MOLECULAR CLONING – SELECTED APPLICATIONS IN MEDICINE AND BIOLOGY Edited by Gregory G. Brown Molecular Cloning – Selected Applications in Medicine and Biology Edited by Gregory G. Brown Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright © 2011 InTech All chapters are Open Access distributed under the Creative Commons Attribution 3.0 license, which permits to copy, distribute, transmit, and adapt the work in any medium, so long as the original work is properly cited. 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. 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Used under license from Shutterstock.com First published October, 2011 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from orders@intechweb.org Molecular Cloning – Selected Applications in Medicine and Biology, Edited by Gregory G. Brown p. cm. 978-953-307-398-9 free online editions of InTech Books and Journals can be found at www.intechopen.com Contents Preface IX Part 1 Technological Advances 1 Chapter 1 Screening of Bacterial Recombinants: Strategies and Preventing False Positives 3 Sriram Padmanabhan, Sampali Banerjee and Naganath Mandi Chapter 2 Non-Viral Vehicles: Principles, Applications, and Challenges in Gene Delivery 21 Abbas Padeganeh, Mohammad Khalaj-Kondori, Babak Bakhshinejad and Majid Sadeghizadeh Part 2 Cancer and Cell Biology 35 Chapter 3 Subcloning and Expression of Functional Human Cathepsin B and K in E. coli: Characterization and Inhibition by Flavonoids 37 Lisa Wen, Soe Tha, Valerie Sutton, Keegan Steel, Franklin Rahman, Matthew McConnell, Jennifer Chmielowski, Kenneth Liang, Roxana Obregon, Jessica LaFollette, Laura Berryman, Ryan Keefer, Michael Bordowitz, Alice Ye, Jessica Hunter, Jenq-Kuen Huang and Rose M. McConnell Chapter 4 Molecular Cloning and Overexpression of WAP Domain of Anosmin-1 (a-WAP) in Escherichia coli 59 Srinivas Jayanthi, Beatrice Kachel, Jacqueline Morris, Igor Prudovsky and Thallapuranam K. Suresh Kumar Chapter 5 Effects of Two Novel Peptides from Skin of Lithobates Catesbeianus on Tumor Cell Morphology and Proliferation 73 Rui-Li ZHAO, Jun-You HAN, Wen-Yu HAN, Hong-Xuan HE and Ji-Fei MA VI Contents Part 3 Immunology/Hematology 81 Chapter 6 Molecular Cloning of Immunoglobulin Heavy Chain Gene Translocations by Long Distance Inverse PCR 83 Takashi Sonoki Chapter 7 Identification of Molecules Involved in the Vulture Immune Sensing of Pathogens by Molecular Cloning 91 Elena Crespo, José de la Fuente and José M. Pérez de la Lastra Chapter 8 Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor 109 Gloria Esteso, Ángeles Jiménez-Marín, Gema Sanz, Juan José Garrido and Manuel Barbancho Chapter 9 Molecular Cloning, Expression, Purification and Immunological Characterization of Proteins Encoded by Regions of Difference Genes of Mycobacterium tuberculosis 141 Shumaila Nida Muhammad Hanif, Rajaa Al-Attiyah and Abu Salim Mustafa Part 4 Toxicology 159 Chapter 10 Molecular Toxinology – Cloning Toxin Genes for Addressing Functional Analysis and Disclosure Drug Leads 161 Gandhi Rádis-Baptista Chapter 11 Molecular Cloning, Expression, Function, Structure and Immunoreactivities of a Sphingomyelinase D from Loxosceles adelaida, a Brazilian Brown Spider from Karstic Areas 197 Denise V. Tambourgi, Giselle Pidde-Queiroz, Rute M. Gonçalves-de-Andrade, Cinthya K. Okamoto, Tiago J. Sobreir, Paulo S. L. de Oliveira, Mário T. Murakami and Carmen W. van den Berg Part 5 Parasitology 219 Chapter 12 Cloning the Ribokinase of Kinetoplastidae: Leishmania Major 221 Patrick Ogbunude, Joy Ikekpeazu, Joseph Ugonabo, Michael Barrett and Patrick Udeogaranya Chapter 13 Genome Based Vaccines Against Parasites 231 Yasser Shahein and Amira Abouelella Contents VII Chapter 14 Phosphagen Kinase System of the Trematode Paragonimus westermani: Cloning and Expression of a Novel Chemotherapeutic Target 247 Blanca R. Jarilla and Takeshi Agatsuma Part 6 Evolutionary Biology 265 Chapter 15 Molecular Cloning, Expression Pattern, and Phylogenetic Analysis of the Lysyl-tRNA Synthetase Gene from the Chinese Oak Silkworm Antheraea pernyi 267 Yan-Qun Liu and Li Qin Chapter 16 Molecular Cloning and Characterization of Fe-Superoxide Dismutase (Fe-SOD) from the Fern Ceratopteris thalictroides 277 Chen Chen and Quanxi Wang Part 7 Plant Biology 289 Chapter 17 Cloning and Characterization of a Candidate Gene from the Medicinal Plant Catharanthus roseus Through Transient Expression in Mesophyll Protoplasts 291 Patrícia Duarte, Diana Ribeiro, Gisela Henriques, Frédérique Hilliou, Ana Sofia Rocha, Francisco Lima, Isabel Amorim and Mariana Sottomayor Chapter 18 Positional Cloning in Brassica napus: Strategies for Circumventing Genome Complexity in a Polyploid Plant 309 Gregory G. Brown and Lydiane Gaborieau Preface The development of technology in the early 1970s for propagating targeted segments of DNA in bacterial plasmids and viruses, molecular cloning, created a revolution in the biological and biomedical sciences that extends to this day. The contributions in this book provide ample evidence of just how extensive the applications of molecular cloning have become. The chapters of this have been organized largely according to the fields this technology is being applied. Two chapters deal with the recent advances in molecular cloning technology per se. Padmanabhan and colleagues review various methods for cloning in E. coli plasmid vectors, emphasizing the shortcomings of various procedures for identifying clones of interest. Abbas Padeganeh et al. provide an interesting discussion of non-viral systems for gene delivery into mammalian cells, with an emphasis on the relatively new “dendrosome” technology. Several chapters deal with the use of molecular cloning techniques for obtaining and characterizing purified animal proteins involved in cancer and aspects of cell biology. The proteins thus characterized include human cathepsins (Wen et al.), a human WAP-like domain (Jayanthi et al.) and potential antibiotic peptides from amphibian skin secretions (Zhao et al.). Three chapters, those of Sonoki, Crespo et al. and Esteso et al., deal with the applications of molecular cloning methodologies to improving our understanding of immune system, while the chapter by Hanif and colleagues deals with the use of the methodology for the production of antigenic peptides and vaccines. Applications in the area of toxicology are reviewed in the chapter by Radis-Baptista, while more specific application of the technology to the purification and characterization of a toxic enzyme from spider venom is covered in the chapter by Tambourgi et al. The contributions of Ogbunude et al. and Jarilla et al. describe the cloning and expression of potential therapeutic targets for trypanosomal and trematode parasites, respectively, while Shahein et al. describe the use of whole genome sequences as a means of developing anti-parasitic vaccines. Liu et al. and Chen et al. describe applications to phylogenetic questions. Finally, two contributions in the area of plant biology are described. Sottomayor et al. describe how molecular cloning technology X Preface can be used to understand the complicated pathway by which the anti-cancer terpenoid indole alkaloids vineblastine and vincristine are synthesized, while Brown and Gaborieau discuss the application of positional cloning with the complex genomes of polyploid plants. There is, in short, something for a wide variety of readers in a truly diverse set of scientific fields. Gregory G. Brown McGill University, Montreal QC, Canada [...]... genes 2 Molecular cloning A recombinant DNA comprises of two entities namely a vector and the gene of interest (GOI) The process of joining vector and any GOI is by making a phosphodiester bond by a 4 Molecular Cloning – Selected Applications in Medicine and Biology process called ligation The ligation reaction is facilitated with the help of T4 DNA ligase in the presence of ATP If a vector and any... zebrafish and in mammalian cells Inouye et al., (1997) have described a bacterial cloning vector with mutated Aequorea GFP protein as an indicator for screening recombinant plasmids The pGREENscript A when expressed in E coli produced colonies showing yellow color in day 10 Molecular Cloning – Selected Applications in Medicine and Biology light and strong green fluorescence under long-UV Inserted foreign... recombinants with rightly oriented gene of interest and to minimize vector background (non-recombinants) Screening for recombinants is one of the most crucial and time-consuming steps in molecular cloning and several approaches available for this purpose include colony PCR screening, blue white screening, screening of recombinants, which have the gene of interest in the MCS region of the cloning vehicle,... single method for achieving fool-proof recombinant clone Conventionally the screening methods employed routinely in academia and industry, for bacterial recombinants include colony hybridization, PCR and plasmid preparations While all the methods involve cloning the gene of interest in a cloning vehicle and then reintroduction of the recombinant clone into another host cell for expression of the interest... 1844-1846 16 Molecular Cloning – Selected Applications in Medicine and Biology Ausubel, F M., R Brent, R E Kingston, D D Moore, J G Seidman, & K Struhl 1988 Current protocols in molecular biology Wiley Interscience, John Wiley and Sons, NY Cambridge, MA Banerjee S, Deshpande AA, Mandi N & Padmanabhan S (2009) A novel cytokine derived fusion tag for over-expression of heterologous proteins in E coli International... transfection (Lewin, 2007; Singleton & Sainsbury, 1995) has been studied widely and various techniques have been developed for it, each possessing its own advantages and shortcomings Generally speaking, gene delivery techniques are classified into viral and 22 Molecular Cloning – Selected Applications in Medicine and Biology non-viral categories Non-viral systems include physical, chemical and biological... containing recombinant plasmids versus those maintaining self-annealed or uncut vector This cloning vector has improved the efficiency of recombinant DNA experiments in gram-positive bacteria Cloning inserts into the multiple cloning region of the pGEM®-Z Vectors disrupts the alpha-peptide coding sequences, and thus inactivates the beta-galactosidase enzyme resulting in white colonies Recombinant plasmids... the vector and insert 2.1.3 Transformation Following ligation, the ligation product (recombinant plasmid) is transformed into bacteria for propagation The transformed bacteria are then plated on selective agar to select for bacteria that have the plasmid of interest Individual colonies are picked up and tested for 6 Molecular Cloning – Selected Applications in Medicine and Biology the desired insert The... systems for the enforcement -cloning method Gene , Vol.137, pp 211-216 18 Molecular Cloning – Selected Applications in Medicine and Biology Horn D (2005) Directional enrichment of directly cloned PCR products BioTechniques, Vol 39, No 1, pp 4 0–4 6 Inouye S, Ogawa H, Yasuda K, Umesono K & Tsuji, F (1997) A bacterial cloning vector using mutated Aequorea green fluorescent protein as an indicator Gene Vol 189,... product resulting in growth of colonies 12 Molecular Cloning – Selected Applications in Medicine and Biology Positive selection has been a powerful method of screening insert containing transformants Here the toxic property of the molecule to the host cells is utilized for recombinant selection The DNA sequence coding for the toxic product is directly cloned under the promoter elements recognized by the . MOLECULAR CLONING – SELECTED APPLICATIONS IN MEDICINE AND BIOLOGY Edited by Gregory G. Brown Molecular Cloning – Selected Applications. vector and any GOI is by making a phosphodiester bond by a Molecular Cloning – Selected Applications in Medicine and Biology 4 process called ligation.