An Introduction to Molecular Biotechnology E-Book

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 1404

Veröffentlichungsjahr: 2013

Beschreibung

Molecular biotechnology continues to triumph, as this textbook testifies - edited by one of the academic pioneers in the field and written by experienced professionals. This completely revised second edition covers the entire spectrum, from the fundamentals of molecular and cell biology, via an overview of standard methods and technologies, the application of the various "-omics", and the development of novel drug targets, right up to the significance of system biology in biotechnology. The whole is rounded off by an introduction to industrial biotechnology as well as chapters on company foundation, patent law and marketing. The new edition features: - Large format and full color throughout - Proven structure according to basics, methods, main topics and economic perspectives - New sections on system biology, RNA interference, microscopic techniques, high throughput sequencing, laser applications, biocatalysis, current biomedical applications and drug approval - Optimized teaching with learning targets, a glossary containing around 800 entries, over 500 important abbreviations and further reading. The only resource for those who are seriously interested in the topic. Bonus material available online free of charge: www.wiley-vch.de/home/molecbiotech

Leseprobe

Contents

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

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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.

Library of Congress Card No.: applied for

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.d-nb.de.

© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Boschstraße 12, 69469 Weinheim

Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical, and Medical business with Blackwell Publishing.

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.

Composition K+V Fotosatz GmbH, BeerfeldenPrinting and Binding betz-druck GmbH, DarmstadtCover Design Formgeber, Eppelheim

ISBN 978-3-527-32637-2

Preface

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

List of Contributors

Michael Breuer BASF SE Fine Chemicals & Biocatalysis Research GVF/B – A030 67056 Ludwigshafen Germany

Benedikt Brors German Cancer Research Center Computational Oncology Im Neuenheimer Feld 580 69120 Heidelberg Germany

Ulrich Deuschle Phenex Pharmaceuticals AG Waldhofer Str. 104 69123 Heidelberg Germany

Stephan Diekmann Leibniz Institute for Age Research Fritz Lipmann Institute Beutenbergstraße 11 07745 Jena Germany

Stefan Dübel Institute for Biochemistry and Biotechnology Technical University of Braunschweig Spielmannstr. 7 38106 Braunschweig Germany

Rainer Fink Institute for Physiology and Pathophysiology University of Heidelberg Im Neuenheimer Feld 326 69120 Heidelberg Germany

Gert Fricker Institute of Pharmacy and Molecular Biotechnology University of Heidelberg Im Neuenheimer Feld 366 69120 Heidelberg Germany

Marcus Frohme Molecular Biology and Functional Genomics Technical University of Applied Sciences Bahnhofstraße 15745 Wildau Germany

Reinhard Gessner Visceral, Transplantation, Thorax and Vascular Surgery University Hospital Leipzig Liebigstr. 20 04103 Leipzig Germany

Ariane Groth General, Visceral and Transplantation Surgery Molecular OncoSurgery University Hospital Heidelberg Im Neuenheimer Feld 365 69120 Heidelberg Germany

Bernhard Hauer Institute of Technical Biochemistry University of Stuttgart Allmandring 31 70569 Stuttgart Germany

Rüdiger Hell Heidelberg Institute of Plant Sciences University of Heidelberg Im Neuenheimer Feld 360 69120 Heidelberg Germany

Ingrid Herr General, Visceral and Transplantation Surgery Molecular OncoSurgery University Hospital Heidelberg Im Neuenheimer Feld 365 69120 Heidelberg Germany

Helke Hillebrand European Molecular Biology Laboratory (EMBL) Meyerhofstr. 1 69117 Heidelberg Germany

Ana Kitanovic Institute for Pharmacy & Molecular Biotechnology University of Heidelberg Im Neuenheimer Feld 364 69120 Heidelberg Germany

Manfred Koegl Boehringer Ingelheim Vienna Oncology Research Dr. Boehringer Gasse 5–11 1121 Vienna Austria

Rainer König Institute of Pharmacy and Molecular Biotechnology University of Heidelberg Bioquant Im Neuenheimer Feld 267 69120 Heidelberg Germany

Robert Kraft Carl Ludwig Institute of Physiology University of Leipzig Liebigstr. 27 04113 Leipzig Germany

Claus Kremoser PheneX Pharmaceuticals AG Im Neuenheimer Feld 515 69120 Heidelberg Germany

Stefan Legewie Institute of Molecular Biology Ackermannweg 4 55128 Mainz Germany

Wolf-Dieter Lehmann German Cancer Research Center Molecular Structure Analysis Mass Spectroscopy Im Neuenheimer Feld 280 69120 Heidelberg Germany

Susanne Lutz Institute of Experimental and Clinical Pharmacology and Toxicology University of Heidelberg Maybachstraße 14 68169 Mannheim Germany

Nils Metzler-Nolte Chair of Inorganic Chemistry I Bioinorganic Chemistry Ruhr-University of Bochum Universitätsstr. 150 44801 Bochum Germany

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 Research Triangle Park, NC 27709 USA

Andreas Schlosser Center for Biological Systems Analysis (ZBSA) University of Freiburg Habsburgerstr. 49 79104 Freiburg Germany

Hannah Schmidt-Glenewinkel German Cancer Research Center Theoretical Systems Biology Im Neuenheimer Feld 280 69120 Heidelberg Germany

Julia Schüler BioMedServices Hecker-Str. 20 68199 Mannheim Germany

Anna Sosniak Chair of Inorganic Chemistry I Bioinorganic Chemistry University of Bochum Universitätsstr. 150 44801 Bochum Germany

Rolf Sprengel Max Planck Institute for Medical Research Jahnstrasse 29 69120 Heidelberg Germany

Ralf Tolle Center for Molecular Biology (ZMBH) University of Heidelberg Im Neuenheimer Feld 282 69120 Heidelberg Germany

Peter Uetz Delaware Biotechnology Institute University of Delaware 15 Innovation Way Newark, DE 19711-5449 USA

Martin Vogel Max Planck Institute of Biophysics Max-von-Laue-Str. 3 60438 Frankfurt Germany

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 Von-Liebig-Str. 20 53359 Rheinbach Germany

Thomas Wieland Institute of Experimental and Clinical Pharmacology and Toxicology University of Heidelberg Maybachstraße 14 68169 Mannheim Germany

Stefan Wiemann German Cancer Research Center Molecular Genome Analysis Im Neuenheimer Feld 580 69120 Heidelberg Germany

Michael Wink Institute of Pharmacy and Molecular Biotechnology University of Heidelberg Im Neuenheimer Feld 364 69120 Heidelberg Germany

Stefan Wölfl Institute for Pharmacy & Molecular Biotechnology University of Heidelberg Im Neuenheimer Feld 364 69120 Heidelberg Germany

Ralf Zwacka National Center for Biomedical Engineering Science National University of Ireland University Road Galway Ireland

Abbreviations

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

Amp

r

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

Ca

2+

/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

EC

50

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

ΔG

d

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

His

6

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

IP

3

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

k

a

second order velocity constant in bimolecular association

Kan

r

kanamycin resistance gene

K

av

specific distribution coefficient

kb

Kilobases

k

d

first order velocity constant in unimolecular dissociation

K

d

=k

d

/k

a

velocity constant in dissociation/K

a

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

P

PH

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

T

C

cytotoxic T-cells

T

C

tetracycline

T-DNA

transfer DNA

TEM

transmission electron-microscope

TEV

Tobacco Etch Virus

T

H

T helper cell

THG

third harmonic generation

TIGR

The Institute for Genome Research

TIM

translocase of inner membrane

T

m

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

V

0

empty volume

VC

venture capital

V

e

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

V

t

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

1

The Cell as the Basic Unit of Lifes

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):

Bacterial cells.

Plant cells.

Animal cells.

Fig. 1.1 Tree of life – phylogeny of life domains. Nucleotide sequences from 16S rRNA, amino acid sequences of cytoskeleton proteins, and characteristics of the cell structure were used to reconstruct this phylogenetic tree. Prokaryotes are divided into Bacteria and Archaea. Archaea form a sister group with eukaryotes; they share important characteristics (Tables 1.1 and 1.2). Many monophyletic groups can be recognized within the eukaryotes (diplomonads/trichomonads, Euglenozoa, Alveolata, Stramenopilata (heterokonts), red algae and green algae/plants, fungi and animals; see Tables 6.3–6.5 for details).

Fig. 1.2 Schematic structure of prokaryotic and eukaryotic cells. (A) Bacterial cell. (B) Plant mesophyll cell. (C) Animal cell.

Table 1.1 Comparison of important biochemical and molecular characteristics of the three domains of life.

Fig. 1.3 Schematic structure of bacteriophages and viruses. (A) Bacteriophage T4. (B) Structure of a retrovirus (human immunodeficiency virus causing AIDS).

Table 1.2 Compartments of animal and plant cells and their main functions.

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.

2

Structure and Function of Cellular Macromolecules

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 (). Among the most important of prokaryotic and eukaryotic cells are , , , and , which are constructed from comparatively few (). The (phospholipids, cholesterol) will also be considered in this context because they spontaneously form supramolecular biomembrane structures in the aqueous environment of a cell.