Over the past decade, our laboratory and others have been concerned with molecular archaeological studies aimed at revealing the origins and evolutionary histories of permeases (1). These studies have revealed that several different families, defined on the basis of sequence similarities, arose independently of each other, at different times in evolutionary history, following different routes. When complete microbial genomes first became available for analysis, we adapted p- existing software and designed new programs that allowed us quickly to identify probable transmembrane proteins, estimate their topologies and determine the likelihood that they function in transport (2). This work allowed us to expand previously-recognized families and to identify dozens of new families. All of this work then led us to attempt to design a rational but comprehensive classification system that would be applicable to the complete complement of transport systems found in all living organisms (3). The classification system that we have devised is based primarily on mode of transport and energy coupling mechanism, secondarily on molecular phylogeny, and lastly on the substrate specificities of the individual permeases (4).

Regenerative medicine, encompassing stem cells and tissue engineering, has attracted huge interest within commercial, clinical and government circles, and promises to change medicine itself. This book provides the first detailed examination and critical assessment of the field to be made by social science.

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."

Designed as an introductory text the authors cover all core strategies in the application of modern recombinant DNA technology. The first chapters directly address the applications of polymerase chain reaction to a variety of problems in DNA cloning that are, or have been, extremely challenging using more traditional approaches and technologies. These include cDNA cloning and transcript mapping, mutagenesis as well as the cloning of very long transcripts and protocols using limiting amounts of total RNA. Further chapters describe approaches to subtractive cloning technologies as well as novel specialized expression cloning and library screening strategies. The handbook contains detailed step-by-step protocols and extensive hands-on advice.

Aspects of genetic engineering research emphasized in this volume are applications to plants (crop plants and grass, both important for human needs) and new methodologies, such as Tar cloning, which make it much easier to isolate specific regions from complex genomes. Another subject discussed is linear DNA replication of prokaryotes.

J. Warren Evans Department of Animal Science Texas A&M University College Station, Texas 77843 In the near future, improvement of domestic animals for the production of food and fiber is poised to undergo a revolution by the utilization of recent breakthroughs and advances in molecular genetics, embryo manipula tions, and gene transfer systems. Utilization of these techniques will have a wide impact on animal agriculture by improvement of production effi ciency via manipulation and control of many physiological systems. The end result will be to decrease production costs, increase food production and quality, and lower food costs. Health and well being of domestic and other animals will be improved as a result of new methods of disease diagnosis, vaccine production, and disease prevention practices. Genetic engineering also offers the possibility of utilizing animals for the development of pharmaceutical products to benefit society. Research progress will be en hanced via manipulation of the gene pool. The objectives of this Conference were to discuss the current status of animal bioengineering and to realistically assess the potential applica tions of current and future genetic technologies for the production of food and fiber to meet the needs of our hungry world, and to provide animal sci entists who may wish to utilize bioengineering in current or future re search programs with current background information regarding concepts, ap plications, and methodologies."

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.

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.

It follows naturally from the widely accepted Darwinian dictum that failures of populations or of species to adapt and to evolve under changing environments will result in their extinction. Population geneti cists have proclaimed a centerstage role in developing conservation biology theory and applications. However, we must critically reexamine what we know and how we can make rational contributions. We ask: Is genetic variation really important for the persistence of species? Has any species become extinct because it ran out of genetic variation or because of inbreeding depression? Are demographic and environmental stochas ticity by far more important for the fate of a population or species than genetic stochasticity (genetic drift and inbreeding)? Is there more to genetics than being a tool for assessing reproductive units and migration rates? Does conventional wisdom on inbreeding and "magic numbers" or rules of thumb on critical effective population sizes (MVP estimators) reflect any useful guidelines in conservation biology? What messages or guidelines from genetics can we reliably provide to those that work with conservation in practice? Is empirical work on numerous threatened habitats and taxa gathering population genetic information that we can use to test these guidelines? These and other questions were raised in the invitation to a symposium on conservation genetics held in May 1993 in pleasant surroundings at an old manor house in southern Jutland, Denmark."

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.

The newest installment in this superb series presents descriptions of the latest DNA recombinants molecule technology. The text combines reports on basic research in genetics with discussions of specific new industrial applications (as well as refinements of older ones) that are likely to prove highly profitable in the years to come.

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.

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.

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 book demonstrates that American agricultural development was far more dynamic than generally portrayed. In the two centuries before World War II, a stream of biological innovations revolutionized the crop and livestock sectors, increasing both land and labor productivity. Biological innovations were essential for the movement of agriculture onto new lands with more extreme climates, for maintaining production in the face of evolving threats from pests, and for the creation of the modern livestock sector. These innovations established the foundation for the subsequent Green and Genetic Revolutions. The book challenges the misconceptions that, before the advent of hybrid corn, American farmers single-mindedly invested in labor-saving mechanical technologies and that biological technologies were static.

This book demonstrates that American agricultural development was far more dynamic than generally portrayed. In the two centuries before World War II, a stream of biological innovations revolutionized the crop and livestock sectors, increasing both land and labor productivity. Biological innovations were essential for the movement of agriculture onto new lands with more extreme climates, for maintaining production in the face of evolving threats from pests, and for the creation of the modern livestock sector. These innovations established the foundation for the subsequent Green and Genetic Revolutions. The book challenges the misconceptions that, before the advent of hybrid corn, American farmers single-mindedly invested in labor-saving mechanical technologies and that biological technologies were static.

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.

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.

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."