The teleost taar family of olfactory receptors [Elektronische Ressource] : from rapidly evolving receptor genes to ligand-induced behavior / Ashiq Hussain

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The teleost taar family of olfactory receptors: From rapidly evolving receptor genes to ligand-induced behavior Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Ashiq Hussain aus Jampur, Pakistan Köln 2010  1  Berichterstatter: Prof. Dr. Sigrun I. Korsching Prof. Dr. Jens C. Brüning Tag der mündlichen Prüfung: 23 November, 2010  2 ERKLÄRUNG Ich versichere, dass ich die von mir vorgelegte Dissertation selbstständig angefertigt, die benutzten Quellen und Hilfsmittel vollständig angegeben und die Stellen in der Arbeit -einschließlich Tabellen, Karten und Abbildungen -, die anderen Werken im Wortlaut oder dem Sinn nach entnommen sind, in jedem Einzelfall als Entlehnung kenntlich gemacht habe; dass diese Dissertation noch keiner anderen Fakultät oder Universität zur Prüfung vorgelegen hat; dass sie - abgesehen von den unten angegebenen Teilpublikationen – noch nicht veröffentlicht worden ist, sowie dass ich eine solche Veröffentlichung vor Abschluss des Promotionsverfahrens nicht vornehmen werde. Die Bestimmungen dieser Promotionsordnung sind mir bekannt. Die von mir vorgelegte Dissertation ist von Prof. Dr. Sigrun. I. Korsching betreut worden. Köln, den 04. Nov, 2010 Teilpublikationen: Hussain, A., Saraiva, L.R., Korsching, S.I.
Publié le : vendredi 1 janvier 2010
Lecture(s) : 140
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Source : D-NB.INFO/1013831349/34
Nombre de pages : 158
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The teleost taar family of olfactory receptors:
From rapidly evolving receptor genes to ligand-induced behavior






Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität zu Köln










vorgelegt von
Ashiq Hussain
aus Jampur, Pakistan



Köln 2010
 





























Berichterstatter: Prof. Dr. Sigrun I. Korsching
Prof. Dr. Jens C. Brüning

Tag der mündlichen Prüfung: 23 November, 2010
 
2 ERKLÄRUNG

Ich versichere, dass ich die von mir vorgelegte Dissertation selbstständig angefertigt, die
benutzten Quellen und Hilfsmittel vollständig angegeben und die Stellen in der Arbeit -
einschließlich Tabellen, Karten und Abbildungen -, die anderen Werken im Wortlaut oder
dem Sinn nach entnommen sind, in jedem Einzelfall als Entlehnung kenntlich gemacht
habe; dass diese Dissertation noch keiner anderen Fakultät oder Universität zur Prüfung
vorgelegen hat; dass sie - abgesehen von den unten angegebenen Teilpublikationen –
noch nicht veröffentlicht worden ist, sowie dass ich eine solche Veröffentlichung vor
Abschluss des Promotionsverfahrens nicht vornehmen werde. Die Bestimmungen dieser
Promotionsordnung sind mir bekannt. Die von mir vorgelegte Dissertation ist von Prof. Dr.
Sigrun. I. Korsching betreut worden.

Köln, den 04. Nov, 2010


Teilpublikationen:

Hussain, A., Saraiva, L.R., Korsching, S.I. (2009)c Positive Darwinian selection and the
birth of an olfactory receptor clade in teleosts. Proc. Natl. Acad. Sci. USA. 106(11):4313-
4318

Faculty of 1000 Biology: evaluations for Hussain A et al. Proc Natl Acad Sci U S A
2009 Mar 17,106 (11):4313-8









 












To my parents






 
 
 
 
 
 
 
 
 
 
4 I. Acknowledgement

This study was conducted in the system neurobiology laboratory of Prof. Dr. Sigrun I.
Korsching at the Institute for Genetics, University of Cologne, Germany. This research
was supported by the generous grant from the International Graduate School of Genetics
and Functional Genomics (IGSGFG) at the Institute for Genetics.

Foremost, I pay my sincere gratitude to Prof. Dr. Sigrun I. Korsching for being a great
mentor. I am very thankful to her for the support, advices and trust she bestowed on me. I
learnt from her the meanings of patience, coordination, candidness and how to tackle
daily life of a science laboratory. I am very contented that I had the opportunity to spend
~4 years in learning, from culture to science, at her laboratory.

I will extend my acknowledgement to Prof. Dr. Jens C. Brüning and to Prof. Dr. Matthias
Hammerschmidt for accepting to be in my thesis committee, for their interest in my
research work, and for kindly serving as my thesis referees.

I greatly appreciate Dr. Stephen D. Liberles at Harvard Medical School for our successful
collaboration and for inviting me to work in his lab for the identification of ligands for Trace
Amine-associated Receptors (TAARs). I am also very thankful to David Ferrero, who
reproduced the TAAR ligands results and did the HPLC analysis, Jamie K. Lemon for her
help regarding methodological aspects of ligands identification assays, and Wayne
Korzan for his technical advices in establishing the behavioral assay. Many thanks to Prof.
Ansgar Büschges and Dr. Matthias Gruhn for providing room for establishing behavior
assay facility and for their technical help in WINANALYZE motion tracking software.

I was humbled and honored for being accepted in the class of 2006 of graduate school.
rdMy family has a special bond of love and respect for Germany. I am the 3 generation of
my family to serve and study in Germany and I am obliged to this country for providing me
the opportunity to learn and to the German people for being so civil and supportive. Many
thanks are to Dr Isabell Witt, Brigitte Wilcken-Bergmann, Cathy Joergens and Joanna
Majczak for their support in all walks of life at graduate school.

I was fortunate to have cooperative and ambitious lab fellows like Dr. Luis R. Saraiva, Dr.
Yuichiro Oka and Dr YenYen Kewan to whom I indebt a great deal of valuable feedback
and collaborations. Sincere thanks to Mehmet Saltürk for taking care of all lab
equipments, fish room, computers and experimental material supplies.

I am in lack of words to thank my father who envision for the education of his children and
migrated to a big city for this sole purpose. I am greatly thankful to him for financially and
morally supporting me in my life. A great deal of thanks to my mother for taking care of the
family, for staying awake with us during exams nights and for her all great sacrifices that
she made with my father for their children´s education.

Very special thanks to my wife Sofia Lyngemark Hussain for being a loving, supportive,
caring and understanding life companion. I am thankful to her for understanding and
accepting me to work in lab during weekends, for moral support in stressful experimental
times (c-Fos and behavioral assays), for staying by herself while I attended international
conferences and for all great things that she did for me. Thank you.
 
















































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6 II. TABLE OF CONTENTS
I. ACKNOWLEDGEMENT ................................................................................................... 5
II. TABLE OF CONTENTS .................................................................................................. 7
III. LIST OF FIGURES ....................................................................................................... 12
IV. LIST OF TABLES......................................................................................................... 13
V. LIST OF SUPPLEMENTARY TABLES ......................................................................... 13
VI. ABSTRACT .................................................................................................................. 14
ZUSAMMENFASSUNG .................................................................................................... 16
CHAPTER 1 ...................................................................................................................... 18
VII. INTRODUCTION ........................................................................................................ 19
1. OLFACTORY SYSTEM ................................................................................................. 19
1.1. Mammalian olfactory system ...................................................................................... 20
1.2. Zebrafish olfactory system ......................................................................................... 22
1.3. OLFACTORY SENSORY NEURON (OSNS) ............................................................. 24
1.3.1. Ciliated olfactory sensory neurons .......................................................................... 24
1.3.2. Microvillous olfactory sensory neurons ................................................................... 25
1.3.3. Crypt sensory neurons ............................................................................................ 25
1.4. OLFACTORY RECEPTOR GENE FAMILY REPERTOIRE ....................................... 26
1.4.1. Odorant receptor family (OR) .................................................................................. 28
1.4.2. Trace amine-associated receptor family (TAAR) .................................................... 30
1.4.3. Vomeronasal receptors family type1 (V1R) ............................................................. 32
 
7 1.4.4. Vomeronasal receptors family type2 (V2R) ............................................................. 34
1.4.5. Formyl peptide receptor family (FPR) ..................................................................... 35
1.5. OLFACTORY SIGNALING TRANSDUCTION ........................................................... 35
1.6. LIGANDS FOR OLFACTORY RECEPTORS ............................................................. 38
1.7. FISH BEHAVIOR........................................................................................................ 39
1.8. ZEBRAFISH AS A MODEL ORGANISM .................................................................... 41
CHAPTER 2 ...................................................................................................................... 44
VIII. RESULTS .................................................................................................................. 45
1. PHYLOGENOMICS, SELECTION PRESSURE, INTRON DYNAMICS AND CELLULAR
EXPRESSION OF TAARS ................................................................................................ 45
1.1. TAAR genes encompass monophyletic origin distinct from aminergic GPCRs ......... 45
1.2. Rapid evolution of TAAR gene as a recurrent species-specific expansions in teleost46
1.3. TAAR genes are an evolutionary young family .......................................................... 50
1.4. TAAR family is characterized by distinctive consensus motifs, despite the overall
heterogeneity. ................................................................................................................... 52
1.5. Genomic arrangement of teleost TAAR genes pinpoints the evolutionary origin of
class III. ............................................................................................................................. 54
1.6. Gene duplication rate and gene divergence are much higher in teleost compared with
mammalian species, suggesting a teleost- restricted rapid evolution of taar genes. ........ 55
1.7. Strong local positive selection in teleost taar genes is masked by global negative
selection. ........................................................................................................................... 57
1.8. Dynamic loss and gain of introns restricted to the class III of neoteleost taar genes. 60
1.9. Most taar genes are expressed in sparse olfactory sensory neurons. ....................... 62
2. LIGANDS FOR ZEBRAFISH TAARS ............................................................................ 65
 
8 2.1. DrTAAR13c recognize volatile diamines .................................................................... 65
2.2. DrTAAR13c activation requires at least 2 amino groups ............................................ 68
2.3. DrTAAR13c is activated by polyamines ..................................................................... 70
2.4. DrTAAR13c recognizes natural activators ................................................................. 71
3. BEHAVIORAL RESPONSE OF ZEBRAFISH TO DIAMINES ....................................... 73
3.1. Zebrafish does not show specific behavior for water, a mock stimulus. ..................... 74
3.2. Food induce attractive behavior in adult zebrafish ..................................................... 76
3.3. Diamines induce avoidance behavior in adult zebrafish ............................................ 77
3.4. Zebrafish aversive behavior towards diamines is concentration dependent .............. 81
4. DIAMINES ACTIVATE A SPARSE SUBSET OF OLFACTORY SENSORY NEURONS
IN ZEBRAFISH OLFACTORY EPITHELIUM .................................................................. 82
CHAPTER 3 ...................................................................................................................... 87
IX. DISCUSSION............................................................................................................... 88
CHAPTER 4 . 94
X. MATERIAL AND METHODS ........................................................................................ 95
1. EXPERIMENTAL MATERIALS ..................................................................................... 95
1.1. Animals ...................................................................................................................... 95
1.2. Chemicals suppliers ................................................................................................... 95
1.3. Plastic ware ................................................................................................................ 96
1.4. Preparation of solutions.............................................................................................. 96
1.5. Laboratory equipment ................................................................................................ 96
1.6. Nucleotides ................................................................................................................ 97
1.7. Bacterial strain............................................................................................................ 97
 
9 1.8. Enzymes .................................................................................................................... 97
1.9. Plasmids and vectors/properties ................................................................................ 97
1.10. Primary antibodies .................................................................................................... 97
1.11. Secondary antibodies ............................................................................................... 98
1.12. Dyes, substrates, embedding media and counter stains .......................................... 98
1.12.1. Alkaline phosphatase substrates ....................................................................... 98
1.12.2. Horseradish peroxidase substrates ................................................................... 98
1.12.3. Embedding media .............................................................................................. 98
1.12.4. Dyes and counterstains ..................................................................................... 98
1.13. Oligonucleotide primers............................................................................................ 98
2. MOLECULAR BIOLOGICAL TECHNIQUES ............................................................... 101
2.1. Isolation, purification and quantification of DNA and RNA ....................................... 101
2.1.1. Isolation of genomic DNA .................................................................................. 101
2.1.2. Genomic DNA PCR ........................................................................................... 101
2.1.3. Quantitation of DNA and RNA ........................................................................... 102
2.1.4. Agarose gel electrophoresis .............................................................................. 102
2.1.5. Isolation of DNA fragments from PCR products or agarose gels ...................... 102
2.1.6. Ligation of DNA fragments and PCR products .................................................. 102
2.1.7. Subcloning of DNA fragments by electroporation .............................................. 103
2.1.8. Subcloning of DNA fragments by DH5 α chemically competent E. coli .............. 103
2.1.9. Colony PCR for identification of positive clones and determination of insert length
..................................................................................................................................... 103
2.1.10. Small scale plasmid DNA preparation (Miniprep) ............................................ 104
2.1.11. Phenol/chloroform extraction ........................................................................... 104
2.1.12. Ethanol precipitation ........................................................................................ 104
2.1.13. Restriction enzyme digestion of DNA .............................................................. 105
2.1.14. Preparation of glycerol stocks .......................................................................... 105
2.1.15. Sequencing of DNA ......................................................................................... 105
3. HISTOLOGICAL STUDIES 105
3.1. Preparation of cover slips 105
3.2. Tissue preparation and sectioning ........................................................................... 106
3.3. Cryosectioning.......................................................................................................... 106
 
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