Preface
List of Contributors
Abbreviations
Part I Fundamentals of Cellular and Molecular Biology
1 The Cell as the Basic Unit of Life
2 Structure and Function of Cellular Macromolecules
2.1 Structure and Function of Sugars
2.2 Structure of Membrane Lipids
2.3 Structure and Function of Proteins
2.4 Structure of Nucleotides and Nucleic Acids (DNA and RNA)
3 Structure and Functions of a Cell
3.1 Structure of a Eukaryotic Cell
3.2 Structure of Bacteria
3.3 Structure of Viruses
3.4 Differentiation of Cells
4 Biosynthesis and Function of Macromolecules (DNA, RNA, and Proteins)
4.1 Genomes, Chromosomes, and Replication
4.2 Transcription: From Gene to Protein
4.3 Protein Biosynthesis (Translation)
5 Distributing Proteins in the Cell (Protein Sorting)
5.1 Import and Export of Proteins via the Nuclear Pore
5.2 Import of Proteins in Mitochondria and Chloroplasts
5.3 Protein Transport into the Endoplasmic Reticulum
5.4 Vesicle Transport from the ER via the Golgi Apparatus to the Cytoplasmic Membrane
6 Evolution and Diversity of Organisms
6.1 Prokaryotes
6.2 Eukaryotes
Part II Standard Methods in Molecular Biotechnology
7 Isolation and Purification of Proteins
7.1 Introduction
7.2 Producing a Protein Extract
7.3 Gel Electrophoretic Separation Methods
7.4 Methods of Protein Precipitation
7.5 Column Chromatography Methods
7.6 Examples
8 Peptide and Protein Analysis with Electrospray Tandem Mass Spectrometry
8.1 Introduction
8.2 Principles of Mass Spectrometry
8.3 Mass Precision, Resolution, and Isotope Distribution
8.4 Principles of ESI
8.5 Tandem Mass Spectrometers
8.6 Peptide Sequencing with MS/MS
8.7 Identifying Proteins with MS/MS Data and Protein Databases
8.8 Determining Protein Molecular Mass
8.9 Analysis of Covalent Protein Modification
8.10 Relative and Absolute Quantification
9 Isolation of DNA and RNA
9.1 Introduction
9.2 DNA Isolation
9.3 RNA Isolation
10 Chromatography and Electrophoresis of Nucleic Acids
10.1 Introduction
10.2 Chromatographic Separation of Nucleic Acids
10.3 Electrophoresis
11 Hybridization of Nucleic Acids
11.1 Significance of Base Pairing
11.2 Experimental Hybridization: Kinetic and Thermodynamic Control
11.3 Analytical Techniques
12 Use of Enzymes in the Modification of Nucleic Acids
12.1 Restriction Enzymes (Restriction Endonucleases)
12.2 Ligases
12.3 Methyltransferases
12.4 DNA Polymerases
12.5 RNA Polymerases and Reverse Transcriptase
12.6 Nucleases
12.7 T4 Polynucleotide Kinase
12.8 Phosphatases
13 Polymerase Chain Reaction
13.1 Introduction
13.2 Techniques
13.3 Areas of Application
14 DNA Sequencing
14.1 Introduction
14.2 DNA Sequencing Methods
14.3 Strategies for Sequencing the Human Genome
14.4 Practical Significance of DNA
15 Cloning Procedures
15.1 Introduction
15.2 Construction of Recombinant Vectors
16 Expression of Recombinant Proteins
16.1 Introduction
16.2 Expression of Recombinant Proteins in Host Organisms
16.3 Expression in Cell-Free Systems
17 Patch Clamp Method
17.1 Biological Membranes and Ion Channels
17.2 Physical Foundations of the Patch Clamp Method
17.3 Patch Clamp Configurations
17.4 Applications of the Patch Clamp Method
18 Cell Cycle Analysis
18.1 Analyzing the Cell Cycle
18.2 Experimental Analysis of the Cell Cycle
19 Microscopic Techniques
19.1 Electron Microscopy
19.2 Atomic or Scanning Force Microscopy
19.3 Light Microscopy
19.4 Microscopy in the Living Cell
20 Laser Applications
20.1 Principles of Laser Technology
20.2 Properties of Laser Radiation
20.3 Types of Lasers and Setups
20.4 Applications
Part III Key Topics
21 Genomics and Functional Genomics
21.1 Introduction
21.2 Technological Developments in DNA Sequencing
21.3 Genome Sequencing
21.4 cDNA Projects
21.5 Functional Genomics
21.6 Identification and Analysis of Individual Genes
21.7 Investigation of Transcriptional Activity
21.8 Cell-based Methods
21.9 Functional Analysis of Entire Genomes
22 Bioinformatics
22.1 Introduction
22.2 Data Sources
22.3 Sequence Analysis
22.4 Evolutionary Bioinformatics
22.5 Gene Prediction
22.6 Bioinformatics in Transcriptome and Proteome Analysis
22.7 Bioinformatic Software
23 Cellular Systems Biology
23.1 Introduction
23.2 Analysis of Cellular Networks by Top-Down Approaches
23.3 Overview of Bottom-Up Modeling of Biochemical Networks
23.4 Biological Examples
24 Protein–Protein and Protein–DNA Interaction
24.1 Protein–Protein Interactions
24.2 Protein–DNA Interactions
25 Drug Research
25.1 Introduction
25.2 Active Compounds and their Targets
25.3 Preclinical Pharmacology and Toxicology
25.4 Clinical Development
25.5 Clinical Testing
26 Drug Targeting and Prodrugs
26.1 Drug Targeting
26.2 Prodrugs
26.3 Penetration of Drugs through Biological Membranes
26.4 Prodrugs to Extend Duration of Effect
26.5 Prodrugs for the Targeted Release of a Drug
26.6 Prodrugs to Minimize Side Effects
27 Molecular Diagnostics in Medicine
27.1 Uses of Molecular Diagnostics
27.2 Which Molecular Variations Should be Detected
27.3 Molecular Diagnostic Methods
27.4 Outlook
28 Recombinant Antibodies and Phage Display
28.1 Introduction
28.2 Why Recombinant Antibodies?
28.3 Obtaining Specific Recombinant Antibodies
28.4 Production of Recombinant Antibodies
28.5 Formats for Recombinant Antibodies
28.6 Applications of Recombinant Antibodies
28.7 Outlook
29 Transgenic and Gene-Targeted Mice and their Impact in Medical Research
29.1 Overview
29.2 Transgenic Mice
29.3 Homologous Recombination: knock-out (-in) mice
29.4 Conditionally Regulated Gene Expression
29.5 Impact of Genetically Modified Mice in Biomedicine
29.6 Outlook
30 Gene Therapy: Strategies and Vectors
30.1 Introduction
30.2 Principles of Somatic Gene Therapy
30.3 Germ Line Therapy
30.4 Setbacks in Gene Therapy
30.5 Vectors for Gene Therapy
30.6 Specific Expression
31 RNA Interference, Modified DNA, Peptide Nucleic Acid, and Applications in Medicine and Biotechnology
31.1 Introduction
31.2 Modified Nucleic Acids
31.3 Interactions of DNA Analogs with Complementary DNA and RNA
31.4 RNAi
31.5 Applications
32 Plant Biotechnology
32.1 Introduction
32.2 Gene Expression Control
32.3 Production of Transgenic Plants
32.4 Selection of Transformed Plant Cells
32.5 Regeneration of Transgenic Plants
32.6 Plant Analysis: Identification and Characterization of Genetically Engineered Plants
33 Biocatalysis in the Chemical Industry
33.1 Introduction
33.2 Bioconversion/Enzymatic Procedures
33.3 Development of an Enzyme for Industrial Biocatalysis
33.4 Fermentative Procedures
Part IV Biotechnology in Industry
34 Industrial Application: Biotech Industry, Markets, and Opportunities
34.1 Historical Overview and Definitions of Concepts
34.2 Areas of Industrial Application of Molecular Biotechnology
34.3 Status Quo of the Biotech Industry World-Wide
35 Patents in the Molecular Biotechnology Industry: Legal and Ethical Issues
35.1 Patent Law
35.2 Ethical and Policy Issues in Biotechnology Patents
35.3 Conclusions
36 Drug Approval in the European Union and United States
36.1 Introduction
36.2 Regulation within the European Union
36.3 Regulation in the United States
36.4 Advent and Regulation of Biosimilars
36.5 International Regulatory Harmonization
37 Emergence of a Biotechnology Industry
38 The 101 of Founding a Biotech Company
38.1 First Steps Towards Your Own Company
38.2 Employees: Recruitment, Remuneration, Participation
39 Marketing
39.1 Introduction
39.2 What Types of Deals are Possible?
39.3 What Milestone or License Fees are Effectively Paid in a Biotech/ Pharma Cooperation?
39.4 PR and IR in Biotech Companies
Appendix
Further Reading
Glossary
Subject Index
Related Titles
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The Editor
Prof. Dr. Michael Wink
Institute of Pharmacy and
Molecular Biotechnology
University of Heidelberg
Im Neuenheimer Feld 364
69120 Heidelberg
Germany
Cover
Pictures courtesy of Michael Knop, EMBL,
Heidelberg (gel chromatography, pipet),
National Human Genome Research Institute,
Bethesda, USA (DNA), Fotolia/Franz Pfluegl
(cereals), PhotoDisc/Getty Images (pills),
Fotolia/SyB (stock exchange charts),
Fotolia/Aintschie (law code)
Limit of Liability/Disclaimer of Warranty: While the publisher and authors have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty can be created or extended by sales representatives or written sales materials. The Advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.
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The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.d-nb.de.
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All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.
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ISBN 978-3-527-32637-2
The term biotechnology was only coined in 1919 by the Hungarian engineer Karl Ereky. He used it as an umbrella term for methods by which microorganisms helped to produce valuable products. Humankind has been using biotechnological methods for thousands of years – think of the use of yeast or bacteria in the production of beer, wine, vinegar, or cheese.
Biotechnology is one of the key technologies of the twenty-first century. It includes established traditional industries such as the production of milk and dairy products, beer, wine, and other alcoholic drinks, as well as the production and biotransformation of enzymes, amino acids, vitamins, antibiotics, and other fine chemicals. This area, including the associated process engineering, is referred to as white or industrial biotechnology. As it is well established, it will only be treated in passing in Chapter 34. Many good books have been written to cover the field.
Breathtaking progress has been made in molecular and cell biology in the past 50 years, particularly in the last 20–30 years. This opens up new exciting perspectives for industrial applications. This area of applied biology is clearly distinguished from the traditional biotechnological fields and is known as molecular biotechnology. In a few years’ time, however, it may well be regarded as another established branch of traditional biotechnology.
Molecular biology and cell biology have revolutionized our knowledge about the function and structure of macromolecules in the cell and the role of the cell itself. Major progress has been made in genomics and proteomics. A historic milestone was the sequencing of the human genome in 2001. At present, more than 1200 genomes of diverse organismal groups (including more than 100 genomes of eukaryotes) have been completely sequenced (http://www.ebi.ac.uk/genomes). As a next milestone it has been proposed to sequence 10000 genomes from species covering the tree of life (http://www.genome10k.org). With the new generation of DNA sequencers it is now possible to sequence the human genome in a matter of weeks. This new knowledge has had direct repercussions on medical science and therapy, as it is now possible for the first time to study the genetic causes of diseases. It should thus be possible in due course to treat the causes rather than the symptoms. High-throughput sequencing will probably become a routine diagnostic, which will allow personalized medical treatment. Opportunities open up for the biotech industry (red biotechnology) to develop new diagnostics and therapeutics such as recombinant hormones, enzymes, antigens, vaccines, and antibodies that were not available before the genetic revolution. In the field of green biotechnology, targeted modification of crop cultivars can improve their properties, such as resistance to pests or the synthesis of new products (including recombinant human proteins). In microbial biotechnology, production processes can be improved and new products can be created through combinatorial biosynthesis.
The term molecular biotechnology also covers state-of-the-art research in genomics, functional genomics, proteomics, transcriptomics, systems biology, gene therapy, or molecular diagnostics. The concepts and methods are derived from cell and molecular biology, structural biology, bioinformatics, and biophysics.
The success of molecular biotechnology has been considerable, if you look at the scientific and economic prowess of companies like Genentech, Biogen, and others. Already today total annual revenues from recombinant drugs exceeds US $ 20 billion. Over 100 recombinant proteins have been approved by the US Food and Drug Administration and several hundred others are in the developmental pipeline.
As textbooks covering this extensive subject are few, a group of experts and university teachers decided to write an introductory textbook that looks at a wide variety of aspects. This is the English language version of the second edition of An Introduction to Molecular Biotechnology, which has been thoroughly updated, a new chapter on systems biology has been added (Chapter 23), and many illustrations are now in color.
The comprehensive introductory chapters (Part I) provide a brief compendium of the essential building blocks and processes in a cell, their structure, and functions. This information is crucial for the understanding of the following chapters, and while it cannot be a substitute for the profound study of more substantial and extensive textbooks on cell and molecular biology (Alberts et al., 2008; Campbell and Reece, 2006), it gives a quick overview and recapitulation.
Part II contains short chapters discussing the most important methods used in biotechnology. Again, for a more thorough approach to the subject, consult the relevant textbooks.
Part III explores the different fields of molecular biotechnology, such as genome research, functional genomics, proteomics, transcriptomics, bioinformatics, systems biology, gene therapy, and molecular diagnostics. It not only gives a summary of current knowledge, but also highlights future applications and developments.
Part IV discusses the industrial environment of molecular biotechnology, including the business environment and difficulties young biotech firms have to cope with and their chances of success.
To give a snapshot of state-of-the-art research in an area where things move faster than anywhere else is next to impossible. Thus, it is inevitable that by the time this book goes into print, some developments will have superseded those described here. Although we have tried to include most relevant issues, the choice of topics must naturally limited in a such a textbook.
Forty-two coauthors worked on this project, and although we tried to find a more or less uniform style, the authors with their different views and values are still recognizable.
The publisher and editors would like to thank all authors for their constructive cooperation. Special thanks go to the team at Wiley-VCH (Dr. A. Sendtko, M. Petersen, H.-J. Schmitt) who gave their enthusiastic support to this project.
Heidelberg, Winter 2011
Michael Wink
Michael Breuer
BASF SE
Fine Chemicals & Biocatalysis
Research
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Benedikt Brors
German Cancer Research Center
Computational Oncology
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Phenex Pharmaceuticals AG
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Leibniz Institute for Age Research
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and Pathophysiology
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Gert Fricker
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Marcus Frohme
Molecular Biology
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General, Visceral and Transplantation
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University of Stuttgart
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70569 Stuttgart
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Rüdiger Hell
Heidelberg Institute of Plant Sciences
University of Heidelberg
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Ingrid Herr
General, Visceral and Transplantation
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Molecular OncoSurgery
University Hospital Heidelberg
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Molecular Biotechnology
University of Heidelberg Bioquant
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Carl Ludwig Institute of Physiology
University of Leipzig
Liebigstr. 27
04113 Leipzig
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PheneX Pharmaceuticals AG
Im Neuenheimer Feld 515
69120 Heidelberg
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55128 Mainz
Germany
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German Cancer Research Center
Molecular Structure Analysis
Mass Spectroscopy
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Institute of Experimental and Clinical
Pharmacology and Toxicology
University of Heidelberg
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Nils Metzler-Nolte
Chair of Inorganic Chemistry I
Bioinorganic Chemistry
Ruhr-University of Bochum
Universitätsstr. 150
44801 Bochum
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Andrea Mohr
National Center for Biomedical
Engineering Science
National University of Ireland
University Road
Galway
Ireland
Ehmke Pohl
Department of Chemistry & School
of Biological and
Biomedical Sciences
Durham University
Durham, DH1 3LE
Great Britain
David B. Resnik
National Institute of Environmental
Health Science
National Institutes of Health
111 T.W. Alexander Drive
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Andreas Schlosser
Center for Biological Systems
Analysis (ZBSA)
University of Freiburg
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79104 Freiburg
Germany
Hannah Schmidt-Glenewinkel
German Cancer Research Center
Theoretical Systems Biology
Im Neuenheimer Feld 280
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BioMedServices
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Anna Sosniak
Chair of Inorganic Chemistry I
Bioinorganic Chemistry
University of Bochum
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Max Planck Institute
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Ralf Tolle
Center for Molecular Biology (ZMBH)
University of Heidelberg
Im Neuenheimer Feld 282
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Peter Uetz
Delaware Biotechnology Institute
University of Delaware
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Martin Vogel
Max Planck Institute of Biophysics
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Gary Walsh
Department of Chemical &
Environmental Sciences
Plassey Park
University of Limerick
Limerick
Ireland
Hans Weiher
Bonn-Rhein-Sieg University
of Applied Sciences
Department of Natural Sciences
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53359 Rheinbach
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Institute of Experimental and Clinical
Pharmacology and Toxicology
University of Heidelberg
Maybachstraße 14
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Germany
Stefan Wiemann
German Cancer Research Center
Molecular Genome Analysis
Im Neuenheimer Feld 580
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Michael Wink
Institute of Pharmacy and Molecular
Biotechnology
University of Heidelberg
Im Neuenheimer Feld 364
69120 Heidelberg
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Stefan Wölfl
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Biotechnology
University of Heidelberg
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Ralf Zwacka
National Center for Biomedical
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National University of Ireland
University Road
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1 Å | =0.1 nm |
aa-tRNA | aminoacyl-tRNA |
AAV | adeno-associated virus |
ABC | ATP binding cassette |
Acetyl-CoA | acetyl coenzyme A |
AcNPV | Autographa californica nuclear polyhedrosis virus |
ACRS | amplification-created restriction sites |
ACTH | adrenocorticotropic hormone |
ADA | adenosine deaminase |
ADEPT | antibody-directed enzyme pro-drug therapy |
ADME-T | absorption, distribution, metabolism, excretion and toxicity |
ADP | adenosine diphosphate |
ADRs | adverse drug reactions |
AEC | aminoethylcysteine |
AFLP | amplified fragment length polymorphism |
AFM | atomic force microscope |
AIDS | acquired immune deficiency syndrome |
ALS | amyotrophic lateral sclerosis |
AMP | adenosine monophosphate |
AMPA | α-amino-3-hydroxyl-5-methyl-4-isoxazol-propionate |
Ampr | ampicillin resistance gene |
AMV | avian myeloblastosis virus |
ANN | artificial neural network |
AO | acridine orange |
AOX1 | alcohol oxidase 1 |
APC | anaphase promoting complex |
ApoB100 | apolipoprotein B100 |
ApoE | apolipoprotein E |
APP | amyloid precursor protein |
ARMS | amplification refractory mutation system |
ARS | autonomously replicating sequence |
ATP | adenosine triphosphate |
att | attachment site |
BAC | bacterial artificial chromosome |
bcl2 | B-cell leukemia lymphoma 2 (protein protecting against apoptosis) |
BfArM | German Bundesinstitut für Arzneimittel und Medizinprodukte |
β-Gal | β-galactosidase |
BHK-21 | baby hamster kidney cells |
BLA | biologics licence application |
BLAST | basic local alignment search tool |
BMP | bone morphogenetic proteins |
bp | base pairs |
BrdU | bromodeoxyuridine |
CA | correspondence analysis |
CAD | coronary artery disease |
CaM-Kinase | Ca2+/calmodulin-dependent protein kinase |
cAMP | cyclic AMP |
cap | AAV gene mediating encapsulation |
CARS | coherent anti-Raman scattering |
CAT | Committee for Advanced Therapies |
CBER | Center for Biologics Evaluation and Research |
CC | chromatin remodelling complex |
CCD | charge-coupled device |
CDER | Center for Drug Evaluation and Research |
CDK | cyclin-dependent kinase |
cDNA | copy DNA |
CDR | complementary determining region |
CDRH | Center for Devices and Radiological Health |
CEO | chief executive officer |
CFP | cyan fluorescent protein |
CFTR | cystic fibrosis transmembrane regulator |
CGAP | cancer genome anatomy project |
CGH | comparative genome hybridization |
CHMP | Committee for Medicinal Products for Human Use |
CHO | Chinese hamster ovary |
CIP | calf intestinal phosphatase |
CML | chronic myeloic leukemia |
CMN | Corynebacterium-Mycobacterium-Nocardia group |
CMV | cauliflower mosaic virus |
CMV | Cytomegalovirus |
CNS | central nervous system |
COMP | Committee on Orphan Medicinal Products |
COS-1 | simian cell line, CV-1, transformed by origin-defective mutant of SV40 |
cpDNA | chloroplast DNA |
CPMV | cowpea mosaic virus |
cPPT-sequence | central polypurine tract – regulatory element in lentiviral vectors that facilitates double strand synthesis and the nuclear import of the pre-integration complex |
CSF | colony-stimulating factor |
CSO | contract service organisation |
CTAB | cetyltrimethylammonium bromide |
CVM | Center for Veterinary Medicine |
CVMP | Committee for Medicinal Products for Veterinary Use |
2D | two-dimensional |
Da | Dalton |
DAG | diacylglycerol |
DAPI | 4,6-diamidino-2-phenylindole |
dATP | deoxyadenosine triphosphate |
DBD | DNA-binding domain |
DAC | divide-and-conquer strategy |
DD | differential display |
DDBJ | DNA Data Bank of Japan |
ddNTP | dideoxynucleotide triphosphate |
DEAE | diethylaminoethyl |
dHPLC | denaturing HPLC |
DIC | differential interference contrast |
DIP | Database of Interacting Proteins |
DNA | deoxyribonucleic acid |
DNAse | deoxyribonuclease |
dNTP | deoxynucleoside triphosphate |
Dox | doxycycline |
ds diabodies | disulfide-stabilized diabodies |
dsDNA | double-stranded DNA |
dsFv-fragment | disulfide-stabilized Fv fragment |
dsRNA | double-stranded RNA |
DtxR | diphtheria toxin repressor |
Ebola-Z | envelope protein of the Ebola-Zaire virus, which has a high affinity to lung epithelial cells |
EC50 | effective concentration, the dose or concentration that produces a 50% effect in the test population within a specified time |
ECD | electron capture dissociation |
EDTA | ethylenediaminetetraacetic acid |
ee | enantiomeric excess |
EF2 | elongation factor 2 |
EF-Tu | elongation factor Tu |
EGF | epidermal growth factor |
EGFP | enhanced green fluorescent protein |
EGTA | ethyleneglycol-bis-(2-aminoethyl)-tetraacetic acid |
EIAV | equine infectious anaemia virus |
ELISA | enzyme-linked immunosorbent assay |
EM | electron microscope |
EMA | European Medicines Agency |
EMBL | European Molecular Biology Laboratory |
EMCV | Encephalomyocarditis virus |
EMSA | electrophoretic mobility shift assay |
EMEA | European Agency for the Evaluation of Medicinal Products |
ENU | N-ethyl-N-nitrosourea |
env | retroviral gene coding for viral envelope proteins |
EPO | European Patent Office |
EPR effect | enhanced permeability and retention effect |
EPC | European Patent Convention |
ER | endoplasmic reticulum |
ESI | electrospray ionization |
EST | expressed sequence tags |
ES cells | embryonic stem cells |
EtBr | ethidium bromide |
Fab-fragment | antigen binding fragment |
FACS | fluorescence-activated cell sorter |
FAD | flavin adenine dinucleotide |
FBA | flux balance analysis |
FCS | fluorescence correlation spectroscopy |
FDA | Food and Drug Administration |
FFL | feed-forward loop |
FGF | fibroblast growth factor |
FISH | fluorescence in situ hybridization |
FIV | feline immunodeficiency virus |
FKBP | FK506-binding protein |
FLIM | fluorescence lifetime imaging microscopy |
FLIPR | fluorescent imaging plate reader |
FMN | flavin mononucleotide |
FPLC | fast performance liquid chromatography |
FRAP | fluorescence recovery after photobleaching |
FRET | fluorescence resonance energy transfer |
FT-ICR | Fourier transformation cyclotron resonance, method in mass spectroscopy |
FtsZ | prokaryotic cell division protein |
Fur | ferric uptake regulator |
Fv-fragment | variable fragment |
FWHM | full width at half maximum |
GABA | gamma aminobutyric acid |
Gag | retroviral gene coding for structural proteins |
Gal | galactose |
GAP | GTPase-activating protein |
GAPDH | glyceraldehyde 3-phosphate-dehydrogenase |
Gb | Gigabases |
GCC | German cDNA consortium |
GCG | genetics computer group |
GCP | good clinical practice |
ΔGd | free enthalpy |
GDH | glutamate dehydrogenase |
GDP | guanosine diphosphate |
GEF | guanine exchange factor |
GEO | gene expression omnibus |
GFP | green fluorescence protein |
GM-CSF | granulocyte/macrophage colony-stimulating factor |
GO | gene ontology |
GOI | gene of interest |
GPCR | G-protein-coupled receptor |
GPI anchor | glycosylphosphatidylinositol anchor |
GRAS | generally regarded as safe |
GST | glutathione-S-transferase |
GTC | guanidinium isothiocyanate |
GTP | guanosine triphosphate |
GUS | glucuronidase |
GMO | genetically modified organism |
HA | hemagglutinin |
HCM | hypertrophic cardiomyopathy |
HCV | Hepatitis C virus |
HEK | human embryonic kidney |
HeLa cells | human cancer cell line (isolated from donor Helene Larsen) |
HER 2 | human epidermal growth factor 2 |
HGH | human growth hormone |
HIC | hydrophobic interaction chromatography |
His6 | hexahistidine tag |
HIV | human immunodeficiency virus, a retrovirus |
HIV 1 | human immunodeficiency virus 1 |
HLA | human leukocyte antigen |
hnRNA | heterogeneous nuclear RNA |
HPLC | high performance liquid chromatography |
HPT | hygromycin phosphotransferase |
HPV | human papilloma virus |
HSP | high-scoring segment pairs |
HSP | heat shock protein |
HSV-1 | Herpes simplex virus |
HTS | high-throughput analysis |
HUGO | Human Genome Organisation |
HV | Herpes virus |
IAS | international accounting standard |
ICDH | isocitric dehydrogenase |
ICH | International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use |
ICL | isocitric lyase |
ICP-MS | inductively coupled-plasma mass spectrometry |
ICR-MS | ion cyclotron resonance mass spectrometer |
IDA | iminodiacetic acid |
IEF | isoelectric focusing |
Ig | immunoglobulin |
IHF | integration host factor |
IMAC | immobilized metal affinity chromatography |
IND-Status | investigational new drug status |
IP3 | inositol-1,4,5-triphosphate |
IPO | initial public offering |
IPTG | isopropyl-b-D-thiogalactoside |
IR | inverted repeats |
IR | investor relations |
IRES | internal ribosome entry site |
ISAAA | International Service for the Acquisition of Agri-Biotech Applications |
ISH | insitu hybridization |
ISSR | inter simple sequence repeats |
ITC | isothermal titration calorimetry |
ITR | inverse terminal repeats – regulatory elements in adenoviruses and AAV |
i.v. | intravenous |
ka | second order velocity constant in bimolecular association |
Kanr | kanamycin resistance gene |
Kav | specific distribution coefficient |
kb | Kilobases |
kd | first order velocity constant in unimolecular dissociation |
Kd=kd/ka | velocity constant in dissociation/Ka in association |
kDa | Kilodalton |
KDEL | amino acid sequence for proteins remaining in the ER |
KDR receptor | kinase insert domain containing receptor |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
Lac | lactose |
LASER | Light Amplification by Stimulated Emission of Radiation |
LB | left border |
LB | Luria-Bertani medium |
LCR | ligation chain reaction |
LDL | low-density-lipoprotein |
LIMS | laboratory information management systems |
LINE | long interspersed elements |
LSC | Laser scanning-cytometer |
LTQ | linear trap quadrupole |
LTQ-FT-ICR | linear trap quadrupole-Fourrier transformation-ion cyclotron resonance |
LTR | long terminal repeats; regulatory elements in retroviruses |
LUMIER | LUMInescence-based mammalian intERactome |
MAC | mammalian artificial chromosome |
mAChR | muscarinic acetylcholine receptor |
MAGE-ML | microarray gene expression markup language |
MALDI | matrix-assisted laser desorption/ionization |
6-MAM | 6-monoacetylmorphine |
MAP | microtubule-associated protein |
MAP | mitosis-activating protein |
Mb | Megabases |
MBP | maltose-binding protein |
MCS | multiple cloning site |
M-CSF | macrophage colony-stimulating factor |
MDR protein | multiple drug resistance protein |
MDS | multidimensional scaling |
MGC | mammalian gene collection |
MHC | major histocompatibility complex |
MIAME | minimum information about a microarray experiment |
miRNA | microRNA |
MIT | Massachusetts Institute of Technology |
MoMLV | moloney murine leukemia virus |
Mowse | molecular weight search |
MPF | M-phase promotion factor |
MPSS | massively parallel signature screening |
Mreb/Mbl | proteins of prokaryotic cytoskeleton |
mRNA | messenger RNA |
MRSA | methicillin-resistant S. aureus |
MS | mass spectrometry |
MSG | monosodium glutamate |
MS-PCR | mutationally separated PCR |
MTA | material transfer agreement |
mtDNA | mitochondrial DNA |
MULVR | Moloney Murine Leukemia Virus |
MW | molecular weight |
μF | μFarad |
nAChR | nicotinic acetylcholine receptor |
NAD | nicotinamide adenine dinucleotide |
NAPPA | nucleic acid programmable protein array |
NCBI | National Center for Biotechnology Information |
NDA | new drug application |
NDP | nucleoside diphosphate |
NDPK | nucleoside diphosphates kinase |
NFjB | nuclear factor jB |
NIH | National Institutes of Health |
NK cell | natural killer cell |
NMDA-receptor | N-methyl-D-aspartate-receptor |
NMR | nuclear magnetic resonance |
NPTII | neomycin phosphotransferase II |
NSAID | non-steroidal anti-inflammatory drug |
NTA | nitrilotriacetic acid |
NTP | nucleoside triphosphate |
OD | optical density |
ODE | ordinary differential equation |
ODHC | 2-oxoglutarate dehydrogenase |
OMIM | online Mendelian inheritance in man |
ORF | open reading frame |
ori | origin of replication |
OXA complex | membrane translocator in mitochondria |
PAC | P1-derived artificial chromosome |
PAGE | polyacrylamide-gel electrophoresis |
PAZ-domain | PIWI Argonaute Zwille domain |
PCA | principal component analysis |
PCR | polymerase chain reaction |
PDB | protein data bank |
PEG | polyethylene glycol |
PFAM | protein families database of alignments and HMMs |
PFG | pulsed-field gel electrophoresis |
PI | propidium iodide |
PIR | protein information resource |
piRNA | piwi-interacting RNA |
PKA | protein kinase A |
PKC | protein kinase C |
PK data | pharmacokinetic data |
Plos | Public Library of Science |
PMSF | phenylmethylsulfonyl fluoride |
PNA | peptide nucleic acid |
PNGaseF | peptide N-glycosidase F |
PNK | T4-polynucleotide kinase |
pol | retroviral gene coding for reverse transcriptase and integrase |
PPH | polyhedrin promoter |
PR | Public Relations |
psi | retroviral packaging signal |
PTGS | posttranscriptional gene silencing |
PTI | pancreatic trypsin inhibitor |
Q-FT-ICR | Q-Fourier transform ion cyclotron resonance |
Q-TOF | Quadrupole-Time-of-Flight |
RACE | rapid amplification of cDNA ends |
Ran | protein involved in nuclear import |
RAPD | random amplification of polymorphic DNA |
RAP-PCR | RNA arbitrary primed PCR |
RB | right border |
RBD | RNA-binding domain |
Rb-gene | retinoblastoma gene |
RBS | ribosome binding site |
RDA | representative difference analysis |
RdRp | RNA-dependent RNA polymerase |
rep | AAV gene, mediating replication |
RES | reticuloendothelial system |
RFLP | restriction fragment length polymorphism |
Rf-value | retention factor |
RGS | regulator of G-protein signaling |
RISC | RNA-induced silencing complex |
RNA | ribonucleic acid |
RNAi | RNA interference |
RNP | ribonucleoprotein |
rpm | revolutions per minute |
RRE | regulatory element in a lentiviral vector, enhancing the nuclear export of viral RNA |
rRNA | ribosomal RNA |
RSV | respiratory syncytial virus |
RSV | promoter of the Rous sarcoma virus |
RT | reverse transcriptase |
rtTA | tetracyclin-sensitive regulatory unit |
SAGE | Serial Analysis of Gene Expression |
SALM | spectrally assigned localization microscopy |
SAM | S-adenosylmethionine |
sc diabodies | single-chain diabodies |
scFab | single-chain Fab-fragment |
scFv/sFv fragment | single-chain Fv fragment |
SCID | severe combined immunodeficiency |
SCOP | structural classification of proteins |
SDS | sodium dodecyl sulfate |
SDS-PAGE | sodium dodecyl sulfate polyacrylamide gel electophoresis |
SELEX | systematic evolution of ligand by exponential enrichment |
SEM | scanning electron microscope |
Sf cells | Spodoptera frugiperda cells |
SFM | scanning force microscope |
SFV | Semliki-Forest virus |
SH1 | Src-homology domain 1=kinase domain |
SH2 | Src-homology domain 2 |
SH3 | Src-homology domain 3 |
SHG | second harmonic generation |
SIM | single input |
SIN | self-inactivating lentiviral vectors, due to a 3′ LTR mutation |
SINE | scattered or short interspersed elements |
siRNA | small interfering RNA |
SIV | simian immunodeficiency virus |
SNARE proteins | SNAP-receptor proteins |
SNP | single nucleotide polymorphism |
snRNA | small nuclear RNA |
snRNP | small nuclear ribonucleoprotein |
SOP | stock option program |
SP function | sum-of-pairs function |
SPA | scintillation proximity assay |
SPDM | spectral precision distance microscopy |
SPF | S-phase promotion factor |
SRP | signal recognition particle |
SSB | single strand binding proteins |
SSCP | single-strand comformation polymorphism |
ssDNA | single-stranded DNA |
SSH | suppressive subtractive hybridization |
SssI methylase | methylase from Spiroplasma |
ssRNA | single-stranded RNA |
STED | stimulated emission depletion |
STEM | scanning transmission electron microscope |
stRNA | small temporal RNA |
STS | sequence-tagged site |
SV40 | Simian-virus-type 40 |
TBP | TATA-binding protein |
TC | cytotoxic T-cells |
TC | tetracycline |
T-DNA | transfer DNA |
TEM | transmission electron-microscope |
TEV | Tobacco Etch Virus |
TH | T helper cell |
THG | third harmonic generation |
TIGR | The Institute for Genome Research |
TIM | translocase of inner membrane |
Tm | melting temperature of dsDNA |
TNF | tumor necrosis factor |
TOF | time of flight |
TOM | translocase of outer membrane |
t-PA | tissue plasminogen activator |
TRE | tetracycline-responsive element |
TRIPs | Trade-Related Aspects of Intellectual Property Rights |
tRNA | transfer RNA |
Trp | tryptophan |
t-SNARE | protein in target membrane to which vSNARE binds |
TSS | transformation and storage solution |
tTA | tetracycline-controlled transactivator |
TY | transposon from yeast |
UPOV | Union for the Protection of New Varieties of Plants |
US-GAAP | US generally accepted accounting principle |
UV | ultraviolet |
V0 | empty volume |
VC | venture capital |
Ve | elution volume |
VEGF | vascular endothelial growth factor |
VIP | vasoactive peptide |
VNTR | variable number tandem repeats |
v-SNARE | protein in vesicular membrane, binding to t-SNARE |
VSV-G | envelope protein of vesicular stomatitis virus, great affinity to a wide range of cells |
Vt | total volume |
wNAPPA | modified nucleic acid programmable protein array |
WPRE | woodchuck hepatitis virus posttranscriptional regulatory element |
X-Gal | 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside |
YAC | yeast artificial chromosome |
YEp | yeast episomal plasmid |
YFP | yellow fluorescence protein |
YIp | yeast-integrating plasmid |
YRp | yeast-replicating plasmid |
Yth | yeast two-hybrid |
Learning Objectives
This chapter offers a short introduction into the structure of prokaryotic and eukaryotic cells, as well as that of viruses.
The base unit of life is the cell. Cells constitute the base element of all prokaryotic cells (cells without a cell nucleus, e.g., Bacteria and Archaea) and eukaryotic cells (or Eukarya) (cells possessing a nucleus, e.g., protozoa, fungi, plants, and animals). Cells are small, membrane-bound units with a diameter of 1–20 μm and are filled with concentrated aqueous solutions. Cells are not created de novo, but possess the ability to copy themselves, meaning that they emerge from the division of a previous cell. This means that all cells, since the beginning of life (around 4 billion years ago), are connected with each other in a continuous lineage. In 1885, the famous cell biologist Virchow conceived the law of omnis cellula e cellulae (all cells arise from cells), which is still valid today.
The structure and composition of all cells are very similar due to their shared evolution and phylogeny (Fig. 1.1). Owing to this, it is possible to limit the discussion of the general characteristics of a cell to a few basic types (Fig. 1.2):
The most important biochemical and cell biological characters of Archaea, Bacteria, and Eukarya are summarized in Table 1.1.
As viruses and bacteriophages (Fig. 1.3) do not have their own metabolism they therefore do not count as organisms in the true sense of the word. They share several macromolecules and structures with cells. Viruses and bacteriophages are dependent on the host cells for reproduction, and therefore their physiology and structure are closely linked to that of the host cell.
Eukaryotic cells are characterized by compartments that are enclosed by biomembranes (Table 1.2). As a result of these compartments, the multitude of metabolic reactions can run in a cell at the same time.
In the following discussion on the shared characteristics of all cells, the diverse differences that appear in multicellular organisms should not be forgotten. The human body has more than 200 different cell types, which show diverse structures and compositions. These differences must be understood in detail if cell-specific disorders, such as cancer, are to be understood and consequently treated.
Before a detailed discussion of cellular structures and their functions (see Chapters 3–5), a short summary of the biochemical basics of cellular and molecular biology is given in Chapter 2.
Learning Objectives
This chapter introduces the structure of polysaccharides, lipids, proteins, and nucleic acids, built from simple monomers (sugars, amino acids, and nucleotides), and illustrates how they are derived from simple monomers. Their most important functions are summarized.
In contrast to the diversity of life forms found in nature with several million species, the cells that make up all of these diverse organisms contain only a limited number of types of ions and molecules (Table 2.1). Among the most important macromolecules of prokaryotic and eukaryotic cells are polysaccharides, lipids, proteins, and nucleic acids, which are constructed from comparatively few monomeric building blocks (Table 2.2). The membrane lipids (phospholipids, cholesterol) will also be considered in this context because they spontaneously form supramolecular biomembrane structures in the aqueous environment of a cell.
Inorganic ions, sugars, amino acids, fatty acids, organic acids, nucleotides, and various metabolites are counted among the low-molecular-weight components and building blocks of the cell. The qualitative composition of cells is similar in prokaryotes and eukaryotes (Table 1.1), even though eukaryote cells generally have a higher protein content, and bacterial cells a higher RNA content. Animal cells have a volume that is 103 times larger than that of bacterial cells.
Owing to their shared evolution, the structure and function of the important cellular molecules is very similar in all organisms, often even identical. Apparently, reliable and functional biomolecules were developed and, if useful for the producer, were selected early in evolution (Table 2.2) and are therefore still used today.
Contents | Bacterium (% of cell mass) | Anim al cell (% of cell mass) |
Water | 70 | 70 |
Inorganic ions | 1 | 1 |
Small molecules (sugars, acids, amino acids) | 3 | 3 |
Proteins | 15 | 18 |
RNA | 6 | 1.1 |
DNA | 1 | 0.25 |
Phospholipids | 2 | 3 |
Other lipids | − | 2 |
Polysaccharides | 2 | 2 |
Cell volume (ml) | 2×10−12 | 4×10−9 |
Relative cell volume | 1 | 2000 |
Basic building blocks | Macromolecule | Function |
Simple sugar | Polysaccharide | Structural substances: composition of the cell walls (cellulose, chitin, peptidoglycan); constituents of connective tissues Storage substances: starch, glycogen |
Amino acids | Proteins | Enzymes: important catalysts for anabolic and catabolic reaction processes Hemoglobin: O2 and CO2 transport Receptors: recognition of external and internal signals Ion channels, ion pumps, transporters: transport of charged molecules across biological membranes Regulatory proteins: signal transduction through protein–protein interactions Transcription factors: regulation of gene activity Antibodies: recognition of antigens Structural proteins: structural organization of supramolecular complexes Cytoskeleton: formation of molecular networks in the cell that are important for shape and function Motor proteins: muscle contraction |
Phospholipids, cholesterol Deoxynucleotide Nucleotide |
DNA RNA |
Elements of biomembranes Storage, replication, and safe transfer of genetic information; recombination rRNA: structural molecules for the construction of ribosomes ribozymes and siRNA: catalytic and regulatory processes tRNA: mediators in translation mRNA: messengers and mediators between genes and proteins snRNA: splicing of mRNA |
Monosaccharides occur in cells either as aldoses or ketoses (Fig. 2.1 A). The most important monosaccharides have a chain length of three, five, and six carbon atoms, and are called trioses, pentoses, and hexoses. Under physiological conditions, pentoses and hexoses can form ring structures through hemiacetal and hemiketal formation (Fig. 2.1 B).
Many important nitrogen-containing derivatives of these monosaccharides (Fig. 2.1 C) use galactose and glucose as a base. Examples include glucosamine, N-acetylglucosamine, and glucuronic acid. These derivatives can be present either as glycosides or as part of a polysaccharide.
Condensation reactions between sugar molecules result in the formation of glycosidic bonds with the elimination of a water molecule. As hydroxyl groups can be present in either the α or β position, the stereochemistry of sugar molecules is of great importance. The condensation of two sugar molecules results in the formation of a disaccharide (Fig. 2.1 D); that of three sugar molecules, correspondingly, is a trisaccharide. Oligosaccharides are built from a few sugar monomers and polysaccharides (e.g., starch, glycogen, cellulose, chitin, etc.) are made up of many sugar monomers.
Sugar molecules can be easily activated through esterification with an acid; one important example being esterification with phosphoric acid. Sugar phosphates are important in glycolysis.
The most important polysaccharide in animal cells is glycogen, which is stored as an energy source in liver and muscle. Glycogen can be quickly transformed into glucose-1-phosphate and then channeled into glycolysis. Glycogen is a branched polysaccharide formed from glucose molecules linked by α-(1→4)-glycosidic bonds or α-(1→6)-glycosidic bonds (Fig. 2.1 D). This results in many free ends on which the enzyme glycogen phosphorylase can begin degradation simultaneously.
Starch or amylose (Fig. 2.1 D) consists of glucose residues linked by α-(1→4)-glycosidic bonds. In amylopectin, additional glucose residues linked by α-(1→6)-glycosidic bonds are built in. Amylopectin, therefore, has a similar structure to glycogen, but is less strongly branched. Starch is formed by photosynthesis in plant cells, where it is stored in amyloplasts. Starch can be broken down easily by animals and is therefore an important part of human nutrition.
Glucose is also used as a building block for cellulose (Fig. 2.1 D), which is necessary for formation of the plant cell wall. Cellulose is an unbranched polymer made from glucose molecules linked by β-(1→4)-glycosidic bonds. Cellulose cannot be broken down in the human digestive tract. Conversely, the rumen (first stomach) of ruminants (animals that chew the cud) contains microorganisms that produce cellulase – an enzyme that makes it is possible for cows, for example, to use cellulose as a nutrient. Additional polymers present in the plant cell wall include polysaccharides, so-called glycans made up of cellulose fibers linked together in a diagonal fashion, pectin (basic unit: galacturonic acid), and lignin (made from the coumaroyl, coniferoyl, and sinapoyl alcohols). Using cellulases, it is possible to digest the cell walls of plant cells. Cells without cell walls are called protoplasts. They are important in plant biotechnology because they are easily transformable by genetic engineering (see Chapter 32). In many plant species it is possible to regenerate intact plant cells from protoplasts. Cell walls of fungi and the exoskeletons of insects are composed of chitin, which has N-acetylglucosamine as a building block in β-(1→4)-glycosidic bonds.