John Wiley & Sons - 2004 - Analysis of Genes and Genomes
.pdfviii CONTENTS
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2.1.3 How do type II restriction enzymes work? |
74 |
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2.2 |
Joining DNA molecules |
76 |
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2.3 |
The basics of cloning |
78 |
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2.4 |
Bacterial transformation |
84 |
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2.4.1 |
Chemical transformation |
86 |
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2.4.2 |
Electroporation |
87 |
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2.4.3 |
Gene gun |
88 |
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2.5 |
Gel electrophoresis |
88 |
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2.5.1 |
Polyacrylamide gels |
89 |
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2.5.2 |
Agarose gels |
89 |
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2.5.3 |
Pulsed-field gel electrophoresis |
95 |
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2.6 |
Nucleic acid blotting |
98 |
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2.6.1 |
Southern blotting |
100 |
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2.6.2 The compass points of blotting |
102 |
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2.7 |
DNA purification |
103 |
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3 |
Vectors |
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109 |
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3.1 |
Plasmids |
112 |
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3.1.1 |
pBR322 |
116 |
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3.1.2 |
pUC plasmids |
119 |
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3.2 |
Selectable markers |
122 |
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3.3 |
λ vectors |
126 |
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3.4 |
Cosmid vectors |
135 |
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3.5 |
M13 vectors |
137 |
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3.6 |
Phagemids |
140 |
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3.7 |
Artificial chromosomes |
142 |
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3.7.1 |
YACs |
143 |
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3.7.2 |
PACs |
146 |
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3.7.3 |
BACs |
148 |
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3.7.4 |
HACs |
149 |
4 |
Polymerase chain reaction |
153 |
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4.1 |
PCR reaction conditions |
159 |
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4.2 |
Thermostable DNA polymerases |
162 |
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4.3 |
Template DNA |
164 |
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4.4 |
Oligonucleotide primers |
165 |
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4.4.1 |
Synthesis of oligonucleotide primers |
167 |
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4.5 |
Primer mismatches |
169 |
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4.6 |
PCR in the diagnosis of genetic disease |
173 |
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4.7 |
Cloning PCR products |
175 |
CONTENTS ix
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4.8 |
RT –PCR |
177 |
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4.9 |
Real-time PCR |
179 |
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4.10 |
Applications of PCR |
181 |
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5 |
Cloning a gene |
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183 |
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5.1 |
Genomic libraries |
185 |
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5.2 |
cDNA libraries |
191 |
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5.3 |
Directional cDNA cloning |
196 |
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5.4 |
PCR based libraries |
199 |
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5.5 |
Subtraction libraries |
200 |
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5.6 |
Library construction in the post-genome era |
204 |
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6 |
Gene identification |
205 |
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6.1 |
Screening by nucleic acid hybridization |
206 |
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6.2 |
Immunoscreening |
211 |
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6.3 |
Screening by function |
216 |
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6.4 |
Screening by interaction |
217 |
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6.5 |
Phage display |
218 |
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6.6 |
Two-hybrid screening |
218 |
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6.6.1 Problems, and some solutions, with two-hybrid |
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screening |
225 |
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6.7 |
Other interaction screens – variations on a theme |
228 |
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6.7.1 |
One hybrid |
229 |
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6.7.2 |
Three hybrid |
229 |
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6.7.3 |
Reverse two hybrid |
229 |
7 |
Creating mutations |
231 |
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7.1 |
Creating specific DNA changes using primer extension |
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mutagenesis |
233 |
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7.2 |
Strand selection methods |
237 |
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7.2.1 |
Phosphorothioate strand selection |
237 |
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7.2.2 |
dut− ung− (or Kunkel) strand selection |
238 |
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7.3 |
Cassette mutagenesis |
240 |
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7.4 |
PCR based mutagenesis |
241 |
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7.5 |
QuikChange mutagenesis |
248 |
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7.6 |
Creating random mutations in specific genes |
250 |
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7.7 |
Protein engineering |
254 |
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8 Protein production and purification |
257 |
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8.1 |
Expression in E. coli |
258 |
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8.1.1 |
The lac promoter |
259 |
xCONTENTS
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8.1.2 |
The tac promoter |
259 |
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8.1.3 |
The λPL promoter |
260 |
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8.1.4 |
The T7 expression system |
261 |
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8.2 |
Expression in yeast |
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265 |
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8.2.1 |
Saccharomyces cerevisiae |
265 |
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8.2.1.1 |
The GAL system |
266 |
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8.2.1.2 |
The CUP1 system |
268 |
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8.2.2 |
Pichia pastoris |
268 |
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8.2.3 |
Schizosaccharomyces pombe |
269 |
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8.3 |
Expression in insect cells |
269 |
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8.4 |
Expression in higher-Eukaryotic cells |
272 |
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8.4.1 |
Tet-on/Tet-off system |
272 |
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8.5 |
Protein purification |
275 |
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8.5.1 |
The His-tag |
276 |
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8.5.2 |
The GST-tag |
279 |
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8.5.3 |
The MBP-tag |
282 |
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8.5.4 |
IMPACT |
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282 |
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8.5.5 |
TAP-tagging |
286 |
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9 |
Genome sequencing projects |
287 |
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9.1 |
Genomic mapping |
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289 |
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9.2 |
Genetic mapping |
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290 |
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9.3 |
Physical mapping |
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293 |
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9.4 |
Nucleotide sequencing |
295 |
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9.4.1 |
Manual DNA sequencing |
296 |
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9.4.2 |
Automated DNA sequencing |
300 |
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9.5 |
Genome sequencing |
303 |
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9.6 |
The human genome project |
305 |
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9.7 |
Finding genes |
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307 |
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9.8 |
Gene assignment |
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309 |
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9.9 |
Bioinformatics |
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311 |
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10 |
Post-genome analysis |
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313 |
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10.1 |
Global changes in gene expression |
314 |
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10.1.1 |
Differential display |
315 |
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10.1.2 |
Microarrays |
317 |
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10.1.3 |
ChIPs with everything |
324 |
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10.2 |
Protein function on a genome-wide scale |
327 |
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10.3 |
Knock-out analysis |
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327 |
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10.4 |
Antisense and RNA interference (RNAi) |
329 |
CONTENTS xi
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10.5 |
Genome-wide two-hybrid screens |
333 |
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10.6 |
Protein detection arrays |
335 |
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10.7 |
Structural genomics |
335 |
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11 |
Engineering plants |
341 |
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11.1 |
Cloning in plants |
341 |
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11.1.1 |
Agrobacterium tumefaciens |
342 |
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11.1.2 |
Direct nuclear transformation |
347 |
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11.1.3 |
Viral vectors |
348 |
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11.1.4 |
Chloroplast transformation |
350 |
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11.2 |
Commercial exploitation of plant transgenics |
354 |
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11.2.1 |
Delayed ripening |
354 |
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11.2.2 |
Insecticidal resistance |
355 |
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11.2.3 |
Herbicidal resistance |
356 |
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11.2.4 |
Viral resistance |
357 |
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11.2.5 |
Fungal resistance |
358 |
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11.2.6 |
Terminator technology |
358 |
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11.3 |
Ethics of genetically engineered crops |
360 |
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12 |
Engineering animal cells |
361 |
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12.1 |
Cell culture |
361 |
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12.2 |
Transfection of animal cells |
362 |
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12.2.1 |
Chemical transfection |
363 |
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12.2.2 |
Electroporation |
364 |
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12.2.3 |
Liposome-mediated transfection |
364 |
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12.2.4 |
Peptides |
366 |
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12.2.5 |
Direct DNA transfer |
366 |
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12.3 |
Viruses as vectors |
367 |
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12.3.1 |
SV40 |
367 |
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12.3.2 |
Adenovirus |
369 |
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12.3.3 |
Adeno-associated virus (AAV) |
371 |
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12.3.4 |
Retrovirus |
372 |
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12.4 |
Selectable markers and gene amplification in animal cells |
375 |
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12.5 |
Expressing genes in animal cells |
378 |
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13 |
Engineering animals |
379 |
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13.1 |
Pronuclear injection |
381 |
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13.2 |
Embryonic stem cells |
384 |
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13.3 |
Nuclear transfer |
390 |
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13.4 |
Gene therapy |
396 |
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13.5 |
Examples and potential of gene therapy |
398 |
xii CONTENTS
Glossary |
401 |
Proteins |
409 |
A1.1 |
409 |
A1.2 |
410 |
A1.3 |
411 |
Nobel prize winners |
413 |
References |
417 |
Index |
459 |
Preface
There are few phrases that can elicit such an emotive response as ‘genetic engineering’ and ‘cloning’. Newspapers and television invariably use these phrases to describe something that is not quite right – even perhaps against nature. Genetic engineering and the modification of genes invariably conjures up images of Frankenstein foods and abnormal animals. During the course of reading this book, however, I hope that readers will appreciate that genetic engineering, and the techniques of molecular biology that underpin it, are essential components to understanding how organisms work. Man has been playing, often unwittingly, with genes for thousands of years through selective breeding to promote certain traits that were seen as desirable. We are currently at a watershed in the way in which we look at genes. Behind us is 50 years of knowledge of the structure of the genetic material, and ahead is the ability to see how every gene that we contain responds to other genes and environmental conditions. Determining the biochemical basis of why certain people respond differently to drug treatments, for example, may not be possible yet, but the techniques to address the appropriate questions are in place. The excitement of entering the post-genome age will go hand-in-hand with concerns over what we have the ability to do – whether we actually do it or not.
The analysis of genes and genomes could easily fall into a list of techniques that can be applied to a particular problem. I have tried to avoid this and, wherever possible, I have used specific examples to illustrate the problem and potential solutions. I have relied heavily on published works and have endeavoured to reference all primary material so that interested readers can explore the topic further. This has also allowed me to place many of the ideas and experiments into a historical context. It seems a common misconception that Watson and Crick were solely responsible for our understanding of how genes work. Their contribution should never be underestimated, but the work of many others should not be discounted. The full sequence of the human genome and, equally or even more importantly, the genomes of experimentally amenable organisms provide exceptional opportunities for advances in biological sciences over the coming years. More and more experiments can now be performed on a genomewide scale and we are just beginning to understand the consequences of this.
One of the main problems that I have encountered during the writing of this text is attaining a balance between depth and coverage. I have purposefully
xiv PREFACE
concentrated on more amenable experimental systems – E. coli for prokaryotes and yeast for eukaryotes. In addition, I have treated higher eukaryotes as being almost exclusively mammals, and especially humans. This is intended to give readers a flavour of the ideas and experiments that are currently being undertaken, but also to give a historical framework onto which today’s experiments may be hung. We ignore the past at our peril. This approach has, however, led to the exclusion of some other systems, e.g. Drosophila and prokaryotes other than E. coli, but is by no means meant as a slight to these neglected fields. Rather than either covering all fields in scant detail or explaining the intricate details and nuances of only a few, I have attempted to provide a broad overview that is punctuated with specific examples. Whether I have succeeded in getting the balance right I will leave to individual readers. I can say for certain, however, that there has never been a more exciting time to study biology, and I hope that this is reflected in this text.
Richard J. Reece
The University of Manchester
October 2003
Acknowledgements
I have had a great deal of help in writing this book. Of course, omissions and inaccuracies are entirely my responsibility, but I thank those who have (hopefully) kept these to a minimum – David Timson, Noel Curtis, Cristina Merlotti, Chris Sellick, Carolyn Byrne, Ray Boot-Handford and Ged Brady. I am also very grateful to Robert Slater (University of Hertfordshire) and to Mick Tuite (University of Kent) for their immensely helpful comments and suggestions. I thank the many friends and colleagues, mentioned in the text, who have so generously provided both figures for the book and for permission to cite their work. I am also deeply indented to Jordi Bella for showing me that molecular graphics programmes are usable by idiots. Nicky McGirr at John Wiley persuaded me that this project was a good idea. Her boundless enthusiasm and encouragement saw me through the times when I was not so sure and, of course, she was right. The ‘guinea pigs’ for many of the ideas presented here have been successive years of Genetic Engineering students at The University of Manchester. I thank the many of them who read parts of the manuscript, and all of them for challenging me, and many of my preconceived ideas. Judith, Daniel and Kathryn have been incredibly patient throughout the inception and writing of this book. Readers who find it useful should be thanking them, not me. Finally, I want to thank my teachers – Tony Maxwell and Mark Ptashne – who, each in his own way, have true passion for science and an insistence that the right experiments are done.
Abbreviations and acronyms
AAT |
α1-antitrypsin |
AAV |
adeno-associated virus |
AD |
activation domain |
BAC |
bacterial artificial chromosome |
CaMV |
cauliflower mosaic virus |
CAP |
catabolite activator protein |
CBD |
chitin binding domain |
CDK |
cyclin-dependent kinase |
cDNA |
complementary DNA |
CFI |
cleavage factor I |
CFII |
cleavage factor II |
CHEF |
contour-clamped homogeneous electric field |
ChIP |
chromatin immunoprecipitation |
CMV |
cytomegalovirus |
CPSF |
cleavage and polyadenylation specificity factor |
CStF |
cleavage stimulation factor |
CTD |
carboxy-terminal repeat domain |
DBD |
DNA binding domain |
DEAE |
diethylaminoethanol |
DHFR |
dihydrofolate reductase |
DNA |
deoxyribonucleic acid |
DTT |
dithiothreitol |
ECM |
extra-cellular matrix |
EMS |
ethyl methane sulphonate |
ER |
endoplasmic reticulum |
ES |
embryonic stem |
EST |
expressed sequence tag |
FIGE |
field inversion gel electrophoresis |
FISH |
fluorescent in situ hybridization |
FRET |
fluorescence resonance energy transfer |
GST |
glutathione S-transferase |
HAC |
human artificial chromosome |
HAT |
histone acetyltransferase |
H-DAC |
histone deacetylase |