Functional analysis of X-chromosomal gene expression in Drosophila melanogaster [Elektronische Ressource] / Claus Kemkemer

Kemkemer, Claus Stefan Oliver

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften an der
Fakultät für Biologie der Ludwig-Maximilians-Universität München








Functional analysis of X-chromosomal gene expression in
Drosophila melanogaster









Claus Kemkemer

aus

Neu-Ulm, Deutschland

2011-03-31Erklärung

Erklärung:

Diese Dissertation wurde im Sinne von § 12 der Promotionsordnung von Prof. Dr. John
Parsch betreut. Ich erkläre hiermit, dass die Dissertation nicht einer anderen
Prüfungskommission vorgelegt worden ist und dass ich mich nicht anderweitig einer
Doktorprüfung ohne Erfolg unterzogen habe.


Ehrenwörtliche Versicherung:

Ich versichere ferner hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir
selbstständig, ohne unerlaubte Hilfe angefertigt wurde.


München, den 2011-03-31



Claus Kemkemer




1. Gutachter: Prof. Dr. John Parsch
2. Gutachter: Prof. Dr. Susanne Renner

Dissertation eingereicht am: 2011-03-31

Datum der Disputation: 2011-05-18 1. Table of contents
1. Table of contents
1. Table of contents ................................................................................................................3

2. Note ...................................................................................................................................5

3. List of abbreviations...........................................................................................................6

4. Figure and table list ............................................................................................................8

5. Zusammenfassung............................................................................................................10

6. Abstract............................................................................................................................13

7. Introduction......................................................................................................................15

7.1 Sex chromosomes.......................................................................................................15

7.2 Sex chromosomes and speciation................................................................................17

7.3 Sex chromosomes and selection..................................................................................18

7.4 Sex chromosome gene expression and gene content....................................................19

7.5 Male germline X inactivation......................................................................................23

7.6 Sex chromosome gene expression variation ................................................................27

8. Material and Methods.......................................................................................................30

8.1 Genome sequences and BLAST search .......................................................................30

8.2 Primer sequences for amplification of putative promoters ...........................................30

8.3 DNA extraction ..........................................................................................................31

8.4 Restriction endonuclease digest ..................................................................................32

8.5 Ligation......................................................................................................................32

8.6 Polymerase chain reaction...........................................................................................32

8.7 Sequencing .................................................................................................................33

8.8 RNA extraction...........................................................................................................33

8.9 Bacterial Transformation ............................................................................................34

8.10 Plasmid extraction ....................................................................................................34

8.11 Agarose gel electrophoresis ......................................................................................35

8.12 LB-media plates........................................................................................................35

8.13 Fly food....................................................................................................................35

8.14 Transformation vector construction for P-element transformation.............................36

8.15 Transformation vector construction for ΦC31 transformation ...................................36

8.16 Germline transformation for ΦC31 transformation....................................................37

8.17 Germline transformation for P-element transformation .............................................38

8.18 Insertion mapping.....................................................................................................39

3 1. Table of contents
8.19 β−galactosidase assay and staining ...........................................................................39

8.20 Quantitative reverse transcription polymerase chain reaction ....................................40

9. Results..............................................................................................................................42

9.1 Fine-scale mapping of additional insertions of the ocnus reporter gene construct ........42

9.2 Comparison of autosomal and X-linked expression of the ocnus construct..................44

9.3 Analysis of male germline X inactivation at cytological band 19.................................47

9.4 Functional analysis of three X-linked, testis-specific promoters ..................................47

9.5 Fine-scale mapping of transgene insertions of three X-linked promoters.....................50

9.6 Comparison of X-linked and autosomal reporter gene insertions for three X-linked
promoters .........................................................................................................................52

9.7 Stage specific expression profiling for three X-linked promoters.................................58

9.8 The expression difference of CG9509 between European and African populations of D.
melanogaster....................................................................................................................60

9.9 Expression profiling of the European and African CG9509 promoter in the malpighian
tubule ...............................................................................................................................65

10. Discussion ......................................................................................................................67

10.1 Global male germline X inactivation.........................................................................67

10.2 The hotspot for new gene evolution at cytological band 19 .......................................69

10.3 X-linked promoters driving testis expression.............................................................71

10.4 Cis-regulatory sequences driving testis expression of X-linked genes, despite male
germline X inactivation ....................................................................................................72

10.5 Stage specific expression profiling of male germline X inactivation..........................73

10.6 The excess of X chromosome to autosome gene movement ......................................75

10.7 The cis-regulatory sequence of the gene CG9509 was positively selected in the
European population of D. melanogaster..........................................................................76

11. Reference list..................................................................................................................79

12. Appendix........................................................................................................................89

13. Curriculum vitae...........................................................................................................102

14. Acknowledgements ......................................................................................................104


4 2. Note
2. Note

In this dissertation I present my doctoral research, all of which has been done by myself. Prof.
Dr. John Parsch assisted with writing the research article cited below that served as the basis
for a portion of this dissertation. In addition, Dr. Winfried Hense provided reagents (plasmids
and Drosophila stocks) that were used in the portion of my research described in the
publication cited below.

The results from my dissertation have contributed to the following publication:

Kemkemer C, Hense W, Parsch J. Fine-scale analysis of X chromosome inactivation in the
male germline of Drosophila melanogaster. Mol Biol Evol. 2010 Dec 30. [Epub ahead of
print]
5 3. List of abbreviations
3. List of abbreviations

Abbreviation Description

A Autosome
BLAST Basic local alignment search tool
bp Base pair
C. elegans Caenorhabditis ele