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