An investigation of two novel genes associated with Wnt signalling in Drosophila melanogaster

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Olivier Hagens
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Last edited by Olivier Hagens
May 17, 2011 | History

An investigation of two novel genes associated with Wnt signalling in Drosophila melanogaster

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MPhil - 2000

Publish Date
Language
English
Pages
204

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Book Details


Table of Contents

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

Edition Notes

M.Phil. 2001.

Published in
Brighton, UK
Series
Sussex theses ; S 5087

The Physical Object

Format
Hardcover
Number of pages
204
Dimensions
30 x 20.6 x 3.1 centimeters

ID Numbers

Open Library
OL18772252M

Work Description

Important segmentation and patterning processes are studied extensively in Drosophila melanogaster, the fruitfly, a model organism in developmental biology. These events are mediated by pathways conserved between invertebrates and vertebrates. The Wnt signal transduction pathway is of major importance in these studies. Furthermore it is related to human diseases like colon cancer, Alzheimer’s and diabetes.
The bristles of Drosophila are external sensory organs in the peripheral nervous system, linking the environment and the behaviour dictated by this environment. The stereo-typed pattern in which the bristles occur is conserved within the Drosophilidae. The ge-netic mechanisms underlying bristle patterning in Drosophila can be used as a model to investigate the Wnt pathway. This dissertation describes work undertaken to elucidate the nature of two previously unknown putative members of the pathway.
l(2)56B-W is a gene of which two alleles with an interesting phenotype were retrieved from a large-scale mutation screen. P[ED17] is an enhancer trap line with a proneural-like expression pattern, recovered from a small-scale insertion screen.
This thesis reports the results from an RNAi-approach to investigate the l(2)56B-W-locus. The mechanics of RNAi itself are also discussed. Both available alleles were par-tially sequenced.
I have confirmed the expression pattern of the P[ED17] construct using both confocal imaging and X-gal staining. I discuss possible genetic models that might form the basis for this expression pattern. Molecular analysis of the genomic DNA flanking the inser-tion site localised this site in the genome and the two nearest transcripts were used to perform in situ hybridisation to test for proneural-like expression. Finally, I report the generation of homozygous ED17 revertant lines and discuss initial steps in their genetic and molecular characterisation.

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May 17, 2011 Edited by Olivier Hagens Edited without comment.
May 17, 2011 Edited by Olivier Hagens Updated TOC & physical characteristics
December 15, 2009 Edited by WorkBot link works
October 19, 2008 Created by ImportBot Imported from Talis record