An Introduction to Molecular Biotechnology E-Book

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

Seitenzahl: 1467

Veröffentlichungsjahr: 2020

Beschreibung

Completely updated in line with the rapid progress made in the field, this new edition of the highly-praised textbook addresses powerful new methods and concepts in biotechnology, such as genome editing, reprogrammed stem cells, and personalized medicine.
An introduction to the fundamentals in molecular and cell biology is followed by a description of standard techniques, including purification and analysis of biomolecules, cloning techniques, gene expression systems, genome editing methods, labeling of proteins and in situ-techniques, standard and high resolution microscopy. The third part focuses on key areas in research and application, ranging from functional genomics, proteomics and bioinformatics to drug targeting, recombinant antibodies and systems biology. The final part looks at the biotechnology industry, explaining intellectual property issues, legal frameworks for pharmaceutical products and the interplay between start-up and larger companies. The contents are beautifully illustrated throughout, with hundreds of full color diagrams and photographs.
Provides students and professionals in life sciences, pharmacy and biochemistry with everything they need to know about molecular biotechnology.

Leseprobe

Table of Contents

Cover

An Introduction toMolecular Biotechnology

Copyright

Abbreviations

Part I: Fundamentals of Cellular and Molecular Biology

1 The Cell as the Basic Unit of Life

References

Further Reading

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)

References

Further Reading

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

3.5 Cell Death

References

Further Reading

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)

Further Reading

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, Chloroplasts, and Peroxisomes

5.3 Protein Transport into the Endoplasmic Reticulum

5.4 Vesicle Transport from the ER via the Golgi Apparatus to the Cytoplasmic Membrane

References

Further Reading

6 Evolution and Diversity of Organisms

6.1 Prokaryotes

6.2 Eukaryotes

References

Further Reading

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

Further Reading

8 Mass Spectrometry and Applications in Proteomics and Microbial Identification

8.1 Principles of ESI and MALDI Mass Spectrometry

8.2 Instrumental Setup

8.3 Intact Protein Analysis

8.4 Protein and Proteome Quantification

8.5 Protein–Protein Interaction Analysis

8.6 Analysis of Posttranslational Modifications

8.7 Microbial Identification and Resistance Detection

References

9 Isolation of DNA and RNA

9.1 Introduction

9.2 DNA Isolation

9.3 RNA Isolation

Reference

10 Chromatography and Electrophoresis of Nucleic Acids

10.1 Introduction

10.2 Chromatographic Separation of Nucleic Acids

10.3 Electrophoresis

Further Reading

11 Hybridization of Nucleic Acids

11.1 Significance of Base Pairing

11.2 Experimental Hybridization: Kinetic and Thermodynamic Control

11.3 Analytical Techniques

References

Further Reading

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

Further Reading

13 Polymerase Chain Reaction

13.1 Introduction

13.2 PCR Methods

13.3 PCR as a Quantitative Method

13.4 Areas of Application

Further Reading

14 DNA Sequencing

14.1 Introduction

14.2 The Sanger Method

14.3 Pyrosequencing

14.4 Second‐Generation Sequencing: Illumina and Ion Torrent

14.5 Third‐Generation Sequencing Techniques

14.6 The Impact of the DNA Sequencing Technology

References

Further Reading

Websites

15 Cloning Procedures

15.1 Introduction

15.2 Construction of Recombinant Vectors

Further Reading

16 Expression of Recombinant Proteins

16.1 Introduction

16.2 Expression of Recombinant Proteins in Host Organisms

16.3 Expression in Cell‐Free Systems

Further Reading

17 Patch Clamp Method

17.1 Ion Channels

17.2 Technical Requirements of the Patch Clamp Method

17.3 Patch Clamp Configurations

17.4 Applications of the Patch Clamp Method

Reference

Further Reading

18 Cell Cycle Analysis

18.1 Introduction

18.2 Analyzing the Cell Cycle

18.3 Experimental Analysis of the Cell Cycle

Acknowledgments

Further Reading

19 Microscopic Techniques

19.1 Introduction

19.2 Electron Microscopy

19.3 Atomic or Scanning Force Microscopy

19.4 Light Microscopy

19.5 Microscopy in the Living Cell

Further Reading

20 Laser Applications

20.1 Laser Development: A Historical Perspective

20.2 Types of Lasers and Setups

20.3 Properties of Laser Radiation

20.4 Applications

Further Reading

Part III: Key Topics

21 Sequencing the Universe of Life

21.1 What to Sequence?

21.2 Sequencing Projects: Human

21.3 Sequencing Other Species, Environments, …

21.4 Sequencing in the Clinics: Personalizing Oncology

21.5 Sequencing in the Private Sector: Direct to Consumer Testing (DTC)

21.6 The Information Content of a Genome Sequence and Ethical Consequences

References

22 Cellular Systems Biology

22.1 Introduction

22.2 Analysis of Cellular Networks by Top‐Down Approaches

22.3 Overview over Bottom‐Up Modeling of Biochemical Networks

Further Reading

References

23 Protein–Protein and Protein–DNA Interactions

23.1 Protein–Protein Interactions

23.2 Protein–DNA Interactions

References

Further Reading

24 Bioinformatics

24.1 Introduction

24.2 Data Sources

24.3 Sequence Analysis

24.4 Evolutionary Bioinformatics

24.5 Gene Prediction

24.6 Bioinformatics in Transcriptome and Proteome Analysis

24.7 Analysis of Ultraparallel Sequencing Data

24.8 Bioinformatic Software

Further Reading

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

Further Reading

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

References

27 Molecular Diagnostics in Medicine

27.1 Introduction

27.2 Uses of Molecular Diagnostics

27.3 Which Molecular Variations Should be Detected

27.4 Molecular Diagnostic Methods

27.5 Outlook

Further Reading

28 Recombinant Antibodies and Phage Display

28.1 Introduction

28.2 Generation of Specific Recombinant Antibodies

28.3 Production and Purification of Recombinant Antibodies

28.4 Features and Applications of Recombinant Antibodies

28.5 Outlook

Further Reading

References

29 Genetically Modified Mice and Their Impact in Medical Research

29.1 Overview

29.2 Transgenic Mice

29.3 Homologous Recombination: Knockout (Knock‐In) Mice

29.4 Endonuclease‐Based Knockout Mice

29.5 Endonuclease‐Based Knock‐In Mice

29.6 Conditionally Regulated Gene Expression

29.7 Gene Transfer to Subpopulations of Cells

29.8 Impact of Genetically Modified Mice in Biomedicine

29.9 Outlook

Reference

Further Reading

30 Plant Biotechnology

30.1 Introduction

30.2 Gene Expression Control and Genome Editing

30.3 Production of Transgenic Plants

30.4 Selection of Transformed Plant Cells

30.5 Regeneration of Transgenic Plants

30.6 Plant Analysis: Identification and Characterization of Genetically Engineered Plants

Further Reading

31 Biocatalysis in the Chemical Industry

31.1 Introduction

31.2 Bioconversion/Enzymatic Procedures

31.3 Development of an Enzyme for Industrial Biocatalysis

31.4 Fermentative Procedures

References

Part IV: Biotechnology in Industry

32 Industrial Application: Biotech Industry, Markets, and Opportunities

32.1 Historical Overview and Definitions of Concepts

32.2 Areas of Industrial Application of Molecular Biotechnology

32.3 Status Quo of the Biotech Industry Worldwide

33 Patents in the Molecular Biotechnology Industry: Legal and Ethical Issues

33.1 Patent Law

33.2 Ethical and Policy Issues in Biotechnology Patents

33.3 Conclusions

Acknowledgments

34 Drug Approval in the European Union and United States

34.1 Introduction

34.2 Regulation Within the European Union

34.3 Regulation in the United States

34.4 The Advent and Regulation of Biosimilars

34.5 International Regulatory Harmonization

References

35 Emergence of a Biotechnology Industry

Reference

Further Reading

36 The 101 of Founding a Biotech Company

36.1 First Steps Toward Your Own Company

36.2 Employees: Recruitment, Remuneration, and Participation

37 Marketing

37.1 Introduction

37.2 What Types of Deals Are Possible?

37.3 What Milestone or License Fees Are Effectively Paid in a Biotech/Pharma Cooperation?

37.4 PR and IR in Biotech Companies

Further Reading

Websites

Glossary

Index

End User License Agreement

List of Tables

Chapter 1

Table 1.1 Comparison of important biochemical and molecular characteristics o...

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

Table 1.3 Important methodological tools of modern biology.

Chapter 2

Table 2.1 Molecular composition of cells.

Table 2.2 Formation and function of the cellular macromolecules.

Table 2.3 Important fatty acids in membrane lipids.

Table 2.4 Compilation and grouping of the proteinogenic amino acids: two type...

Table 2.5 Important classes of enzymes.

Table 2.6 Many vitamins serve as essential coenzymes for enzyme reactions.

Table 2.7 Nomenclature of DNA and RNA building blocks.

Table 2.8 Enzymes that use DNA as a substrate and are used in genetic enginee...

Chapter 3

Table 3.1 Ion concentrations inside mammalian cells and in the extracellular ...

Table 3.2 Most important hormones in humans.

Table 3.3 The role of adenylyl cyclase and phospholipase C

‐

β in signal ...

Table 3.4 Some functions of trimeric G‐proteins.

Table 3.5 Signal proteins that act via receptor tyrosine kinases.

Table 3.6 Prokaryotic properties of plastids and mitochondria.

Table 3.7 Classification of major animal and human pathogenic viruses.

Table 3.8 Viral oncogenes that may play a part in the emergence of tumors.

Table 3.9 Overview of important cell types in plants and animals.

Chapter 4

Table 4.1 Overview of a few of the genomes that are already sequenced and pub...

Table 4.2 Relation between genome size and the number of genes of a few selec...

Table 4.3 A few characteristics of the human genome.

Table 4.4 Spontaneous DNA damage in a single diploid mammalian cell within 24...

Table 4.5 Genetic diseases, which are associated with defective DNA repair sy...

Table 4.6 Consensus sequences in eukaryotic promoter regions.

Table 4.7 Protein biosynthesis in bacterial ribosomes as a target for antibio...

Chapter 5

Table 5.1 Examples of typical recognition sequences.

Table 5.2 Occurrence of some Rab proteins.

Chapter 6

Table 6.1 Important groups of protists (model organisms or diseases caused by...

Table 6.2 Systematic classification of the land plants.

Table 6.3 Systematic classification of multicellular animals (important phyla...

Chapter 7

Table 7.1 Commonly used protease inhibitors in protein purification.

Table 7.2 Suggestions for choosing ion exchangers when enriching proteins of ...

Table 7.3 Commonly used lectins for the enrichment of glycoproteins.

Chapter 12

Table 12.1 Features of different polymerases.

Chapter 15

Table 15.1 Vectors, heterologous DNA uptake capacity, and host organisms.

Table 15.2 Properties of enzymes for turning sticky into blunt DNA ends.

Table 15.3 Commonly used cytostatic or cytotoxic selection markers.

Table 15.4 Commonly used antigenic fusion components (tags).

Table 15.5 Viral expression systems for mammal cells.

Chapter 16

Table 16.1 Comparison of prokaryotic and eukaryotic host organisms for the ex...

Table 16.2 Comparison of the characteristics of the two most common modificat...

Table 16.3 Overview of problems for protein expression in

E. coli

.

Table 16.4 Typical properties of some yeast expression systems.

Table 16.5 Typical properties of some important mammalian cell lines.

Chapter 23

Table 23.1 Selected databases and Internet resources.

Table 23.2 Important biochemical and biophysical methods to examine protein–D...

Table 23.3 Selected DNA‐ and RNA‐binding domains in the human genome and in t...

Chapter 25

Table 25.1 Diagram of the position of common sequence motifs in members of th...

Table 25.2 Target validation.

Table 25.3 Screening methods that can be accomplished in high throughput.

Table 25.4 Overview of preclinical and clinical drug development.

Chapter 26

Table 26.1 Example antibodies in therapeutic use.

Chapter 28

Table 28.1 Clinically approved monoclonal antibody products.

Chapter 31

Table 31.1 Biocatalytic processes.

Table 31.2 The 15 top‐selling recombinant proteins (million US$) in Aggarwal ...

Table 31.3 Selected bioconversions.

Table 31.4 Annual production volumes of different bioconversions.

Chapter 32

Table 32.1 Selected examples of in Germany/EU in 2019 newly registered biopha...

Table 32.2 Selected therapeutic antibodies by highest rank of sales (US$ mill...

Table 32.3 Selected examples of therapeutic RNAs on the market or under devel...

Table 32.4 Selected biotech companies with gene therapy programs.

Table 32.5 Selected biotech companies with T‐cell therapy programs.

Chapter 34

Table 34.1 The volumes comprising the rules governing medicinal products with...

Table 34.2 Product categories regulated by the FDA.

Table 34.3 Major biotechnology/biological‐based drug types regulated by CDER ...

Table 34.4 ICH guidelines that specifically focus upon products of pharmaceut...

Chapter 35

Table 35.1 Differences between biotech and big pharma companies.

Chapter 36

Table 36.1 Business attitude and experience.

Table 36.2 Fields of competence and necessary qualifications in a startup com...

Chapter 37

Table 37.1 Potential revenue for the sale of developed compounds.

List of Illustrations

Chapter 1

Figure 1.1 Tree of life – phylogeny of life domains.

Figure 1.2 Schematic structure of prokaryotic and eukaryotic cells. (a) Bact...

Figure 1.3 Schematic structure of bacteriophages and viruses. (a) Bacterioph...

Chapter 2

Figure 2.1 Composition and structure of sugar molecules. (a) Structures of t...

Figure 2.2 Structure of the cytoplasmic membrane. Schematic diagram of the l...

Figure 2.3 Structures of important phospholipids. Phosphatidylcholine, phosp...

Figure 2.4 Chemical structure of cerebrosides (glycolipids). (a) Galactocere...

Figure 2.5 Cholesterol and related sterols. Cholesterol;

β

‐sitosterol r...

Figure 2.6 General structure of amino acids and peptides.

Figure 2.7 Structures of proteinogenic amino acids. (Cysteine muss zu den am...

Figure 2.8 Important hydrogen bonds in biomolecules.

Figure 2.9 Noncovalent bonds and disulfide bridges lead to a spatial folding...

Figure 2.10 Folding of peptide chains under aqueous conditions leads to a co...

Figure 2.11 Importance of hydrogen bonds for the construction of

α

‐heli...

Figure 2.12 Size of proteins in yeast (

Saccharomyces cerevisiae

). The yeast ...

Figure 2.13 Structure of Src protein with four domains. The four domains are...

Figure 2.14 Occurrence of domains in different proteins.

Figure 2.15 Structure of binding sites within proteins. (a) Schematic illust...

Figure 2.16 Reversible activation and inactivation of enzymes and regulatory...

Figure 2.17 Structure of nucleotides. (a) Structures of purine and pyrimidin...

Figure 2.18 Linear structure of DNA and RNA. In nucleic acid biosynthesis, t...

Figure 2.19 Structure of the DNA double helix. The spatial orientation of th...

Figure 2.20 Structure of RNA molecules. (A) Yeast tRNA. The base sequence is...

Figure 2.21 Structure and function of a hammerhead ribozyme.

Chapter 3

Figure 3.1 Mobility of phospholipids in a biomembrane. Three types of moveme...

Figure 3.2 Vesicle and liposome formation. (a) In an aqueous environment, li...

Figure 3.3 Asymmetric structure of biomembranes.

Figure 3.4 Permeability of artificial lipid membranes for biologically relev...

Figure 3.5 Important membrane proteins and transport processes. (a) Schemati...

Figure 3.6 Glucose transporters in an intestinal cell. Glucose is pumped fro...

Figure 3.7 Schematic view of communication pathways between cells. (a) Endoc...

Figure 3.8 Schematic representation of receptor classes on the cell surface....

Figure 3.9 Activation of adenylyl cyclase and formation from cAMP as second ...

Figure 3.10 Role of phospholipase C‐

β

in the production of second messe...

Figure 3.11 Signal transduction after activation of G‐protein and enzyme‐lin...

Figure 3.12 Schematic representation of the endomembrane system of the cell:...

Figure 3.13 Similarities of lysosomes and plant vacuoles. (a) Schematic stru...

Figure 3.14 Composition of a mitochondrion. (a) Electron microscope photogra...

Figure 3.15 Function of mitochondrion: metabolism and respiratory chain. (a)...

Figure 3.16 Schematic overview of the arrangement of genes in the mtDNA of m...

Figure 3.17 Development of an early eucyte and origin of mitochondria. α‐Pur...

Figure 3.18 Structure of a chloroplast. (a) Electron microscope photo of a c...

Figure 3.19 Essential steps in photosynthesis. (a) Overview of photosyntheti...

Figure 3.20 Overview of the arrangement of genes in chloroplast genomes.

Figure 3.21 Development of chloroplasts through phagocytosis of cyanobacteri...

Figure 3.22 Synopsis of the breakdown pathways and energy‐producing pathways...

Figure 3.23 Importance of glycolysis and the citric acid cycle as a point of...

Figure 3.24 Schematic composition of actin filaments (microfilaments).

Figure 3.25 Mechanism of muscle contraction. (a) Molecular mechanism of musc...

Figure 3.26 Schematic view of microtubules and cilia structures. Tubulin dim...

Figure 3.27 Schematic view of bacterial cell walls. (a) Gram‐positive bacter...

Figure 3.28 Infection cycle and genome of retroviruses. (a) Genome compositi...

Figure 3.29 Schematic outline of apoptotic pathways.

Chapter 4

Figure 4.1 Number of nucleotides in the haploid genomes of important groups ...

Figure 4.2 Composition of eukaryotic genomes and a fraction of a few DNA ele...

Figure 4.3 Schematic illustration of human chromosomes. The indentations ind...

Figure 4.4 Important structural elements of chromosomes necessary for the re...

Figure 4.5 Principle of telomere replication. The telomerase exhibits an RNA...

Figure 4.6 From the nucleosome to the condensed metaphase chromosome. The DN...

Figure 4.7 Schematic overview of mitosis and meiosis.

Figure 4.8 Schematic summary of DNA replication.

SSB

,

single‐strand binding

...

Figure 4.9 Asymmetric composition of replication bubbles. DNA is unwound at ...

Figure 4.10 Depurination, deamination, oxidation, and dimerization as exampl...

Figure 4.11 Consequences of deamination, depurination, and oxidation. Cytidi...

Figure 4.12 Base pairing of tautomeric DNA bases. The correct base pairings ...

Figure 4.13 Consequences of gene mutations.

Figure 4.14 Inheritance of mutations leading to the loss of protein function...

Figure 4.15 From gene to protein: comparison of prokaryotes and eukaryotes. ...

Figure 4.16 Schematic overview of the function of RNA polymerase and transcr...

Figure 4.17 Simplified schematic illustration of the control of gene express...

Figure 4.18 Structure of a eukaryotic gene. NCS, noncoding sequence.

Figure 4.19 Schematic representation of alternative splicing processes. The ...

Figure 4.20 Differences between genetic and epigenetic inheritance.

Figure 4.21 Structure of RNA cassettes and synthesis of rRNA.

ITS

s,

internal

...

Figure 4.22 Structure of (a) prokaryotic and (b) eukaryotic ribosomes. For t...

Figure 4.23 Schematic illustration of protein biosynthesis in ribosomes. Thr...

Figure 4.24 Loading tRNA with an amino acid. First the amino acid is activat...

Figure 4.25 rRNA‐catalyzed peptide transfer in ribosomes. (a) Possible react...

Chapter 5

Figure 5.1 Schematic overview of protein transport inside a cell.

Figure 5.2 Structure of a nuclear pore (reconstructed from electron microsco...

Figure 5.3 Simplified model of the import and export of proteins via the nuc...

Figure 5.4 Schematic overview of the uptake of a precursor protein by the mi...

Figure 5.5 Simplified scheme of the import of a protein into the ER lumen.

Figure 5.6 Simplified scheme of the integration of a membrane protein into t...

Figure 5.7 Assembly of glycoproteins in the ER. The oligosaccharide exists a...

Figure 5.8 Vesicle transport pathways in the cell.

Figure 5.9 Structure of clathrin‐coated vesicles: (a) electron micrograph an...

Figure 5.10 Schematic progression of receptor‐mediated endocytosis of LDL....

Chapter 6

Figure 6.1 A phylogenetic tree of life, showing the relationship between spe...

Figure 6.2 Phylogenetic relationships between protists and transition to pla...

Figure 6.3 Phylogeny of land plants.

Figure 6.4 Phylogeny of Deuterostomia and vertebrates.

Figure 6.5 Evolutionary trends in animal phylogeny.

Chapter 7

Figure 7.1 SDS gel electrophoresis. (a) Denaturing effect of SDS. (b) Setup ...

Figure 7.2 Size exclusion chromatography. (a) Time course of size exclusion ...

Figure 7.3 Anion exchange chromatography. Illustration of the time course of...

Figure 7.4 Purification of NDPK with a Cibacron Blue‐Sepharose column. Plot ...

Figure 7.5 Purification of His

6

‐RGS16: Coomassie Blue R‐250 stain of a 15% S...

Chapter 8

Figure 8.1 Key features of a mass spectrum: (a) natural isotope pattern of a...

Figure 8.2 Setup of a tandem mass spectrometer allowing the recording of MS1...

Figure 8.3 Collision‐induced fragment ion spectrum of the peptide FSGSGSGTSY...

Figure 8.4 Metabolic stable isotope labeling. (a) Schematic setup of a SILAC...

Figure 8.5 Label‐based quantification strategies in quantitative proteomics ...

Figure 8.6 Identification of specific protein interaction partners by Co‐IP,...

Figure 8.7 MALDI‐TOF fingerprinting of microorganisms. (a) Generation and an...

Chapter 9

Figure 9.1 Mammalian chromosomal DNA in solution (right) precipitated after ...

Figure 9.2 Separated plasmid DNA after ultracentrifugation in a CsCl–EtBr gr...

Figure 9.3 Scheme of DNA purification for prokaryotes or eukaryotes using a ...

Chapter 10

Figure 10.1 Agarose gel electrophoresis of plasmid DNA in the presence of Et...

Chapter 11

Figure 11.1 Classical setup of a Southern blot after.

Figure 11.2 Genetic analysis of transgenic mice by Southern blotting. Genomi...

Figure 11.3 Analysis of gene expression in two strains of transgenic mice (2...

Figure 11.4 Result of the expression screening of thousands of genes using l...

Figure 11.5 FISH in chromosome preparations. (a) Detection of a deletion in ...

Figure 11.6 ISH of two developmental genes (

even skipped

[blue] and

fushi ta

...

Chapter 12

Figure 12.1 The discovery of restriction endonucleases such as HindIII was a...

Figure 12.2 Palindromic sequence recognized by a restriction enzyme. The sym...

Figure 12.3 Restriction sites of the restriction enzymes XbaI, AluI, and Pst...

Figure 12.4 In order to incorporate nucleotides, a polymerase requires a DNA...

Chapter 13

Figure 13.1 Schematic outline of PCR. (a) Basic principle: double‐stranded D...

Figure 13.2 Increase in DNA copies, determined by using quantitative real‐ti...

Figure 13.3 Schematic representation of the quantitative real‐time detection...

Chapter 14

Figure 14.1 Schematic representation of the Sanger sequencing technique. The...

Figure 14.2 Schematic representation of the pyrosequencing technique. (a) Nu...

Figure 14.3 Schematic representation of the Illumina sequencing system. (a) ...

Figure 14.4 Schematic representation of the Ion Torrent sequencing system. (...

Chapter 15

Figure 15.1 Cloning, amplification, and selection of heterologous DNA in hos...

Chapter 16

Figure 16.1 Which organism for recombinant protein expression?

Figure 16.2 Growth and protein induction in an

E. coli

culture using an...

Figure 16.3 Life cycle of wild‐type and recombinant baculoviruses. (a) After...

Chapter 17

Figure 17.1 Patch clamp setup. Motorized micromanipulators (a) are mounted o...

Figure 17.2 Working principle of a patch clamp amplifier and the effect of t...

Figure 17.3 Patch clamp configurations. When the patch pipette touches the c...

Figure 17.4 Paired whole‐cell recording of pyramidal neurons from mouse cere...

Chapter 18

Figure 18.1 The cell cycle and its phases in

S. cerevisiae

.

Figure 18.2 Regulation of the cell cycle in the yeast

S. cerevisiae

. Ad...

Figure 18.3 Elutriation – schematic view.

Figure 18.4 Mating cycle of

S. cerevisiae

. The presence of a‐ and α‐fac...

Figure 18.5 DAPI staining and

differential interference contrast

(

DIC

) micro...

Figure 18.6 Cell cycle profiles after DNA staining and

fluorescence‐activate

...

Figure 18.7 Schematic configuration of a laser scanning microscope.

PMT

,

pho

...

Figure 18.8 Plotting the progression throughout the cell cycle in yeast cell...

Figure 18.9 Visualization of cell cycle phases using the FUCCI expression an...

Chapter 19

Figure 19.1 Layout of optical components in a basic TEM.

Figure 19.2 Functional principle of the AFM. The scan table moves the sample...

Figure 19.3 Functional principle of the confocal microscope. Through the bea...

Chapter 20

Figure 20.1 Setup of a ruby laser.

Figure 20.2 Effect of optical tweezers or trap on an object.

Chapter 21

Figure 21.1 Cost estimate for sequencing of a single human genome and its pr...

Figure 21.2 Just a minor fraction of the human genome encodes proteins (i.e....

Figure 21.3 ENCODE encyclopedia of DNA Elements. The goal of ENCODE is to bu...

Figure 21.4 Major types of variation found in genomes. A lot of such variati...

Chapter 22

Figure 22.1 The three networks of a cell.

Figure 22.2 A linear regression function (red line) is fitted to the express...

Figure 22.3 The machine learning system needs features of the network, the g...

Figure 22.4 (a) An example of a simple network. Knocking out reaction (22.3)...

Figure 22.5 TCA cycle and glyoxylate shunt of

E. coli

for the example in the...

Figure 22.6 Numerical simulation of the Michaelis–Menten equations, (a) fast...

Figure 22.7 Stimulus response of the Hill equation for increasing Hill expon...

Figure 22.8 Model of the MAPK signaling pathway. (a) Schematic representatio...

Figure 22.9 Euler integration scheme for two consecutive time steps. Note ho...

Figure 22.10 Linear regression and parameter estimation. (a) The true output...

Figure 22.11 Schematic representation of the signaling pathway leading to ca...

Figure 22.12 Caspase‐3 levels can reach two different steady states, dependi...

Figure 22.13 Phase‐space plot of Eq. (22.21). The rate of change

d

C3/

dt

is p...

Figure 22.14 The steady states (stable, solid line; unstable, dotted line) o...

Figure 22.15 Architecture of an autocatalytic positive feedback of the rtTA...

Figure 22.16 Mutual inhibition of two molecules on transcription (a) and pro...

Figure 22.17 Simulation of the mutual inhibition mechanism (Eq. (22.23)). Pa...

Chapter 23

Figure 23.1 Protein domains of the Src oncoprotein. The Src protein has thre...

Figure 23.2 RNA polymerase II, a multimeric protein complex. (a) Crystal str...

Figure 23.3 Protein interaction network of

Helicobacter pylori

. This map was...

Figure 23.4 Selected methods for the study of protein–protein interactions. ...

Figure 23.5 Predicting protein–protein interactions using docking and evolut...

Figure 23.6 The NF‐κB signaling pathway as an example for protein–protein an...

Figure 23.7 Crystal structure of the Zinc uptake regulator (Zur) in complex ...

Figure 23.8 Watson–Crick pairing and hydrogen bond pattern of the 2 bp A–T a...

Figure 23.9 ChIP‐Seq, a global method to map binding sites of DNA‐binding pr...

Figure 23.10 Network representation of transcriptional regulation (a) transc...

Figure 23.11 Crystal structure of EthR from

Mycobacterium tuberculosis

. This...

Figure 23.12 Schematic representation of the Cas9 endonuclease with its gui...

Chapter 24

Figure 24.1 Kyte–Doolittle plot of bacteriorhodopsin from

Halobacterium

spp....

Figure 24.2 Part of a multiple alignment of sequences of the a subunit of ca...

Figure 24.3 Jukes–Cantor model. Each single nucleotide changes to any other ...

Figure 24.4 Consequences of time reversibility. Two actual sequences, 1 and ...

Figure 24.5 Multiple substitutions. Several types of multiple substitutions ...

Figure 24.6 Operating principle of a simple HMM. In this case, there are onl...

Figure 24.7 Results of a classification experiment. Bone marrow samples from...

Chapter 25

Figure 25.1 Distribution of targets of known therapeutic agents over protein...

Figure 25.2 Domain structure of GPCRs. H1–H7 are the seven

α

‐helices, e...

Figure 25.3 Target validation pyramid. Genomic methods (sequence analysis, e...

Figure 25.4 Filter‐binding and FRET assays. The top part shows a FRET assay ...

Figure 25.5 Potency and efficiency. Compounds A and B are similar in potency...

Chapter 26

Figure 26.1 EPR effect. Drug carriers permeate through the pathologically ch...

Figure 26.2 Schematic diagram to illustrate physical targeting. The active s...

Figure 26.3 Structure of liposomes that can be used for drug targeting. (a) ...

Figure 26.4 Prodrug principle. The free drug cannot cross a membrane barrier...

Figure 26.5 Phenytoin and fosphenytoin. Fosphenytoin is around 40 times more...

Figure 26.6 Prodrugs of ampicillin. The functional acid group makes it diffi...

Figure 26.7 Pivaloyloxyethyl ester of methyldopa. Esterification greatly imp...

Figure 26.8 Dipivefrin, a dipivalyl ester of epinephrine, is used to treat g...

Figure 26.9 Targeting of the CNS using a redox‐based prodrug system. The dru...

Figure 26.10 Azo prodrugs of aminosalicylic acid, used to treat inflammatory...

Figure 26.11 Dexamethasone‐21‐β‐

D

‐glucoside. Following administration, up to...

Figure 26.12 Ftorafur [1‐(2‐tetrahydrofuranyl)‐5‐fluorouracil]. This has an ...

Chapter 27

Figure 27.1 Overview of mutations in a protein‐coding gene that can influenc...

Figure 27.2 Mutation of a single nucleotide. A given nucleic acid sequence (...

Figure 27.3 Mutations through repeat expansion or reduction. The repeat of a...

Figure 27.4 Gene duplication. In a few cases the duplication of an entire ge...

Figure 27.5 Epigenetics: DNA methylation. The expression of a gene can also ...

Figure 27.6 Overview of PCR‐based approaches for the detection of target seq...

Figure 27.7 DNA microarrays: the principle. DNA microarrays are a further de...

Figure 27.8 PCR detection of a length polymorphism. Length insertions and de...

Figure 27.9 RFLP. If a mutation disrupts a given restriction enzyme recognit...

Figure 27.10 ARCS. If the mutation of interest does not alter a restriction ...

Figure 27.11 ARMS. PCR analysis of the negative influence of a mismatch on t...

Figure 27.12 Minisequencing. If, in a sequence reaction, instead of a mixtur...

Chapter 28

Figure 28.1 Schematic (a) and crystal (b) structure of an immunoglobulins ga...

Figure 28.2 Experimental flowchart for the production of an antibody gene li...

Figure 28.3 Different selection systems for human antibodies based on recomb...

Figure 28.4 Phage display using M13K07 or Hyperphage. After electroporating ...

Figure 28.5 From people to people: the human antibody generation cycle. With...

Figure 28.6 Modes of action of various antibody‐based anticancer therapies. ...

Figure 28.7 Numbers of antibody‐based therapeutics approved by FDA and

Europ

...

Chapter 29

Figure 29.1 First example of a human gene (growth hormone gene) expressed in...

Figure 29.2 Experimental flowchart. All gene manipulations are performed in ...

Figure 29.3 Gene manipulations in early mouse embryos. Holding pipette (H), ...

Figure 29.4 Examples for strategies to make a gene accessible for Cre‐mediat...

Figure 29.5 Chimeric founders. The efficiency of ES cell integration into C5...

Figure 29.6 Gene editing. Gene editing by CRISPR/Cas9 works in every strain ...

Figure 29.7 Conditional gene expression in “compound transgenic” mice. (a, C...

Figure 29.8 Virus‐mediated Venus expression in the mouse brain. Expression o...

Chapter 30

Figure 30.1 Expression cassette for plant transformation and examples for pl...

Figure 30.2 Binary vector for plant transformation. The binary vector system...

Figure 30.3 Cre/lox‐based DNA excision. The loxP (locus of excision) system ...

Chapter 31

Figure 31.1 Biotechnological processes can differentiate between fermentatio...

Figure 31.2 Screening of strain collections can make new enzymes available. ...

Figure 31.3 In addition to classical screening of culturable microorganisms,...

Figure 31.4 Nitrilases are suitable biocatalysts for the production of optic...

Figure 31.5 Reaction mechanism of lipase.

Figure 31.6 Lipase‐catalyzed racemic resolution of amines gives access to an...

Figure 31.7 Directed evolution increases the stability of pyruvate decarboxy...

Figure 31.8

n

‐Butanol. Using metabolic engineering, pathways for the synthes...

Figure 31.9 Systematic representation of glutamate biosynthesis in

C. glutam

...

Figure 31.10 Influence of penicillin on glutamate formation and on the enzym...

Figure 31.11 Selection of feedback‐deregulated mutants with antimetabolites....

Chapter 34

Figure 34.1 Simplified structural overview of the EMA.

Figure 34.2 Overview of the EU centralized procedure. Refer to text for deta...

Figure 34.3 Partial organizational structure of the FDA.

Figure 34.4 Summary overview of the main points during a drug's lifetime at ...

Chapter 35

Figure 35.1 From descriptive biology towards microbiology.

Figure 35.2 Medical and genetic discoveries in the first half of the twentie...

Figure 35.3 1953–1976: from molecular genetics toward genetic engineering....

Figure 35.4 From genetic engineering toward biotechnology.

Figure 35.5 From biotechnology toward genomics.

Guide

Glossary

Cover Page

Title Page

Copyright

Abbreviations

Table of Contents

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Index

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An Introduction toMolecular Biotechnology

Fundamentals, Methods and Applications

Edited by MichaelWink

Copyright

Editor

Michael Wink

Universität Heidelberg

Institut für Pharmazie und Molekulare

Biotechnologie (IPMB)

Im Neuenheimer Feld 329

69120 Heidelberg

Germany

Cover credits

Shutterstock #287352494 / paulista

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© 2021 WILEY‐VCH GmbH, Boschstr. 12, 69469 Weinheim, Germany

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Cover Design Formgeber, Mannheim, Germany

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 prodrug 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 immunodeficiency 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 remodeling complex

CCD

charge‐coupled device

CDER

Center for Drug Evaluation and Research

CDK

cyclin‐dependent kinase

cDNA

copy DNA

CDR

complementarity‐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 myeloid leukemia

CMN

Corynebacterium–Mycobacterium–Nocardia

group

CaMV

cauliflower mosaic virus

CMV

cytomegalovirus

CNS

central nervous system

COMP

Committee for 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 organization

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

ethylene glycol bis(2‐aminoethyl)tetraacetic acid

EIAV

equine infectious anemia 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

Δ

G

d

free enthalpy

GDH

glutamate dehydrogenase

GDP

guanosine diphosphate

GEF

guanine exchange factor

GEO

gene expression omnibus

GFP

green fluorescent 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 papillomavirus

HSP

high‐scoring segment pairs

HSP

heat shock protein

HSV‐1

herpes simplex virus

HTS

high‐throughput analysis

HUGO

Human Genome Organization

HV

herpesvirus

IAS

international accounting standard

ICDH

isocitric dehydrogenase

ICH

International Council for Harmonization of Technical Requirements for 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‐β‐

D

‐thiogalactoside

IRs

inverted repeats

IR

investor relations

IRES

internal ribosome entry site

ISAAA

International Service for the Acquisition of Agri‐biotech Applications

ISH

in situ

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–Fourier 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

multidrug 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 Sequencing

Mreb/Mbl

proteins of prokaryotic cytoskeleton

mRNA

messenger RNA

MRSA

methicillin‐resistant

Staphylococcus 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

nonsteroidal 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 arbitrarily 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

R

f

‐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

tetracycline‐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 electrophoresis

SELEX

systematic evolution of ligands 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 conformation polymorphism

ssDNA

single‐stranded DNA

SSH

suppression 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

Tc

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 v‐SNARE 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‐β‐

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

Part IFundamentals of Cellular and Molecular Biology

1The Cell as the Basic Unit of Life

Michael Wink

Heidelberg University, Institute of Pharmacy and Molecular Biotechnology (IPMB), Im Neuenheimer Feld 329, 69120 Heidelberg, Germany

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 Rudolf Virchow conceived the law of omnis cellula e cellula (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 (Figure 1.1). We see an astonishing constancy in fundamental structures and mechanisms. Owing to this, it is possible to limit the discussion of the general characteristics of a cell to a few basic types (Figure 1.2):

Bacterial cells

Plant cells

Animal cells

Figure 1.1 Tree of life – phylogeny of life domains.

Figure 1.2 Schematic structure of prokaryotic and eukaryotic cells. (a) Bacterial cell, (b) plant mesophyll cell, and (c) animal cell.

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).

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

Character

Prokaryotes

Eukaryotes

Archaea

Bacteria

Organization

Unicellular

Unicellular

Unicellular or multicellular

Cytology

Internal membranes

Rare

Rare

Always (Table 

1.2

)

Compartments

Only cytoplasm

Only cytoplasm

Several (Table 

1.2

)

Organelles

No

No

Mitochondria; plastids

Ribosomes

70S

70S

80S (mt, cp: 70S)

Membrane lipids

Ether lipids

Ester lipids, hopanoids

Ester lipids, sterols

Cell wall

Pseudopeptidoglycan, polysaccharides, glycoproteins

Murein (peptidoglycan), polysaccharides, proteins

PL: polysaccharides, cellulose F: chitin A: no

Cytoskeleton

FtsZ and MreB protein

FtsZ and MreB protein

Tubulin, actin, intermediary filaments

Cell division

Binary fission

Binary fission

Mitosis

Genetics

Nuclear structure

Nucleoid

Nucleoid

Membrane‐enclosed nucleus with chromosomes

Recombination

Similar to conjugation

Conjugation

Meiosis, syngamy

Chromosome

Circular, single

Circular, single

Linear, several

Introns

Rare

Rare

Frequent

Noncoding DNA

Rare

Rare

Frequent

Operon

Yes

Yes

No

Extrachromosomal

DNA plasmids (linear)

Plasmids (circular)

mtDNA, cpDNA, plasmids in fungi

Transcription/translation

Concomitantly

Concomitantly

Transcription in nucleus, translation in cytoplasm

Promotor structure

TATA box

−35 and −10 sequences

TATA box

RNA polymerases

Several (8–12 subunits)

1 (4 subunits)

3 (with 12–14 subunits)

Transcription factors

Yes

No (sigma factor)

Yes

Initiator tRNA

Methionyl‐tRNA

N

‐Formylmethionyl‐tRNA

Methionyl‐tRNA

Cap structure of mRNA polyadenylation

No

No

Yes

PL, plants; F, fungi; A, animals; mt, mitochondria; cp, plastid.

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

Compartment

Occurrence

Functions

Nucleus

A

P

Harbors chromosomes, site of replication, transcription, and assembly of ribosomal subunits

Endoplasmic reticulum

(

ER

)

   Rough ER

A

P

Posttranslational modification of proteins

   Smooth ER

A

P

Synthesis of lipids and lipophilic substances

Golgi apparatus

A

P

Posttranslational modification of proteins, modification of sugar chains

Lysosome

A

Harbors hydrolytic enzymes, degrades organelles and macromolecules, macrophages eat invading microbes

Vacuole

P

Sequestration of storage proteins, defense and signal molecules, contains hydrolytic enzymes, degrades organelles and macromolecules

Mitochondrium

A

P

Organelle derived from endosymbiotic bacteria; contains circular DNA, own ribosomes; enzymes of citric acid cycle, β‐oxidation, and respiratory chain (ATP generation)

Chloroplast

P

Organelle derived from endosymbiotic bacteria; contains circular DNA, own ribosomes; chlorophyll and proteins of photosynthesis, enzymes of CO

2

fixation and glucose formation (Calvin cycle)

Peroxisome

A

P

Contains enzymes that generate and degrade H

2

O

2

Cytoplasm

A

P

Harbors all compartments, organelles, and the cytoskeleton of a cell; many enzymatic pathways (e.g. glycolysis) occur in the cytoplasm

A, animal; P, plant.

A highly resolved tree of life is based on completely sequenced genomes (Ciccarelli 2006). The image was generated using Interactive Tree Of Life (iTOL) (Letunic 2007), an online phylogenetic tree viewer and Tree of Life resource. Eukaryotes are colored red, archaea green, and bacteria blue.

The most important biochemical and cell biological characters of Archaea, Bacteria, and Eukarya are summarized in Table 1.1.

As viruses and bacteriophages (Figure 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 structures are closely linked to that of the host cell.

Figure 1.3 Schematic structure of bacteriophages and viruses. (a) Bacteriophage T4 and (b) structure of a retrovirus (human immunodeficiency virus causing AIDS).

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.