Genetic Engineering, Volume 24 contains discussions of contemporary and relevant topics in genetics, including: -Gene silencing: principles and applications, -Integrins and the myocardium, -Plant virus gene vectors: biotechnology and applications in agriculture and medicine, -Novel approaches to controlling transcription, -Use of DNA polymorphisms in genetic mapping, -Application of FLP/FRT site-specific DNA recombination system in plants. This principles and methods approach to genetics and genetic engineering is essential reading for all academics, bench scientists, and industry professionals wishing to take advantage of the latest and greatest in this continuously emerging field.

Under the expert guidance of Bernd H.A. Rehm, the authors of this book provide a survey of the most striking and successful approaches for the production of biogenic nanodevices considering not only living organisms as manufacturer but also in vitro processes that utilise the self-assembly of isolated biomolecules.

th We compiled this volume mostly from presentations at the 6 International Plant Cold Hardiness Seminar (PCHS) after consulting with Professor Tony H. H. Chen, Oregon State University, USA, Professor Pekka Heino, University of Helsinki, Finland, th and Dr. Gareth J. Warren, University of London, Surrey, UK. The 6 International PCHS was held at the Unitas Congress Center, Helsinki, Finland from July 1-5, 2001. There were 110 registered scientists at the serttinar representing 20 countries: Australia, Belgium, Canada, Chile, the Czech Republic, Denmark, Estonia, Finland, Gennany, Hungary, Iceland, Italy, Japan, Norway, Poland, Spain, Sweden, Taiwan, United Kingdom, and United States of America. The infonnation compiled represents the state of the art of research in phmt cold hardiness in tenns of gene regulation, gene expression, signal transduction, the physiology of cold hardiness and, ultimately, the genetic engineering for cold tolerant plants. The International PCHS was initiated in 1977 at the University of Minnesota, St. Paul, Minnesota. It has been traditionally held at 5-year intervals at various locations. th Because of the rapid advances of research in plant cold hardiness, attendees at the 6 meeting unanimously adopted a resolution to hold the seminar in 3-year intervals instead of 5 in the future. Consequently, the next seminar will be held in 2004 in Sapporo, Japan, and Professor Seizo Fujikawa from Hokkaido University will serve as the host.

Genetic Engineering, Volume 25 contains discussions of contemporary and relevant topics in genetics, including: - Genotyping by Mass Spectrometry; - Development of Targeted Viral Vectors for Cardiovascular Gene Therapy; - Practical Applications of Rolling Circle Amplification of DNA Templates; - Bacterial ION Channels; - Applications of Plant Antiviral Proteins; - The Bacterial Scaffoldin: Structure, Function and Potential Applications in the Nanosciences. This principles and methods approach to genetics and genetic engineering is essential reading for all academics, bench scientists, and industry professionals wishing to take advantage of the latest and greatest in this continuously emerging field.

Due to the possibility that petroleum supplies will be exhausted in the next decades to come, more and more attention has been paid to the production of bacterial pl- tics including polyhydroxyalkanoates (PHA), polylactic acid (PLA), poly(butylene succinate) (PBS), biopolyethylene (PE), poly(trimethylene terephthalate) (PTT), and poly(p-phenylene) (PPP). These are well-studied polymers containing at least one monomer synthesized via bacterial transformation. Among them, PHA, PLA and PBS are well known for their biodegradability, whereas PE, PTT and PPP are probably less biodegradable or are less studied in terms of their biodegradability. Over the past years, their properties and appli- tions have been studied in detail and products have been developed. Physical and chemical modifications to reduce their cost or to improve their properties have been conducted. PHA is the only biopolyester family completely synthesized by biological means. They have been investigated by microbiologists, molecular biologists, b- chemists, chemical engineers, chemists, polymer experts, and medical researchers for many years. PHA applications as bioplastics, fine chemicals, implant biomate- als, medicines, and biofuels have been developed. Companies have been est- lished for or involved in PHA related R&D as well as large scale production. It has become clear that PHA and its related technologies form an industrial value chain in fermentation, materials, feeds, and energy to medical fields.

Experience in laboratory experiments in molecular biology is important to students, researchers and practioners in the biolo gical and medical sciences. This book is designed to introduce the reader to basic methods used in the isolation, cloning and analysis of genetic material. The text includes theory and fundamental methods in suffi cient experimental detail which have been successfully used in our studies of viral genome and eukaryotic gene regulation. Spe cific protocols together with troubleshooting tips are given for RT-PCR amplification, gene cloning, hybridization analysis and sequencing of nucleic acids, PCR-based site-specific mutagene sis, analysis of protein DNA-specific interaction, cell-free protein synthesis and product electrophoretic and immunological analy sis. The methods chosen have been approved in several interna tional Molecular Biology Courses and without doubt can be used as a manual in basic molecular studies of DNA and RNA. The editor is grateful to all the contributors for their coopera tion and would like to express his thanks to the publishers, espe cially Dr. J. Lindenborn from Springer-Verlag, for their coopera tion, patience and assistance during the preparation of this book. Riga, Latvia V. BERZINS Contents Chapter 1 cDNA Synthesis and Cloning ... . . . . . . . . . . . . . . . . . . . . . . 1 JURIS STEINBERGS AND ALEKSANDER TSIMANIS Chapter 2 DNA Sequencing. . . . . . . . . . . . . . . . . . . . 22 . . . . . . . . . . . . . . . . ERIKS JANKEVICS Chapter 3 RNA In Vitro Synthesis by Phage T7 DNA-Dependent RNA Polymerase. . . . . . . . . . . . . . . . . . . . 43 . . . . . . . . . . . . . . . . ELITA AVOTA AND NORMUNDS LICIS Chapter 4 PCR-Based Site-Specific Mutagenesis. . . . . . . . . . . 53 . . . . . . . . JANIS KLOVINS AND VALDIS BERZINS Chapter 5 Analysis of Specific Protein-DNA Interactions. . . . . . . . . 68 . ."

Like many genetic engineers, I have recently been receiving the atten tion of various venture capital companies, international drug houses and Members of Parliament. I will not discuss which of these approaches are most welcome, but it did cause me to consider the speed of advance in genetic engineering, and the implications of this rapid growth. There were few who anticipated it - only five years ago, most scientists thought applications would come at the end of the century, yet we see products such as insulin and interferon already available for clinical testing. In Europe in general and Britain in particular, this explosive growth in our own field has coincided with a general industrial depression and a marked reduction in funding for biomedical research. The brain drain from Britain is a serious matter, for we are losing the best of our younger scientists, on whom we would rely to train the next generation of molecular biologists. These volumes have come from British labs (mostly because I happen to be based in London, and my contacts and friends are here), and I feel that the quality of the con tributions also shows that our current research is of a high standard.

This volume is the first of a series concerning a new tech nology which is revolutionizing the study of biology, perhaps as profoundly as the discovery of the gene. As pointed out in the introductory chapter, we look forward to the future impact of the technology, but cannot see where it might take us. The purpose of these volumes is to follow closely the explosion of new tech niques and information that is occurring as a result of the newly acquired ability to make particular kinds of precise cuts in DNA molecules. Thus we are particularly committed to rapid publication. Jane K. Setlow Alexander Hollaender v INTRODUCTION AND HISTORICAL BACKGROUND 1 Maxine F. Singer CLONING OF DOUBLE-STRANDED cDNA . . 15 Argiris Efstratiadis and Lydia Vi11a-Komaroff GENE ENRICHMENT . . . . . . . 37 M. H. Edgell, S. Weaver, Nancy Haigwood and C. A. Hutchison III 51 TRANSFORMATION OF MAMMALIAN CELLS . . . . . M. Wig1er, A. Pe11icer, R. Axel and S. Silverstein CONSTRUCTED MUTANTS OF SIMIAN VIRUS 40 73 D. Short1e, J. Pipas, Sondra Lazarowitz, D. DiMaio and D. Nathans STRUCTURE OF CLONED GENES FROM XENOPUS: A REVIEW 93 R. H. Reeder TRANSFORMATION OF YEAST 117 Christine lIgen, P. J. Farabaugh, A. Hinnen, Jean M. Walsh and G. R. Fink THE USE OF SITE-DIRECTED MUTAGENESIS IN REVERSED GENETICS 133 C. Weissmann, S. Nagata, T. Taniguchi, H. Weber and F. Meyer AGROBACTERIUM TUMOR INDUCING PLASMIDS: POTENTIAL VECTORS FOR THE GENETIC ENGINEERING OF PLANTS . 151 P. J. J. Hooykaas, R. A. Schi1peroort and A."

Plant biotechnology offers important opportunities for agriculture, horticul ture, and the food industry by generating new transgenic crop varieties with altered properties. This is likely to change farming practices, improve the quality of fresh and processed plant products, and reduce the impact of food production on the environment. The purpose of this series is to review the basic science that underpins plant biotechnology and to show how this knowledge is being used in directed plant breeding. It is intended for those involved in fundamental and applied research on transgenic plants in the academic and commercial sectors. The first volume deals with plant genes, how they work, and their transfer from one organism to another. Authors discuss the production and evaluation of the first generation of transgenic crops resistant to insects, viruses and herbicides, and consider aspects of gene regulation and targeting of their protein products to the correct cellular location. All the contributors are actively engaged in research in plant biotechnology and several are concerned directly with its commercial applications. Their chapters highlight the importance of a fundamental understanding of plant physiology, biochemistry, and cell and molecular biology for the successful genetic engineering of plants. This interdisciplinary approach, which focuses research from traditionally separate areas, is the key to further developments which are considered in subsequent volumes. Don Grierson Contributors Alan B. Bennett Mann Laboratory, Department of Vegetable Crops, University of California, Davis, CA 95616 John W. s."

Gene therapy was conceived during the early and mid part of the 20th century. At first, it was considered a revolutionary biomedical procedure, which could potentially cure any disease for which the molecular bases were understood. Since then, gene therapy has gone through many stages and has evolved from a nearly unrealistic perspective to a real life application. Clinical efficacy in humans was demonstrated at the beginning of this century after its successful application in small-scale clinical trials to cure severe immunodeficiency in children. However, their successes were overshadowed some time later by the occurrence of vector-related leukaemia in a number of treated children. It is in this context that lentiviral vectors have appeared, with improved efficiency and, possibly, increased biosafety. Very recently, the first clinical trials with lentivectors have been carried out with some success.

This Brief firstly defines gene therapy, and places lentivectors within this fascinating therapeutic strategy. Then follows a comprehensive description of the development of retroviral and lentiviral vectors and how to specifically target distinct cell types and tissues. The authors also discuss the application of lentivector gene therapy for the treatment of cancer and autoimmune diseases, ending with the application of lentivectors in human gene therapy clinical trials.

Mammalian cell lines command an effective monopoly for the production of therapeutic proteins that require post-translational modifications. This unique advantage outweighs the costs associated with mammalian cell culture, which are far grater in terms of development time and manufacturing when compared to microbial culture. The development of cell lines has undergone several advances over the years, essentially to meet the requirement to cut the time and costs associated with using such a complex hosts as production platforms.

This book provides a comprehensive guide to the methodology involved in the development of cell lines and the cell engineering approach that can be employed to enhance productivity, improve cell function, glycosylation and secretion and control apoptosis. It presents an overall picture of the current topics central to expression engineering including such topics as epigenetics and the use of technologies to overcome positional dependent inactivation, the use of promoter and enhancer sequences for expression of various transgenes, site directed engineering of defined chromosomal sites, and examination of the role of eukaryotic nucleus as the controller of expression of genes that are introduced for production of a desired product. It includes a review of selection methods for high producers and an application developed by a major biopharmaceutical industry to expedite the cell line development process. The potential of cell engineering approch to enhance cell lines through the manipulation of single genes that play important roles in key metabolic and regulatory pathways is also explored throughout.

Over 20 years have elapsed since publication of the seminal two volume series entitled Q Fever: The Biology of Coxiella burnetii (edited by J. C. Williams and H. A. Thompson) and Q fever: The Disease (edited by T. J. Marrie) that described the current state of Coxiella burnetii research. The ensuing years have brought the post-genomic era and accompanying technologies that have catalyzed major advances in the field, including milestones discoveries of genetic transformation and host cell-free growth of this former obligate intracellular bacterium. Understanding how the bacterium resists the degradative functions of vacuole, and the host cell functions coopted for successful parasitism, are central to understanding Q fever pathogenesis. Recent achievements in glycomics and proteomics are guiding development of enhanced detection schemes for the bacterium in addition to shedding light on the host immune response to the pathogen. The book covers the current state-of-the-art knowledge in the selected fields of C. burnetii/Q fever research. Coxiella has matured from a niche organism, investigated by a handful of laboratories worldwide, to a model system to study macrophage parasitism, developmental biology, host-pathogen interactions, and immune evasion/modulation.

Plant biotechnology offers important opportunities for agriculture, horticul ture, and the food industry by generating new transgenic crop varieties with altered properties. This is likely to change farming practices, improve the quality of fresh and processed plant products, and reduce the impact of food production on the environment. The purpose of this series is to review the basic science that underpins plant biotechnology and to show how this knowledge is being used in directed plant breeding. It is intended for those involved in fundamental and applied research on transgenic plants in the academic and commercial sectors. The first volume deals with plant genes, how they work, and their transfer from one organism to another. Authors discuss the production and evaluation of the first generation of transgenic crops resistant to insects, viruses and herbicides, and consider aspects of gene regulation and targeting of their protein products to the correct cellular location. All the contributors are actively engaged in research in plant biotechnology and several are concerned directly with its commercial applications. Their chapters highlight the importance of a fundamental understanding of plant physiology, biochemistry, and cell and molecular biology for the successful genetic engineering of plants. This interdisciplinary approach, which focuses research from traditionally separate areas, is the key to further developments which are considered in subsequent volumes. Don Grierson Contributors Alan B. Bennett Mann Laboratory, Department of Vegetable Crops, University of California, Davis, CA 95616 John W. s."

This book, published by Springer since 1979, presents state-of-the-art discussions in modern genetics and genetic engineering. This focus affirms a commitment to publish important reviews of the broadest interest to geneticists and their colleagues in affiliated disciplines. Recent volumes have covered gene therapy research, genetic mapping, plant science and technology, transport protein biochemistry, and viral vectors in gene therapy, among other topics.

The present volume presents essential information on advancements in oilseed production, processing and utilization. Advances in the technology of seed processing to produce oil and oil quality for edible and industrial applications are well presented, followed by hybrid technology, biotechnology, oil technology and meal quality for animal nutrition. The following areas are also covered: the potential for oil in developing biodiesel markets, fatty acid long chains and their derivative, pollination management, and safety of pollinators from harmful effects of pesticides. This volume also includes an economic assessment of oilseed integrated pest management (IPM) programs in different regions of the world. Dr. Surinder Kumar Gupta is Professor/Chief Scientist (Oilseeds) Plant Breeding & Genetics and Nodal officer in School of Biotechnology, S K University of Agricultural Sciences & Technology, Faculty of Agriculture, Chatha, Jammu-India. He holds a distinguished academic and service record and has been devoted primarily to research on oilseed Brassicas for nearly two decades. He has written two books on plant breeding and edited three volumes, one on 'Recent Advances in Oilseed Brassicas', Kalyani Publishers, New Delhi, India, second on 'Rapeseed Breeding-Advances in Botanical Research', Vol. 45, Academic Press, Elsevier Publishers and third on Biology and Breeding of crucifers, CRC Publishers, Taylor and Francis Group.

Systems Metabolic Engineering is changing the way microbial cell factories are designed and optimized for industrial production.
Integrating systems biology and biotechnology with new concepts from synthetic biology enables the global analysis and engineering of microorganisms and bioprocesses at super efficiency and versatility otherwise not accessible. Without doubt, systems metabolic engineering is a major driver towards bio-based production of chemicals, materials and fuels from renewables and thus one of the core technologies of global green growth. In this book, Christoph Wittmann and Sang-Yup Lee have assembled the world leaders on systems metabolic engineering and cover the full story from genomes and networks via discovery and design to industrial implementation practises.
This book is a comprehensive resource for students and researchers from academia and industry interested in systems metabolic engineering. It provides us with the fundaments to targeted engineering of microbial cells for sustainable bio-production and stimulates those who are interested to enter this exiting research field.

Plant molecular biology came to the fore in the early 1980s and there has been tremendous growth in the subject since then. The study of plant genes and genomes and the development of techniques for the incorporation of novel or modified genes into plants eventually led to the commercialisation of genetically modified (GM) crops in the mid-1990s. This was seen as the start of a biotechnological revolution in plant breeding. However, plant biotechnology has become one of the hottest debates of the age and, in Europe at least, one of the greatest challenges that plant scientists have ever faced.This book covers the history and development of the science and techniques that underpin plant biotechnology. It describes the GM crops that are or have been grown commercially around the world, including failures as well as successes, and the new varieties that are being developed. The safety record of GM crops is reviewed together with the legislation that has been adopted to cover their use. The book also deals with the concerns of consumers, the GM crop debate and the prospects for the technology. In the second edition, sections on current GM crops and future developments in plant biotechnology have been greatly expanded, while those on techniques, legislation and the GM crop debate have also been updated.The book is a concise, comprehensive and readable study that is accessible to a general readership with a scientific background but also provides useful information for the specialist.

Induced pluripotent stem (iPS) cell technology has paved new ways for disease modelling and drug discovery. Disease modelling with the differentiated neuronal cells from patient-specific iPS cells partially recapitulated the phenotypes of spinal muscular atrophy, familial dysautonomia and Rett syndrome. In this book, the authors present current research in the study of induced stem cells, including the cryopreservation of pluipotent stem cells; tissue engineering approaches using bioactive ceramics towards bone regeneration; induced pluripotent stem cell-derived hepatocytes as an alternative to human adult hepatocytes; iPS technology for studying neurodegenerative diseases and iPS from cord blood cells.

Classical methods for microbial strain engineering, used to improve the production of bioproducts, have serious drawbacks and have been found to be unsuitable for complex strain development applications. In Strain Engineering: Methods and Protocols, powerful new genetic engineering-based strain engineering methods are presented for rational modification of a variety of model organisms. These methods are particularly powerful when utilized to manipulate microbes for which sequenced and annotated genomes are available. Collectively, these methods systematically introduce genome alterations in a precise manner, allowing the creation of novel strains carrying only desired genome alterations. In the first section, E. coli-based bacterial strain engineering strategies are reviewed, while the second section presents analogous microbial engineering strategies for eukaryotic cells using the yeast Saccharomyces cerevisiae as a model. The third section covers examples of the proliferative adaptations of these base technologies to strain engineer industrially important prokaryotic or eukaryotic microbial systems. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Strain Engineering: Methods and Protocols serves as an ideal guide to scientists in academia, pharmaceutical science, and biotechnology who perform microbial strain engineering.

With the rapid proliferation of RNAi applications in basic and clinical sciences, the challenge has now become understanding how components of RNAi machinery function together in a regulated manner. Argonaute proteins are the central effectors of RNAi and are highly conserved among eukaryotes and some archaebacteria. These RNA-binding proteins use small guide RNAs to silence expression of genes at the mRNA and DNA levels. In Argonaute Proteins: Methods and Protocols, expert researchers in this burgeoning field provide detailed, up-to-date methods to study Argonaute protein functions and interactions in a wide variety of cell types ranging from yeast to mammalian systems, as well as in vitro. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Practical and authoritative, Argonaute Proteins: Methods and Protocols serves as a vital reference for both experienced and novice scientists approaching the vast complexities of RNAi research.

The rapid progression of genetics and molecular biology has turned chromosomal engineering from science fiction to reality, with the successful production of transgenic animals with engineered chromosomes and chromosomes developed for pharmaceutical protein production which are now ready for the medical industry. Mammalian Chromosome Engineering: Methods and Protocols provides the reader with up-to date information on this rapidly evolving field and strives to take the reader into the exciting realm of chromosomal engineering from the basic principles to the practical applications of these new technologies. The five overview and ten protocol chapters cover the engineering of chromosomes with extrachromosomal vectors and transposon systems, the manipulation of naturally occurred minichromosomes, the generation and engineering of synthetic artificial chromosomes, and the induced de novo platform artificial chromosome system. Written in the highly successful Methods in Molecular Biology (TM) series format, protocols chapters contain brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Mammalian Chromosome Engineering: Methods and Protocols serves as a bench-side resource for current protocols and aims to help scientists to explore the many prospects for future research and vital applications.