THE REGULATION AND FUNCTION OF THE DROSOPHILA MELANOGASTER CYTOCHROME P450 GENE, Cyp12d1

Boey Hui Kuang Adrian

Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy

September 2011

Department of Genetics

The University of Melbourne

Abstract

Cytochrome P450s are an important family of monooxygenase enzymes implicated in numerous xenobiotic detoxification events as well as in essential endogenous functions. The vinegar fly Drosophila melanogaster has 85 P450 genes; however, the large majority of them remain uncharacterised in terms of their function and regulation. Cyp12d1 is arguably the most xenobiotic inducible P450 gene in the D. melanogaster genome. It has been suggested that Cyp12d1 is an excellent candidate gene to study

Drosophila xenobiotic induction pathways as it responds to a wide range of chemical inducers, indicating that it contains most if not all of the cis-regulatory elements needed for xenobiotic induction in

Drosophila. Hence, the transcriptional regulation of Cyp12d1 was investigated to identify novel P450 induction pathways in D. melanogaster. Cyp12d1 basal transcriptional regulators were found in Cyp12d1 upstream and downstream regulatory regions, while enhancers for Phenobarbital and caffeine induction were located upstream. Site-directed mutagenesis experiments identified GATA family transcription factors as important Cyp12d1 midgut expression regulatory proteins. However, their role in xenobiotic induction remains unclear. Biochemical sequencing of electomobility-shift assay protein bands, and genetic RNAi screens of genes encoding other candidate transcription factors, failed to identify any other potential xenobiotic regulatory proteins. Cyp12d1 function was also investigated in this study.

Cyp12d1 overexpression has been shown to confer resistance to the insecticides DDT and dicyclanil, but other functions have not been identified prior to this study. Adult Cyp12d1 functions were investigated through Cyp12d1 RNAi and overexpression studies. Cyp12d1 was found to be involved in adult longevity and oxidative stress resistance, suggesting other potential functions in addition to known detoxification functions. Cyp12d1 has been tandemly duplicated in D. melanogaster, and this duplication exists as a polymorphism in field populations. The geographical distribution of the Cyp12d1 duplication was examined in flies collected along the eastern coastline of Australia. The frequency of the duplicated

Cyp12d1 gene was found to vary spatially, with flies in lower latitudes being more likely to possess the

I

Cyp12d1 duplication and flies in higher latitudes being less likely. Cyp12d1 tissue-specific embryonic expression and mRNA transcript length was different in Cyp12d1-duplicated lines when compared to non-Cyp12d1 duplicated lines. These results indicate the Cyp12d1 duplication confers changes in

Cyp12d1 expression patterns and suggest that Cyp12d1 may be involved in local adaptation to the microenvironment.

II

Preface

Certain portions of this thesis were contributed by others. Their help is greatly appreciated and listed below:

Chapter 2

Henry Chung

 Made the Cyp12d1 mRNA in situ hybridisation probe  Contributed Cyp6g1 Phenobarbital induction data  Provided in situ hybridisation images

Dr. Phillip Daborn

 Contributed Cyp6g1 Phenobarbital induction data

Professor Carl Thummel

 Provided HR96 null mutant fly lines

Lee Willoughby

 Provided in situ hybridisation images

Paul O’Donnell

 Assisted with the electromobility shift assay protein band sequencing

Chapter 3

Christopher Lumb

 Made the Cyp12d1-overexpression fly lines  Designed the Cyp12d1 Realtime Primers

Chapter 4

Dr. Charles Robin

 Provided Cyp12d1 population genetics data

Robert Good

 Provided Cyp12d1 population genetics data

III

Chapter 5

Dr. Charles Robin

 Provided Cyp12d1 population genetics data

Robert Good

 Provided Cyp12d1 population genetics data  Provided Cyp12d1 allele status in different Drosophila species image

Stephen Pearce

 Provided midgut RNA-sequencing data

IV

Declaration

This is to certify that; i) the thesis comprises only my original work towards the PhD except where indicated in the Preface ii) due acknowledgement has been made in the text to all other material used iii) the thesis is less than 100,000 words in length, exclusive of tables, figures and bibliographies

Boey Hui Kuang Adrian

September 2011

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Acknowledgements

No man is an island, and this doctorate would not have been completed without the assistance and support of many other people. To quote Sir Isaac Newton, esteemed scientist: “If I have seen further, it is because I have been standing on the shoulders of giants.”

Firstly, I would like to thank Dr Phillip Daborn and Professor Philip Batterham for their excellent guidance and tutelage. They have always demonstrated great kindness and patience towards me, and I owe much to their supervision and sage advice.

My family have been a great source of comfort and encouragement all these years. In particular, I want to express my sincerest gratitude to my parents for their unquestioning support during this time. Words cannot describe the great debt I owe to them. I also would like to thank my family in Melbourne for their care during my stay here. I am also grateful for the love and support from my many cousins, both in Melbourne, Sydney and elsewhere.

I have to acknowledge the help provided by Dr Charles Robin, Associate Professor Alex Andrianopoulos,

Robert Good, Lee Willoughby, Stephen Pearce and Paul O’Donnell. Charles Robin performed the initial experiments examining the Cyp12d1 gene, and together with Robert Good, have kindly led me through the population genetics of Cyp12d1. Lee Willoughby studied the xenobiotic induction of D. melanogaster, thereby laying the framework for this study. Alex Andrianopoulos provided much technical advice for the biochemical aspects of this project. Stephen Pearce provided midgut section

RNA-sequencing data, and Paul O’Donnell kindly assisted with the mass spectroscopy sequencing.

I would also like to thank my friends, both inside and outside of the Batterham lab. In particular, Chan

Jianxiong, Tom Harrop, Trent Perry, Emily Remnant, Kirsten Allen, Joshua Schmidt, Chris Lumb, Wee Tek

Tay, Adam Williams, Janice Chan, Tinna Yang, and many others in the Genetics department have been

VI great friends and colleagues over the years. There have been many hours of fruitful, and fruitfly, discussion with them in matters both related and unrelated to Science. I feel extremely fortunate to have been in such a fantastic environment, and can only hope I have contributed in turn to the spirit of the department.

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Table of Contents

Abstract ...... I

Preface ...... III

Declaration ...... V

Acknowledgements ...... VI

List of Figures ...... XVI

List of Tables ...... XVIII

List of Abbreviations ...... XIX

Chapter 1: Introduction ...... 1

1.1 The Cytochrome P450s: A brief overview ...... 3

1.1.1 Intracellular localisation ...... 3

1.1.2 P450 Reactions ...... 5

1.1.3 P450 nomenclature ...... 5

1.2 P450 Functions ...... 7

1.2.1 Endogenous P450 Functions ...... 7

1.2.1.1 Cholesterol and steroid hormone synthesis ...... 9 1.2.2 Endogenous Insect P450 functions ...... 12

1.2.2.1 Endogenous P450 functions in D. melanogaster ...... 12 1.2.3 Xenobiotic Metabolism ...... 17

1.2.3.1 Dietary tolerance ...... 17

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1.2.3.2 Insecticide resistance ...... 18 1.2.3.3 Drosophila melanogaster P450s and Insecticide resistance ...... 18 1.2.3.5 P450 Alleles and Drug Metabolism ...... 21 1.2.3.6 Bioactivation of toxic metabolites ...... 23 1.2.4 Mitochondrial P450s ...... 24

1.3 Cytochrome P450 transcriptional regulation ...... 26

1.3.1 P450 Xenobiotic Induction ...... 26

1.3.1.1 Transcription Factors regulating mammalian P450 induction ...... 28

1.3.1.2.The PAS-bHLH family ...... 29 1.3.1.3 The Nuclear receptor family ...... 35 1.3.2 Phenobarbital induction pathways in mammals ...... 38

1.3.2.1 Phenobarbital Induction enhancers ...... 38 1.3.2.2 CAR and CYP2B genes ...... 39 1.3.2.3 PXR and CYP3A genes...... 40 1.4 Insect P450 induction transcriptional regulation...... 41

1.4.1 Papillio polyxenes P450 Induction ...... 42

1.4.2 D. melanogaster xenobiotic induction ...... 46

1.4.2.1 D. melanogaster xenobiotic induction microarray studies ...... 46 1.4.2.2 Promoter sequence analysis ...... 50 1.4.2.3 Drosophila Induction Regulatory Proteins ...... 55 1.5 Cyp12d1 induction as a tool to study D. melanogaster xenobiotic induction ...... 58

1.6 Aims for this Thesis ...... 59

Chapter 2: Transcriptional Regulation of Cyp12d1 ...... 62

2.1 Introduction ...... 63

2.1.1 Cyp12d1 regulation ...... 63

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2.1.2 Chapter aims ...... 64

2.2 Materials and Methods ...... 65

2.2.1 Fly rearing conditions ...... 65

2.2.2 Reporter constructs ...... 65

2.2.3 Transgenic Drosophila line generation ...... 67

2.2.4 GFP expression and Image capture...... 68

2.2.5 Exposure of larvae to Xenobiotics ...... 68

2.2.6 Bioinformatic analysis ...... 69

2.2.7 ELectromobility Shift Assays (EMSA) ...... 69

2.2.8 EMSA Protein-DNA band Sequencing ...... 70

2.2.9 Genetic Screen for Cyp12d1-regulating Transcription factors ...... 71

2.2.9.1 Transcription factor RNAi...... 71 2.3 Results ...... 76

2.3.1 Cyp12d1 Tissue specific expression ...... 76

2.3.2 Cyp12d1 tissue-specific native enhancers ...... 76

2.3.2.1 5’ constructs and tissue-specific native expression ...... 76 2.3.2.2 Investigating the downstream region for Cyp12d1 enhancers ...... 86 2.3.2.3 CG30490 regulation in 3rd instar larvae ...... 86 2.3.2.4 3’ constructs investigating the +1917/+2207bp region ...... 90 2.3.2.5 Combined 5’ and 3’ Constructs ...... 90 2.3.2.6 Native Enhancers summary ...... 92 2.3.3 Xenobiotic Induced expression ...... 92

2.3.3.1 Investigating the downstream region for phenobarbital induction enchancers ...... 94

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2.3.3.2 Investigating the Cyp12d1 upstream promoter region for phenobarbital-induction enhancers ...... 94 2.3.3.3 Caffeine induction ...... 107 2.3.3.4 Cyp12d1 xenobiotic induction regulation Summary...... 109 2.3.4 Bioinformatic analysis ...... 109

2.3.4.1 Dotplot analysis of the -1100/-1bp region ...... 109 2.3.4.2 Matinspector analysis for putative transcription factor binding sites ...... 113 2.3.4.3 Identifying conserved blocks of sequence using EvoPrinterHD ...... 113 2.3.5 Transcription factor identification ...... 115

2.3.5.1 Electromobility shift assays ...... 115 2.3.5.2 Protein Band Sequencing ...... 116 2.3.5.3 Transcription factor reduction and Cyp12d1 phenobarbital induction ...... 119 2.3.5.4 Transcription Factor Identification Summary ...... 130 2.4 Discussion ...... 131

2.4.1 Cyp12d1 induction is tissue-specific ...... 131

2.4.2 Cyp12d1 model of regulation ...... 132

2.4.2.1 Investigating the -288/-168bp region further ...... 132 2.4.2.2 Cyp12d1 Repressive region ...... 134 2.4.3 GATA factors regulate P450 midgut expression ...... 135

2.4.4 P450 Induction Pathways in D. melanogaster ...... 136

2.5 Chapter Conclusion ...... 140

Chapter Three: Cyp12d1 Functional studies ...... 141

3.1 Introduction ...... 142

3.1.1 Chapter aims ...... 142

3.2 Materials and Methods ...... 144

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3.2.1 Cyp12d1 RNAi construction ...... 144

3.2.2 Cyp12d1 RNAi impact on viability ...... 145

3.2.3 Longevity Assay ...... 145

3.2.4 Hydrogen Peroxide assay ...... 146

3.3 Results ...... 149

3.3.1 Cyp12d1 Overexpression ...... 149

3.3.2 Cyp12d1 RNAi quantification ...... 149

3.3.2.1 Cyp12d1 mRNA quantification in Cyp12d1 ubiquitously-reduced flies ...... 149 3.3.2 Viability assay ...... 151

3.3.3 Longevity Assays ...... 151

3.3.3.1 Cyp12d1-RNAi transgenic fly longevity ...... 156 3.3.3.2 Cyp12d1-overexpression transgenic fly longevity ...... 156 3.3.4 Oxidative stress ...... 156

3.4 Discussion ...... 160

3.4.1 Mitochondrial Cyp12d1 and possible roles in longevity and oxidative stress ...... 160

3.4.2 Drosophila Lifespan ...... 160

3.4.3 P450s and oxidative stress ...... 162

3.5 Chapter conclusion ...... 166

Chapter 4: Investigating the Cyp12d1 duplication ...... 167

4.1 Introduction ...... 168

4.1.1 Chapter aims ...... 171

4.2 Materials and Methods ...... 172

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4.2.1 Cyp12d1 Duplication assay ...... 172

4.2.2 Temporal lifestage sample collection ...... 173

4.2.3 Exposure of larvae to phenobarbital ...... 173

4.2.4 RNA isolation and cDNA synthesis ...... 173

4.2.5 3’ Rapid Amplification of cDNA Ends (3’ RACE) ...... 174

4.2.6 mRNA in situ Hybridisation ...... 174

4.2.7 Embryo mRNA in situ hybridisation ...... 175

4.3 Results ...... 176

4.3.1 The Cyp12d1 Duplication in y; cn bw sp ...... 176

4.3.2 Geographical distribution of the Cyp12d1 duplication ...... 176

4.3.3 Identifying single Cyp12d1 copy and double Cyp12d1 copy strains for further study ...... 180

4.3.4 Two Cyp12d1 amino acid substitutions are found in substrate recognition sites ...... 180

4.3.5 Cyp12d1 mRNA expression in duplicated and non-duplicated lines ...... 184

4.3.5.1 Cyp12d1 3’UTR length differences ...... 184 4.3.5.2 Developmental Cyp12d1 expression ...... 184 4.3.5.3 Cyp12d1 induction in single copy and double copy lines...... 187 4.3.6 Tissue specific expression in single copy and double copy lines ...... 190

4.3.6.1 Cyp12d1 mRNA in situ hybridisations in 3rd instar larvae ...... 190 4.3.6.2 Cyp12d1 mRNA in situ hybridisations in embryos ...... 190 4.4 Discussion ...... 197

4.4.1 The Cyp12d1 duplication haplotype was found to vary in frequency spatially ...... 197

4.4.2 Is Cyp12d1 involved in adaptation to physical environmental stresses? ...... 200

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4.4.3 Do individual differences between Cyp12d1-d’ and Cyp12d1-p’ contribute to Cyp12d1

duplication selection? ...... 201

4.4.3.1 Cyp12d1-d’ and Cyp12d1-p’ may have different substrate specificities ...... 202 4.4.3.2 Do tissue-specific expression differences contribute to Cyp12d1 duplication selection? ...... 203 4.4.3.3 Is Cyp12d1 induction and detoxification a selective agent for the Cyp12d1 cline? .... 205 4.5 Chapter conclusion ...... 206

Chapter 5: Final Discussion and Conclusion...... 208

5.1 Introduction ...... 209

5.2 This study ...... 209

5.3 Cyp12d1 is an evolutionary unstable mitochondrial P450 ...... 210

5.4 Cyp12d1 is expressed at low levels in the midgut when uninduced ...... 211

5.4.1 Cyp12d1 expression compared to a group of P450 genes ...... 211

5.4.2 Cyp12d1 basal expression compared to non-P450 genes ...... 215

5.4.3 Cyp6g1 expression compared to Cyp12d1 expression ...... 216

5.5 How is Cyp12d1 induction linked to Cyp12d1 function? ...... 217

5.5.1 Is low Cyp12d1 basal expression linked to Cyp12d1 function? ...... 217

5.5.2 Is Cyp12d1 part of an inducer-specific or generalised induction response? ...... 217

5.5.2.1 Substrate specificity of Cyp12d1 ...... 218 5.5.2.2 Could Cyp12d1 be part of an nonspecific stress response? ...... 219 5.5.2.3 Are environmental conditions an inducer of Cyp12d1 expression? ...... 219 5.6 Xenobiotic induction in Drosophila melanogaster ...... 220

5.6.1 D. melanogaster responds to prototypical P450 inducers ...... 221

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5.6.2 Drosophila respond differently to caffeine induction ...... 221

5.7 Evolution of cis-regulatory elements involved in D. melanogaster xenobiotic induction ...... 222

5.7.1 Do direct repeat elements regulate phenobarbital induction in Drosophila? ...... 223

5.7.2 Induction modulator elements in D. melanogaster ...... 224

5.8 Concluding remarks ...... 225

Chapter 6: Bibliography ...... 226

Appendix I: List of fly lines used ...... 243

Appendix II: List of Primers used ...... 246

Appendix III: EMSA protein band sequencing protocol ...... 248

Appendix IV: Cyp12d1 duplication survey gels ...... 252

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List of Figures

Figure 1.1: General Cytochrome P450 Structural motifs.

Figure 1.2: Cytochrome P450 nomenclature.

Figure 1.3: An overview of Cyp51 activity in the sterol synthesis pathway.

Figure 1.4: The Ecdysone synthesis and catabolism pathway in D. melanogaster.

Figure 1.5 Conservation between D. melanogaster and A. gambiae mitochondrial P450s.

Figure 1.6: Xenobiotic induction-regulating transcription factor modes of action.

Figure 1.7: cis-regulatory regions of Xenobiotic-inducible P450s.

Figure 1.8: P. polyxenes Cyp6B1 cis- and trans -regulation.

Figure 1.9: cis-regulatory regions of D. melanogaster Phenobarbital-inducible genes.

Figure 2.1 GFP reporter vectors used in this Study.

Figure 2.2: -1540/-1bp pStinger/5HR-GAL4 driver line generation.

Figure 2.3: Crossing scheme for transcription factor knockdown.

Figure 2.4: Crossing scheme to generate -1540/-1bp pStinger; HR96-/- lines.

Figure 2.5: Cyp12d1 3rd instar mRNA in situ hybridisation expression patterns.

Figure 2.6: Cyp12d1 promoter constructs.

Figure 2.7: Uninduced expression from 5’ constructs covering the -9653 to -1100bp region.

Figure 2.8: Uninduced expression from 5’ constructs covering the -670bp to -1bp region.

Figure 2.9: Uninduced expression from 3’ constructs covering the +1917bp/+2207bp region.

Figure 2.10: CG30490 expression in 3rd instar w1118 larvae.

Figure 2.11: Combined 5’ and 3’ constructs uninduced expression.

Figure 2.12: A model for the regulation of Cyp12d1 uninduced native expression.

Figure 2.13: +1917bp/2207 pH-Stinger does not respond to Phenobarbital induction.

Figure 2.14: Constructs investigating the -9653/-945bp region for Phenobarbital induction enhancers.

Figure 2.15 Constructs investigating the -1540/-1bp region for Phenobarbital induction enhancers.

Figure 2.16: -1100/-1bp pStinger and -670/-1bp pStinger Phenobarbital inductions.

Figure 2.17: -288/-168bp pStinger Phenobarbital-induced and basal expression.

XVI

Figure 2.18: -288/-168bp GATA mutant pStinger Phenobarbital-induced and basal expression.

Figure 2.19: Caffeine induction of -1560/-1bp pStinger and -288/-168bp pH-Stinger.

Figure 2.20: A model for the regulation of Cyp12d1 xenobiotic induction.

Figure 2.21: -1100/-1bp sequence Dot Plot.

Figure 2.22: Cyp12d1 5’ promoter conserved regions identified using EvoPrinterHD.

Figure 2.23: Cyp12d1 electromobility shift assay gels.

Figure 2.24: Gelshifts prepared for Protein sequencing.

Figure 2.25: Nuclear receptor transcription factor expression reduction inductions.

Figure 2.26: -1560/-1bp pStinger;HR96 null lines Phenobarbital-induced GFP expression .

Figure 2.27: PAS-bHLH Transcription factor knockdown inductions.

Figure 2.28: GATA transcription factor knockdown inductions.

Figure 2.29: Model of Cyp12d1 cis-regulation.

Figure 3.1: Crossing scheme to test for the impact of Cyp12d1 knockdown on viability.

Figure 3.2: Cyp12d1 Tub-GAL4/UAS-RNAi viability.

Figure 3.3: Cyp12d1-RNAi and Cyp12d1-overexpression longevity survival curves.

Figure 3.4: Cyp12d1-RNAi and Cyp12d1-overexpression survival on 50mM hydrogen peroxide.

Figure 4.1: A Comparison of Cyp12d1 Single Copy and Double Copy Loci.

Figure 4.2: Geographical distribution of the duplicated Cyp12d1 gene frequencies along the east Coast of Australia. Figure 4.3: Cyp12d1 duplication status of several laboratory and natural population lines.

Figure 4.4: CYP12D1-d’ and CYP12D1-p’ alignments with other P450s.

Figure 4.5: y; cn bw sp Cyp12d-d’ and Cyp12d1-p’ transcript length differences.

Figure 4.6: Cyp12d1 temporal expression patterns in Cyp12d1 single copy and double copy lines.

Figure 4.7: Early 3rd instar Cyp12d1 mRNA in situ staining in single and double copy lines.

Figure 4.8: Embryonic Cyp12d1 expression in single and double copy lines.

Figure 4.9: Cyp12d1 base pair substitution frequency along the east coast of Australia.

Figure 5.1: Cyp12d1 duplication and loss in the Melanogaster group.

Figure 5.2: Cyp12d1 midgut section expression compared to the expression of a selected subset of genes.

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List of Tables

Table 1.1: Alternate P450 Reactions

Table 1.2: Some Endogenous P450 Functions

Table 1.3: Characterised Drosophila melanogaster P450 functions

Table 1.4: Drosophila P450 Microarray induction

Table 1.5: Cyp12d1 induction by various chemicals

Table 2.1: Matinspector-identified putative binding site in the -288/-168bp sequence.

Table 2.2: Top protein hits from EMSA protein band sequencing

Table 3.1: Cyp12d1 mRNA expression in Tub-GAL4/Cyp12d1-RNAi lines and controls

Table 3.2: Mean lifespan of Cyp12d1-RNAi and Cyp12d1-overexpression flies

Table 4.1: Cyp12d1 expression in Phenobarbital-exposed w1118 and y; cn bw sp 3rd instar larvae

XVIII

List of Abbreviations

3’ UTR 3' Untranslated Region 5HR-GAL4 Cyp6g1HR-GAL4-6c GAL4 Driver line AE anterior endoderm ARE Antioxidant response element ARNT AhR-nuclear transporter protein AS aminoserosa ATP Adenosine Triphosphate BR-C Broad-Complex CAR Constitutive Andostane Receptor chico Insulin receptor substrate CITCO 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime Con non-xenobiotic exposed control DDT dichlorodiphenyltrichloroethane DR Direct-repeat element DT Digestive tissue sample EcR Ecdysone Receptor EcRE Ecdysone-response element eGFP/GFP Green Fluorescent Protein EMSA Electromobility shift assays ER Everted Repeat element FE Foregut primordium FF-MAS Folical fluid-meoisis activating factor GR Glucocorticoid Receptor GST Glutathione S-Transferase HAH Halogenated hydrocarbons HNF4α Hepatic Nuclear Factor 4alpha ICZ Indolo[2,,3-b]carbazole Indy I’m not dead yet Inr Initiator element InR Insulin Receptor LTR Long Terminal Repeat NF1 Nuclear-factor 1 NRSE neuron-restrictive silencing element Oct-1 Octamer-1 ORF Open reading frame CYP/P450 Cytochrome P450 PAH Polycyclic Aromatic Hydrocarbons PAS-bHLH Per, ARNT and Sim (PAS)-basic-Helix-Loop-Helix (bHLH) transcription factor family PB Phenobarbital PBRE Phenobarbital-reponse enhancers

XIX

PBREM Phenobarbital response module PCB polychlorobiphenyl mixture PCN pregnenolone 16α-carbonitrile PCR Polymerase Chain Reaction PE Positive element PD Posterior endoderm PXR Pregnane X Receptor PXRE Proximal PXR responsive element RE1 repressor element 1 RNAi RNA interference RNA-Seq RNA sequencing ROS Reactive Oxygen Species RT-PCR Quantitative Real-Time Polymerase Chain Reaction RXR Retinoid X Receptor Sf9 Spodoptera frugiperda 9 cell line SL-2 D. melanogaster Schneider's Line S2 cell line SRS Substrate Recognition Sites Ss Spineless Sxe-1 Sex-specific enzyme 1 TCDD 2’,3’,7’,8’-Tetrachlorodibenzo-p-dioxin TCOBOP 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene Tgo Tango Tub-GAL4 Tubulin-GAL4/TM3,Sb GAL4 driver line VDR Vitamin D receptor WIS1 Drosophila melanogaster Wisconsin-1 strain WIS1LAB Drosophila melanogaster Wisconsin-1 Lab-selected strain WL Whole Larvae sample XREM Xenobiotic regulatory module XRE-xan Xanthotoxin-responsive element

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Chapter 1: Introduction

“The wanton boy that kills the fly

Shall feel the spider's enmity.”

Auguries of Innocence

- William Blake

1

The Cytochrome P450s (P450s) are a large family of enzymes found across every taxa. P450s perform crucial xenobiotic detoxification roles by metabolizing toxic substances and preventing cellular damage.

They also carry out essential endogenous functions and participate in many key metabolic and synthetic pathways. The large size of this family is a reflection of their functional importance, with many organisms possessing numerous P450s in their genomes (LEWIS et al. 1998).

P450s involved in xenobiotic detoxification can be rapidly transcriptionally upregulated after an organism is exposed to toxic xenobiotics, in a process known as xenobiotic induction. Organisms use xenobiotic induction to keep P450 expression at low levels of basal expression and only induce them when necessary. This process has been extensively studied in mammals (reviewed in (TOLSON and WANG

2010)), and several regulatory pathways for P450 xenobiotic induction are now well characterised.

The model organism Drosophila melanogaster uses P450s for insecticide metabolism, tissue development, behavior modification and hormone synthesis amongst other functions (reviewed in

(FEYEREISEN 2005)). D. melanogaster induces multiple P450s in response to xenobiotic inducing agents such as the pharmaceutical drug Phenobarbital (KING-JONES et al. 2006; LE GOFF et al. 2006; SUN et al.

2006; WILLOUGHBY et al. 2006). However, xenobiotic induction is poorly understood in insects. In this study, the transcriptional regulation of the xenobiotic-inducible D. melanogaster Cyp12d1 gene was investigated to identify novel D. melanogaster xenobiotic induction regulatory pathways. Cyp12d1 functions were examined to better understand why Cyp12d1 is induced in response to many xenobiotic chemicals. Cyp12d1 has been tandemly duplicated, and this duplication is found as a polymorphism in certain strains. The frequency of the Cyp12d1 duplication was also investigated to examine the importance of Cyp12d1 in the wild.

2

1.1 The Cytochrome P450s: A brief overview

Cytochrome P450s were named after the characteristic Soret absorption maximum at 450 nm in the UV light spectrum of their carbon monoxide adduct (ORTIZ DE MONTELLANO 2005). All P450 proteins share several common motifs and features to varying degrees (FEYEREISEN 2005; ORTIZ DE MONTELLANO 2005) .

The signature P450 motif is the haem-ligand binding pocket, but there are other P450 structural motifs which are essential for their function (Figure 1.1).

P450s in extant species are hypothesised to have arisen from a common prokaryotic ancestor approximately 3.85 billion years ago (LEWIS et al. 1998). P450s then spread throughout both prokaryotes and eukaryotes via a process of gene duplication and divergence, possibly due to changes in their nucleotide and amino acid sequences that allowed the acquisition of new reactions and thus new functions. Positive selection for these new functions could have driven the diversification of these enzymes and, hence, evolution of new family members (LEWIS et al. 1998; NEBERT and GONZALEZ 1987;

ORTIZ DE MONTELLANO 2005).

P450s are usually present in sizable numbers in genomes of eukatyotes. Humans have 58 P450s (NELSON et al. 2004b), mice have 105 P450 genes (HRYCAY and BANDIERA 2009), while the plant Arabidopsis thaliana has 246 genes (NELSON et al. 2004a) and the rice plant Oryza sativa has 356 P450 genes(NELSON et al. 2004a). The fungus Aspergillus nidulans has 111 genes (KELLY et al. 2009) and the vinegar fly

Drosophila melanogaster has 85 (TIJET et al. 2001) while the mosquito Aedes aegypti has 173 P450s

(RANSON et al. 2002).

1.1.1 Intracellular localisation

In eukaryotes the majority of Cytochrome P450s are found in the smooth section of endoplasmic reticulum, but can also be found in the inner membranes of mitochondria or plant plastids . In bacteria they exist as soluble cytosolic bacterial enzymes (ORTIZ DE MONTELLANO 2005). Cellular localisation of

3

Figure 1.1: General Cytochrome P450 Structural motifs. (A) Microsomal P450. A membrane targeting hydrophobic region allows anchoring to microsomal membranes. Several basic residues and proline residues form a hinge region between the anchor region and the globular domain. Substrate recognition sites (SRSs) are thought to recognise substrates and consequently are extremely variable in sequence. The I-helix loop forms the distal face of the haem involved in catalysis. The GLU-X-X-ARG is absolutely conserved between P450s. The haem binding loop is the main characteristic of P450s. (B) Mitochondrial P450s. Mitochondrial P450s have similar features, but differ by having a Mitochondrial targeting sequence at the amino terminal. They also have an additional two positive charges needed to interact with the ferrodoxin electron-transfer protein in the mitochondria. Figure adapted from (WERCK-

REICHHART and FEYEREISEN 2000).

4

P450s usually depends on the presence of organelle targeting sequences. P450s targeted to the mitochondria have an N-terminal mitochondrial targeting sequence, while P450s targeted to the endoplasmic reticulum and other microsomal fractions have an N-terminal microsomal targeting sequence (Figure 1.1) (ORTIZ DE MONTELLANO 2005).

1.1.2 P450 Reactions

P450s are classically perceived as monooxygenases. P450s firstly bind substrates to form enzyme- substrate complexes, following which oxygen binds to the haem molecule. This complex then recruits electron donors such as microsomal P450 reductases or mitochondrial adrenodoxin reductase which provide reducing agents NADPH or NADH to the complexes. The dioxygen molecule is cleaved by the input of NADPH or NADH and single oxygen atoms are generated. This single oxygen molecule is then inserted into the substrate, oxygenising the substrate, making it more hydrophilic. This reaction can be summarised as such:

+ + NADPH /NADH + H2 + O2 + R NADP /NAD + H20 +RO, where R is the substrate to be oxygenated. However, P450s are capable of a multitude of chemical reactions other than oxygenation (Table 1.1).

1.1.3 P450 nomenclature

P450 genes follow a set nomenclature (Figure 1.2 ) (FEYEREISEN 2005) . P450s are organized into families

(>40% homology at least) and subfamilies (>55% homology) by sequence homology. Genes are prefixed by the word “Cyp” (short for cytochrome P450), followed by (in order): a numeral denoting the P450 family, a letter designating the subfamily, and finally an individual gene numeral identifier (Figure 1.2).

P450 protein names are in uppercase and are not italised. For instance, Cyp12d1 belongs to the Cyp12 family, subfamily ‘d’, and the protein is called CYP12D1.

5

Figure 1.2: Cytochrome P450 nomenclature. P450 genes follow a set guideline for P450 nomenclature as outlined in the figure. Figure taken from (FEYEREISEN 2005).

6

1.2 P450 Functions

The wide range of possible P450 reactions, coupled with the broad substrate specificities of most P450s, means that P450s are capable of a vast number of potential functions. P450s can be loosely grouped by their functions into two large subsets: ones that metabolise foreign substances (xenobiotic metabolism), and ones that perform endogenous functions. However, this distinction is blurred when we consider that many P450s have both xenobiotic detoxification and endogenous functions. As noted elsewhere:

‘There are many cases where P450 act as anything but “detoxification enzymes”. The easy dichotomy between biosynthetic and detoxification functions of P450s reflects more the teleological tendencies of the observer than the phylogeny or biochemistry of the enzymes.’ (FEYEREISEN 1999)

For example, human Cyp3A4 is responsible for slightly less than half of all initial drug metabolism events

(INGELMAN-SUNDBERG 2004), but also has other important endogenous roles in oxidative testosterone metabolism, where it catalyses the 11β-hydroxylation of testosterone in the liver (CHOI et al. 2005). It is clear that this division of P450 functions into xenobiotic metabolising and endogenous is sophistic.

Nevertheless, for ease of discussion this division will be maintained.

Despite our increasing knowledge of P450 characteristics, the functions of many P450s remain unknown. Most P450s were identified based on sequence homology and virtually nothing is known about their functions. It would seem that our reach has exceeded our grasp in terms of identifying P450 genes faster than we can characterise them.

1.2.1 Endogenous P450 Functions

P450s perform many important endogenous functions, and loss of these P450s often results in lethality.

In mammals, P450s are involved in multiple processes including cholesterol biosynthesis (PIKULEVA 2006) and hormone synthesis (BISHOP 2007; HUANG et al. 2008; SCHUSTER 2011). Plant P450s participate in the

7

Table 1.1: Alternate P450 Reactions

P450 Species Reaction Example involved Reference

Reduction of N-Oxides (SETO and (deoxygenation/demethylation of GUENGERICH Reduction N-alkylarylamine to N-oxides) Cyp2B1 Human 1993)

Reduction of nitro-glycerin to Cyp3A Rat and (DELAFORGE et Reduction nitric oxide Family Human al. 1997)

Oxidative Ester Cleavage of Hanzsch pyridine (GUENGERICH et Cleavage esters Cyp2C11 Rat al. 1988)

Dehydration of aldoximes to (BOUCHER et al. Dehydration nitriles Cyp3A4 Human 1994)

Rearrangement of Fatty Acid and Dehydration of a 9- Prostaglandin hydroperoxydiene to colnelieic (ITOH and HOWE hyperoxides acid Cyp74D1 Tomato 2001)

Table adapted from (GUENGERICH 2001)

8 synthesis of many plant hormones, defence compounds, and flower pigments amongst others (BISHOP

2007). A brief summary of P450 endogenous functions is presented in Table 1.2.

1.2.1.1 Cholesterol and steroid hormone synthesis

The biosynthesis of sterols requires P450-catalyzed oxidation reactions, which means that P450s are essential for eukaryotic life (WERCK-REICHHART AND FEYEREISEN,2000). The cholesterol steroidogenesis pathway and its subsequent conversion into steroid hormones provide a good example of essential P450 endogenous functions, and how multiple P450s perform different steps in a metabolic cycle.

CYP51 encodes the enzyme Sterol 14α-demethylase, and it catalyses a three-step reaction of sterol 14α- demethylation, where the 14α-methyl subgroup of substrates are removed (FISCHER et al. 1991). CYP51 participates in the cholesterol biosynthesis pathway (Figure 1.3). CYP51 also converts lanosterol into the hormone Folical fluid-meoisis activating factor (4,4 dimethyl-cholest-8,14,24-trien-3β-ol, or FF-MAS), necessary for sex determination in mammals (ROZMAN 2000).

Cholesterol is converted into steroid hormones by P450s. Vitamin D is a fat soluble steroid hormone that regulates uptake of dietary calcium and phosphate, controlling calcium and phosphate homeostasis. The

Vitamin D activation pathway requires several microsomal and mitochondrial P450 inputs to convert

Vitamin D into the circulatory form, 25-OH-D3 and the final active form 1,25-(OH)2-D3. Initial vitamin D is converted into circulatory 25-OH-D3 by hepatic 25-OHase. Human 25-OHase activity was found to be shared between two P450s, Cyp27A1 and Cyp2R1 (CHENG et al. 2004; GUO et al. 1993). The mitochondrial Cyp27A1 performs 70% of the total 25-hydroxylation reactions and the microsomal

Cyp2R1 responsible for 30%, showing functional redundancy between both P450s (CHENG et al. 2004).

The active 1,25-(OH)2-D3 form is created from the 1-hydroxylation of 25-OH-D3 by the Cyp27B1 enzyme. Patients with nonsense mutations in Cyp27b1 consequently develop the disease Vitamin D-

dependent Rickets Type 1 due to insufficient 1,25-(OH)2-D3 levels (FU et al. 1997). Transgenic Cyp27B1

9

Table 1.2: Some Endogenous P450 Functions

Organism P450 Function Pathway References

1 cockroach Cyp15a1 Juvenile Hormone synthesis Hormone Metabolism (HELVIG et al. 2004a)

Cyp307a1 Cyp307a2 Cyp306a1 Reviewed in (HUANG et Ecdysone steroid Hormone 2 Drosophila Cyp302a1 Hormone Metabolism al. 2008) synthesis and activation Cyp315a1 Cyp314a1 Cyp18a1 (RAHIER and TATON 3 Plants Cyp51 obtusifoliol 14-demethylation Sterol metabolism 1986)

Auxin (plant growth (DURST and O'KEEFE 4 Plants Cyp71B15 Indole acid hydroxylation hormone) metabolism 1995)

Secondary Plant (DURST and O'KEEFE 5 Plants Cyp73 Cinnamic acid hydroxylation Metabolites synthesis 1995)

Plant pigment DURST and O'KEEFE 6 Plants Cyp75 Flavonoid 3',5' hydroxylation metabolism (1995) (DURST and O'KEEFE 7 Plants Cyp94a1 Oleic acid epoxidation Fatty Acid Metabolism 1995) Fungi and 8 Cyp51 Lanosterol 14-demethylation Sterol metabolism (ROZMAN 2000) Bacteria

Fungi and Palmitoleic acid; pentadecanoic 9 Cyp102 Fatty Acid Metabolism (MUNRO et al. 2002) Bacteria acid; Arachidonic acid Fungi and 10 Cyp109b1 Fatty acid oxidation Fatty Acid Metabolism (GIRHARD et al. 2010) Bacteria Cyp1A1, 11 Mammal Cyp1A2, Estrogen hydroxylation Hormone Metabolism (TSUCHIYA et al. 2005) Cy3A4 Cyp2C9, 12 Mammal Cyp2C19, Testosterone hydroxylation Hormone Metabolism (CHOI et al. 2005) Cyp3A4

13 Mammal Cyp4b1 Lauric acid metabolism Fatty Acid Metabolism (BAER and RETTIE 2006)

14 Mammal Cyp51 Lanosterol 14-demethylation Sterol metabolism (ROZMAN 2000)

10

Figure 1.3: An overview of Cyp51 activity in the sterol synthesis pathway. CYP51 (14DM) is active in the sterol biogenesis pathway where it removes the 14-methyl sidegroup of lanosterol in animals and fungi and obtusifoliol in plants. In mammals, lanosterol is also demethylated during the synthesis of meiosis- activating sterols (MAS) in the gonads.

11 knockout mice also develop Vitamin D-dependent Rickets, confirming the importance of this enzyme

(DARDENNE et al. 2001). Finally, Cyp24A1 catabolises 1,25-(OH)2-D3 and 25-OH-D3 into non-active byproducts, reducing the amount of cellular active 1,25-(OH)2-D3 and attenuating 1,25-(OH)2-D3 levels

(PROSSER and JONES 2004).

1.2.2 Endogenous Insect P450 functions

Although some insect endogenous P450 functions have been described, the functions of the majority of insect P450s remain uncharacterised. CYP15 stereospecifically epoxidises the precursor of the insect steroid hormone juvenile hormone in several insects (HELVIG et al. 2004a). The steroid hormone ecdysone synthesis and metabolism pathway in athropods requires the input of several P450s (discussed further below in Section 1.2.2.1), and deletion of any of these results in lethality.

1.2.2.1 Endogenous P450 functions in D. melanogaster

D. melanogaster has been the organism of choice insect P450 studies. D. melanogaster has 85 recognised P450 sequences (TIJET et al. 2001), 18 of which have been functionally characterised (Table

1.3). Most of our initial knowledge of Drosophila P450s came from insecticide resistance studies, but in recent years more P450s with endogenous functions have been identified. D. melanogaster P450s have been shown to have an wide range of endogenous functions (Table 1.3). Many functionally characterised D. melanogaster P450s were initially identified based on the phenotypes that result from the loss of specific P450s, highlighting the importance of wild-type P450 functions to D. melanogaster.

For example, investigations into the Halloween class of D. melanogaster mutants led to the identification of the ecdysone pathway P450s. Cyp302a1, Cyp306a1, Cyp307a1, Cyp314a1 and Cyp315a1 have been shown to catalyse several steps in the synthesis and activation of the moulting hormone ecdysone (Table 1.3, Figure 1.4) (WARREN et al. 2002; WILLINGHAM and KEIL 2004; NIWA et al. 2004). Cyp18a1 involvement in the ecdysone metabolism pathway was first suspected after Cyp18a1 was reported to be

12

Table 1.3: Characterised Drosophila melanogaster P450 functions Gene Function (s) Reference (FUJII et al. Cyp4d21 Also named Sxe-1; necessary for efficient male mating 2008) (GUTIERREZ et Cyp4g1 lipid storage al. 2007) (DUNKOV et al. 1997; SPIEGELMAN et Cyp6a2 Ecdysone inducible; able to metabolise some insecticides in vitro al. 1997) Able to metabolise lauric acid in vitro, able to metabolise the insecticide (HELVIG et al. Cyp6a8 alderin in vitro 2004b) (DIERICK and GREENSPAN Behaviour modification through modulating male aggression during mating 2006; WANG et Cyp6a20 and feeding al. 2008) (DABORN et al. 2007; DABORN Resistance to the insecticides DDT, nitenpyram, dicyclanil, lufenuron, et al. 2002; imidacloprid. JOUSSEN et al. Cyp6g1 Able to metabolise the insecticides DDT and imidacloprid in vitro 2008) (DABORN et al. Cyp6g2 Resistance to the insecticides diazanon and nitenpyram 2007) (DABORN et al. 2007; FESTUCCI- BUSELLI et al. Cyp12d1 Resistance to the insecticides DDT and dicyclanil 2005) (BOGWITZ et Cyp12a4 Resistance to the insecticide lufeneron al. 2005) (GUITTARD et Cyp18a1 20-hydroxyecdysone catabolism al. 2011) (WARREN et al. Cyp302a1 Ecdysone synthesis 2002) (WILLINGHAM Cyp303a1 Sensory bristle development and KEIL 2004) (NIWA et al. Cyp306a1 Ecdysone synthesis 2004) (NAMIKI et al. Cyp307a1 Ecdysone synthesis 2005) (ONO et al. 2006; SZTAL et Cyp307a2 Ecdysone synthesis al. 2007) (PETRYK et al. Cyp314a1 Ecdysone activation 2003) (WARREN et al. Cyp315a1 Ecdysone synthesis 2002) (MOHIT et al. Cyp310a1 Wing development 2006)

13 ecdysone-inducible (DAVIES et al. 2006; HURBAN and THUMMEL 1993). Cyp18a1 has since been found to participate in this pathway through breakdown of the activated 20-hydroxyecdysone (GUITTARD et al.

2011). Interestingly, Cyp6a2 expression is also regulated by ecdysone (SPIEGELMAN et al. 1997); (WHITE et al. 1999). Using temperature sensitive mutants, it was shown that interrupting ecdysone synthesis led to a decrease in the amount of Cyp6a2 mRNA compared to flies raised at permissive temperatures. This suggested that Cyp6a2 is an ecdysone response gene involved in ecdysone-mediated activities, although no further investigation into this has been reported.

Cyp6a20 has been found to be involved in behaviour modification through regulation of male aggression in two independent studies (DIERICK and GREENSPAN 2006; WANG et al. 2008). Microarray analysis of aggressive male flies revealed differences in Cyp6a20, with aggressive flies expressing lower amounts of

Cyp6a20 mRNA compared to non-aggressive flies when adult-head specific Cyp6a20 expression was compared. Environmental factors such as the conditions the flies were reared in were found to be a factor in determining the amount of Cyp6a20 expression. Cyp6a20 was found to be expressed in antenna and maxillary palps, suggesting that it modulated aggressiveness through metabolism of pheromones. Flies reared alone in isolated conditions expressed lower Cyp6a20 mRNA and were more aggressive than flies reared in groups (DIERICK and GREENSPAN 2006; WANG et al. 2008). This aggressiveness and correlated lower Cyp6a20 expression was reversed when isolated flies were placed in group housing, showing that Cyp6a20 mediates the suppressive effect of aggressiveness when flies are housed in groups.

Analysis of male and female head specific gene expression revealed enrichment of several male specific genes. One such gene, Sxe-1 (sex-specific enzyme 1) was found to be Cyp4d21, and has been shown to be necessary for efficient male mating (FUJII et al. 2008).

14

Figure 1.4: The Ecdysone synthesis and activation pathway in D. melanogaster. Plant sterols are converted into Ecdysone and eventually into activated 20-Hydroxyecdysone with the input of several

P450s (Named). The “Black Box” refers to a still-uncharacterised series of reactions in this pathway.

15

A screen for sensory mutants led to the identification of Cyp303a1 as being essential for the development of sensory bristles (WILLINGHAM and KEIL 2004). The NompH mutant lacked proper bristle development and subsequent mapping found that a disrupted Cyp303a1 gene was responsible for this.

Cyp310a1 is expressed in the dorsal and ventral parts of the wing pouch, and is thought to specify the mature wing blade (BAKER 2007). Wingless signalling negatively regulates Cyp310a1 expression (BUTLER et al. 2003) and supports a role for Cyp310a1 in wing development (BAKER 2007).

Cyp4g1 is an extremely well conserved gene with clear one to one orthologues in many insect species.

Cyp4g1 is highly expressed in the lipid-storing oenocyte cells, and was thought to be involved in lipid storage (GUTIERREZ et al. 2007).

Cyp6g2 is expressed in the corpus alletum of the ring gland, where the steroid hormone juvenile hormone is synthesised. RNAi experiments established that reducing Cyp6g2 expression ubiquitously resulted in lethality, but the cause of this lethality is still unclear (CHUNG et al. 2009). Cyp6g2 has been proposed to be involved in the juvenile hormone synthesis pathway (TAMAR SZTAL, unpublished results).

Finally, studying regulatory pathways may also indicate P450 function. The nuclear hormone receptor

Hr39 regulates Drosophila female reproductive tract development and function, and is thought to achieve this in part through regulation of P450 activity (ALLEN and SPRADLING 2008). Transgenic expression of Hr39 in spermathecae upregulated Cyp4p2, Cyp312a1 and Cyp6a14 expression. Knocking out Hr39 resulted in upregulation of the previously-discussed male-mating enzyme Cyp4d21 in the reproductive tract, Cyp6a17, Cyp4g1, Cyp6w1 and Cyp305a1 in the spermathecae while Cyp4p2 is downregulated in the reproductive tract(ALLEN and SPRADLING 2008). This suggests that these P450s play roles in Drosophila reproduction.

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1.2.3 Xenobiotic Metabolism

Organisms rely on P450s to oxidise hydrophobic toxic substances, rendering them more hydrophilic and easier to export out of the cell. Historically, the processing of xenobiotes has been the more-recognised function of P450s, and most of our current knowledge of P450s comes from investigating drug metabolism in humans. Other xenobiotic functions of P450s include providing tolerance to dietary toxins and metabolic inactivation of insecticides by insects (FEYEREISEN 2005; SCHULER 2011).

1.2.3.1 Dietary tolerance

Xenobiotic functions in insect P450s take place primarily in the form of tolerance to toxic secondary plant metabolites present in their diet, and resistance to insecticides applied to control insect pests.

The recent expansion in P450 gene numbers over the past 800 million years has been attributed in part to being driven by adaptation to dietary plant toxins (LEWIS et al. 1998). For instance, certain Drosophila species feed on rotting and decayed cactus in the Sonoran desert, which contain high amounts of toxic isoquinoline alkaloids. Cyp4D10 and Cyp28A1 have been shown to be induced by these alkoids, and have been suggested to mediate resistance to these toxins thereby allowing D. mettleri to feed safely

(DANIELSON et al. 1998; DANIELSON et al. 1995).

Larvae of the butterfly Papillio polyxenes require P450 detoxification to feed safely. P. polyxenes Cyp6B1 and Cyp6B3 are able to recognise and metabolise the plant toxins linear furancoumarins xanothotoxin and bergapten, as well as to a lesser extent, angular furancoumarins such as angelicin and (COHEN et al.

1992; HUNG et al. 1997). Helicoverpa zea uses the P450 Cyp6B8 for resistance to a wide variety of toxins including xanthotoxin, flavone, chlorogenic acid, indole-3-carbinol, quercetin, and rutin (LI et al. 2004;

RUPASINGHE et al. 2007).

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1.2.3.2 Insecticide resistance

Insecticide resistance is thought to have arisen through adaptation of this dietary tolerance to insecticide challenge. P450-based Insecticide resistance often relies on the overexpression of certain

P450s to selectively detoxify the insecticide. In natural populations, this overexpression is often caused by regulatory changes that result in the constitutive overexpression of target P450s and subsequent insecticide resistance (PERRY et al.). Transgenic methods such as the UAS-GAL4 system can also be used to drive P450 overexpression to study insecticide resistance (DABORN et al. 2007).

1.2.3.3 Drosophila melanogaster P450s and Insecticide resistance

Many of the commercially important crop pests are difficult to maintain and study in laboratory conditions, so researchers have turned to Drosophila melanogaster as a model organism to study insecticide resistance (PERRY et al. ; SCHNEIDER 2000).

The use of Drosophila to study insecticide resistance is important in three areas: firstly, to learn how the insecticide works and which molecular process they target; secondly, by determining what resistance mechanisms can develop; and lastly, providing knowledge that could lead to the development of new insecticides (SCHNEIDER 2000). In general, increasing our knowledge of Drosophila P450s could help in the design of new insecticides that avoid inactivation by P450s. New insecticides could even be designed to target P450s themselves, inhibiting their activity and thereby killing the insect (PERRY ET AL.).

D. melanogaster P450 insecticide resistance research began when resistance to the insecticide dichlorodiphenyltrichloroethane (DDT) was investigated in D. melanogaster lines (KING 1954; OGITA

1958). Mapping experiments using DDT-resistant natural population lines identified the 64.5cM region on chromosome 2R as being responsible for significant and dominant DDT resistance (HALLSTROM 1985;

KIKKAWA 1961). Subsequent studies established that this region contained several P450s (Cyp6g1,

18

Cyp6g2 and Cyp6t3) and that only Cyp6g1 was consistently overexpressed in multiple resistant strains

(DABORN et al. 2002; LE GOFF et al. 2003).

Resistance was closely linked to an insertion of a partial Long Terminal Repeat (LTR) sequence from the transposable element Accord in the promoter region of Cyp6g1 (DABORN et al. 2002). It was also shown that this insertion also gave resistance to other insecticides (lufenuron, nitenpyram and imidacloprid)

(DABORN et al. 2002). Subsequently, it was found that the presence of enhancers on the Accord LTR fragment drove higher Cyp6g1 expression in more digestive tissues, and led to greater levels of insecticide resistance (CHUNG et al. 2007). Transgenic expression studies eventually established Cyp6g1 to be able to confer resistance to numerous insecticides (DABORN et al. 2007; LE GOFF et al. 2003).

CYP6G1 was heterologously expressed in tobacco cells and shown to dechlorinate DDT into the metabolic byproduct DDD (JOUSSEN et al. 2008). Imidacloprid was also converted by hydroxylation into 4- hydroxyimidacloprid and 5-hydroxyimidacloprid (JOUSSEN et al. 2008), thereby confirming that Cyp6g1 was able to metabolise DDT and imidacloprid. Cyp6g1 has been found to be tandemly duplicated in certain lines (SCHMIDT et al. 2010). Since the discovery of the Accord LTR insertion, additional transposable element insertions have also been found in duplicated loci, and it has been found that having both a dulplicated Cyp6g1 locus and multiple transposable element insertions confer greater resistance to DDT than unduplicated Cyp6g1 loci (SCHMIDT et al. 2010).

The overexpression of several other D. melanogaster P450s has been shown to be associated with insecticide resistance (Table 1.3). Cyp12d1 was found to be overexpressed jointly with Cyp6g1 in the

DDT-selected 91R strain and the wild-caught DDT-resistant Wisconsin-1 (WIS1) strains (BRANDT et al.

2002; FESTUCCI-BUSELLI et al. 2005; LE GOFF et al. 2003). Cyp12d1 has since been shown to confer resistance independently of Cyp6g1 to DDT and dicyclanil when overexpressed transgenically (DABORN et al. 2007). The WIS1lab strain was derived from the same wild-caught population as the WIS1 strain and

19 the Cyp6g1-containing region in this strain was removed by recombination. The WIS1LAB strain was also selected for DDT resistance, and microarray analysis found Cyp6a8 was overexpressed which implied that Cyp6a8 was the cause of the DDT resistance observed. However, a separate heterologous expression study did not find CYP6A8 capable of metabolising DDT in vitro (HELVIG et al. 2004b), indicating that another mechanism was responsible for DDT resistance in the WIS1lab strain. Both

CYP6A8 and CYP6A2 however can convert aldrin into dieldrin in vitro when heterolgously expressed

(DUNKOV et al. 1997; HELVIG et al. 2004b), although CYP6A2 has been shown to do so at a 10-fold higher rate (DUNKOV et al. 1997).

The lufenuron-resistant NB16 strain was found to constitutively overexpress Cyp12a4, and subsequently transgenic overexpression experiments found that Cyp12a4 was able to confer resistance to lufenuron when overexpressed (BOGWITZ et al. 2005).

Another P450, Cyp6g2, was found to be able to provide resistance to diazanon and nitenpyram when transgenically overexpressed in larval digestive tissues (DABORN et al. 2007).

Despite these transgenic experiments, strong evidence for P450 overexpression conferring resistance in field D. melanogaster populations is limited to Cyp6g1 and to a lesser extent Cyp12a4 and Cyp12d1.

While transgenic expression may show that certain P450s are able to confer resistance when overexpressed under laboratory conditions, without data from naturally-isolated field populations, it is unclear if these P450s contribute to insecticide resistance in wild populations.

In the past, it was assumed that all Drosophila P450s had xenobiotic detoxification functions to some extent and could provide some degree of insecticide resistance (SCOTT 2008). However, when a small group of D. melanogaster P450s were overexpressed, it was found that some of these P450s failed to confer resistance to any of a range of four insecticides tested (DABORN et al. 2007). For instance,

Cyp6a17 and Cyp6a23 did not confer resistance to nitenpyram, DDT, dicyclanil and diazanon when

20 overexpressed. Furthermore, none of the P450s studied gave resistance to all of the insecticides tested

(DABORN et al. 2007), again suggesting that P450s may not have as broad substrate recognition as previously thought. It seems that contrary to previous thought, P450s can have very specific functions and substrate specificities instead of all P450s broadly having similar functions as previously thought.

1.2.3.4 Human Drug Metabolism

Human P450s and the metabolism of pharmaceutical drugs represents the best-known example of P450 xenobiotic metabolism. There are approximately 59 P450 genes in humans (RENDIC 2002). Ten of these genes have been implicated in metabolising xenobiotic substrates. However, only six of these genes are considered to actively participate in pharmaceutical drug metabolism events (INGELMAN-SUNDBERG 2004).

Cyp2C9, Cyp2C19, Cyp2D6, Cyp2E1, Cyp3A4 and Cyp3A5 are responsible for 70-80% of all initial pharmaceutical drug metabolism events (INGELMAN-SUNDBERG 2004).

1.2.3.5 P450 Alleles and Drug Metabolism

Drugs need to be within a defined therapeutic serum concentration range to achieve the desired clinical effect. Excessively high concentrations can cause adverse reactions, while concentrations that are too low will not have the desired effects. There can be tremendous individual variation in serum concentrations even with the same drug dose, necessitating individual treatments for patients. An important contribution to this variation is the ability of the P450s to metabolise drugs. P450- hydroxylation inactivation is part of drug metabolism, and is haplotype-dependant (INGELMAN-SUNDBERG et al. 2007). All six P450s genes listed above are highly polymorphic, with many allelic variants already characterised. The allelic haplotype of an individual can account for much of the variation in initial drug reactions. Poor drug metabolism results in excessive drug levels in plasma accumulating dangerously, as opposed to a good metaboliser having expected drug levels in plasma by clearing surplus drug

(INGELMAN-SUNDBERG et al. 2007). Similarly, having an overactive metaboliser haplotype might result in

21 premature clearance of the drugs. Therefore, individual allelic variation should be taken into account when prescribing drugs to avoid any unwanted adverse drug reactions.

Cyp3A4 Haplotype

Cyp3A4 is one of the most important drug metabolising P450s in the human body, and is responsible for approximately 50% of all drug metabolism events (INGELMAN-SUNDBERG et al. 2007). Cyp3A4 is the most highly expressed human P450 accounting for 70% of all human P450 expression. Cyp3A4 recognises over

80 individual drug substrates (For a list of subtrates, please see (FLOCKHART 2007)). As such, correct

Cyp3A4 expression and activity is obviously crucial for proper drug metabolism and dosage. Cyp3A4 expression and activity depends markedly on a range of factors including haplotype, diet, ethnicity, environmental factors and individual variability. There are 40 major Cyp3A4 haplotypes considered to be important for drug metabolism (SHULDINER et al. 2007). Therefore, it is clear that Cyp3A4 activity is of supreme importance when planning drug regimes. Certain combinations of drugs should be avoided because they induce higher Cyp3A4 expression and could result in adverse drug reactions. For instance, hyperforin, a component of the popular plant herbal remedy St John’s Wort, is the most potent inducer of Cyp3A4 identified to date (MANNEL 2004). As such, this might interact unfavourably with other pharmaceutical drugs and should be avoided when embarking on an intensive drug regime. Conversely, grapefruit contains potent inhibitors of P450 activity, and also should be avoided when regularly consuming pharmaceutical drugs (SEDEN et al. 2010).

Cyp2C9 and Warfarin

Another example of how different P450 alleles could have potentially fatal drug metabolic side effects is

Cyp2C9 and its metabolism of warfarin. The drug warfarin is an anticoagulant taken orally to reduce blood clotting. Warfarin acts by antagonising and inhibiting vitamin-K dependant coagulation factors

22

(MAJERUS et al. 1996). Overdoses of warfarin causes uncontrollable bleeding that can result in death

(MAJERUS et al. 1996).

Warfarin used in clinical applications is a racemic mixture consisting of two isoforms, R and S. The S form has five times more potency, and is primarily metabolised by the CYP2C9 metabolic pathway into the inactive 6-hydroxy and 7-hydroxy metabolites (KAMINSKY and ZHANG 1997). Mutant alleles of Cyp2c9 have been shown to have impaired rates of warfarin metabolism. For example, the R144C allele of Cyp2c9

(now called Cyp2c9*2) was found to have reduced activity for S-warfarin when compared to the wildtype allele, Cyp2c9*1 (FURUYA et al. 1995). A clinical study found that patients possessing this variant allele required lower doses of warfarin and had increased risks of bleeding complications compared to clinic controls (AITHAL et al. 1999), again emphasising the importance of P450 polymorphism in xenobiotic functions.

1.2.3.6 Bioactivation of toxic metabolites

P450s do not only detoxify xenobiotic substances, and they have been implicated in the bioactivation of substrates into more toxic metabolites. For instance, Cyp3A4 and Cyp2D6 have been implicated in the activation of organophosphate insecticides, which inhibited cholinesterase activity in human microsomes. Cyp1A1 has roles in activating many environmental procarcinogens, including polycyclic aromatic hydrocarbons (PAH) (SHIMADA et al. 1992). Cyp1A1 activation of PAH compounds in cigarette smoke is considered one of the major contributory factors to lung cancer (SHIMADA et al. 1992). Cyp2E1 is involved in the metabolism of ethanol in the liver, and is induced by chronic ethanol consumption.

Cyp2E1 bioactivates many procarcinogens in alcoholic beverages, tobacco smoke and diet, and having constantly high alcohol-induced CYP2E1 expression leads to highly detrimental effects on health

(MCKILLOP and SCHRUM 2005).

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1.2.4 Mitochondrial P450s

Cyp12d1 is a mitochondrially-located P450, and this section will concentrate on mitochondrial P450s.

Mitochondrial P450s are characterised by a mitochondrial targeting sequence and two conserved residues that interact with ferrodoxin, and are a minor group when compared to the considerably more numerable microsomal P450s (FEYEREISEN 2011; OMURA 2006). Mitochondrial P450s are hypothesised to have arisen from mutations in the microsomal targeting sequence of an ancestral P450, which resulted in the microsomal P450 being mistargeted to the mitochondria (FEYEREISEN 2011; OMURA 2010).

Interestingly, plants have not been found to have mitochondrial P450s, suggesting that they arose after the split of the plant and animal kingdom (OMURA 2010).

Vertebrate mitochondrial P450s comprise of three families (CYP11, CYP24 and CYP27). These have well conserved roles in the synthesis of hormones such as steroid hormones, adrenal cortex hormones and sex steroid hormones, but do not appear to have xenobiotic detoxification capabilities (OMURA 2006).

Cyp11A1 the catalyses side chain cleavage of cholesterol, which is the first step of all steroid hormone genesis (OMURA 2006). Cyp11B1 and Cyp11B2 are involved in cortisol and aldosterone synthesis respectively (BUREIK et al. 2002). CYP24A, CYP27A and CYP27B participate in the vitamin D synthesis pathway and were discussed in greater detail in Section 1.2.1.1.

Insect mitochondrial P450s have similar roles in steroid hormone synthesis. In direct contrast to vertebrate mitochondrial P450s, some have been shown to have xenobiotic functions. Insect mitochondrial P450s can be separated into well-conserved and less well-conserved groups (Figure 1.5).

Ones that have essential roles, like the mitochondrial Halloween P450s (Cyp301A1, Cyp302A1, Cyp314A1 and Cyp315a1) heavily involved in the ecdysone synthesis pathway, are well conserved with 1:1 orthologues readily recognisable in other insect species (CLAUDIANOS et al. 2006; FEYEREISEN 2006;

FEYEREISEN 2011).

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Figure 1.5: Conservation between D. melanogaster and A. gambiae mitochondrial P450s. D. melanogaster (black) and A. gambiae (red) mitochondrial P450 amino acid sequences were compared using a phylogenetic tree. It can be seen that the CYP12 family genes of each species cluster together

(with the only exception of CYP12B2), with no clear cross-species orthologues. This indicates that members of the CYP12 family in each species arose through species-specific duplications and divergences. In contrast, the second group of well conserved genes have clear one to one orthologues between each species. Three of the genes listed have known critical functions in ecdysone synthesis, while CYP303A1 and CYP49A1 has been described as highly conserved (FEYEREISEN 2011). Figure adapted from (RANSON et al. 2002).

25

Other less conserved mitochondrial P450s seem to be species-specific and closely-related sequences are not found in other species. Cyp12A1 is a housefly-specific mitochondrial P450 that can metabolise the insecticide diazanon (FEYEREISEN et al. 1997; GUZOV et al. 1998). D. melanogaster Cyp12a4 confers resistance to lufenuron when overexpressed in resistant strains (BOGWITZ et al. 2005). D. melanogaster

Cyp12d1 overexpression confers resistance to DDT and dicyclanil (DABORN et al. 2007; LE GOFF et al.

2003), and Cyp12d1 is extremely inducible by xenobiotics (WILLOUGHBY et al. 2006). Cyp12f1 is an

Anopheles gambiae mitochondrial P450 and is one of several P450s overexpressed in a DDT-resistant strain and a permethrin-resistant strain (WONDJI et al. 2009).

1.3 Cytochrome P450 transcriptional regulation

There must be correct spatial and temporal expression for P450s to perform their specified functions.

P450 transcription can be induced by various stimuli. Xenobiotic transcriptional induction will be the main topic discussed in this section, but it must be noted that P450s are also regulated by non- xenobiotic signals like hormones (MODE et al. 1992; SPIEGELMAN et al. 1997; WHITE et al. 1997), epigenetic interactions (BEEDANAGARI et al. 2010) and Reactive Oxidant Species (ROS) signalling (MOREL et al. 1999).

P450 induction can also take place through other methods such as mRNA processing, mRNA stabilisation, increased translation or even enzyme stabilisation (Reviewed in LEWIS 2001).

1.3.1 P450 Xenobiotic Induction

Exposure to xenobiotic substances results in large changes in cellular gene expression, in a process known as xenobiotic induction. Many genes are transcriptionally upregulated in this global response. For instance, rats fed phenobarbital for 13 weeks upregulated genes from eight gene ontology classes

(biotransformation; cell cycle and growth regulation and apoptosis; signal transduction; cellular metabolism; membrane transport and clearance; stress response; cytoskeletal and structural; and novel sequences and genes of unknown function) (Elrick et al. 2005). P450s from multiple families (the CYP2A,

26

CYP2B, CYP2C, CYP2H, and CYP3A subfamilies) are induced after phenobarbital exposure in rats (Tolson and Wang 2010). Likewise in D. melanogaster 3rd instar larvae, a global response occurs, with the induction of approximately 500 genes and the downregulation of approximately 400 genes in response to phenobarbital exposure (King-Jones et al. 2006), showing that large transcriptional changes after xenobiotic induction are also found in vertebrates.

P450 induction is mediated by cellular regulatory networks. Xenobiotic sensing transcription factors detect incoming xenobiotics and upregulate various target genes, including P450s, in response. In general, P450 induction ensures detoxification genes are expressed only when necessary, reducing the metabolic cost of constitutively expressing these genes at appropriately high levels. Constantly high

P450 protein levels may also result in intracellular damage from promiscuous enzymatic activity or excessive levels of reactive oxygen species (ROS) from increased P450 activity (Zangar et al. 2004). High levels of P450s can also result in the unnecessary bioactivation of xenobiotics, resulting in potentially deleterious effects to the organism. Expressing these enzymes selectively also reduces the energy burden of constantly producing high P450 levels.

Historically, P450s were postulated to only be induced by their substrates, allowing for the expression of genes capable of detoxifying the incoming toxins (Guengerich 1999; Whitlock 1999). For instance, it has been noted that in insects, plant secondary metabolites induce P450 expression to detoxify dietary toxins, leading to speculation that induction has evolved as a response. Although this may be true in a few select examples, there is little solid evidence that this applies in most situations.

An example against the theory that P450s are only induced by their substrates is the lack of induction in

D. melanogaster when exposed to insecticides. A study examining the induction reponse of D. melanogaster to six different insecticides found that only one insecticide, DDT evoked an induction response (WILLOUGHBY et al. 2007). Even so, DDT only induced two genes, Cyp12d1 and the glutathione

27

S-transferase GSTD2 gene, in the y; cn bw sp strain (WILLOUGHBY et al. 2007). Cyp12d1 has been shown to possibly play a role in DDT resistance (DABORN et al. 2007), but it has never been shown to directly metabolise DDT. GSTD2 was not able to metabolise DDT when expressed heterologously, showing that

DDT was not a substrate for GSTD2 (TANG and TU 1994). Cyp6g1 has been shown to be able to convert

DDT into the metabolite DDE, but Cyp6g1 was not found to be induced by DDT in adult flies or larvae(WILLOUGHBY et al. 2007). Induction and insecticides are covered in greater detail in section 1.4.2.

Furthermore, feeding organisms with artificial compounds unlikely to be encountered in the wild, such as phenobarbital, still results in P450 xenobiotic induction. This shows that P450 induction is not necessarily substrate-specific, as these organism are unlikely to have been exposed to these artificial compounds long enough to have evolved the precise P450 substrate recognition to detoxify it.

P450 induction may instead have evolved through the appearance and continued selection of transcription factors able to recognise various classes of inducing chemicals, rather than the specific compounds. These transcription factors upregulate target genes, some of which are able to recognise and metabolise the xenobiotic chemical. It would seem that induction may have evolved at a very early point after the emergence of P450s and the mechanisms of responding to classes of inducing chemical has been well conserved.

1.3.1.1 Transcription Factors regulating mammalian P450 induction

Mammalian P450 xenobiotic induction has been a well-studied phenomenon. This has resulted in several regulatory pathways for P450s being well mapped out and understood. Xenobiotic regulatory transcription factors have been identified, and the cis-regulatory regions of many mammalian P450s have been comprehensively characterised.

Induction of P450s is controlled through xenobiotic-sensing transcription factors termed xenosensors.

These transcription factors are often cytosolic, and translocate into the nucleus upon activation by the

28 binding of a xenobiotic ligand. These transcription factors subsequently bind to cis-enhancer elements

(xenobiotic-responsive elements or XREs) in P450 regulatory regions and increases transcription, leading to elevated levels of intracellular P450 enzymes. This results in the detoxification and clearance of xenobiotics (Figure 1.6).

Mammals have several transcription factors that contribute to regulating xenobiotic induction.

However, only the three main xenosensors will be discussed in detail. The Per, ARNT and Sim basic-

Helix-Loop-Helix (PAS-bHLH) family Aryl Hydrocarbon Receptor (AhR) gene and the Nuclear receptor family Pregnane X receptor (PXR) and Constitutive andostane receptor (CAR) genes collectively regulate most of the xenobiotic induction response in mammals. These genes and their mechanisms of regulation for P450s will be discussed in this section.

1.3.1.2.The PAS-bHLH family

The Aryl Hydrocarbon receptor (AhR) is a member of the PAS-bHLH family (BURBACH et al. 1992). This is a large family of transcription factors that regulate a variety of functions including hypoxia, retinal cell development and xenobiotic response (HATAKEYAMA and KAGEYAMA 2004). AhR and its orthologues remain thus far the only known PAS-bHLH genes capable of inducing xenobiotic genes, and AhR is considered one of the major xenobiotic regulatory proteins in mammals.

AhR substrates are more likely to be environmental substances rather than pharmaceutical drugs. For example, AhR recognises carcinogens like polyaromatic hydrocarbons (PAH) and halogenated hydrocarbons (HAH) aromatic, commonly found in tobacco smoke (KAWAJIRI and FUJII-KURIYAMA 2007).

The classical inducers that have been defined for AhR are 2’,3’,7’,8’-Tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) and indolo[2,,3-b]carbazole (ICZ).

29

Figure 1.6: Xenobiotic induction-regulating transcription factor modes of action.

30

Figure 1.6: Xenobiotic induction-regulating transcription factor modes of action. (A) AhR mode of action.

An AhR agonist, in this case dioxin, enters the cell (I). Inactive cytosolic AhR binds to dioxin and dissociates from its inactive complex (II). Activated AhR dimerises with the Aryl hydrocarbon nuclear transport protein (III) and enters the nucleus (IV). The activated AhR/ARNT dimer binds to enhancer sites in target genes and induces expression (V). (B) PXR mode of action. The PXR ligand phenobarbital enters the cell (I) and binds to inactive PXR bound in an inactive cytosolic complex (II). Activated PXR dissociates and partners with RXR (III). The PXR/RXR complex translocates into the nucleus (IV) and binds to target gene promoter enhancer sequences to upregulate target gene expression. (C) CAR ligand-independent activity. (I) Phenobarbital enters the cell and possibly the nucleus as well (II), although it is not clear if this is the case. CAR associates with RXR and enters the nucleus independent of any ligand binding, to bind to enhancers located in target gene promoter regions. Unknown repressing factors suppress

CAR/RXR activity and target genes are not upregulated (III). Phenobarbital indirectly stimulates unknown factors to relieve this repressive effect (IV). Transcription of target genes now takes place after this inhibitory factor has been removed (V). (D) CAR ligand-dependent activity. A known CAR ligand, 1,4- bis[2-(3,5-dichloropyridyloxy)]benzene (TCOBOP), enters the cell (I). Cytosolic CAR binds to it and dissociates from an inactive complex (II). TCOBOP-activated CAR dimmerises with RXR (III) and translocates into the nucleus (IV) to bind to enhancer sequences and induce target genes (V).

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The mode of action for AhR has been established (Figure 1.6A). Non-induced AhR is held in an inactive cytoplasmic complex bound to co-factor proteins including Hsp90, p23 and ARA9 (FUJII-KURIYAMA and

MIMURA 2005). Upon ligand binding, AhR is activated and dissociates from its cytoplasmic partners and translocates into the nucleus to heterodimerise with the AhR-nuclear transporter protein (ARNT). The

AhR-ARNT heterodimer binds to XREs in promoter regions of several P450 genes, activating transcription and expression. The repressor protein OCT-1 binds to the AhR binding site when the gene is quiescent, and is only displaced when the AhR-ARNT complex binds to the Ahr binding site to activate transcription

(FUJII-KURIYAMA and MIMURA 2005).

Cyp1A1 and AhR

AhR regulates several P450 genes, of which Cyp1A1 has been the best studied. Cyp1A1 participates in the metabolism of PAHs, and is upregulated by AhR as part of the cell’s response to PAH exposure

(WHITLOCK 1999). Cyp1A1 has several distinct domains which regulate Cyp1A1 basal and induced expression (Figure 1.7A). AhR binds to a conserved TNGCGTG target sequence which is intolerant of nucleotide substitution (FUJII-KURIYAMA and MIMURA 2005).

There is a cluster of four AhR-binding sites located 1kb upstream of rat Cyp1A1 that regulates dioxin induction (Figure 1.7A) (KOBAYASHI et al. 1996). Two distinct sets of enhancers, each containing two overlapping AhR binding sites, were identified, with the first set located from -1007 to -1021bp and the second set in -1088 to -1092bp (FUJISAWA-SEHARA et al. 1987). This cluster is conserved in human Cyp1A1, although there were as many as twelve predicted AhR-binding sites identified in the Cyp1A1 promoter region (NUKAYA and BRADFIELD 2009). A Basal Transcription element (BTE) containing GC box binding sites for the transcription factor Sp1 was needed for maximum Cyp1A1 induction (KOBAYASHI et al. 1996).

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Figure 1.7: cis-regulatory regions of Xenobiotic-inducible P450s.

33

Figure 1.7: cis-regulatory regions of xenobiotic-inducible P450s. (A) Human Cyp1A1 is regulated by the

AhR transcription factor. Four AhR binding sites are located approximately 1kb upstream of the start codon, and a basic transcription element (BTE) unit is located in the upstream region as well. An additional ER-8 CAR binding site adjacent to the AhR binding sites has been recently discovered. (B)

Cyp2B2 induction by phenobarbital relies primarily on the phenobarbital response module (PBREM) located approximately 2.2kb upstream. The PBREM contains two DR-4 sequences and one NF1 sequence. An overlapping Barbie box/Postive Element (PE) sequence is found in the -150/-50bp region.

Figure modified from (SUEYOSHI and NEGISHI 2001). (C) Cyp3A4 relies on both the Proximal PXR responsive element (PXRE) and a distal Xenobiotic regulatory module (XREM) for phenobarbital induction. Figure modified from (SUEYOSHI and NEGISHI 2001).

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1.3.1.3 The Nuclear receptor family

Nuclear receptors are a family of transcription factors which regulate the expression of genes involved in many important developmental, homeostatic and xenobiotic functions. Nuclear receptors regulate P450 xenobiotic induction through sensing incoming toxic substances and subsequently upregulating various

P450s to detoxify the incoming xenobiotic.

The Pregnane X Receptor (PXR) and the Constitutive Andostane Receptor (CAR) are two of the main regulators of xenobiotic genes in mammals. Together, they regulate the CYP2, CYP3 and CYP4 families, effectively controlling a large portion of cellular xenobiotic response (TOLSON and WANG 2010). However, while they regulate many genes in common, there are still clear differences in the lists of PXR and CAR activated genes (MAGLICH et al. 2002). It appears that PXR is more important for CYP3 family P450 induction, while CAR is crucial for CYP2B family induction (WEI et al. 2002).

PXR and CAR recognise similar types of xenobiotic chemicals, and both mediate response to a wide variety of known ligands. These ligands include the classical P450 inducers phenobarbital, Rifampicin, pregnenolone 16α-carbonitrile (PCN) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), and thus PXR and CAR co-ordinate genetic responses to many pharmaceutical drugs (WILLSON and KLIEWER

2002). However, although they have overlapping lists of recognised ligands, they still have differences in ligand specificities (WILLSON and KLIEWER 2002).

PXR and CAR knockout mice confirmed their central roles in regulating xenobiotic responses in mice.

Removal of PXR abolished PXN and dexamethasone-induced Cyp3A11 expression (XIE et al. 2000).

Expressing human PXR in these mice resulted in previously-unseen Cyp3A11 induction by the human- specific PXR agonist rifampicin, showing that PXR orthologues are highly functional conserved. Finally, mice that expressed constitutively-active PXR had increased resistance to effects of the anaesthetics tribromoethanol and zoxazolamine, due to elevated amounts of induced P450s (XIE et al. 2000).

35

Targeted disruption of CAR results in a loss of phenobarbital and TCPOBOP-induced Cyp2B10 expression, establishing that CAR is the main regulator of phenobarbital induction in mice (WEI et al. 2000) (KODAMA and NEGISHI 2006a). CAR null mice are also hypersensitive to zoxazolamine-induced paralysis because of a decreased ability to detoxify zoxazolamine stemming from a lack of P450 induction (WEI et al. 2000).

PXR and CAR Modes of action

PXR and CAR have different preferred binding sites, although they are both able to bind to the same binding sites to activate transcription (WILLSON and KLIEWER 2002). Both genes require the binding of the

Retinoid X receptor (RXR) as a heterodimeric binding partner in order to translocate into the nucleus and bind to enhancer sites to induce expression (WILLSON and KLIEWER 2002). The mode of CAR and PXR activity is also similar, but with crucial differences (Figure 1.6). PXR can only affect transcription in a ligand-dependent mode of action. Inactive cytosolic PXR is held in an inactive complex and requires the binding of a ligand before it is able to dissociate from the complex and dimerise with RXR. The PXR/RXR heterodimer is then able to enter the nucleus and upregulate transcription of target genes.

CAR in contrast has a more complicated mode of action, with CAR being able to induce transcription in a ligand-dependent and ligand-independent manner (Figure 1.6(C-D)). Ligand-dependent CAR activity behaves similarly to PXR, with cytosolic CAR requiring the binding of a CAR-ligand before dimerising with

RXR and translocating into the nucleus to binding to CAR binding sites (TOLSON and WANG 2010). CAR ligand-independent activity is considerably less well understood, but seems to be the more predominant form of CAR activity. In fact, most of the chemicals (including phenobarbital) that CAR mediates the response to do not actually bind directly to CAR but instead activate CAR activity through unknown indirect mechanisms (TOLSON and WANG 2010). In the CAR ligand-independent mode of action, CAR is able to dimerise with RXR and enter the nucleus without CAR binding to a ligand, reflecting the

“constitutive” portion of its name. The CAR/RXR heterodimer binds to enhancer sites but does not

36 induce transcription because of some poorly-understood form of repression. The appearance of a xenobiotic chemical like phenobarbital results in the removal of this repression and the transcriptional upregulation of target genes.

Interestingly, while mammals have both PXR and CAR, non-mammals seem to have only one gene copy belonging to the PXR/CAR class of nuclear receptors (LINDBLOM et al. 2001; MAGLICH et al. 2001). The C. elegans NHR-8 gene is necessary for wild-type levels of resistance to the toxins colchicine and chloroquine, suggesting it potentially regulates genes that confer resistance to xenobiotics (LINDBLOM et al. 2001). However, no evidence for the direct regulation of P450s by NHR-8 has been reported. The D. melanogaster orthologue, HR96 has been shown to regulate some phenobarbital-induced P450 expression (KING-JONES et al. 2006). Two fish species, the zebrafish Danio rerio and the pufferfish Fugu rubripes also have only one PXR/CAR-like gene (MAGLICH et al. 2003; MOORE et al. 2002). The chicken genome has the Chicken X Receptor (CXR) gene, which was shown to be the primary regulator of xenobiotic induction (HANDSCHIN et al. 2000). This indicates that mammals are unique in having two xenobiotic-sensing nuclear receptor genes with different ligand specificities and activity, and that this duplication and divergence likely occurred after the split of mammals from birds.

CXR was shown to have aspects of both PXR and CAR in its activity, having a CAR-like mode of nuclear translocation, but PXR-like promiscuity in having a wide ligand specificity (HANDSCHIN et al. 2004). CXR,

PXR and CAR were shown to recognise the same phenobarbital-responsive enhancer motifs in the chicken Cyp2H1 and Cyp3A37 genes in transactivation and electromobility shift assay experiments, showing a high degree of functional conservation during evolution (HANDSCHIN et al. 2004). Together, these data suggest that there is a high degree of functional conservation for PXR and CAR across taxa.

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Other induction regulating Nuclear receptors

Other non-PXR/CAR Nuclear receptors are also involved in xenobiotic induction. The Hepatic Nuclear

Factor 4alpha (HNF4α), the Glucocorticoid Receptor (GR) and the Vitamin D receptor (VDR) have been shown to be needed for some xenobiotic induction. HNF4α has been shown to interact with PXR/CAR to enable maximum response to the drugs rifampicin and phenobarbital (FERGUSON et al. 2005; TIRONA et al.

2003). GR mediates response to dexamethasone for Cyp2C19 (CHEN et al. 2003; HUSS and KASPER 2000), and together with PXR also is needed for glucocorticoid induction of Cyp3A23 (HUSS and KASPER 2000).

Cyp2C9 is under the direct control of GR, and of PXR and CAR which activate the same response element

(GERBAL-CHALOIN et al. 2002; GERBAL-CHALOIN et al. 2001). VDR is able to interact with the PXR and CAR- response elements in Cyp2B6, Cyp2C9 and Cyp3A4 and activate the transcription of these genes

(DROCOURT et al. 2002).

1.3.2 Phenobarbital induction pathways in mammals

Phenobarbital was the main inducing agent used in this study, and will be discussed in greater detail.

Phenobarbital is a barbiturate widely used as a sedative and an anaesthetic to treat epilepsy and convulsions (KWAN and BRODIE 2004). Phenobarbital is considered one of the prototypical P450 inducers, and has been used widely to study the mechanisms of xenobiotic induction in mammals as a substitute for other pharmaceutical drugs. Phenobarbital induces 138 genes in wildtype mice (UEDA et al. 2002).

Phenobarbital also induces P450 expression in other organisms, and has been used to study P450 induction in microorganisms (LIANG et al. 1995), insects (WILLOUGHBY et al. 2006), reptiles (BANI et al.

1998) and fish (SMITH and WILSON 2010; WILLIAMS et al. 1998) amongst others.

1.3.2.1 Phenobarbital Induction enhancers

Several classes of enhancers that mediate phenobarbital induction have been characterised. The first sequences found to regulate phenobarbital induction were discovered in the bacterium Bacillus

38 megaterium (SHAW and FULCO 1992). These “Barbie box” sequences were found to regulate phenobarbital induction of the P450BM1 and P450BM2 genes (LIANG et al. 1995). Barbie boxes are repressive elements and bind the Bm3R1 repressing protein during a basal uninduced state, repressing transcription of target P450s. This repressive Barbie box sequence was found to overlap with another

Positive Element (PE) sequence, which conferred positive phenobarbital-induced transcriptional upregulation. The repression effect is alleviated when phenobarbital is introduced and the repressing

Bm3R1 protein is replaced by unknown positive-factors upregulating P450 expression (LIANG et al. 1995).

It appears that the two motifs regulate phenobarbital of target genes in a competitive binding scenario between the two motifs. Mutation of a conserved core AAAG sequence in Barbie boxes reduced affinity for Bm3R1 binding and alleviated normal repression, allowing 10-fold higher P450 expression under basal conditions (LIANG et al. 1995).

Putative Barbie boxes were also identified in vertebrate P450 promoter sequences. However, these sequences were not found to be as important for vertebrate CYP2B family phenobarbital induction as for B. megaterium induction, although the Positive element sequence has been found to have some phenobarbital induction activity (SUEYOSHI and NEGISHI 2001). Mammalian P450s have since been shown to use different enhancer sequences and the PXR/CAR Nuclear receptor regulatory pathways for phenobarbital induction.

1.3.2.2 CAR and CYP2B genes

Studies examining mice with their CAR gene deleted established CAR as the main regulator of phenobarbital induction in mice, with PXR regulating a smaller amount of the induction response

(KODAMA and NEGISHI 2006b; WEI et al. 2000). CYP2B phenobarbital induction is primarily regulated by

CAR. The CYP2B family rely on a cluster of PXR and CAR binding sites known as the phenobarbital- response module (PBREM) located in their promoter regions to regulate induction by phenobarbital

39

(KODAMA and NEGISHI 2006b) (Figure 1.7B). The PBREM was first mapped to a 176bp module in the

Cyp2B2 promoter (TROTTIER et al. 1995) before being reduced to a 51bp sequence in the Cyp2B10 gene

(HONKAKOSKI and NEGISHI 1997). Further investigations revealed two Direct Repeat binding sequences (DR repeats, where the repeat sequences are in the same orientation). The canonical sequence is 5′-TGACCT

((N)6 TGACCT-3′), termed the NR1 and NR2 motifs, which flank a nuclear-factor 1 site (HONKAKOSKI et al.

1998). Only the NR sites are essential for the phenobarbital response activity, although the NF1 site may be required to confer full PBREM activity. Singly mutating either of the DR-4 motifs in the 51bp PBREM leads to a reduction of phenobarbital induction, while mutating both sites resulting in the complete loss of induction, confirming that this region was necessary for phenobarbital induction (RAMSDEN et al.

1999).

Interestingly, a recent report found that CAR was also able to induce Cyp1A1 and Cyp1A2 (YOSHINARI et al. 2010). An ER-8 (Everted-repeat 8, where the repeat sequences are everted, in different orientations and separated by 8 nucleotides) CAR binding site was found adjacent to the XRE sites in the joint 50kb promoter region between Cyp1A1 and Cyp1A2 [Figure 1.7A]. Phenobarbital exposure induced low levels

of Cyp1A1 expression in wild type mice, and this induction was lost in CAR null mice (YOSHINARI et al.

2010). Taken together, these point to a well-co-ordinated and complicated xenobiotic detoxification response gene network in the liver.

1.3.2.3 PXR and CYP3A genes

Phenobarbital induction of the CYP3A family is mostly regulated by the PXR transcription factor using a different set of enhancers. Instead of a common PBREM module, in the CYP3A genes there are a variety of repeats including DR-3 repeats, Everted-6 repeats (ER-6, where the repeat sequences are everted and in different orientations) and DR-4 repeats (SUEYOSHI and NEGISHI 2001).

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The best characterised example of PXR CYP3A family regulation is the Cyp3A4 gene (Figure 1.7C).

Phenobarbital-induced regulation of Cyp3A4 has been comprehensively studied and the enhancer sites finely mapped. The proximal PXR responsive element (PXRE) contains an ER-6 PXR binding site located

150p upstream of the Cyp3A4 transcriptional start site (BOMBAIL et al. 2004). Promoter constructs comprised of only this binding site gave considerably less induction than the full length sequence when the inducer rifampicin was used (approximately 2-4 fold, compared to approximately 50 fold when the distal XREM is included) (OGG et al. 1999), suggesting that additional elements were important for full induction.

Further investigation found a cluster of NR binding sites located approximately 8kb away from the

Cyp3A4 transcriptional start site (GOODWIN et al. 1999). This cluster, called the Cyp3A4 distal XREM

(Xenobiotic Response Element Module), contains a DR-3 and an ER-6 binding site, as well as an HNF4α binding site. The XREM module alone was not able to drive induction, but placing the distal XREM directly 5’ of the PXRE produced much higher Cyp3A4 induction (approximately 50-fold), showing that these two sites were needed for maximum phenobarbital induction (GOODWIN et al. 1999). An additional

HNF4α binding site in the distal XREM module was also found to be necessary for maximum induction

(GOODWIN et al. 1999).

1.4 Insect P450 induction transcriptional regulation

As with mammals, the promoter regions of several xenobiotic-inducible insect P450s have been characterised and the locations of tissue-specific and xenobiotic induction enhancers have been mapped. Several transcription factors have been proposed to regulate insect xenobiotic induction, and will be discussed below.

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1.4.1 Papillio polyxenes P450 Induction

The best characterised insect P450 induction system is the black swallowtail butterfly Papillio polyxenes

Cyp6B1 gene. P. polyxenes caterpillars constantly encounter dietary toxins while feeding and consequently induce Cyp6B1 and Cyp6B3 to feed safely (COHEN et al. 1992; HUNG et al. 1995).

The cis-regulatory regions of the Cyp6B1 promoter have been well studied (Figure 1.8A). Peterson et al showed that the 380bp upstream region of Cyp6B1 was able to regulate induction to a variety of xenobiotic agents including Xanthotoxin (8.35X induction), Flavone (7.7X induction), Angelicin (6.52X induction) and Dexamethasone (1.92X induction) amongst others (PETERSEN et al. 2003). 5’ promoter constructs in Sf9 cells established that there were separate regions which controlled positive and negative expression of Cyp6B1. The -228 to -146 region was found to repress basal transcription of

Cyp6B1 promoter constructs by approximately 7-fold, while induction and basal expression was mediated by multiple elements within the first 147bp upstream of Cyp6B1 (PETERSEN et al. 2003).

The -137 to -118bp region upstream of Cyp6B1 contained an overlapping Ecdysone-response element/Antioxidant response element/Xanthotoxin-responsive element (EcRE/ARE/XRE-xan) (Figure

1.8A) (PETERSEN BROWN et al. 2004). Mutation of the shared four basepairs between all three constructs abolished all induced and basal expression, showing that this region was necessary for Cyp6B1 promoter expression. Further testing revealed that a central single G residue at position -135 which was shared by all three elements was necessary for basal and xanthotoxin-induced expression (PETERSEN BROWN et al.

2005).

Adding 20-hydroxyecdysone severely reduced basal and xanthotoxin-induced expression, showing that

Cyp6B1 was also under ecdysone control to some degree (PETERSEN BROWN et al. 2004). Mutation of the nucleotides unique to EcRE but not the ARE or XRE-xan in this element reduced the absolute levels of basal and xanthotoxin-induced expression but not the relative fold-change increase of xanthotoxin-

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Figure 1.8: P. polyxenes Cyp6B1 cis and trans -regulation. (A) A schematic image of the Cyp6B1 promoter and identified cis-regulatory regions. See text for details. (B) A model of Cyp6B1 regulation by transcription factors proposed by (PETERSEN BROWN et al. 2005). Basal and induced Cyp6B1 require the input of both the EcR/Are/XRE-xan and XRE-AhR binding sites. A cytosolic xanthotoxin induction- regulating transcription factor is activated by Xanthotoxin binding before entering the cell to bind to the

EcR/ARE/XRE-xan binding site to induce Cyp6B1. The Spineless-tango dimer is able to enter the cell and bind to the XRE-AhR site to effect basal expression. There may be possible interaction between the

Xanthotoxin-transcription factor and the Ss-Tgo dimer. Figure adapted from (PETERSEN BROWN et al.

2005).

43 induced expression, indicating that the EcRE element contributes to positive regulation of Cyp6B1

(PETERSEN et al. 2003).

Later work showed that other elements in the 5’ untranslated region, namely the -57 to +22 region, contributed to induction and basal expression. Bioinformatic analysis identified putative binding sites including an Aryl hydrocarbon receptor response element (XRE-AhR) at -57 to -48bp, an Octamer-1 binding site (Oct-1) from -47 to -38, a C/EBP binding site at -21 to -14 and an arthropod Initiator element

(Inr) at +2 to +8 (PETERSEN BROWN et al. 2004; PETERSEN BROWN et al. 2005; PETERSEN et al. 2003).

Mutagenesis experiments using a full length -146/+22 construct revealed that the XRE-AHR element was necessary for basal, xanthotoxin-induced and the AhR-specific xenobiotic ligand benzo[α]pyrene- induced expression (PETERSEN BROWN et al. 2005). The C/EBP binding site and the InR element were again found to be important for basal expression and xanthotoxin-induction, with mutation of these sites reducing expression to a level indistinguishable from controls (PETERSEN BROWN et al. 2004).

However, the putative Oct-1 binding site did not affect basal or induced expression when mutated, suggesting that this site was not involved in any Cyp6B1 regulatory activity (PETERSEN BROWN et al. 2005).

This highlights the importance of experimentally testing putative binding sites, and not relying on bioinformatic analyses alone to predict enhancer sites.

A systematic approach to investigating the -13 to +20 bp region was undertaken. Sequential mutagenesis of a series of six basepairs revealed a surprising degree of cis-regulation around the 5’- untranslated region. Mutating the +9/+15 region abolished all basal and induced expression and showed that this region was crucial for basal expression and xanthotoxin-induction (PETERSEN BROWN et al. 2004).

Mutating the -13 to -7 region increased xanthotoxin-induced expression significantly to a level even greater than wild-type -146/+22 constructs, suggesting that this region repressed xanthotoxin-induction

(PETERSEN BROWN et al. 2004). It is unclear how these regions control expression. It may be that they

44 contain enhancers for expression, or that they interacted with distal enhancer regions and are required to initiate transcription.

What are the transcription factors regulating Cyp6B1 expression?

To investigate the functionality of the XRE-AhR site in Cyp6B1, the Drosophila genes spineless (Ss), single-minded (Sim) and tango (Tgo) genes were co-transfected into Sf9 cells with the -146/+22 reporter construct to measure their influence on Cyp6B1 expression (PETERSEN BROWN et al. 2005). Spineless is the

Drosophila orthologue of AhR, while tango is the orthologue of the AhR nuclear transport protein ARNT.

Sim/Tgo did not alter either basal or xanthotoxin-induced or benzo[α]pyrene-induced expression, showing that Sim/Tgo did not regulate Cyp6B1 expression in Sf9 cells (PETERSEN BROWN et al. 2005).

Ss/Tgo co-transfection increased the level of xanthotoxin-induced expression by 1.7 fold, while increasing basal expression 2.6-fold. Benzo[α]pyrene-induced expression increased markedly to the same level as observed for xanthotoxin-induced expression in Ss/Tgo co-transfected cells. When Ss and

Tgo were individually transfected, both genes were able to increase basal expression on their own.

However, only Ss was able to increase xanthotoxin-induced expression when transfected individually.

Tgo was only able to induce higher xanthotoxin-induced expression when co-transfected with Ss

(PETERSEN BROWN et al. 2005). This suggested that in this case, Ss and Tgo operated differently to mammalian AhR/ARNT. It would seem that either Ss and Tgo operated independently to regulate expression on their own, or that Ss was able to utilise endogenous Sf9 Tgo-like partners to induce higher xanothoxin-induced expression (Figure 1.8B].

Taken together, these Cyp6B1 regulatory studies revealed an high degree of genetic control over basal and xenobiotic-induced expression, with multiple loci seemingly necessary for either positive or negative regulation of Cyp6B1. This was a level of complexity in cis-regulation had not be reported for an insect

P450 system prior to these studies, easily comparable to mammalian P450 induction systems. Strikingly,

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Cyp6B4 in the closely related P. glaucus species has also been reported to have the overlapping

EcRE/ARE/Xre-XAN motif (MCDONNELL et al. 2004). This suggests that this mechanism for xanthotoxin detoxification has been conserved since these two species diverged.

1.4.2 D. melanogaster xenobiotic induction

Xenobiotic induction in D. melanogaster has been primarily studied using two techniques. Microarray experiments carried out by several groups on xenobiotic-exposed larvae and adult flies have been used to detect and generate lists of induced P450s. The second technique has been to use promoter constructs and transgenic flies to study the cis-regulatory architecture of selected P450 genes to identify promoter regions controlling xenobiotic induction and to characterise transcription factor binding sites.

1.4.2.1 D. melanogaster xenobiotic induction microarray studies

The use of microarrays to investigate xenobiotic induction has allowed the rapid discovery of large numbers of genes inducible by xenobiotics (Table 1.4). This has been especially useful in D. melanogaster P450 research and has contributed to our knowledge of Drosophila P450s. An excellent summary of these studies can be found in (GIRAUDO et al. 2010).

Microarrays probing phenobarbital-induced expression

Phenobarbital has been so far the most common inducing agent used in D. melanogaster, with four groups having reported studies from phenobarbital arrays. Phenobarbital induction in D. melanogaster activated global changes in gene transcription. The Canton-S strain was used by King-Jones and colleagues to investigate phenobarbital induction (KING-JONES et al. 2006). Using whole genome arrays,

503 genes were found to be upregulated in total, with 29 P450s among them. 484 other genes were also downregulated in the phenobarbital-challenged samples compared to control unexposed flies.

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Willoughby et al. examined the genetic response to phenobarbital in the y; cn bw sp strain using a custom “detox chip” microarray slide containing only P450, GST and esterase genes (WILLOUGHBY et al.

2006). 3rd instar y; cn bw sp larvae upregulated 21 P450 genes while adult male y; cn bw sp flies only upregulated 10 P450s, showing distinct differences in phenobarbital induction between lifestages (Table

1.4). There was an overlap of 10 P450s and 4 GSTs being induced by phenobarbital in both larvae and adults, suggesting that these P450s possibly had shared functions at larval and adult stages.

A separate study identified eleven P450s which were transcriptionally responsive to phenobarbital induction. Using a detox microarray chip similar to Willoughby et al., Le Goff and co-workers induced adult male and female OregonR flies and found sex-specific differences in induced P450 genes (LE GOFF et al. 2006). Male flies induced seven P450s while females induced twelve P450s, inclusive of the five induced in males. P450 sex-specific expression differences have already been reported in other studies

(KASAI and TOMITA 2002), and it appears that P450 xenobiotic induction in D. melanogaster is under a certain degree of sex-specific control.

Sun et al. investigated 3rd instar w1118 larvae for phenobarbital-induced genes. They detected six P450 genes upregulated by phenobarbital using a whole genome array and confirmed that another gene

(Cyp6g1) was transcriptionally upregulated by quantitative real time PCR analysis (SUN et al. 2006).

It is obvious from these datasets that a wide range of factors play an important role in P450 xenobiotic induction. Xenobiotic induction has been previously found to be background-specific (TERRIERE and YU

1974; HALLSTROM et al. 1982). The differences in numbers of induced P450s seen in these papers confirm this, and seem to reflect the variances in induction protocol, gender, lifestage tested and fly strain used.

For instance, Cyp6a8 was induced 677-fold in adult CantonS flies (KING-JONES et al. 2006), but only 21- fold in adult male y; cn bw sp flies (WILLOUGHBY et al. 2006), and not at all in male and female OregonR flies (LE GOFF et al. 2006). While this striking difference may be attributed partly to different

47 experimental protocols, such as the concentration of the inducing chemical and the length of the exposure, multiple lines should be investigated before any final conclusions drawn about inducer- specific responding P450.

Despite this, several common P450s were found to be upregulated in all four studies (Table 1.4). This suggests that these genes are genuinely phenobarbital-inducible, and possibly are involved in some direct detoxification response of phenobarbital. Appropriately, these genes are expressed in the digestive tissues where the majority of detoxification takes place (CHUNG et al. 2009).

Transcriptional responses to other xenobiotic chemicals

The response to several other inducing agents has been investigated using microarrays (Table 1.4).

Based on investigations using multiple inducers, it appears that P450 induction was inducer-specific.

Different inducers appeared to induce their own pattern of P450 genes, usually comprised of a common battery of P450s which responded to any xenobiotic challenge, along with other more selective P450s which were only induced by specific chemicals. Again though, multiple lines should be investigated before any firm conclusions drawn about lists of induced P450s.

Insecticides do not induce most P450s

One surprising result was that most insecticides did not induce any D. melanogaster P450 expression.

Willoughby et al. tested six different insecticides, and only DDT was found to induce a slight transcriptional response (WILLOUGHBY et al. 2006). Even so, only two genes (Cyp12d1 and GSTD2) were found to respond. Known DDT resistance genes such as Cyp6g1 and Cyp6a2, with the exception of

Cyp12d1, were not induced, contrary to the theory that P450s are induced by their substrates.

Moreover, while Cyp6a8 did not confer resistance to DDT when transgenically overexpressed

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Table 1.4: Drosophila P450 Microarray induction

Piper 1 2 3 Piperonyl 7 8 9 10 11 Phenobarbital Atrazine Caffeine nigrum 5 Pyrethrum Ethanol DDT Paraquat Tunicamycin Neem Neem 4 Butoxide Pyrethrum6 extract oil12 oil12 Cyp4d4 Cyp6a2 Cyp4ae1 Cyp6a8 Cyp4ae1 Cyp305a1 Cyp9f2 Cyp6a8 Cyp12d1 Cyp4ac3 Cyp4e1 Cyp12a4 Cyp4d20

Cyp4e2 Cyp6d5 Cyp4d14 Cyp6d4 Cyp4e2 Cyp12a4 Cyp12d1 Cyp4p1 Cyp4p1 Cyp12d1 Cyp28d1

Cyp6a2 Cyp6g1 Cyp4e2 Cyp6d5 Cyp4p1 Cyp12d1 Cyp6a13 Cyp6a23 Cyp4s3 Cyp6a2

Cyp6a8 Cyp6w1 Cyp6a2 Cyp9b2 Cyp6a2 Cyp4s3 Cyp6a22 Cyp6d4 Cyp9f2 Cyp314a1

Cup6a17 Cyp12d1 Cyp6a8 Cyp12d1 Cyp6a8 Cyp4e3 Cyp6a23 Cyp9b2 Cyp4e2 Cyp309a1

Cyp6a23 Cyp304a1 Cyp6a21 Cyp6a21 Cyp28d1 Cyp6d4 CYp28a5 Cyp4e3 Cyp6w1

Cyp6d5 Cyp6d4 Cyp6a23 Cyp4p1 Cyp6d5 Cyp313a1 Cyp6a13 Cyp12e1

Cyp6g1 Cyp6d5 Cyp6d5 Cyp4p2 Cyp28a5 Cyp28a5 Cyp18a1

Cyp6w1 Cyp6g1 Cyp6w1 Cyp314a1 Cyp309a1 Cyp9b2 Cyp6a17

Cyp9b2 Cyp6w1 Cyp12b2 Cyp12e1 Cyp6a21 Cyp6a9

Cyp12d1 Cyp12d1 Cyp12c1 Cyp18a1 Cyp6g1 Cyp4ac3

Cyp12d1 Cyp6a17 Cyp4p2 Cyp6a20

Cyp6a9 Cyp6a14 Cyp4c3

Cyp4ac3 Cyp4p1 Cyp6a23

Cyp6a20 Cyp6d4

Cyp6a23 Cyp9b1

Cyp12a5 Cyp12a5 1: 6 datasets, adult male, female and larval stages tested; 2: 2 data sets, Adult Male and Female lifestages tested; 3: 1 dataset, Larval lifestage tested; 4: 1 dataset, Adult Female lifestage tested; 5: 2 Datasets, Male lifestage tested; 6: 1 dataset, larvae lifestage; 7: 1 dataset, Adult Female lifestage tested; 8: 1 dataset, Adult Male lifestage tested; 9: 1 dataset, Adult Male lifestage tested; 10: 2 datasets, Adult Male lifestage tested;

11: 1 dataset, Adult Male lifestage tested, 12: 1 dataset, larvae lifestage tested. Table adapted from (GIRAUDO et al. 2010).

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(DABORN et al. 2007), it was found to be induced 3-fold when 5’ promoter constructs were exposed to

DDT in a SL-2 cell assay (MORRA et al. 2010). It appears that there is no link between induction and insecticide resistance in D. melanogaster, and that induction may not accurately predict resistance. This agrees with previous findings that most P450-mediated resistance is mostly due to constitutively overexpressed P450s.

The speed of induction also may be a factor in determining if induction plays an important role in insecticide resistance. A peak in phenobarbital-induced induction was only observed after four hours of exposure (WILLOUGHBY et al. 2006), whereas insecticides can kill their targets within minutes. It may be that induction may simply be too slow to respond to lethal doses of insecticides.

However, other studies have reported P450s from non- D. melanogaster species responding to insecticide induction. Fifth instar silkworm larvae (Bombyx mori) induced several P450s in response to diazinon and imidacloprid (YAMAMOTO et al. 2010). Larvae of the mosquito Aedes aegypti have also been shown to induce various P450s when exposed to temephos and fluorenthane (POUPARDIN et al. 2008).

This may reflect divergences in xenobiotic response pathways, and that D. melanogaster have lost certain components needed to respond to insecticide challenge.

1.4.2.2 Promoter sequence analysis

The cis-transcriptional regulation of three D. melanogaster P450 genes has been reported (Figure 1.9).

Cyp6a2, Cyp6a8 and Cyp6g1 have been studied using nested promoter region deletions to identify promoter regions controlling xenobiotic inductions.

Cyp6a2 Regulation

Cyp6a2 expression and regulation has been extensively studied by several groups. 3rd instar larvae express basal Cyp6a2 in the Midgut (M1-M3 and M13 sections) and Malpighian tubules

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Figure 1.9: cis-regulatory regions of D. melanogaster phenobarbital-inducible genes. (A) Cyp6a2 (B)

Cyp6a8 (C) Cyp6g1. Refer to text for details.

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(CHUNG et al. 2009). In adults, basal Cyp6a2 expression is found in the hemocytes, male accessory glands, adult fat body, intestinal wall, central and peripheral nervous system sensory cells and their axons, adult brain neuropile and optic lobe lamina (GIRAUDO et al. 2010).

Cyp6a2 is inducible by multiple xenobiotics including phenobarbital (BRUN et al. 1996; DUNKOV et al.

1997; WILLOUGHBY et al. 2006), caffeine (BHASKARA et al. 2008; BHASKARA et al. 2006; WILLOUGHBY et al.

2006), polychlorobiphenyl mixture (PCB), Aroclor 1254 (GIRAUDO et al. 2010). Cyp6a2 is induced by phenobarbital in larval 3rd instar Malpighian tubules, muscle fibres, weak larval brain and ganglion, and heart tissues (GIRAUDO et al. 2010).

Cyp6a2 native expression

5’ promoter construct experiments established that Cyp6a2 expression was controlled by enhancers in the 5’ region of Cyp6a2 (Figure 1.9A). Dunkov and co-workers transfected Drosophila SL-2 cell lines with the 5’ promoter sequence linked to a luciferase reporter gene and found the first 428bp sufficient to drive Cyp6a2 native expression, but additional elements found further upstream were required to give higher basal expression (DUNKOV et al. 1997).

Cyp6a2 promoter constructs xenobiotic induction

Dunkov found that phenobarbital induction was regulated by the -428bp promoter region (DUNKOV et al.

1997). Drombrowski (1998) used transgenic 5’ promoter construct flies to show that the first 129 basepairs upstream of the start codon did not contribute to phenobarbital induction, whereas 3.5-fold induction was still achieved with -985bp and full length Cyp6a2 upstream region -1335bp constructs

(DOMBROWSKI et al. 1998). This served to narrow the phenobarbital inducible region to the -129 to -

428bp sequence (Figure 1.9A).

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Caffeine induction was also found to be regulated by the same sequences that regulated phenobarbital induction (BHASKARA et al. 2006), although it is unclear if the Cyp6a2 PBRE also regulates caffeine induction. 5’ promoter constructs were transfected into Drosophila SL-2 cells and exposed to caffeine

(BHASKARA et al. 2006). Luciferase quantifications indicated that sequences from -120 up to -265bp were able to regulate caffeine induction and induce 4-fold luciferase expression. Unexpectedly, the -520bp construct showed less induction than the -265bp construct with only 2-fold induction, indicating that the additional 260 basepairs reduced the level of induction and suggesting the presence of a repressive element in this region. The largest construct tested (-980bp/-1bp) showed the greatest level of induction at six-fold induction, indicating that there were other sequences upstream that were able to alleviate the repression found in the -265 to -520bp region.

Cyp6a8 regulation

rd Cyp6a8 is natively expressed in 3 instar Malpighian tubules (CHUNG et al. 2009). Cyp6a8 is induced by several xenobiotics including phenobarbital, DDT and caffeine (KING-JONES et al. 2006; WILLOUGHBY et al.

2006); (BHASKARA et al. 2006; MAITRA et al. 2002). Adult females expressed caffeine–induced luciferase expression in the head, ovaries, gut, cuticle plus fat body and Malpighian tubules (BHASKARA et al. 2006).

Cyp6a8 basal expression in SL-2 cells and adult transgenic flies is controlled by elements within the -

199/-761bp (Figure 1.9B) (BHASKARA et al. 2006; MAITRA et al. 2002). Phenobarbital, caffeine and DDT induction was found to be regulated by the -109/-199bp region (Figure 1.9B) (MORRA et al. 2010). The -

199/-761bp was found to contain repressive sequences that reduced the magnitude of caffeine induction (MORRA et al. 2010). However, no attempt at further defining this region to identify which sequences directly regulate induction to these three chemicals was reported, and thus it is unclear which sequences specifically regulate xenobiotic-induced and basal expression.

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Putative AP-1, Barbie box, CREB, CREB-P, Broad-complex and Oct-1 binding sites were identified using a bioinformatic screen (MAITRA et al. 2002). The functionality of the AP-1 site was partially addressed through manipulating Jun protein levels which suggested Jun proteins contributed to the regulation of caffeine induction (BHASKARA et al. 2008). The functionality of the rest of the proposed sites was not investigated, and therefore it cannot be concluded if these sites play any roles in Cyp6a8 regulation.

Cyp6g1 regulation

The Cyp6g1 regulatory region was also investigated to identify specific tissue enhancers (Figure 1.9C)

(CHUNG et al., SUBMITTED). Two separate and distinct tissue specific enhancer blocks were found.

Malpighian tubule expression was controlled by an enhancer located in 300bp of the 5’ region while midgut expression was regulated by a 250bp region in the first intron of Cyp6g1 (CHUNG et al.,

SUBMITTED).

Further analysis found that the midgut expression-regulating region contained three putative GATA factor binding sites (CHUNG et al., SUBMITTED). Site directed mutagenesis experiments found that while mutating one of these sites did not affect expression, mutating a pair of overlapping GATA sites disrupted midgut and fat body expression. Feeding 3rd instar larvae lost midgut and fat body expression, while wandering 3rd instar larvae only lost fat body expression. This showed that this overlapping GATA binding site was necessary for midgut and fat body expression, and suggested that GATA transcription factors are crucial for Cyp6g1 midgut and fat body expression (CHUNG et al., SUBMITTED).

Cyp6g1 shows 2-4 fold induction upon phenobarbital exposure (WILLOUGHBY et al. 2006). 5’ promoter constructs were exposed to phenobarbital and a 149bp element (-1048 to -898bp) was found to regulate Cyp6g1 induction by phenobarbital (Figure 1.9C) (CHUNG et al., SUBMITTED). The Cyp6g1 PBREM element was found to be adjacent to the Malpighian tubule enhancer and is able to direct induction independently when attached to a HSP70 TATA box without any Cyp6g1 elements (CHUNG et al.,

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SUBMITTED). Several putative phenobarbital-regulating motifs were identified in this region based on sequence analysis and homology to known PXR/CAR binding sites, and these sites were mutated using site directed mutagenesis to test for functionality.

Two DR sequences were mutated with no effect on phenobarbital-induced expression being seen, suggesting that these two sites did not play any role in phenobarbital induction of Cyp6g1. However, the mutation of another Cyp6g1 sequence in this region, which was homologous to the PBREM NR1 sequence, reduced Cyp6g1 induction (CHUNG et al., SUBMITTED). This showed that this site was important for attenuation of the Cyp6g1 transcriptional response to phenobarbital, and suggested the presence of other phenobarbital-inducing sequences in this region. It remains unknown where they lie and what proteins bind to them to activate higher Cyp6g1 transcription.

1.4.2.3 Drosophila Induction Regulatory Proteins

Two D. melanogaster transcription factors have been found to regulate P450 phenobarbital induction,

HR96 and Broad-complex. HR96 is the closest D. melanogaster orthologue to PXR and CAR (KING-JONES et al. 2006) . HR96 was reported to regulate phenobarbital response in D. melanogaster (HORNER et al.

2009; KING-JONES et al. 2006).

A HR96 null mutant was created through ends-in targeted gene knockout and exposed to phenobarbital

(HORNER et al. 2009; KING-JONES et al. 2006). Comparative microarray analysis against wild-type CantonS flies showed that only half of all induced genes in the WT flies are still inducible in the HR96 mutant, suggesting that HR96 regulates half of all phenobarbital -induced genes. Closer examination of these genes has revealed that most of the genes affected are involved in energy homeostasis.

Only five P450s were no longer induced by phenobarbital in the HR96 null mutant. Cyp9h1, Cyp4s3,

Cyp49a1, Cyp6a14 and Cyp309a2 failed to respond to phenobarbital induction in the HR96 null mutant, suggesting an alternative regulatory mechanism for phenobarbital induction for other P450s.

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The majority of the phenobarbital-responsive P450s are still induced in the phenobarbital-exposed HR96 mutant, implying that HR96 is not the main transcription factor regulating phenobarbital induction

(HORNER et al. 2009; KING-JONES et al. 2006). PXR and CAR null mice still continue to induce at least half of their normal gene response (UEDA et al. 2002), suggesting that other regulatory mechanisms compensate for the loss of these genes. Because of this, other transcription factors could be involved in the response to phenobarbital induction. To date, these additional regulatory proteins have not been conclusively identified.

The HR96 ligand-binding domain was able to bind the CAR-specific ligand 6-(4-

Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO), but not the CAR-specific ligand 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or the PXR-specific ligand pregnenolone 16α-carbonitrile (PCN) (PALANKER et al. 2006). This indicated that while HR96 was able to recognise some CAR-specific ligands, it was not able to recognise others and suggested that HR96 had different substrate specificity to CAR and PXR. Unfortunately, Palanker and co-workers did not report if the HR96 ligand-binding domain was able to recognise phenobarbital.

HR96 has since been shown to be involved in maintaining dietary lipid levels by monitoring and regulating cholesterol and triacylglyceride homeostasis, in part at least through regulation of genes necessary for cholesterol homeostasis (BUJOLD et al. 2010; HORNER et al. 2009; SIEBER and THUMMEL 2009).

This finding agrees with reports that the mammalian orthologues PXR and CAR are also involved in nutritional monitoring (WADA et al. 2009), again showing that these functions are conserved between mammals and insects. This finding also suggests that the five P450s affected by the loss of HR96 are involved in lipid metabolism events.

It is worth noting that while King-Jones et al. found that flies still survived to adulthood after knocking out HR96, Giraudo et al. reported that targeted knockdown of HR96 using RNAi resulted in

56 developmental lethality (KING-JONES et al. 2006); (GIRAUDO et al. 2010). This striking difference between these reports casts some confusion upon King-Jones et al.’s findings. If the knockout is an incomplete one and low levels of HR96 expression is still present, this might be sufficient to ensure that the majority of P450s are still induced in the HR96 null mutant. However, King-Jones et al. failed to detect any HR96 protein in the null mutant using a HR96 antibody, providing strong evidence for this line being a complete null mutant. It is also possible that the RNAi construct may have unknown off-target effects which knocked down other important gene(s) and resulted in developmental lethality.

Do HR96 and Broad-complex interact together to regulate xenobiotic expression?

Studies into transfected Musca domestica Cyp6D1 promoter regions in D. melanogaster SL-2 cells found that HR96 and Broad-complex regulated phenobarbital induction (LIN et al. 2011). dsRNAi experiments knocking down HR96 and Broad-complex established that HR96 acted as a positive regulator of induction as phenobarbital induction magnitude decreased after RNAi treatment. Conversely, Broad- complex RNAi resulted in an amplification of phenobarbital induction, showing that Broad-complex negatively regulated induction. However, no evidence for the direct binding of Broad-complex or HR96 was presented. It may well be possible that these genes operate further upstream, and do not directly contact DNA and upregulate Cyp6D1. Furthermore, this was an in vitro experiment involving components from multiple species, and hence it is unclear if this finding was biologically relevant in vivo in D. melanogaster.

Is SL-2 cell culture appropriate for studying Drosophila induction?

Several papers have used SL-2 cell culture to investigate insect P450 induction. While cell culture assays offer considerable benefits, such as considerably easier experimental protocols and a standardised genetic background line across different labs, it must be noted that SL-2 cells were derived from embryonic D. melanogaster cells and consequently has a different expression profile to D. melanogaster

57 digestive tract cells (NORGATE et al. 2007). Tissue-specific spatial expression as well as developmentally regulated expression also cannot be determined using cell culture.

For instance, Cyp12d1 was found to be only induced 3-fold in SL-2 cells by phenobarbital, while 3rd instar larvae induced 24-fold higher expression (Adam Southern, personal communication). This suggests that SL-2 cell culture induction may lack induction components present in 3rd instar larva.

Interestingly, both Cyp6g1 Accord+ and Accord- promoters did not show any induction in SL-2 cells when

rd exposed to phenobarbital (MORRA et al. 2010). 3 instar larval phenobarbital induction enhancers have already shown to be present in this upstream Cyp6g1 region (CHUNG et al., SUBMITTED), and this difference in results further indicates that SL-2 cells are not the best system to study induction in D. melanogaster. Using transgenic flies expressing reporter constructs allows for accurate demarcation of induction enhancers in a biologically relevant context and also permits the detection of tissue-specific expression patterns.

1.5 Cyp12d1 induction as a tool to study D. melanogaster xenobiotic induction

The model organism D. melanogaster is used to study xenobiotic induction in insects. D. melanogaster offers many advantages to investigating xenobiotic induction compared to other insect species, such as a relatively easy in vivo approach to studying gene regulation in whole animals with the right biological context, simple rearing conditions compared to other insects, a sequenced genome, short generation time, and highly conserved regulatory mechanisms with other metazoan species that ensures that results generated can be compared directly to other organisms.

Our understanding of D. melanogaster xenobiotic induction has grown greatly in recent years. Some D. melanogaster P450s have already been identified, with promoter regulatory elements known. Some

58 transcription factors that regulate xenobiotic induction have been identified, but large gaps remain in our knowledge still and there is more to discover and learn.

The D. melanogaster gene Cyp12d1 stands out as the most xenobiotic inducible P450 in the Drosophila genome, appearing in most lists of induced genes to date regardless of inducing agent, strain or lifestage tested (Table 1.5). Cyp12d1 also consistently ranked as one of the highest responding P450s when expression fold-change is compared across studies. Cyp12d1 was also one of the few Drosophila P450s known to be DDT-inducible (FESTUCCI-BUSELLI et al. 2005; WILLOUGHBY et al. 2006). This DDT induction effect was noted in several lines by microarray and Northern blot experiments, showing that this induction was not line specific and supporting that it was a genuine effect. These findings point to

Cyp12d1 being extremely inducible by xenobiotics, and that Cyp12d1 has essential functions that D. melanogaster requires after xenobiotic exposure.

Based on this, it was reasoned that Cyp12d1 is an excellent gene to use to investigate xenobiotic induction in Drosophila melanogaster. Since Cyp12d1 is such a highly inducible gene, characterisation of

Cyp12d1 regulation will allow us to characterise transcription factors regulating xenobiotic induction in

D. melanogaster.

1.6 Aims for this Thesis

The aims of this thesis were to identify new xenobiotic regulatory pathways in D. melanogaster.

Cyp12d1 regulation was investigated by identifying phenobarbital enhancers, and subsequently using these to isolate transcription factors that bound to them and regulated Cyp12d1 phenobarbital induction. Knowledge of these proteins would then allow the characterisation of more D. melanogaster induction pathways. Cyp12d1 function was studied to identify novel Cyp12d1 functions, and perhaps

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Table 1.5: Cyp12d1 induction by various chemicals

Fold- Reference Inducer Strain Lifestage tested Rank Change King-Jones et al 2006 phenobarbital 14.3 CantonS Adult flies 55 out of 988 Willoughby et al 2006 phenobarbital 30.9 y cn bw sp 3rd instar larvae 1 Willoughby et al 2006 phenobarbital 16.2 y cn bw sp Adult flies 3 1118 Sun et al 2006 phenobarbital 32.67 w 3rd instar larvae ? Le Goff et al 2006 phenobarbital 9.75 OregonR Female Adult flies 1

Le Goff et al 2006 phenobarbital 1.72 OregonR Male Adult flies 4 Willoughby et al 2006 DDT 3 y cn bw sp 3rd instar larvae 1 Willoughby et al 2006 Caffeine 11.4 y cn bw sp 3rd instar larvae 2 Kee et al (Unpublished results) Caffeine 30.6 y cn bw sp 3rd instar larvae 4 Jensen et al 2005 piper nigrum 2.51 OregonR Female Adult flies 3

Jensen et al 2006 Pyrethrum 1.53 OregonR Female Adult flies 4 Kee et al (Unpublished results) Pyrethrum 10.1 y cn bw sp 3rd instar larvae 7 Le Goff et al 2006 Atrazine 4.56 OregonR Male Adult flies 1 Le Goff et al 2006 Atrazine 2.56 OregonR Female Adult flies 1 Kee et al (Unpublished results) Neem oil 24.8 y cn bw sp 3rd instar larvae 7 Willoughby et al 2007 Piperonyl butoxide 18.6 y cn bw sp Male Adult flies 3 Chahine and O’Donnell 2011 Piperonyl butoxide 4 OregonR Adult flies 1st out of 4 genes tested Chahine and O’Donnell 2011 MTX 18 OregonR Adult flies 1st out of 4 genes tested

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better understand why Cyp12d1 is so inducible. Cyp12d1 was overexpressed or Cyp12d1 expression was ubiquitously reduced using the UAS-GAL4 system, and the phenotypes of these mutants studied.

Cyp12d1 duplication was also investigated in this thesis. Cyp12d1 has been tandomly duplicated in certain lines to give two copies of Cyp12d1. A survey of the duplication frequency was performed in flies collected from different locations along the Australian east coast. Cyp12d1 mRNA expression was also studied in multiple laboratory and natural population lines to look for possible expression differences between single copy and double copy lines.

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Chapter 2: Transcriptional Regulation of Cyp12d1

The great tragedy of Science -- the slaying of a beautiful hypothesis by an ugly fact.

- Thomas Henry Huxley

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2.1 Introduction

Organisms have evolved many different defence systems against toxic compounds (MEYER 2007). The ability to detoxify foreign compounds (xenobiotics), in part, comes from the increased production of detoxification enzymes, which include members of the cytochrome P450 (P450) family (LEWIS et al.

1998; NEBERT et al. 1989). Although some P450 induction pathways in mammals are well defined (refer to Section 1.3.1), the transcriptional induction of P450 genes in response to xenobiotics in insects is less well understood. Drosophila melanogaster has been used to study the pathways and transcription factors involved in xenobiotic responses in insects. The transcription factor HR96 has been shown to regulate the induction of some P450s in response to phenobarbital (KING-JONES et al. 2006). However, out of the 29 phenobarbital inducible P450s, only five P450s were found to be induced in a HR96 dependent fashion, with the majority of phenobarbital-inducible P450s continuing to be induced in a

HR96 null mutant strain. This suggested that other unknown factors were also involved in mediating response to phenobarbital.

2.1.1 Cyp12d1 regulation

The D. melanogaster cytochrome P450 gene Cyp12d1 is highly inducible by many xenobiotic chemicals.

Cyp12d1 is transcriptionally induced approximately 24-30 fold upon exposure to phenobarbital (KING-

JONES et al. 2006; LE GOFF et al. 2006; WILLOUGHBY et al. 2006). Studying how Cyp12d1 is induced by phenobarbital may help in understanding induction responses and mechanisms of induction responses to xenobiotics in general.

The D. melanogaster Cyp12d1 gene is located on Chromosome 2R, at cytological location 54D. This chapter will concentrate on the transcriptional regulation of Cyp12d1 in the w1118 strain, which has a single copy of Cyp12d1. Cyp12d1 induction was first noted when strains exposed to DDT showed stronger Cyp12d1 expression than unexposed flies (BRANDT et al. 2002). Since then, other studies have

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found that Cyp12d1 is arguably the most inducible Drosophila P450 gene as it is induced by a wide range of chemicals. It was reasoned that the regulatory region of Cyp12d1 would thus contain most if not all of the elements required for xenobiotic induction of this gene. Characterising the Cyp12d1 regulatory region and discovering the factors regulating Cyp12d1 induction would allow the gene networks regulating Cyp12d1 induction to be determined.

2.1.2 Chapter aims

This chapter investigates the transcriptional regulation of Cyp12d1. The upstream and downstream

Cyp12d1 promoter region were investigated to find enhancer regions that controlled native tissue enhancers and xenobiotic inducible enhancers, to understand the transcriptional mechanisms that regulate Cyp12d1 native and induced expression. These enhancers were then used to identify protein binding partners in order to discover regulatory proteins that controlled Cyp12d1 induction, and ultimately Drosophila induction. Characterising these binding partners may also allow the discovery of other P450 induction regulatory pathways.

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2.2 Materials and Methods

2.2.1 Fly rearing conditions

Flies were reared at 25°C in constant light. Lines used in this study are listed in Appendix I.

2.2.2 Reporter constructs

All promoter constructs were constructed using the pStinger or pH-Stinger vectors (Figure 2.1,

1118 (BAROLO et al. 2000)). Promoter fragments were amplified from w genomic DNA using the BD

Advantage 2 Polymerase kit (BD Biosciences). All forward primers used were designed to include the

BglII restriction site, while all reverse primers incorporated the XbaI restriction site. Primers used are listed in Appendix II. Amplified PCR products were first cloned into the pGEM-Teasy vector

(Promega) and sequenced to check for correct sequence. Inserts were then digested using BglII and

XbaI (Promega) and then ligated into BglII and XbaI pre-digested linear pStinger or pH-Stinger vector using the 2X LigaFast system (Promega). Clones were again sequenced to check correct orientation and sequence.

-9653/-2932bp pH-Stinger construct generation

The large size of the Cyp12d1 upstream region (approximately 10kb) to the next gene, BBS4, prohibited the use of PCR to amplify the entire 5’ region due to the possibility of incorporating numerous PCR errors. Instead, the Bacterial Artificial Chromosome (BAC) clone 98RP-13D20 which contains the Cyp12d1 locus region was obtained from the Drosophila Genome Resource Centre

(DGRC). Two BglII sites located in the Cyp12d1 5’ region at bases -9948 and -2931 allowed the isolation of the -9948 to -2931 genomic region (“-9653/-2932bp”) which was cloned into the pH- stinger vector.

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Figure 2.1 GFP reporter vectors used in this study. The pStinger vector was used for 5’ constructs with a Cyp12d1 TATA box. The pH-Stinger construct was used for constructs without an endogenous

TATA box. Both vectors contain the Su(Hw) insulators (marked with an ‘I’) which theoretically reduce position effects from nearby enhancers. There is a SpeI cut site located behind the eGFP gene.

Combined 5’and 3’ constructs used a pStinger vector with a 5’ construct cloned in the multi-cloning site (MCS) and a 3’ fragment cloned in this SpeI site. Note that these vectors have the nuclear eGFP gene, where a nuclear localization signal has been cloned behind the eGFP gene, meaning that GFP is visualised as discreet dots representing the nucleus of each cell.

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-168/-1bp pStinger +1917/+2207bp SpeI construct generation

The downstream +1917/2207bp sequence was cloned into the SpeI site downstream of the eGFP gene in the-168/-1bp pStinger to create the -168/-1bp pStinger +1917/+2207bp SpeI (Figure 2.1). To achieve this, The +1917/2207bp sequence was amplified with primers incorporating the SpeI recognition cut site (+9175bp FspeI and +2207bp RspeI primers; see Appendix II for primer sequences). Amplified PCR products were first cloned into the pGEM-Teasy vector (Promega) and sequenced to check for the correct sequence. Inserts were then digested using SpeI (Promega) and then ligated into SpeI pre-digested linear -168/-1bp pStinger vector using the 2X LigaFast system

(Promega) to create the -168/-1bp pStinger +1917/+2207bp SpeI. Clones were again sequenced to check correct orientation and sequence.

-288/-168bp GATA mutant pH-Stinger

The canonical GATA binding site in the -288/-168bp region was mutated using site directed mutagenesis to generate the -288/-168bp GATA mutant pH-Stinger. The -288/168bp region was amplified from w1118 genomic DNA using -288bp FBglII and -168bp RXbaI primers (Appendix II) with the BD Advantage 2 Polymerase kit (BD Biosciences). Amplified PCR products were cloned into the pGEM-Teasy vector (Promega) and sequenced to check for correct sequence. The GATA binding site was then mutated using the QuikChange II Site-directed Mutagenesis Kit (Stratagene) (GATA mutant

F and GATA mutant R, Appendix I). Inserts were then digested using BglII and XbaI (Promega) and then ligated into BglII and XbaI pre-digested linear pStinger or pH-Stinger vector using the 2X

LigaFast system (Promega).

2.2.3 Transgenic Drosophila line generation pStinger and pH-Stinger constructs (0.3 μg/μl) were microinjected into <1 hour old w1118 embryos with a Δ2-3 transposase source (0.1 μg/μl) as per standard procedures (RUBIN and SPRADLING

1982; SPRADLING and RUBIN 1982) to generate transgenic Drosophila lines. Transformed lines were

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identified through expression of the w+ gene and made homozygous using the If/CyO;

TM6b,Tb/MKRS (Gift from Gary Hime, The University of Melbourne).

2.2.4 GFP expression and Image capture

At least two independent homozygous viable P-element insertion lines were examined for each construct, with a minimum of 5-6 larvae examined over multiple experiments. All GFP images were captured using the SZX12 stereomicroscope system (Olympus).

2.2.5 Exposure of larvae to Xenobiotics

rd The exposure protocol described in Willoughby et al. was used (WILLOUGHBY et al. 2006). Briefly, 3 instar larvae were collected at 108 hours after embryo laying and placed on standard flyfood agar plates supplemented with 10mM of phenobarbital (Sigma) or 15mg/ml Caffeine (Sigma). Larvae were left to feed for 4 hours at 25C before three replicate biological samples consisting of 10 larvae each were collected from xenobiotic-exposed and unexposed samples.

Malpighian tubule dissection

Phenobarbital exposed and unexposed -288/1168bp pStinger and -288/1168bp GATA mutant pStinger larvae were dissected in phosphate buffered saline and Malpighian tubules were removed, taking care to avoid including any midgut or fat body tissue. Tubules were snap frozen immediately on dry ice.

RNA extraction and cDNA synthesis

RNA extracted using Trizol Reagent (Invitrogen) and quantified using the Qubit Quantification

Platform (Invitrogen). The Superscript II Reverse Transcriptase system (Invitrogen) was used to make cDNA from 1µg of RNA.

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Real-time PCR

Real-time PCR (RT-PCR) was performed using the QuantiTect SYBR Green PCR kit (Qiagen) on a

RotorGene-3000 (Corbett Research, Sydney) to quantify the amount of GFP and RPL11 transcript produced. Annealing temperature was set at 55°C and runs set to 45 cycles. Three biological replicates were analysed. The amount of GFP and the housekeeping gene RPL11 mRNA was quantified using the Standard curve method of quantification with the Corbett RotorGene-3000

Realtime analysis program (Corbett Research, Sydney). GFP mRNA expression was normalised to the

RPL11 expression levels for comparison between induced and uninduced samples.

2.2.6 Bioinformatic analysis

The -1100/-1 sequence was investigated for repetitive elements with a Dot Plot algorithm using the

Macvector program (DOUGLAS 1994). This sequence was also analysed for putative binding sites using the Matinspector program (CARTHARIUS et al. 2005). The -1802/-1bp sequence was analysed for conserved sequences across several Drosophila genomes using the EvoPrinterHD program (YAVATKAR et al. 2008).

2.2.7 ELectromobility Shift Assays (EMSA)

Radioactive DNA probes

DNA sequences identified as containing phenobarbital-reponse enhancers (PBREs) from the 5’ upstream region of Cyp12d1 were used as DNA probes for Electromobility shift assays (EMSA). These probes were amplified with primers containing BglII or XbaI recognition cut sites incorporated using

GoTaq (Promega). PCR products were purified using the Wizard SV Gel and PCR Clean-Up System

(Promega) and then restriction enzyme digested with BglII and XbaI (Promega) to give overhanging

DNA ends. Klenow polymerase (New England Biolabs) was used to fill in and end-label the probes with dCTP-P32 (Promega). Probes were then purified again using the QIAquick PCR Purification Kit

(Qiagen). The radioactivity of the Probes was assayed using the Model 3 Survey Meter (Ludlum

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Measurements Inc., Texas) and diluted to 20,000 counts per second before use for EMSA experiments.

Nuclear Protein Preparation

Nuclear Proteins for use in EMSA experiments were extracted from phenobarbital-exposed w1118 larvae. Two types of nuclear protein samples were used. The DT (digestive tissues) sample was made from whole midguts, Malpighian tubules and fat bodies dissected in phosphate-buffered saline (PBS) from 50 larvae and snap-frozen on dry ice. The whole larvae sample comprised of 50 3rd instar larvae snap-frozen on dry ice. Nuclear proteins were then isolated from both tissue types using the protocol and buffers listed in (PETERSEN et al. 1999). Protein sample concentrations were measured using the Qubit Protein Quantification system (Invitrogen).

EMSA Gel running

20 μg/μl Protein samples and 20,000 cps radioactive probes were combined and allowed to incubate at room temperature for 30 minutes before being mixed with loading dye and loaded into the gel.

5% Polyacrimalide gels (Sigma) were cast and run in a Mini-Protean II tank (Biorad) at 100V for

90minutes at 4C. Gels were then dried and exposed to a PhosphorImager screen (Molecular

Dynamics) in a PhosphorImager Cassette (Molecular Dynamics) overnight at room temperature.

Images were scanned and processed on a Typhoon Phosphoimager 6000 (GE Lifesciences).

2.2.8 EMSA Protein-DNA band Sequencing

A radiolabelled -288/-168bp probe with the whole larvae sample and a non-radiolabelled -288/-

168bp probe were loaded in adjacent lanes in a 5% Polyacrylamide gel. The radiolabelled lane was cut away from the rest of the gel and developed to identify the approximate location of the desired protein band. The non-radioactive lane was commassie stained and the corresponding location was isolated on the cold lane. A band from an empty lane was also analysed as a negative control. Bands were trypsin-digested overnight to give peptide fragments which were sequenced using mass

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spectroscopy (Protocol listed in Appendix III). All analyses were done on an Agilent 6220 ESI-TOF

LC/MS Mass Spectrometer coupled to an Agilent 1100 LC system (Agilent, Palo Alto, CA). All data were acquired and reference mass corrected via a dual-spray electrospray ionisation (ESI) source.

Each data point on the Total Ion Chromatogram (TIC) is an average of 15,000 transients, producing a spectrum every second. Mass spectra were created by averaging the scans across each peak and background subtracted against the first 10 seconds of the TIC. Acquisition was performed using the

Agilent Mass Hunter Acquisition software version B.02.01 (B2116.30) and analysis was performed using Mass Hunter version B.03.01. Protein sequences were compared against the SwissProt

Drosophila-specific protein database to identify the top hitting fragments.

2.2.9 Genetic Screen for Cyp12d1-regulating Transcription factors

Candidate phenobarbital-response-regulating transcription factors were chosen based on homology to known mammalian xenobiotic-regulating transcription factors. RNAi lines targeting candidate transcription factors were obtained from the Vienna Drosophila Resource Centre (VDRC) (DIETZL et al. 2007). See Appendix I for a list of fly lines used in this study.

-1560/-1bp; 5HR GAL4 driver

The -1560/-1bp pStinger transgenic line was crossed into the Cyp6g1HR-GAL4-6c line (5HR-GAL4) to generate the -1560/-1bp;5HR-GAL4 line (Figure 2.3).

2.2.9.1 Transcription factor RNAi

Four day-old virgin GAL4-driver females were crossed to UAS-RNAi males and allowed to mate and lay eggs overnight (Figure 2.2). Larvae were allowed to develop for approximately 108 hours before

3rd instar larvae were collected and placed on standard flyfood agar plates supplemented with

10mM of phenobarbital or control plates without phenobarbital. Larvae were then dissected and examined under the SZX12 stereomicroscope system (Olympus) to look for a detectable loss of fluorescence.

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Figure 2.2: -1540/-1bp pStinger/5HR-GAL4 driver line generation. (A) Homozygous 2nd chromosome insertion -1540/-1bp pStinger adult males were crossed to virgin If/CyO;MKRS/TM6b,Tb females.

Male -1540/-1bp pStinger/CyO;+/TM6b,Tb flies were collected. (B) Homozygous 3rd chromosome insertion 5HR-GAL4 adult males were crossed to virgin If/CyO;MKRS/TM6b,Tb females and

+/CyO;5HR-GAL4/TM6b,Tb virgin females collected. (C) Male 1540/-1bp pStinger/CyO;+/TM6b,Tb flies were crossed to virgin +/CyO;5HR-GAL4/TM6b,Tb females. -1540/-1bp pStinger/CyO;5HR-

GAL4/TM6b,Tb flies were collected. (D) -1540/-1bp pStinger/CyO;5HR-GAL4/TM6b,Tb flies were selfed and homozygous-1540/-1bp pStinger;5HR-GAL4 flies collected.

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Figure 2.3: Crossing scheme for transcription factor knockdown. Male homozygous transcription factor RNAi lines are crossed to -1540/-1bp pStinger;HR-GAL4 virgin females to give heterozygous transcription factor-RNAi/-1540/-1bp pStinger;HR-GAL4 F1 progeny.

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-1560/-1bp pStinger;HR96-/- lines

The -1560/-1bp pStinger was crossed into two different HR96 null lines (HR96e25 and HR9616A) (Gift from Professor Carl Thummel, University of Utah) to make the 1560/-1bp pStinger;HR96e25 and

1560/-1bp pStinger;Hr9616A lines (Figure 2.5). 3rd instar larvae were collected at approximately 108 hours old post-laying and exposed to phenobarbital using the standard phenobarbital-induction assay. Larvae were dissected and examined under the SZX12 stereomicroscope system (Olympus) to look for a detectable loss of fluorescence.

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Figure 2.4: Crossing scheme to generate -1540/-1bp pStinger; HR96-/- lines. (A) Homozygous 2nd chromosome insertion -1540/-1bp pStinger adult males were crossed to virgin

If/CyO;MKRS/TM6b,Tb females to generate Male 1540/-1bp pStinger/CyO;+/TM6b,Tb flies. (B)

Homozygous 3rd chromosome insertion HR96-/- adult males were crossed to virgin

If/CyO;MKRS/TM6b,Tb females. Virgin +/CyO; HR96-/-/TM6b,Tb females were collected. (C) Male

1540/-1bp pStinger/CyO;+/TM6b,Tb flies were crossed to virgin +/CyO; HR96-/- /TM6b,Tb females to generate -1540/-1bp pStinger/CyO;5HR-GAL4/TM6b,Tb flies. (D) -1540/-1bp pStinger/CyO; HR96-/-

/TM6b,Tb flies were self-crossed and homozygous -1540/-1bp pStinger; HR96-/- flies collected.

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2.3 Results

2.3.1 Cyp12d1 Tissue specific expression

The tissue-specific expression of Cyp12d1 in third instar larvae of the y; cn bw sp strain has previously been described (CHUNG et al. 2009; WILLOUGHBY et al. 2006)(Figure 2.5). In this strain,

Cyp12d1 mRNA is detected in the anterior (M1-M4) and posterior midgut (M6-M13) regions (CHUNG et al. 2009). mRNA in situ hybridisation performed on y; cn bw sp third instar larvae exposed to phenobarbital showed no change in spatial expression of Cyp12d1, with darker staining suggesting more Cyp12d1 transcript present. Cyp12d1 expression was induced in the anterior (M1-M4) and mid

(M6-M13) midgut, Malpighian tubules and fat body (Figure 2.5) (WILLOUGHBY et al. 2006).

2.3.2 Cyp12d1 tissue-specific native enhancers

The upstream and downstream regions of the Cyp12d1 genomic locus from the w1118 strain (which has a single copy of Cyp12d1) were investigated for regulatory elements driving basal Cyp12d1 expression. Figure 2.6 is a composite figure showing all the promoter constructs used in this study and their locations in the Cyp12d1 single copy genomic locus.

2.3.2.1 5’ constructs and tissue-specific native expression

The -1 to -10kb region was first investigated for Cyp12d1 basal enhancers. 3rd instar larvae have native Cyp12d1 expression in the midgut, Malpighian tubules and fat body. Three constructs, -9653/-

2932bp pH-Stinger, -3155/-1841bp pH-Stinger and -2043/-945bp pH-Stinger were first examined for basal GFP expression. GFP could not be detected in the midgut, fat body and Malpighian tubules in transgenic third instar larvae for any of these three constructs, indicating that they lacked required enhancer(s) for Cyp12d1 expression in these tissues (Figure 2.7).

The -9653/-2932bp pH-Stinger construct showed strong peripheral nervous system expression

(Figure 2.7), which was not observed when Cyp12d1 expression was determined by in situ

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Figure 2.5: Cyp12d1 3rd instar mRNA in situ hybridisation expression patterns

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Figure 2.5: Cyp12d1 3rd instar mRNA in situ hybridisation expression patterns. (A) Cyp12d1 basal uninduced expression. Basal expression is seen throughout the midgut in sections M1-M4 and M6-

M13. Expression was also observed in the fat body and the Malpighian tubules. (B) Caffeine induces

12-fold higher expression in the M1-M4 and M6-M13 sections of the midgut, the Malpighian tubules and the fat body (WILLOUGHBY et al. 2006). (C) Phenobarbital induces 24-fold higher expression in the same M1-M4, M6-M13, fat body and Malpighian tubule tissues also. (A), (B) and (C) taken from

(WILLOUGHBY et al. 2006). Note that the staining in (B) and (C) is much darker compared to (A), which is indicative of more Cyp12d1 signal detected. (D) Schematic diagram of the different sections of the midgut. Image taken from (MURAKAMI et al. 1999).

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Figure 2.6: Cyp12d1 promoter constructs.

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Figure 2.6: Cyp12d1 promoter constructs. A master image of all Cyp12d1 promoter constructs is presented here. The Cyp12d1 genomic locus (top) was divided into several smaller reporter constructs which were cloned in front of eGFP. Names of the constructs are on the right hand side.

5’ constructs have a black line showing the extent of the insert. 3’ constructs have a red line and were cloned in front of or behind the nuclear eGFP gene depending on the construct. Constructs with a +1917/+2207bp fragment cloned behind are denoted with an additional SpeI label to signify the +1917/+2207bp fragment being cloned into the SpeI restriction enzyme site behind nuclear eGFP. Consensus GATA binding sites [(T/A)TATA(T/A)] are marked with a blue star, while non- consensus GATA binding sites with a recognisable GATA central motif are marked with a green star.

A red star denotes a mutated GATA binding site (GATA mutated to CATA). Glossary: None= no GFP expression seen in any tissue; NA= Induction was not tested on this construct; Ant MG= Anterior midgut M1-M3 sections, Post MG= Posterior midgut M6-M13 sections; MT= Malpighian Tissues; FB=

Fat body.

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Figure 2.7: Uninduced expression from 5’ constructs covering the -9653 to -1100bp region.

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Figure 2.7: Uninduced expression from 5’ constructs covering the -9653 to -1100bp region. (A) -

9653/-2932bp pH-Stinger shows expression in discreet cells in the peripheral nervous system along the larval torso. (B) Transgenic -9653/-2932bp pH-Stinger 3rd instar larvae midgut and Malpighian tubules. Nuclear GFP was not seen in any of the tissues examined. (C)-(E) -3155/-1841bp pH-Stinger midgut, Malpighian tubules and fat body. Again, GFP expression was not observed in these tissues.

(F)-(H) -2043/-945bp pH-Stinger midgut, Malpighian tubules and fat body. These tissues did not express GFP, suggesting Cyp12d1 enhancers necessary for midgut, Malpighian tubules and fat body were not present in the -2043/-945bp region. (I)-(K). -1560/-1bp pStinger. GFP expression was seen in the midgut (I, arrowed) and the Malpighian tubules (J, arrowed) but not the fat body (K). This indicated that Cyp12d1 basal enhancers were present in the -945 to -1bp region. (L)-(N) -1100/-1bp pStinger. No GFP expression was observed in any digestive tract tissues, in contrast to the -1560/-

1bp pStinger construct despite overlapping sequences between the two constructs. (O) Schematic showing the approximate positions of the constructs listed in this figure.

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hybridisation. The -9653/-2932bp region extends to the 5’ region of the upstream gene BBS4 (Figure

2.6). BBS4 is expressed in the peripheral nervous system and this was shown to be driven by elements in the -1000bp region of BBS4 (AVIDOR-REISS et al. 2004). This region was encompassed in the -9653/-2932bp fragment and subsequently this expression pattern was replicated in the -9653/-

2932bp construct (Figure 2.7A), suggesting that the expression seen in this construct is driven by

BBS4 enhancers and not Cyp12d1 enhancers.

The -1560/-1bp pStinger construct showed expression in the anterior midgut (M1-M3) and

Malpighian tubules, with no GFP fluorescence detected in the fat body (Figure 2.6). As the -2043/-

945bp pH-Stinger construct already includes the region from -1100 to -1560bp, making it unlikely that these enhancers were located in this sequence, this result suggested that the Cyp12d1 regulatory elements necessary for uninduced Cyp12d1 midgut, Malpighian tubule and fat body expression were present in first 1100bp upstream of Cyp12d1.

GFP fluorescence was not seen in any tissues with the -1100/-1bp pStinger (Figure 2.7) and -670/-

1bp pStinger constructs (Figure 2.8). This was inconsistent with previous data, as anterior midgut

(M1-M3) GFP, Malpighian tubule and fat body GFP expression was seen with the -288/-1bp pStinger construct and also with the -492/-168bp pH-Stinger constructs, which supportedthe finding that

Cyp12d1 basal enhancers were located in the -1100bp sequence (Figure 2.8). These fragments overlap the -1100bp region and would theoretically contain enhancers found in the first -1100bp of sequence.

This implies that the -670 to -492bp region contains a region which represses anterior midgut and

Malpighian tubule expression. Once this region was removed, the smaller constructs were able to express GFP in these tissues, as seen in the -288/-1bp pStinger construct and -492/-168bp pH-Stinger construct. The -168/-1bp pStinger construct did not show any GFP expression in the midgut,

Malpighian tubues and fat body, suggesting that it does not contain any Cyp12d1 enhancers for expression in these tissues.

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Figure 2.8: Uninduced expression from 5’ constructs covering the -670bp to -1bp region.

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Figure 2.8: Uninduced expression from 5’ constructs covering the -670bp to -1bp region. (A)-(C). -

670/-1bp pStinger. GFP fluorescence was not seen in the midgut (A) or fat body (C). -670/-1bp pStinger lines show strong autofluoresence in the Malpighian tubules (B), masking any nuclear GFP.

(D)-(F) -492/-168bp pH-Stinger. This construct showed midgut (D), Malpighian tubule (E) and fat body expression(F). (G)-(I) -288/-1bp pStinger. Midgut GFP expression was observed (arrowed) (G), along with with Malpighian tubule (H) and fat body expression (arrowed)(I). (J)-(L) -168bp pStinger.

No expression was observed in the midgut (J), Malpighian tubules (J) and fat body (L), suggesting that Cyp12d1 enhancers necessary for midgut, Malpighian tubules and fat body uninduced expression were not present in this sequence. (M) Schematic showing the approximate positions of the constructs listed in this figure.

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2.3.2.2 Investigating the downstream region for Cyp12d1 enhancers

The downstream region of Cyp12d1 was cloned into the pH-stinger vector to investigate the possibility of downstream Cyp12d1 enhancer elements. The +1917/2207bp pH-Stinger construct is

400bp in size and consists of the intergenic region between Cyp12d1 and the neighbouring gene

CG30490. The +1917/2207bp pH-Stinger construct showed expression in the proventriculous, the anterior and middle midgut, Malpighian tubules and fat body, showing that it contained enhancers capable of driving expression in a Cyp12d1-like manner (Figure 2.9A). As this region was also the

CG30490 5’ promoter region, the expression and regulation of CG30490 in 3rd instar larval digestive tissues was investigated in Section 2.3.2.3.

2.3.2.3 CG30490 regulation in 3rd instar larvae

As the +1917/2207bp fragment lies 5’ of the CG30490 gene, it is possible that CG30490 regulatory enhancers, instead of Cyp12d1 enhancers, might be driving the expression observed in the

1917/2207bp pH-Stinger construct. The expression of the downstream gene CG30490 was investigated in 3rd instar larvae to determine if expression seen in +1917/2207bp was due to

CG30490 enhancers and not Cyp12d1 enhancers. Primers specific to the CG30490 coding region were used to check for expression in w1118 3rd instar larvae cDNA (Figure 2.10). Multiple PCR experiments failed to detect any CG30490 expression, implying that CG30490 was not expressed in w1118 3rd instar larvae. Additionally, Flyatlas reported that CG30490 expression was not detected in rd 3 instar digestive tissues by microarray (CHINTAPALLI et al. 2007). These data suggest that CG30490 is not expressed in 3rd instar larvae, and any GFP expression seen in the Cyp12d1 1917/2207bp construct was unlikely to be driven by CG30490 enhancers. The annotated CG30490 gene has since been removed from the Drosophila genome [Drosophila genome release (FB2011_02) (TWEEDIE et al.

2009)], strongly indicating that these enhancers are Cyp12d1-specific.

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Figure 2.9: Uninduced expression from 3’ constructs covering the +1917bp/+2207bp region.

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Figure 2.9: Uninduced expression from 3’ constructs covering the +1917bp/+2207bp region. (A)

+1917/2207bp pH-Stinger gave strong expression in the proventriculous (PV), midgut, Malpighian tubule and fat body. (B) +1917bp/2049bp pH-Stinger showed expression in the proventriculous section 2 (PV2) region only. (C) – (E) +1917/+2116bp pH-Stinger. Strong GFP expression was seen throughout the proventriculous and midgut (C), the Malpighian tubules (E) and the fat body (D). (F)

+2116/+2207bp pH-Stinger. No GFP expression was observed in the proventriculous, midgut,

Malpighian tubules and fat body. The salivary gland (SG) expression seen is a non-specific byproduct of the pH-Stinger vector (ZHU and HALFON 2007). (G) Schematic showing the approximate positions of the constructs listed in this figure.

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1.2kb

517bp

350bp 222bp

M 1 2 3 4 M 5 6 7 8

Figure 2.10: CG30490 expression in 3rd instar w1118 larvae. Reverse transcriptase PCR was performed to detect CG30490 expression. Lanes 1-4 are PCRs carried out using CG30490 specific primers. Lanes

1 (dissected w1118 3rd instar gut tissue cDNA) and 2 (whole w1118 larvae cDNA) did not show a band while lane 3 (w1118 genomic DNA positive control) did. The no-template control lane 4 did not show any contamination. Lanes 5-8 used RPL11 primers as a control testing the integrity of DNA used with sample order remaining the same. All samples tested for RPL11 expression showed strong bands, showing that the cDNA template used was of good quality and suggesting that CG30490 is not expressed in 3rd instar larvae.

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2.3.2.4 3’ constructs investigating the +1917/+2207bp region

To pinpoint the location of the enhancers, several other constructs were generated to dissect the

408bp 3’ region (+1917/2207bp) further (Figure 2.9). The 1917/2207bp pH-Stinger construct was found to drive expression in the proventriculous, throughout the midgut, the fat body and the

Malpighian tubules, which replicated the known Cyp12d1 uninduced mRNA in situ hybdridisation pattern. This expression was replicated in the +1917/2207bp pH-Stinger construct but not the

+2116bp/+2207bp pH-Stinger construct, suggesting that the enhancers driving expression seen in the intact +1917/2207bp fragment are in the first 200bp (the +1917/+2116bp region).

The +1917bp/2049bp pH-Stinger only showed expression in a very discreet ring of cells surrounding the circumference of the proventriculous (the PV2 section) (Figure 2.9B). GFP expression was not seen in the midgut, Malpighian tubules or fat body, indicating that expression in the +1917/2207bp fragment was driven by enhancers located in the +2049/2116bp region. This result narrowed the downstream midgut, fat body and Malpighian tubule enhancers to a 68bp region, while the PV2 enhancers were located in the first 133bp of the 3’ region (the +2049bp/2116bp region).

2.3.2.5 Combined 5’ and 3’ Constructs

The 3’ constructs showed that enhancers capable of driving expression in a Cyp12d1-like manner were present in the Cyp12d1 3’ region. It was still unclear if these enhancers were actually driving

Cyp12d1 expression. To demonstrate this, the +1917/2207bp fragment was cloned into the -168bp pStinger construct 3’ of the eGFP gene (-168/-1bp pStinger +1917/2207bp SpeI) to investigate if the

+1917/2207bp enhancers were able to operate with the Cyp12d1 TATA box in a 3’ to 5’ direction to drive GFP transcription. The -168/-1bp pStinger +1917/2207bp SpeI showed strong GFP expression in the midgut and the Malpighian tubules, with fainter expression in the fat body (Figure 2.11). As the -

168/-1bp pStinger construct was found not to have any endogenous GFP

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Figure 2.11: Combined 5’ and 3’ constructs uninduced expression. (A)-(C) -168/-1bp pStinger did not show any GFP expression in the midgut, Malpighian tubules and fat body. (D) -168/-1bp pStinger

+1917/2207bp SpeI displayed midgut expression but no proventriculous expression. (E) 168/-1bp pH-Stinger +1917/2207bp SpeI exhibited Malpighian tubule expression. (D) -168/-1bp pStinger

+1917bp/2207bp SpeI showed Fat body expression. (F) -168/-1bp pStinger +1917bp/2207bp SpeI had fat body expression. (G) Schematic showing the approximate positions of the constructs listed in this figure.

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expression (Figure 2.8), this signified that the additional expression seen in the -168bp pStinger

+1917/2207bp SpeI construct was directed from the +1917/2207bp fragment. These data suggests that basal Cyp12d1 expression required the input of enhancers located 3’ of the Cyp12d1 gene for full expression. Interestingly the proventriculous expression seen in the 3’ constructs was no longer observed, implying that the proventriculous enhancers were possibly not functional with the

Cyp12d1 enhancer or that other unknown effects combined to suppress proventriculous expression.

Nonetheless, these data are consistent with the in situ hybridisation expression pattern in which no proventriculous expression was seen. This expression pattern also recapitulates the 3rd instar

Cyp12d1 expression seen in mRNA in situ hybridisations, and indicates that most if not all of the native uninduced expression enhancers were found in this construct.

2.3.2.6 Native Enhancers summary

A summary of basal Cyp12d1 regulation is presented in Figure 2.12. 5’ elements drive uninduced expression in the anterior midgut, Malpighian tubules and fat body, while 3’ elements contributed additional anterior and posterior midgut, Malpighian tubules and fat body expression.

2.3.3 Xenobiotic Induced expression

Phenobarbital (PB) is a barbiturate compound that has been extensively used to study induction of

P450s in mammals, and is used to study P450 induction in insects (Section 1.3.2). Phenobarbital

rd induces approximately 24-30-fold higher Cyp12d1 expression in 3 instar larvae (KING-JONES et al.

2006; WILLOUGHBY et al. 2006). mRNA in situ hybridisation experiments found that induction occurs in the midgut, Malpighian tubule and fat body, sites where Cyp12d1 is already basally expressed

rd (Figure 2.5; WILLOUGHBY et al. 2006). Cyp12d1 5’ promoter and 3’promoter construct transgenic 3 instar feeding larvae were exposed to 10mM phenobarbital to identify enhancer regions controlling phenobarbital induction.

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Figure 2.12: A model for the regulation of Cyp12d1 uninduced native expression. A model for

Cyp12d1 basal expression is presented. Basal anterior midgut (M1-M3), Malpighian tubules and fat body expression were driven by elements in the -288/-168bp region. Anterior (M1-M3) and posterior (M6-M13) midgut, Malpighian tubules and fat body expression were driven by enhancers in the downstream +2049/+2116bp region. The -670/-492bp region contained a repressing region that repressed transcription, which was alleviated by removing this region entirely or by the insertion of additional factors in the -1570/-1100bp region.

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2.3.3.1 Investigating the downstream region for phenobarbital induction enchancers

The +1917/2207bp region was shown to contain tissue specific enhancers for basal levels of Cyp12d1 expression. +1917/2207bp pH-Stinger lines were exposed to phenobarbital to check if the

+1917/2207bp region contained any phenobarbital responsive elements (PBREs). As strong basal

GFP expression was already present in the midgut, Malpighian tubules and fat body, it was not possible to visually detect any further increase in fluorescence. RT- PCR was therefore performed to quantify the relative amount of GFP mRNA induction in phenobarbital exposed larvae compared to controls (Figure 2.13). No significant increase in GFP expression was found, suggesting that the

+1917/2207bp region does not contain Cyp12d1 PBREs.

2.3.3.2 Investigating the Cyp12d1 upstream promoter region for phenobarbital-induction enhancers

No induced GFP expression was observed in the midgut, fat body and Malpighian tubules when third instar larvae containing the -9653/-2932bp, -3155/-1841bp and -2043/-945bp pStinger constructs were exposed to phenobarbital (Figure 2.14). This indicates that the -9653 to -945 bp regions of the

5’ Cyp12d1 promoter region do not contain any enhancers capable of driving phenobarbital-induced expression in these tissues. In contrast, the -1560/-1bp pStinger construct yielded higher GFP mRNA expression in phenobarbital-exposed larvae compared to controls, suggesting that Cyp12d1 phenobarbital xenobiotic response elements (PBREs) were present in this construct (Figure 2.15(A-

D)). The phenobarbital exposed larvae demonstrated stronger GFP fluorescence in the midgut and fat body compared to unexposed controls. This indicated that the Cyp12d1 5’ region was important for Cyp12d1 phenobarbital induction. Malpighian tubule GFP expression was already strong in the uninduced -1560/-1bp pStinger construct, and no difference in expression intensity could be visually detected upon phenobarbital exposure.

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Figure 2.13: +1917/2207bp pH-Stinger does not respond to phenobarbital induction. The

+1917/2207bp pH-Stinger 3A and the +1917/2207bp pH-Stinger 4A line were exposed to phenobarbital and the amount of GFP mRNA measured. No significant difference was found between exposed and unexposed (Control, or Con) samples for the +1917/2207bp pH-Stinger 3A line

(Paired T-test, P=0.303). The +1917/2207bp pH-Stinger 4A line also did not show any induced GFP expression after phenobarbital induction when compared to controls (Paired T-test, P=0.98). These results indicated that the +1917/2207bp region did not carry any phenobarbital-induction enhancers. RT-PCR data is from three biological replicates, with error bars representing Standard error of the mean (SEM).

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Figure 2.14: Constructs investigating the -9653/-945bp region for phenobarbital induction enhancers. (A)-(C). Phenobarbital exposed -9652/-2932bp pH-Stinger constructs. No GFP fluorescence was seen in the midgut (A), the Malpighian tubules (B) and the Fat Body (C). (D)-(F).

Phenobarbital-exposed -3155/-1841bp pH-stinger construct. Phenobarbital did not induce GFP expression in the midgut (D), Malpighian tubules (B) and fat body (C). (G)-(I) Phenobarbital induced -

2043/-945bp pH-Stinger. No induced GFP fluorescence was seen in the midgut, Malpighian tubules and fat body. (K) Schematic showing the approximate positions of the constructs listed in this figure.

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Figure 2.15 Constructs investigating the -1540/-1bp region for phenobarbital induction enhancers.

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Figure 2.15 Constructs investigating the -1540/-1bp region for phenobarbital induction enhancers.

(A)-(D) -1560/-1bp pStinger showed strong expression after phenobarbital exposure in the midgut

(A), Malpighian tubules (B) and fat body (C). (D) RT-PCR showed higher GFP mRNA expression in phenobarbital-exposed -1560/-1bp pStinger larvae. (E)-(H). Phenobarbital-induced -492/-168bp pStinger showed strong midgut (E), Malpighian tubule (F) and fat body (G) expression. (H) RT-PCR showed induced GFP mRNA expression in phenobarbital-exposed -492/-168bp pStinger larvae. (I)-(L)

Phenobarbital -Induced -288/-1bp pStinger. This construct also showed strong midgut expression (I),

Malpighian tubule (J) and fat body (K) expression when induced by phenobarbital. (L) RT-PCR detected higher GFP mRNA expression in phenobarbital–exposed -288/-1bp pStinger larvae compared to control larvae. (M)-(P) -168/-1bp pStinger. This construct did not express GFP after phenobarbital induction in the midgut (M), Malpighian tubule (N) and fat body (O). RT-PCR could only detect extremely low levels of GFP mRNA in both phenobarbital–exposed and unexposed -168/-

1bp pStinger larvae. (Q) Schematic showing the approximate positions of the constructs listed in this figure. RT-PCR data is from three biological replicates, with error bars representing Standard error of the mean (SEM).

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Figure 2.16: -1100/-1bp pStinger and -670/-1bp pStinger phenobarbital inductions.

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Figure 2.16: -1100/-1bp pStinger and -670/-1bp pStinger phenobarbital inductions. (A)-(D) 4 hour phenobarbital exposure did not appear to increase GFP fluoresence in the -1100/-1bp pStinger midgut, Malpighian tubules and fat body. (D) RT-PCR however showed significant induction of GFP mRNA compared to unexposed controls in phenobarbital -exposed -1100/-1bp pStinger larvae. (E)-

(G) In contrast, after 24hours of phenobarbital exposure, -1100/-1bp pStinger larvae showed strong

GFP expression in their midgut, Malpighian tubules and fat body. (H) RT-PCR showed also strong GFP mRNA induction as well compared to unexposed controls. (I)-(K) -670/-1bp pStinger showed the same pattern of expression, with little to no GFP observed in the midgut (I), Malpighian tubules (J) and fat body (K) of four hour-exposed larvae. (L) GFP mRNA was highly expressed in larvae that fed on phenobarbital-supplemented food for four hours compared to controls. (M)-(O) -670/-1bp pStinger larvae showed higher GFP expression when observed again after 24 hours in the midgut

(M), Malpighian tubules (N) and fat body (O). (P) RT-PCR still showed that GFP mRNA continued to be expressed at higher levels compared to controls in phenobarbital-exposed larvae. (Q) Schematic showing the approximate positions of the constructs listed in this figure. RT-PCR data is from three biological replicates, with error bars representing Standard error of the mean (SEM).

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The -1100/-1bp and -670/-1bp pStinger constructs were next tested for phenobarbital-induced GFP transcription and fluorescence (Figure 2.16). RT-PCR showed higher GFP mRNA levels in phenobarbital -exposed -1100/-1bp pStinger and -670/-1bp pStinger constructs when compared to uninduced controls. Contrary to expectations, no GFP fluorescence was seen in the tissues of -1100/-

1bp pStinger and -670/-1bp pStinger constructs after phenobarbital exposure for four hours.

However when larvae were left to feed on the phenobarbital plates for an additional 20 hours (for a total of 24 hours), -1100/-1bp and -670/-1bp showed strong GFP fluorescence in the midgut,

Malpighian tubules and fat body (Figure 2.16), demonstrating that these constructs were able to drive GFP expression. RT-PCR found that GFP mRNA in samples exposed for 24 hours was expressed at similar levels to samples exposed for four hours. The -1100bp to -492bp region has already been hypothesised to contain a repressor region for basal expression (Section 2.3.2.1, Figure 2.7 and 2.8).

However, as RT-PCR data showed GFP mRNA continued to be induced after four hours of phenobarbital exposure while fluorescence was only seen 24 hours later, it appears that this region delays phenobarbital-induced GFP expression, rather than completely inhibiting induction, and insufficient GFP mRNA accumulation occurred for enough visible GFP protein fluorescence observable after four hours. This also suggests that the -1560/-1bp pStinger construct contains additional elements in the -1100/-1bp to -1560/-1bp region that is able to alleviate the basal and phenobarbital-induced expression suppression.

RT-PCR found that the -492/-168bp pH-Stinger and -288/-1bp pStinger constructs also showed higher GFP mRNA expression after phenobarbital exposure (Figure 2.15). Strong GFP fluorescence was also observed in the midgut and fat body of induced -492/-168bp pH-Stinger and -288/-1bp pStinger (Figure 2.15). Similar to the -1560/-1b construct, strong endogenous Malpighian tubule expression masked the detection of any further induced expression (Figure 2.15). These constructs also demonstrated that the repressing effect seen in the -1100/-1bp pStinger and -670/-1bp pStinger constructs was relieved once the -1100bp to -492bp region is removed.

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Figure 2.17: -288/-168bp pStinger phenobarbital-induced and basal expression.

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Figure 2.17: -288/-168bp pStinger phenobarbital-induced and basal expression. (A)-(C). -288/-168bp pStinger showed basal uninduced expression in the midgut (A), Malpighian tubules (B) and fat body

(C). (D) RT-PCR showed higher GFP mRNA expression in phenobarbital-exposed -288/-168bp pStinger larvae compared to unexposed larvae. (E)-(G) -288/-168bp pStinger phenobarbital induction increased GFP expression in the midgut (E), Malpighian tubules (F) and fat body (F). (G) 288/-168bp pStinger showed strong GFP mRNA phenobarbital induction in Malpighian tubules compared to control unexposed larvae Malpighian tubules. (I) Schematic showing the approximate positions of the constructs listed in this figure. RT-PCR data is from three biological replicates, with error bars representing Standard error of the mean (SEM).

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The -168/-1bp pStinger construct did not show any induced GFP mRNA expression in the midgut after phenobarbital exposure when GFP mRNA was measured using RT-PCR (Figure 2.15). No GFP fluorescence was also observed when dissected -168/-1bp pStinger 3rd instar larvae midgut were examined. The -168bp region therefore was found not to contain any Cyp12d1 basal and xenobiotic- inducible enhancers for expression in the midgut, Malpighian tubules and fat body, and suggested that the Cyp12d1 PBREs were located in the overlapping section between the -492/-168bp and -

288/-1bp fragments, i.e. the -288/-168bp region.

The -288/-168 region contains Cyp12d1 basal and phenobarbital-induction enhancers

The -288/-168bp pH-Stinger construct was designed to investigate this region upstream of Cyp12d1.

-288/-168bp pH-Stinger demonstrated uninduced midgut, Malpighian tubule and fat body GFP expression and showed higher GFP mRNA expression after phenobarbital exposure (Figure 2.17).

Phenobarbital induced GFP fluorescence was observed in the midgut and fat body, confirming that

Cyp12d1 PBREs are found in this region.

This construct showed strong basal GFP expression in the Malpighian tubules which masked induced

Malpighian tubule fluorescence. To investigate Malpighian tubule induction, tubules were dissected from phenobarbital-induced and uninduced larvae before RNA extracted and RT-PCR performed

(Figure 2.17H). Malpighian tubules showed induced GFP mRNA expression compared to unexposed tubules, demonstrating that the -288/-168bp pStinger was capable of inducing GFP in Malpighian tubules. This region thus constituted the smallest region containing Cyp12d1 PBREs that were capable of inducing GFP expression upon phenobarbital exposure.

GATA factor binding site

A canonical GATA binding site was found in the -288/-168bp region at position -186bp. GATA family members regulate a variety of processes including gut tissue development and immune system response in Drosophila (BROWN and CASTELLI-GAIR HOMBRIA 2000; SENGER et al. 2006). GATA members

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Figure 2.18: -288/-168bp GATA mutant pStinger phenobarbital-induced and basal expression.

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Figure 2.18: -288/-168bp GATA mutant pStinger phenobarbital-induced and basal expression. 288/-

168bp pStinger was included as a comparison for -288/-168bp GATA mutant pStinger (A)-(C). -288/-

168bp GATA mutant pStinger. This construct did not express GFP in the midgut (A) but had strong basal expression in the Malpighian tubules (B) with no expression in the fat body (C). (D) RT-PCR still showed induction of GFP mRNA in phenobarbital-exposed -288/-168bp GATA mutant pStinger whole larvae. (E)-(G) -288/-168bp GATA mutant pStinger phenobarbital induced expression. € No GFP fluoresence was seen in the induced midgut, while the Malpighian tubules (F) and Fat body (G) showed strong GFP expression. (H) RT-PCR showed that GFP mRNA was still being induced in

Malpighian tubules in the -288/-168bp pH-stinger GATA mutant lines compared to controls. (I)

Schematic showing the approximate positions of the constructs listed in this figure. RT-PCR data is from three biological replicates, with error bars representing Standard error of the mean (SEM).

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bind to a conserved (T/A)GATA(T/A) DNA motif (TSAI et al. 1989). This GATA site was mutated using site-directed mutagenesis from a TGATAA site to a TCTAA site and the mutant -288/-168bp sequence cloned into the pH-Stinger vector to create the -288/-168bp GATA mutant pH-Stinger. Mutating the

Guanine residue to a Cytosine residue has been shown to abolish GATA transcription factor binding

(LUDLOW et al. 1996).

The intact -288/-168bp pStinger demonstrated basal midgut, Malpighian tubule and fat body GFP expression, and phenobarbital-induced midgut and fat body expression( Figure 2.17). The -288/-

168bp GATA mutant pH-Stinger did not show uninduced midgut or fat body expression (Figure 2.18).

This indicates that uninduced midgut and fat body expression was lost after the GATA binding site was altered and that the GATA binding site was crucial for Cyp12d1 midgut and fat body expression in this construct. RT-PCR showed that GFP was still being induced in phenobarbital-exposed larvae

(2.19D). Midgut GFP expression was again not seen when transgenic larvae were exposed to phenobarbital, but induced fat body expression was now observed (Figure 2.18). Basal Malpighian tubule GFP fluorescence again masked visual determination of induction in the tubules.

To investigate if Malpighian tubule Cyp12d1 expression was induced by phenobarbital, tubules were dissected from phenobarbital exposed and unexposed -288/-168bp GATA mutant pH-Stinger 3rd instar larvae and RT-PCR performed to quantify the amount of GFP mRNA expressed (Figure 2.18H).

The -288/-168bp GATA mutant was still able to induce Malpighian tubule GFP mRNA in response to phenobarbital exposure. This indicated that the GATA site was not critical for Malpighian tubule induction. Taken together, this suggested that Cyp12d1 requires an intact GATA site in the -288/-

169bp region for basal midgut and fat body expression, and for induced midgut expression.

2.3.3.3 Caffeine induction

Caffeine has been shown to induce 11 P450s in 3rd instar y; cn bw sp larvae, with Cyp12d1 being induced 12-fold by caffeine (WILLOUGHBY et al. 2006). Cyp12d1 -1560/-1bp pStinger and -288/-168bp pH-Stinger constructs were exposed to caffeine to test if these constructs were also able to regulate

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Figure 2.19: Caffeine induction of -1560/-1bp pStinger and -288/-168bp pH-Stinger. Caffeine induced stronger GFP fluorescence in the -1560/-1bp pStinger midgut (A), Malpighian tubules (B) and fat body (C). The -288/-168bp pH-Stinger line also induced brighter GFP fluorescence in response to caffeine in the (D) midgut, (E) Malpighian tubules and (F) fat body.

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caffeine induction in addition to phenobarbital induction.

The -1560/-1bp pStinger construct showed higher GFP expression in the midgut and fat body after caffeine exposure when these tissues were examined for GFP fluorescence (Fig 2.19). Again, basal

Malpighian tubule expression masked any induced higher GFP fluorescence in the tubules.

Nevertheless, this indicated that the -1560/-1bp region contained regulatory elements capable of driving caffeine induction in the midgut, Malpighian tubules and fat body. The -288/-168bp region also expressed more GFP in the midgut, Malpighian tubules and fat body when exposed to caffeine, showing that the smallest region mapped for phenobarbital induction also controlled caffeine induction.

It seems that the enhancer sites regulating caffeine induction are located in the same region as phenobarbital enhancers. This suggested that either the same mechanisms regulated both caffeine and phenobarbital induction, or that this sequence contained separate enhancers for both caffeine and phenobarbital induction.

2.3.3.4 Cyp12d1 xenobiotic induction regulation Summary

A model of Cyp12d1 xenobiotic induction regulation is presented in Figure 2.20. Phenobarbital and caffeine enhancer sequences were found in the -288/-168bp region. The -670/-492bp repressive region displayed delayed induction, with GFP fluorescence only observed after a 24 hour incubation.

Again, the -1570/-1100bp region was able to alleviate this delaying effect.

2.3.4 Bioinformatic analysis

Bioinformatic approaches were used in an effort to identify enhancers for tissue specific expression and phenobarbital induction in the Cyp12d1 region.

2.3.4.1 Dotplot analysis of the -1100/-1bp region

Transcription factor binding sites can consist of direct repeat elements. To examine this, dotplots were performed using the -1100bp sequence data. Dotplots compare sample sequences against

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Figure 2.20: A model for the regulation of Cyp12d1 xenobiotic induction. Cyp12d1 phenobarbital and caffeine induction was driven by enhancer sequences in the -288/-168bp region. This induction was delayed by repressing elements in the -670/-492bp region. Again, the -1570/-1100bp region contained additional factors that were able to remove the repression effect from the -670/-492bp region.

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100 200 300 400 500 600 700 800 900 1000 1100

100 100

200 200

300 300

400 400

500 500

600 600

700 700

800 800

900 900

1000 1000

1100 1100

100 200 300 400 500 600 700 800 900 1000 1100

Figure 2.21: -1100/-1bp sequence Dotplot. No repetitive sequences were observed when the -

1100bp sequence were analysed using a Dotplot program. This suggested that no repetitive sequences were present in the -1100bp sequence, and that any binding sites present were not direct repeat sequences. A sliding window of 3 basepairs was used and stringency was set at 3.

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Table 2.1: Matinspector-identified putative binding site in the -288/-168bp sequence.

Gene Site

1 Trithorax group protein binding site aaattgAGAGcgttg

2 Iroquois transcription factors agaaaAACA

3 Drosophila fork head factors aaaTAAAcaaatgaacg

4 Drosophila broad-complex tgaaaaaATAAACaaatga

5 Zeste transvection gene product agttGAGTgag

6 Iroquois transcription factors acaaaAACA

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themselves in a sliding window, and any repetitive sequences are denoted by diagonal lines in a dot plot figure. Dot plots are a common tool to identify repetitive sequences (HARE et al. 2008; KIM et al.

2004). No repetitive sequences were found in the -1100bp sequence, suggesting that direct repeat binding sites were not present (Figure 2.21).

2.3.4.2 Matinspector analysis for putative transcription factor binding sites

The Matinspector motif-finding program (CARTHARIUS et al. 2005) was used to analyse the -288/-

168bp sequence for known transcription factor binding sites to help identify potential regulatory proteins. Several putative binding sites for transcription factors were found (Table 2.1). Binding sites for known P450-regulating transcription factor families such as the Nuclear Receptor family or the

PAS-bHLH family were not identified. The Broad-complex transcription factor Z4 isoform was found to regulate Cyp6d1 phenobarbital induction in Drosophila Sl-2 cells (LIN et al. 2011). Broad-complex has also been shown to regulate some ecdysone response (KARIM et al. 1993). A canonical Broad- complex Z4 isoform binding site (ATAAACA) (KARIM et al. 1993) was found at position -142/-136bp upstream of Cyp12d1, suggesting that Broad-complex could also be involved in Cyp12d1 phenobarbital induction regulation (Table 2.1).

2.3.4.3 Identifying conserved blocks of sequence using EvoPrinterHD

EvoPrinterHD is a transcription factor binding site analysis program that compares promoter sequences between sequenced Drosophila species with sequenced genomes to identify blocks of conserved nucleotides (YAVATKAR et al. 2008). These nucleotides would be more likely to have important regulatory elements, as they have been conserved after divergence of Drosophila species.

The -1879/-1bp sequence of the upstream region of Cyp12d1 was analysed using EvoPrinterHD to identify conserved blocks of sequences between D. melanogaster, D. simulans, D. sechellia and D. erecta (Figure 2.22). These species were chosen because the upstream region of the next closest

Drosophila species, D. yakuba, did not hold any significant homology to the upstream region of the four species chosen. Attempts to align the upstream regions of D. yakuba and other more distant

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Figure 2.22: Cyp12d1 5’ promoter conserved regions identified using EvoPrinterHD. Bold capital letters indicate conserved sequences between D. melanogaster, D. simulans, D. sechellia and D. erecta. It can be seen that the -288/-168bp sequence (boxed) contains a large amount of conserved bases. The GATA binding site (solid underline in red) and the putative BR-C Z4 binding site (red dotted line underneath) are conserved, suggesting that they contribute to Cyp12d1 regulation in all four species tested.

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sequenced Drosophila species with the upstream regions from D. melanogaster, D. simulans, D. sechellia and D. erecta resulted in the complete loss of all conserved homology blocks (Data not shown), and thus only the D. melanogaster, D. simulans, D. sechellia and D. erecta regions were chosen.

It can be seen that several large blocks of conserved nucleotides are observed in the sequence. The

-288/-168 sequence was well conserved with 67.5% of all nucleotides conserved between species

(81 out of 120bp). The GATA binding site was conserved between species which suggesting that it may have been conserved because of its functional significance. Intriguingly, the putative Broad complex binding site was also conserved and suggested that this site may be important for regulation of Cyp12d1.

2.3.5 Transcription factor identification

Experiments were performed to attempt to identify the transcription factor(s) binding to the

Cyp12d1 PBRE site. Two approaches were used: (1) a biochemical approach where gel-shifted protein-bound DNA bands identified from Electromobility shift assays (EMSA) were sequenced using mass spectroscopy to identify bound proteins, and (2) a genetic screen where candidate transcription factors factors were knocked down using RNA interference (RNAi) and larvae screened for any differences in GFP expression in reporter constructs.

2.3.5.1 Electromobility shift assays

The first step was to establish if these DNA regions were able to bind proteins in order to identify probes which contained phenobarbital and caffeine induction enhancers. Gel Shifts were performed using previously identified 5’ promoter DNA probes which contained phenobarbital and caffeine induction enhancers. DNA-Protein bound complexes were subsequently to be sequenced using mass spectroscopy to identify the transcription factor binding and ultimately regulating Cyp12d1 basal and phenobarbital induced expression.

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Initial gel shift experiments

The -492/-168bp and -288/-168bp regions were chosen for investigation as they were shown to contain Cyp12d1 phenobarbital and caffeine induction response elements. The -168bp region was used as a negative control as this region was not able to drive Cyp12d1 expression and presumably did not include any Cyp12d1 enhancers. An initial gel shift testing these three probes was performed

(Figure 2.23). A smear was seen in every phenobarbital-exposed Whole Larvae (WL) protein lane, suggesting a large number of bound proteins retarding probe migration down the gel compared to the free probe control which was not challenged with protein.

A shifted band was seen when the -288/-168bp probe was mixed with the phenobarbital-exposed digestive tissue protein source (DT) (Figure 2.23A). The -288/-168bp construct was the smallest fragment capable of driving xenobiotic induction in transgenic larvae. This was a more discrete band compared to the smears seen with the WL protein extract, implying a smaller number of proteins binding to this probe. The -492/-168bp probe did not show this same shift despite this region overlapping the -288/-168bp fragment. Unfortunately, subsequent experiments did not achieve this result with the -288/-168bp probe and dissected gut tissue nuclear proteins, despite multiple attempts under several different experimental conditions (Figure 2.23 (C-D)). The -492/-168bp fragment was also tested to see if the larger fragment could bind proteins, but no band was seen in numerous attempts (Figure 2.23B).

2.3.5.2 Protein Band Sequencing

The -492/-168bp probe was used for the protein band sequencing experiments. Non-radioactive -

492/-168bp probe was combined with phenobarbital-exposed whole larvae nuclear proteins and an

EMSA experiment performed (Figure 2.24). The combined DNA-protein complex was then removed from the acrylamide gel and trypsin digested to obtain peptides for mass spectroscopy peptide sequencing. Table 2.2 shows the top hits generated by the WL sample. No transcription factors were

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Figure 2.23: Cyp12d1 Electromobility shift assay gels

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Figure 2.23: Cyp12d1 Electromobility shift assay gels. (A) Initial gel shift showing a discrete shift with

DT-specific nuclear extract challenging the -288/-168bp probe (arrowed). Both the -492/-168bp and the -288/-168bp showed large smears in the WL lanes, suggesting many proteins binding to the probes. (B) -492/-168bp probe EMSA gel testing different experimental parameters. Lane 1 unexposed dissected gut tissue nuclear protein source. Lane 2 used 10µg of phenobarbital-exposed dissected gut tissue nuclear extract. Lane 3 used 50µg of phenobarbital-exposed dissected gut tissue nuclear extract. Lane 4 used 10 µg of nuclear proteins extracted from the gut tissues of larvae subjected to a 24hour exposure. Lane 5 was free probe without any protein source. Lane 6 was unexposed whole larvae nuclear protein extract. It can be seen that the shift observed in this lane was lower than the following lanes which used phenobarbital-exposed larvae. Lane 7 used phenobarbital-exposed whole larvae nuclear protein extract. Lane 8 combined phenobarbital- exposed whole larvae nuclear protein extract with 2µg of poly dI/dC. Lane 9 combined phenobarbital-exposed whole larvae nuclear protein extract with 4µg of poly dI/dC. It can be seen that no change was observed in the intensity of lanes 8 and 9 which used poly dI/dC compared to lane 7 without poly dI/dC. Lane 10 used 24 hours phenobarbital -exposed larvae nuclear protein extract. (C)-(D) -298/-168bp probe gel shift using different experimental conditions. (C) 5X HI binding buffer (AUSUBEL et al. 1994) was used instead of the standard 5X binding buffer. (D) Binding buffer from (UVELL and ENGSTROM 2003) was used instead of the standard 5X binding buffer. In both

(C) and (D), the shift seen with the -288/-168bp in (A) was not reproduced. Glossary: FP= Free probe;

WL= Whole larvae nuclear proteins; DT= dissected midgut, Malpighian tubules and fat body nuclear proteins.

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identified in this list. Instead, the proteins identified were all highly expressed proteins in larvae

(Flyatlas, (CHINTAPALLI et al. 2007)), suggesting binding was non-specific. This implies that the large smear seen during Gel shifts performed with the whole larvae samples may be non-specific protein binding with the probes. It is possible that any transcription factors binding to the probe may have been swamped by non-specific noise from other more numerous protein signals and not been detected. The empty lane negative control showed no hits (Data not shown).

2.3.5.3 Transcription factor reduction and Cyp12d1 phenobarbital induction

A genetic screen was performed using the UAS-GAL4 system to reduce expression of candidate transcription factors and observe the effect of reducing the expression of the transcription factors on the induction of Cyp12d1 by phenobarbital. Several TF families were identified from the literature as being involved in P450 regulation and were subsequently investigated as potential candidates for binding to Cyp12d1 xenobiotic response elements (Section 1.3.1.1). The Cyp6g1 5’ Hikone R-6c (5HR-

GAL4) driver, which drives GAL4 expression in the midgut, Malpighian tubules and fat body was used to knock down transcription factor expression in these tissues. The -1560/-1bp pStinger construct was crossed into the 5HR-GAL4 background to generate the -1560/-1bp; 5HR-GAL4 driver strain. The

-1560/-1bp pStinger construct would act as a reporter gene and allow the detection of any reduction or complete loss of fluorescence when the -1560/-1bp; 5HR-GAL4 driver was crossed to transcription factor RNAi lines. -1560/-1bp;5HR-GAL4 was crossed to w1118 and exposed to phenobarbital to establish a control for subsequent experiments. -1560/-1bp ;5HR-GAL4/ w1118 showed weak midgut expression and no fat body expression, similar to the -1560/-1bp construct. However, very strong fluorescence was seen in the Malpighian tubules (Data not shown). As this fluorescence did not resemble nuclear-localised GFP, it seemed likely that this was autofluoresence. This fluorescence unfortunately masked any visible nuclear GFP expression in the Malpighian tubules. All lines crossed to -1560/-1bp pStinger;5HR-GAL4 showed this strong autofluorescence and as such, Malpighian tubule data was excluded from the rest of this section. phenobarbital-induced -1560/-1bp; 5HR-

GAL4/w1118 larvae showed strong expression in the midgut and fat body (Figure 2.25(A-B)).

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A B

Neg control FP WL

FP WL

Phosphoimage Commassie stain

Figure 2.24: Gelshifts prepared for Protein sequencing (A) -288/-168bp gelshift phosphoimage to detect where the largest concentration of proteins were (Boxed, arrowed). (B) The corresponding area was cut out from the non-radioactive lane run next to the radioactive lane. This lane was commassie stained to visualise the lanes better. Note that the area where the shift is concentrated

(Boxed, arrowed) does not match the darkest staining on the commassie stained gel. A equivalently- sized band was cut out from an empty lane to serve as a negative control.

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Table 2.2: Top protein hits from EMSA protein band sequencing

Rank Identity

1 Larval serum protein 1 beta chain

2 60s acidic ribosomal protein PO

3 Glyceraldehyde-3-phosphate dehydrogenase 1

4 Tubulin alpha-1 chain

5 Protein disulfide-isomerase

6 L-lactate dehydrogenase

7 14-3-3 protein zeta

8 Alpha-actinin

9 Translationally-controlled tumour protein homolog

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Nuclear Receptors

The nuclear receptor family is extensively involved in xenobiotic induction, with the Constitutive

Androstane Receptor (CAR) gene considered the primary transcription factor regulating P450 phenobarbital induction in mammals (TOLSON and WANG 2010). The Drosophila orthologue of CAR,

HR96, has been shown to be involved in phenobarbital induction, but it is unclear how much of the response it regulates (KING-JONES et al. 2006). D. melanogaster has 14 other nuclear receptor genes, and several of these were investigated if they regulated phenobarbital induction.

Ecdysone receptor (EcR), HNF4, E75, Hr39 and HR78 were knocked down with -1560/-1bp, 5HR-GAL4 and F1 larvae exposed to phenobarbital. GFP was still able to be induced in these lines (Figure 2.25), suggesting that these transcription factors were not involved in xenobiotic induction with the levels of knockdown achieved.

EcR UAS-RNAi/-1560/-1bp ; pStinger 5HR-GAL4 larvae were unable to eclose and died in their pupal cases (Data not shown). This suggested that the amount of knockdown achieved with the -1560/-1bp

; 5HR-GAL4 driver was sufficiently high to cause lethality in this line. The GAL4/UAS system has been shown to have spatial and temporal variation in expression, and this may have caused the amount of reduction caused by EcR RNAi in pupae and larvae to be different (MARKSTEIN et al. 2008). As such, the effect of knocking down Ecdysone receptor on xenobiotic induction is still uncertain.

HR96

HR96 is a nuclear receptor gene reported to be involved in xenobiotic induction. However, HR96 was not reported to regulate Cyp12d1 induction (KING-JONES et al. 2006). To confirm this, The -1560/-

1bp pStinger 6A line was crossed into two separate HR96 null (HR96e25 and HR9616A) lines homozygous 2nd chromosome -1560/-1bp insert and 3rd chromosome HR96 null stocks established

(i.e. -1560/-1bp HR96e25 and -1560/-1bp;HR9616A, Figure 2.26). These lines were then characterised for phenobarbital induced and uninduced expression.

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Figure 2.25: Nuclear Receptor transcription factor knockdowns inductions

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Figure 2.25: Nuclear Receptor transcription factor knockdowns inductions. (A) - (B) Phenobarbital - induced -1560/-1bp; 5HR-GAL4/ w1118 control showed strong midgut (A) and fat midgut (B) GFP expression. (C)-(D) Ecdysone UAS-RNAi/ -1560/-1bp;5HR-GAL4 progeny still showed midgut induction and fat body induction (D). (E)-(F) HNF4 UAS-RNAi/-1560/-1bp; 5HR-GAL4 F1 larvae induced midgutexpression (F) and fat bodyexpression (G)-(H). E75 UAS-RNAi/-1560/-1bp pStinger;

5HR-GAL4. Larvae still showed midgut (E) and fat body (F) induction. (I)-(J) HR39 UAS-RNAi/-1560/-

1bp ; 5HR-GAL4 had induction in the midgut (I) and fat body (J). (K)-(L) HR78 UAS-RNAi/-1560/-1bp pStinger ;5HR-GAL4 larvae showed midgut induction (K) and fat body induction (L).

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Figure 2.26: -1560/-1bp pStinger;HR96 null lines phenobarbital-induced GFP expression.

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Figure 2.26: -1560/-1bp pStinger;HR96 null lines phenobarbital-induced GFP expression. (A)-(C) -1560/-

1bp pStinger;HR9616A. GFP expression is still seen in the Midgut (A), Malpighian tubules (B) and fat body

(C). (D) RT-PCR showed GFP mRNA induction in -1560/-1bp pStinger;HR9616A larvae compared to unexposed controls. (E)-(G) -1560/-1bp pStinger ; HR96e25. GFP Induction was present in the Midgut (F),

Malpighian tubules (G) and fat body (I) when larvae are exposed to phenobarbital. (H) It can be seen from RT-PCR data that GFP is still being induced in -1560/-1bp pStinger; HR96e25 mutants. (I) A schematic description of the HR96 alleles in the lines used. HR96e25 has a cassette inserted in the HR96 coding region through targeted recombination, disrupting the gene. HR9616A is essentially the same allele, but with the GFP gene excised. RT-PCR data is from three biological replicates, with error bars representing

Standard error of the mean (SEM).

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Both -1560/-1bp pStinger;HR96 null lines still showed GFP induction in the midgut, Malpighian tubules and fat body, showing that Cyp12d1 induction was not affected by the loss of HR96. This confirmed microarray data published in King-Jones et al. that found that HR96 null mutants did not affect Cyp12d1 induction, and indicated that other unknown transcription factors mediated Cyp12d1 phenobarbital induction.

PAS-bHLH Family

The Aryl hydrocarbon receptor (AhR) regulates xenobiotic induction in the CYP1 family. The D. melanogaster orthologue Spineless (Ss) has been shown to regulate antennal development (EMMONS et al. 1999). The Drosophila Ss gene was investigated for Cyp12d1 xenobiotic induction regulation.

A Spineless RNAi line was crossed to -1560/-1bp pStinger;5HR GAL4 driver lines and offspring exposed to phenobarbital. Induced expression was still observed in the midgut and fat body (Figure 2.27), implying that the Ss pathway is not important for Cyp12d1 phenobarbital induction at the levels of knockdown achieved.

GATA family

The GATA family regulates a wide range of developmental and immune processes in Drosophila (BROWN and CASTELLI-GAIR HOMBRIA 2000; QIAN and BODMER 2009; SENGER et al. 2006). Five GATA factors are present in Drosophila. Pannier and GATAe expression were knocked down using the -1560/-1bp ; 5HR

GAL4 driver. GFP was still induced in the midgut and fat body (Figure 2.28), suggesting that these genes did not regulate Cyp12d1 phenobarbital induction at the levels of knockdown achieved with this cross.

Unfortunately due to time constraints three other GATA factors (Serpent, Grain and GATAd) were not tested, and it is not known if they are able to regulate Cyp12d1 phenobarbital induction.

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Figure 2.27: PAS-bHLH transcription factor knockdown inductions. (A)-(B) Phenobarbital-induced -1560/-

1bp ; 5HR-GAL4/ w1118 control showed strong midgut (A) and fat body (B) GFP expression. (C)-(D) Ss

UAS-RNAi/ -1560/-1bp ; 5HR-GAL4 showed midgut induction [arrowed, (C)] and fat body induction (D) when exposed to phenobarbital. (E)-(F) Ss UAS-RNAi/-1560/-1bp ; 5HR-GAL4. Larvae still were able to induce midgut GFP (E) and fat body GFP (F) expression upon phenobarbital induction.

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Figure 2.28: GATA transcription factor knockdown inductions. (A)-(B) Phenobarbital-induced -1560/-

1bp;5HR-GAL4/ w1118 control showed strong midgut (A) and fat body (B) GFP expression. (C)-(D) GATAe

UAS-RNAi/ -1560/-1bp ; 5HR-GAL4 showed midgut induction (C) and fat body induction (D) when exposed to phenobarbital. (E)-(F) Pannier UAS-RNAi/-1560/-1bp ; 5HR-GAL4. Larvae still were able to induce midgut GFP (E) and fat body GFP (F) expression upon phenobarbital induction.

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2.3.5.4 Transcription Factor Identification Summary

Three methods were used to identify Cyp12d1-regulating transcription factors. Site-directed mutagenesis identified GATA factor binding as being crucial for Cyp12d1 midgut expression. Targeted knockdown of transcription factors by RNAi did not find any Cyp12d1-regulating transcription factors.

Several of the transcription factors investigated here have been shown to regulate genes involved in developmental processes. With the exception of EcR, none of the transcription factors tested showed lethal phenotypes when knocked down, strongly suggesting that this series of experiments may be misleading, due to insufficient knockdown of transcription factors achieved. Electromobility-shift assay protein band sequencing also failed to identify any candidate transcription factors, but it appears that this was due to an insufficiently optimised EMSA protocol, rather than inherent flaws in this technique.

This approach perhaps would be more successful when repeated with shorter DNA probes and better- optimised protocols.

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2.4 Discussion

Cyp12d1 is arguably the most inducible P450 in the D. melanogaster genome, and Cyp12d1 regulation was used study Drosophila P450 induction. This study was the first to examine the regulation of the

Cyp12d1 gene and identify enhancers responding to phenobarbital and caffeine- induction. 3rd instar phenobarbital- and caffeine- responsive elements were mapped to a 120bp 5’ promoter sequence, while basal enhancers were found both upstream and downstream of the Cyp12d1 coding sequence.

GATA factors were crucial for basal midgut and fat body Cyp12d1 expression, but not for basal

Malpighian tubule or phenobarbital-induced Malpighian tubule and fat body Cyp12d1 expression.

2.4.1 Cyp12d1 induction is tissue-specific

This study found that phenobarbital-induced GFP fluorescence was only seen in natively Cyp12d1- expressing tissues. This showed that induction was tissue specific and emphasised the importance of using an in vivo whole Drosophila approach which allows the determination of tissue-specific expression patterns. Cell culture systems do not permit the investigation of tissue specific expression, and are also limited to only identifying enhancer regions which respond to xenobiotic induction and the magnitude of the response. Using an in vivo Drosophila approach also allows the examination of induction in a proper biological context and it can be safely assumed that all the biological components required for phenobarbital induction are in place. Schneider's L-2 (Sl-2) cells are the most common Drosophila cell culture system used to study induction. Sl-2 cells are derived from Oregon R late embryonic stage

rd embryos (SCHNEIDER 1972), and as such would have an inherently different expression to 3 instar larval digestive tract cells, especially after decades of continuous cell passages.

Cyp12d1 was only induced 1.7-fold in SL-2 cells after a 24 hour incubation with added 1mM phenobarbital (Adam Southern, personal communication) while in 3rd instar y; cn bw sp larvae it is induced 30 fold after a four hour exposure to 10mM phenobarbital-supplemented fly food plates. While

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this difference in fold-change may be due to different induction experimental protocols and different background strains, it also suggests that SL-2 cells lack essential components needed for induction.

2.4.2 Cyp12d1 model of regulation

A model of Cyp12d1 transcriptional regulation of Cyp12d1 in the w1118 strain is presented in Figure 2.29.

Cyp12d1 has 5’ and 3’ regulatory regions that work in concert to regulate Cyp12d1 induced and uninduced expression. 3rd instar Cyp12d1 anterior (M1-M3 sections) and posterior midgut (M6-M13 sections) basal expression is mostly controlled by downstream +2049/+2116bp enhancers, with some basal anterior midgut expression driven by the upstream -288/-168bp region GATA site. Malpighian tubule expression is likely to be jointly driven by elements in both the -288/-168bp region and the

+2049/+2116bp region as the -288/-168bp and +2049/+2116bp constructs each showed strong uninduced Malpighian tubule expression. Basal fat body expression was observed in both the -288/-

168bp and the +2049/+2116bp constructs and indicated that basal fat body enhancers are found in both regions. Negative regulation was also observed. The -670/-492bp region repressed basal midgut, fat body and Malpighian tubule expression and delayed phenobarbital-induced GFP expression.

2.4.2.1 Investigating the -288/-168bp region further

The -288/-168bp region was identified as the smallest fragment capable of inducing Cyp12d1 xenobiotic expression as well as regulating uninduced anterior midgut, fat body and Malpghian tubule expression. phenobarbital and caffeine responsive elements were mapped to the -288/-168bp region. These enhancers drove induced GFP expression in the midgut (M1-M3, M6-M13 sections), Malpighian tubules. and fat body. At this stage, it is still uncertain if the induction enhancers and basal enhancers are separate sequences, or if the same enhancers regulate both induced and basal expression. Further narrowing down of this region by smaller constructs might resolve this issue by testing if it was possible to separate the induction enhancers from basal enhancers. This might also help identify the exact

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Figure 2.29: Model of Cyp12d1 cis-regulation. A single-copy Cyp12d1 locus is presented here. Basal midgut, Malpighian tubule and Fat body expression is driven by 3’ +2049/+2116bp and 5’-288/-168bp enhancers working in concert together. Phenobarbital and caffeine induction is only controlled by the upsteam -288/-168bp region. The -670/-492bp repressing region represses both basal and induced expression, which is alleviated by additional unknown factors in the -1560/-1100bp region.

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location of the Cyp12d1 PBRE sequences. Several putative transcription factor binding sites were identified using the Matinspector program. Perhaps the best way to investigate these binding sites would be to utilise site-directed mutagenesis to show that these binding sites are important for Cyp12d1 regulation.

The -288/-168bp pH-Stinger construct, as well as other constructs, should be tested for response to other chemicals like those performed for Cyp6a2 in (GIRAUDO et al. 2010). This will serve to check if the

288/-168bp region mediated induction to other chemicals such as DDT, Aroclor 1254, and Limonene, besides phenobarbital and caffeine, or if additional sequences were important for response to other chemicals.

2.4.2.2 Cyp12d1 Repressive region

The -670/-492bp region repressed basal midgut and Malpighian tubule expression and delayed phenobarbital-induced GFP expression. Other P450s have also been reported to have repressors in upstream promoter regions. The P. polyxenes Cyp6B1 gene has been shown to have an upstream region repressing transcription (PETERSEN BROWN et al. 2004). Analysis of the Cyp1A1, Cyp2A2, Cyp2E1 and

Cyp3A2 promoter regions found a repressor element 1/neuron-restrictive silencing element (RE1/NRSE) sequence in their 5’ regions (GARCÍA-SÁNCHEZ et al. 2003). The RE1 silencing transcription factor/neuron- restrictive silencing factor was shown to bind to these RE1/NRSE sequences in vitro, indicating that these sites were able to repress P450 repression (GARCÍA-SÁNCHEZ et al. 2003). The D. melanogaster Cyp6a2 gene also has a repressive region in its upstream region (BHASKARA et al. 2006). This suggests that negative control of P450 transcription is another component used by organisms to finetune their P450 expression.

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2.4.3 GATA factors regulate P450 midgut expression

Drosophila has five GATA family members. Grain regulates adult leg development through controlling epithelial cell rearrangements (BROWN and CASTELLI-GAIR HOMBRIA 2000). Pannier is a well-known GATA transcription factor regulating a wide range of tissue development including heart development and function (ALVAREZ et al. 2003; QIAN and BODMER 2009) and dorsal closure (HEITZLER et al. 1996) amongst others. GATAd and GATAe are essential for correct endoderm and hence midgut formation (MURAKAMI et al. 2005; OKUMURA et al. 2005). Serpent is involved in immune responses (SENGER et al. 2006), and also activates GATAe expression in the early endoderm (SENGER et al. 2006).

Basal midgut and fat body, and induced midgut expression was lost when the GATA binding site in the -

288/-168bp kb construct was mutated. This mutation has been shown to invoke a loss of GATA binding

(LUDLOW et al. 1996). Presumably, the loss of basal and phenobarbital-induced midgut expression was due to the loss of GATA transcription factor binding after the GATA site was mutated. Malpighian tubule basal expression, Malpighian tubule and fat body induction was still observed in the mutant. This suggested that while GATA factors were important for midgut expression, they were not involved in

Malpighian tubule expression and fat body induction, and that some other known transcription factor(s) was controlling Cyp12d1 expression in these tissues.

Other studies also implicate GATA factors in regulating D. melanogaster P450 expression. Cyp6g1 midgut expression is also dependent on GATA binding sites. Mutation of two overlapping GATA sites in the Cyp6g1 promoter leads to a loss of basal and phenobarbital-induced midgut expression, similar to

Cyp12d1 (CHUNG et al., SUBMITTED). Cyp6a2 expression and induction was also lost when a 5’ GATA binding site was deleted (Carl Thummel, personal communication). Subsequent protein expression and gel shift assay experiments found that GATAe bound to the Cyp6a2 promoter (Thummel, pers comm).

Ectopic expression of GATAe in the fat body resulted in the upregulation of several P450s including

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Cyp6g1, Cyp6a2, but importantly not Cyp12d1 (SENGER et al. 2006). This suggests that Cyp12d1 is not regulated by GATAe, and that the Cyp12d1 GATA binding site did not bind GATAe but instead attracted a different GATA factor protein.

Mammalian GATA transcription factors have also been reported to regulate P450 expression. Cyp2C9 and Cyp2c19 both require the input of GATA-4 and GATA-6 transcription factors binding to GATA binding sites in their 5’ promoter region in order to for proper expression (CAI et al. 2007; MWINYI et al. 2010).

Based on this, P450 regulation by GATA transcription factors seems to be crucial in both vertebrates and invertebrates.

The question remains: which GATA factor is regulating this expression? Although GATAe has already been found to be involved in P450 regulation in several studies, Cyp12d1 was not found to be regulated by GATAe. Perhaps the answer to this question has to wait till the elusive Cyp12d1-regulating transcription factors have been identified either through the protein sequencing method used here or other approaches.

2.4.4 P450 Induction Pathways in D. melanogaster

Cyp12d1 induction was used to identify xenobiotic induction pathways in Drosophila. Candidate transcription factor families were chosen based on Drosophila orthologs of mammalian transcription factors identified as being involved in P450 induction.

The first candidate is the nuclear receptor family. Members of the nuclear receptor family regulate a variety of essential processes in mammals, insects and birds among others. Cyp6g1 phenobarbital induction was reduced when a putative CAR-like binding site was mutated (CHUNG et al., SUBMITTED), pointing to nuclear factors regulating Cyp6g1 induction. However, direct evidence for binding of this site by proteins has not been found and the identity of the transcription factor binding to and presumably upregulating Cyp6g1 expression is not known. The mammalian nuclear receptor CAR ios considered the

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main regulators of the transcriptional response to phenobarbital. The nuclear receptor genes tested did not show any loss of induction when knocked down, suggesting these genes were not essential for phenobarbital induction at the levels of knockdown achieved in this screen. As only six of the 18

Drosophila nuclear receptor genes were tested in this study, it is possible that an untested member might be responsible for regulating phenobarbital induction. However, it is not possible to conclude that these genes are definitively regulating Cyp12d1 induction without any quantitative data on Cyp12d1 and nuclear receptor transcription factor expression. HR96 has been identified as a transcription factor regulating some phenobarbital induction through the use of targeted gene knockout (KING-JONES et al.

2006). HR96 is the closest Drosophila orthologue of PXR and CAR (KING-JONES and THUMMEL 2005), showing a high level of conservation between mammalian induction systems and Drosophila induction networks. Five P450s lost induction when HR96 was knocked out, suggesting that the majority of P450s were regulated by other transcription factors (KING-JONES et al. 2006). Induced GFP transcription was still observed in Cyp12d1-reporter constructs crossed into a HR96 null background, suggesting that Cyp12d1 is not regulated by HR96. Taken together, this suggests that HR96 may not be the main regulatory transcription factor for phenobarbital induction and that another unknown transcription factor may be responsible for the majority of the response.

However, mice studies indicated that more than half of all xenobiotic-inducible genes still continued to be induced when PXR or CAR were knocked out (UEDA et al. 2002). This points to the complexity of the induction response and the possibility of functional redundancy between xenobiotic induction transcription factors. It is still possible that HR96 still plays a large role in the regulation of phenobarbital and that other unknown transcription factors compensate for the loss of HR96.

HR96 has been implicated together with the Broad-complex (BR-C) gene to regulate Musca domestica

Cyp6d1 phenobarbital induction in Drosophila Sl-2 cell culture (LIN et al. 2011). Putative HR96 and BR-C

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binding sites were identified in the smallest phenobarbital-inducible promoter fragment of Cyp6d1.

Using dsRNAi to knock down HR96 and BR-C expression individually abolished and increased phenobarbital -induction respectively. These data supports HR96 as a positive controller of induction and identifies BR-C as a novel induction negative regulator. However, it is not clear from the data if BR-C is directly binding to and regulating the Cyp6d1 promoter, or if BR-C is affecting some other upstream component in the gene network. The limitations of the Sl-2 cell culture system for studying induction have already been discussed in Section 1.4.2.3. Furthermore, this was a study investigating the induction of a M. domestica P450 in a D. melanogaster embryonic cell line. It is unclear from these results if BR-C and HR96 regulate phenobarbital induction in whole M. domestica adults and larvae. BR-C and HR96 protein functions should be further investigated in M. domestica flies to determine if these proteins regulate Cyp6b1 phenobarbital induction in whole M. domestica flies. Cyp12d1 has a canonical BR-C Z4 isoform binding site located at -142/-136bp. Site-directed mutation of this site in the -288/-168bp sequence would help understand the role of BR-C better in the phenobarbital-mediated induction of

Cyp12d1.

Another possible nuclear receptor gene involved in Drosophila xenobiotic induction is HNF4. HNF4α in mammals is needed for the full induction of several P450s (GOODWIN et al. 1999; JOVER et al. 2009;

TIRONA et al. 2003). The Drosophila orthologue HNF4 has been shown to be engaged in lipid homeostasis through the regulation of lipid mobilisation from fat reserves (PALANKER et al. 2009), a function which has also been described in vertebrates (YIN et al. 2011). HNF4’s role in Drosophila xenobiotic induction has not been examined. Given that the Drosophila and mammalian orthologues already share a degree of functional conservation, it is possible that HNF4 is involved in xenobiotic induction of Drosophila P450s.

HNF4 is expressed in the midgut, Malpighian tubules and fat body, sites where P450 induction takes place. The phenobarbital induction response of HNF4 null mutants should be examined to determine if

HNF4 is involved in phenobarbital induction.

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Other transcription factors involved in mammalian P450 xenobiotic induction have Drosophila orthologues. The PAS-bHLH family member Aryl Hyrdocarbon receptor regulates CYP1 family induction

(FUJII-KURIYAMA and MIMURA 2005). The Drosophila orthologue, Spineless, was shown to be crucial for P. polyxenes Cyp6B1 xenobiotic induction in Sf9 cells (PETERSEN BROWN et al. 2005). Spineless was not found to regulate Cyp12d1 phenobarbital induction in this screen when knocked down with the -1560/-1bp pStinger;5HR driver. However, this does not rule out Spineless‘s involvement in xenobiotic induction in response to other inducing agents, particularly hydrocarbons. as the Aryl hydrocarbon receptor has been shown to regulate xenobiotic response to hydrocarbons. Spineless’s role in hydrocarbon induction should be tested before it is possible to conclude Spineless involvement in xenobiotic induction.

Of the transcription factors tested in this screen, none gave any positive results at the levels of reduction achieved. However, it must be noted that the RNAi technique does not completely abolish all gene expression, and it allows a small amount of residual expression. This may be particularly important in transcription factor studies, where reduced levels of transcription factor may still be able to mediate gene regulation. It would be perhaps a better test if null alleles of transcription factors were tested for

Cyp12d1 induction. However, this could be complicated by redundancy between transcription factors, which might compensate for the loss of a deleted transcription factor, thereby leading to false positive results. Pleiotropic effects from inactivating these genes, such as developmental lethality, may also confound any results obtained.

If a candidate is strongly implicated, it is vital to perform further experiments. One approach would be to compare transcriptional responses in genetic null mutants against wild-type flies. This approach has been used with some success when HR96 was investigated (HORNER et al. 2009; KING-JONES et al. 2006).

Overexpressing the candidate transcription factor to look for an altered induction response may reveal roles in induction.

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Biochemical assays should also be performed. Electromobility shift assays can be performed nuclear protein extracts from transcription factor null mutants and wild type flies to check for any loss of protein binding to Cyp12d1 promoter regions in null mutant protein extracts. Chromatin immunopercipitation assays and EMSA supershift experiments using antibodies specific to the transcription factor may confirm if the protein is indeed involved in phenobarbital induction.

2.5 Chapter Conclusion

The regulation of the D.melanogaster Cyp12d1 gene was investigated in this chapter. Cyp12d1 basal expression enhancers were mapped to a 120bp upstream and a 67bp downstream Cyp12d1 genomic regions. A repressive region potentially involved in negative regulation of Cyp12d1 was also identified. phenobarbital and caffeine induction enhancers were detected in the 120bp upstream region that was found to contain some basal enhancers. It was unclear if the same enhancers were responsible for basal and induced expression, or that separate elements in this 120bp region regulated induction and basal expression independently. Finally, a GATA factor binding site was found to be crucial for regulating induced and basal midgut expression in addition to basal fat body expression.

Cyp12d1 is arguably the most xenobiotic inducible Cytochrome P450 gene in the Drosophila genome, and studying how Cyp12d1 is regulated has helped in our understanding of Drosophila induction.

Investigating Cyp12d1 functions will also help understand why Cyp12d1 is so inducible, and allow us to better understand xenobiotic induction in Drosophila melanogaster.

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Chapter Three: Cyp12d1 Functional studies

A dying drosophila exclaimed,

"A geneticist has poisoned my brain!"

The cause of his sorrow

Was para-dichloro-

Diphenyl-trichloroethane.

- unknown

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3.1 Introduction

In Drosophila, different members of the P450 family have been shown to play important roles in hormone biosynthesis and catabolism (BECKSTEAD et al. 2005; GUITTARD et al. 2011; HUANG et al. 2008;

NIWA et al. 2004; ONO et al. 2006), tissue development (MOHIT et al. 2006; WILLINGHAM and KEIL 2004), insecticide resistance (DABORN et al. 2007; DABORN et al. 2002) and behaviour modification (DIERICK and

GREENSPAN 2006) (Refer to Section 1.2.2). In this chapter, the function of Cyp12d1 is investigated.

Cyp12d1 is also able to confer insecticide resistance when overexpressed. The first evidence for this emerged when DDT resistant lines derived from natural populations were found to overexpress Cyp12d1

(BRANDT et al. 2002). Subsequently, transgenic over-expression of Cyp12d1 was shown to confer resistance to the insecticides DDT and dicyclanil (DABORN et al. 2007). These studies suggest that

Cyp12d1 has a role in xenobiotic detoxification function.

In addition to roles in insecticide detoxification, Cyp12d1 has been found to respond at the transcriptional level to different environmental stresses. For example, Cyp12d1 is downregulated in D. melanogaster lines selected for rapid chill coma recovery (TELONIS-SCOTT et al. 2009). Flies transgenically overexpressing the antioxidant enzyme Manganese Superoxide Dismutase (MnSOD) upregulate Cyp12d1 expression (CURTIS et al. 2007). These flies were found to have a significantly longer lifespan and increased resistance to oxidative stress, indicating that Cyp12d1 may be involved in these functions.

3.1.1 Chapter aims

Cyp12d1 function was investigated in this chapter. The GAL4-UAS system was used to drive overexpression of Cyp12d1 as well as the selective reduction of Cyp12d1 expression to identify functions for Cyp12d1. The lifespan and oxidative stress resistance of these lines were investigated to discover if altering Cyp12d1 mRNA levels in these flies affected their phenotypes. This may help explain the role

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Cyp12d1 plays in flies, and to determine if Cyp12d1 is only required when responding to chemical induction, or if it is involved in other essential functions in Drosophila.

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3.2 Materials and Methods

3.2.1 Cyp12d1 RNAi construction

Cyp12d1 RNAi lines were constructed using the pWiz vector (SIK LEE and CARTHEW 2003). A 236bp

Cyp12d1 coding sequence from +1316 to +1551 with <18bp-long homology to other genes was identified. Primers flanking this 236bp region were designed with additional NheI restriction sites incorporated. These primers were used to amplify the 236bp fragment from w1118 3rd instar cDNA. This was then cloned into the pWiz vector in both sense and anti-sense directions to form a complementary hairpin loop. The clones were sequenced to check for correct sequence and orientation. The completed

Cyp12d1 UAS-RNAi pWiz vector was then injected into w1118 embryos using standard transformation techniques and two independent homozygous transgenic RNAi lines established (Tub-GAL4/Cyp12d1-

RNAi-4A, a 2nd chromosome insertion line and Tub-GAL4/Cyp12d1-RNAi-7A, a 3rd chromosome insertion line).

Virgin female four day old Tub-GAL4/Cyp12d1-RNAi-4A and Tub-GAL4/Cyp12d1-RNAi-7A RNAi females were crossed to homozygous Tubulin-GAL4/TM3,Sb driver males (Tub-GAL4) and heterozygous F1 four day old males collected. Male w1118 flies were crossed to four day-old virgin Tub-GAL4/Cyp12d1-RNAi-4A or Tub-GAL4/Cyp12d1-RNAi-7A to form an Cyp12d1-RNAi-4A/w1118 control or an Cyp12d1-RNAi-4A/w1118 control. Tubulin-GAL4/TM3,Sb driver males were crossed to virgin female four day old w1118 flies to create the Tub-GAL4/w1118 control. RNA was extracted and RT-PCR done to quantify the amount of

Cyp12d1 expression compared to control lines.

RNA was extracted using Trizol reagent (Invitrogen). RNA was quantified using the Qubit Quantification

Platform (Invitrogen). cDNA was made using the Superscript II Reverse Transcriptase system

(Invitrogen). Cyp12d1 real time primers which recognised a shared conserved region between Cyp12d1- d’ and Cyp12d1-p’ were used to quantify the total amount of Cyp12d1 transcript (Primer sequences in

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Appendix I). The housekeeping gene RPL11 was used as an internal control for comparison between samples.

Real-time PCR (RT-PCR) was performed using a QuantiTect SYBR Green PCR kit (Qiagen) on a RotorGene-

3000 (Corbett Research, Sydney) to quantify the amount of GFP transcript produced. Annealing temperature was set at 55°C and runs set to 45 cycles. Three biological replicates were analysed. The amount of GFP and the housekeeping gene RPL11 mRNA was quantified using the Standard curve method of quantification with the Corbett RotorGene-3000 Realtime analysis program (Corbett

Research, Sydney).

3.2.2 Cyp12d1 RNAi impact on viability

Viability at 25°C

Three replicate vials each containing five four-day old virgin females from Cyp12d1-RNAi-4A or Cyp12d1-

RNAi-7A Cyp12d1 RNAi lines were crossed to five four day old Tubulin-GAL4/ TM3,Sb driver males and allowed to mate and lay eggs for three days at 25°C to test for progeny survival at 25°C (Figure 3.2).

Adults were then transferred to another vial and left to lay for another three days at 29°C to examine survival of offspring raised at 29°C. F1 adults were sorted between Cyp12d1-RNAi/Tub-GAL4 flies and

Cyp12d1-RNAi/ TM3,Sb lines and numbers of each genotype recorded.

3.2.3 Longevity Assay

Virgin females from two Cyp12d RNAi lines (Cyp12d1-RNAi-4A and Cyp12d1-RNAi-7A) were crossed to

Tub-GAL4/TM3,Sb flies to ubiquitously reduce Cyp12d1 expression. Tub-GAL4/TM3,Sb males were crossed to four-day old w1118 virgin females to establish the Tub-GAL4/ w1118 control line. w1118 males were crossed to virgin Cyp12d1-RNAi-4A or Cyp12d1-RNAi-7A four-day old females to generate

Cyp12d1-RNAi-4A / w1118 or Cyp12d1-RNAi-7A / w1118 flies. For each cross, approximately 100 virgin four-

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day old females were crossed to 30 male Tub-GAL4/TM3,Sb driver males in individual population cages and allowed to lay eggs on grapefruit agar plates. Plates were changed every day and embryos were collected. An equal amount of embryos from each genotype were deposited in bottles for rearing at

25°C. Bottles were cleared once a day and F1 flies allowed to intermate before 50 F1 four-day old mated male flies were collected from each genotype. Flies were divided into five groups of ten flies and placed into replicate empty plastic vials. A 1cm2 piece of folded kimwipe paper (Kimberly-Clark) was placed into each vial. 300ul of 250mM hydrogen peroxide (Ajax Chemicals) diluted in 1% sucrose solution was pipetted onto each square. The kimwipe squares were changed every 48 hours to prevent flies dying from desiccation. The assay was ended at 96 hours and the numbers of living flies recorded. Mortality was analysed with the log-rank statistical test using GraphPad Prism statistical software (GraphPad,

California).

3.2.4 Hydrogen Peroxide assay

Virgin females from two Cyp12d1 RNAi lines (Cyp12d1-RNAi-4A and Cyp12d1-RNAi-7A) were crossed to

Tub-GAL4/TM3,Sb flies to ubiquitously reduce Cyp12d1 expression. Tub-GAL4/TM3,Sb males were crossed to four-day old w1118 virgin females to establish the Tub-GAL4/ w1118 control line. w1118 males were crossed to virgin Cyp12d1-RNAi-4A or Cyp12d1-RNAi-7A four-day old females to generate the

Cyp12d1-RNAi-4A / w1118 or Cyp12d1-RNAi-7A / w1118 flies. For each cross, approximately 100 virgin four-day old females were crossed to 30 male Tub-GAL4/TM3,Sb driver males in individual population cages and allowed to lay eggs on grapefruit agar plates. Plates were changed every day and embryos were collected. An equal amount of embryos from each genotype were deposited in bottles for rearing at 25°C. Bottles were cleared once a day and F1 flies allowed to intermate before 50 F1 four-day old mated male flies were collected from each genotype. Flies were divided into five groups of ten flies and placed into replicate empty plastic vials. A 1cm2 piece of folded kimwipe tissue paper (Kimberly-Clark)

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Figure 3.1: Crossing scheme to test for the impact of Cyp12d1 knockdown on viability. Male Tub-

GAL4//TM3, Sb flies were crossed to virgin Cyp12d1-RNAi females and offspring allowed to develop.

Emergent flies were counted and the ratio of Tub-GAL4/Cyp12d1-RNAi to Cyp12d1-RNAi/ TM3,Sb chromosome offspring was determined. An even ratio would be expected if a reduction in Cyp12d1 expression reduction did not impact viability at all. At the other extreme, if Cyp12d1 knockdown is lethal, then no Tub-GAL4/Cyp12d1-RNAi offspring would be observed.

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was placed into each vial. 300ul of 250mM hydrogen peroxide (Ajax Chemicals) diluted in 1% sucrose solution was pipetted onto each square. The kimwipe paper squares were changed every 48 hours to prevent flies dying from desiccation. The assay was ended at 96 hours and the numbers of living flies recorded. Mortality was analysed with the one-way ANOVA statistical program using GraphPad Prism statistical software (GraphPad, California).

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3.3 Results

Cyp12d1 functions were investigated using the UAS-GAL4 system. Two approaches were used, Cyp12d1 overexpression and Cyp12d1 underexpression using RNAi. All experiments were conducted in a Cyp12d1 single-copy w1118 background. For consistency, four-day old male flies were used in all experiments.

3.3.1 Cyp12d1 Overexpression

Two UAS-Cyp12d1 overexpression lines previously characterised by Daborn et al. were used in this study

(DABORN et al. 2007). The Cyp12d1-UAS-11c line overexpresses Cyp12d1 67-fold higher compared to

Cyp12d1-UAS-11c/w1118 controls when driven by the 5HR-GAL4 driver, which drives expression in the midgut, fat body and Malpighian tubules compared to Cyp12d1-UAS-11c/w1118 controls while the

Cyp12d1-UAS-41A line overexpresses 37-fold compared to the Cyp12d1-UAS-41A/w1118 control. These two lines were also selected to investigate the possible significance of the three amino acid substitutions between the two alleles.

3.3.2 Cyp12d1 RNAi quantification

The level of Cyp12d1 expression in the Cyp12d1-RNAi lines was quantified by quantitative real-time PCR

(RT-PCR) experiments.

3.3.2.1 Cyp12d1 mRNA quantification in Cyp12d1 ubiquitously-reduced flies

Table 3.1 shows the mean levels of Cyp12d1 expression in the Tub-GAL4/Cyp12d1-RNAi lines compared to the Tub-GAL4/w1118 control. Both the Tub-GAL4/Cyp12d1-RNAi-4A and Tub-GAL4/Cyp12d1-RNAi-7A lines showed a reduction in Cyp12d1 expression of approximately 70% compared to the Tub-GAL4/w1118 control. These lines were then phenotyped to determine the effects of ubiquitously reducing Cyp12d1 expression.

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Table 3.1: Cyp12d1 mRNA expression in Tub-GAL4/Cyp12d1- RNAi lines and controls

Cyp12d1 mRNA level relative to Expression ratio* Significance^ RPL11 mRNA level

Tub-GAL4/w1118 0.154 1.00

Tub-GAL4/UAS-RNAi 4A 0.043 0.28 P<0.05

Tub-GAL4/UAS-RNAi 7A 0.048 0.31 P<0.05

*normalised to Tub-GAL4/w1118 expression

^ One-way ANOVA

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3.3.2 Viability assay

Cyp12d1-RNAi-4A and Cyp12d1-RNAi-7A were crossed to the Tub-GAL4 driver and offspring raised at

25°C. Both Cyp12d1-RNAi-4A and Cyp12d1-RNAi-7A lines showed normal viability when crossed to the

Tub-GAL4 driver (Figure 3.2).

GAL4 protein has greater activity at higher temperatures (DUFFY 2002), which is predicted to result in more reduction of Cyp12d1 mRNA in flies raised at 29°C compared to 25°C. However, again there was no difference in viability between either of the RNAi lines and controls (Figure 3.2). The Tub-GAL4/UAS-

RNAi flies reared at both 25°C and 29°C were carefully examined under the microscope and no abnormal phenotypes were observed.

Decreasing Cyp12d1 expression ubiquitously did not cause developmental lethality. However, the transgenic Cyp12d1-reduced lines did not totally abolish all Cyp12d1 expression (Table 3.1). This showed that a residual amount of Cyp12d1 expression is still present in these flies. The best way to test this would be to delete the Cyp12d1 gene through homologous gene targeting (MAGGERT et al. 2008) thereby removing all Cyp12d1 expression and studying the progeny for any developmental defects.

3.3.3 Longevity Assays

The lifespan of Cyp12d1-overexpression and Cyp12d1–RNAi down flies was tested to examine the effects of perturbing normal Cyp12d1 expression on the longevity of transgenic flies (Figure 3.3).

Cyp12d1-RNAi-4A and Cyp12d1-RNAi-7A lines and Cyp12d1-UAS-11c and Cyp12d1-UAS-41A lines were crossed to Tub-GAL4 and offspring reared at 25°C. Tub-GAL4/w1118 lines were used as control lines.

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Figure 3.2: Cyp12d1 Tub-GAL4/UAS-RNAi viability. Emergence was compared to the number of Cyp12d1-

RNAi/ TM3,Sb / lines which served as an internal control for the cross used (see Figure 3.1), and hence may be greater than 100%. (A) Tub-GAL4/Cyp12d1 UAS-RNAi at 25°C. No significant difference from the

Tub-GAL4/w1118 control was found when Cyp12d1-reduced offspring were raised at 25°C for both Tub-

GAL4/Cyp12d1-RNAi-4A and Tub-GAL4/Cyp12d1-RNAi-7A. (B) Tub-GAL4/Cyp12d1 UAS-RNAi at 29°C.

Again, no difference was observed between the control and tested lines when lines were raised at 29°C.

Error bars represent the Standard error of the mean (SEM).

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Figure 3.3: Cyp12d1-RNAi and Cyp12d1-overexpression longevity survival curves.

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Figure 3.3: Cyp12d1-RNAi and Cyp12d1-overexpression longevity survival curves. (A) Tub-GAL4/

Cyp12d1-RNAi-4A vs. Tub-GAL4/w1118. Tub-GAL4/ Cyp12d1-RNAi-4A had a median death of 34 days while the Tub-GAL4/w1118 line had a media death of 45 days. Tub-GAL4/ Cyp12d1-RNAi-4A showed a significantly shorter lifespan to the Tub-GAL4/w1118control when both curves were compared (Log-rank test P>0.0001). (D) Tub-GAL4/ Cyp12d1-RNAi-7A vs. Tub-GAL4/w1118. Tub-GAL4/ Cyp12d1-RNAi-7A also had a shorter lifespan compared to the Tub-GAL4/w1118 control with a median death of 34 days (Log-rank test P>0.0001). (C) Tub-GAL4/ Cyp12d1-UAS-11c vs. Tub-GAL4/w1118 survival curves. Tub-GAL4/ Cyp12d1-

UAS-11c had a median death of 42 days while the Tub-GAL4/ w1118 control line had a median death of 45 days. Tub-GAL4/UAS-11c was found to have a significantly shorter lifespan when both survival curves were compared statistically (Log-Rank test, P>0.0001) (D) Tub-GAL4/ Cyp12d1-UAS-41A vs. Tub-

GAL4/w1118. Tub-GAL4/ Cyp12d1-UAS-41A had a median death age of 34 days and had a statistically shorter lifespan compared to the Tub-GAL4/w1118 control (Log-Rank test, P>0.0001).

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Table 3.2: Mean lifespan of Cyp12d1-RNAi and Cyp12d1-overexpression flies

Line Mean Lifespan (days) Ratio*

Tub-GAL4/w1118 45 1.00

Tub-GAL4/ Cyp12d1-RNAi-4A 34 0.75

Tub-GAL4/ Cyp12d1-RNAi-7A 34 0.75

Tub-GAL4/ Cyp12d1-UAS-11c 42 0.93

Tub-GAL4/ Cyp12d1-UAS-41A 34 0.75

*Normalised to Tub-GAL4/w1118 lifespan.

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3.3.3.1 Cyp12d1-RNAi transgenic fly longevity

When Cyp12d1 expression was reduced ubiquitously, F1 progeny lifespan was shortened significantly compared to controls (Figure 3.3, Table 3.2). The Tub-GAL4/ Cyp12d1-RNAi-4A and Tub-GAL4/ Cyp12d1-

RNAi-7A survival curves were nearly identical with both having a median survival age of 34 days, compared to 45 days for the Tub-GAL4/w1118 control.

This suggests that reducing Cyp12d1 expression in a ubiquitous manner shortens lifespan, implying that

Cyp12d1 may be involved either directly or indirectly in some function which contributes to lifespan determination.

3.3.3.2 Cyp12d1-overexpression transgenic fly longevity

Cyp12d1 was overexpressed ubiquitously and the effect on lifespan measured (Figure 3.6).

Overexpressing Cyp12d1 ubiquitously significantly also shortened the lifespan of both lines tested

(Figure 3.3, Table 3.2). Both Tub-GAL4/Cyp12d1-UAS-41A and Tub-GAL4/Cyp12d1-UAS-11c survival curves were significantly shorter than the control Tub-GAL4/w1118 (Log-rank test, P>0.0001). Tub-

GAL4/Cyp12d1-UAS-41A had a median survival age of 34 days, while Tub-GAL4/Cyp12d1-UAS-11c had a median survival age of 42 days. The control Tub-GAL4/w1118 had a median survival of 45 days.

3.3.4 Oxidative stress

The Tubulin-GAL4 driver was used to decrease Cyp12d1 expression ubiquitously to test for Cyp12d1 hydrogen peroxide resistance (Figure 3.4). Tub-GAL4/Cyp12d1-RNAi-7A flies showed significant resistance to hydrogen peroxide (T-test, P<0.05). Tub-GAL4/Cyp12d1-RNAi-4A had a lower survival rate than Tub-GAL4/UAS-RNAi 7A, but was significantly different to the Tub-GAL4/w1118 control (T-test,

P=0.0161). The data suggested that ubiquitously reducing Cyp12d1 expression conferred resistance to hydrogen peroxide. As both Cyp12d1-RNAi lines showed the same amount of Cyp12d1 expression when

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measured with RT-PCR, there seemed to be a line-specific effect, with Cyp12d1-RNAi-7A more resistant than Cyp12d1-RNAi-4A.

Tub-GAL4/ Cyp12d1-UAS-11c was more resistant to hydrogen peroxide compared to the Tub-GAL4/w1118 control (T-Test, P=0.0044). Tub-GAL4/Cyp12d1-UAS-41A did not show any significant difference in survival compared to the Tub-GAL4/w1118 control (T-test, P=0.42). This suggested that overexpressing

Cyp12d1-p’ gave resistance to oxidative stress. However, given that only one Cyp12d1-p’ overexpression line was significantly resistant, it cannot be concluded from this dataset if Cyp12d1 overexpression confers resistance to hydrogen peroxide.

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**

*

**

Figure 3.4: Cyp12d1-RNAi and Cyp12d1-overexpression survival on 50mM hydrogen peroxide.

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Figure 3.4: Cyp12d1-RNAi and Cyp12d1-overexpression survival on 50mM hydrogen peroxide. (A) Tub-

GAL4/ Cyp12d1-RNAi-4A. Moderate resistance to oxidative stress was seen compared to Tub-GAL4/w1118

(T-Test, P=0.0161). (B) Tub-GAL4/ Cyp12d1-RNAi-7A. Strong resistance was seen in the Tub-GAL4/UAS-

RNAi 7A compared to the Tub-GAL4/ Cyp12d1-RNAi-4A line (T-Test, P<0.05). (C) Tub-GAL4/Cyp12d1-

UAS-11c. Significant resistance was seen in the Tub-GAL4/Cyp12d1-UAS-11c compared to Tub-GAL4/ w1118 (T-Test, P=0.0044). (D) Tub-GAL4/Cyp12d1-UAS-41A. No significant resistance was seen in Tub-

GAL4/Cyp12d1-UAS-41A compared to the Tub-GAL4/ w1118 line (T-Test, P=0.42). Error bars represent the

Standard error of the mean (SEM).

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3.4 Discussion

The xenobiotic detoxification functions of Cyp12d1 have been investigated through transgenic overexpression insecticide resistance studies (DABORN et al. 2007). This chapter investigated other possible Cyp12d1 functions in adult males.

3.4.1 Mitochondrial Cyp12d1 and possible roles in longevity and oxidative stress

The mitochondria generate large amounts of energy in the form of adenosine triphosphate (ATP), and are considered the principle source of energy in the cell (ORRENIUS et al. 2007). Mitochondria also have other essential roles and are involved in a range of other processes, such as signalling, cell death, hormone synthesis (BUTOW and AVADHANI 2004; JEZEK and HLAVATA 2005; MAYER and OBERBAUER 2003).

Mitochondria are the largest source of Reactive Oxygen Species (ROS) production in the cell, and it has been shown that reducing the expression of individual mitochondrial proteins involved in various mitochondrial processes leads to changes in lifespan and aging (COPELAND et al. 2009).

Cyp12d1 is a mitochondrial P450. It was reasoned that because of its mitochondrial localisation and that some mitochondrial P450s have been shown to be involved in mitochondrial biosynthetic pathways,

Cyp12d1 could be involved in some mitochondrial processes. The phenotype of Cyp12d1-RNAi and

Cyp12d1-overexpression down flies was therefore examined to uncover any potential Cyp12d1 involvement in mitochondrial processes, focusing specifically on the longevity of these flies and their tolerance to oxidative stress.

3.4.2 Drosophila Lifespan

Longevity can be determined by both non-genetic and genetic factors. For instance, environmental factors such as keeping flies at a cooler ambient temperature, or subjecting flies to mild stresses before assaying for lifespan, is known to increased lifespan. Dietary restriction is a major factor in determining lifespan. Caloric restriction extends lifespan in many organisms (reviewed in PARTRIDGE et al. 2005), and

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Drosophila have been shown to live longer and females produce fewer eggs when their dietary food is diluted.

The genetics of lifespan determination have been partially dissected and several pathways are known to increase lifespan in Drosophila when modified. Mutating various components of the insulin/IGF-like signalling pathway have been shown to extend lifespan. The first study to find that mutating a specific gene could extend lifespan was when the Daf-2 gene in Caenorhabditis elegans was mutated (KIMURA et al. 1997). Daf-2 is the insulin receptor gene gene in C.elegans, and the inactivation of Daf-2 doubles lifespan in worms. Similarly, mutating the Insulin receptor gene (InR) in D. melanogaster extended lifespan by up to 85% (TATAR et al. 2001), while mutating the Insulin receptor substrate (chico) extended female lifespan by 48% in homozygotes and 38% in heterozygotes (CLANCY et al. 2001). The I’m not dead yet gene (Indy) has functions in transport and storage of Krebs cycle intermediates (ROGINA et al. 2000).

Indy P-element insertion mutants have a doubling in lifespan without any loss of activity or fertility

(ROGINA et al. 2000). It has been shown that Indy interacts with caloric restriction to further extend

Drosophila lifespan (WANG et al. 2009).

Despite these, the leading accepted hypothesis explaining aging and senescence is the oxidative stress hypothesis. The free radical theory of aging states that the organism is unable to repair damage to cellular components and that over time, damage accumulates to cellular macromolecues so that organism subsequently ages and dies. Aging and oxidative stress provoked similar gene expression patterns in Drosophila, suggesting that flies react to both stresses in a common manner (LANDIS et al.

2004). Indeed, overexpressing antioxidant enzymes such as Superoxide dismutase (SOD) extends lifespan in Drosophila (CURTIS et al. 2007).

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3.4.3 P450s and oxidative stress

The mitochondria is considered a primary source of ROS production. Oxidative stress has been implicated in a variety of pathological diseases in humans including Alzheimer’s disease, cancer, diabetes and atherosclerosis (MATÉS et al. 1999), and is considered the primary cause of aging. P450s contribute to intracellular oxidative stress through uncoupling of the P450 catalytic cycle and

- subsequent ROS (superoxide anion radical, O2 and hydrogen peroxide) formation and escape (ZANGAR et al. 2004). The Cyp2E1 gene in humans has been shown to play important roles in oxidative stress and liver cirrhosis through its roles in ethanol metabolism (GONZALEZ 2005). Cyp2E1 converts ethanol to acetaldehyde and 1-hydroxyethyl radical and generates large amounts of ROS in the process, and this process is considered one of the main contributors to liver damage in alcoholism (GONZALEZ 2005).

Increased P450-generated ROS production has been noted in xenobiotic-induced rodents, presumably from large quantities of P450 enzymes being expressed (PREMEREUR et al. 1986).

Reducing Cyp12d1 expression ubiquitously significantly shortened adult male lifespan and increased tolerance to hydrogen peroxide-induced oxidative stress. One explanation for the resistance seen would be that reducing Cyp12d1 expression decreased P450 activity in the mitochondria, which reduced the amount of endogenous ROS production and hence freed up some ROS detoxification capacity in the cell

(FINKEL and HOLBROOK 2000). Ascertaining the endogenous oxidative stress load of Cyp12d1-reduced flies would indicate if these flies were under a lower amount of oxidative stress and if the extra unused ROS detoxification capacity was used to help alleviate the effects of the extraneous hydrogen peroxide.

Reducing Cyp12d1 expression should theoretically reduce the ROS production in these transgenic flies as the total P450 enzyme content is decreased. If oxidative stress was the major determinant of lifespan in

Cyp12d1-reduced flies, this lower level of oxidative stress should result in a prolonged lifespan. It was therefore surprising to find that these flies still had a significantly shorter lifespan compared to control

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flies, and indicated that reasons other than oxidative stress could be responsible for this effect. It has been suggested that genetic interventions that shorten lifespan are less likely to be related to the aging process, but instead may be due to other pathological reasons (HELFAND and ROGINA 2003). It may be that Cyp12d1 shortens lifespan through indirect means, rather than directly playing a role in Drosophila longevity. For example, Cyp12d1 has been shown to be involved in xenobiotic detoxification. It may be that reducing the amount of CYP12D1 enzyme may leave flies vulnerable to toxic substances and shorten their lifespan.

It is also possible that Cyp12d1 performs non-detoxification functions that were needed for a normal lifespan. Adult CantonS flies express Cyp12d1 in the adult head, midgut, Malpighian Tubules, fat body, heart, male accessory glands, virgin spermatheca, mated spermatheca and adult carcass (minimum expression threshold >100 mRNA signal, (CHINTAPALLI et al. 2007)). Of these, the head and heart expression immediately stand out as possible tissues where Cyp12d1 could be performing important functions necessary for a normal lifespan, but it is also possible that the other tissues where Cyp12d1 is expressed may be involved in determining lifespan.

It must be noted that these results were achieved through RNAi. While strong efforts were expended to avoid the unintended off target effects by careful design of the RNAi Cyp12d1 target fragment, it is still possible that expression of other genes may be reduced which has complicated these results. RNAi has been used to study longevity in other studies, notably in a large RNAi screen of mitochondrial complex proteins. Copeland and co-workers found that reducing the expression of certain proteins increased lifespan, while decreasing the amount of other mitochondrial complex proteins shortened lifespan

(COPELAND et al. 2009). It has been proposed that using an gene-inducing system (like the RU-486 method or the GAL-80 system) together with the GAL4/UAS system of inducing RNAi activity is a more accurate method of measuring longevity than the standard GAL4/UAS system alone (HELFAND and ROGINA

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2003). The induced Tub-GAL4/Cyp12d1-RNAi flies can be compared directly against the uninduced sample, thus eliminating any genetic background effects. Incorporating this system into future work will allow more accurate measurement of longevity in Cyp12d1-RNAi flies.

Overexpressing Cyp12d1 expression also resulted in a decreased lifespan. Overexpressing Cyp12d1 would theoretically increase the amount of ROS produced, and this might account for the decrease in lifespan. Again, testing the total amount of ROS produced in these flies would help determine if these flies have increased amounts of ROS and hence are under oxidative stress. Alternatively, other reasons for the shortened lifespan phenotype observed could be due to the high metabolic cost of producing increased amounts of CYP12D1 protein, or that cellular damage from expressing Cyp12d1 in non-native

Cyp12d1 expressing tissues, could lead to the shortened lifespan seen.

Mitochondria function could be measured in Cyp12d1-RNAi and Cyp12d1-overexpression to test if it was impaired. Parameters such as respiratory complex activities, ATP synthesis rates and concentrations of respiratory subunits could be investigated. As mitochondrial P450s are located on the inner mitochondrial membrane (ORTIZ DE MONTELLANO 2005), the lipid composition of the inner mitochondrial membrane should also be measured.

Aged flies show distinctly different mitochondria functional profiles to young flies (DUBESSAY et al. 2007).

The activities of mitochondrial complexes I, III and IV were found to be lower in 8 and 12 week old flies compared to 6-8 day old flies. ATP synthesis rates were also impaired in older flies. Inner mitochondrial membrane lipid content has been shown to be important for the activities of respiratory complexes.

Mitochondrial P450s have been shown to recognise phospholipids and cholesterol as substrates

(MURTAZINA et al. 2004). If Cyp12d1 was involved in lipid metabolism in flies, reducing Cyp12d1 expression may alter the mitochondrial membrane composition and hence affect mitochondrial complex

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activity. Cyp12d1-RNAi and Cyp12d1-overexpression flies should be examined for their mitochondrial state to investigate if their mitochondria appeared to be prematurely aged.

This was one of the first studies looking at P450 oxidative stress and longevity in D. melanogaster.

Previous studies in Drosophila have focused on the induction of P450s in response to challenge with known oxidative-stress inducing agents such as hydrogen peroxide, paraquat and Tunamycin (GIRARDOT et al. 2004). It is uncertain at this juncture if the oxidative stress phenotype observed with Cyp12d1 was specific only to Cyp12d1 or if other P450s have the same effects when overexpressed or are reduced in expression. Cyp12d1 was not found to be induced by hydrogen peroxide, tunamycin or paraquat

(GIRARDOT et al. 2004), suggesting that Cyp12d1 was not involved in the cellular response to these chemicals. Other P450s including Cyp6a23 were induced by hydrogen peroxide, tunamycin and paraquat

(GIRARDOT et al. 2004). The endoplasmic reticulum-located Cyp6a23 has been found to confer resistance to hydrogen peroxide stress when overexpressed in Sl-2 cell culture studies, while reduction in Sl-2 cells and whole flies showed no difference in survival when challenged with hydrogen peroxide (Adam

Southon, Personal communication). The effect of overexpressing Cyp6a23 in whole flies however has not been examined. More studies using other mitochondrial P450s might help confirm if this was a general P450 effect or a genuine Cyp12d1 function.

This study found that overexpressing Cyp12d1 was detrimental for flies. This may partially explain why

Cyp12d1 is so inducible, as flies would want to keep CYP12D1 protein levels under tight regulatory control and at a low basal expression level. Constitutive overexpression would be harmful for flies, and it would be beneficial to only induce Cyp12d1 when necessary to reduce harm from excessive CYP12D1 protein.

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3.5 Chapter conclusion

The function of Cyp12d1 was investigated in this chapter. Male Cyp12d1 mRNA expression was reduced ubiquitously by 70% using RNAi and the effects examined. Cyp12d1 was found not to be essential for development, although a full null mutant should be generated to thoroughly test this.

Decreasing Cyp12d1 expression ubiquitously significantly shortened lifespan in both RNAi lines tested.

The shortened lifespan may be due to several reasons, such as Cyp12d1 having essential functions which maintained a normal lifespan or that reducing Cyp12d1 left flies vulnerable to xenobiotic stresses.

Reducing Cyp12d1 expression also gave resistance to hydrogen peroxide-induced oxygen stress.

Reducing the amount of Cyp12d1 may have lowered the P450-generated ROS load, leaving flies with some unused antioxidant capacity and making them more resistant to hydrogen peroxide.

Overexpressing Cyp12d1 also reduced lifespan, whether through increased oxidative stress load, the metabolic costs of maintaining high amounts of Cyp12d1, or the unwelcome pleiotropic effects of expressing Cyp12d1 in non-natively expressing tissues. This reduction of lifespan may help explain the selective inducibility of Cyp12d1, where the fly has to maintain Cyp12d1 expression at low levels and only upregulate it when necessary.

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Chapter 4: Investigating the Cyp12d1 duplication

The joy of discovery is certainly the liveliest that the mind of man can ever feel.

- Claude Bernard

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4.1 Introduction

Gene duplication is one of the driving forces for the creation of new genes and provides the raw material for the evolution of novel proteins with new functions. In a process known as birth-death evolution, frequent gene duplication events have resulted in proliferation and diversification in large gene families (NEI and ROONEY 2005; THOMAS 2007).

The cytochrome P450 gene family had a large increase over the past 800 million years (LEWIS et al. 1998;

NEBERT and GONZALEZ 1987). This explosion in numbers has been fed by numerous gene duplications and subsequent functional divergence, resulting in new gene members with different substrate specificities.

P450s can be categorised as either evolutionary stable or evolutionary unstable, based on genome comparisons between related species (THOMAS 2007). Phylogenetically stable P450s generally have well conserved functions, and selection maintains orthologues in a wide range of distantly related species.

The strong selective pressure means that these genes are less likely to undergo duplications and divergences. In fact, copy number variation in endogenously important P450s can be detrimental. For instance, CNV polymorphism in the Cyp21A2 gene causes congenital adrenal hyperplasia (WILSON et al.

2007).

D. melanogaster has 85 P450s (TIJET et al. 2001). Approximately one third of these are evolutionary stable P450s, with recognisable 1:1 orthologues in the twelve sequenced Drosophila species (Lydia

Gramzow, Robert Good and Charles Robin, unpublished data). These conserved P450s include genes that have key roles in important functions, such as the Halloween P450s involved in ecdysone synthesis and catabolism. This functional importance is underscored by the finding that a high proportion (36.4%, compared to 1.2% for evolutionary unstable D. melanogaster P450s) of these genes are lethal when their expression is reduced or abolished (CHUNG et al. 2009).

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Evolutionary unstable P450s undergo numerous gene duplications are more likely to be involved in xenobiotic detoxification functions and are often found in gene clusters (THOMAS 2007). Duplicated genes are under less constraint, allowing divergence and paralogous expansion of family members. For instance, copy number variation (CNV) in human drug metabolising P450s have been well documented.

Cyp2D6 accounts for only 4% of all hepatic P450 expression, but is responsible for approximately 25% of all drug metabolism events (MEIJERMAN et al. 2007). The Cyp2D6 2xn allele is a CNV polymorphic haplotype that has undergone gene duplication (with up to 13 different copies) through unequal crossover events, leading to extremely high Cyp2D6 activity (MEIJERMAN et al. 2007). The D. melanogaster gene Cyp6g1 confers resistance to numerous insecticides when overexpressed, and has been shown to directly metabolise insecticides (DABORN et al. 2001; DABORN et al. 2007; DABORN et al.

2002; JOUSSEN et al. 2008). Cyp6g1 has undergone at least one round of duplication in many natural populations (SCHMIDT et al. 2010). Interestingly, this locus attracts transposable element insertions, with three separate recognised insertions to date (CATANIA et al. 2004; DABORN et al. 2002; SCHMIDT et al.

2010). These transposable element insertions mostly serve to increase Cyp6g1 expression (CHUNG et al.

2007; SCHMIDT et al. 2010), and coupled with the Cyp6g1 duplication, means that the more evolutionary derived alleles of Cyp6g1 can have much higher expression than an ancestral unduplicated Cyp6g1 gene

(SCHMIDT et al. 2010).

The mitochondrial P450s are a good example illustrating the grouping of P450s by their conserved status. Mitochondrial P450s comprise a distinct clade within the P450 family, and can also be separated into evolutionary stable and unstable P450s (FEYEREISEN 2006; FEYEREISEN 2011).

Several mitochondrial P450s form an evolutionary stable clade where members have important endogenous functions in conserved pathways. These P450s are therefore under strong selection with easily discernable orthologues found in other species. Mammalian mitochondrial P450s have essential

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conserved functions in steroid hormone genesis, adrenal cortex hormone genesis and bile acid synthesis

(OMURA 2006). The insect mitochondrial P450s Cyp301A1, Cyp302A1, Cyp314A1, and Cyp315A1 have conserved 1:1 orthologues between D. melanogaster, Anopheles gambiae and Apis mellifera which likely reflects their conserved functions in ecdysone synthesis in insects (Figure 1.5) (CLAUDIANOS et al.

2006).

The second group of mitochondrial P450s form a less conserved clade where the sequence of member

P450s are more variable and no obvious 1:1 orthologues in other species are present. For example, the

CYP12 family is marked by species-specific expansions in numbers (Figure 1.5) (CLAUDIANOS et al. 2006).

A. gambiae has four paralogous genes, while D. melanogaster has six. Genes in this class may have copy number variation and are more likely to be involved in xenobiotic detoxification than conserved metabolic functions. Musca domestica Cyp12A1 has been shown to metabolise xenobiotic substances but not endogenous compounds in vitro, and is overexpressed in certain insecticide resistant strains of

M. domestica, showing that it has a xenobiotic detoxification function in addition to possibly having unknown endogenous functions (FEYEREISEN et al. 1997; GUZOV et al. 1998).

Cyp12d1 is a xenobiotic-inducible mitochondrial P450 that is able to confer insecticide resistance when overexpressed. Cyp12d1 is not lethal when expression is reduced ubiquitously throughout development, suggesting that reducing Cyp12d1 expression did not reduce viability (Chapter 3). The Cyp12d1 gene has been tandemly duplicated giving rise to Cyp12d1-d’ and Cyp12d1-p’. The duplication exists as a polymorphism as some lines from natural populations have two copies of the gene (double copy lines), while others have only one copy (single copy lines). In the sequenced y; cn bw sp line, this 3.7 kb duplication of Cyp12d1 is characterised by only three base pair differences between Cyp12d1-d’ and

Cyp12d1-p’, each base pair difference giving rise to an amino acid replacement in the CYP12D1 protein

(TWEEDIE et al. 2009). The significance and benefits of the Cyp12d1 duplication are as yet unknown.

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4.1.1 Chapter aims

This chapter investigates the Cyp12d1 duplication and the possible outcome of possessing copy number variation. Firstly, the distribution of the Cyp12d1 duplication was examined in wild populations to discover the allelic frequency and geographical spread of this duplication. It was felt that knowing the pattern of how the Cyp12d1 duplication was distributed would help better understand the function and benefits, if any, that possessing this duplication confers. The level of Cyp12d1 expression in duplicated and non-duplicated lines was also investigated to test if having two copies of Cyp12d1 resulted in different amounts of Cyp12d1 being expressed. Finally, the tissue specific expression of Cyp12d1 in embryos and larvae from double copy and single copy lines was investigated to determine if double copy lines expressed Cyp12d1 in different expression patterns to single copy lines.

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4.2 Materials and Methods

4.2.1 Cyp12d1 Duplication assay

Parental Drosophila melanogaster females were caught from ten separate locations along the east coast of Australia extending from Northern Queensland to Tasmania (Flies were a gift from Professor Ary

Hoffman, The University of Melbourne). The locations were: Cooktown (15.67°S); Innisfail (17.6°S);

Mackay (21.15°S); Gladstone (24.02°S); Maryborough (25.53°S); Coffs Harbour (30.32°S); Bega (36.47°S);

Yering Station (37.67°S); Spreytons Ayers (41.23°S) and Sorrell (42.78°S). The flies were collected in

2005, within 30 km of the coast and at low altitude (< 100 m), to ensure that latitudinal variation was not confounded by effects of altitude. Isofemale lines were established from wild-caught females from each of these geographical locations. F1 progeny were collected from these isofemale lines and used in this study.

For each geographical location, three isofemale lines were selected for further analysis. Three replicate flies were taken from each of these isofemale lines to characterise their Cyp12d1 duplication status. In total, nine flies were tested from each geographical location. Altogether, 90 flies (nine flies each from ten geographical locations) were tested in this assay.

Genomic DNA was extracted using a Chelex extraction protocol (Roche). Individual flies were ground up in a 5% Chelex solution, before being incubated at 37°C for an hour. 1ul of 20mg/ul of Proteinase K

(Biorad) was added and the plate incubated at 37°C for another hour. The Cyp12d1 duplication PCR protocol described in (SCHMIDT et al. 2010) was used to determine the duplication status of these flies

(Figure 4.1). However, it must be noted that this assay is not a codominant assay and is unable to detect individuals heterozygous for the duplication, instead scoring them as homozygous duplicated.

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To test the duplication status of the natural populations line used in this study, 20 flies from each population were collected and DNA was extracted using a CTAB extraction protocol. Briefly, 20 flies were homogenized in 400 μL of Cetyltrimethylammonium Bromide (CTAB) buffer (Sigma). 10 μL of

Proteinase K (8 mg/mL) was added per sample and samples incubated at 55 for 1-3 hours. DNA was then extracted by adding an equal volume of phenol-chloroform, and precipitated using an equal volume of 100% isopropanol before being cleaned with 70% ethanol and resuspended in water.

4.2.2 Temporal lifestage sample collection

Samples for RNA isolation were collected at the developmental stages Early (feeding) 3rd Instar larvae,

Late (wandering) 3rd Instar larvae, White prepupae, Early pupae (Stage P2-P9), Late pupae (Stage P10-

P15), four-day old mated Adult males and four-day old mated Adult females with three biological replicates at each time point for both strains.

4.2.3 Exposure of larvae to phenobarbital

rd Xenobiotic induction as performed as per the protocol listed in (WILLOUGHBY et al. 2006). 3 instar larvae from the single copy w1118 line and the double copy y; cn bw sp line were reared at 25C and collected at

108 hours old post-hatching. Larvae were placed on standard flyfood agar plates supplemented with

10mM of phenobarbital (Sigma). Larvae were left to feed for 4 hours at 25C before three replicate biological samples consisting of 10 larvae each were collected from phenobarbital-exposed and unexposed samples. RNA was extracted from these larvae and the amount of Cyp12d1 mRNA quantified.

4.2.4 RNA isolation and cDNA synthesis

RNA was extracted using Trizol reagent (Invitrogen). RNA was quantified using the Qubit Quantification

Platform (Invitrogen). cDNA was made using the Superscript II Reverse Transcriptase system

(Invitrogen). Cyp12d1 real time primers which recognised a shared conserved region between Cyp12d1-

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d’ and Cyp12d1-p’ were used to quantify the total amount of Cyp12d1 transcript (Primer sequences in

Appendix I). The housekeeping gene RPL11 was used as an internal control for comparison between samples.

Real-Time PCR

Real-time PCR (RT-PCR) was performed using the QuantiTect SYBR Green PCR kit (Qiagen) on a

RotorGene-3000 (Corbett Research, Sydney) to quantify the amount of GFP transcript produced.

Annealing temperature was set at 55°C and runs set to 45 cycles. Three biological replicates were run and the total amount of Cyp12d1 and RPL11 transcript was quantified using the Standard curve method of quantification with the Corbett RotorGene-3000 Realtime analysis program (Corbett Research,

Sydney). Total Cyp12d1 mRNA expression was normalised to the RPL11 expression levels for comparison between induced and uninduced samples.

4.2.5 3’ Rapid Amplification of cDNA Ends (3’ RACE)

3’ RACE was performed using late 3rd instar larvae y; cn bw sp RNA. The SMARTtm RACE cDNA amplification kit (Clontech, California) was used to synthesise cDNA. Two sets of forward primers,

3’RACEF1 and 3’RACEF2 were used in conjunction with the supplied reverse primer to obtain Cyp12d1 3’

RACE amplicons (Primer sequences are listed in Appendix II). These were then cloned into the pGEM-

Teasy (Promega) and sequenced to obtain 3’UTR sequences.

4.2.6 mRNA in situ Hybridisation

Flies from the single Cyp12d1 copy lines w1118 and BG and the double copy lines, y; cn bw sp, RK146 and

TR2 were placed in cages and allowed to lay for 4 hours on grapefruit agar plates and embryos collected.

Embryos were transferred to plates containing standard cornmeal diet food and allowed to develop into feeding 3rd instar larvae. After approximately 108 hours of development at 25°C, larvae from the single

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Cyp12d1 copy lines w1118 and BG and the double copy lines, y; cn bw sp, RK146 and TR2 were collected for mRNA in situ hybridisation experiments.

The Cyp12d1 open reading frame (ORF) antisense RNA probe (Gift from Henry Chung) was used to was used to probe dissected larvae for Cyp12d1 tissue specific expression. The 3rd instar mRNA in situ hybridisation protocol outlined in (CHUNG et al. 2009) was used. At least six individual larvae were examined in each line. Images were taken using the SZX12 stereomicroscope system (Olympus).

4.2.7 Embryo mRNA in situ hybridisation

Flies from the single Cyp12d1 copy lines w1118 and BG and the double copy lines, y; cn bw sp, RK146 and

TR2 were raised in cages and allowed to lay for 24 hours on grapefruit agar plates before embryos were collected. Embryos were prepared and probed for Cyp12d1 expression using the embryo mRNA in situ hybridisation protocol described in (CHUNG et al. 2009). Cyp12d1 ORF antisense RNA probe was used to probe embryos for Cyp12d1 tissue specific expression (Gift from Henry Chung). Approximately 50-100 embryos from all stages of embryonic development were examined in each line. Images were taken using the SZX12 stereomicroscope system (Olympus).

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4.3 Results

4.3.1 The Cyp12d1 Duplication in y; cn bw sp

A schematic figure of the Cyp12d1 duplicated locus derived from the published y; cn bw sp sequence data (TWEEDIE et al. 2009) is presented in Figure 4.1. A tandem duplication event has occurred resulting in two Cyp12d1 genes, Cyp12d1-d’ and Cyp12d1-p’, where d’ stands for distal from centromere and p’ stands for proximal to centromere. The duplication unit is 3935bp (Figure 4.1). This includes the 1916bp

Cyp12d1 coding region, 133bp of the downstream region (+1917/+2049bp) and an 1886bp 5’ region (-

1886/-1bp). There are only three basepair changes in the entire duplication unit, including non-coding

DNA regions, which give rise to three amino acid replacements. These changes are discussed further in

Section 4.3.4.

4.3.2 Geographical distribution of the Cyp12d1 duplication

A survey was performed to determine the frequency of the Cyp12d1 duplication in natural D. melanogaster populations using wild-caught flies sampled from along the eastern coast of Australia.

Single flies were tested for their duplication status using a Cyp12d1 PCR duplication assay (SCHMIDT et al.

2010). Gel photos are presented in Appendix IV.

The percentage of flies found to have two copies of Cyp12d1 or one copy of Cyp12d1 was calculated for each location. However, given that the duplication PCR assay is unable to distinguish heterozygotes from one class of homozygotes, the duplication haplotype frequency can only be inferred by assuming Hardy- weinberg equilibrium. The gene frequency of the duplicated Cyp12d1 gene for every location was calculated using the Hardy-weinberg equation (p2 + 2pq +q2 = 1). This method was performed as it would give a more precise estimate of the frequency of the Cyp12d1 duplication by estimating the frequency of the duplicated gene (q), rather than simply calculating the total duplication

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Figure 4.1: A comparison of Cyp12d1 single copy and double copy haplotypes.

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Figure 4.1: A comparison of Cyp12d1 single copy and double copy haplotypes. A schematic diagram of a single copy Cyp12d1 and a double copy Cyp12d1 locus is presented. The duplication assay and approximate primer locations described in (SCHMIDT et al. 2010) are also presented (Fin, Rout, Fout, Rin).

Fin, Rout, Fout were used in the PCR duplication assay, while the Rin primer was only used with Fout to sequence the Fout/Rin region to search for polymorphisms. (A) A single copy haplotype. The duplicated

1.8kb region in double copy lines is also present in single copy loci. Fin binds to a region approximately

2.5kb from Cyp12d1 gene, while Rout binds within the 1.8kb region. The Fout primer binds within the

Cyp12d1 coding region and is unable to form a product with the Rout reverse primer. Single copy lines therefore give a single 769bp band in the duplication assay. (B) The double copy haplotype from y; cn bw sp. The Cyp12d1-p’ start codon is marked as position -1. The duplication is approximately 3.7kb in size, and has duplicated 1.8kb of the 5’ region and a 133bp fragment 3’ of Cyp12d1. There are three

SNPs between Cyp12d1-d’ and Cyp12d1-p’ (yellow and purple bars respectively) which constitute the only polymorphism in the entire 7.4kb region. Note that the 3’ region of Cyp12d1-d’ is significantly different from Cyp12d1-p’, and now consists of the +1917/+2049bp, -1886/-1bp and Cyp12d1-p’ gene.

The Fin/Rout primer is still able to amplify a 769bp band, but now the Fout and Rout primers are able to amplify an additional 391bp band to give a 769bp and a 391bp band, signifying that this locus is duplicated.

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Spatial distribution of the Cyp12d1 duplicated gene frequency

100%

) q 90% 80% 70% 60% 50% 40% 30% 20%

10% Estimate of the percentage gene frequency ( 0% Cooktown QLD Innisfail QLD Mackay QLD Gladstone QLD Maryborough Coff's Harbour Bega NSW 36° Yerring Station Speytone TAS Sorrell TAS 15° 17° 22° 23° QLD 25° NSW 30° VIC 37° 41° 42° Latitude

Figure 4.2: Geographical distribution of the duplicated Cyp12d1 gene frequencies along the east Coast of

Australia. The estimated percentage frequency of the duplicated Cyp12d1 gene (q) was calculated for flies from each location and plotted. It can be seen that Cyp12d1 duplication frequencies rises as latitude increases, indicating that flies from higher latitudes are more likely to have the duplicated

Cyp12d1 gene (R2=0.602). N= 18 alleles for each location (Nine diploid individual flies from each locations). 95% confidence interval bars are shown.

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frequency per location (Figure 4.2). The duplicated Cyp12d1 gene was present more often in the higher latitudes compared to the lower latitudes (r2 = 0.602). However, this sample size was too small to draw any definite conclusions about geographical trends in duplication frequencies.

4.3.3 Identifying single Cyp12d1 copy and double Cyp12d1 copy strains for further study

Three natural isofemale lines derived from wild-caught flies (BG4, RK146 and TR2) and three laboratory strains (w1118, y; cn bw sp and CantonS) were investigated using the Cyp12d1 duplication PCR assay to determine their Cyp12d1 duplication status (Figure 4.3). The reference sequenced strain y; cn bw sp is a predicted double copy line based on sequence data, and it was included as a positive control. It can be seen that BG4 and w1118 were single copy lines, while RK146, TR2, CantonS and y; cn bw sp are double copy lines. BG4, w1118, RK146, TR2 and y; cn bw sp were used for subsequent experiments examining the expression of Cyp12d1 in these lines.

4.3.4 Two Cyp12d1 amino acid substitutions are found in substrate recognition sites

The three base pair changes are within Cyp12d1 coding regions and lead to three replacement amino acid changes (Figure 4.4). Two of these three changes (F392L and L505V) appear to be located in the

Substrate Recognition Sites (SRS), which are hypervariable regions hypothesised to recognise and bind substrates (GOTOH 1992). The F392L substitution occurs in the SRS5 region, while the L505V substitution is found in the SRS6 region. The A277S site change is predicted to be located near the G’ helix (Figure

4.4) These changes could potentially alter substrate specificity and may mean the copies encode proteins that differ in their substrate recognition. Despite this, both copies have been shown to confer a similar level of resistance to DDT and dicyclanil when overexpressed (DABORN et al. 2007), showing that some degree of shared substrate recognition is retained.

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Figure 4.3: Cyp12d1 duplication status of several laboratory and natural population lines. The Cyp12d1 duplication PCR assay was used to investigate the Cyp12d1 duplication status in several lines. Single copy lines should only show one 769bp band, while double copy lines the same 769bp and an additional

391bp band should be amplified. However, in practice the smaller 391bp band in double copy lines is preferentially amplified and the 769bp band is rarely observed. It can be seen that w1118 and BG4 have only one copy, while RK146, TR2, CantonS and y; cn bw sp lines have two copies. The no template control (neg) lane is clear, indicating no contamination of the PCR assay.

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Figure 4.4: CYP12D1-d’ and CYP12D1-p’ alignments with other P450s.

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Figure 4.4: CYP12D1-d’ and CYP12D1-p’ alignments with other P450s. An alignment of D. melanogaster

CYP6G1, CYP6A2, CYP12D1-d’, CYP12D1-p’ and human CYP3A4 protein sequences is presented here.

Predicted SRS sites, inferred from known CYP3A4 SRS sites, are boxed. Sequences corresponding to predicted P450 protein helixes are underlined. CYP12D1 amino acid substitutions are circled. It can be seen that the amino acid replacements F392L and L505V lie within the SRS5 and SRS6 sites respectively, while the A277S replacement is found two bases downstream of the G-helix. Figure adapted from (JONES et al. 2010).

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4.3.5 Cyp12d1 mRNA expression in duplicated and non-duplicated lines

Total Cyp12d1 expression was investigated in duplicated and non-duplicated lines to look for possible differences in expression between duplicated and non-duplicated lines.

4.3.5.1 Cyp12d1 3’UTR length differences

The Cyp12d1-d’ transcript in the reference D. melanogaster strain y; cn bw sp is predicted to have a

214bp 3’ untranslated region (3’ UTR), while the Cyp12d1-p’ transcript was not reported to have a 3’UTR sequence (TWEEDIE et al. 2009). To confirm this, 3’ Rapid Amplification of cDNA ends (3’RACE) was performed using RNA from the double copy y; cn bw sp line to characterise the 3’ UTR region of the

Cyp12d1-p’ and Cyp12d1-d’ transcripts (Figure 4.5A). Sequenced clones revealed two consistently differently-sized 3’UTR isoforms, the 133bp and 214bp isoforms. These data also indicated that y; cn bw sp expresses both copies of Cyp12d1 in 3rd instar larvae.The 133bp form was always found together with

Cyp12d1-p’-specific SNPs. This indicated that the y; cn bw sp Cyp12d1-p’ transcript had a 133bp-long

3’UTR. Interestingly, this 133bp sequence is part of the duplication unit (Figure 4.1) and can also be found 3’ of the Cyp12d1-d’ gene.

The 214bp isoform was also only associated with Cyp12d1-d’ SNPs, and confirms Flybase data that the

Cyp12d1-d’ transcript has a 214bp 3’ UTR sequence (Figure 4.5B) (TWEEDIE et al. 2009). This region contains the 133bp-long sequence that comprises the Cyp12d1-p’ 3’ UTR, but now contains an additional 81bp that is part of the duplicated 5’ UTR sequence. More lines should be investigated to see if this transcript difference was found in both double copy and single copy lines.

4.3.5.2 Developmental Cyp12d1 expression

The developmental expression profile of Cyp12d1 was next investigated in Cyp12d1 single copy and double copy lines using quantitative real-time PCR.

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Figure 4.5: y; cn bw sp Cyp12d-d’ and Cyp12d1-p’ transcript length differences.

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Figure 4.5: y; cn bw sp Cyp12d-d’ and Cyp12d1-p’ transcript length differences. (A) Gel image showing amplification of partial Cyp12d1 transcripts from y; cn bw sp 3’ RACE-derived cDNA. 3’ RACE was performed using y; cn bw sp 3rd instar larvae RNA. PCR amplification was performed using 3’RACEF1

(lane F1) and 3’RACEF2 (lane F2). Note that lanes F1 and F2 had two bands which were consistently approximately 80bp different in size, indicative of differently-sized transcripts for Cyp12d1-p’ and

Cyp12d1-d’. The amplified PCR products were sequenced to investigate this size difference. F1neg and

F2neg are the no template controls for 3’RACEF1 and 3’RACEF2 primer sets respectively. No bands were observed indicating no contamination in the PCR. (B) A schematic view of the transcript length difference between Cyp12d-d’ and Cyp12d1-p’ in the y; cn bw sp line. The positions of the 3’RACE forward primers and where they bind relative to Cyp12d1 SNPs are shown. It can be seen that both genes have a shared 133bp sequence in their 3’UTR, but Cyp12d1-d’ has an additional unique 81bp sequence.

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The total Cyp12d1 mRNA level was determined at several developmental stages in the single copy

(w1118) and double copy (y; cn bw sp) laboratory lines (Figure 4.6A). Three field isolated lines, BG4 (single

Cyp12d1 copy) and RK146 and TR2 (double Cyp12d1 copy) were also examined at the several developmental lifestages for the amount of total Cyp12d1 mRNA (Figure 4.6A). For the double copy lines the combined amount of Cyp12d1-p’ and Cyp12d1-d’ mRNA was quantitated whereas in single copy lines Cyp12d1 mRNA was quantitated.

For all lines, Cyp12d1 mRNA is detected throughout all lifestages tested. Crucially, double copy lines did not always express more uninduced total Cyp12d1 mRNA (combined Cyp12d1-d’ and Cyp12d1-p’ expression combined) (Figure 4.6). For instance, the single copy line BG4 was the highest expresser of total Cyp12d1 while the single copy w1118 line had the 3rd highest level at the white prepuparium stage.

BG4 also had the second most Cyp12d1 mRNA at the adult male stage (Figure 4.6B). It seems that

Cyp12d1 mRNA expression is line-specific and it is not always clear that having two copies of Cyp12d1 gives more Cyp12d1 expression than having a single copy.

A previous study found that male OregonR flies expressed three-fold more Cyp12d1 mRNA than females

(KASAI and TOMITA 2002). This trend did not hold in many of the lines examined in the current study

(Figure 4.6B). Males did not always express three-fold more Cyp12d1, and only the y; cn bw sp line expresses roughly three-fold more Cyp12d1 (Figure 4.6B). Again, this supports the finding that Cyp12d1 expression is line-specific and that having the duplication is not always correlated with increased expression.

4.3.5.3 Cyp12d1 induction in single copy and double copy lines w1118 and y; cn bw sp 3rd instar feeding larvae were exposed to phenobarbital to examine their induction response. w1118 induced 189-fold higher Cyp12d1 expression after phenobarbital exposure. y; cn bw sp induced 36-fold higher expression, in agreement with other studies (WILLOUGHBY et al. 2006).

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Figure 4.6: Cyp12d1 temporal expression patterns in Cyp12d1 single copy and double copy lines.

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Figure 4.6: Cyp12d1 temporal expression patterns in Cyp12d1 single copy and double copy lines. (A)

Total Cyp12d1-d’/Cyp12d1-p’ mRNA expression in single copy and double copy lines at different life stages. Single copy lines (w1118, BG4) are shown in blue while double copy lines (y; cn bw sp, RK146 and

TR2) are in red. Cyp12d1 expression varies across lines at different lifestages. Double copy lines do not always express more Cyp12d1 than single copy lines, suggesting that expression is line-specific and subject to more factors other than having multiple copies of Cyp12d1. (B) The ratio of adult male-female

Cyp12d1 expression in the lines tested. The ratio of male and female adult Cyp12d1 expression was calculated and graphed.Male to female expression ratios were generally relatively equal, with only y; cn bw sp approaching the three-fold more expression in males compared to females reported in (KASAI and

TOMITA 2002). RT-PCR data is from three biological replicates, with error bars representing Standard error of the mean (SEM).

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RT-PCR showed that y; cn bw sp had five-fold more induced Cyp12d1 mRNA compared to w1118 (Table

4.1). This result suggested that double copy lines had significantly more induced Cyp12d1 expression than single copy lines.

4.3.6 Tissue specific expression in single copy and double copy lines

Real time PCR data showed line-specific variability in Cyp12d1 mRNA. To examine if these lines also showed variability in tissue-specific expression patterns, mRNA in situ experiments were performed on

3rd instar larvae and embryos .

4.3.6.1 Cyp12d1 mRNA in situ hybridisations in 3rd instar larvae

Feeding 3rd instar larvae were collected and mRNA in situ hybridisations performed to examine Cyp12d1 tissue specific expression in individuals from the single copy lines w1118 and BG4, and the double copy lines y; cn bw sp, RK146 and TR2 (Figure 4.7). All lines examined showed uninduced basal tissue-specific expression in the midgut, fat body and Malpighian tubules, agreeing with previous mRNA in situ experiments with y; cn bw sp. This indicated that single copy and double copy lines both had the same uninduced Cyp12d1 tissue-specific expression patterns.

4.3.6.2 Cyp12d1 mRNA in situ hybridisations in embryos

Embryonic Cyp12d1 expression was investigated using mRNA in situ hybridisation (Figure 4.8). Double copy lines were observed to express Cyp12d1 in a different pattern during embyonic development to single copy lines. Single copy lines only expressed Cyp12d1 in the aminoserosa at stage 11-12, while double copy lines expressed Cyp12d1 at stages 7-12 in the developing foregut, midgut, aminoserosa and the clypeo-labral primordium. The Berkeley Drosophila Genome Project (BDGP) has published Cyp12d1

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Table 4.1: Cyp12d1 expression in phenobarbital-exposed w1118 and y; cn bw sp 3rd instar larvae

Mean Cyp12d1 expression Fold- (relative to RPL11 expression) change Ratio w1118 phenobarbital 0.62 189 5.2*

w1118 unexposed 0.003 - - y; cn bw sp phenobarbital 3.18 36 1 y; cn bw sp unexposed 0.09 - -

*ratio of y; cn bw sp phenobarbital-induced Cyp12d1 expression compared to w1118 phenobarbital- induced Cyp12d1 expression.

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Figure 4.7: Early 3rd instar Cyp12d1 mRNA in situ staining in single and double copy lines.

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Figure 4.7: Early 3rd instar Cyp12d1 mRNA in situ staining in single and double copy lines. (A)-(B) Staining was observed in the Midgut, fat body and Malpighian tubules of both single copy lines w1118 (A) and BG4

(B). (C)-(D) Staining was seen in the Midgut, fat body and Malpighian tubules in the natural population double copy lines RK146 (C) and TR2 (D). (E) Midgut, fat body and Malpighian tubules staining in the y; cn bw sp line (Image obtained from WILLOUGHBY et al. 2006). The level of staining cannot be used as an indication of Cyp12d1 mRNA level, as in situ hybridisation conditions may have varied between experiments. These experiments were therefore not quantitative, and can only be used to ascertain the expression pattern of Cyp12d1 in these lines.

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Figure 4.8: Embryonic Cyp12d1 expression in single and double copy lines.

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Figure 4.8: Embryonic Cyp12d1 expression in single and double copy lines. (A) The published Berkley

Drosophila Genome Project (BDGP) Cyp12d1 expression profile in the double copy CantonS line

(TOMANCAK et al. 2007). Stage 7-8 embryos express Cyp12d1 in the developing anterior endoderm anlage

(AE) and posterior endoderm primordium (PD) (2). Stage 9-10 embryos express Cyp12d1 in the anterior endoderm primordium, posterior endoderm primordium and foregut primordium (FE). Stage 11-12 embryos express Cyp12d1 in the anterior endoderm primordium, foregut primordium, clypeo-labral primordium and aminoserosa (AS). (B) Single copy w1118 lines only showed expression in the aminoserosa. (C) The single copy BG4 lines also only showed expression in the aminoserosa. (D) Double copy y; cn bw sp embryos showed expression in the anterior endoderm primordium, posterior endoderm primordium, foregut primordium , clypeo-labral primordium and aminoserosa, similar to the

BDGP pattern. (E) Double copy RK146 also had Cyp12d1 expression in the same BDGP double copy expression pattern. (F) Double copy TR2 lines again showed Cyp12d1 expression in the same BDGP double copy expression pattern. Glossary: AE, anterior endoderm; PD, posterior endoderm; FE, foregut primordium; AS, aminoserosa.

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staining patterns in the double copy CantonS line embryos (TOMANCAK et al. 2007) (Figure 4.7). In this line, Cyp12d1 mRNA was expressed at Stages 7-8 in the developing anterior endoderm analage and posterior endoderm primordium, while Stage 9-10 expressed Cyp12d1 mRNA in the the anterior endoderm primordium, posterior endoderm primordium and foregut primordium. Stage 11-12 embryos were found to express Cyp12d1 mRNA in the anterior endoderm primordium, foregut primordium, clypeo-labral primordium and aminoserosa (TOMANCAK et al. 2007). This same pattern was consistently observed in three double copy lines, suggesting that Cyp12d1 was expressed in a fixed pattern in double copy lines. This indicates that expression of Cyp12d1 in single copy and double copy lines is different, and was the only identified tissue-specific expression difference between single copy and double copy lines.

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4.4 Discussion

4.4.1 The Cyp12d1 duplication haplotype was found to vary in frequency spatially

The Cyp12d1 duplication is postulated to be a recent duplication. In the sequenced double copy y; cn bw sp line, this duplicated region is extremely well conserved, and only three basepairs are different between the two 3.7kb duplication units (Figure 4.1). Sequencing of the shared Fin/Rout and Fout/Rout genomic regions (Figure 4.1) from four single copy and four double copy natural population lines found that the four single copy lines had 20 different SNPs in approximately 1kb of flanking sequence (Charles

Robin, personal communication). In contrast, no polymorphisms when the same region was sequenced in double copy lines (Charles Robin, personal communication). This suggests that this duplication is very recent as random mutation has not resulted in any variation between the surveyed flanking regions in the duplicate lines.

In a small sample of genomes tested in this study, the Cyp12d1 duplication was observed to be distributed unevenly along the eastern coast of Australia, with higher latitudes having higher frequencies of the duplications and lower latitudes having lower frequencies. This effect was also observed in a separate larger study examining copy number variation along the same geographical region (Charles Robin, unpublished results). Flies were again collected from 19 locations along the eastern coast of Australia, and part of the Cyp12d1 open reading frame (ORF) sequenced in these flies using 454 sequencing (Figure 4.9). This partial Cyp12d1 ORF sequence contained the A277S and F392L mutations, and allowed the determination of the relative frequencies of these residues. The A/F haplotype was almost fixed in the northern most populations, declining to 50% in the south. This would indicate that the S/L haplotype is also at 50% frequency. Assuming that the S/L haplotype is only associated with the duplication, this 50:50 ratio between A/F and S/L haplotypes indicates that the duplication is fixed in the southern populations, agreeing with the data seen in this study (Charles Robin,

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Figure 4.9: Cyp12d1 base pair substitution frequency along the east coast of Australia. An approximately

490bp region from a common region of Cyp12d1-d’ and Cyp12d1-p’ was sequenced using 454 sequencing in both directions in flies from 19 locations spread along the eastern coast of Australia

(n=307 reads and n=407 reads from the 5’ and 3’ sides of the region respectively). This sequence encompassed the A277S and F392L mutations, which allowed the assessment of the frequencies of these nucleotide substitutions. Each vertical black line represents a basepair change, and the red dot marks the r2 value of this change, denoting how strongly this change is associated with latitudinal changes. It can be seen that numerous nucleotide substitutions compared to the reference y; cn bw sp sequence were present, which showed that variation in Cyp12d1 coding sequence existed in natural populations. However, it is unclear if this variation was associated with single copy Cyp12d1 loci or double-copy Cyp12d1 loci. The A/S (i) substitution and the F/L substitution (ii) frequencies were found to be differentiated with latitude (r2 > 0.4). Assuming that the S and L variants were only associated with the Cyp12d1-d’ gene copy and hence the Cyp12d1 duplication, this indicates that the Cyp12d1 duplication varied with latitude. Figure provided by Charles Robin (unpublished results).

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(Charles Robin, unpublished results). Assuming that the S and L variants were only associated with the

Cyp12d1-d’ gene copy and hence the Cyp12d1 duplication, this indicates that the Cyp12d1 duplication varied with latitude.

Turner et al. used tiling microarrays to map genetic polymorphisms that varied in geographical regions in

D. melanogaster wild-caught populations (TURNER et al. 2008). They examined wild-caught populations from northern and southern locations from the east coasts of North America and Australia, and found

104 probes which showed highly significant latitudinal differentiation between both north and south populations in both countries. One of these ‘golden probes’ was located in the coding region of

Cyp12d1, and this probe is predicted to be able to hybridise to both the proximal and distal copies of

Cyp12d1 (TURNER et al. 2008). This finding suggested that the Cyp12d1 coding region corresponding to this golden probe was varying in sequence with latitude, and that the nucleotide differences between both Cyp12d1 copies are driving this geographical variation in frequency. This report also demonstrated that spatial variation was found in other geographical areas (America in the Northern Hemisphere), rather than just only along the eastern coast of Australia, and gave further evidence of Cyp12d1 duplicated gene allele frequencies having spatial differentiation .

Subsequently, this group has sequenced 20 northern isofemale lines (from Queensland) and 19 southern isofemale lines (from Tasmania) to further investigate genetic differentiation between these populations

(KOLACZKOWSKI et al. 2011). These locations are at opposite ends of the proposed cline, and based on previous findings, the Cyp12d1 duplication is predicted to be fixed in the southern Tasmania populations while being present in very low frequencies in the northern Queensland populations. The Cyp12d1-d’ coding region was found to demonstrate significant population frequency differences between these two locations. This showed that Cyp12d1 sequence was varying between northern and southern

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locations, further supporting that the Cyp12d1 duplication was present in different frequencies along the eastern coast of Australia (KOLACZKOWSKI et al. 2011).

Altogether, these studies point to the existence of a cline for Cyp12d1 duplication along the Australian

Eastern Coast.

4.4.2 Is Cyp12d1 involved in adaptation to physical environmental stresses?

It is unknown what advantages having a duplicated Cyp12d1 locus confers. No phenotypic differences have been observed between single and double copy lines, but it is possible that differences may be observed under the appropriate (yet to be discovered) stress conditions.

The geographical regions sampled in this survey undertake a range of environments, from cool temperate climates in higher latitudes (Sorrell, TAS; Spreytone, TAS, Yerring Station, VIC) to milder temperate climates (Bega, NSW, Coff’s Harbour, NSW), and finally warm and humid tropical climates

(Cooktown, QLD; Innisfail, QLD). As such, flies collected in this survey can have experienced vastly different environmental conditions and adapted accordingly.

Various studies have examined the transcriptional response of D. melanogaster to environmental stresses like heat, cold, starvation, longevity and desiccation. SØrensen et al. published a large scale study examining the transcriptional response of D. melanogaster after being selected for seven different ecologically-relevant traits (Starvation tolerance, Longevity, Heat knockdown, Heat survival, Desiccation tolerance, rearing at constant 30°C and cold survival) (SØRENSEN et al. 2007). Flies originating from a mass bred starting population were selected for these different traits, and after 10 selection events, their gene expression profiles were determined by microarray experiments. Cyp12d1 expression was not found to be significantly changed in any of the lines selected for the seven traits listed above, suggesting that Cyp12d1 was not involved in the response to these traits and therefore these traits were not part of the selective forces acting on the Cyp12d1 duplication cline.

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Other studies have investigated separate aspects of cold tolerance. One such study examining chill coma resistance found Cyp12d1 expression to be down-regulated in lines selected for chill-coma resistance

(TELONIS-SCOTT et al. 2009). Another report did not find Cyp12d1 transcription to be significantly different between flies subjected to a cold-hardening process (0°C for 2hours before recovery at 25°C for 30 minutes) and control lines (QIN et al. 2005). It appears that the role of Cyp12d1 in cold tolerance is complex, and may be only associated with certain aspects of cold tolerance.

It is unknown at this stage if the Cyp12d1 duplication is involved in an adaptation response to changes in microenvironment. The finding that Cyp12d1 was not selected for in a majority of studies investigating multiple ecologically relevant traits suggests that Cyp12d1 is not involved in the adaptation response to environmental factors. However, more experiments need to be done before this can be conclusively ruled out. Multivariate analysis examining all possible environmental factors along this Cyp12d1 duplication cline should be performed in order to further investigate if the Cyp12d1 duplication is involved in adaptation to physical environmental stresses.

4.4.3 Do individual differences between Cyp12d1-d’ and Cyp12d1-p’ contribute to Cyp12d1 duplication selection?

This study found that Cyp12d1 double copy lines expressed Cyp12d1 differently to single copy lines.

Cyp12d1-d’ and Cyp12d1-p’ sequences are extremely conserved in sequence, reflecting their recent duplication. Despite this, there are still crucial differences between the two genes, and it is unclear whether these differences contribute to selection for the duplication.

There was a difference in transcript lengths between y; cn bw sp Cyp12d1-d’ and Cyp12d1-p’ because of differences in their 3’ UTR sequences. This difference in transcript length reveals an intriguing aftereffect of the duplication. Some 3’ UTRs have been shown to play important roles in transcript stability and protein translation (AMRANI et al. 2004), and longer 3’UTR sequences are postulated to bring decreased

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mRNA stability in humans and E. coli (FENG and NIU 2007). 3’UTR also participate in gene regulation through binding of microRNAs, and having different terminal sequences may result in differential post transcriptional regulation of both copies (YANG et al. 2005). Ultimately, it may be that having different

3’UTR between Cyp12d1-d’ and Cyp12d1-p’ results in differences in the way Cyp12d1 transcripts are translated between single copy and double copy lines, and which ultimately affects CYP12D1 expression in these lines.

4.4.3.1 Cyp12d1-d’ and Cyp12d1-p’ may have different substrate specificities

The Substrate Recognition Sites (SRS1-SRS6) are hypervariable regions hypothesised to recognise and bind substrates and were inferred from sequence alignments with substrate recognition regions identified from crystal modelling of the P450cam structure (GOTOH 1992). SRS are considered one of the principle determining factors for substrate specificity. Three nonsynonomous substitutions exist between Cyp12d1-p’ and Cyp12d1-d’, and two of these three amino acid residues occur in the predicted substrate recognition sites (SRS). These residues were not predicted to interact with DDT, diazanon and dicyclanil when bound to the CYP12D1 protein active site (JONES et al. 2010), suggesting they do not contribute to substrate specificity for these insecticides.

These amino acid substitutions suggest substrate specificity and functional differences between the two copies. Alterations of even single amino acid residues in SRS sites have been shown to change substrate recognition. For instance, mutating a single isoleucine residue to a leucine in the SRS1 region of P. polyxenes Cyp6B1 led to a change in substrate retention (WEN et al. 2005). The I115L mutant had a lower turnover rate for xanthotoxin than the wild type, possibly due to a more constricted channel from the I115L catalytic site to the P450 surface (WEN et al. 2005). Equally, changing key residues in the rat

Cyp2B2 gene shifted its total activity and regioselectivity to match the rat Cyp2B1 gene (STROBEL and

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HALPERT 1997). It is therefore possible that these two amino acid substitutions may have changed the substrate specificity between Cyp12d1-d’ and Cyp12d1-p’.

Amino acid changes outside of the SRS sites have also been shown to change substrate specificity. Three residues in the Cyp2C19 gene, Ile99, Pro220 and Thr221 were found to control binding specificity for

99 220 221 omeprazole (IBEANU et al. 1996). Only Ile was found to lie within the SRS1 region. Pro and Thr lie outside the SRS sites, and were found to be important for omeprazole through amino acid substitution experiments with the homologous Cyp2C9 gene. These two residues were hypothesised to lie in the F-G helix region, which forms part of the substrate access channel to the substrate–binding pocket (IBEANU

220 221 et al. 1996; WADE et al. 2004). The Pro and Thr residues may therefore contribute to substrate specificity through controlling access to or egress from the active site, rather than directly recognising substrates. in silico modelling of the CYP12D1 protein predicted that CYP12D1 contains a pw2-type substrate acess channel (JONES et al. 2010). In this channel, substrates pass between the F/G loop, B’ helix/BB’ loop/BC loop and the B1 sheet (WADE et al. 2004). The A277S site change in CYP12D1 is found two residues downstream of the G helix, and may be a factor in controlling access to the substrate- binding pocket.

Cyp12d1-d’ and Cyp12d1-p’ could be heterologously expressed to perform substrate binding and metabolism assays to look for substrate recognition differences between both copies. Insecticide resistance assays indicated that both copies already show a degree of function overlap because both copies conferred resistance to the same insecticides (DABORN et al. 2007). Testing more substrates may uncover variations in substrate specificities.

4.4.3.2 Do tissue-specific expression differences contribute to Cyp12d1 duplication selection?

Single copy and double copy lines appear to have the same tissue expression pattern in 3rd instar larvae.

However, embryonic in situ hybridisation experiments found a striking difference between single copy

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and double copy Cyp12d1 expression. This expression difference was consistently identified across several lines, and points to possible embryonic development differences between single copy and double copy lines because of this additional expression in double copy lines. At this stage, it is not clear which gene copy, or possibly even both copies, is responsible for this double copy expression pattern. As the duplication has resulted in changes in the genetic architecture of the double copy locus compared to the single copy, it is possible that changes in cis-regulatory elements through the gain/loss of enhancer elements or the removal of insulator elements results in the altered expression now seen. Cyp12d1 was shown to require the input of both 5’ and 3’ enhancer sequences for native uninduced expression

(Chapter 2). The Cyp12d1-d’ downstream sequence is drastically changed compared to the Cyp12d1-p’ sequence (Figure 4.1). It may be that enhancer sequences located in this 3’ region now drive Cyp12d1— d’ expression in new tissues. 5’ enhancers also drove uninduced Cyp12d1 expression (Chapter 2), and it is also possible that the changed upstream region of Cyp12d1-p’ drives its expression in a new embryonic expression pattern.

This change in expression after duplication is akin to the Zebrafish CYP19 aromatase, which has also undergone gene duplication through a whole chromosome duplication, and has resulted in changes in tissue distribution for both copies (CHIANG et al. 2001; TCHOUDAKOVA et al. 2001). One copy is now expressed in the brain only and is estrogen-inducible (KISHIDA et al. 2001), while the other is expressed primarily in the ovary (CHIANG et al. 2001). This change in expression is possibly due to differences of putative cis-regulatory elements between both gene copies (CALLARD et al. 2001; TCHOUDAKOVA et al.

2001; TONG and CHUNG 2003), although no functional assays of predicted binding sites have been performed. This gene duplication event has been noted in other teleost fish species (TCHOUDAKOVA et al.

2001; ZHANG et al. 2004), and has been proposed to give rise to possible subfunctionalisation in different tissues (FEYEREISEN 2011). In Drosophila, The Cyp307a2 has duplicated twice to give a second copy in the

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Drosophila subgenus and a second copy in the Sophophoran subgenus (SZTAL et al. 2007), with the expression patterns of the paralogs in both cases having diverged.

It is unclear what benefits, if any, this difference in embryonic expression causes. Cyp12d1 expression in the developing digestive tissue (endoderm) regions suggest possible roles in the development of these tissues in double copy flies.

This was the only solid evidence for tissue-specific expression differences between single copy and double copy lines, and while the significance of it remains unclear, future work examining the developmental phenotypes of these embryos may provide further clues as to the function of the

Cyp12d1 duplication.

4.4.3.3 Is Cyp12d1 induction and detoxification a selective agent for the Cyp12d1 cline?

Cyp12d1 induction by xenobiotic substances has been extensively investigated (Chapter 2). When the double copy y; cn bw sp line and single copy w1118 line was exposed to phenobarbital, Cyp12d1 was induced to approximately five times higher than in the single copy w1118 line in the same amount of time, showing that double copy lines may have markedly higher induced expression than single copy lines.

While this may be again due to inherent line-specific differences in Cyp12d1 regulation, this effect should not be discounted. Cyp12d1 was found to be induced by the natural compounds caffeine, pepper and pyrethrum (JENSEN et al. 2006; WILLOUGHBY et al. 2006), suggesting that the induction of Cyp12d1 by natural substances might be responsible for the variances in Cyp12d1 duplication observed.

Cyp12d1 transgenic overexpression gives resistance to DDT and dicyclanil, but resistance to other natural compounds has not been investigated. The Cyp12d1 duplication was not found to be selected for in a screen of DDT resistance. Schmidt et al. assessed a large population of wild-caught flies and determined that the frequency of the Cyp12d1 duplication did not differ in flies that were highly resistant to DDT when compared to DDT-susceptible flies. In contrast, flies that survived the DDT assay

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were found to be twelve times more likely to have the most resistant allelic status of Cyp6g1 (SCHMIDT et al. 2010). This suggested that the Cyp12d1 duplication did not affect survival rates when flies were exposed to DDT. The large sample size of this experiment (n= approximately 2500) suggests that if the

Cyp12d1 duplication truly contributes an effect to DDT resistance, it must be small (or non-additive).

Given that D. melanogaster is not a pest species, DDT is unlikely to be encountered normally in its environment and is unlikely to be the selective agent for the Cyp12d1 duplication cline. It seems more probable that some other more relevant chemical toxins, like a naturally occurring plant toxin, may be the selective agent.

4.5 Chapter conclusion

The duplication of Cyp12d1 was investigated in this chapter. The duplicated gene frequency was observed to associate with latitude (r2=0.602) when a small group of samples were examined from different locations along the eastern coast of Australia, with flies more likely to possess the duplication when captured from a lower latitude and flies less likely to have the duplication when caught from a higher latitude . Taken together with other published and unpublished reports, there is strong evidence for a Cyp12d1 duplication cline in existence along the eastern coast of Australia. It is unknown what the selective agent that is driving this duplication. Further investigation may reveal this agent, and provide better understanding of the function of this duplication.

Studying Cyp12d1-d’ and Cyp12d1-p’ mRNA expression in y; cn bw sp line revealed mRNA transcript length differences, with Cyp12d1-d’ having an extra 81bp in its 3’UTR than Cyp12d1-p’. This extra sequence may contribute to mRNA stability and regulation and ultimately affect CYP12D1 protein translation, leading to differences in CYP12D1 protein expression between duplicated and non- duplicated lines.

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Embryonic expression patterns were found to be different between multiple single copy and double copy lines, and provides the first indication of tissue-specific expression differences between duplicated and non-duplicated lines. It is unknown as yet what effect this has on single copy and double copy lines, and further work should look into this embryonic expression differences. The double copy y; cn bw sp line induced Cyp12d1 expression after phenobarbital exposure to greater amounts compared to phenobarbital induction of the single copy w1118 line. This suggested that double copy lines induce higher amounts of Cyp12d1 compared to single copy lines and points to induction being a possible driving force for the selection of the duplication.

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Chapter 5: Final Discussion and Conclusion

Men love to wonder, and that is the seed of science.

- Ralph Waldo Emerson

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5.1 Introduction

Cytochrome P450s are a large superfamily of enzymes found in most organisms, involved in a variety of functions. P450s have important functions in xenobiotic metabolism (INGELMAN-SUNDBERG 2004), steroidogenesis (BISHOP 2007; SCHUSTER 2011), behaviour modification (DIERICK and GREENSPAN 2006) and development (HUANG et al. 2008) amongst other functions. The multitude of functions P450s perform in many organisms highlight their critical importance as enzymes needed for growth and development, organism homeostasis and survival when exposed to environmental or dietary toxins.

Organisms transcriptionally upregulate some P450s when they encounter toxic substances in a process called xenobiotic induction. In mammals, this process has been extensively investigated as part of studies into the metabolism of pharmaceutical drugs and two well-characterised P450 xenobiotic induction regulatory pathways are now known.

The process of xenobiotic induction is still poorly understood in insects compared to mammals.

Drosophila melanogaster upregulates various P450s in response to various xenobiotic inducers to metabolise incoming toxins. In Drosophila, the PXR and CAR orthologue HR96 has been shown to regulate some response to the classical P450 inducer phenobarbital by regulating the induction of some

P450s, but not all the P450s known to be induced by phenobarbital (KING-JONES et al. 2006). The promoter regions of some P450s have been mapped for the locations of their phenobarbital-induction enhancers, but no protein binding experiments identifying the transcription factors binding to them have been reported.

5.2 This study

This study examined the Cyp12d1 gene to better understand xenobiotic induction in Drosophila melanogaster. Cyp12d1 is arguably the most xenobiotic inducible P450 in the D. melanogaster genome, and studying how Cyp12d1 is induced will help characterise Drosophila P450 induction systems and

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ultimately discover novel P450 regulatory pathways. The Cyp12d1 promoter region was investigated using promoter::GFP constructs and a 120bp region found to control Cyp12d1 phenobarbital induction.

This region was subsequently used for further experiments to identify Cyp12d1 enhancer protein binding partners. Cyp12d1 functions were investigated to discover the biological role of Cyp12d1 in flies and to help explain why Cyp12d1 is so inducible. The UAS-GAL4 system was used to ubiquitously reduce and overexpress Cyp12d1 transcription and the phenotypes of these transgenic mutants examined. The

Cyp12d1 gene has been tandemly duplicated in certain lines to give two copies of Cyp12d1. The geographical distribution and frequency of this duplication along the east coast of Australian was studied to identify any possible roles this duplication played in adaptation to localised environments.

The expression of both Cyp12d1 genes in duplicated D. melanogaster lines was also examined to ascertain the consequences of possessing a duplicated Cyp12d1 genes.

5.3 Cyp12d1 is an evolutionary unstable mitochondrial P450

Cyp12d1 is a mitochondrially-localised P450. Mitochondrial P450s can be divided into two clades

(FEYEREISEN 2011). Evolutionary stable mitochondrial P450s are well conserved among species and carry out key endogenous functions (CLAUDIANOS et al. 2006; OMURA 2006). Evolutionary stable P450s are more likely to be lethal when expression is reduced during development, in direct contrast to unstable

P450s (CHUNG et al. 2009; THOMAS 2007). Conversely, evolutionary unstable mitochondrial P450s are poorly conserved, and are more likely to have xenobiotic functions. They are also more likely to undergo copy number variation (CNV) compared to evolutionary stable P450s. Cyp12d1 mRNA reduction during development did not affect viability, suggesting that Cyp12d1 expression was not essential for development.

The Cyp12d1 gene has also been duplicated in some other Drosophila species (Charles Robin, Personal communication) (Figure 5.1). D. simulans has Cyp12d1 and an additional Cyp12d1-like copy (termed

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Cyp12d2), which has been lost in D. melanogaster. D. ananassae, D. sechellia and D. erecta also have

Cyp12d1 and Cyp12d2, while D. yakuba has only the Cyp12d1 gene, having lost the Cyp12d2 gene

(Figure 5.1). In fact, evidence of four Cyp12d1 genes has been found in sequenced lines recently derived from natural populations of D. melanogaster. This indicates that an additional duplication event has occurred in the field (Lydia Gramzow, Robert Good and Charles Robin, unpublished data).

Taken together, the xenobiotic induction and possible detoxification function, high degree of gene gain and gene loss in certain Drosophila lineages, and non-essentiality of Cyp12d1 expression during development indicates that Cyp12d1 is a member of the evolutionary unstable mitochondrial P450 clade.

5.4 Cyp12d1 is expressed at low levels in the midgut when uninduced

The midgut is comprised of several different gut sections (MURAKAMI et al. 1999). The transcriptome profile of these sections were investigated in 3rd instar larvae using direct RNA-sequencing (RNA-seq) of

RNA samples isolated from dissected midgut sections (Stephen Pearce, unpublished data). This method quantifies the amount of mRNA for each gene, and it therefore allows the direct comparison of expression levels for all of the P450s expressed in the different sections of the midgut (Figure 5.2). A small group of P450s and transcription factors, as well as a housekeeping gene, were selected and their expression in the midgut examined.

5.4.1 Cyp12d1 expression compared to a group of P450 genes

Cyp12d1 had the lowest uninduced expression of all P450s in this subset (Figure 5.2). Cyp6a2, Cyp6a8 and Cyp6g1 have been shown to be inducible by xenobiotics, and provide the most obvious comparisons for Cyp12d1 expression when the functions of these P450s are considered. Cyp6a2 was higher expressed than Cyp12d1 in all sections, while Cyp6a8 is higher in the M1-M5 and M9-M13 regions and not

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Figure 5.1: Cyp12d1 duplication and loss in the Melanogaster group. D. sechellia has an intact Cyp12d1 copy and a Cyp12d2 psuedogene, while D. simulans has Cyp12d1 and Cyp12d2 genes. Certain D. melanogaster lines have two copies of Cyp12d1, with Cyp12d2 having been lost in D. melanogaster. D. erecta has an intact Cyp12d1 and Cyp12d2 genes, while D. yakubu only has Cyp12d1. D. ananassae has

Cyp12d1 and Cyp12d2. Figure from Robert Good (unpublished results).

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Figure 5.2: Cyp12d1 midgut section expression compared to the expression of a selected subset of genes.

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Figure 5.2: Cyp12d1 midgut section expression compared to the expression of a selected subset of genes. The RNA-sequencing method was used to determine the amount of expression of a small subset of midgut-expressed genes (six P450 genes, Cyp12d1, Cyp6a2, Cyp6a8, Cyp12a4, Cyp214a1; two transcription factors, HR96, EcR; and the housekeeping gene RPL11). All y-axis are presented in log scale. (A) Gene expression in the M1 region. Cyp12d1 expression is the third lowest expressed gene in this subset. (B) Gene expression in the M2 region. Cyp12d1 has the second lowest expression of the genes in this tissue section. (C) Gene expression in the M3-4-5 regions. Cyp12d1 has the second lowest expression of this subset of genes in this tissue section. (D) Gene expression in the M6 region. Cyp6a8 expression was not detected in this tissue section, which leaves Cyp12d1 as having the lowest expression in this subset. (E) Gene expression in the M7-M8 regions. Again, Cyp6a8 expression was not detected, which suggests that Cyp12d1 has the lowest expression of the genes tested in these tissue sections. (F) Gene expression in the M9 region. Cyp12d1 expression is the lowest detected among all the genes in this subset in this tissue.(G) Gene expression in the M10-11 regions. Cyp12d1 was the second lowest expressed gene after EcR in this dataset in this tissue section. (H) Gene expression in the M12-

M13 regions. Cyp12d1 has the lowest expression levels detected of this subset of genes in this region. (I)

Cyp6g1 vs. Cyp12d1 midgut expression. Both genes are similarly expressed in very low quantities in the

M1 section, but Cyp6g1 is expressed more highly in the M2, m3-4-5, M9, M10-11 and M12-13, but appear to be expressed at similar levels in the M6 and M7-8 sections. (F) A schematic diagram showing the location of the midgut sections. Diagram taken from (MURAKAMI et al. 1999). Glossary: FKPM =

Fragments Per Kilobase of Transcript per Million reads.

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expressed in the M6-M8 regions. Cyp6g1 was expressed at a lower level than Cyp12d1 in the M1 region, although it is unclear if this was significant, particularly given how lowly expressed both genes were in this region. Cyp6g1 expression was however higher in the M2-M13 domains. Cyp12d1 was consistently ranked among the lowest expressed P450s in all midgut sections tested (Figure 5.2).

Cyp12d1 is a mitochondrial P450, and might be expressed in lower quantities compared to microsomal

P450s because of its intracellular location. To investigate this possibility, other mitochondrial P450s

(Cyp12a4 and Cyp314a1) were included in this dataset to determine if mitochondrial P450s were in general expressed in lower quantities compared to microsomal P450s. Cyp314a1 has been shown to participate in the ecdysone pathway (PETRYK et al. 2003). Cyp314a1 was included to provide a basis for comparing Cyp12d1 expression to a P450 with an important endogenous function in the midgut. It was found that Cyp12a4 had the highest expression of all the P450s investigated while Cyp12d1 had the lowest (Figure 5.2), showing that the cellular location of a P450 was not a factor in determining the amount of expression in this dataset. Cyp314a1 was expressed in higher quantities than Cyp12d1 in all midgut sections, but was not among the highest expressed P450s. This however could be due to the age of the larvae, as these larvae were at the feeding 3rd instar larvae stage, and thus the ecdysone synthesis pathway would not be highly active. Cyp12d1 was again much lower expressed than either Cyp12a4 and

Cyp314a1 (Figure 5.2).

5.4.2 Cyp12d1 basal expression compared to non-P450 genes

Two transcription factors (HR96 and Ecdysone receptor) and one housekeeping gene (RPL11) were included to provide a basis for comparision of Cyp12d1 expression to non-P450 gene expression. In general, transcription factors are regarded as some of the lowest expressed genes. It can be seen that

HR96 is consistently higher expressed in all tissue sections compared to Cyp12d1. In contrast, Cyp12d1 and EcR were expressed at similarly low levels in the M1-M5 and M10-M11 regions, with EcR being

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higher expressed in the M6, M9 and M12-13 regions. These results showed that Cyp12d1 had mostly lower levels of expression than some transcription factors, and emphasised that Cyp12d1 is expressed at low levels when not induced.

The housekeeping gene RPL11 was used in this study as a housekeeping gene control during RT-PCR analysis. RPL11 showed the highest expression of all genes tested in the RNA-seq experiment, and again highlighted the low level of uninduced Cyp12d1 expression seen.

5.4.3 Cyp6g1 expression compared to Cyp12d1 expression

Cyp6g1 perhaps offers the best example to consider the relevance of low Cyp12d1 basal expression.

High Cyp6g1 expression confers resistance to multiple insecticides when transgenically overexpressed or in wild-caught natural populations. Cyp6g1 has been shown to be induced by fewer chemicals and to lower levels of induction compared to Cyp12d1. However, the role induction plays in insecticide resistance is limited (Section 1.4.2.1). Cyp6g1 insecticide resistance has been shown to require constitutively high expression of Cyp6g1. This may showcase a fundamental difference in the functions of Cyp6g1 and Cyp12d1, as Cyp6g1 does not require the same degree of induction Cyp12d1 demonstrates but instead relies on higher levels of uninduced expression. It must be noted that while

Cyp6g1 was expressed in lower amounts than Cyp6a2 and Cyp6a8 in this study, these experiments were performed using w1118 flies. Cyp6g1 expression has been shown to be dependent on the allelic status of

Cyp6g1. This line has the ancestral form of the Cyp6g1 allele (Joshua Schmidt, personal communication) and hence the lowest Cyp6g1 expression of all the alleles. The discrepancy between Cyp12d1 and

Cyp6g1 basal expression observed in this study may be larger when a line with a more derived Cyp6g1 allele is examined.

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5.5 How is Cyp12d1 induction linked to Cyp12d1 function?

It is still unclear what role induction plays in the functions of Cyp12d1. But given that Cyp12d1 is induced by numerous xenobiotic compounds, it seems reasonable to assume that Cyp12d1 function is linked to

Cyp12d1 induction.

5.5.1 Is low Cyp12d1 basal expression linked to Cyp12d1 function?

The RNA-seq dataset showed that in the absence of induction, Cyp12d1 is expressed at relatively low levels in the midgut. This may explain why Cyp12d1 has higher fold-change levels when induced compared to other xenobiotic inducible P450s like Cyp6a2, as they are already expressed at higher levels and would require a comparatively smaller increase in expression. As Cyp12d1 expression is present in such low quantities, it would require a much larger fold-change to reach an equivalently high expression level.

The low basal expression and high Cyp12d1 inducibility seen in this study provides an insight into the functions of Cyp12d1. In contrast to highly expressed genes like Cyp12a4 or RPL11, Cyp12d1 does not seem to require high levels of basal expression in the midgut to perform its function but instead need to be induced first. In short, Cyp12d1 midgut function may only be achieved after induction has occurred, and by extending this argument, implies that Cyp12d1 midgut function is only needed when Drosophila encounters substances that require xenobiotic induction.

5.5.2 Is Cyp12d1 part of an inducer-specific or generalised induction response?

To better understand the relationship between Cyp12d1 induction and Cyp12d1 function, the question of why Cyp12d1 is induced by numerous compounds must first be addressed. Two explanations for this come to mind: 1) that it is induced to detoxify xenobiotic chemicals, 2) it is part of a general stress response that responds to environmental and chemical stress inducers, and is upregulated when this response is activated.

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5.5.2.1 Substrate specificity of Cyp12d1

Investigating Cyp12d1 substrate specificity may help determine if Cyp12d1 may be induced as part of a whole cell detoxification response to xenobiotic challenge, or if it is part of an indirect generalised stress response that is upregulated during induction but not directly involved in detoxification. Cyp12d1 could be expressed in a heterologous system and investigated to see if it was able to metabolise the chemicals known to induce it. It would be interesting to test if Cyp12d1 was able to break down phenobarbital initially before testing other chemicals such as caffeine. Cyp12d1 is induced by the natural compounds pepper, pyrethrum and caffeine (JENSEN et al. 2006; WILLOUGHBY et al. 2006), but it is unknown if other naturally occurring environmental or dietary toxins also induce it. Environmental and dietary toxins were proposed to be a selective agent for the Cyp12d1 geographical cline (Chapter 4), and further investigation should be undertaken to determine if Cyp12d1 is induced by toxins commonly found in these microenvironments.

Using transgenic Cyp12d1-overexpressing flies to determine resistance to substances will also help establish substrate specifies for Cyp12d1. Determining which compounds Cyp12d1-overexpressing flies are resistant to will provide evidence for Cyp12d1 being able to metabolise these compounds.

Homology modelling is a third method which could be used to investigate potential Cyp12d1 substrates.

For instance, the insecticide DDT is a known Cyp12d1 substrate and inducer. Homology modelling of

Cyp12d1 has indicated that Cyp12d1 was able to bind DDT reasonably well (JONES et al. 2010), suggesting that in the case of DDT, Cyp12d1 is directly induced in response to DDT in order to detoxify DDT.

However, the accuracy of using homology modelling to determine substrate specificity has been questioned (DOMANSKI and HALPERT 2001). For instance, Cyp12d1 was predicted to strongly bind to the insecticide nitenpyram, implying that Cyp12d1 was able to metabolise nitenpyram (JONES et al. 2010).

Transgenic fly assays showed Cyp12d1 overexpressing flies had increased susceptibility to nitenpyram,

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not resistance (DABORN et al. 2007). It may be that CYP12D1 activates nitenpyram into a more toxic form, similar to how CYP3A4 and CYP2D6 activate organophosphate insecticides, making flies overexpressing Cyp12d1 more susceptible to nitenpyram (SAMS et al. 2000). This highlights the danger of relying solely on in silico results without experimental validation.

5.5.2.2 Could Cyp12d1 be part of an nonspecific stress response?

Xenobiotic induction results in the upregulation of many pathways, such as stress response pathways and energy metabolism pathways (KING-JONES et al. 2006; UEDA et al. 2002). It is possible that Cyp12d1 participates in these other pathways, and is upregulated accordingly as part of the general response, rather than being upregulated specifically to detoxify xenobiotic chemicals. This would help explain why

Cyp12d1 is inducible by many different chemicals, as it may be involved in crucial mitochondrial pathways and activities required to support the induction response. Given that induction is accompanied by an increase in mitochondrial activity (LUNDGREN and DEPIERRE 1987; MARONPOT et al.

2010) as cells cope with the xenobiotic challenge, it is possible that Cyp12d1 is upregulated because of its participation in these networks.

Generating a complete Cyp12d1 null mutant and looking at xenobiotic-induced gene expression using microarrays would help determine what pathways if any Cyp12d1 is involved in. Toxicology and chemical stress tests looking at the resistance profile of these mutants may also indicate if Cyp12d1 is also involved in mediating resistance to the chemicals used, or if Cyp12d1 is involved indirectly in xenobiotic induction.

5.5.2.3 Are environmental conditions an inducer of Cyp12d1 expression?

Cyp12d1 has been shown to be induced by numerous xenobiotic substances, indicating that it responds to chemical stress. However, environmental stress has not been reported to be an inducer of higher

Cyp12d1 transcription.

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Cyp12d1 was not induced when flies were stressed by cold, heat or desiccation (QIN et al. 2005;

SØRENSEN et al. 2009; SØRENSEN et al. 2007). This suggested that Cyp12d1 induction is not needed for adaptation to changes in microenvironment factors such as temperature or humidity. In fact, Cyp12d1 was downregulated in chill-coma selected lines (TELONIS-SCOTT et al. 2009), further evidence that

Cyp12d1 is not involved in the response to cold stress. These findings perhaps indicates that environmental factors were not the selective agent for the Cyp12d1 duplication cline discussed in

Chapter 4.

5.6 Xenobiotic induction in Drosophila melanogaster

Many cytochrome P450s require the input of multiple transcription factors in order to respond to different inducing agents and to achieve maximum induction. For instance, the Cyp3A4 gene requires

PXR (TOLSON and WANG 2010), HNF4α (JOVER et al. 2009) and the Vitamin D receptor (DROCOURT et al.

2002) in order to respond to a variety of inducing agents. Studies in mammals have indicated the presence of regulatory cascades controlling the activation of mammalian CAR and PXR in response to phenobarbital (SUEYOSHI and NEGISHI 2001; TIMSIT and NEGISHI 2007).

These studies suggest complex genetic networks could be regulating Drosophila P450 induction. Rather than one master transcription factor regulating all transcriptional induction in cells, a cascade of genes may be acting in concert to control xenobiotic induction. The role of HR96 in phenobarbital induction appears limited, and other possible transcription factors regulating phenobarbital induction have been proposed in this study (See Section 2.4.4). Once additional phenobarbital-regulating transcription factors have been identified, other known chemical inducers should be used to investigate if these transcription factors also regulate response to these inducers. This will also serve to identify other upstream regulatory elements that could take part in the xenobiotic induction, and help improve our knowledge of Drosophila induction.

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5.6.1 D. melanogaster responds to prototypical P450 inducers

Insect P450 expression has been shown to be induced by classical P450 inducing chemicals. D. melanogaster responds to classical P450 inducers like phenobarbital and rifampicin (AMICHOT et al.

1998). This demonstrates that D. melanogaster has conserved induction mechanisms that respond to the same classes of inducing chemicals as mammals. An extensive assay investigating different known mammalian P450 inducers found that Cyp6a2 was found to respond to mostly CYP2B agonists rather that CYP1A-type inducers (GIRAUDO et al. 2010). Although this experiment has not been repeated using other D. melanogaster P450s, taken together with the finding that phenobarbital induces numerous D. melanogaster CYP6 genes, it seems to suggest that the xenobiotic-inducible CYP6 family in D. melanogaster is regulated in a CYP2 and CYP3 family-like manner. Despite this, differences still exist between mammalian and Drosophila xenobiotic induction, and will be discussed below.

5.6.2 Drosophila respond differently to caffeine induction

Ten Drosophila P450s are induced by both phenobarbital and caffeine in 3rd instar y; cn bw sp larvae

(WILLOUGHBY et al. 2006). This is in direct contrast to mammals, where phenobarbital and caffeine have been shown to induce different xenobiotic induction pathways and P450s. phenobarbital induces the

CYP2 and CYP3 P450 family genes primarily via the PXR/CAR pathway (KODAMA and NEGISHI 2006b;

TOLSON and WANG 2010), while caffeine induces the Cyp1A1 and Cyp1A2 gene via the AhR pathway

(GOASDUFF et al. 1996). This suggests that Drosophila induction pathways and mammalian induction pathways have diverged in the recognition and transcriptional response to the CYP1A agonist caffeine.

The zebrafish Cyp3A65 gene has also been shown to respond to 2’,3’,7’,8’-Tetrachlorodibenzo-p-dioxin

(TCDD), which induces CYP1A gene expression but not CYP3A expression in mammals (TSENG et al. 2005).

These results demonstrate that the xenobiotic induction is species specific, and different species can induce markedly different P450s in response to various chemicals.

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Work on Cyp6a8 indicated that phenobarbital and caffeine were regulated by different pathways

(BHASKARA et al. 2008; BHASKARA et al. 2006), as exposing larvae to both compounds simultaneously induced Cyp6a8 expression to higher expression than either alone. The authors explained this by suggesting two separate proteins binding to the Cyp6a8 enhancer and synergistically inducing expression. However, no evidence was presented for this not simply being an additive effect from greater stimulation of a phenobarbital/caffeine-sensing transcription factor. Different ligands have been shown to provoke different receptor activity through conformational changes in the ligand binding domain of nuclear receptor (GRONEMEYER and LAUDET 1995; WEATHERMAN et al. 1999), and it is also possible that caffeine does not stimulate P450 expression as much as phenobarbital.

Caffeine and phenobarbital provoke different fold-change responses for Cyp12d1, with caffeine only inducing 12-fold higher Cyp12d1 expression compared to 24-30 fold Cyp12d1 upregulation for phenobarbital, depending on the strain used. This may be due to caffeine and phenobarbital being regulated by different pathways as proposed by (BHASKARA et al. 2008) or that caffeine is not as potent an inducer as phenobarbital of the same regulatory transcription factor. Work in this thesis concentrated on identifying phenobarbital response elements and proteins, but in future caffeine induction should also be studied to determine if similar networks regulate Cyp12d1 caffeine and phenobarbital induction. It would be interesting to find out which Drosophila P450s responded to other

CYP1A family-type inducers, to establish if two distinct pathways of induction exist in Drosophila like in mammals.

5.7 Evolution of cis-regulatory elements involved in D. melanogaster xenobiotic induction cis-regulatory elements are essential for ensuring correct temporal and spatial expression of genes. It has been proposed that cis-regulatory elements and transcription factors co-evolve, and it is the overall functionality of this complex which is preserved by selection and helps to conserve gene expression

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patterns with little discernable similarities in enhancer sequences between species (RUVINSKY and

RUVKUN 2003). As a result, promoter sequences and gene expression patterns could be markedly different between species. This divergence in gene expression patterns has been said to be one of the driving factors for speciation (WRAY et al. 2003).

Studies into the evolution of gene regulatory systems have found varying levels of functional conservation between evolutionary distant species. A study examining promoter conservation between

C. elegans and D. melanogaster found that only two promoter sequences from D. melanogaster genes were able to drive GFP expression in C. elegans in similar tissue-specific patterns when the promoter regions of 22 D. melanogaster genes were cloned and injected into C. elegans (RUVINSKY and RUVKUN

2003). Phenobarbital-response elements from human CYP2B6, rat CYP2B2, and mouse Cyp2b10 were able to be activated by various inducers in chicken LMH cells, showing that chicken xenobiotic induction machinery were able to recognise and initiate xenobiotic induction with Cis-elements from other species

(HANDSCHIN et al. 2001).

5.7.1 Do direct repeat elements regulate phenobarbital induction in Drosophila?

The cis-regulatory enhancer sequences of multiple mammalian P450s have been extensively characterised (see Section 1.3). There is evidence that D. melanogaster uses direct-repeat elements, similar to mammalian CYP2B family cis-regulatory motifs, to regulate phenobarbital induction. CYP2B family members use the defined 51-bp PBREM module to respond to phenobarbital challenge (SUEYOSHI and NEGISHI 2001). The PBREM sequence contains several repeat elements including a DR6 sequence that has been shown to bind CAR and upregulate CYP2B expression (SUEYOSHI and NEGISHI 2001). An imperfect DR6 match element was found to modulate some Cyp6g1 phenobarbital induction when mutated through site-directed mutagenesis (CHUNG et al., SUBMITTED). This suggests that induction mechanisms in D. melanogaster and mammals may use similar classes of cis-regulatory elements to

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control phenobarbital induction. No repeated elements were found in the Cyp12d1 -1100/-1bp regulatory sequence, suggesting that Cyp12d1 was not regulated by enhancers with direct repeat elements. However, given the wide plasticity of enhancer sites (WRAY et al. 2003), it is possible that

Cyp12d1 may still be regulated by the same mechanisms that control Cyp6g1 induction.

5.7.2 Induction modulator elements in D. melanogaster

Promoters consist of two distinct components. The basal transcriptional machinery operate to turn on gene transcription, but usually can only do this with the help of other enhancers. These enhancers, such as tissue-specific enhancers or developmentally-regulated enhancers co-operate with the basal transcriptional elements to upregulate target gene expression.

A Cyp6g1 149bp minimal fragment, termed a “modulator element” by the authors, was able to drive induced expression using the HSP70 TATA box (CHUNG et al., SUBMITTED). This element contains the DR-6 element discussed above, and was found to operate independently of other Cyp6g1 tissue specific enhancers, and was able to interact with a non-Cyp6g1 TATA box (the HSP70 TATA box) to drive phenobarbital-induced expression. It was postulated that this element has evolved to operate with existing tissue-specific enhancers to selectively increase the output of Cyp6g1 in the presence of an inducing agent (e.g. phenobarbital).

This separation of Induction enhancers and tissue-specific enhancers has been noted in other P450 induction systems. Cyp3A4 has two distinct and necessary enhancer regions needed to fully respond to inducing chemicals. Cyp3A4 tissue-specific enhancer regions were also localised to different upstream regions, showing the separation of induction and tissue-specific enhancers (GOODWIN et al. 1999;

MARTÍNEZ-JÍMENEZ et al. 2007). The Cyp2B family uses the PBRE element to upregulate CYP2B genes, but these PBRE elements require the presence of other basal transcriptional elements in order to induce expression (SUEYOSHI and NEGISHI 2001).

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The induction modulator elements have not been defined for other Drosophila P450s. Cyp6a2 PBREs and native tissue-specific enhancers were mapped to a 299bp region (-129/-428bp sequence) but this region has never been further narrowed to separate Cyp6a2 induction and tissue-specific enhancers, if possible (DUNKOV et al. 1997). Similarly, Cyp6a8 promoter elements are located in a 562bp region (-199/-

761bp) and also have never been further defined (BHASKARA et al. 2006; MAITRA et al. 2002). Cyp12d1 shows some degree of separation, with 3’ tissue-specific enhancers found in the +2049/+2116bp region, and induction enhancers located together with some tissue-specific enhancers in the 120bp -188/168bp sequence. Further work separating the two types of enhancers may discover if Cyp12d1 induction is also regulated by an induction modulator element similar to Cyp6g1.

A P450 could theoretically accumulate multiple modulator elements which would allow it to respond to multiple forms of stimuli, and allow participation in numerous gene regulatory networks. This may even partially explain why Cyp12d1 is so inducible. Cyp12d1 could have multiple modulator elements located in its promoter region which allow it to be induced by different transcription factors and different inducing chemicals.

5.8 Concluding remarks

The Cytochrome P450s are a fascinating subject to study. P450s perform many diverse functions and organisms have evolved intricate mechanisms to control their expression. This study examined the induction of Cyp12d1 as a medium for understanding how Drosophila induces P450s in response to xenobiotic challenge. Our knowledge of Drosophila xenobiotic induction is still sparse, and I hope that this study has contributed to expanding this knowledge.

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Chapter 6: Bibliography

Happy is he who gets to know the reasons for things.

Virgil (70-19 BCE) Roman poet

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AITHAL, G. P., C. P. DAY, P. J. L. KESTEVEN AND A. K. DALY, 1999 ASSOCIATION OF POLYMORPHISMS IN THE CYTOCHROME P450 CYP2C9 WITH WARFARIN DOSE REQUIREMENT AND RISK OF BLEEDING COMPLICATIONS. LANCET 353: 717-719. ALLEN, A. K., AND A. C. SPRADLING, 2008 THE SF1-RELATED NUCLEAR HORMONE RECEPTOR HR39 REGULATES DROSOPHILA FEMALE REPRODUCTIVE TRACT DEVELOPMENT AND FUNCTION. DEVELOPMENT 135: 311-321. ALVAREZ, A. D., W. SHI, B. A. WILSON AND J. B. SKEATH, 2003 PANNIER AND POINTEDP2 ACT SEQUENTIALLY TO REGULATE DROSOPHILA HEART DEVELOPMENT. DEVELOPMENT 130: 3015-3026. AMICHOT, M., A. BRUN, A. CUANY, G. DE SOUZA, T. LE MOUÉL ET AL., 1998 INDUCTION OF CYTOCHROME P450 ACTIVITIES IN DROSOPHILA MELANOGASTER STRAINS SUSCEPTIBLE OR RESISTANT TO INSECTICIDES. COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY PART C: PHARMACOLOGY, TOXICOLOGY AND ENDOCRINOLOGY 121: 311-319. AMRANI, N., R. GANESAN, S. KERVESTIN, D. A. MANGUS, S. GHOSH ET AL., 2004 A FAUX 3[PRIME]-UTR PROMOTES ABERRANT TERMINATION AND TRIGGERS NONSENSE- MEDIATED MRNA DECAY. NATURE 432: 112-118. AUSUBEL, F., R. BRENT, R. KINGSTON, D. MOORE, J. SEIDMAN ET AL. (EDITORS), 1994 CURRENT PROTOCOLS IN MOLECULAR BIOLOGY. JOHN WILEY & SONS, NEW YORK. AVIDOR-REISS, T., A. M. MAER, E. KOUNDAKJIAN, A. POLYANOVSKY, T. KEIL ET AL., 2004 DECODING CILIA FUNCTION: DEFINING SPECIALIZED GENES REQUIRED FOR COMPARTMENTALIZED CILIA BIOGENESIS. CELL 117: 527-539. BAER, B. R., AND A. E. RETTIE, 2006 CYP4B1: AN ENIGMATIC P450 AT THE INTERFACE BETWEEN XENOBIOTIC AND ENDOBIOTIC METABOLISM. DRUG METABOLISM REVIEWS 38: 451-476. BAKER, N. E., 2007 PATTERNING SIGNALS AND PROLIFERATION IN DROSOPHILA IMAGINAL DISCS. CURRENT OPINION IN GENETICS & DEVELOPMENT 17: 287-293. BANI, M.-H., M. FUKUHARA, M. KIMURA AND F. USHIO, 1998 MODULATION OF SNAKE HEPATIC CYTOCHROME P450 BY 3-METHYLCHOLANTHRENE AND PHENOBARBITAL. COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY PART C: PHARMACOLOGY, TOXICOLOGY AND ENDOCRINOLOGY 119: 143-148. BAROLO, S., L. A. CARVER AND J. W. POSAKONY, 2000 GFP AND B-GALACTOSIDASE TRANSFORMATION VECTORS FOR PROMOTER/ENHANCER ANALYSIS IN DROSOPHILA. BIOTECHNIQUES 29: 726-732. BECKSTEAD, R. B., G. LAM AND C. S. THUMMEL, 2005 THE GENOMIC RESPONSE TO 20-HYDROXYECDYSONE AT THE ONSET OF DROSOPHILA METAMORPHOSIS. GENOME BIOLOGY 6. BEEDANAGARI, S. R., R. T. TAYLOR, P. BUI, F. WANG, D. W. NICKERSON ET AL., 2010 ROLE OF EPIGENETIC MECHANISMS IN DIFFERENTIAL REGULATION OF THE DIOXIN-INDUCIBLE HUMAN CYP1A1 AND CYP1B1 GENES. MOLECULAR PHARMACOLOGY 78: 608-616. BHASKARA, S., M. B. CHANDRASEKHARAN AND R. GANGULY, 2008 CAFFEINE INDUCTION OF CYP6A2 AND CYP6A8 GENES OF DROSOPHILA MELANOGASTER IS MODULATED BY CAMP AND D-JUN PROTEIN LEVELS. GENE 415: 49-59. BHASKARA, S., E. D. DEAN, V. LAM AND R. GANGULY, 2006 INDUCTION OF TWO CYTOCHROME P450 GENES, CYP6A2 AND CYP6A8, OF DROSOPHILA MELANOGASTER BY CAFFEINE IN ADULT FLIES AND IN CELL CULTURE. GENE 377: 56-64. BISHOP, G. J., 2007 REFINING THE PLANT STEROID HORMONE BIOSYNTHESIS PATHWAY. TRENDS IN PLANT SCIENCE 12: 377-380. BOGWITZ, M. R., H. CHUNG, L. MAGOC, S. RIGBY, W. WONG ET AL., 2005 CYP12A4 CONFERS LUFENURON RESISTANCE IN A NATURAL POPULATION OF DROSOPHILA MELANOGASTER. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102: 12807-12812. BOMBAIL, V., K. TAYLOR, G. G. GIBSON AND N. PLANT, 2004 ROLE OF SP1, C/EBP{ALPHA}, HNF3, AND PXR IN THE BASAL- AND XENOBIOTIC-MEDIATED REGULATION OF THE CYP3A4 GENE. DRUG METAB DISPOS 32: 525-535. BOUCHER, J.-L., M. DELAFORGE AND D. MANSUY, 1994 DEHYDRATION OF ALKYL- AND ARYLALDOXIMES AS A NEW CYTOCHROME P450-CATALYZED REACTION: MECHANISM AND STEREOCHEMICAL CHARACTERISTICS. BIOCHEMISTRY 33: 7811-7818. BRANDT, A., M. SCHARF, J. H. F. PEDRA, G. HOLMES, A. DEAN ET AL., 2002 DIFFERENTIAL EXPRESSION AND INDUCTION OF TWO DROSOPHILA CYTOCHROME P450 GENES NEAR THE RST(2)DDT LOCUS. INSECT MOLECULAR BIOLOGY 11: 337- 341.

227

BROWN, S., AND J. CASTELLI-GAIR HOMBRIA, 2000 DROSOPHILA GRAIN ENCODES A GATA TRANSCRIPTION FACTOR REQUIRED FOR CELL REARRANGEMENT DURING MORPHOGENESIS. DEVELOPMENT 127: 4867-4876. BRUN, A., A. CUANY, T. LEMOUEL, J. BERGE AND M. AMICHOT, 1996 INDUCIBILITY OF THE DROSOPHILA MELANOGASTER CYTOCHROME P450 GENE, CYP6A2, BY PHENOBARBITAL IN INSECTICIDE SUSCEPTIBLE OR RESISTANT STRAINS. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 26: 697-703. BUJOLD, M., A. GOPALAKRISHNAN, E. NALLY AND K. KING-JONES, 2010 NUCLEAR RECEPTOR DHR96 ACTS AS A SENTINEL FOR LOW CHOLESTEROL CONCENTRATIONS IN DROSOPHILA MELANOGASTER. MOL. CELL. BIOL. 30: 793-805. BURBACH, K. M., A. POLAND AND C. A. BRADFIELD, 1992 CLONING OF THE AH-RECEPTOR CDNA REVEALS A DISTINCTIVE LIGAND-ACTIVATED TRANSCRIPTION FACTOR. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES 89: 8185- 8189. BUREIK, M., M. LISUREK AND R. BERNHARDT, 2002 THE HUMAN STEROID HYDROXYLASES CYP11B1 AND CYP11B2. BIOLOGICAL CHEMISTRY 383: 1537-1551. BUTLER, M. J., T. L. JACOBSEN, D. M. CAIN, M. G. JARMAN, M. HUBANK ET AL., 2003 DISCOVERY OF GENES WITH HIGHLY RESTRICTED EXPRESSION PATTERNS IN THE DROSOPHILA WING DISC USING DNA OLIGONUCLEOTIDE MICROARRAYS. DEVELOPMENT 130: 659-670. BUTOW, R. A., AND N. G. AVADHANI, 2004 MITOCHONDRIAL SIGNALING: THE RETROGRADE RESPONSE. MOLECULAR CELL 14: 1-15. CAI, Z., J. KWINTKIEWICZ, M. E. YOUNG AND C. STOCCO, 2007 PROSTAGLANDIN E2 INCREASES CYP19 EXPRESSION IN RAT GRANULOSA CELLS: IMPLICATION OF GATA-4. MOLECULAR AND CELLULAR ENDOCRINOLOGY 263: 181-189. CALLARD, G. V., A. V. TCHOUDAKOVA, M. KISHIDA AND E. WOOD, 2001 DIFFERENTIAL TISSUE DISTRIBUTION, DEVELOPMENTAL PROGRAMMING, ESTROGEN REGULATION AND PROMOTER CHARACTERISTICS OF CYP19 GENES IN TELEOST FISH. THE JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 79: 305-314. CARTHARIUS, K., K. FRECH, K. GROTE, B. KLOCKE, M. HALTMEIER ET AL., 2005 MATLNSPECTOR AND BEYOND: PROMOTER ANALYSIS BASED ON TRANSCRIPTION FACTOR BINDING SITES. BIOINFORMATICS 21: 2933-2942. CATANIA, F., M. O. KAUER, P. J. DABORN, J. L. YEN, R. H. FFRENCH-CONSTANT ET AL., 2004 WORLD-WIDE SURVEY OF AN ACCORD INSERTION AND ITS ASSOCIATION WITH DDT RESISTANCE IN DROSOPHILA MELANOGASTER. MOLECULAR ECOLOGY 13: 2491-2504. CHEN, Y. P., S. S. FERGUSON, M. NEGISHI AND J. A. GOLDSTEIN, 2003 IDENTIFICATION OF GLUCOCORTICOID RECEPTOR (GR) AND CONSTITUTIVE ANDROSTANE RECEPTER (CAR) BINDING SITES IN THE PROMOTER OF CYP2C19. FASEB JOURNAL 17: A240-A240. CHENG, J. B., M. A. LEVINE, N. H. BELL, D. J. MANGELSDORF AND D. W. RUSSELL, 2004 GENETIC EVIDENCE THAT THE HUMAN CYP2R1 ENZYME IS A KEY VITAMIN D 25-HYDROXYLASE. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101: 7711-7715. CHIANG, E. F.-L., Y.-L. YAN, Y. GUIGUEN, J. POSTLETHWAIT AND B.-C. CHUNG, 2001 TWO CYP19 (P450 AROMATASE) GENES ON DUPLICATED ZEBRAFISH CHROMOSOMES ARE EXPRESSED IN OVARY OR BRAIN. MOLECULAR BIOLOGY AND EVOLUTION 18: 542-550. CHINTAPALLI, V. R., J. WANG AND J. A. T. DOW, 2007 USING FLYATLAS TO IDENTIFY BETTER DROSOPHILA MELANOGASTER MODELS OF HUMAN DISEASE. NAT GENET 39: 715-720. CHOI, M., P. SKIPPER, J. WISHNOK AND S. TANNENBAUM, 2005 CHARACTERIZATION OF TESTOSTERONE 11-HYDROXYLATION CATALYZED BY HUMAN LIVER MICROSOMAL CYTOCHROMES P450. DRUG METABOLISM AND DISPOSITION 33: 714-718. CHUNG, H., M. R. BOGWITZ, C. MCCART, A. ANDRIANOPOULOS, R. H. FFRENCH-CONSTANT ET AL., 2007 CIS- REGULATORY ELEMENTS IN THE ACCORD RETROTRANSPOSON RESULT IN TISSUE-SPECIFIC EXPRESSION OF THE DROSOPHILA MELANOGASTER INSECTICIDE RESISTANCE GENE CYP6G1. GENETICS 175: 1071-1077. CHUNG, H., T. SZTAL, S. PASRICHA, M. SRIDHAR, P. BATTERHAM ET AL., 2009 CHARACTERIZATION OF DROSOPHILA MELANOGASTER CYTOCHROME P450 GENES. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES 106: 5731-5736.

228

CLANCY, D. J., D. GEMS, L. G. HARSHMAN, S. OLDHAM, H. STOCKER ET AL., 2001 EXTENSION OF LIFE-SPAN BY LOSS OF CHICO, A DROSOPHILA INSULIN RECEPTOR SUBSTRATE PROTEIN. SCIENCE 292: 104-106. CLAUDIANOS, C., H. RANSON, R. M. JOHNSON, S. BISWAS, M. A. SCHULER ET AL., 2006 A DEFICIT OF DETOXIFICATION ENZYMES: PESTICIDE SENSITIVITY AND ENVIRONMENTAL RESPONSE IN THE HONEYBEE. INSECT MOLECULAR BIOLOGY 15: 615-636. COHEN, M., M. BERENBAUM AND M. SCHULER, 1992 A HOST-INDUCIBLE CYTOCHROME P-450 FROM A HOST-SPECIFIC CATERPILLAR: MOLECULAR CLONING AND EVOLUTION. PROC NATL ACAD SCI USA 89: 10920 - 10924. COPELAND, J. M., J. CHO, T. LO, J. H. HUR, S. BAHADORANI ET AL., 2009 EXTENSION OF DROSOPHILA LIFE SPAN BY RNAI OF THE MITOCHONDRIAL RESPIRATORY CHAIN. CURRENT BIOLOGY : CB 19: 1591-1598. CURTIS, C., G. LANDIS, D. FOLK, N. WEHR, N. HOE ET AL., 2007 TRANSCRIPTIONAL PROFILING OF MNSOD-MEDIATED LIFESPAN EXTENSION IN DROSOPHILA REVEALS A SPECIES-GENERAL NETWORK OF AGING AND METABOLIC GENES. GENOME BIOLOGY 8: R262. DABORN, P., S. BOUNDY, J. YEN, B. PITTENDRIGH AND R. FFRENCH-CONSTANT, 2001 DDT RESISTANCE IN DROSOPHILA CORRELATES WITH CYP6G1 OVER-EXPRESSION AND CONFERS CROSS-RESISTANCE TO THE NEONICOTINOID IMIDACLOPRID. MOLECULAR GENETICS AND GENOMICS 266: 556-563. DABORN, P. J., C. LUMB, A. BOEY, W. WONG, R. H. FFRENCH-CONSTANT ET AL., 2007 EVALUATING THE INSECTICIDE RESISTANCE POTENTIAL OF EIGHT DROSOPHILA MELANOGASTER CYTOCHROME P450 GENES BY TRANSGENIC OVER- EXPRESSION. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 37: 512-519. DABORN, P. J., J. L. YEN, M. R. BOGWITZ, G. LE GOFF, E. FEIL ET AL., 2002 A SINGLE P450 ALLELE ASSOCIATED WITH INSECTICIDE RESISTANCE IN DROSOPHILA. SCIENCE 297: 2253-2256. DANIELSON, P. B., J. L. M. FOSTER, M. M. MCMAHILL, M. K. SMITH AND J. C. FOGLEMAN, 1998 INDUCTION BY ALKALOIDS AND PHENOBARBITAL OF FAMILY 4 CYTOCHROME P450S IN DROSOPHILA : EVIDENCE FOR INVOLVEMENT IN HOST PLANT UTILIZATION. MOLECULAR AND GENERAL GENETICS MGG 259: 54-59. DANIELSON, P. B., J. A. LETMAN AND J. C. FOGLEMAN, 1995 ALKALOID METABOLISM BY CYTOCHROME P-450 ENZYMES IN DROSOPHILA MELANOGASTER. COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY PART B: BIOCHEMISTRY AND MOLECULAR BIOLOGY 110: 683-688. DARDENNE, O., J. PRUD'HOMME, A. ARABIAN, F. H. GLORIEUX AND R. ST-ARNAUD, 2001 TARGETED INACTIVATION OF THE 25-HYDROXYVITAMIN D-3-1 ALPHA-HYDROXYLASE GENE (CYP27B1) CREATES AN ANIMAL MODEL OF PSEUDOVITAMIN D-DEFICIENCY RICKETS. ENDOCRINOLOGY 142: 3135-3141. DAVIES, L., D. R. WILLIAMS, P. C. TURNER AND H. H. REES, 2006 CHARACTERIZATION IN RELATION TO DEVELOPMENT OF AN ECDYSTEROID AGONIST-RESPONSIVE CYTOCHROME P450, CYP18A1, IN LEPIDOPTERA. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 453: 4-12. DELAFORGE, M., F. ANDRE, M. JAOUEN, H. DOLGOS, H. BENECH ET AL., 1997 METABOLISM OF TENTOXIN BY HEPATIC CYTOCHROME P-450 3A ISOZYMES. EUROPEAN JOURNAL OF BIOCHEMISTRY 250: 150-157. DIERICK, H. A., AND R. J. GREENSPAN, 2006 MOLECULAR ANALYSIS OF FLIES SELECTED FOR AGGRESSIVE BEHAVIOR. NATURE GENETICS 38: 1023-1031. DIETZL, G., D. CHEN, F. SCHNORRER, K.-C. SU, Y. BARINOVA ET AL., 2007 A GENOME-WIDE TRANSGENIC RNAI LIBRARY FOR CONDITIONAL GENE INACTIVATION IN DROSOPHILA. NATURE 448: 151-156. DOMANSKI, T. L., AND J. R. HALPERT, 2001 ANALYSIS OF MAMMALIAN CYTOCHROME P450 STRUCTURE AND FUNCTION BY SITE-DIRECTED MUTAGENESIS. CURRENT DRUG METABOLISM 2: 117-137. DOMBROWSKI, S. M., R. KRISHNAN, M. WITTE, S. MAITRA, C. DIESING ET AL., 1998 CONSTITUTIVE AND BARBITAL- INDUCED EXPRESSION OF THE CYP6A2 ALLELE OF A HIGH PRODUCER STRAIN OF CYP6A2 IN THE GENETIC BACKGROUND OF A LOW PRODUCER STRAIN. GENE 221: 69-77. DOUGLAS, S. E., 1994 DNA STRIDER, PP. 181-194 IN COMPUTER ANALYSIS OF SEQUENCE DATA. DROCOURT, L., J.-C. OURLIN, J.-M. PASCUSSI, P. MAUREL AND M.-J. VILAREM, 2002 EXPRESSION OF CYP3A4, CYP2B6, ANDCYP2C9 IS REGULATED BY THE VITAMIN D RECEPTOR PATHWAY IN PRIMARY HUMAN HEPATOCYTES. JOURNAL OF BIOLOGICAL CHEMISTRY 277: 25125-25132.

229

DUBESSAY, P., I. GARREAU-BALANDIER, A.-S. JARROUSSE, A. FLEURIET, B. SION ET AL., 2007 AGING IMPACT ON BIOCHEMICAL ACTIVITIES AND GENE EXPRESSION OF DROSOPHILA MELANOGASTER MITOCHONDRIA. BIOCHIMIE 89: 988-1001. DUFFY, J. B., 2002 GAL4 SYSTEM IN DROSOPHILA: A FLY GENETICIST'S SWISS ARMY KNIFE, PP. 1-15. DUNKOV, B. C., V. M. GUZOV, G. MOCELIN, F. SHOTKOSKI, A. BRUN ET AL., 1997 THE DROSOPHILA CYTOCHROME P450 GENE CYP6A2: STRUCTURE, LOCALIZATION, HETEROLOGOUS EXPRESSION, AND INDUCTION BY PHENOBARBITAL. DNA AND CELL BIOLOGY 16: 1345-1356. DURST, F., AND D. P. O'KEEFE, 1995 PLANT CYTOCHROMES P450: AN OVERVIEW. DRUG METABOLISM AND DRUG INTERACTIONS 12: 171-187. EMMONS, R. B., D. DUNCAN, P. A. ESTES, P. KIEFEL, J. T. MOSHER ET AL., 1999 THE SPINELESS-ARISTAPEDIA AND TANGO BHLH-PAS PROTEINS INTERACT TO CONTROL ANTENNAL AND TARSAL DEVELOPMENT IN DROSOPHILA. DEVELOPMENT 126: 3937-3945. ÉRIC LE, B., 2001 OXIDATIVE STRESS, AGING AND LONGEVITY IN DROSOPHILA MELANOGASTER. FEBS LETTERS 498: 183- 186. FENG, L., AND D.-K. NIU, 2007 RELATIONSHIP BETWEEN MRNA STABILITY AND LENGTH: AN OLD QUESTION WITH A NEW TWIST. BIOCHEMICAL GENETICS 45: 131-137. FERGUSON, S. S., Y. P. CHEN, E. L. LECLUYSE, M. NEGISHI AND J. A. GOLDSTEIN, 2005 HUMAN CYP2C8 IS TRANSCRIPTIONALLY REGULATED BY THE NUCLEAR RECEPTORS CONSTITUTIVE ANDROSTANE RECEPTOR, PREGNANE X RECEPTOR, GLUCOCORTICOID RECEPTOR, AND HEPATIC NUCLEAR FACTOR 4 ALPHA. MOLECULAR PHARMACOLOGY 68: 747-757. FESTUCCI-BUSELLI, R. A., A. S. CARVALHO-DIAS, M. DE OLIVEIRA-ANDRADE, C. CAIXETA-NUNES, H. M. LI ET AL., 2005 EXPRESSION OF CYP6G1 AND CYP12D1 IN DDT RESISTANT AND SUSCEPTIBLE STRAINS OF DROSOPHILA MELANOGASTER. INSECT MOLECULAR BIOLOGY 14: 69-77. FEYEREISEN, R., 1999 INSECT P450 ENZYMES. ANNUAL REVIEW OF ENTOMOLOGY 44: 507-533. FEYEREISEN, R., 2005 INSECT CYTOCHROME P450., PP. 1-77 IN COMPREHENSIVE MOLECULAR INSECT SCIENCE., EDITED BY L. GILBERT, K. LATROU AND S. GILL. ELSEVIER OXFORD. FEYEREISEN, R., 2006 EVOLUTION OF INSECT P450. BIOCHEMICAL SOCIETY TRANSACTIONS 34: 1252-1255. FEYEREISEN, R., 2011 ARTHROPOD CYPOMES ILLUSTRATE THE TEMPO AND MODE IN P450 EVOLUTION. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1814: 19-28. FEYEREISEN, R., V. M. GUZOV, A. A. CHERNOGOLOV AND G. C. UNNITHAN, 1997 CYP12A1, AN INSECT MITOCHONDRIAL P450 THAT METABOLIZES XENOBIOTICS. FASEB JOURNAL 11: P249. FINKEL, T., AND N. J. HOLBROOK, 2000 OXIDANTS, OXIDATIVE STRESS AND THE BIOLOGY OF AGEING. NATURE 408: 239- 247. FISCHER, R. T., J. M. TRZASKOS, R. L. MAGOLDA, S. S. KO, C. S. BROSZ ET AL., 1991 LANOSTEROL 14 ALPHA-METHYL DEMETHYLASE. ISOLATION AND CHARACTERIZATION OF THE THIRD METABOLICALLY GENERATED OXIDATIVE DEMETHYLATION INTERMEDIATE. JOURNAL OF BIOLOGICAL CHEMISTRY 266: 6124-6132. FLOCKHART, D., 2007 DRUG INTERACTIONS: CYTOCRHOME P450 DRUG INTERACTION TABLE HTTP://MEDICINE.IUPUI.EDU/CLINPHARM/DDIS/TABLE.ASP., PP. INDIANNA UNIVERSITY SCHOOL OF MEDICINE. FU, G. K., D. LIN, M. Y. H. ZHANG, D. D. BIKLE, C. H. L. SHACKLETON ET AL., 1997 CLONING OF HUMAN 25- HYDROXYVITAMIN D-1 ALPHA-HYDROXYLASE AND MUTATIONS CAUSING VITAMIN D-DEPENDENT RICKETS TYPE 1. MOLECULAR ENDOCRINOLOGY 11: 1961-1970. FUJII-KURIYAMA, Y., AND J. MIMURA, 2005 MOLECULAR MECHANISMS OF AHR FUNCTIONS IN THE REGULATION OF CYTOCHROME P450 GENES. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS CELEBRATING 50 YEARS OF OXYGENASES 338: 311-317.

FUJII, S., A. TOYAMA AND H. AMREIN, 2008 A MALE-SPECIFIC FATTY ACID {OMEGA}-HYDROXYLASE, SXE1, IS NECESSARY FOR EFFICIENT MALE MATING IN DROSOPHILA MELANOGASTER. GENETICS 180: 179-190.

230

FUJISAWA-SEHARA, A., K. SOGAWA, M. YAMANE AND Y. FUJII-KURIYAMA, 1987 CHARACTERIZATION OF XENOBIOTIC RESPONSIVE ELEMENTS UPSTREAM FROM THE DRUG-METABOLIZING CYTOCHROME P-450C GENE: A SIMILARITY TO GLUCOCORTICOID REGULATORY ELEMENTS. NUCL. ACIDS RES. 15: 4179-4191. FURUYA, H., P. FERNANDEZ-SALGUERO, W. GREGORY, H. TABER, A. STEWARD ET AL., 1995 GENETIC POLYMORPHISM OF CYP2C9 AND ITS EFFECT ON WARFARIN MAINTENANCE DOSE REQUIREMENT IN PATIENTS UNDERGOING ANTICOAGULATION THERAPY. PHARMACOGENETICS AND GENOMICS 5: 389-392. GARCÍA-SÁNCHEZ, R., J. AYALA-LUJÁN, A. HERNÁNDEZ-PERÉZ, T. MENDOZA-FIGUEROA AND J. TAPIA-RAMÍREZ, 2003 IDENTIFICATION OF REPRESSOR ELEMENT 1 IN CYTOCHROME P450 GENES AND THEIR NEGATIVE REGULATION BY RE1 SILENCING TRANSCRIPTION FACTOR/NEURON-RESTRICTIVE SILENCER FACTOR. BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - GENERAL SUBJECTS 1620: 39-46. GERBAL-CHALOIN, S., M. DAUJAT, J.-M. PASCUSSI, L. PICHARD-GARCIA, M.-J. VILAREM ET AL., 2002 TRANSCRIPTIONAL REGULATION OF CYP2C9 GENE. JOURNAL OF BIOLOGICAL CHEMISTRY 277: 209-217. GERBAL-CHALOIN, S., J.-M. PASCUSSI, L. PICHARD-GARCIA, M. DAUJAT, F. WAECHTER ET AL., 2001 INDUCTION OF CYP2C GENES IN HUMAN HEPATOCYTES IN PRIMARY CULTURE. DRUG METABOLISM AND DISPOSITION 29: 242- 251. GIRARDOT, F., V. MONNIER AND H. TRICOIRE, 2004 GENOME WIDE ANALYSIS OF COMMON AND SPECIFIC STRESS RESPONSES IN ADULT DROSOPHILA MELANOGASTER. BMC GENOMICS 5. GIRAUDO, M., G. C. UNNITHAN, G. LE GOFF AND R. FEYEREISEN, 2010 REGULATION OF CYTOCHROME P450 EXPRESSION IN DROSOPHILA: GENOMIC INSIGHTS. PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 97: 115-122. GIRHARD, M., T. KLAUS, Y. KHATRI, R. BERNHARDT AND V. B. URLACHER, 2010 CHARACTERIZATION OF THE VERSATILE MONOOXYGENASE CYP109B1 FROM BACILLUS SUBTILIS. APPL MICROBIOL BIOTECHNOL. GOASDUFF, T., Y. DRÉANO, B. GUILLOIS, J.-F. MENEZ AND F. BERTHOU, 1996 INDUCTION OF LIVER AND KIDNEY CYP1A1/1A2 BY CAFFEINE IN RAT. BIOCHEMICAL PHARMACOLOGY 52: 1915-1919. GONZALEZ, F. J., 2005 ROLE OF CYTOCHROMES P450 IN CHEMICAL TOXICITY AND OXIDATIVE STRESS: STUDIES WITH CYP2E1. MUTATION RESEARCH/FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS 569: 101- 110. GOODWIN, B., E. HODGSON AND C. LIDDLE, 1999 THE ORPHAN HUMAN PREGNANE X RECEPTOR MEDIATES THE TRANSCRIPTIONAL ACTIVATION OF CYP3A4 BY RIFAMPICIN THROUGH A DISTAL ENHANCER MODULE. MOLECULAR PHARMACOLOGY 56: 1329-1339. GOTOH, O., 1992 SUBSTRATE RECOGNITION SITES IN CYTOCHROME P450 FAMILY 2 (CYP2) PROTEINS INFERRED FROM COMPARATIVE ANALYSES OF AMINO ACID AND CODING NUCLEOTIDE SEQUENCES. JOURNAL OF BIOLOGICAL CHEMISTRY 267: 83-90. GRONEMEYER, H., AND V. LAUDET, 1995 TRANSCRIPTION FACTORS 3: NUCLEAR RECEPTORS. PROTEIN PROGILE 2: 1173- 1308. GUENGERICH, F. P., 2001 COMMON AND UNCOMMON CYTOCHROME P450 REACTIONS RELATED TO METABOLISM AND CHEMICAL TOXICITY. CHEMICAL RESEARCH IN TOXICOLOGY 14: 611-650. GUENGERICH, F. P., L. A. PETERSON AND R. H. BÖCKER, 1988 CYTOCHROME P-450-CATALYZED HYDROXYLATION AND CARBOXYLIC ACID ESTER CLEAVAGE OF HANTZSCH PYRIDINE ESTERS. JOURNAL OF BIOLOGICAL CHEMISTRY 263: 8176-8183. GUITTARD, E., C. BLAIS, A. MARIA, J. P. PARVY, S. PASRICHA ET AL., 2011 CYP18A1, A KEY ENZYME OF DROSOPHILA STEROID HORMONE INACTIVATION, IS ESSENTIAL FOR METAMORPHOSIS. DEVELOPMENTAL BIOLOGY 349: 35-45. GUO, Y. D., S. STRUGNELL, D. W. BACK AND G. JONES, 1993 TRANSFECTED HUMAN LIVER CYTOCHROME-P-450 HYDROXYLATES VITAMIN-D ANALOGS AT DIFFERENT SIDE-CHAIN POSITIONS. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 90: 8668-8672. GUTIERREZ, E., D. WIGGINS, B. FIELDING AND A. P. GOULD, 2007 SPECIALIZED HEPATOCYTE-LIKE CELLS REGULATE DROSOPHILA LIPID METABOLISM. NATURE 445: 275-280. GUZOV, V. M., G. C. UNNITHAN, A. A. CHERNOGOLOV AND R. FEYEREISEN, 1998 CYP12A1, A MITOCHONDRIAL CYTOCHROME P450 FROM THE HOUSE FLY. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 359: 231-240.

231

HALLSTROM, I., 1985 GENETIC REGULATION OF THE CYTOCHROME P-450 SYSTEM IN DROSOPHILA MELANOGASTER. II. LOCALIZATION OF SOME GENES REGULATING CYTOCHROME P-450 ACTIVITY. CHEM BIOL INTERACT 56: 173-184. HALLSTROM, I., J. MAGNUSSON AND C. RAMEL, 1982 RELATION BETWEEN THE SOMATIC TOXICITY OF DIMETHYLNITROSAMINE AND A GENETICALLY-DETERMINED VARIATION IN THE LEVEL AND INDUCTION OF CYTOCHROME-P450 IN DROSOPHILA-MELANOGASTER. Mutation Research 92: 161-168. HANDSCHIN, C., S. BLATTLER, A. ROTH, R. LOOSER, M. OSCARSON ET AL., 2004 THE EVOLUTION OF DRUG-ACTIVATED NUCLEAR RECEPTORS: ONE ANCESTRAL GENE DIVERGED INTO TWO XENOSENSOR GENES IN MAMMALS. NUCLEAR RECEPTOR 2: 7. HANDSCHIN, C., M. PODVINEC AND U. A. MEYER, 2000 CXR, A CHICKEN XENOBIOTIC-SENSING ORPHAN NUCLEAR RECEPTOR, IS RELATED TO BOTH MAMMALIAN PREGNANE X RECEPTOR (PXR) AND CONSTITUTIVE ANDROSTANE RECEPTOR (CAR). PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES 97: 10769-10774. HANDSCHIN, C., M. PODVINEC, J. STOCKLI, K. HOFFMANN AND U. A. MEYER, 2001 CONSERVATION OF SIGNALING PATHWAYS OF XENOBIOTIC-SENSING ORPHAN NUCLEAR RECEPTORS, CHICKEN XENOBIOTIC RECEPTOR, CONSTITUTIVE ANDROSTANE RECEPTOR, AND PREGNANE X RECEPTOR, FROM BIRDS TO HUMANS. MOL ENDOCRINOL 15: 1571-1585. HARE, E. E., B. K. PETERSON AND M. B. EISEN, 2008 A CAREFUL LOOK AT BINDING SITE REORGANIZATION IN THE EVEN- SKIPPED ENHANCERS OF DROSOPHILA AND SEPSIDS. PLOS GENET 4: E1000268. HATAKEYAMA, J., AND R. KAGEYAMA, 2004 RETINAL CELL FATE DETERMINATION AND BHLH FACTORS. SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY 15: 83-89. HEITZLER, P., M. HAENLIN, P. RAMAIN, M. CALLEJA AND P. SIMPSON, 1996 A GENETIC ANALYSIS OF PANNIER, A GENE NECESSARY FOR VIABILITY OF DORSAL TISSUES AND BRISTLE POSITIONING IN DROSOPHILA. GENETICS 143: 1271- 1286. HELFAND, S. L., AND B. ROGINA, 2003 GENETICS OF AGING IN THE FRUIT FLY, DROSOPHILA MELANOGASTER. ANNUAL REVIEW OF GENETICS 37: 329-348. HELVIG, C., J. F. KOENER, G. C. UNNITHAN AND R. FEYEREISEN, 2004A CYP15A1, THE CYTOCHROME P450 THAT CATALYZES EPOXIDATION OF METHYL FARNESOATE TO JUVENILE HORMONE III IN COCKROACH CORPORA ALLATA. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101: 4024-4029. HELVIG, C., N. TIJET, R. FEYEREISEN, F. A. WALKER AND L. L. RESTIFO, 2004B DROSOPHILA MELANOGASTER CYP6A8, AN INSECT P450 THAT CATALYSES LAURIC ACID (-1)-HYDROXYLATION. BIOCHEMICAL BIOPHYSICAL RESEARCH COMMUNICATIONS 325: 1495-1502. HONKAKOSKI, P., R. MOORE, K. A. WASHBURN AND M. NEGISHI, 1998 ACTIVATION BY DIVERSE XENOCHEMICALS OF THE 51-BASE PAIR PHENOBARBITAL-RESPONSIVE ENHANCER MODULE IN THE CYP2B10 GENE. MOLECULAR PHARMACOLOGY 53: 597-601. HONKAKOSKI, P., AND M. NEGISHI, 1997 CHARACTERIZATION OF A PHENOBARBITAL-RESPONSIVE ENHANCER MODULE IN MOUSE P450 CYP2B10 GENE. JOURNAL OF BIOLOGICAL CHEMISTRY 272: 14943-14949. HORNER, M. A., K. PARDEE, S. LIU, K. KING-JONES, G. LAJOIE ET AL., 2009 THE DROSOPHILA DHR96 NUCLEAR RECEPTOR BINDS CHOLESTEROL AND REGULATES CHOLESTEROL HOMEOSTASIS. GENES & DEVELOPMENT 23: 2711-2716. HRYCAY, E. G., AND S. M. BANDIERA, 2009 EXPRESSION, FUNCTION AND REGULATION OF MOUSE CYTOCHROME P450 ENZYMES: COMPARISON WITH HUMAN P450 ENZYMES. CURRENT DRUG METABOLISM 10: 1151-1183. HUANG, X., J. T. WARREN AND L. I. GILBERT, 2008 NEW PLAYERS IN THE REGULATION OF ECDYSONE BIOSYNTHESIS. JOURNAL OF GENETICS AND GENOMICS 35: 1-10. HUNG, C. F., M. R. BERENBAUM AND M. A. SCHULER, 1997 ISOLATION AND CHARACTERIZATION OF CYP6B4, A FURANOCOUMARIN-INDUCIBLE CYTOCHROME P450 FROM A POLYPHAGOUS CATERPILLAR (LEPIDOPTERA: PAPILIONIDAE). INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 27: 377-385. HUNG, C. F., H. PRAPAIPONG, M. R. BERENBAUM AND M. A. SCHULER, 1995 DIFFERENTIAL INDUCTION OF CYTOCHROME-P450 TRANSCRIPTS IN PAPILIO POLYXENES BY LINEAR AND ANGULAR FURANOCOUMARINS. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 25: 89-99.

232

HURBAN, P., AND C. S. THUMMEL, 1993 ISOLATION AND CHARACTERIZATION OF FIFTEEN ECDYSONE-INDUCIBLE DROSOPHILA GENES REVEAL UNEXPECTED COMPLEXITIES IN ECDYSONE REGULATION. MOLECULAR AND CELLULAR BIOLOGY 13: 7101-7111. HUSS, J. M., AND C. B. KASPER, 2000 TWO-STAGE GLUCOCORTICOID INDUCTION OF CYP3A23 TTHROUGH BOTH THE GLUCOCORTICOID AND PREGNANE X RECEPTORS. MOLECULAR PHARMACOLOGY 58: 48-57. IBEANU, G. C., B. I. GHANAYEM, P. LINKO, L. LI, L. G. PEDERSEN ET AL., 1996 IDENTIFICATION OF RESIDUES 99, 220, AND 221 OF HUMAN CYTOCHROME P450 2C19 AS KEY DETERMINANTS OF OMEPRAZOLE HYDROXYLASE ACTIVITY. JOURNAL OF BIOLOGICAL CHEMISTRY 271: 12496-12501. INGELMAN-SUNDBERG, M., 2004 PHARMACOGENETICS OF CYTOCHROME P450 AND ITS APPLICATIONS IN DRUG THERAPY: THE PAST, PRESENT AND FUTURE. TRENDS IN PHARMACOLOGICAL SCIENCES 25: 193-200. INGELMAN-SUNDBERG, M., S. SIM, A. GOMEZ AND C. RODRIGUEZ-ANTONA, 2007 INFLUENCE OF CYTOCHROME P450 POLYMORPHISMS ON DRUG THERAPIES: PHARMACOGENETIC, PHARMACOEPIGENETIC AND CLINICAL ASPECTS. PHARMACOLOGY & THERAPEUTICS 116: 496-526. ITOH, A., AND G. A. HOWE, 2001 MOLECULAR CLONING OF A DIVINYL ETHER SYNTHASE. JOURNAL OF BIOLOGICAL CHEMISTRY 276: 3620-3627. JENSEN, H. R., I. A. SCOTT, S. SIMS, V. L. TRUDEAU AND J. T. ARNASON, 2006 GENE EXPRESSION PROFILES OF DROSOPHILA MELANOGASTER EXPOSED TO AN INSECTICIDAL EXTRACT OF PIPER NIGRUM. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 54: 1289-1295. JEZEK, P., AND L. HLAVATA, 2005 MITOCHONDRIA IN HOMEOSTASIS OF REACTIVE OXYGEN SPECIES IN CELL, TISSUES, AND ORGANISM. THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY 37: 2478-2503. JONES, R. T., S. E. BAKKER, D. STONE, S. N. SHUTTLEWORTH, S. BOUNDY ET AL., 2010 HOMOLOGY MODELLING OF DROSOPHILA CYTOCHROME P450 ENZYMES ASSOCIATED WITH INSECTICIDE RESISTANCE. PEST MANAGEMENT SCIENCE 66: 1106-1115. JOUSSEN, N., D. G. HECKEL, M. HAAS, I. SCHUPHAN AND B. SCHMIDT, 2008 METABOLISM OF IMIDACLOPRID AND DDT BY P450 GYP6G1 EXPRESSED IN CELL CULTURES OF NICOTIANA TABACUM SUGGESTS DETOXIFICATION OF THESE INSECTICIDES IN CYP6G1-OVEREXPRESSING STRAINS OF DROSOPHILA MELANOGASTER, LEADING TO RESISTANCE. PEST MANAGEMENT SCIENCE 64: 65-73. JOVER, R., M. MOYA AND M. J. GOMEZ-LECHON, 2009 TRANSCRIPTIONAL REGULATION OF CYTOCHROME P450 GENES BY THE NUCLEAR RECEPTOR HEPATOCYTE NUCLEAR FACTOR 4-ALPHA. CURRENT DRUG METABOLISM 10: 508- 519. KAMINSKY, L., AND Z. ZHANG, 1997 HUMAN P450 METABOLISM OF WARFARIN. PHARMACOLOGY & THERAPEUTICS 3: 67-74. KARIM, F. D., G. M. GUILD AND C. S. THUMMEL, 1993 THE DROSOPHILA BROAD-COMPLEX PLAYS A KEY ROLE IN CONTROLLING ECDYSONE-REGULATED GENE EXPRESSION AT THE ONSET OF METAMORPHOSIS. DEVELOPMENT 118: 977-988. KASAI, S., AND T. TOMITA, 2002 MALE SPECIFIC EXPRESSION OF A CYTOCHROME P450 (CYP312A1) IN DROSOPHILA MELANOGASTER. BIOCHEMICAL BIOPHYSICAL RESEARCH COMMUNICATIONS 300: 894-9000. KAWAJIRI, K., AND Y. FUJII-KURIYAMA, 2007 CYTOCHROME P450 GENE REGULATION AND PHYSIOLOGICAL FUNCTIONS MEDIATED BY THE ARYL HYDROCARBON RECEPTOR. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 464: 207-212. KELLY, D. E., N. KRASEVEC, J. MULLINS AND D. R. NELSON, 2009 THE CYPOME (CYTOCHROME P450 COMPLEMENT) OF ASPERGILLUS NIDULANS. FUNGAL GENETICS AND BIOLOGY 46: S53-S61. KIKKAWA, H., 1961 GENETICAL STUDIES ON THE RESISTANCE TO PARATHION IN DROSOPHILA MELANOGASTER. ANN. REP. SCI. WORKS FAC. SCI, OSAKA UNIV. 9: 1-20. KIM, J.-W., K. I. ZELLER, Y. WANG, A. G. JEGGA, B. J. ARONOW ET AL., 2004 EVALUATION OF MYC E-BOX PHYLOGENETIC FOOTPRINTS IN GLYCOLYTIC GENES BY CHROMATIN IMMUNOPRECIPITATION ASSAYS. MOL. CELL. BIOL. 24: 5923-5936. KIMURA, K. D., H. A. TISSENBAUM, Y. LIU AND G. RUVKUN, 1997 DAF-2, AN INSULIN RECEPTOR-LIKE GENE THAT REGULATES LONGEVITY AND DIAPAUSE IN CAENORHABDITIS ELEGANS. SCIENCE 277: 942-946.

233

KING-JONES, K., M. A. HORNER, G. LAM AND C. S. THUMMEL, 2006 THE DHR96 NUCLEAR RECEPTOR REGULATES XENOBIOTIC RESPONSES IN DROSOPHILA. CELL METABOLISM 4: 37-48. KING-JONES, K., AND C. S. THUMMEL, 2005 NUCLEAR RECEPTORS- A PERSPECTIVE FROM DROSOPHILA. NATURE REVIEWS GENETICS 6: 311-323. KING, J. C., 1954 THE GENETICS OF RESISTANCE TO DDT IN DROSOPHILA-MELANOGASTER. JOURNAL OF ECONOMIC ENTOMOLOGY 47: 387-393. KISHIDA, M., M. MCLELLAN, J. A. MIRANDA AND G. V. CALLARD, 2001 ESTROGEN AND XENOESTROGENS UPREGULATE THE BRAIN AROMATASE ISOFORM (P450AROMB) AND PERTURB MARKERS OF EARLY DEVELOPMENT IN ZEBRAFISH (DANIO RERIO). COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY PART B: BIOCHEMISTRY AND MOLECULAR BIOLOGY 129: 261-268. KOBAYASHI, A., K. SOGAWA AND Y. FUJII-KURIYAMA, 1996 COOPERATIVE INTERACTION BETWEEN AHRARNT AND SP1 FOR THE DRUG-INDUCIBLE EXPRESSION OF CYP1A1 GENE. JOURNAL OF BIOLOGICAL CHEMISTRY 271: 12310- 12316. KODAMA, S., AND M. NEGISHI, 2006A PHENOBARBITAL CONFERS ITS DIVERSE EFFECTS BY ACTIVATING THE ORPHAN NUCLEAR RECEPTOR CAR. DRUG METABOLISM REVIEWS 38: 75-87. KODAMA, S., AND M. NEGISHI, 2006B PHENOBARBITAL CONFERS ITS DIVERSE EFFECTS BY ACTIVATING THE ORPHAN NUCLEAR RECEPTOR CAR. DRUG METABOLISM REVIEWS 38: 75-87. KOLACZKOWSKI, B., A. D. KERN, A. K. HOLLOWAY AND D. J. BEGUN, 2011 GENOMIC DIFFERENTIATION BETWEEN TEMPERATE AND TROPICAL AUSTRALIAN POPULATIONS OF DROSOPHILA MELANOGASTER. GENETICS 187: 245- 260. KWAN, P., AND M. J. BRODIE, 2004 PHENOBARBITAL FOR THE TREATMENT OF EPILEPSY IN THE 21ST CENTURY: A CRITICAL REVIEW. EPILEPSIA 45: 1141-1149. LANDIS, G. N., D. ABDUEVA, D. SKVORTSOV, J. YANG, B. E. RABIN ET AL., 2004 SIMILAR GENE EXPRESSION PATTERNS CHARACTERIZE AGING AND OXIDATIVE STRESS IN DROSOPHILA MELANOGASTER. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101: 7663-7668. LE GOFF, G., S. BOUNDY, P. J. DABORN, J. L. YEN, L. SOFER ET AL., 2003 MICROARRAY ANALYSIS OF CYTOCHROME P450 MEDIATED INSECTICIDE RESISTANCE IN DROSOPHILA. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 33: 701- 708. LE GOFF, G., F. HILLIOU, B. D. SIEGFRIED, S. BOUNDY, E. WAJNBERG ET AL., 2006 XENOBIOTIC RESPONSE IN DROSOPHILA MELANOGASTER: SEX DEPENDENCE OF P450 AND GST GENE INDUCTION. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 36: 674-682. LEWIS, D. F. V., 2001 GUIDE TO CYTOCHROMES P450: STRUCTURE AND FUNCTION. TAYLOR & FRANCIS. LEWIS, D. F. V., E. WATSON AND B. G. LAKE, 1998 EVOLUTION OF THE CYTOCHROME P450 SUPERFAMILY: SEQUENCE ALIGNMENTS AND PHARMACOGENETICS. MUTATION RESEARCH 410: 245-270. LI, X. C., J. BAUDRY, M. R. BERENBAUM AND M. A. SCHULER, 2004 STRUCTURAL AND FUNCTIONAL DIVERGENCE OF INSECT CYP6B PROTEINS: FROM SPECIALIST TO GENERALIST CYTOCHROME P450. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101: 2939-2944. LIANG, Q., J.-S. HE AND A. J. FULCO, 1995 THE ROLE OF BARBIE BOX SEQUENCES AS CIS-ACTING ELEMENTS INVOLVED IN THE BARBITURATE-MEDIATED INDUCTION OF CYTOCHROMES P450BM−1 AND P450BM−3 IN BACILLUS MEGATERIUM. JOURNAL OF BIOLOGICAL CHEMISTRY 270: 4438-4450. LIN, G. G. H., T. KOZAKI AND J. G. SCOTT, 2011 HORMONE RECEPTOR-LIKE IN 96 AND BROAD-COMPLEX MODULATE PHENOBARBITAL INDUCED TRANSCRIPTION OF CYTOCHROME P450 CYP6D1 IN DROSOPHILA S2 CELLS. INSECT MOLECULAR BIOLOGY 20: 87-95. LINDBLOM, T. H., G. J. PIERCE AND A. SLUDER, 2001 A C.ELEGANS ORPHAN NUCLEAR RECEPTOR CONTRIBUTES TO XENOBIOTIC RESISTANCE. CURRENT BIOLOGY 11: 864-868. LINFORD, N. J., S. E. SCHRINER AND P. S. RABINOVITCH, 2006 OXIDATIVE DAMAGE AND AGING: SPOTLIGHT ON MITOCHONDRIA. CANCER RESEARCH 66: 2497-2499.

234

LUDLOW, L. B., B. P. SCHICK, M. L. BUDARF, D. A. DRISCOLL, E. H. ZACKAI ET AL., 1996 IDENTIFICATION OF A MUTATION IN A GATA BINDING SITE OF THE PLATELET GLYCOPROTEIN IB BETA PROMOTER RESULTING IN THE BERNARD- SOULIER SYNDROME. JOURNAL OF BIOLOGICAL CHEMISTRY 271: 22076-22080. LUNDGREN, B., AND J. W. DEPIERRE, 1987 INDUCTION OF XENOBIOTIC-METABOLIZING ENZYMES AND PEROXISOME PROLIFERATION IN RAT LIVER CAUSED BY DIETARY EXPOSURE TO DI(2-ETHYLHEXYL)PHOSPHATE. XENOBIOTICA 17: 585-593. MAGGERT, K. A., W. J. GONG AND K. G. GOLIC, 2008 METHODS FOR HOMOLOGOUS RECOMBINATION IN DROSOPHILA. METHODS IN MOLECULAR BIOLOGY 420: 155-174. MAGLICH, J. M., J. A. CARAVELLA, M. H. LAMBERT, T. M. WILLSON, J. T. MOORE ET AL., 2003 THE FIRST COMPLETED GENOME SEQUENCE FROM A TELEOST FISH (FUGU RUBRIPES) ADDS SIGNIFICANT DIVERSITY TO THE NUCLEAR RECEPTOR SUPERFAMILY. NUCLEIC ACIDS RESEARCH 31: 4051-4058. MAGLICH, J. M., A. SLUDER, X. GUAN, Y. SHI, D. D. MCKEE ET AL., 2001 COMPARISON OF COMPLETE NUCLEAR RECEPTOR SETS FROM THE HUMAN, CAENORHABDITIS ELEGANS AND DROSOPHILA GENOMES. GENOME BIOLOGY 2: 0029.0021-0029.0027. MAGLICH, J. M., C. M. STOLTZ, B. GOODWIN, D. HAWKINS-BROWN, J. T. MOORE ET AL., 2002 NUCLEAR PREGNANE X RECEPTOR AND CONSTITUTIVE ANDROSTANE RECEPTOR REGULATE OVERLAPPING BUT DISTINCT SETS OF GENES INVOLVED IN XENOBIOTIC DETOXIFICATION. MOLECULAR PHARMACOLOGY 62: 638-646. MAITRA, S., C. PRICE AND R. GANGULY, 2002 CYP6A8 OF DROSOPHILA MELANOGASTER: GENE STRUCTURE, AND SEQUENCE AND FUNCTIONAL ANALYSIS OF THE UPSTREAM DNA. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 32: 859-870. MAJERUS, P., G. BROZE, J. MILETICH AND D. TOLLEFSEN, 1996 ANTICOAGULANT THROMBOLYTIC, AND ANTIPLATELET DRUGS., PP. 1347-1351 IN GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, EDITED BY J. HARDMAN AND L. LIMBIRD. MCGRAW-HILL, NEW YORK. MANNEL, M., 2004 DRUG INTERACTIONS WITH ST JOHN'S WORT - MECHANISMS AND CLINICAL IMPLICATIONS. DRUG SAFETY 27: 773-797. MARKSTEIN, M., C. PITSOULI, C. VILLALTA, S. E. CELNIKER AND N. PERRIMON, 2008 EXPLOITING POSITION EFFECTS AND THE GYPSY RETROVIRUS INSULATOR TO ENGINEER PRECISELY EXPRESSED TRANSGENES. NAT GENET 40: 476-483. MARONPOT, R. R., K. YOSHIZAWA, A. NYSKA, T. HARADA, G. FLAKE ET AL., 2010 HEPATIC ENZYME INDUCTION: HISTOPATHOLOGY. TOXICOLOGIC PATHOLOGY 38: 776-795. MARTÍNEZ-JÍMENEZ, C. P., R. JOVER, M. TERESA DONATO, J. V. CASTELL AND M. JOSE GOMEZ-LECHON, 2007 TRANSCRIPTIONAL REGULATION AND EXPRESSION OF CYP3A4 IN HEPATOCYTES. CURRENT DRUG METABOLISM 8: 185-194. MATÉS, J. M., C. PÉREZ-GÓMEZ AND I. N. DE CASTRO, 1999 ANTIOXIDANT ENZYMES AND HUMAN DISEASES. CLINICAL BIOCHEMISTRY 32: 595-603. MAYER, B., AND R. OBERBAUER, 2003 MITOCHONDRIAL REGULATION OF APOPTOSIS. PHYSIOLOGY 18: 89-94. MCDONNELL, C. M., R. P. BROWN, M. R. BERENBAUM AND M. A. SCHULER, 2004 CONSERVED REGULATORY ELEMENTS IN THE PROMOTERS OF TWO ALLELOCHEMICAL-INDUCIBLE CYTOCHROME P450 GENES DIFFERENTIALLY REGULATE TRANSCRIPTION. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 34: 1129-1139. MCKILLOP, I. H., AND L. W. SCHRUM, 2005 ALCOHOL AND LIVER CANCER. ALCOHOL 35: 195-203. MEIJERMAN, I., L. M. SANDERSON, P. H. M. SMITS, J. H. BEIJNEN AND J. H. M. SCHELLENS, 2007 PHARMACOGENETIC SCREENING OF THE GENE DELETION AND DUPLICATIONS OF CYP2D6. DRUG METABOLISM REVIEWS 39: 45-60. MEYER, U. A., 2007 ENDO-XENOBIOTIC CROSSTALK AND THE REGULATION OF CYTOCHROMES P450. DRUG METABOLISM REVIEWS 39: 639-646. MODE, A., P. TOLLET, A. STRÖM, C. LEGRAVEREND, C. LIDDLE ET AL., 1992 GROWTH HORMONE REGULATION OF HEPATIC CYTOCHROME P450 EXPRESSION IN THE RAT. ADVANCES IN ENZYME REGULATION 32: 255-263. MOHIT, P., K. MAKHIJANI, M. B. MADHAVI, V. BHARATHI, A. LAL ET AL., 2006 MODULATION OF AP AND DV SIGNALING PATHWAYS BY THE HOMEOTIC GENE ULTRABITHORAX DURING HALTERE DEVELOPMENT IN DROSOPHILA. DEVELOPMENTAL BIOLOGY 291: 356-367.

235

MOORE, L. B., J. M. MAGLICH, D. D. MCKEE, B. WISELY, T. M. WILLSON ET AL., 2002 PREGNANE X RECEPTOR (PXR), CONSTITUTIVE ANDROSTANE RECEPTOR (CAR), AND BENZOATE X RECEPTOR (BXR) DEFINE THREE PHARMACOLOGICALLY DISTINCT CLASSES OF NUCLEAR RECEPTORS. MOL ENDOCRINOL 16: 977-986. MOREL, Y., N. MERMOD AND R. BAROUKI, 1999 AN AUTOREGULATORY LOOP CONTROLLING CYP1A1 GENE EXPRESSION: ROLE OF H2O2 AND NFI. MOL. CELL. BIOL. 19: 6825-6832. MORRA, R., S. KURUGANTI, V. LAM, J. C. LUCCHESI AND R. GANGULY, 2010 FUNCTIONAL ANALYSIS OF THE CIS-ACTING ELEMENTS RESPONSIBLE FOR THE INDUCTION OF THE CYP6A8 AND CYP6G1 GENES OF DROSOPHILA MELANOGASTER BY DDT, PHENOBARBITAL AND CAFFEINE. INSECT MOLECULAR BIOLOGY 19: 121-130. MUNRO, A. W., D. G. LEYS, K. J. MCLEAN, K. R. MARSHALL, T. W. B. OST ET AL., 2002 P450 BM3: THE VERY MODEL OF A MODERN FLAVOCYTOCHROME. TRENDS IN BIOCHEMICAL SCIENCES 27: 250-257. MURAKAMI, R., T. OKUMURA AND H. UCHIYAMA, 2005 GATA FACTORS AS KEY REGULATORY MOLECULES IN THE DEVELOPMENT OF DROSOPHILA ENDODERM. DEVELOPMENT, GROWTH & DIFFERENTIATION 47: 581-589. MURAKAMI, R., S. TAKASHIMA AND T. HAMAGUCHI, 1999 DEVELOPMENTAL GENETICS OF THE DROSOPHILA GUT: SPECIFICATION OF PRIMORDIA, SUBDIVISION AND OVERT-DIFFERENTATION. CELLULAR AND MOLECULAR BIOLOGY 45: 661–676. MURTAZINA, D. A., U. ANDERSSON, I.-S. HAHN, I. BJORKHEM, G. A. S. ANSARI ET AL., 2004 PHOSPHOLIPIDS MODIFY SUBSTRATE BINDING AND ENZYME ACTIVITY OF HUMAN CYTOCHROME P450 27A1. JOURNAL OF LIPID RESEARCH 45: 2345-2353. MWINYI, J., J. NEKVINDOVÁ, I. CAVACO, Y. HOFMANN, R. S. PEDERSEN ET AL., 2010 NEW INSIGHTS INTO THE REGULATION OF CYP2C9 GENE EXPRESSION: THE ROLE OF THE TRANSCRIPTION FACTOR GATA-4. DRUG METABOLISM AND DISPOSITION 38: 415-421. NAMIKI, T., R. NIWA, T. SAKUDOH, K. SHIRAI, H. TAKEUCHI ET AL., 2005 CYTOCHROME P450CYP307A1/SPOOK: A REGULATOR FOR ECDYSONE SYNTHESIS IN INSECTS. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 337: 367-374. NEBERT, D. W., AND F. J. GONZALEZ, 1987 P450 GENES - STRUCTURE, EVOLUTION, AND REGULATION. ANNUAL REVIEW OF BIOCHEMISTRY 56: 945-993. NEBERT, D. W., D. R. NELSON AND R. FEYEREISEN, 1989 EVOLUTION OF THE CYTOCHROME P450 GENES. XENOBIOTICA 19: 1149-1160. NEI, M., AND A. P. ROONEY, 2005 CONCERTED AND BIRTH-AND-DEATH EVOLUTION OF MULTIGENE FAMILIES*. ANNUAL REVIEW OF GENETICS 39: 121-152. NELSON, D. R., M. A. SCHULER, S. M. PAQUETTE, D. WERCK-REICHHART AND S. BAK, 2004A COMPARATIVE GENOMICS OF RICE AND ARABIDOPSIS. ANALYSIS OF 727 CYTOCHROME P450 GENES AND PSEUDOGENES FROM A MONOCOT AND A DICOT. PLANT PHYSIOLOGY 135: 756-772. NELSON, D. R., D. C. ZELDIN, S. M. HOFFMAN, L. J. MALTAIS, H. M. WAIN ET AL., 2004B COMPARISON OF CYTOCHROME P450 (CYP) GENES FROM THE MOUSE AND HUMAN GENOMES, INCLUDING NOMENCLATURE RECOMMENDATIONS FOR GENES, PSEUDOGENES AND ALTERNATIVE-SPLICE VARIANTS. PHARMACOGENETICS AND GENOMICS 14: 1-18. NIWA, R., T. MATSUDA, T. YOSHIYAMA, T. NAMIKI, K. MITA ET AL., 2004 CYP306A1, A CYTOCHROME P450 ENZYME, IS ESSENTIAL FOR ECDYSTEROID BIOSYNTHESIS IN THE PROTHORACIC GLANDS OF BOMBYX AND DROSOPHILA. JOURNAL OF BIOLOGICAL CHEMISTRY 279: 35942-35949. NORGATE, M., A. SOUTHON, S. ZOU, M. ZHAN, Y. SUN ET AL., 2007 COPPER HOMEOSTASIS GENE DISCOVERY IN DROSOPHILA MELANOGASTER. BIOMETALS 20: 683-697. NUKAYA, M., AND C. A. BRADFIELD, 2009 CONSERVED GENOMIC STRUCTURE OF THE CYP1A1 AND CYP1A2 LOCI AND THEIR DIOXIN RESPONSIVE ELEMENTS CLUSTER. BIOCHEMICAL PHARMACOLOGY 77: 654-659. OGG, M. S., J. M. WILLIAMS, M. TARBIT, P. S. GOLDFARB, T. J. GRAY ET AL., 1999 A HUMAN REPORTER GENE ASSAY TO ASSESS THE MOLECULAR MECHANISMS OF XENOBIOTIC-DEPENDENT INDUCITON OF THE HUMAN CYP3A4 GENE IN VITRO. XENOBIOTICA 29: 269-279. OGITA, Z., 1958 A NEW TYPE OF INSECTICIDE. NATURE 182: 1529-1530.

236

OKUMURA, T., A. MATSUMOTO, T. TANIMURA AND R. MURAKAMI, 2005 AN ENDODERM-SPECIFIC GATA FACTOR GENE, DGATAE, IS REQUIRED FOR THE TERMINAL DIFFERENTIATION OF THE DROSOPHILA ENDODERM. DEVELOPMENTAL BIOLOGY 278: 576-586. OMURA, T., 2006 MITOCHONDRIAL P450S. CHEMICO-BIOLOGICAL INTERACTIONS MITOCHONDRIAL TOXICITY 163: 86- 93. OMURA, T., 2010 STRUCTURAL DIVERSITY OF CYTOCHROME P450 ENZYME SYSTEM. JOURNAL OF BIOCHEMISTRY 147: 297-306. ONO, H., K. F. REWITZ, T. SHINODA, K. ITOYAMA, A. PETRYK ET AL., 2006 SPOOK AND SPOOKIER CODE FOR STAGE- SPECIFIC COMPONENTS OF THE ECDYSONE BIOSYNTHETIC PATHWAY IN DIPTERA. DEVELOPMENTAL BIOLOGY 298: 555-570. ORRENIUS, S., A. GOGVADZE AND B. ZHIVOTOVSKY, 2007 MITOCHONDRIAL OXIDATIVE STRESS: IMPLICATIONS FOR CELL DEATH. ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY 47: 143-183. ORTIZ DE MONTELLANO, P. R., 2005 CYTOCHROME P450 : STRUCTURE, MECHANISM, AND BIOCHEMISTRY KLUWER ACADEMIC/PLENUM PUBLISHERS, NEW YORK. PALANKER, L., A. S. NECAKOV, H. M. SAMPSON, R. NI, C. HU ET AL., 2006 DYNAMIC REGULATION OF DROSOPHILA NUCLEAR RECEPTOR ACTIVITY IN VIVO. DEVELOPMENT 133: 3549-3562. PALANKER, L., J. M. TENNESSEN, G. LAM AND C. S. THUMMEL, 2009 DROSOPHILA HNF4 REGULATES LIPID MOBILIZATION AND ²-OXIDATION. CELL METABOLISM 9: 228-239. PARTRIDGE, L., M. D. W. PIPER AND W. MAIR, 2005 DIETARY RESTRICTION IN DROSOPHILA. MECHANISMS OF AGEING AND DEVELOPMENT 126: 938-950. PERRY, T., P. BATTERHAM AND P. J. DABORN, THE BIOLOGY OF INSECTICIDAL ACTIVITY AND RESISTANCE. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY IN PRESS, CORRECTED PROOF. PETERSEN BROWN, R., M. R. BERENBAUM AND M. A. SCHULER, 2004 TRANSCRIPTION OF A LEPIDOPTERAN CYTOCHROME P450 PROMOTER IS MODULATED BY MULTIPLE ELEMENTS IN ITS 5′ UTR AND REPRESSED BY 20- HYDROXYECDYSONE. INSECT MOLECULAR BIOLOGY 13: 337-347. PETERSEN BROWN, R., C. M. MCDONNELL, M. R. BERENBAUM AND M. A. SCHULER, 2005 REGULATION OF AN INSECT CYTOCHROME P450 MONOOXYGENASE GENE (CYP6B1) BY ARYL HYDROCARBON AND XANTHOTOXIN RESPONSE CASCADES. GENE (AMSTERDAM) 358: 39-52. PETERSEN, R. A., H. NIAMSUP, M. R. BERENBAUM AND M. A. SCHULER, 2003 TRANSCRIPTIONAL RESPONSE ELEMENTS IN THE PROMOTER OF CYP6B1, AN INSECT P450 GENE REGULATED BY PLANT CHEMICALS. BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1619: 269-282. PETERSEN, U.-M., L. KADALAYIL, K.-P. REHORN, D. K. HOSHIZAKI, R. REUTER ET AL., 1999 SERPENT REGULATES DROSOPHILA IMMUNITY GENES IN THE LARVAL FAT BODY THROUGH AN ESSENTIAL GATA MOTIF. EMBO J 18: 4013-4022. PETRYK, A., J. T. WARREN, G. MARQUES, M. P. JARCHO, L. I. GILBERT ET AL., 2003 SHADE IS THE DROSOPHILA P450 ENZYME THAT MEDIATES THE HYDROXYLATION OF ECDYSONE TO THE STEROID INSECT MOLTING HORMONE 20- HYDROXYECDYSONE. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100: 13773-13778. PIKULEVA, I. A., 2006 CYTOCHROME P450S AND CHOLESTEROL HOMEOSTASIS. PHARMACOLOGY & THERAPEUTICS 112: 761-773. POUPARDIN, R., S. REYNAUD, C. STRODE, H. RANSON, J. VONTAS ET AL., 2008 CROSS-INDUCTION OF DETOXIFICATION GENES BY ENVIRONMENTAL XENOBIOTICS AND INSECTICIDES IN THE MOSQUITO AEDES AEGYPTI: IMPACT ON LARVAL TOLERANCE TO CHEMICAL INSECTICIDES. INSECT BIOCHEM MOL BIOL 38: 540 - 551. PREMEREUR, N., C. VAN DEN BRANDEN AND F. ROELS, 1986 CYTOCHROME P45O-DEPENDENT H202 PRODUCTION DEMONSTRATED IN VIVO. INFLUENCE OF PHENOBARBITAL AND ALLYL-ISOPROPYLACETAMIDE. FEBS LETT 199: 19- 22. PROSSER, D. E., AND G. JONES, 2004 ENZYMES INVOLVED IN THE ACTIVATION AND INACTIVATION OF VITAMIN D. TRENDS IN BIOCHEMICAL SCIENCES 29: 664-673.

237

QIAN, L., AND R. BODMER, 2009 PARTIAL LOSS OF GATA FACTOR PANNIER IMPAIRS ADULT HEART FUNCTION IN DROSOPHILA. HUMAN MOLECULAR GENETICS 18: 3153-3163. QIN, W., S. J. NEAL, R. M. ROBERTSON, J. T. WESTWOOD AND V. K. WALKER, 2005 COLD HARDENING AND TRANSCRIPTIONAL CHANGE IN DROSOPHILA MELANOGASTER. INSECT MOLECULAR BIOLOGY 14: 607-613. RAHIER, A., AND M. TATON, 1986 THE 14[ALPHA]-DEMETHYLATION OF OBTUSIFOLIOL BY A CYTOCHROME P-450 MONOOXYGENASE FROM HIGHER PLANTS MICROSOMES. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 140: 1064-1072. RAMSDEN, R., N. B. BECK, K. M. SOMMER AND C. J. OMIECINSKI, 1999 PHENOBARBITAL RESPONSIVENESS CONFERRED BY THE 5'-FLANKING REGION OF THE RAT CYP2B2 GENE IN TRANSGENIC MICE. GENE 228: 169-179. RANSON, H., C. CLAUDIANOS, F. ORTELLI, C. ABGRALL, J. HEMINGWAY ET AL., 2002 EVOLUTION OF SUPERGENE FAMILIES ASSOCIATED WITH INSECTICIDE RESISTANCE. SCIENCE 298: 179-181. RENDIC, S., 2002 SUMMARY OF INFORMATION ON HUMAN CYP ENZYMES: HUMAN P450 METABOLISM DATA. DRUG METABOLISM REVIEWS 34: 83-448. ROGINA, B., R. A. REENAN, S. P. NILSEN AND S. L. HELFAND, 2000 EXTENDED LIFE-SPAN CONFERRED BY COTRANSPORTER GENE MUTATIONS IN DROSOPHILA. SCIENCE 290: 2137-2140. ROZMAN, D., 2000 LANOSTEROL 14A-DEMETHYLASE (CYP51) - A CHOLESTEROL BIOSYNTHETIC ENZYME INVOLVED IN PRODUCTION OF MEIOSIS ACTIVATING STEROLS IN OOCYTES AND TESTIS - A MINIREVIEW. PFLÜGERS ARCHIV EUROPEAN JOURNAL OF PHYSIOLOGY 439: R056-R057. RUPASINGHE, S. G., Z. WEN, T.-L. CHIU AND M. A. SCHULER, 2007 HELICOVERPA ZEA CYP6B8 AND CYP321A1: DIFFERENT MOLECULAR SOLUTIONS TO THE PROBLEM OF METABOLIZING PLANT TOXINS AND INSECTICIDES. PROTEIN ENGINEERING DESIGN AND SELECTION 20: 615-624. RUVINSKY, I., AND G. RUVKUN, 2003 FUNCTIONAL TESTS OF ENHANCER CONSERVATION BETWEEN DISTANTLY RELATED SPECIES. DEVELOPMENT 130: 5133-5142. SAMS, C., H. J. MASON AND R. RAWBONE, 2000 EVIDENCE FOR THE ACTIVATION OF ORGANOPHOSPHATE PESTICIDES BY CYTOCHROMES P450 3A4 AND 2D6 IN HUMAN LIVER MICROSOMES. TOXICOLOGY LETTERS 116: 217-221. SCHMIDT, J. M., R. T. GOOD, B. APPLETON, J. SHERRARD, G. C. RAYMANT ET AL., 2010 COPY NUMBER VARIATION AND TRANSPOSABLE ELEMENTS FEATURE IN RECENT, ONGOING ADAPTATION AT THE CYP6G1 LOCUS. PLOS GENET 6: E1000998. SCHNEIDER, D., 2000 USING DROSOPHILA AS A MODEL INSECT. NATURE REVIEWS GENETICS 1: 218-226. SCHNEIDER, I., 1972 CELL LINES DERIVED FROM LATE EMBRYONIC STAGES OF DROSOPHILA MELANOGASTER. JOURNAL OF EMBRYOLOGY AND EXPERIMENTAL MORPHOLOGY 27: 353-365. SCHULER, M. A., 2011 P450S IN PLANT-INSECT INTERACTIONS. BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - PROTEINS & PROTEOMICS 1814: 36-45. SCHUSTER, I., 2011 CYTOCHROMES P450 ARE ESSENTIAL PLAYERS IN THE VITAMIN D SIGNALING SYSTEM. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1814: 186-199. SCOTT, J. G., 2008 INSECT CYTOCHROME P450S: THINKING BEYOND DETOXIFICATION, PP. 117-124 IN RECENT ADVANCES IN INSECT PHYSIOLOGY, TOXICOLOGY AND MOLECULAR BIOLOGY, EDITED BY N. LIU. RESEARCH SIGNPOST, KERALA, INDIA. SEDEN, K., L. DICKINSON, S. KHOO AND D. BACK, 2010 GRAPEFRUIT-DRUG INTERACTIONS. DRUGS 70: 2373-2407. SENGER, K., K. HARRIS AND M. LEVINE, 2006 GATA FACTORS PARTICIPATE IN TISSUE-SPECIFIC IMMUNE RESPONSES IN DROSOPHILA LARVAE. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES 103: 15957-15962. SETO, Y., AND F. P. GUENGERICH, 1993 PARTITIONING BETWEEN N-DEALKYLATION AND N-OXYGENATION IN THE OXIDATION OF N,N-DIALKYLARYLAMINES CATALYZED BY CYTOCHROME-P450-2B1. JOURNAL OF BIOLOGICAL CHEMISTRY 268: 9986-9997. SHAW, G. C., AND A. J. FULCO, 1992 BARBITURATE-MEDIATED REGULATION OF EXPRESSION OF THE CYTOCHROME-P450BM-3 GENE OF BACILLUS-MEGATERIUM BY BM3R1 PROTEIN. JOURNAL OF BIOLOGICAL CHEMISTRY 267: 5515-5526.

238

SHIMADA, T., C. H. YUN, H. YAMAZAKI, J. C. GAUTIER, P. H. BEAUNE ET AL., 1992 CHARACTERIZATION OF HUMAN LUNG MICROSOMAL CYTOCHROME P-450 1A1 AND ITS ROLE IN THE OXIDATION OF CHEMICAL CARCINOGENS. MOLECULAR PHARMACOLOGY 41: 856-864. SHULDINER, A., M. W. CARILLO AND J. M. HEBERT, 2007 IMPORTANT HAPLOTYPE INFORMATION FOR CYP3A4 HTTP://WWW.PHARMGKB.ORG/SEARCH/ANNOTATEDGENE/CYP3A4/HAPLOTYPE.JSP, PP. SIEBER, M. H., AND C. S. THUMMEL, 2009 THE DHR96 NUCLEAR RECEPTOR CONTROLS TRIACYLGLYCEROL HOMEOSTASIS IN DROSOPHILA. CELL METABOLISM 10: 481-490. SIK LEE, Y., AND R. W. CARTHEW, 2003 MAKING A BETTER RNAI VECTOR FOR DROSOPHILA: USE OF INTRON SPACERS. METHODS 30: 322-329. SMITH, E. M., AND J. Y. WILSON, 2010 ASSESSMENT OF CYTOCHROME P450 FLUOROMETRIC SUBSTRATES WITH RAINBOW TROUT AND KILLIFISH EXPOSED TO DEXAMETHASONE, PREGNENOLONE-16[ALPHA]-CARBONITRILE, RIFAMPICIN, AND [BETA]-NAPHTHOFLAVONE. AQUATIC TOXICOLOGY 97: 324-333. SØRENSEN, J. G., M. M. NIELSEN, M. KRUHØFFER, J. JUSTESEN AND V. LOESCHCKE, 2009 FULL GENOME GENE EXPRESSION ANALYSIS OF THE HEAT STRESS RESPONSE IN DROSOPHILA MELANOGASTER. CELL STRESS & CHAPERONES 10: 312-328. SØRENSEN, J. G., M. M. NIELSEN AND V. LOESCHCKE, 2007 GENE EXPRESSION PROFILE ANALYSIS OF DROSOPHILA MELANOGASTER SELECTED FOR RESISTANCE TO ENVIRONMENTAL STRESSORS. JOURNAL OF EVOLUTIONARY BIOLOGY 20: 1624-1636. SPIEGELMAN, V. S., S. Y. FUCHS AND G. A. BELITSKY, 1997 THE EXPRESSION OF INSECTICIDE RESISTANCE-RELATED CYTOCHROME P450 FORMS IS REGULATED BY MOLTING HORMONE IN DROSOPHILA MELANOGASTER. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 232: 304-307. STROBEL, S. M., AND J. R. HALPERT, 1997 REASSESSMENT OF CYTOCHROME P450 2B2: CATALYTIC SPECIFICITY AND IDENTIFICATION OF FOUR ACTIVE SITE RESIDUES†. BIOCHEMISTRY 36: 11697-11706. SUEYOSHI, T., AND M. NEGISHI, 2001 PHENOBARBITAL RESPONSE ELEMENTS OF CYTOCHROME P450 GENES AND NUCLEAR RECEPTORS. ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY 41: 123-143. SUN, W., V. M. MARGAM, L. SUN, G. BUCZKOWSKI, G. W. BENNETT ET AL., 2006 GENOME-WIDE ANALYSIS OF PHENOBARBITAL-INDUCIBLE GENES IN DROSOPHILA MELANOGASTER. INSECT MOLECULAR BIOLOGY 15: 455-464. SZTAL, T., H. CHUNG, L. GRAMZOW, P. J. DABORN, P. BATTERHAM ET AL., 2007 TWO INDEPENDENT DUPLICATIONS FORMING THE CYP307A GENES IN DROSOPHILA. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 37: 1044- 1053. TANG, A. H., AND C. P. TU, 1994 BIOCHEMICAL CHARACTERIZATION OF DROSOPHILA GLUTATHIONE S-TRANSFERASES D1 AND D21. JOURNAL OF BIOLOGICAL CHEMISTRY 269: 27876-27884. TATAR, M., A. KOPELMAN, D. EPSTEIN, M.-P. TU, C.-M. YIN ET AL., 2001 A MUTANT DROSOPHILA INSULIN RECEPTOR HOMOLOG THAT EXTENDS LIFE-SPAN AND IMPAIRS NEUROENDOCRINE FUNCTION. SCIENCE 292: 107-110. TCHOUDAKOVA, A., M. KISHIDA, E. WOOD AND G. V. CALLARD, 2001 PROMOTER CHARACTERISTICS OF TWO CYP19 GENES DIFFERENTIALLY EXPRESSED IN THE BRAIN AND OVARY OF TELEOST FISH. THE JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 78: 427-439. TELONIS-SCOTT, M., R. HALLAS, S. W. MCKECHNIE, C. W. WEE AND A. A. HOFFMANN, 2009 SELECTION FOR COLD RESISTANCE ALTERS GENE TRANSCRIPT LEVELS IN DROSOPHILA MELANOGASTER. JOURNAL OF INSECT PHYSIOLOGY 55: 549-555. TERRIERE, L. C., AND S. J. YU, 1974 INDUCTION OF DETOXIFYING ENZYMES IN INSECTS. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 22: 366-373. THOMAS, J. H., 2007 RAPID BIRTH–DEATH EVOLUTION SPECIFIC TO XENOBIOTIC CYTOCHROME P450 GENES IN VERTEBRATES. PLOS GENET 3: E67. TIJET, N., C. HELVIG AND R. FEYEREISEN, 2001 THE CYTOHROME P450 GENE SUPERFAMILY IN DROSOPHILA MELANOGASTER: ANNOTATION, INTRON-EXON ORGANISATION AND PHYLOGENY. GENETICA 262: 189-198. TIMSIT, Y. E., AND M. NEGISHI, 2007 CAR AND PXR: THE XENOBIOTIC-SENSING RECEPTORS. STEROIDS 72: 231-246.

239

TIRONA, R. G., W. LEE, B. F. LEAKE, L. B. LAN, C. B. CLINE ET AL., 2003 THE ORPHAN NUCLEAR RECEPTOR HNF4 ALPHA DETERMINES PXR- AND CAR-MEDIATED XENOBIOTIC INDUCTION OF CYP3A4. NATURE MEDICINE 9: 220-224. TOLSON, A. H., AND H. WANG, 2010 REGULATION OF DRUG-METABOLIZING ENZYMES BY XENOBIOTIC RECEPTORS: PXR AND CAR. ADVANCED DRUG DELIVERY REVIEWS 62: 1238-1249. TOMANCAK, P., B. BERMAN, A. BEATON, R. WEISZMANN, E. KWAN ET AL., 2007 GLOBAL ANALYSIS OF PATTERNS OF GENE EXPRESSION DURING DROSOPHILA EMBRYOGENESIS. GENOME BIOLOGY 8: R145. TONG, S.-K., AND B.-C. CHUNG, 2003 ANALYSIS OF ZEBRAFISH CYP19 PROMOTERS. THE JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 86: 381-386. TROTTIER, E., L. A. BELZI, C. STOLTZ AND A. ANDERSON, 1995 LOCALIZATION OF A PHENOBARBITAL-RESPONSIVE ELEMENT (PBRE) IN THE 5'-FLANKING REGION OF THE RAT CYP2B2 GENE. GENE 158: 263-268. TSAI, S.-F., D. I. K. MARTIN, L. I. ZON, A. D. D'ANDREA, G. G. WONG ET AL., 1989 CLONING OF CDNA FOR THE MAJOR DNA-BINDING PROTEIN OF THE ERYTHROID LINEAGE THROUGH EXPRESSION IN MAMMALIAN CELLS. NATURE 339: 446-451. TSENG, H.-P., T.-H. HSEU, D. R. BUHLER, W.-D. WANG AND C.-H. HU, 2005 CONSTITUTIVE AND XENOBIOTICS-INDUCED EXPRESSION OF A NOVEL CYP3A GENE FROM ZEBRAFISH LARVA. TOXICOLOGY AND APPLIED PHARMACOLOGY 205: 247-258. TSUCHIYA, Y., M. NAKAJIMA AND T. YOKOI, 2005 CYTOCHROME P450-MEDIATED METABOLISM OF ESTROGENS AND ITS REGULATION IN HUMAN. CANCER LETTERS 227: 115-124. TURNER, T. L., M. T. LEVINE, M. L. ECKERT AND D. J. BEGUN, 2008 GENOMIC ANALYSIS OF ADAPTIVE DIFFERENTIATION IN DROSOPHILA MELANOGASTER. GENETICS 179: 455-473. TWEEDIE, S., M. ASHBURNER, K. FALLS, P. LEYLAND, P. MCQUILTON ET AL., 2009 FLYBASE: ENHANCING DROSOPHILA GENE ONTOLOGY ANNOTATIONS. NUCLEIC ACIDS RESEARCH 37: D555-D559. UEDA, A., H. K. HAMADEH, H. K. WEBB, Y. YAMAMOTO, T. SUEYOSHI ET AL., 2002 DIVERSE ROLES OF THE NUCLEAR ORPHAN RECEPTOR CAR IN REGULATING HEPATIC GENES IN RESPONSE TO PHENOBARBITAL. MOLECULAR PHARMACOLOGY 61: 1-6. UVELL, H., AND Y. ENGSTROM, 2003 FUNCTIONAL CHARACTERIZATION OF A NOVEL PROMOTER ELEMENT REQUIRED FOR AN INNATE IMMUNE RESPONSE IN DROSOPHILA. MOL. CELL. BIOL. 23: 8272-8281. WADA, T., J. GAO AND W. XIE, 2009 PXR AND CAR IN ENERGY METABOLISM. TRENDS IN ENDOCRINOLOGY AND METABOLISM: TEM 20: 273-279. WADE, R. C., P. J. WINN, I. SCHLICHTING AND SUDARKO, 2004 A SURVEY OF ACTIVE SITE ACCESS CHANNELS IN CYTOCHROMES P450. JOURNAL OF INORGANIC BIOCHEMISTRY 98: 1175-1182. WANG, L., H. DANKERT, P. PERONA AND D. J. ANDERSON, 2008 A COMMON GENETIC TARGET FOR ENVIRONMENTAL AND HERITABLE INFLUENCES ON AGGRESSIVENESS IN DROSOPHILA. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES 105: 5657-5663. WANG, P.-Y., N. NERETTI, R. WHITAKER, S. HOSIER, C. CHANG ET AL., 2009 LONG-LIVED INDY AND CALORIE RESTRICTION INTERACT TO EXTEND LIFE SPAN. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES 106: 9262-9267. WARREN, J. T., A. PETRYK, G. MARQUES, M. JARCHO, J. P. PARVY ET AL., 2002 MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF TWO P450 ENZYMES IN THE ECDYSTEROIDOGENIC PATHWAY OF DROSOPHILA MELANOGASTER. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 99: 11043-11048. WEATHERMAN, R. V., R. J. FLETTERICK AND T. S. SCANLAN, 1999 NUCLEAR-RECEPTOR LIGANDS AND LIGAND- BINDING DOMAINS. ANNUAL REVIEW OF BIOCHEMISTRY 68: 559-581. WEI, P., J. ZHANG, D. H. DOWHAN, Y. HAN AND D. D. MOORE, 2002 SPECIFIC AND OVERLAPPING FUNCTIONS OF THE NUCLEAR HORMONE RECEPTORS CAR AND PXR IN XENOBIOTIC RESPONSE. PHARMACOGENOMICS J 2: 117-126. WEI, P., J. ZHANG, M. EGAN-HAFLEY, S. LIANG AND D. D. MOORE, 2000 THE NUCLEAR RECEPTOR CAR MEDIATES SPECIFIC XENOBIOTIC INDUCTION OF DRUG METABOLISM. NATURE 407: 920-923.

240

WEN, Z., J. BAUDRY, M. R. BERENBAUM AND M. A. SCHULER, 2005 ILE115LEU MUTATION IN THE SRS1 REGION OF AN INSECT CYTOCHROME P450 (CYP6B1) COMPROMISES SUBSTRATE TURNOVER VIA CHANGES IN A PREDICTED PRODUCT RELEASE CHANNEL. PROTEIN ENGINEERING DESIGN AND SELECTION 18: 191-199. WERCK-REICHHART, D., AND R. FEYEREISEN, 2000 CYTOCHROMES P450: A SUCCESS STORY. GENOME BIOLOGY 1: REVIEWS3003.3001 - REVIEWS3003.3009. WHITE, K. P., P. HURBAN, T. WATANABE AND D. S. HOGNESS, 1997 COORDINATION OF DROSOPHILA METAMORPHOSIS BY TWO ECDYSONE-INDUCED NUCLEAR RECEPTORS. SCIENCE 276: 114-117. WHITE, K. P., S. A. RIFKIN, P. HURBAN AND D. S. HOGNESS, 1999 MICROARRAY ANALYSIS OF DROSOPHILA DEVELOPMENT DURING METAMORPHOSIS. SCIENCE 286: 2179-2184. WHITLOCK, J., 1999 INDUCTION OF CYTOCHROME P4501A1. ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY 39: 103-125. WILLIAMS, D. E., J. J. LECH AND D. R. BUHLER, 1998 XENOBIOTICS AND XENOESTROGENS IN FISH: MODULATION OF CYTOCHROME P450 AND CARCINOGENESIS. MUTATION RESEARCH/FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS 399: 179-192. WILLINGHAM, A. T., AND T. KEIL, 2004 A TISSUE SPECIFIC CYTOCHROME P450 REQUIRED FOR THE STRUCTURE AND FUNCTION OF DROSOPHILA SENSORY ORGANS. MECHANISMS OF DEVELOPMENT 121: 1289-1297. WILLOUGHBY, L., P. BATTERHAM AND P. J. DABORN, 2007 PIPERONYL BUTOXIDE INDUCES THE EXPRESSION OF CYTOCHROME P450 AND GLUTATHIONE S-TRANSFERASE GENES IN DROSOPHILA MELANOGASTER. PEST MANAGEMENT SCIENCE 63: 803-808. WILLOUGHBY, L., H. CHUNG, C. LUMB, C. ROBIN, P. BATTERHAM ET AL., 2006 A COMPARISON OF DROSOPHILA MELANOGASTER DETOXIFICATION GENE INDUCTION RESPONSES FOR SIX INSECTICIDES, CAFFEINE AND PHENOBARBITAL. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 36: 934-942. WILLSON, T. M., AND S. A. KLIEWER, 2002 PXR, CAR AND DRUG METABOLISM. NATURE REVIEWS DRUG DISCOVERY 1: 259-266. WILSON, R. C., S. NIMKARN, M. DUMIC, J. OBEID, M. AZAR ET AL., 2007 ETHNIC-SPECIFIC DISTRIBUTION OF MUTATIONS IN 716 PATIENTS WITH CONGENITAL ADRENAL HYPERPLASIA OWING TO 21-HYDROXYLASE DEFICIENCY. MOLECULAR GENETICS AND METABOLISM 90: 414-421. WONDJI, C. S., H. IRVING, J. MORGAN, N. F. LOBO, F. H. COLLINS ET AL., 2009 TWO DUPLICATED P450 GENES ARE ASSOCIATED WITH PYRETHROID RESISTANCE IN ANOPHELES FUNESTUS, A MAJOR MALARIA VECTOR. GENOME RESEARCH 19: 452-459. WRAY, G. A., M. W. HAHN, E. ABOUHEIF, J. P. BALHOFF, M. PIZER ET AL., 2003 THE EVOLUTION OF TRANSCRIPTIONAL REGULATION IN EUKARYOTES. MOL BIOL EVOL 20: 1377-1419. XIE, W., J. L. BARWICK, M. DOWNES, B. BLUMBERG, C. M. SIMON ET AL., 2000 HUMANIZED XENOBIOTIC RESPONSE IN MICE EXPRESSING NUCLEAR RECEPTOR SXR. NATURE 406: 435-439. YAMAMOTO, K., H. ICHINOSE, Y. ASO AND H. FUJII, 2010 EXPRESSION ANALYSIS OF CYTOCHROME P450S IN THE SILKMOTH, BOMBYX MORI. PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 97: 1-6. YANG, M. C., Y. LI AND R. W. PADGETT, 2005 MICRORNAS: SMALL REGULATORS WITH A BIG IMPACT. CYTOKINE & GROWTH FACTOR REVIEWS 16: 387-393. YAVATKAR, A., Y. LIN, J. ROSS, Y. FANN, T. BRODY ET AL., 2008 RAPID DETECTION AND CURATION OF CONSERVED DNA VIA ENHANCED-BLAT AND EVOPRINTERHD ANALYSIS. BMC GENOMICS 9: 106. YIN, L., H. MA, X. GE, P. A. EDWARDS AND Y. ZHANG, 2011 HEPATIC HEPATOCYTE NUCLEAR FACTOR 4{ALPHA} IS ESSENTIAL FOR MAINTAINING TRIGLYCERIDE AND CHOLESTEROL HOMEOSTASIS. ARTERIOSCLER THROMB VASC BIOL 31: 328-336. YOSHINARI, K., N. YODA, T. TORIYABE AND Y. YAMAZOE, 2010 CONSTITUTIVE ANDROSTANE RECEPTOR TRANSCRIPTIONALLY ACTIVATES HUMAN CYP1A1 AND CYP1A2 GENES THROUGH A COMMON REGULATORY ELEMENT IN THE 5'-FLANKING REGION. BIOCHEMICAL PHARMACOLOGY 79: 261-269. ZANGAR, R. C., D. R. DAVYDOV AND S. VERMA, 2004 MECHANISMS THAT REGULATE PRODUCTION OF REACTIVE OXYGEN SPECIES BY CYTOCHROME P450. TOXICOLOGY AND APPLIED PHARMACOLOGY 199: 316-331.

241

ZHANG, Y., W. ZHANG, L. ZHANG, T. ZHU, J. TIAN ET AL., 2004 TWO DISTINCT CYTOCHROME P450 AROMATASES IN THE ORANGE-SPOTTED GROUPER (EPINEPHELUS COIOIDES): CDNA CLONING AND DIFFERENTIAL MRNA EXPRESSION. THE JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 92: 39-50. ZHU, Q., AND M. HALFON, 2007 VECTOR-DEPENDENT GENE EXPRESSION DRIVEN BY INSULATED P-ELEMENT REPORTER VECTORS. FLY 1: 55-56.

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Appendix I: List of fly lines used

243

Stock name Comments Cyp12d1 -9653/-2932bp pH-Stinger 1A Cyp12d1 -9653/-2932bp pH-Stinger 2A Cyp12d1 -3155/-1841bp pH-Stinger 2D Cyp12d1 -3155/-1841bp pH-Stinger 12C Cyp12d1 -2043/-945bp pH-Stinger 1A Cyp12d1 -2043/-945bp pH-Stinger 3A Cyp12d1 -1540/-1bp pStinger 6A Cyp12d1 -1540/-1bp pStinger 8A Cyp12d1 -1100/-1bp pStinger 3C Cyp12d1 -1100/-1bp pStinger 8A Cyp12d1 -670/-1bp pStinger 2A Cyp12d1 -670/-1bp pStinger 3A Cyp12d1 -492/-168bp pH-Stinger 1A Cyp12d1 -492/-168 bp pH-Stinger 3A Cyp12d1 -288/-1bp pStinger 1A Cyp12d1 -288/-1bp pStinger 3A Cyp12d1 -288/-168bp pH-Stinger 1A Cyp12d1 -288/-168 bp pH-Stinger 3A Cyp12d1 -288/-168bp GATA mutant pH-Stinger 4A Cyp12d1 -288/-168bp GATA mutant pH-Stinger 5A Cyp12d1 -168/-1bp pStinger 1A Cyp12d1 -168/-1bp pStinger 5A Cyp12d1 +1917/+2207bp pH-Stinger 3A Cyp12d1 +1917/+2207bp pH-Stinger 4A Cyp12d1 +1917/+2049bp pH-Stinger 2A Cyp12d1 +1917/+2049bp pH-Stinger 3A Cyp12d1 +2116/+2207 pH-Stinger 6A Cyp12d1-168/-1bp pStinger +1917/2207bp SpeI 1A Cyp12d1 -168/-1bp pStinger +1917/2207bp SpeI XY 12692 VDRC RNAi GD line HNF4 (CG33007) VDRC RNAi GD line Ecdysone Receptor 37058 (CG1765) VDRC RNAi GD line Spineless-Aristapedia 10715 (CG6993) 37694 VDRC RNAi GD line Hr39 (CG8676) 44951 VDRC RNAi GD line E75 (CG8127) 10396 VDRC RNAi GD line E78 (CG18023) 48979 VDRC RNAi GD line HR78 (CG7199) 48979 10418 VDRC RNAi GD line GATAe

6224 VDRC RNAi GD line Pannier

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Stock name Comments Cyp12d1 1540/-1bp pStinger 6A; Hr96e25 Cyp12d1 1540/-1bp pStinger 6A; Hr9616A Cyp12d1 1540/-1bp pStinger6A; Cyp6g1-5'HR GAl4 Cyp12d1-RNAi-4A Cyp12d1-RNAi-7A Cyp12d1-UAS-11c Cyp12d1-UAS-41A Tubulin-GAL4 w1118 y; cn bw sp BG4 Single Cyp12d1 copy natural population TR2 Double Cyp12d1 copy natural population RK146 Double Cyp12d1 copy natural population

245

Appendix II: List of Primers used

246

Primer Sequence 1 -168bp FBglII GAAAAAGATCTTCATGCTTTGTTTGCTGGC 2 -288bp FBglII GTATTAGATCTAAGTTCCAACGCTCTCAATTTG 3 -492bp FBglII GCAAAAGATCTAAGGATCAACTCTAAGAATTG 4 -670bp FBglII CAATTAGATCTCAGCCATCAATGTGGAGATTT 5 -1100bp FBglII ATTTCAGATCTTGGAGAAAGTGTGGGTCTGA 6 -1540bp FBglII GTGTAAGATCTAAGCGAGATCCTATAAGAACATTCC 7 -2043bp FBglII GATATAGATCTACAATGCCGCCAAGATTTAC 8 -3155bp FBglII ACATAAGATCTGGAGGTTGTTGCATTCCAAG 9 -1bp RXbaI CCA TGT CTA GAT TCT ATT TTT GGT GAT 10 -168bp RXbaI TGCAATCTAGACAAACAAAAACAAAAGCC 11 -1100bp RXbaI CTCATTCTAGATTTTCAATCCTTTGGGTTGC 12 -2043bp RXbaI TTTATTCTAGATTCTGTCGGAATGCCTTTTA 13 +1917bp FBglII TCGAAAGATCTTTTGCTTCTCATTTGTGGC 14 +2207bp RXbaI GCAAATCTAGACCTCTCGGACTAGTGTTCT 15 +1917bp FSpeI TCGAAACTAGTTTTGCTTCTCATTTGTGGC 16 +2207bp RSpeI GCAAAGGACATCCTCTCGGACTAGTGTTCT 17 +878bp RSpeI AATCTTCTAGACGTATTAAACATTTTGCTTATCA 18 +2116bp FSpeI CGCTTAGATCTGGCATCCCTATCGCTTTTTTTCG 19 GATA mut F GATTATACCGTTCAGCTCATAAGAAAATGAAACTCATGCT 20 GATA mut R AGCATGAGTTTCATTTTCTTATGAGCTGAACGGTATAATC 21 Cyp12d1 RT F AGGAACACAAGTAAAGGCCAC 22 Cyp12d1 RT R GTCCATTCAAGACCATGTTCC 23 Cyp12d1 RNAi F AGGGCTCTAGACAAGGAGACACTGCGATAC 24 Cyp12d1 RNAi R ATATCTCTAGACACCTACAGGAAAATGAAG 25 CG30490 ORF F ATGTCCTTTGCCGCGCCAC 26 CG30490 ORF R TTAGCCGACAAAGTGGCAACACAC 27 RPL11F CGATCCCTCCATCGGTATCT 28 RPL11R AACCACTTCATGGCATCCTC 29 GFPF GGACGACGGCAACTACAAGAC 30 GFPR TGC TCA GGT AGT GGT TGT CGG 31 RACE F1 TATGCCATATTTGCGGGCTG 32 RACE F2 TGATTCGCAATTTCCATGTG

247

Appendix III: EMSA protein band sequencing protocol

248

In-gel Digestion of Coomassie Stained Proteins For greater sequence coverage (with DTT Reduction and Iodoacetamide Alkylation)

Protocol supplied by Mr Paul O’Donnell, Bio21 Institute, University of Melbourne

1. Always wear gloves and work in the hood to reduce keratin contamination.

2. Excise gel bands or spots from acrylamide gel.

3. It is highly recommended that a positive and negative control be conducted in parallel to all

analyses.

4. Wash twice with 300µL of 18 Mohm water for approx 15 minutes.

5. Wash 2 times with 50 µL of 40% acetonitrile / 200mM ammonium bicarbonate to remove all

stain from gel plug. Each wash should be approximately 30 minutes in length. Two washes

should be sufficient for moderately intensity bands.

6. Dehydrate with 100 µL of acetonitrile until gel plugs turn opaque, generally 20minutes.

7. Decant all liquid from opaque gel plugs.

8. Dry sample plugs in vacuum centrifuge or in protective hood.

9. Rehydrate and incubate samples in 10mM dithiothreitol (DTT) in 25mM ammonium bicarbonate

at 56oC for 1 hour. Solution volume should be sufficient to cover gel plugs or bands, i.e. 20-50

mL (10mM DTT is approximately a 1.5mg/µL solution of DTT).

10. Allow samples to cool to room temperature

11. Replace 10mM DTT solution with an equal volume of 55mM iodoacetamide in 25 mM

ammonium bicarbonate and incubate in the dark for 45 minutes.

12. Decant iodoacetamide solution.

13. Wash with 25 mM ammonium bicarbonate solution for 10 min followed by 10 min dehydration

with 100% acetonitrile.

249

14. Rehydrate with 25 mM ammonium bicarbonate solution for 15 min. Decant 25 mM ammonium

bicarbonate solution and dehydrate for 10 min with 100% acetonitrile.

15. Remove all liquid and dry sample plugs in vacuum centrifuge or in protective hood.

16. Prepare trypsin solution while drying samples using sequence grade, modified bovine or porcine

Trypsin.

Prepare stock trypsin solution by dissolving 20 to 25 µg in 200µL 1mM HCl or 1% Acetic acid so

that final concentration is between 100ng/µL to 125 ng/µL.

17. Prepare working trypsin solution by diluting stock solution 1 to 10 with 25 mM ammonium

bicarbonate such that final concentration = 10 to 12.5 ng/µL.

Note the pH difference in stock solution (~2.9) versus working solution (pH~8.0). Trypsin activity

is pH dependent. At low pH, trypsin has low activity and can be stored for approximately 2

weeks. Trypsin working solutions must be used shortly after increasing pH to 8, i.e. 2 hrs. If not

autolytic digestion products are observed at higher levels than desirable.

18. Rehydrate dried gel plug/bands in approximately 20 µL trypsin for 20 minutes at 4°C or on ice.

This allows the trypsin to infuse into the gel plug for "in-gel" digestion.

19. Remove excess trypsin solution with pipette.

20. Add 10 to 20 µL of 25mM ammonium bicarbonate to sample vial to ensure proper hydration

during digestion at elevated temperatures.

21. Digest at 37oC for 4 to 6 hrs. Overnight digestions are possible but yield higher levels of autolytic

trypsin products which may decrease sensitivity for low level protein digests.

22. Stop digestion by adding 25 µL of 5 % formic acid (note pH change) allow to stand for 15

minutes.

250

23. Recover supernatant and add to clean eppendorf tube. One can sample this solution directly for

peptide mass mapping or continue extracting additional peptides. We suggest further

extraction.

24. Extract with 50% acetonitrile / 5% formic acid and sonicate for 15 minutes to recover additional

peptides. Pool with supernatant from step 23.

25. Concentrate to 10 µL or dryness in vacuum centrifuge. Concentration to dryness is more

convenient but may result in some loss of peptides which will not resolubilize. . Need to reduce

acetonitrile concentration below 5% so peptides bind to reversed phase column.

26. Dried peptides can be stored at -20°C or -80°C for long periods of time (1yr).

27. Immediately prior to mass analysis dissolve dried peptides in 1% formic acid.

Comments: great care should be taken not to touch gels, allow hairs to fall on gels, or allow wools to come into contact with gels prior to staining or digestion. All these can contribute protein contaminants which will appear and interfere with the final peptide mass map.

Mass Spectrometer Conditions:

Ionisation mode: Electrospray Ionisation

Drying gas flow: 7 L/min;

Nebuliser: 35 psi;

Drying gas temperature: 325°C;

Capillary Voltage (Vcap): 4000 V;

Fragmentor: 250 V;

Skimmer: 65 V;

OCT RFV: 250 V;

Scan range acquired: 100–3200 m/z

Internal Reference ions: Positive Ion Mode = m/z = 121.050873 & 922.009798

251

Appendix IV: Cyp12d1 duplication survey gels

252

*Samples 34a and 61a failed to amplify and were subsequently re-tested to give samples 34b and 61b.

253

Cyp12d1 Cyp12d1 No. Location population locus No. Location population locus

1 Cooktown, QLD IX 1 SC 46 Bega, NSW BG 35 SC

2 Cooktown, QLD IX 1 SC 47 Bega, NSW BG 35 DC

3 Cooktown, QLD IX 1 SC 48 Bega, NSW BG 35 DC 4 Cooktown, QLD IW 8 DC 49 Bega, NSW BG 43 SC

5 Cooktown, QLD IW 8 DC 50 Bega, NSW BG 43 DC 6 Cooktown, QLD IW 8 DC 51 Bega, NSW BG 43 SC

7 Cooktown, QLD IV5 48 SC 52 Bega, NSW BG 28 DC

8 Cooktown, QLD IV5 48 SC 53 Bega, NSW BG 28 DC

9 Cooktown, QLD IV5 48 SC 54 Bega, NSW BG 28 DC

10 Innisfail, QLD 117 SC 55 Yerring, VIC 304 DC 11 Innisfail, QLD 117 SC 56 Yerring, VIC 304 DC

12 Innisfail, QLD 117 SC 57 Yerring, VIC 304 DC

13 Innisfail, QLD 157 DC 58 Yerring, VIC 206 SC

14 Innisfail, QLD 157 DC 59 Yerring, VIC 206 DC

15 Innisfail, QLD 157 SC 60 Yerring, VIC 206 DC 16 Innisfail, QLD 35 SC 61 Yerring, VIC 16 SC

17 Innisfail, QLD 35 SC 62 Yerring, VIC 16 SC 18 Innisfail, QLD 35 SC 63 Yerring, VIC 16 DC

19 Mackay, QLD IE 13 SC 64 Gladstone QLD IC 37 SC

20 Mackay, QLD IE 13 SC 65 Gladstone QLD IC 37 SC

21 Mackay, QLD IE 13 SC 66 Gladstone QLD IC 37 SC

22 Mackay, QLD IE 2 SC 67 Gladstone QLD IC 30 SC 23 Mackay, QLD IE 2 SC 68 Gladstone QLD IC 30 SC

24 Mackay, QLD IE 2 SC 69 Gladstone QLD IC 30 DC

25 Mackay, QLD IE 15 SC 70 Gladstone QLD IC 39 SC

26 Mackay, QLD IE 15 SC 71 Gladstone QLD IC 39 SC

27 Mackay, QLD IE 15 SC 72 Gladstone QLD IC 39 DC 28 Maryborough, QLD IB 2 SC 73 Spreytone, TAS Sa 9 DC

29 Maryborough, QLD IB 2 SC 74 Spreytone, TAS Sa 9 DC 30 Maryborough, QLD IB 2 SC 75 Spreytone, TAS Sa 9 DC

31 Maryborough, QLD IB 5 SC 76 Spreytone, TAS Sa 27 DC

32 Maryborough, QLD IB 5 SC 77 Spreytone, TAS Sa 27 DC

33 Maryborough, QLD IB 5 SC 78 Spreytone, TAS Sa 27 DC

34 Maryborough, QLD IB 1 SC 79 Spreytone, TAS Sa 28 DC 35 Maryborough, QLD IB 1 SC 80 Spreytone, TAS Sa 28 DC

36 Maryborough, QLD IB 1 SC 81 Spreytone, TAS Sa 28 DC

37 Coff's Harbour, NSW KJ 67 DC 82 Sorrell, TAS S 6 SC

38 Coff's Harbour, NSW KJ 67 DC 83 Sorrell, TAS S 6 DC

39 Coff's Harbour, NSW KJ 67 DC 84 Sorrell, TAS S 6 DC 40 Coff's Harbour, NSW KJ 16 DC 85 Sorrell, TAS S 38 DC

41 Coff's Harbour, NSW KJ 16 SC 86 Sorrell, TAS S 38 SC 42 Coff's Harbour, NSW KJ 16 SC 87 Sorrell, TAS S 38 DC

43 Coff's Harbour, NSW KJ26 DC 88 Sorrell, TAS S 28 DC

44 Coff's Harbour, NSW KJ 26 SC 89 Sorrell, TAS S 28 DC

45 Coff's Harbour, NSW KJ 26 SC 90 Sorrell, TAS S 28 DC

254

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Boey, Hui Kuang Adrian

Title: The regulation and function of the Drosophila melanogaster Cytochrome P450 gene, Cyp12d1

Date: 2011

Citation: Boey, H. K. A. (2011). The regulation and function of the Drosophila melanogaster Cytochrome P450 gene, Cyp12d1. PhD thesis, Department of Genetics, The University of Melbourne.

Persistent Link: http://hdl.handle.net/11343/36501

File Description: The regulation and function of the Drosophila melanogaster Cytochrome P450 gene, Cyp12d1

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