Studies of Wnt Signalling in Drosophila:

Screens for Candidate Transcripts of a New Gene Involved in Transduction and Genetic and Molecular Analysis of a Potential Target

  • 0 Ratings
  • 0 Want to read
  • 0 Currently reading
  • 0 Have read
Not in Library

My Reading Lists:

Create a new list

Check-In

×Close
Add an optional check-in date. Check-in dates are used to track yearly reading goals.
Today

  • 0 Ratings
  • 0 Want to read
  • 0 Currently reading
  • 0 Have read

Buy this book

Last edited by Olivier Hagens
May 16, 2011 | History

Studies of Wnt Signalling in Drosophila:

Screens for Candidate Transcripts of a New Gene Involved in Transduction and Genetic and Molecular Analysis of a Potential Target

  • 0 Ratings
  • 0 Want to read
  • 0 Currently reading
  • 0 Have read

MSc - 1999

Publish Date
Language
English
Pages
84

Buy this book

Book Details


Table of Contents

Dedication.
Table of Contents.
Abbreviations. 1
Nederlandse Samenvatting. 2
English Summary. 6
Introduction. 7
I. Peripheral sensory development. 7
1. Introduction. 7
2. The genetic mechanisms underlying bristle patterns. 8
A. Overview. 8
B. Expression of achaete and scute in the proneural clusters. 10
C. Selection of the SMC in the proneural cluster. 10
D. Asymmetric division of the SMC. 12
E. Development of the proneural clusters relies on a prepattern. 12
II. Wingless in Drosophila development. 13
III. Wnt signal transduction pathway. 15
1. Wg/Wnt-1 pathway. 15
2. Conservation of the Wg/Wnt-1 pathway. 17
IV. Wnts in vertebrate development. 18
V. Wnts in human disease. 19
1. Wnt signalling and colon cancer. 19
2. Wnt signalling and Alzheimer’s Disease. 20
3. GSK-3 and diabetes. 21
VI. Objectives. 22
Materials and Methods. 24
I. Kiwi. 24
1. General. 24
A. Preparing ampicillin (kanamycin) solutions. 24
B. Pouring plates. 24
C. Overnights. 24
D. Small-scale preparations of plasmid DNA. 25
E. Glycerol stocks. 25
F. Competent cells and transformation. 26
a. Competent cells. 26
b. Transformations. 26
G. Electrophoresis. 26
H. Fixing DNA to membranes. 27
I. Hybridisation. 27
a. Introduction. 27
b. Prehybridisation. 28
c. Preparing radioactive probe. 28
d. Assay the radioactive incorporation. 28
e. Hybridisation. 29
f. Washes. 29
g. Mounting of membrane. 29
2. Screening a library. 30
3. Tn1000 approach. 31
A. Mating. 31
B. Identification of inserts. 32
a. Overnights. 32
b. Cosmid preps. 32
c. Digest. 33
d. Primer synthesis. 33
e. Annealing of the vectorette. 34
f. Ligation. 35
g. PCR. 35
h. Running a gel. 38
i. The blotting conditions. 38
i1. Type of blotting procedure. 38
i2. Denaturation step and blotting buffer. 39
i3. Brand of membrane. 39
i4. Optimised blotting protocol. 39
II. ED17. 40
1. Plasmid rescue. 40
A. Isolation of DNA from adult flies. 40
B. Plasmid rescue. 41
2. Fly crosses. 41
3. Staining. 43
A. X-gal staining and light microscopy. 43
B. Ab-staining and epifluorescence/confocal microscopy. 44
Results. 45
I. Kiwi. 45
1. Introduction. 45
2. Mapping of kiwi. 45
A. Deletion mapping. 45
B. Inversion. 47
C. Contigs. 47
3. Screening of a cDNA library from 0h - 4h embryos. 48
4. Tn1000 approach. 50
II. ED17. 55
1. Enhancer trapping. 55
2. X-gal and antibody staining of larval imaginal wing discs. 55
3. Excision lines. 58
Discussion. 60
I. The kiwi locus. 60
1. Improving the transposon approach. 60
2. Alternative approaches to retrieve coding region. 60
3. Information from in vitro transcribed RNA. 61
II. The ED17 locus. 62
1. Study of the phenotype of excision lines of P[ED17]. 62
2. Mapping of the P[ED17] insertion site. 62
3. Genetic model explaining the ED17 expression pattern. 63
Acknowledgements. 66
References. 67
Appendix A. 71
Appendix B. 73
Appendix C. 78

Edition Notes

Published in
Antwerp, Belgium

The Physical Object

Format
Paperback
Number of pages
84
Dimensions
29.7 x 21 x 0.9 centimeters

ID Numbers

Open Library
OL24647813M

Work Description

The work presented is based on Drosophila genetics, a model organism in developmental biology. Several important segmentation and patterning processes are studied extensively in Drosophila. These processes are mediated by a few very conserved pathways. Because of its relation to human diseases like cancer, Alzheimer’s and diabetes, one of these pathways is of major importance in this studies, the Wnt signal transduction pathway.
The bristles of Drosophila are the extremes of the peripheral nervous system, linking the outside to the inside. It is therefore that the stereotyped pattern in which the bristles occur is conserved throughout evolution. A successful model to investigate the Wnt pathway are the genetic mechanisms underlying bristle patterning in Drosophila. Two previous uncharacterised genes that are strong candidates to be members of the pathway under investigation form the objects of study in this thesis. kiwi, is a gene retrieved from a big scale mutation screen with an interesting phenotype, whereas P[ED17] is an enhancer trap line that was recovered from a small scale insertion screen.
This account reports the results from different strategies to obtain coding regions from the kiwi gene. It also describes the design and fine tuning of a novel combination of molecular techniques to obtain such coding sequence. Finally, it indicates directions for future research on the kiwi locus.
Concerning the P[ED17] locus this thesis confirms the proposed expression pattern of the P[ED17] construct by means of both a double antibody and an X-gal staining and discusses possible genetic models that might form the basis for this expression pattern. Besides a molecular analysis of the genomic DNA adjacent to the putative insertion site, it reports the generation of homozygous ED17 revertant fly-lines. Finally, directions for future approaches are given.

Community Reviews (0)

Feedback?
No community reviews have been submitted for this work.

Lists

This work does not appear on any lists.

History

Download catalog record: RDF / JSON / OPDS | Wikipedia citation
May 16, 2011 Edited by Olivier Hagens Corrected two spelling mistakes
May 16, 2011 Edited by Olivier Hagens Edited without comment.
May 16, 2011 Edited by Olivier Hagens Updated some fields
May 16, 2011 Edited by Olivier Hagens Initial addition of record
May 16, 2011 Created by Olivier Hagens Added new book.