Principles of Gene Manipulation and Genomics

Contents
......Page 6
Preface......Page 19
Abbreviations......Page 21
Gene manipulation involves the creation and cloning of recombinant DNA......Page 24
Recombinant DNA has opened new horizons in medicine......Page 26
Mapping and sequencing technologies formed a crucial link between gene manipulation and genomics......Page 27
The genomics era began in earnest in 1995 with the complete sequencing of a bacterial genome......Page 29
The post-genomics era aims at the complete characterization of cells at all levels......Page 30
Outline of the rest of the book......Page 31
Part I Fundamental Techniques of Gene Manipulation......Page 36
A number of basic techniques are common to most gene-cloning experiments......Page 38
Gel electrophoresis is used to separate different nucleic acid molecules on the basis of their size......Page 39
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......Page 41
Western blotting is used to transfer proteins from acrylamide gels to membranes......Page 42
The ability to transform E. coli with DNA is an essential prerequisite for most experiments on gene manipulation......Page 47
The ability to transform organisms other than E. coli with recombinant DNA enables genes to be studied in different host backgrounds
......Page 48
The polymerase chain reaction (PCR) has revolutionized the way that biologists manipulate and analyze DNA......Page 49
The principle of the PCR is exceedingly simple......Page 50
The basic PCR is not efficient at amplifying long DNA fragments......Page 51
The success of a PCR experiment is very dependent on the choice of experimental variables......Page 52
By using special instrumentation it is possible to make the PCR quantitative......Page 53
There are a number of different ways of generating fluorescence in quantitative PCR reactions......Page 54
It is now possible to amplify whole genomes as well as gene segments......Page 57
Understanding the biological basis of host-controlled restriction and modification of bacteriophage DNA led to the identification of restriction endonucleases
......Page 59
Four different types of restriction and modification (R-M) system have been recognized but only one is widely used in gene manipulation
......Page 60
Restriction enzymes cut DNA at sites of rotational symmetry and different enzymes recognize different sequences......Page 62
Simple DNA manipulations can convert a site for one restriction enzyme into a site for another enzyme......Page 64
Methylation can reduce the susceptibility of DNA to cleavage by restriction endonucleases and the efficiency of DNA transformation......Page 65
The success of a cloning experiment is critically dependent on the quality of any restriction enzymes that are used......Page 66
The enzyme DNA ligase is the key to joining DNA molecules in vitro......Page 67
Adaptors and linkers are short double-stranded DNA molecules that permit different cleavage sites to be interconnected......Page 71
Special methods are often required if DNA produced by PCR amplification is to be cloned......Page 72
Amplified DNA can be cloned using in vitro recombination......Page 73
Plasmid biology and simple plasmid vectors......Page 78
The number of copies of a plasmid in a cell varies between plasmids and is determined by the regulatory mechanisms controlling replication
......Page 80
The purification of plasmid DNA......Page 82
Good plasmid cloning vehicles share a number of desirable features......Page 84
pBR322 is an early example of a widely used, purpose-built cloning vector......Page 85
A large number of improved vectors have been derived from pBR322......Page 87
Bacteriophage λ has sophisticated control circuits......Page 89
A number of phage λ vectors with improved properties have been described......Page 92
By packaging DNA into phage λ in vitro it is possible to eliminate the need for competent cells of E. coli......Page 93
DNA cloning with single-stranded DNA vectors......Page 94
Phage M13 has been modified to make it a better vector......Page 95
Cosmids are plasmids that can be packaged into bacteriophage λ particles......Page 98
BACs and PACs are vectors that can carry much larger fragments of DNA than cosmids because they do not have packaging constraints......Page 99
Recombinogenic engineering (recombineering) simplifies the cloning of DNA, particularly with high-molecular-weight constructs......Page 102
Expression vectors enable a cloned gene to be placed under the control of a promoter that functions in E. coli......Page 104
Specialist vectors have been developed that facilitate the production of RNA probes and interfering RNA......Page 105
Vectors with strong, controllable promoters are used to maximize synthesis of cloned gene products......Page 108
Purification of a cloned gene product can be facilitated by use of purification tags......Page 110
Vectors are available that promote solubilization of expressed proteins......Page 115
Proteins that are synthesized with signal sequences are exported from the cell......Page 116
Putting it all together: vectors with combinations of features......Page 117
Introduction......Page 119
The first genomic libraries were cloned in simple plasmid and phage vectors......Page 120
More sophisticated vectors have been developed to facilitate genomic library construction......Page 122
The PCR can be used as an alternative to genomic DNA cloning......Page 124
cDNA is representative of the mRNA population, and therefore reflects mRNA levels and the diversity of splice isoforms in particular tissues
......Page 125
The first stage of cDNA library construction is the synthesis of double-stranded DNA using mRNA as the template......Page 128
Obtaining full-length cDNA for cloning can be a challenge......Page 130
The PCR can be used as an alternative to cDNA cloning......Page 133
Both genomic and cDNA libraries can be screened by hybridization......Page 134
Probes are designed to maximize the chances of recovering the desired clone......Page 136
The PCR can be used as an alternative to hybridization for the screening of genomic and cDNA libraries......Page 138
Immunological screening uses specific antibodies to detect expressed gene products......Page 139
Southwestern and northwestern screening are used to detect clones encoding nucleic acid binding proteins......Page 140
Functional cloning exploits the biochemical or physiological activity of the gene product......Page 142
Difference cloning exploits differences in the abundance of particular DNA fragments......Page 144
Differentially expressed genes can also be identified using PCR-based methods......Page 145
Representational difference analysis is a PCR-based subtractive-cloning procedure......Page 147
The commonest method of DNA sequencing is Sanger sequencing (also known as chain-terminator or dideoxy sequencing)......Page 149
The original Sanger method has been greatly improved by a number of experimental modifications......Page 151
It is possible to automate DNA sequencing by replacing radioactive labels with fluorescent labels......Page 153
The accuracy of automated DNA sequencing can be determined with basecalling algorithms......Page 154
Different strategies are required depending on the complexity of the DNA to be sequenced......Page 155
Pyrosequencing permits sequence analysis in real time......Page 157
It is possible to sequence DNA by hybridization using microarrays......Page 159
Methods are being developed for sequencing single DNA molecules......Page 163
Primer extension (the single-primer method) is a simple method for site-directed mutation......Page 164
The single-primer method has a number of deficiencies......Page 165
Methods have been developed that simplify the process of making all possible amino acid substitutions at a selected site......Page 166
The PCR can be used for site-directed mutagenesis......Page 167
Methods are available to enable mutations to be introduced randomly throughout a target gene......Page 169
Altered proteins can be produced by inserting unusual amino acids during protein synthesis......Page 170
Phage display can be used to facilitate the selection of mutant peptides......Page 171
Cell-surface display is a more versatile alternative to phage display......Page 172
Protein engineering......Page 173
A number of different methods of gene shuffling have been developed......Page 176
Chimeric proteins can be produced in the absence of gene homology......Page 177
Introduction......Page 180
SWISS-PROT and TrEMBL are databases of annotated protein sequences......Page 181
Secondary sequence databases pull out common features of protein sequences and structures......Page 183
Sequence analysis is based on alignment scores......Page 186
Algorithms for pairwise similarity searching find the best alignment between pairs of sequences......Page 187
Genes in prokaryotic DNA can often be found by six-frame translation......Page 189
Algorithms have been developed that find genes automatically......Page 191
Additional algorithms are necessary to find non-coding RNA genes and regulatory elements......Page 194
Several in silico methods are available for the functional annotation of genes......Page 196
Sequencing also provides new data for molecular phylogenetics......Page 198
Part II Manipulating DNA in Microbes, Plants, and Animals......Page 200
Many bacteria are naturally competent for transformation......Page 202
Recombinant DNA can integrate into the chromosome in different ways......Page 206
Vectors derived from the IncQ-group plasmid RSF1010 are not self-transmissible......Page 208
Mini-versions of the IncP-group plasmids have been developed as conjugative broad-host-range vectors......Page 209
Vectors derived from the broad-host-range plasmid Sa are used mostly with Agrobacterium tumefaciens......Page 210
Proper transcriptional analysis of a cloned gene requires that it is present on the chromosome......Page 211
Cloning in Gram-positive bacteria......Page 212
Many of the cloning vectors used with Bacillus subtilis and other low-GC bacteria are derived from plasmids found in Staphylococcus aureus
......Page 213
The mode of plasmid replication can affect the stability of cloning vectors in B. subtilis......Page 214
Specialist vectors have been developed that permit controlled expression in B. subtilis and other low-GC hosts......Page 217
As an aid to understanding gene function in B. subtilis , vectors have been developed for directed gene inactivation......Page 218
A variety of different methods can be used to transform high-GC organisms such as the streptomycetes......Page 219
Most of the vectors used with streptomycetes are derivatives of endogenous plasmids and bacteriophages......Page 222
Cloning in Archaea......Page 223
Fungi are not naturally transformable and special methods are required to introduce exogenous DNA......Page 225
Exogenous DNA that is not carried on a vector can only be maintained by integration into a chromosome......Page 226
Different kinds of vectors have been developed for use in S. cerevisiae......Page 227
The availability of different kinds of vector offers yeast geneticists great flexibility......Page 228
Recombinogenic engineering can be used to move genes from one vector to another......Page 230
Yeast promoters are more complex than bacterial promoters......Page 231
Promoter systems have been developed to facilitate overexpression of recombinant proteins in yeast......Page 232
A number of specialist multi-purpose vectors have been developed for use in yeast......Page 234
The methylotrophic yeast Pichia pastoris is particularly suited to high-level expression of recombinant proteins......Page 235
Classical YACs have a number of deficiencies as vectors......Page 236
Transformation-associated recombination (TAR) cloning in yeast permits selective isolation of large chromosomal fragments......Page 237
There are four major strategies for gene transfer to animal cells......Page 241
The calcium phosphate method involves the formation of a co-precipitate which is taken up by endocytosis......Page 242
Transfection with polyplexes is more efficient because of the uniform particle size......Page 243
Electroporation and ultrasound create transient pores in the cell......Page 245
Cells can be transfected with either replicating or non-replicating DNA......Page 246
Endogenous selectable markers are already present in the cellular genome, and mutant cell lines are required when they are used......Page 247
There is no competing activity for dominant selectable markers......Page 248
Some marker genes facilitate stepwise transgene amplification......Page 249
Non-replicating plasmid vectors persist for a short time in an extrachromosomal state......Page 251
Runaway polyomavirus replicons facilitate the accumulation of large amounts of protein in a short time......Page 253
BK and BPV replicons facilitate episomal replication, but the plasmids tend to be structurally unstable......Page 254
DNA can be delivered to animal cells using bacterial vectors......Page 259
Adenovirus vectors are useful for short-term transgene expression......Page 261
Adeno-associated virus vectors integrate into the host-cell genome......Page 262
Baculovirus vectors promote high-level transgene expression in insect cells, but can also infect mammalian cells......Page 263
Retrovirus vectors integrate efficiently into the host-cell genome......Page 266
Retroviral vectors are often replication-defective and self-inactivating......Page 267
There are special considerations for the construction of lentiviral vectors......Page 268
Sindbis virus and Semliki forest virus vectors replicate in the cytoplasm......Page 269
Vaccinia and other poxvirus vectors are widely used for vaccine delivery......Page 271
Summary of expression systems for animal cells......Page 272
Three major methods have been developed for the production of transgenic mice......Page 274
Pronuclear microinjection involves the direct transfer of DNA into the male pronucleus of the fertilized mouse egg......Page 275
Recombinant retroviruses can be used to transduce early embryos prior to the formation of the germline......Page 276
Transgenic mice can be produced by the transfection of ES cells followed by the creation of chimeric embryos......Page 277
ES cells can be used for gene targeting in mice......Page 278
Gene-targeting vectors may disrupt genes by insertion or replacement......Page 279
Two rounds of gene targeting allow the introduction of subtle mutations......Page 280
Applications of transgenic mice......Page 281
Applications of gene targeting......Page 285
Standard transgenesis methods are more difficult to apply in other mammals and birds......Page 286
Nuclear transfer technology can be used to clone animals......Page 287
Xenopus oocytes can be used for functional expression cloning......Page 289
Transgenic Xenopus embryos can be produced by restriction enzyme-mediated integration......Page 290
Gene transfer to fish is generally carried out by microinjection, but other methods are emerging......Page 291
Natural P elements have been developed into vectors for gene transfer......Page 292
Gene targeting in Drosophila has been achieved using a combination of homologous and site-specific recombination......Page 294
Callus cultures are established under conditions that maintain cells in an undifferentiated state......Page 297
Callus cultures can be broken up to form cell suspensions, which can be maintained in batches......Page 298
Regeneration of fertile plants can occur through organogenesis or somatic embryogenesis......Page 299
Agrobacterium tumefaciens is a plant pathogen that induces the formation of tumors......Page 300
The ability to induce tumors is conferred by a Ti-plasmid found only in virulent Agrobacterium strains......Page 301
A short segment of DNA, the T-DNA, is transferred to the plant genome......Page 303
Disarmed Ti-plasmid derivatives can be used as plant gene-transfer vectors......Page 304
Binary vectors separate the T-DNA and the genes required for T-DNA transfer, allowing transgenes to be cloned in small plasmids......Page 308
Agrobacterium-mediated transformation can be achieved using a simple experimental protocol in many dicots......Page 310
Monocots were initially recalcitrant to Agrobacterium-mediated transformation, but it is now possible to transform certain varieties of many cereals using this method
......Page 311
Agrobacterium rhizogenes is used to transform plant roots and produce hairy-root cultures......Page 312
Transgenic plants can be regenerated from transformed protoplasts......Page 313
Particle bombardment can be used to transform a wide range of plant species......Page 314
Direct DNA transfer is also used for chloroplast transformation......Page 315
In planta transformation minimizes or eliminates the tissue culture steps usually needed for the generation of transgenic plants
......Page 316
The first plant viral vectors were based on DNA viruses because of their small and simple genomes......Page 317
Most plant virus expression vectors are based on RNA viruses because they can accept larger transgenes than DNA viruses......Page 319
Some naturally occurring inducible promoters can be used to control transgene expression......Page 322
Recombinant inducible systems are built from components that are not found in the host animal or plant......Page 323
The lac and tet repressor systems are based on bacterial operons......Page 324
The tet activator and reverse activator systems were developed to circumvent some of the limitations of the original tet system......Page 325
Steroid hormones also make suitable heterologous inducers......Page 326
Chemically induced dimerization exploits the ability of a divalent ligand to bind two proteins simultaneously......Page 327
Site-specific recombination allows precise manipulation of the genome in organisms where gene targeting is inefficient......Page 329
Site-specific recombination can be used to delete unwanted transgenes......Page 330
Site-specific recombination can be used to activate transgene expression or switch between alternative transgenes......Page 331
Inducible site-specific recombination allows the production of conditional mutants and externally regulated transgene excision......Page 332
Antisense RNA blocks the activity of mRNA in a stoichiometric manner......Page 335
Ribozymes are catalytic molecules that destroy targeted mRNAs......Page 336
Cosuppression is the inhibition of an endogenous gene by the presence of a homologous sense transgene......Page 337
RNA interference is a potent form of silencing caused by the direct introduction of double-stranded RNA into the cell......Page 341
Intracellular antibodies and aptamers bind to expressed proteins and inhibit their assembly or activity......Page 342
Active proteins can be inhibited by dominant-negative mutants in multimeric assemblies......Page 343
Part III Genome Analysis, Genomics, and Beyond......Page 344
The genomes of cellular organisms vary in size over five orders of magnitude......Page 346
Increases in genome complexity sometimes are accompanied by increases in the complexity of gene structure......Page 349
Viruses and bacteria have very simple genomes......Page 351
Chloroplast DNA structure is highly conserved......Page 353
Mitochondrial genome architecture varies enormously, particularly in plants and protists......Page 354
Telomeres play a critical role in the maintenance of chromosomal integrity......Page 355
Tandemly repeated sequences can be detected in two ways......Page 356
Tandemly repeated sequences can be subdivided on the basis of size......Page 358
Dispersed repeated sequences are composed of multiple copies of two types of transposable elements......Page 361
Retrotransposons can be divided into two groups on the basis of transposition mechanism and structure......Page 362
Repeated DNA is non-randomly distributed within genomes......Page 363
Segmental duplications are very large, low-copy-number repeats......Page 364
The human Y chromosome has an unusual chro-mosomes.
structure......Page 365
Summary of structural elements of eukaryotic chromosomes......Page 367
The first physical map of an organism made use of restriction fragment length polymorphisms (RFLPs)......Page 369
Sequence tags are more convenient markers than RFLPs because they do not use Southern blotting......Page 371
Single nucleotide polymorphisms (SNPs) are the most favored physical marker......Page 372
Polymorphic DNA can be detected in the absence of sequence information......Page 374
AFLPs resemble RFLPs and can be detected in the absence of sequence information......Page 375
Padlock probes allow different alleles to be examined simultaneously......Page 376
Optical mapping is undertaken on single DNA molecules......Page 377
Radiation hybrid (RH) mapping involves screening of randomly broken fragments of DNA for specific markers......Page 381
It is essential that the different mapping methods are integrated......Page 383
High-throughput sequencing is an essential prerequisite for genome sequencing......Page 385
There are two different strategies for sequencing genomes......Page 386
Gaps in sequences occur with all genome-sequencing methodologies and need to be closed......Page 391
The quality of genome-sequence data needs to be determined......Page 393
The formation of orthologs and paralogs are key steps in gene evolution......Page 396
Protein evolution occurs by exon shuffling......Page 397
The minimal gene set consistent with independent existence can be determined using comparative genomics......Page 398
Larger microbial genomes have more paralogs than smaller genomes......Page 399
Horizontal gene transfer may be a significant evolutionary force but is not easy to detect......Page 401
The comparative genomics of closely related bacteria gives useful insights into microbial evolution......Page 402
Comparative genomics can be used to analyze physiological phenomena......Page 404
Mitochondrial genomes exhibit an amazing structural diversity......Page 405
Gene transfer has occurred between mtDNA and nuclear DNA......Page 406
Horizontal gene transfer has been detected in mitochondrial genomes......Page 407
Comparative genomics can be used to identify genes and regulatory elements......Page 408
The evolution of species can be analyzed at the genome level......Page 410
Analysis of dipteran insect genomes permits analysis of evolution in multicellular organisms......Page 411
A number of mammalian genomes have been sequenced and the data is facilitating analysis of evolution......Page 413
Comparative genomics can be used to uncover the molecular mechanisms that generate new gene structures......Page 414
Genome-wide gene targeting is the systematic approach to large-scale mutagenesis......Page 417
It is unlikely that systematic gene targeting will be achieved in higher eukaryotes in the foreseeable future......Page 418
Insertional mutagenesis leaves a DNA tag in the interrupted gene, which facilitates cloning and gene identification......Page 419
Genome-wide insertional mutagenesis in yeast has been carried out with endogenous and heterologous transposons......Page 421
Genome-wide insertional mutagenesis in vertebrates has been facilitated by the development of artificial transposon systems......Page 422
T-DNA mutagenesis requires gene transfer by A. tumefaciens
......Page 423
Transposon mutagenesis in plants can be achieved using endogenous or heterologous transposons......Page 425
Insertional mutagenesis in invertebrates......Page 426
Libraries of knock-down phenocopies can be created by RNA interference......Page 427
The first genome-wide RNAi screens in other organisms have been carried out......Page 428
Traditional approaches to expression profiling allow genes to be studied singly or in small groups......Page 430
The transcriptome is the collection of all messenger RNAs in the cell......Page 432
Serial analysis of gene expression uses concatemerized sequence tags to identify each gene......Page 433
Massively parallel signature sequencing involves the parallel analysis of millions of DNA-tagged microbeads......Page 434
DNA microarray technology allows the parallel analysis of thousands of genes on a convenient miniature device......Page 435
Spotted DNA arrays are produced by printing DNA samples on treated microscope slides......Page 436
There are numerous printing technologies for spotted arrays......Page 440
Oligonucleotide chips are manufactured by in situ oligonucleotide synthesis......Page 441
Spotted arrays and oligo chips have similar sensitivities......Page 442
As transcriptomics technology matures, standardization of data processing and presentation become important challenges......Page 444
Global profiling of microbial gene expression......Page 445
Applications of expression profiling in human disease......Page 446
Protein expression analysis is more challenging than mRNA profiling because proteins cannot be amplified like nucleic acids......Page 448
Two-dimensional electrophoresis produces a visual display of the proteome......Page 449
The sensitivity, resolution, and representation of 2D gels need to be improved......Page 450
Multidimensional liquid chromatography is more sensitive than 2DGE and is directly compatible with mass spectrometry......Page 451
High-throughput protein annotation is achieved by mass spectrometry and correlative database searching......Page 454
Specialized strategies are used to quantify proteins directly by mass spectrometry......Page 457
Protein modifications can also be detected by mass spectrometry......Page 458
Antibody arrays contain immobilized antibodies or antibody derivatives for the capture of specific proteins......Page 461
Other molecules may be arrayed instead of proteins......Page 462
Solution arrays are non-planar microarrays......Page 463
Sequence analysis alone is not sufficient to annotate all orphan genes......Page 464
Protein structures are more highly conserved than sequences......Page 465
Protein structures are determined experimentally by X-ray crystallography or nuclear magnetic resonance spectroscopy......Page 467
Protein structures can be modeled on related structures......Page 469
Protein structures can be aligned using algorithms that carry out intramolecular and intermolecular comparisons......Page 470
The annotation of proteins by structural comparison has been greatly facilitated by standard systems for the structural classification of proteins
......Page 471
International structural proteomics initiatives have been established to solve protein structures on a large scale......Page 472
Protein interactions can be inferred by a variety of genetic approaches......Page 476
New methods based on comparative genomics can also infer protein interactions......Page 477
Traditional biochemical methods for protein interaction analysis cannot be applied on a large scale......Page 480
The yeast two-hybrid system is an in vivo interaction screening method......Page 481
In the matrix approach, defined clones are generated for each bait and prey......Page 483
In the random library method, bait and/or prey are represented by random clones from a highly complex expression library......Page 484
Robust experimental design is necessary to increase the reliability of two-hybrid interaction screening data......Page 485
Systematic analysis of protein complexes can be achieved by affinity purification and mass spectrometry......Page 488
Protein localization is an important component of interaction data......Page 489
Interaction screening produces large data sets which require extensive bioinformatic support......Page 490
Introduction......Page 495
Metabolomics studies in humans are different from those in other organisms......Page 496
Compromises have to be made in choosing analytical methodology for metabolomics studies......Page 497
Sample selection and sample handling are crucial stages in metabolomics studies......Page 500
Metabolomics produces complex data sets......Page 502
A good reference database is an essential prerequisite for preparing global biochemical networks but currently is missing......Page 504
Part IV Applications of Gene Manipulation and Genomics......Page 506
Investigating discrete traits in outbreeding populations (genetic diseases of humans)......Page 508
Model-free (nonparametric) linkage analysis looks at the inheritance of disease genes and selected markers in several generations of the same family
......Page 510
Linkage disequilibrium (association) studies look at the co-inheritance of markers and the disease at the population level......Page 515
Once a disease locus is identified, all the ’omics can be used to analyze it in detail......Page 516
The integration of global information about DNA, mRNA, and protein can be used to facilitate disease-gene identification......Page 517
The existence of haplotype blocks should simplify linkage disequilibrium analysis......Page 518
Particular kinds of genetic cross are necessary if QTLs are to be mapped......Page 520
Identifying QTLs involves two challenging steps......Page 521
Chromosome substitution strains make the identification of QTLs easier......Page 524
Genetic variation accounts for the different responses of individuals to drugs......Page 526
Pharmacogenomics is being used by the pharmaceutical industry......Page 527
Personalized medicine involves matching genotypes to therapy......Page 529
Recombinant therapeutic proteins are produced commercially in bacteria, yeast, and mammalian cells......Page 531
Transgenic animals and plants can also be used as bioreactors to produce recombinant proteins......Page 541
Metabolic engineering provides new routes to small molecules......Page 547
Combinatorial biosynthesis can produce completely novel compounds......Page 549
Metabolic engineering can also be achieved in plants and plant cells to produce diverse chemical structures......Page 550
Production of vinblastine and vincristine in Catharanthus cell cultures is a challenge because of the many steps and control points in the pathway......Page 551
The production of vitamin A in cereals is an example of extending an endogenous metabolic pathway......Page 552
The enhancement of plants to produce more vitamin E is an example of balancing several metabolic pathways and directing flux in the preferred direction
......Page 555
Herbicide resistance is the most widespread trait in commercial transgenic plants......Page 556
Virus-resistant crops can be produced by expressing viral or non-viral transgenes......Page 558
Resistance to fungal pathogens is often achieved by manipulating natural plant defense mechanisms......Page 559
The bacterium Bacillus thuringiensis provides the major source of insect-resistant genes......Page 560
Drought resistance provides a good example of how plants can be protected against abiotic stress......Page 561
Plants can be engineered to cope with poor soil quality......Page 562
Transgenic animals can be created as models of human disease......Page 563
Gene medicine is the use of nucleic acids to prevent, treat, or cure disease......Page 564
DNA vaccines are expression constructs whose products stimulate the immune system......Page 566
Gene augmentation therapy for recessive diseases involves transferring a functional copy of the gene into the genome......Page 567
Gene-therapy strategies for cancer may involve dominant suppression of the overactive gene or targeted killing of the cancer cells
......Page 568
References......Page 570
Appendix: The genetic code and single-letter amino acid designations......Page 650
Index......Page 651