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 . ."

In the past few decades there has been incredible growth in "bionano"-related research, which has been accompanied by numerous publications in this field. Although various compilations address topics related to deoxyribonucleic acid (DNA) and protein, there are few books that focus on determining the structure of ribonucleic acid (RNA) and using RNA as building blocks to construct nanoarchitectures for biomedical and healthcare applications.

RNA Nanotechnology is a comprehensive volume that details both the traditional approaches and the latest developments in the field of RNA-related technology. This book targets a wide audience: a broad introduction provides a solid academic background for students, researchers, and scientists who are unfamiliar with the subject, while the in-depth descriptions and discussions are useful for advanced professionals.

The book opens with reviews on the basic aspects of RNA biology, computational approaches for predicting RNA structures, and traditional and emerging experimental approaches for probing RNA structures. This section is followed by explorations of the latest research and discoveries in RNA nanotechnology, including the design and construction of RNA-based nanostructures. The final segment of the book includes descriptions and discussions of the potential biological and therapeutic applications of small RNA molecules, such as small/short interfering RNAs (siRNAs), microRNAs (miRNAs), RNA aptamers, and ribozymes.

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.

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.

Ethanol as an alternative fuel is receiving a lot of attention because it addresses concerns related to dwindling oil supplies, energy independence, and climate change. The majority of the ethanol in the US is produced from corn starch. With the US Department of Energy's target that 30% of the fuel in the US is produced from renewable resources by 2030, the anticipated demand for corn starch will quickly exceed the current production of corn. This, plus the concern that less grain will become available for food and feed purposes, necessitates the use of other feedstocks for the production of ethanol. For the very same reasons, there is increasing research activity and growing interest in many other biomass crops. Genetic Improvement of Bio-Energy Crops focuses on the production of ethanol from lignocellulosic biomass, which includes corn stover, biomass from dedicated annual and perennial energy crops, and trees as well as a number of important biomass crops. The biomass is typically pretreated through thermochemical processing to make it more amenable to hydrolysis with cellulolytic enzymes. The enzymatic hydrolysis yields monomeric sugars that can be fermented to ethanol by micro-organisms. While much emphasis has been placed on the optimization of thermo-chemical pretreatment processes, production of more efficient hydrolytic enzymes, and the development of robust microbial strains, relatively little effort has been dedicated to the improvement of the biomass itself.

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.

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."

These Proceedings evolved from the OECD Co-operative Research Programme workshop on "Potential ecological impact of transgenic plants expressing viral sequencies," held at the Agricultural Biotechnology Center in Godollo, Hungary on 24-26 April 1997. The OECD Co-operative Research Programme At the Directorate for Agrieulture of the Organisation for Economie Co-operation and Development (OECD) a co-operative research programme for "Biological Resource Management" has existed since 1990. It foeuses on work in four specific topie areas, one of whieh is "Ecology and utilisation of new organisms" (Theme 3). The activities promoted by this programme are post-doctoral fellowships (announced annually), and the organisation of expert workshops (1-2 workshops per Theme per year). The 26 OECD member countries participating in this programme are: Australia, Austria, Belgium, Canada, Czech Republic, Denmark, Finland, France, Germany, Greece, Hungary, Ireland, ltaly, Korea, Japan, the NetherIands, New Zealand, Norway, Poland, Portugal, Spain, Sweden, SwitzerIand, Turkey, the UK, and the USA."

SpringerBriefs in Biotech Patents presents timely reports on intellectual properties (IP) issues and patent aspects in the field of biotechnology. In this volume the limits of patentability are addressed, a question that is often raised when it comes to biotechnological inventions: The first section addresses current issues in the patentability of plants produced by essentially biological processes including the controversy between farmer's privilege and patent exhaustion with respect to seeds in the US. The second section examines the patentability of human embryonic stem cells in Europe and the US, also considering alternative technologies with respect to their practicability and patentability. The third section focuses on the patentability of genes and nucleic acids, especially the issue of patenting of encoding genes and nucleic acids.

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.

PCR s simplicity as a molecular technique is, in some ways, responsible for the huge amount of innovation that surrounds it, as researchers continually think of new ways to tweak, adapt, and re-formulate concepts and applications. PCR Technology Current Innovations, Third Edition is a collection of novel methods, insights, and points of view that provides a critical and timely reference point for anyone wishing to use this technology.

Topics in this forward-thinking volume include:

• The purification and handling of PCR templates
• The effect of the manufacture and purification of the oligonucleotide on PCR behavior
• Optimum buffer composition
• Probe options
• The design and optimization of qPCR assays
• Issues surrounding the development and refinement of instrumentation
• Effective controls to protect against uncertainties due to reaction variability

Covering all aspects of PCR and real-time PCR, the book contains detailed protocols that make it suitable as both a reference and an instruction manual. Each chapter presents detailed guidelines as well as helpful hints and tips supplied by authors who are recognized experts in their fields. In addition to descriptions of current technology and best practices, the book also provides information about new developments in the PCR arena.

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."

Changing environmental conditions substantially affect genetic variation and its dynamics in forest ecosystems and various systems of plantations. In response to these challenges, the present book focuses on the response to stress in terms of case studies which address physiological and genetic characters as well as various metric traits. Furthermore a choice of studies is presented which refers to diversity and geographic variation of various species and site conditions, respectively. In addition, genetic resources are characterised and a variety of studies is compiled which address reproduction and migration as well as management aspects. Finally, a set of studies is presented which focus on forest tree breeding with respect to uncertain climatic futures.

"Leonardo's Choice: Genetic Technologies and Animals" is an edited collection of twelve essays and one dialogue focusing on the profound affect the use of animals in biotechnology is having on both humans and other species. Communicating crucial understandings of the integrated nature of the human and non-human world, these essays, unlike the majority of discussions of biotechnology, take seriously the impact of these technologies on animals themselves. This collection's central questions revolve around the disassociation Western ideas of creative freedom have from the impacts those ideas and practices have on the non-human world.

"This transdisciplinary collection includes perspectives from the disciplines of philosophy, cultural theory, art and literary theory, history and theory of science, environmental studies, law, landscape architecture, history, and geography. Included authors span three continents and four countries."

"Included essays contribute significantly to a growing scholarship surrounding "the question of the animal" emanating from philosophical, cultural and activist discourses. Its authors are at the forefront of the growing number of theorists and practitioners across the disciplines concerned with the impact of new technologies on the more-than-human world."

Genome Mapping and Molecular Breeding in Plants presents the current status of the elucidation and improvement of plant genomes of economic interest. The focus is on genetic and physical mapping, positioning, cloning, monitoring of desirable genes by molecular breeding and the most recent advances in genomics. The series comprises seven volumes: Cereals and Millets; Oilseeds; Pulses, Sugar and Tuber Crops; Fruits and Nuts; Vegetables; Technical Crops; and Forest Trees.

Technical Crops includes plants of great agricultural importance. One chapter is devoted to cotton, the most important fiber crop on which significant progress in molecular genetic research has been made. Reviews on oil palm, coffee, tea, cocoa and rubber describe traditional breeding and preliminary molecular results. Chapters on forage crops, ornamentals, and medicinal and aromatic plants each cover a large number of crops and may serve as road maps for further molecular research.

Recent advances in plant cell and molecular biology have opened new avenues for the improvement of crop plants in the genus "Brassica" - oilseeds and vegetables of worldwide economic importance. This volume reviews advances in various areas of "Brassica" biotechnology. It covers the use of rapid-cycle brassicas, tissue culture and gene transfer, molecular genetics, biotic and abiotic stress resistance, and molecular farming. Contributors are world-leading international "Brassica" researchers. The volume is an invaluable reference for plant breeders, researchers and graduate students in the fields of plant biotechnology, agronomy, horticulture, genetics, and cell and molecular biology.

This volume collects new information on the genomics of saprophytic soil Pseudomonas, as well as functions related to genomic islands. It explores life styles in different settings and sheds further insights on the wide metabolic potential of this microbe for the removal of pollutants and production of added-value products. This volume also explores how Pseudomonas responds and reacts to environmental signals, including detection of cell density.

This book covers trends in modern biotechnology. All aspects of this interdisciplinary technology, where knowledge, methods and expertise are required from chemistry, biochemistry, microbiology, genetics, chemical engineering and computer science, are treated. More information as well as the electronic version is available at springer.com.

Louis-Marie Houdebine and Jianglin Fan The study of biological functions of proteins and their possible roles in the pathogenesis of human diseases requires more and more relevant animal m- els. Although mice including genetically modified mice offer many possibilities, other non-murine species are absolutely required in some circumstances. Rabbit is one of these species, which has been widely used in biomedical studies. This animal is genetically and physiologically closer to humans including cardiov- cular system and metabolism characteristics. Rabbit is thus more appropriate than mice to study some diseases such as atherosclerosis and lipid metabolism. Because of its larger size, surgery manipulation, bleeding, and turn-over studies are much easier performed in rabbits than in mice. Furthermore, transgenic rabbits can be produced using microinjection and other methods such as lentiviral v- tors. Cloning in rabbits has been proved possible, even though still laborious and time-consuming. Hopefully, functional rabbit ES cell lines will be available in the coming years. Gene deletion or knock-out in rabbits will then become possible.

Molecular Methods of Plant Analysis Concept of the Series The powerful recombinant DNA technology and related developments have had an enormous impact on molecular biology. Any treatment of plant analy sis must make use of these new methods. Developments have been so fast and the methods so powerful that the editors of Modern Methods of Plant Analy sis have now decided to rename the series Molecular Methods of Plant Analy sis. This will not change the general aims of the series, but best describes the thrust and content of the series as we go forward into the new millennium. This does not me an that all chapters apriori deal only with the methods of molecular biology, but rather that these methods are to be found in many chapters together with the more traditional methods of analysis which have seen re cent advances. The numbering of the volumes of the series therefore continues on from 20, which is the most recently published volume under the title Modern Methods of Plant Analysis."