Principles of Gene Manipulation and Genomics

, by Primrose, Sandy B.; Twyman, Richard

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ISBN: 9781405135443 | 1405135441
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Copyright: 2/17/2006

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There has been rapid progress in recombinant DNA technology over the last few years, particularly in the area of large-scale biology (genomics and related disciplines). The increasing integration between gene manipulation and genomics is embraced in this book, Principles of Gene Manipulation and Genomics, which brings together for the first time the subjects covered by the best-selling books Principles of Gene Manipulation and Principles of Genome Analysis and Genomics. This important new text has been comprehensively revised, updated, and rewritten to encompass within one volume, basic and advanced gene-manipulation techniques, genome analysis, genomics, transcriptomics, proteomics, and metabolomics, as well as the applications of these fascinating technologies. Principles of Gene Manipulation and Genomics is an accessible and up-to-date guide to this exciting field, and is the essential reference for upper-level undergraduate and graduate students of genetics, genomics, molecular biology, and recombinant DNA technology.

Preface

xviii

Abbreviations

Gene manipulation in the post-genomics era

(14)

Introduction

(1)

Gene manipulation involves the creation and cloning of recombinant DNA

(5)

Recombinant DNA has opened new horizons in medicine

(1)

Mapping and sequencing technologies formed a crucial link between gene manipulation and genomics

(2)

The genomics era began in earnest in 1995 with the complete sequencing of a bacterial genome

(2)

Genome sequencing greatly increases our understanding of basic biology

(1)

The post-genomics era aims at the complete characterization of cells at all levels

(1)

Recombinant DNA technology and genomics form the foundation of the biotechnology industry

(1)

Outline of the rest of the book

(7)

Part I Fundamental Techniques of Gene Manipulation

Basic techniques

(21)

Introduction

(1)

Three technical problems had to be solved before in vitro gene manipulation was possible on a routine basis

(1)

A number of basic techniques are common to most gene-cloning experiments

(1)

Gel electrophoresis is used to separate different nucleic acid molecules on the basis of their size

(2)

Blotting is used to transfer nucleic acids from gels to membranes for further analysis

(1)

Southern blotting is the method used to transfer DNA from agarose gels to membranes so that the compositional properties of the DNA can be analyzed

(1)

Northern blotting is a variant of Southern blotting that is used for RNA analysis

(1)

Western blotting is used to transfer proteins from acrylamide gels to membranes

(5)

A number of techniques have been devised to speed up and simplify the blotting process

(1)

The ability to transform E. coli with DNA is an essential prerequisite for most experiments on gene manipulation

(1)

Electroporation is a means of introducing DNA into cells without making them competent for transformation

(1)

The ability to transform organisms other than E. coli with recombinant DNA enables genes to be studied in different host backgrounds

(1)

The polymerase chain reaction (PCR) has revolutionized the way that biologists manipulate and analyze DNA

(1)

The principle of the PCR is exceedingly simple

(1)

RT-PCR enables the sequences on a mRNA molecule to be amplified as DNA

(1)

The basic PCR is not efficient at amplifying long DNA fragments

(1)

The success of a PCR experiment is very dependent on the choice of experimental variables

(1)

By using special instrumentation it is possible to make the PCR quantitative

(1)

There are a number of different ways of generating fluorescence in quantitative PCR reactions

(3)

It is now possible to amplify whole genomes as well as gene segments

(2)

Cutting and joining DNA molecules

(19)

Cutting DNA molecules

(8)

Understanding the biological basis of host-controlled restriction and modification of bacteriophage DNA led to the identification of restriction endonucleases

(1)

Four different types of restriction and modification (R-M) system have been recognized but only one is widely used in gene manipulation

(2)

The naming of restriction endonueleases provides information about their source

(1)

Restriction enzymes cut DNA at sites of rotational symmetry and different enzymes recognize different sequences

(2)

The G+C content of a DNA molecule affects its susceptibility to different restriction endonucleases

(1)

Simple DNA manipulations can convert a site for one restriction enzyme into a site for another enzyme

(1)

Methylation can reduce the susceptibility of DNA to cleavage by restriction endonucleases and the efficiency of DNA transformation

(1)

It is important to eliminate restriction systems in E. coli strains used as hosts for recombinant DNA

(1)

The success of a cloning experiment is critically dependent on the quality of any restriction enzymes that are used

(1)

Joining DNA molecules

(11)

The enzyme DNA ligase is the key to joining DNA molecules in vitro

(4)

Adaptors and linkers are short double-stranded DNA molecules that permit different cleavage sites to be interconnected

(1)

Homopolymer tailing is a general method for joining DNA molecules that has special uses

(1)

Special methods are often required if DNA produced by PCR amplification is to be cloned

(1)

DNA molecules can be joined without DNA ligase

(1)

Amplified DNA can be cloned using in vitro recombination

(5)

Basic biology of plasmid and phage vectors

(20)

Plasmid biology and simple plasmid vectors

(11)

The host range of plasmids is determined by the replication proteins that they encode

(1)

The number of copies of a plasmid in a cell varies between plasmids and is determined by the regulatory mechanisms controlling replication

(2)

The stable maintenance of plasmids in cells requires a specific partitioning mechanism

(1)

Plasmids with similar replication and partitioning systems cannot be maintained in the same cell

(1)

The purification of plasmid DNA

(2)

Good plasmid cloning vehicles share a number of desirable features

(1)

pBR 322 is an early example of a widely used, purpose-built cloning vector

(2)

Example of the use of plasmid pBR 322 as a vector: isolation of DNA fragments which carry promoters

(1)

A large number of improved vectors have been derived from pBR 322

(2)

Bacteriophage λ

(5)

The genetic organization of bacteriophage λ favors its subjugation as a vector

(1)

Bacteriophage λ has sophisticated control circuits

(3)

There are two basic types of phage λ vectors: insertional vectors and replacement vectors

(1)

A number of phage λ vectors with improved properties have been described

(1)

By packaging DNA into phage λ in vitro it is possible to eliminate the need for competent cells of E. coli

(1)

DNA cloning with single-stranded DNA vectors

(4)

Filamentous bacteriophages have a number of unique properties that make them suitable as vectors

(1)

Vectors with single-stranded DNA genomes have specialist uses

(1)

Phage M13 has been modified to make it a better vector

(3)

Cosmids, phasmids, and other advanced vectors

(21)

Introduction

(1)

Vectors for cloning large fragments of DNA

(6)

Cosmids are plasmids that can be packaged into bacteriophage λ particles

(1)

BACs and PACs are vectors that can carry much larger fragments of DNA than cosmids because they do not have packaging constraints

(3)

Recombinogenic engineering (recombineering) simplifies the cloning of DNA, particularly with high-molecular-weight constructs

(2)

A number of factors govern the choice of vector for cloning large fragments of DNA

(1)

Specialist-purpose vectors

(15)

M13-based vectors can be used to make single-stranded DNA suitable for sequencing

(1)

Expression vectors enable a cloned gene to be placed under the control of a promoter that functions in E. coli

(1)

Specialist vectors have been developed that facilitate the production of RNA probes and interfering RNA

(3)

Vectors with strong, controllable promoters are used to maximize synthesis of cloned gene products

(2)

Purification of a cloned gene product can be facilitated by use of purification tags

(5)

Vectors are available that promote solubilization of expressed proteins

(1)

Proteins that are synthesized with signal sequences are exported from the cell

(1)

The Gateway® system is a highly efficient method for transferring DNA fragments to a large number of different vectors

(1)

Putting it all together: vectors with combinations of features

(2)

Gene-cloning strategies

(30)

Introduction

(1)

Genomic DNA libraries are generated by fragmenting the genome and cloning overlapping fragments in vectors

(4)

The first genomic libraries were cloned in simple plasmid and phage vectors

(2)

More sophisticated vectors have been developed to facilitate genomic library construction

(2)

Genomic libraries for higher eukaryotes are usually constructed using high-capacity vectors

101

(1)

The PCR can be used as an alternative to genomic DNA cloning

101

(1)

Long PCR uses a mixture of enzymes to amplify long DNA templates

102

(1)

Fragment libraries can be prepared from material that is unsuitable for conventional library cloning

102

(1)

Complementary DNA (cDNA) libraries are generated by the reverse transcription of mRNA

102

(8)

cDNA is representative of the mRNA population, and therefore reflects mRNA levels and the diversity of splice isoforms in particular tissues

102

(3)

The first stage of cDNA library construction is the synthesis of double-stranded DNA using mRNA as the template

105

(2)

Obtaining full-length cDNA for cloning can be a challenge

107

(3)

The PCR can be used as an alternative to cDNA cloning

110

(1)

Full-length cDNA cloning is facilitated by the rapid amplification of cDNA ends (RACE)

111

(1)

Many different strategies are available for library screening

111

(10)

Both genomic and cDNA libraries can be screened by hybridization

111

(2)

Probes are designed to maximize the chances of recovering the desired clone

113

(2)

The PCR can be used as an alternative to hybridization for the screening of genomic and cDNA libraries

115

(1)

More diverse strategies are available for the screening of expression libraries

116

(1)

Immunological screening uses specific antibodies to detect expressed gene products

116

(1)

Southwestern and northwestern screening are used to detect clones encoding nucleic acid binding proteins

117

(2)

Functional cloning exploits the biochemical or physiological activity of the gene product

119

(2)

Positional cloning is used when there is no biological information about a gene, but its position can be mapped relative to other genes or markers

121

(1)

Difference cloning exploits differences in the abundance of particular DNA fragments

121

(5)

Library-based approaches may involve differential screening or the creation of subtracted libraries enriched for differentially represented clones

122

(1)

Differentially expressed genes can also be identified using PCR-based methods

122

(2)

Representational difference analysis is a PCR-based subtractive-cloning procedure

124

(2)

Sequencing genes and short stretches of DNA

126

(15)

The commonest method of DNA sequencing is Sanger sequencing (also known as chain-terminator or dideoxy sequencing)

126

(2)

The original Sanger method has been greatly improved by a number of experimental modifications

128

(2)

It is possible to automate DNA sequencing by replacing radioactive labels with fluorescent labels

130

(1)

DNA sequencing throughput can be greatly increased by replacing slab gels with capillary array electrophoresis

131

(1)

The accuracy of automated DNA sequencing can be determined with basecalling algorithms

131

(1)

Different strategies are required depending on the complexity of the DNA to be sequenced

132

(2)

Alternatives to Sanger sequencing have been developed and are particularly useful for resequencing of DNA

134

(1)

Pyrosequencing permits sequence analysis in real time

134

(2)

It is possible to sequence DNA by hybridization using microarrays

136

(4)

Massively parallel signature sequencing can be used to monitor RNA abundance

140

(1)

Methods are being developed for sequencing single DNA molecules

140

(1)

Changing genes: site-directed mutagenesis and protein engineering

141

(16)

Introduction

141

(1)

Primer extension (the single-primer method) is a simple method for site-directed mutation

141

(1)

The single-primer method has a number of deficiencies

142

(1)

Methods have been developed that simplify the process of making all possible amino acid substitutions at a selected site

143

(1)

The PCR can be used for site-directed mutagenesis

144

(2)

Methods are available to enable mutations to be introduced randomly throughout a target gene

146

(1)

Altered proteins can be produced by inserting unusual amino acids during protein synthesis

147

(1)

Phage display can be used to facilitate the selection of mutant peptides

148

(1)

Cell-surface display is a more versatile alternative to phage display

149

(1)

Protein engineering

150

(7)

A number of different methods of gene shuffling have been developed

153

(1)

Chimeric proteins can be produced in the absence of gene homology

154

(3)

Bioinformatics

157

(22)

Introduction

157

(1)

Databases are required to store and cross-reference large biological datasets

158

(5)

The primary nucleotide sequence databases are repositories for annotated nucleotide sequence data

158

(1)

SWISS-PROT and TrEMBL are databases of annotated protein sequences

158

(2)

The Protein Databank is the main repository for protein structural information

160

(1)

Secondary sequence databases pull out common features of protein sequences and structures

160

(3)

Other databases cover a variety of useful topics

163

(1)

Sequence analysis is based on alignment scores

163

(3)

Algorithms for pairwise similarity searching find the best alignment between pairs of sequences

164

(2)

Multiple alignments allow important features of gene and protein families to be identified

166

(1)

Sequence analysis of genomic DNA involves the de novo identification of genes and other features

166

(7)

Genes in prokaryotic DNA can often be found by six-frame translation

166

(2)

Algorithms have been developed that find genes automatically

168

(3)

Additional algorithms are necessary to find non-coding RNA genes and regulatory elements

171

(2)

Several in silico methods are available for the functional annotation of genes

173

(2)

Caution must be exercised when using purely in silico methods to annotate genomes

175

(1)

Sequencing also provides new data for molecular phylogenetics

175

(4)

Part II Manipulating DNA in Microbes, Plants, and Animals

Cloning in bacteria other than Escherichia coli

179

(23)

Introduction

179

(1)

Many bacteria are naturally competent for transformation

179

(4)

Recombinant DNA needs to replicate or be integrated into the chromosome in new hosts

183

(1)

Recombinant DNA can integrate into the chromosome in different ways

183

(2)

Cloning in Gram-negative bacteria other than E. coli

185

(4)

Vectors derived from the IncQ-group plasmid RSF1010 are not self-transmissible

185

(1)

Mini-versions of the IncP-group plasmids have been developed as conjugative broad-host-range vectors

186

(1)

Vectors derived from the broad-host-range plasmid Sa are used mostly with Agrobacterium tumefaciens

187

(1)

pBBR 1 is another plasmid that has been used to develop broad-host-range cloning vectors

188

(1)

Cloned DNA can be shuttled between high-copy-number and low-copy-number vectors

188

(1)

Proper transcriptional analysis of a cloned gene requires that it is present on the chromosome

188

(1)

Cloning in Gram-positive bacteria

189

(11)

Many of the cloning vectors used with Bacillus subtilis and other low-GC bacteria are derived from plasmids found in Staphylococcus aureux

190

(1)

The mode of plasmid replication can affect the stability of cloning vectors in B. subtilis

191

(3)

Compared with E. coli, B. subtilis has additional requirements for efficient transcription and translation and this can prevent the expression of genes from Gram-negative organisms in ones that are Gram-positive

194

(1)

Specialist vectors have been developed that permit controlled expression in B. subtilis and other low-GC hosts

194

(1)

Vectors have been developed that facilitate secretion of foreign proteins from B. subtilis

195

(1)

As an aid to understanding gene function in B. subtilis, vectors have been developed for directed gene inactivation

195

(1)

The mechanism whereby B. subtilis is transformed with plasmid DNA facilitates the ordered assembly of dispersed genes

196

(1)

A variety of different methods can be used to transform high-GC organisms such as the streptomycetes

196

(3)

Most of the vectors used with streptomycetes are derivatives of endogenous plasmids and bacteriophages

199

(1)

Cloning in Archaea

200

(2)

Cloning in Saccharomyces cerevisiae and other fungi

202

(16)

There are a number of reasons for cloning DNA in S. cerevisiae

202

(1)

Fungi are not naturally transformable and special methods are required to introduce exogenous DNA

202

(1)

Exogenous DNA that is not carried on a vector can only be maintained by integration into a chromosome

203

(1)

Different kinds of vector have been developed for use in S. cerevisiae

204

(1)

The availability of different kinds of vector offers yeast geneticists great flexibility

205

(2)

Recombinogenic engineering can be used to move genes from one vector to another

207

(1)

Yeast promoters are more complex than bacterial promoters

208

(1)

Promoter systems have been developed to facilitate overexpression of recombinant proteins in yeast

209

(2)

A number of specialist multi-purpose vectors have been developed for use in yeast

211

(1)

Heterologous proteins can be synthesized as fusions for display on the cell surface of yeast

212

(1)

The methylotrophic yeast Pichia pastoris is particularly suited to high-level expression of recombinant proteins

212

(1)

Cloning and manipulating large fragments of DNA

213

(5)

Yeast artificial chromosomes can be used to clone very large fragments of DNA

213

(1)

Classical YACs have a number of deficiencies as vectors

213

(1)

Circular YACs have a number of advantages over classical YACs

214

(1)

Transformation-associated recombination (TAR) cloning in yeast permits selective isolation of large chromosomal fragments

214

(4)

Gene transfer to animal cells

218

(33)

Introduction

218

(1)

There are four major strategies for gene transfer to animal cells

218

(1)

There are several chemical transfection techniques for animal cells but all are based on similar principles

219

(3)

The calcium phosphate method involves the formation of a co-precipitate which is taken up by endocytosis

219

(1)

Transfection with polyplexes is more efficient because of the uniform particle size

220

(2)

Transfection can also be achieved using liposomes and lipoplexes

222

(1)

Physical transfection techniques have diverse mechanisms

222

(1)

Electroporation and ultrasound create transient pores in the cell

222

(1)

Other physical transfection methods pierce the cell membrane and introduce DNA directly into the cell

223

(1)

Cells can be transfected with either replicating or non-replicating DNA

223

(1)

Three types of selectable marker have been developed for animal cells

224

(4)

Endogenous selectable markers are already present in the cellular genome, and mutant cell lines are required when they are used

224

(1)

There is no competing activity for dominant selectable markers

225

(1)

Some marker genes facilitate stepwise transgene amplification

226

(2)

Plasmid vectors for the transfection of animal cells contain modules from bacterial and animal genes

228

(8)

Non-replicating plasmid vectors persist for a short time in an extrachromosomal state

228

(2)

Runaway polymavirus replicons facilitate the accumulation of large amounts of protein in a short time

230

(1)

BK and BPV replicons facilitate episomal replication, but the plasmids tend to be structurally unstable

231

(5)

Replicons based on Epstein-Barr virus facilitate long-term transgene stability

236

(1)

DNA can be delivered to animal cells using bacterial vectors

236

(2)

Viruses are also used as gene-transfer vectors

238

(11)

Adenovirus vectors are useful for short-term transgene expression

238

(1)

Adeno-associated virus vectors integrate into the host-cell genome

239

(1)

Baculovirus vectors promote high-level transgene expression in insect cells, but can also infect mammalian cells

240

(3)

Herpesvirus vectors are latent in many cell types and may promote long-term transgene expression

243

(1)

Retrovirus vectors integrate efficiently into the host-cell genome

243

(1)

Retroviral vectors are often replication-defective and self-inactivating

244

(1)

There are special considerations for the construction of lentiviral vectors

245

(1)

Sindbis virus and Semliki forest virus vectors replicate in the cytoplasm

246

(2)

Vaccinia and other poxvirus vectors are widely used for vaccine delivery

248

(1)

Summary of expression systems for animal cells

249

(2)

Genetic manipulation of animals

251

(23)

Introduction

251

(1)

Three major methods have been developed for the production of transgenic mice

251

(4)

Pronuclear microinjection involves the direct transfer of DNA into the male pronucleus of the fertilized mouse egg

252

(1)

Recombinant retroviruses can be used to transduce early embryos prior to the formation of the germline

253

(1)

Transgenic mice can be produced by the transfection of ES cells followed by the creation of chimeric embryos

254

(1)

ES cells can be used for gene targeting in mice

255

(3)

Gene-targeting vectors may disrupt genes by insertion or replacement

256

(1)

Sophisticated selection strategies have been developed to isolate rare gene-targeting events

257

(1)

Two rounds of gene targeting allow the introduction of subtle mutations

257

(1)

Recent advances in gene-targeting technology

258

(1)

Applications of genetically modified mice

258

(4)

Applications of transgenic mice

258

(4)

Yeast artificial chromosome (YAC) transgenic mice

262

(1)

Applications of gene targeting

262

(1)

Standard transgenesis methods are more difficult to apply in other mammals and birds

263

(1)

Intracytoplasmic sperm injection uses sperm as passive carriers of recombinant DNA

264

(1)

Nuclear transfer technology can be used to clone animals

264

(2)

Gene transfer to Xenopus can result in transient expression or germline transformation

266

(2)

Xenopus oocytes can be used as a heterologous expression system

266

(1)

Xenopus oocytes can be used for functional expression cloning

266

(1)

Transient gene expression in Xenopus embryos is achieved by DNA or mRNA injection

267

(1)

Transgenic Xenopus embryos can be produced by restriction enzyme-mediated integration

267

(1)

Gene transfer to fish is generally carried out by microinjection, but other methods are emerging

268

(1)

Gene transfer to fruit flies involves the microinjection of DNA into the pole plasma

269

(5)

P elements are used to introduce DNA into the Drosophila germline

269

(1)

Natural P elements have been developed into vectors for gene transfer

269

(2)

Gene targeting in Drosophila has been achieved using a combination of homologous and site-specific recombination

271

(3)

Gene transfer to plants

274

(25)

Introduction

274

(1)

Plant tissue culture is required for most transformation procedures

274

(3)

Callus cultures are established under conditions that maintain cells in an undifferentiated state

274

(1)

Callus cultures can be broken up to form cell suspension, which can be maintained in batches

275

(1)

Protoplasts are usually derived from suspension cells and can be ideal transformation targets

276

(1)

Cultures can be established directly from the rapidly dividing cells of meristematic tissues or embryos, or from haploid cells

276

(1)

Regeneration of fertile plants can occur through organogenesis or somatic embryogenesis

276

(1)

There are four major strategies for gene transfer to plant cells

277

(1)

Agrobacterium-mediated transformation

277

(13)

Agrobacterium tumefaciens is a plant pathogen that induces the formation of tumors

277

(1)

The ability to induce tumors is conferred by a Ti-plasmid found only in virulent Agrobacterium strains

278

(2)

A short segment of DNA, the T-DNA, is transferred to the plant genome

280

(1)

Disarmed Ti-plasmid derivatives can be used as plant gene-transfer vectors

281

(4)

Binary vectors separate the T-DNA and the genes required for T-DNA transfer, allowing transgenes to be cloned in small plasmids

285

(2)

Agrobacterium-mediated transformation can be achieved using a simple experimental protocol in many dicots

287

(1)

Monocots were initially recalcitrant to Agrobacterium-mediated transformation, but it is now possible to transform certain varieties of many cereals using this method

288

(1)

Binary vectors have been modified to transfer large segments of DNA into the plant genome

289

(1)

Agrobacterium rhizogenes is used to transform plant roots and produce hairy-root cultures

289

(1)

Direct DNA transfer to plants

290

(3)

Transgenic plants can be regenerated from transformed protoplasts

290

(1)

Particle bombardment can be used to transform a wide range of plant species

291

(1)

Other direct DNA transfer methods have been developed for intact plant cells

292

(1)

Direct DNA transfer is also used for chloroplast transformation

292

(1)

Gene targeting in plants

293

(1)

In planta transformation minimizes or eliminates the tissue culture steps usually needed for the generation of transgenic plants

293

(1)

Plant viruses can be used as episomal expression vectors

294

(5)

The first plant viral vectors were based on DNA viruses because of their small and simple genomes

294

(2)

Most plant virus expression vectors are based on RNA viruses because they can accept larger transgenes than DNA viruses

296

(3)

Advanced transgenic technology

299

(24)

Introduction

299

(1)

Inducible expression systems allow transgene expression to be controlled by physical stimuli or the application of small chemical modulators

299

(1)

Some naturally occurring inducible promoters can be used to control transgene expression

299

(1)

Recombinant inducible systems are built from components that are not found in the host animal or plant

300

(6)

The lac and tet repressor systems are based on bacterial operons

301

(1)

The tet activator and reverse activator systems were developed to circumvent some of the limitations of the original tet system

302

(1)

Steroid hormones also make suitable heterologous inducers

303

(1)

Chemically induced dimerization exploits the ability of a divalent ligand to bind two proteins simultaneously

304

(2)

Not all inducible expression systems are transcriptional switches

306

(1)

Site-specific recombination allows precise manipulation of the genome in organisms where gene targeting is inefficient

306

(6)

Site-specific recombination can be used to delete unwanted transgenes

307

(1)

Site-specific recombination can be used to activate transgene expression or switch between alternative transgenes

308

(1)

Site-specific recombination can facilitate precise transgene integration

309

(1)

Site-specific recombination can facilitate chromosome engineering

309

(1)

Inducible site-specific recombination allows the production of conditional mutants and externally regulated transgene excision

309

(3)

Many strategies for gene inactivation do not require the direct modification of the target gene

312

(7)

Antisense RNA blocks the activity of mRNA in a stoichiometric manner

312

(1)

Ribozymes are catalytic molecules that destroy targeted mRNAs

313

(1)

Cosuppression is the inhibition of an endogenous gene by the presence of a homologous sense transgene

314

(4)

RNA interference is a potent form of silencing caused by the direct introduction of double-stranded RNA into the cell

318

(1)

Gene inhibition is also possible at the protein level

319

(4)

Intracellular antibodies and aptamers bind to expressed proteins and inhibit their assembly or activity

319

(1)

Active proteins can be inhibited by dominant-negative mutants in multimeric assemblies

320

(3)

Part III Genome Analysis, Genomics, and Beyond

The organization and structure of genomes

323

(23)

Introduction

323

(1)

The genomes of cellular organisms vary in size over five orders of magnitude

323

(3)

Increases in genome complexity sometimes are accompanied by increases in the complexity of gene structure

326

(2)

Viruses and bacteria have very simple genomes

328

(2)

Organelle DNA is a repetitive sequence

330

(1)

Chloroplast DNA structure is highly conserved

330

(1)

Mitochondrial genome architecture varies enormously, particularly in plants and protists

331

(1)

The organization of nuclear DNA in eukaryotes

332

(14)

The gross anatomy of chromosomes is revealed by Giemsa staining

332

(1)

Telomeres play a critical role in the maintenance of chromosomal integrity

332

(1)

Tandemly repeated sequences can be detected in two ways

333

(2)

Tandemly repeated sequences can be subdivided on the basis of size

335

(3)

Dispersed repeated sequences are composed of multiple copies of two types of transposable elements

338

(1)

Retrotransposons can be divided into two groups on the basis of transposition mechanism and structure

339

(1)

DNA transposons are simpler than retrotransposons

340

(1)

Transposon activity is highly variable across eukaryotes

340

(1)

Repeated DNA is non-randomly distributed within genomes

340

(1)

Eukaryotic genomes are very plastic

341

(1)

Pseudogenes are derived from repeated DNA

341

(1)

Segmental duplications are very large, low-copy-number repeats

341

(1)

The human Y chromosome has an unusual structure

342

(2)

Centromeres are filled with tandem repeats and retroelements

344

(1)

Summary of structural elements of eukaryotic chromosomes

344

(2)

Mapping and sequencing genomes

346

(27)

Introduction

346

(1)

The first physical map of an organism made use of restriction fragment length polymorphisms (RFLPs)

346

(2)

Sequence tags are more convenient markers than RFLPs because they do not use Southern blotting

348

(1)

Single nucleotide polymorphisms (SNPs) are the most favored physical marker

349

(2)

Polymorphic DNA can be detected in the absence of sequence information

351

(1)

AFLPs resemble RFLPs and can be detected in the absence of sequence information

352

(1)

Physical markers can be placed on a cytogenetic map using in situ hybridization

353

(1)

Padlock probes allow different alleles to be examined simultaneously

353

(1)

Physical mapping is limited by the cloning process

354

(1)

Optical mapping is undertaken on single DNA molecules

354

(4)

Radiation hybrid (RH) mapping involves screening of randomly broken fragments of DNA for specific markers

358

(2)

HAPPY mapping is a more versatile variation on RH mapping

360

(1)

It is essential that the different mapping methods are integrated

360

(2)

Sequencing genomes

362

(11)

High-throughput sequencing is an essential prerequisite for genome sequencing

362

(1)

There are two different strategies for sequencing genomes

363

(5)

A combination of shotgun sequencing and physical mapping now is the favored method for sequencing large genomes

368

(1)

Gaps in sequences occur with all genome-sequencing methodologies and need to be closed

368

(2)

The quality of genome-sequence data needs to be determined

370

(3)

Comparative genomics

373

(21)

Introduction

373

(1)

The formation of orthologs and paralogs are key steps in gene evolution

373

(1)

Protein evolution occurs by exon shuffling

374

(1)

Comparative genomics of bacteria

375

(6)

The minimal gene set consistent with independent existence can be determined using comparative genomics

376

(1)

Larger microbial genomes have more paralogs than smaller genomes

376

(2)

Horizontal gene transfer may be a significant evolutionary force but is not easy to detect

378

(1)

The comparative genomics of closely related bacteria gives useful insights into microbial evolution

379

(2)

Comparative analysis of phylogenetically diverse bacteria enables common structural themes to be uncovered

381

(1)

Comparative genomics can be used to analyze physiological phenomena

381

(1)

Comparative genomics of organelles

381

(4)

Mitochondrial genomes exhibit an amazing structural diversity

381

(2)

Gene transfer has occurred between mtDNA and nuclear DNA

383

(1)

Horizontal gene transfer has been detected in mitochondrial genomes

384

(1)

Comparative genomics of eukaryotes

385

(9)

The minimal eukaryotic genome is smaller than many bacterial genomes

385

(1)

Comparative genomics can be used to identify genes and regulatory elements

385

(2)

Comparative genomics gives insight into the evolution of key proteins

387

(1)

The evolution of species can be analyzed at the genome level

387

(1)

Analysis of dipteran insect genomes permits analysis of evolution in multicellular organisms

388

(2)

A number of mammalian genomes have been sequenced and the data is facilitating analysis of evolution

390

(2)

Comparative genomics can be used to uncover the molecular mechanisms that generate new gene structures

392

(2)

Large-scale mutagenesis and interference

394

(13)

Introduction

394

(1)

Genome-wide gene targeting is the systematic approach to large-scale mutagenesis

394

(2)

The only organism in which systematic gene targeting has been achieved is the yeast Saccharomyces cerevisiae

395

(1)

It is unlikely that systematic gene targeting will be achieved in higher eukaryotes in the foreseeable future

395

(1)

Genome-wide random mutagenesis is a strategy applicable to all organisms

396

(7)

Insertional mutagenesis leaves a DNA tag in the interrupted gene, which facilitates cloning and gene identification

396

(2)

Genome-wide insertional mutagenesis in yeast has been carried out with endogenous and heterologous transposons

398

(1)

Genome-wide insertional mutagenesis in vertebrates has been facilitated by the development of artificial transposon systems

399

(2)

Insertional mutagenesis in plants can be achieved using Agrobacterium T-DNA or plant transposons

401

(1)

T-DNA mutagenesis requires gene transfer by A. tumefaciens

401

(1)

Transposon mutagenesis in plants can be achieved using endogenous or heterologous transposons

402

(1)

Insertional mutagenesis in invertebrates

403

(1)

Chemical mutagenesis is more efficient than transposon mutagenesis, and generates point mutations

403

(1)

Libraries of knock-down phenocopies can be created by RNA interference

404

(3)

RNA interference has been used to generate comprehensive knock-down libraries in Caenorhabditis elegans

404

(1)

The first genome-wide RNAi screens in other organisms have been carried out

405

(2)

Analysis of the transcriptome

407

(18)

Introduction

407

(2)

Traditional approaches to expression profiling allow genes to be studied singly or in small groups

403

(6)

The transcriptome is the collection of all messenger RNAs in the cell

409

(1)

Steady-state mRNA levels can be quantified directly by sequence sampling

410

(2)

The first large-scale gene expression studies involved the sampling of ESTs from cDNA libraries

410

(1)

Serial analysis of gene expression uses concatemerized sequence tags to identify each gene

410

(1)

Massively parallel signature sequencing involves the parallel analysis of millions of DNA-tagged microbeads

411

(1)

DNA microarray technology allows the parallel analysis of thousands of genes on a convenient miniature device

412

(9)

Spotted DNA arrays are produced by printing DNA samples on treated microscope slides

413

(4)

There are numerous printing technologies for spotted arrays

417

(1)

Oligonucleotide chips are manufactured by in situ oligonucleotide synthesis

418

(1)

Spotted arrays and oligo chips have similar sensitivities

419

(2)

As transcriptomics technology matures, standardization of data processing and presentation become important challenges

421

(1)

Expression profiling with DNA arrays has permeated almost every area of biology

422

(3)

Global profiling of microbial gene expression

422

(1)

Applications of expression profiling in human disease

423

(2)

Proteomics I - Expression analysis and characterization of proteins

425

(16)

Introduction

425

(1)

Protein expression analysis is more challenging than mRNA profiling because proteins cannot be amplified like nucleic acids

425

(1)

There are two major technologies for protein separation in proteomics

426

(5)

Two-dimensional electrophoresis produces a visual display of the proteome

426

(1)

The sensitivity, resolution, and representation of 2D gels need to be improved

427

(1)

Multiplexed analysis allows protein expression profiles to be compared on single gels

428

(1)

Multidimensional liquid chromatography is more sensitive than 2DGE and is directly compatible with mass spectrometry

428

(3)

Mass spectrometry is used for protein characterization

431

(7)

High-throughput protein annotation is achieved by mass spectrometry and correlative database searching

431

(3)

Specialized strategies are used to quantify proteins directly by mass spectrometry

434

(1)

Protein modifications can also be detected by mass spectrometry

435

(3)

Protein microarrays can be used for expression analysis

438

(3)

Antibody arrays contain immobilized antibodies or antibody derivatives for the capture of specific proteins

438

(1)

Antigen arrays are used to measure antibodies in solution

439

(1)

General protein arrays can be used for expression profiling and functional analysis

439

(1)

Other molecules may be arrayed instead of proteins

439

(1)

Some biochips bind to particular classes of protein

440

(1)

Solution arrays are non-planar microarrays

440

(1)

Proteomics II - Analysis of protein structures

441

(12)

Introduction

441

(3)

Sequence analysis alone is not sufficient to annotate all orphan genes

441

(1)

Protein structures are more highly conserved than sequences

442

(2)

Structural proteomics has required developments in structural analysis techniques and bioinformatics

444

(5)

Protein structures are determined experimentally by X-ray crystallography or nuclear magnetic resonance spectroscopy

444

(2)

Protein structures can be modeled on related structures

446

(1)

Protein structures can be aligned using algorithms that carry out intramolecular and intermolecular comparisons

447

(1)

The annotation of proteins by structural comparison has been greatly facilitated by standard systems for the structural classification of proteins

448

(1)

Tentative functions can be assigned based on crude structural features

449

(1)

International structural proteomics initiatives have been established to solve protein structures on a large scale

449

(4)

Proteomics III - Protein interactions

453

(19)

Introduction

453

(1)

Protein interactions can be inferred by a variety of genetic approaches

453

(1)

New methods based on comparative genomics can also infer protein interactions

454

(3)

Traditional biochemical methods for protein interaction analysis cannot be applied on a large scale

457

(1)

Library-based screening methods allow the large-scale analysis of binary interactions

458

(7)

In vitro expression libraries are of limited use for interaction screening

458

(1)

The yeast two-hybrid system is an in vivo interaction screening method

458

(2)

In the matrix approach, defined clones are generated for each bait and prey

460

(1)

In the random library method, bait and/or prey are represented by random clones from a highly complex expression library

461

(1)

Robust experimental design is necessary to increase the reliability of two-hybrid interaction screening data

462

(3)

Systematic analysis of protein complexes can be achieved by affinity purification and mass spectrometry

465

(2)

Protein localization is an important component of interaction data

466

(1)

Interaction screening produces large data sets which require extensive bioinformatic support

467

(5)

Metabolomics and global biochemical networks

472

(13)

Introduction

472

(1)

There are different levels of metabolite analysis

473

(1)

Metabolomics studies in humans are different from those in other organisms

473

(1)

Compromises have to be made in choosing analytical methodology for metabolomics studies

474

(1)

Sample selection and sample handling are crucial stages in metabolomics studies

475

(4)

Metabolomics produces complex data sets

479

(2)

A good reference database is an essential prerequisite for preparing global biochemical networks but currently is missing

481

(4)

Part IV Applications of Gene Manipulation and Genomics

Applications of genomics: understanding the basis of polygenic disorders and identifying quantitative trait loci

485

(23)

Introduction

485

(1)

Investigating discrete traits in outbreeding populations (genetic diseases of humans)

485

(12)

Model-free (nonparametric) linkage analysis looks at the inheritance of disease genes and selected markers in several generations of the same family

487

(5)

Linkage disequilibrium (association) studies look at the co-inheritance of markers and the disease at the population level

492

(1)

Once a disease locus is identified, all the 'omics can be used to analyze it in detail

493

(1)

The integration of global information about DNA, mRNA, and protein can be used to facilitate disease-gene identification

494

(1)

The existence of haplotype blocks should simplify linkage disequilibrium analysis

495

(2)

Investigating quantitative trait loci (QTLs) in inbred populations

497

(6)

Particular kinds of genetic cross are necessary if QTLs are to be mapped

497

(1)

Identifying QTLs involves two challenging steps

498

(3)

Various factors influence the ability to isolate QTLs

501

(1)

Chromosome substitution strains make the identification of QTLs easier

501

(2)

The level of gene expression can influence the phenotype of a QTLs

503

(1)

Understanding responses to drugs (pharmacogenomics)

503

(5)

Genetic variation accounts for the different responses of individuals to drugs

503

(1)

Pharmacogenomics is being used by the pharmaceutical industry

504

(2)

Personalized medicine involves matching genotypes to therapy

506

(2)

Applications of recombinant DNA technology

508

(39)

Introduction

508

(1)

Theme 1: Producing useful molecules

508

(25)

Recombinant therapeutic proteins are produced commercially in bacteria, yeast, and mammalian cells

508

(10)

Transgenic animals and plants can also be used as bioreactors to produce recombinant proteins

518

(6)

Metabolic engineering allows the directed production of small molecules in bacteria

524

(1)

Metabolic engineering provides new routes to small molecules

524

(2)

Combinatorial biosynthesis can produce completely novel compounds

526

(1)

Metabolic engineering can also be achieved in plants and plant cells to produce diverse chemical structures

527

(1)

Production of vinblastine and vincristine in Catharanthus cell cultures is a challenge because of the many steps and control points in the pathway

528

(1)

The production of vitamin A in cereals is an example of extending an endogenous metabolic pathway

529

(3)

The enhancement of plants to produce more vitamin E is an example of balancing several metabolic pathways and directing flux in the preferred direction

532

(1)

Theme 2: Improving agronomic traits by genetic modification

533

(7)

Herbicide resistance is the most widespread trait in commercial transgenic plants

533

(2)

Virus-resistant crops can be produced by expressing viral or non-viral transgenes

535

(1)

Resistance to fungal pathogens is often achieved by manipulating natural plant defense mechanisms

536

(1)

Resistance to blight provides an example of how plants can be protected against bacterial pathogens

537

(1)

The bacterium Bacillus thuringiensis provides the major source of insect resistant genes

537

(1)

Drought resistance provides a good example of how plants can be protected against abiotic stress

538

(1)

Plants can be engineered to cope with poor soil quality

539

(1)

One of the most important goals in plant biotechnology is to increase food yields

540

(1)

Theme 3: Using genetic modification to study, prevent, and cure disease

540

(7)

Transgenic animals can be created as models of human disease

540

(1)

Gene medicine is the use of nucleic acids to prevent, treat, or cure disease

541

(2)

DNA vaccines are expression constructs whose products stimulate the immune system

543

(1)

Gene augmentation therapy for recessive diseases involves transferring a functional copy of the gene into the genome

544

(1)

Gene-therapy strategies for cancer may involve dominant suppression of the overactive gene or targeted killing of the cancer cells

545

(2)

References

547

(80)

Appendix: the genetic code and single-letter amino acid designations

627

(1)

Index

628

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Principles of Gene Manipulation and Genomics

Summary

Author Biography

Table of Contents

Supplemental Materials