An investigation of two novel genes associated with Wnt signalling in Drosophila melanogaster
By: [by] Olivier Hagens.
Series: Sussex theses ; S 5087
Publisher: University of Sussex
Published: 2000
Place of publication: Brighton, UK
Language: eng
Format: Hardcover
Number of pages: 204
Description: MPhil - 2000
Notes: M.Phil. 2001.
Table of contents: title: i
label: Table of Contents; title: vii
label: Abbreviations; title: x
label: Index of Figures; title: xiii
label: Index of Tables; title: 1
label: Chapter I: Introduction; title: 2
label: 1. Hedgehog and Wnt signalling pathways; title: 2
label: A. The Hedgehog pathway; title: 2
label: a. Vertebrate Hh signalling; title: 3
label: b. Drosophila Hh signalling; title: 4
label: c. Drosophila Hh protein biochemistry; title: 5
label: d. The Hh pathway in Drosophila; title: 7
label: B. The Wnt pathway; title: 8
label: a. Vertebrate Wnt signalling; title: 8
label: b. Drosophila Wnt signalling; title: 9
label: c. The Wnt pathway; title: 14
label: d. Wnts in human disease; title: 15
label: d1.Wnts and cancer; title: 16
label: d2. Wnt-signalling and Alzheimer’s Disease; title: 18
label: d3. GSK-3 and diabetes; title: 20
label: 3. Signal transduction in patterning processes; title: 20
label: A. DV patterning in Xenopus; title: 21
label: B. Limb patterning in vertebrates; title: 22
label: C. Segmentation in Drosophila; title: 27
label: D. Wg in the Drosophila wing imaginal disc; title: 30
label: E. Bristle pattern in Drosophila; title: 34
label: 4. Objectives; title: 36
label: Chapter II: l(2)56B-W - RNAi Approach; title: 36
label: 1. Introduction; title: 36
label: A. Mapping of l(2)56B-W; title: 37
label: a. The FLP-FRT system; title: 40
label: b. Mapping by complementation with translocation fragments; title: 40
label: c. Mapping by complementation with deletions and inversions; title: 41
label: B. RNA interference: dsRNA mystery; title: 44
label: C. The larval denticle pattern in Drosophila melanogaster; title: 46
label: 2. Materials and Methods; title: 46
label: A. Competent Cells; title: 46
label: B. Transformation; title: 47
label: C. Phenol:Chloroform extraction; title: 47
label: a. Plasmid DNA; title: 47
label: b. PCR products, fly-genomic DNA and RNA; title: 47
label: D. Ethanol precipitation; title: 47
label: a. Plasmid DNA; title: 47
label: b. PCR products, fly-genomic DNA and RNA; title: 48
label: E. Small-scale preparations of plasmid DNA; title: 48
label: F. Electrophoresis; title: 49
label: G. PCR; title: 52
label: H. Transcription – Preparation of dsRNA; title: 53
label: I. Microinjection of dsRNA; title: 54
label: 3. Results; title: 57
label: A. dsRNA injections; title: 57
label: a. Control injections; title: 57
label: a1. Injection Buffer; title: 57
label: a2. pNeural; title: 58
label: a3. Enhanced Green Fluorescent Protein (EGFP); title: 58
label: a4. Wingless; title: 59
label: a5. Fz, DFz2 and Fz + DFz2; title: 59
label: b. dsRNA injections of transcripts located in the l(2)56B-W region; title: 59
label: b1. LD23409/SD08336; title: 60
label: b2. CK00367; title: 60
label: b3. LD31969; title: 60
label: b4. LD21192; title: 60
label: b5. LP04016; title: 60
label: b6. R01N09; title: 61
label: 4. Discussion; title: 65
label: Chapter III: Molecular Analysis of the l(2)56B-W Transcription Unit; title: 65
label: 1. Introduction; title: 66
label: A. Molecularly characterised putative candidates for l(2)56B-W; title: 66
label: a. 5-HT1AR; title: 67
label: b. enabled; title: 68
label: B. Putative candidates for l(2)56B-W based on ESTs and computational predictions; title: 69
label: a. CG15119; title: 69
label: b. CG15118; title: 70
label: c. CG15110; title: 72
label: d. GC15117; title: 73
label: 2. Materials and Methods; title: 73
label: A. General; title: 73
label: B. Maintenance of fly stocks; title: 73
label: C. Sequencing of l(2)56B-W mutant flies; title: 73
label: a. Preparation of genomic DNA; title: 75
label: b. PCR amplification of the CG15111 transcription unit; title: 76
label: c. Compiling of a CG15111 cDNA; title: 76
label: d. Mutation analysis of the l(2)56B-W mutants; title: 77
label: 3. Results; title: 77
label: A. Compiling of a CG15111 cDNA; title: 80
label: B. Genomic organisation of the CG15111 gene; title: 82
label: C. Coding sequence and putative CG15111 protein; title: 85
label: D. Molecular characterisation of the two recovered l(2)56B-W alleles; title: 86
label: 4. Discussion; title: 94
label: Chapter IV: RNA Network Hypothesis; title: 94
label: 1. Introduction; title: 94
label: A. Discovery of RNAi; title: 96
label: B. Molecular mechanism of RNAi; title: 99
label: C. RNA network hypothesis; title: 101
label: 2. Materials and Methods; title: 101
label: A. General; title: 101
label: B. Reannealing of RNA networks; title: 101
label: a. Tm measurement; title: 101
label: b. Generation of completely dsRNA; title: 102
label: C. Evaluation of reannealed RNA networks; title: 102
label: a. Reaction with formaldehyde; title: 102
label: b. Terbium quenching of ethidium bromide stained RNA networks; title: 103
label: c. RNase A assay; title: 103
label: d. Injection of reannealed RNA constructs; title: 104
label: 3. Results; title: 105
label: A. Optimisation of the measurement of the melting temperature of nucleic acids; title: 106
label: B. Estimation of the Tm of wg and EGFP dsRNA preparations; title: 108
label: C. Monitoring the ss – ds transition; title: 108
label: a. Reaction with formaldehyde; title: 112
label: b. Terbium quenching of ethidium bromide stained RNA networks; title: 114
label: c. RNase A assay; title: 116
label: d. Functional assay; title: 118
label: 4. Discussion; title: 122
label: Chapter V: ED17 – Expression; title: 122
label: 1. Introduction; title: 122
label: A. Enhancer detection; title: 123
label: B. Plasmid rescue; title: 123
label: a. CG15149; title: 123
label: b. CG15150; title: 125
label: C. P[ED17] enhancer detection construct; title: 126
label: 2. Materials and Methods; title: 126
label: A. General; title: 126
label: B. X-gal staining; title: 126
label: C. Ab-staining and epifluorescence/confocal microscopy; title: 127
label: D. Plasmid rescue; title: 127
label: a. Isolation of genomic DNA from adult flies; title: 128
label: b. Plasmid rescue; title: 129
label: E. In situ hybridisation to wing imaginal discs; title: 129
label: a. Production of DIG labelled RNA probe; title: 130
label: b. Estimation of the efficiency of the labelling reaction; title: 131
label: c. In situ hybridisation to Drosophila wing imaginal discs; title: 132
label: 3. Results; title: 132
label: A. X-gal staining of larval imaginal discs; title: 134
label: B. Antibody staining; title: 135
label: C. Mapping of the P[ED17] insertion site; title: 137
label: 4. Discussion; title: 141
label: Chapter VI: ED17 – Excision; title: 141
label: 1. Introduction; title: 141
label: 2. Materials and Methods; title: 141
label: A. General; title: 142
label: B. Generation of P[ED17] excision lines; title: 143
label: C. Complementation analysis of the P[ED17] revertants; title: 143
label: D. Deletion mapping of the ED17 locus; title: 144
label: 3. Results; title: 144
label: A. Generation of P[ED17] homozygous revertants; title: 145
label: B. Establishing of P[ED17] complementation groups; title: 147
label: C. Initial deletion mapping of the ED17 locus; title: 148
label: 4. Discussion; title: 150
label: Acknowledgements; title: 151
label: References