analysis of Halophilic Dehydrogenases
Altered by Site-Directed Mutagenesis 17
J. Esclapez, M. Camacho, C. Pire and M.J. Bonete
Chapter 3 Targeted Mutagenesis in the Study of the Tight Adherence
(tad) Locus of Aggregatibacter actinomycetemcomitans 43
David H. Figurski, Daniel H. Fine, Brenda A. Perez-Cheeks,
Valerie W. Grosso, Karin E. Kram, Jianyuan Hua,
Ke Xu
and Jamila Hedhli
Chapter 4 Directed Mutagenesis of Nicotinic Receptors
to Investigate Receptor Function 71
Jürgen Ludwig, Holger Rabe, Anja Höffle-Maas,
Marek Samochocki, Alfred Maelicke and Titus Kaletta
Chapter 5 Site-Directed Mutagenesis as a Tool to Characterize
Specificity in Thiol-Based Redox Interactions
Between Proteins and Substrates 91
Luis Eduardo S. Netto and Marcos Antonio Oliveira
Chapter 6 Protein Engineering in Structure-Function Studies
of Viper's Venom Secreted Phospholipases A2 107
Toni Petan, Petra Prijatelj Žnidaršič and Jože Pungerčar
Chapter 7 Site-Directed Mutagenesis in the Research of Protein Kinases
- The Case of Protein Kinase CK2 133
Ewa Sajnaga, Ryszard Szyszka and Konrad Kubiński
VI Contents
Chapter 8 Directed Mutagenesis in Structure Activity Studies
of Neurotransmitter Transporters 167
Jane E. Carland, Amelia R. Edington, Amanda J. Scopelliti,
Renae M. Ryan and Robert J. Vandenberg
Chapter 9 Site-Directed Mutagenesis as a Tool for Unveiling
Mechanisms of Bacterial Tellurite Resistance 185
José Manuel Pérez-Donoso and Claudio C. Vásquez
Section 2 Molecular Genetics in Disease-Related Research 201
Chapter 10 A Mutagenesis Approach for the Study of
the Structure-Function Relationship of Human
Immunodeficiency Virus Type 1 (HIV-1) Vpr 203
Kevin Hadi, Oznur Tastan, Alagarsamy Srinivasan
and Velpandi Ayyavoo
Chapter 11 New Insights into the Epithelial Sodium Channel
Using Directed Mutagenesis 221
Ahmed Chraibi and Stéphane Renauld
Chapter 12 Use of Site-Directed Mutagenesis in the Diagnosis,
Prognosis and Treatment of Galactosemia 233
M. Tang, K.J. Wierenga and K. Lai
Chapter 13 Inherited Connective Tissue Disorders of Collagens:
Lessons from Targeted Mutagenesis 253
Christelle Bonod-Bidaud and Florence Ruggiero
Section 3 Molecular Genetics in Applied Research 271
Chapter 14 Biological Activity of Insecticidal Toxins: Structural Basis,
Site-Directed Mutagenesis and Perspectives 273
Silvio Alejandro López-Pazos and Jairo Cerón
Chapter 15 Site-Directed Mutagenesis as Applied to Biocatalysts 303
Juanita Yazmin Damián-Almazo and Gloria Saab-Rincón
Section 4 New Tools or Approaches for Molecular Genetics 331
Chapter 16 Using Cys-Scanning Analysis Data
in the Study of Membrane Transport Proteins 333
Stathis Frillingos
Chapter 17 Site-Directed Mutagenesis Using
Oligonucleotide-Based Recombineering 361
Roman G. Gerlach, Kathrin Blank and Thorsten Wille
Contents VII
Chapter 18 Studying Cell Signal Transduction
with Biomimetic Point Mutations 381
Nathan A. Sieracki and Yulia A. Komarova
Chapter 19 Using Genetic Reporters to Assess Stability and Mutation
of the Yeast Mitochondrial Genome 393
Shona A. Mookerjee and Elaine A. Sia
Chapter 20 Site-Directed and Random Insertional Mutagenesis
in Medically Important Fungi 417
Joy Sturtevant
Chapter 21 Recombineering and Conjugation as Tools
for Targeted Genomic Cloning 437
James W. Wilson, Clayton P. Santiago,
Jacquelyn Serfecz and Laura N. Quick
Preface
This diverse collection of research articles is united by the enormous power of modern
molecular genetics. The current period is an exciting time both for researchers and the
curious who want to know more about genetic approaches to solving problems.
This volume is noteworthy. Every author accomplished two important objectives: (1)
making the field and the particular research described accessible to a large audience
and (2) explaining fully the genetic tools and approaches that were used in the
research. One fact stands out – the importance of a genetic approach to addressing a
problem. I encourage you to read several chapters. You will feel the excitement of the
scientists, and you will learn about an area of research with which you may not be
familiar. Perhaps most importantly, you will understand the genetic approaches; and
you will appreciate their importance to the research.
Anyone can benefit from reading these chapters – even those of you who have a solid
foundation in modern molecular genetics. This is an eclectic mix of topics (only the
surface has been scratched). These chapters are valuable, not only because they reflect
the current state of the art and are easy to read, but also because they are concise
reviews. The variety will provide you with new knowledge to be sure, but it may also
affect your own thoughts about a problem. Thinking about a topic very different from
the one you are considering can stimulate fresh and often unconventional ideas.
We all know that the code for all life on the planet is in DNA and RNA. The purpose
of genetics is to decipher life’s information – to understand why the genome codes for
its various functions. Much of the work in this volume is geared to manipulating DNA
with that knowledge, not only to provide clues about a function, but also to test an
idea or to change a protein to learn how it works or to make it work better.
For a time, the field of molecular genetics was concerned with a few manipulable
model organisms. This was necessary to answer basic questions like “How does a gene
work?” Now modern molecular genetics has given us the confidence to explore the
unknowns in the diversity of life, including complex organisms, like humans. We may
need to adapt or develop genetic tools (see the contents section on tools). We have
already learned that many of the “paradigms” of the model organisms do not apply to
other organisms.
X Preface
“Manipulate” is a problem word in genetics for some people. This volume has another
purpose - to be accessible to those who fear the power of genetics. Those of us who
know modern genetics understand that the current precision of genetically modified
food, for example, is far safer than the unknowns of genetic crosses, a technology that
is strangely acceptable. We have ourselves to blame for the apparent mystery and the
public’s misperceptions. Too often we discuss our work with our colleagues but fail to
explain our work to the public.
By making these chapters freely available to everyone and by the authors clearly
describing the question being asked and the approach taken to answer it, this book is
partly addressing that concern. People who fear genetics should take comfort in the
dissemination of knowledge about this science. Scientists have the same concerns as
the public. The more who understand genetics, the more there will be vigilance.
This collection of research articles is testimony to the optimism in the field. Both major
and minor problems can be solved. For example, genetics will likely be a part of the
solution to hunger, and genetically engineered microorganisms may help solve the
problem of global warming. Basic research (see the contents sections on basic research
and the development of approaches and tools) is difficult to explain, but it is vitally
important for any progress. Genetics will help alleviate suffering by leading to new
therapies for disease (see the contents section on disease-related research), and it can
generate improved or new molecular activities (see the contents section on applied
research).
With a complete understanding of genetics, humankind will reach an important new
stage. Humans will be able to change their own genes. Of course, evolution will
continue to be an agent of genetic change; but it is slow in humans, and it acts on
populations. With the knowledge of genetics, humans will be able to direct change
(like the curing of a disease) to an individual; and it can be rapid.
You will be exposed to investigations on bacteria, archaea, fungi, mitochondria, and
higher eukaryotes, including humans. You will learn about various genetic
approaches, including specific alteration of amino acid residues in proteins, gene
fusions, cysteine- and alanine-scanning mutagenesis, recombineering, cloning by
“capturing” large segments of DNA, transposable elements, and allelic exchange. The
chapters are all very readable, and again I encourage you to sample more than one.
David Figurski
Professor of Microbiology & Immunology at Columbia University,
USA
[...]... generated by recombination (Madabhushi and Marians, 2009) 10 Genetic Manipulation of DNA and Protein – Examples from Current Research Fig 4 A substrate designed to separate temporally and spatially DnaB translocation from DNA unwinding A 5’ tailed DNA with otherwise a blunt end on the complementary strand enters the substrate and then slides over the dsDNA until it meets the fork like structure (in blue) and. .. transcriptional activation domain of Gal4 of yeast (pGAD424-X) A suspected interacting 6 Genetic Manipulation of DNA and Protein – Examples from Current Research Fig 2 Crystal structure of Tus-Ter complex of E coli and RTP apoprotein of B subtilis A, crystal structure of Tus-Ter complex showing the blocking face with the L1 loop shown in red Three residues, namely P42, E47 and E49, when mutated (see lower... (e.g., Leu) and an ars Using this protocol, we extensively mutagenized Fob1 and were able to identify many of its functional domains, such as its 12 Genetic Manipulation of DNA and Protein – Examples from Current Research DNA binding domain and a domain for its interaction with the silencing linker protein called Net1 Net1 recruits the histone deacetylase Sir2 onto Fob1 by direct protein- protein interaction... identified and sequenced (Bastia et al., 1981) and subsequently shown to consist of a pair of Ter sites with opposite polarity (Hidaka et al., 1988) An in vitro replication system was 4 Genetic Manipulation of DNA and Protein – Examples from Current Research developed in which host cell extracts initiated replication of a plasmid DNA template and the moving forks were arrested at the Ter sites (Germino and. .. (1996) The relationship between sequence-specific termination of DNA replication and transcription EMBO J 15, 2530-2539 16 Genetic Manipulation of DNA and Protein – Examples from Current Research Mohanty, B.K., Sahoo, T., and Bastia, D (1998) Mechanistic studies on the impact of transcription on sequence-specific termination of DNA replication and vice versa J Biol Chem 273, 3051-3059 Mulcair, M.D., Schaffer,... a rate of 60 ºC/h using 50 mM potassium phosphate buffer pH 7.3 containing 1 mM EDTA and 0.5 M or 2.0 M KCl, which also served 22 Genetic Manipulation of DNA and Protein – Examples from Current Research for baseline measurements Prior to scanning, all samples of protein and buffer were degassed under vacuum using a ThermoVac unit (MicroCal) The protein concentrations were in the range of 5 0–8 0 M (approximately... resistance of ICDH from Hfx volcanii to chemical denaturation has also been found This study strongly suggests that Hfx volcanii ICDH might be seen as a type of halophilic protein never described before: an oligomeric halophilic protein devoid of intersubunit anion-binding sites (Madern et al., 2004) 20 Genetic Manipulation of DNA and Protein – Examples from Current Research 2 Materials and methods... encountering a linear DNA with a 5’ tail and 3’ blunt end, DnaB enters DNA with both strands passing through the central channel of DnaB (Kaplan, 2000) The translocation of DnaB on double-stranded DNA (dsDNA) requires ATP hydrolysis We constructed the DNA substrate shown in Fig 4 The DnaB helicase enters the substrate from the left by riding the 5’-single-stranded tail, slides over dsDNA containing a Ter... Brewer, B.J., and Fangman, W.L (1987) The localization of replication origins on ARS plasmids in S cerevisiae Cell 51, 463-471 14 Genetic Manipulation of DNA and Protein – Examples from Current Research Brewer, B.J., and Fangman, W.L (1988) A replication fork barrier at the 3' end of yeast ribosomal RNA genes Cell 55, 637-643 Brewer, B.J., Lockshon, D., and Fangman, W.L (1992) The arrest of replication... that the side-chain carboxyl of D172 is involved in interactions with a cluster of surface water molecules near a bound potassium counter-ion In contrast, the side-chain carboxyl of D216 forms interactions with surface waters in a region in which no counter-ions can be 24 Genetic Manipulation of DNA and Protein – Examples from Current Research seen The side-chain carboxyl of D344 lies on the surface, . GENETIC MANIPULATION
OF DNA AND PROTEIN –
EXAMPLES FROM
CURRENT RESEARCH
Edited by David Figurski
Genetic Manipulation of DNA and. activation domain of Gal4 of yeast (pGAD424-X). A suspected interacting
Genetic Manipulation of DNA and Protein – Examples from Current Research
6
Ngày đăng: 17/03/2014, 21:20
Xem thêm: Genetic Manipulation of DNA and Protein – Examples from Current Research Edited by David Figurski potx, Genetic Manipulation of DNA and Protein – Examples from Current Research Edited by David Figurski potx