Model Organisms in Drug Discovery

More documents

Recommendations

Info

130 DROSOPHILA – A MODEL SYSTEM Nature of mutations Table 5.1 Comparison of different mutagens used in Drosophila EMS X-ray P-element EP element Point mutations Small deletions Advantages Saturation screens Disadvantages No molecular anchor Special features Most frequently used Deletions Chromosome breaks Inversions Translocations Fast Big deletions cytologically visible Several genes deleted Mainly null alleles Loss-of-function mutations Molecular anchor Hot spots for insertion Reporter assays Gain-of-function mutations Molecular anchor Hot spots for insertion Only method of efficiently inducing ectopic activation mutations, the appropriate mutagen is chosen (see Table 5.1). Transposable elements such as P and EP insert into the genome almost randomly (Spradling et al., 1999). At the insertion site, they have any of the following effects. When they integrate into coding regions, they destroy the corresponding gene, leading to a loss-of-function (LOF) mutation. This case, however, is rare because transposable elements have a preference to insert into non-coding sequences in the 5’ region of the gene. When P elements with a reporter gene driven by a minimal promoter come under the influence of an endogenous enhancer, the reporter will be expressed in the same way as the corresponding gene (enhancer trap) (O’Kane and Gehring, 1987). In this way, genes are identified based on their expression pattern rather than their LOF phenotype. Finally, EP elements containing an enhancer/promoter that is activated by the yeast transcription factor Gal4 are used to activate ectopically the nearby genes to produce a GOF mutation (Rorth et al., 1998) (see later section on EP overexpression screens). Obviously, transposable elements have the advantage that they serve as a molecular tag to isolate flanking sequences. This makes the gene identification process very rapid. However, they are not suitable as a mutagen for saturation screens because the frequency with which the genes inactivate is low due to their preferential jumping into non-coding 5’ regions. Furthermore, not all the genes
TARGET IDENTIFICATION/TARGET VALIDATION STRATEGIES 131 are targeted with P elements at the same frequency. There are hotspots and coldspots in the genome (Berg and Spradling, 1991). The second widely used mutagen is EMS. Chemical mutagens induce mutations randomly. The frequency of mutations depends on the concentration of the mutagen and can reach one lethal hit per chromosome arm at high concentration (Lewis and Bacher, 1968). Using EMS, genome-wide saturation is reached readily. The degree of genome saturation is measured by the number of mutations (alleles) identified at different loci. For example, in a screen for suppressors of the rough eye phenotype caused by expression of an activated form of the Raf kinase, we identified, in a total of 300 000 flies, 45 individual mutations in rolled, encoding the Drosophila homolog of MAP kinase (Dickson et al., 1996). For functional studies of a particular gene it is important to have several alleles with different effects on the final protein. In addition, when the identified mutations cluster in certain parts of the coding regions, this points to functionally important protein domains (see earlier section on WNT pathway, lgs alleles; Kramps et al., 2002). The fact that it is not trivial to detect the mutations induced by EMS molecularly has been a disadvantage for a long time. However, with the availability of the entire genome sequence, new precise mapping strategies have become available (see later section on gene mapping strategies). For target identification, chemical mutagenesis using EMS is therefore the method of choice. In the following sections we will discuss the art of designing screens: dominant or recessive screens, screens for LOF or GOF mutations, screens for null-alleles or hypomorphs, tissue-specific screens and modifier screens. Screens for recessive mutations Christiane Nüsslein-Volhard and Eric Wieschaus pioneered the recessive screens. Their work was revolutionary because it was the first mutagenesis in any multicellular organism that attempted to find most or all the genes that affect a given process (saturation screen). They identified most of the essential patterning genes that are required throughout embryonic development (Nu¨sslein and Wieschaus, 1980). Their groundbreaking work was honored with the Nobel Prize in 1995. For several reasons, however, screens for recessive mutations are limited to certain aspects of development and to special classes of genes: 1. Mutations in essential genes are homozygous lethal. The phenotypic classification is restricted to phenotypes that are visible during embryogenesis or larval development. 2. Only the first essential function of a particular gene can be identified. However, many genes are used several times during development. The wg
Page 1 and 2:
Model Organisms in Drug Discovery M
Page 3 and 4:
Copyright u 2003 John Wiley & Sons
Page 5 and 6:
Contents List of contributors .....
Page 7 and 8:
CONTENTS ix 7 Genetics and Genomics
Page 9 and 10:
List of Contributors Hector Beltran
Page 11 and 12:
LIST OF CONTRIBUTORS xiii Stefan Sc
Page 13 and 14:
1 Introduction to Model Systems in
Page 15 and 16:
Organism INTEGRATING MODEL ORGANISM
Page 17 and 18:
INTEGRATING MODEL ORGANISM RESEARCH
Page 19 and 20:
INTEGRATING MODEL ORGANISM RESEARCH
Page 21 and 22:
10 GROWING YEAST FOR FUN AND PROFIT
Page 23 and 24:
Page 25 and 26:
Page 27 and 28:
Page 29 and 30:
Page 31 and 32:
Page 33 and 34:
Page 35 and 36:
Page 37 and 38:
Page 39 and 40:
Page 41 and 42:
Page 43 and 44:
Page 45 and 46:
Page 47 and 48:
Page 49 and 50:
Page 51 and 52:
3 Caenorhabditis elegans Functional
Page 53 and 54:
THE DRUG DISCOVERY PROCESS 43 thoug
Page 55 and 56:
multivulva phenotype of Ras gain-of
Page 57 and 58:
disease. These molecular targets ar
Page 59 and 60:
FROM DISEASE TO TARGET 49 Figure 3.
Page 61 and 62:
FROM DISEASE TO TARGET 51 Figure 3.
Page 63 and 64:
signaling pathway. The third catego
Page 65 and 66:
FROM DISEASE TO TARGET 55 resistant
Page 67 and 68:
phenomenon was first observed in C.
Page 69 and 70:
profiles. The C. elegans gene expre
Page 71 and 72:
emerging technologies. Forward gene
Page 73 and 74:
The compound library LEAD DISCOVERY
Page 75 and 76:
LEAD DISCOVERY 65 previous section,
Page 77 and 78:
LEAD DISCOVERY 67 Figure 3.5 Distri
Page 79 and 80:
Compound learning set for assay val
Page 81 and 82:
10 000-15 000 human drug targets ar
Page 83 and 84:
transporter itself. The SSRIs that
Page 85 and 86:
REFERENCES 75 de Montigny, C., Blie
Page 87 and 88: REFERENCES 77 Lewis, J. A., Wu, C.
Page 89 and 90: REFERENCES 79 Tissenbaum, H. A. and
Page 91 and 92: 82 DROSOPHILA AS A TOOL FOR DRUG DI
Page 109 and 110: 100 DROSOPHILA AS A TOOL FOR DRUG D
Page 127 and 128: 5 Drosophila - a Model System for T
Page 129 and 130: EVOLUTIONARY CONSERVATION OF DISEAS
Page 137: TARGET IDENTIFICATION/TARGET VALIDA
Page 141 and 142: TARGET IDENTIFICATION/TARGET VALIDA
Page 151 and 152: CHEMICAL GENETICS 143 acid identity
Page 153 and 154: 3. A hit identifies a biologically
Page 155 and 156: about their function in a multicell
Page 157 and 158: REFERENCES 149 Han, Z. S., Enslen,
Page 159 and 160: REFERENCES 151 Rintelen, F., Stocke
Page 161 and 162: 6 Mechanism of Action in Model Orga
Page 163 and 164: INTRODUCTION TO COMPOUND DEVELOPMEN
Page 165 and 166: MODEL ORGANISMS ARRIVE ON THE SCENE
Page 167 and 168: ELUCIDATING THE MECHANISM OF COMPOU
Page 169 and 170: ELUCIDATING THE MECHANISM OF COMPOU
Page 171 and 172: A CASE STUDY FOR ALZHEIMER’S DISE
Page 179 and 180: NEW CHEMICAL GENETIC STRATEGIES 171
Page 181 and 182: A CASE STUDY FOR INNATE IMMUNITY 17
Page 183 and 184: GLOBAL GENE EXPRESSION STUDIES IN M
Page 185 and 186: As described above, the extensive i
Page 187 and 188: REFERENCES 179 Austin, J. and Kimbl
Page 189 and 190:
REFERENCES 181 Hughes, T. R., Marto
Page 191 and 192:
REFERENCES 183 Sin, N., Meng, L., W
Page 193 and 194:
186 GENETICS AND GENOMICS IN THE ZE
Page 195 and 196:
Page 197 and 198:
Page 199 and 200:
Page 201 and 202:
Page 203 and 204:
Page 205 and 206:
Page 207 and 208:
Page 209 and 210:
8 Lipid Metabolism and Signaling in
Page 211 and 212:
FISH AS A MODEL ORGANISM 205 genes
Page 213 and 214:
LIPID METABOLISM SCREEN 207 transpo
Page 215 and 216:
LIPID METABOLISM SCREEN 209 Figure
Page 217 and 218:
the embryo media containing radioac
Page 219 and 220:
ZEBRAFISH AS A MODEL SYSTEM 213 Fig
Page 221 and 222:
ZEBRAFISH AS A MODEL SYSTEM 215 Reg
Page 223 and 224:
aspirin, which has potent inhibitor
Page 225 and 226:
REFERENCES 219 Chau, I. and Cunning
Page 227 and 228:
REFERENCES 221 Patrono, C., Patrign
Page 229 and 230:
224 CHEMICAL MUTAGENESIS IN THE MOU
Page 231 and 232:
Page 233 and 234:
Page 235 and 236:
Page 237 and 238:
Page 239 and 240:
Page 241 and 242:
Page 243 and 244:
Page 245 and 246:
Page 247 and 248:
Page 249 and 250:
Page 251 and 252:
Page 253 and 254:
Page 255 and 256:
Page 257 and 258:
252 SATURATION SCREENING OF DRUGGAB
Page 259 and 260:
Page 261 and 262:
Page 263 and 264:
Page 265 and 266:
Page 267 and 268:
Page 269 and 270:
Page 271 and 272:
Page 273 and 274:
Page 275 and 276:
Page 277 and 278:
Page 279 and 280:
Page 281 and 282:
Page 283 and 284:
Page 285 and 286:
280 INDEX bupropion 92 busulfan 143
Page 287 and 288:
282 INDEX Dscam 171 dual-energy X-r
Page 289 and 290:
284 INDEX L-685,818 16 lead discove
Page 291 and 292:
286 INDEX protein function 19-22 pr
Page 293:
288 INDEX yeast (continued) functio
show all

Model Organisms in Drug Discovery

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?