THE APICOMPLEXAN PLASTID DNA: AN EVOLUTIONARY AND MOLECULAR STUDY Paul William Denny Ph.D . University College London, University of London 1997 ProQuest Number: 10106922 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10106922 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT The discovery and characterisation in this laboratory of a 35 kilobase plastid genome from the malaria parasite, Plasmodium, has led to intense speculation concerning its origins and function. This thesis describes how sequence data were garnered from malaria's distant apicomplexan cousins, the coccidians and piroplasms, and used to make an evolutionary analysis of their corresponding plastid DNAs. From the sequence data produced it appears highly likely that the plastid DNA was gained by an ancient progenitor of the Apicomplexa, and singularly reorganised upon the adoption of a parasitic lifestyle. Previously, the identification in this laboratory of a Plasmodium plastid encoded open reading frame with homologues in rhodoplast genomes led to the suggestion that the apicomplexan organelle has a red algal origin. However, phylogenetic analysis of the tuf gene, which encodes the ubiquitous plastid and prokaryotic elongation factor Tu, provides support for the hypothesis that the organelle was derived through endosymbiosis of a green alga. The highly conserved nature of the extrachromosomal DNA found across the range of parasites studied here suggests that the organelle within which the genome is housed performs a vital cellular role. However, the results presented provide no indication of its particular function. The sequence data generated also led to the speculation that certain plastid encoded products may provide targets for novel anti-apicomplexan chemotherapeutics. Finally, it is inferred from the sequence of the plastid DNA of the coccidians. Toxoplasma gondii and Eimeria tenella, that opal stop codons are occasionally used as tryptophan codons. Such a system would be the first identified in a plastid organelle. ACKNOWLEDGEMENTS Grateful thanks to Dr. Iain Wilson for his thoughtful and supportive supervision through the practical and cerebral aspects of this thesis. Gratitude also to Dr. Don Wilhamson for his helpful insight into an array of matters. Great appreciation must also be expressed towards Dr. Andrea Whyte for patient thesis advice; and Malcolm Strath, Dr. Peter Preiser, Dr. Kaveri Rangachari, Dr. Barbara Clough, Peter Moore, Daphne Moore, Dr. Anjana Roy, Kate Roberts, Irene Ling, Terry Scott-Finnigan, Anna Law and all the other 'parasitology people' who, over the years, have provided sound advice, ready smiles and even the occasional pint. Thanks also to Dr. Nick Goldman for evolutionary endeavours, and to the Photographies section of the National Institute for Medical Research for their expert assistance in the preparation of the figures. AU the work described was performed at the National Institute for Medical Research whilst in receipt of a Medical Research Council post-graduate studentship. A University CoUege London travel grant is also gratefully acknowledged. CONTENTS ABSTRACT I ACKNOWLEDGEMENTS II CONTENTS III FIGURES AND TABLES VII ABBREVIATIONS IX AMINO ACID CODES XII ACCESSION NUMBERS XIII CHAPTER 1: INTRODUCTION 1.1.0 The Apicomplexa 1 1.2.0 The Parasites Studied 2 1.2.1 The coccidians: Toxoplasma gondii and Eimeria tenella 2 1.2.2 The piroplasm: Theileria annulata 3 1.3.0 Apicomplexans have two Extrachromosomal DNAs 4 1.4.0 The Malarial DNA Circle: A Vestigial Plastid Genome 5 1.4.1 The RNA polymerase genes 5 1.4.2 The ribosomal protein genes 6 1.4.3 The m/gene 6 1.4.4 The inverted repeat 7 1.5.0 Provenance of the Apicomplexan Plastid 8 1.5.1 A dinoflagellate origin? 10 1.5.2 A chromistan origin? 10 1.5.3 A 'green' origin? 12 1.5.4 The 'unitary' hypothesis 13 1.6.0 Further Indications of a 'Green' Origin for the Apicomplexan Plastid 14 1.7.0 The AT Content of the Apicomplexan Plastid DNA 16 1.8.0 Is the Parasite Plastid Functional? 17 1.9.0 Susceptibly of Apicomplexans to Antibiotics and Herbicides 19 1.10.0 Aims 22 111 CHAPTER 2: MATERIALS AND METHODS 2.1.0 Parasite Culture 27 2.2.0 Parasite Purification 28 2.3.0 DNA Extraction 28 2.3.1 Toxoplasma 28 2.3.2 Other parasites 28 2.4.0 Caesium Chloride/D API Gradients 29 2.5.0 Southern Blots 29 2.6.0 Slot Blots 30 2.7.0 Copy Number Determination 30 2.8.0 Synthesis of Oligonucleotides 31 2.9.0 Polymerase Chain Reaction 31 2.10.0 Cloning PCR Products 32 2.11.0 Sequencing 32 2.12.0 Sequence Analysis 33 2.13.0 RNA Extraction 34 2.14.0 Northern Blots 35 2.15.0 Reverse Transcription PCR 35 2.16.0 Production of Fusion Proteins 36 2.17.0 Polyclonal Antibody Production 37 2.18.0 Western Blotting 37 CHAPTER 3: ISOLATION OF THE PLASTID DNA FROM TOXOPLASMA 3.1.0 Introduction 45 3.2.0 Fractionation of Toxoplasma DNA 45 3.3.0 Identification of the Fractions 46 3.4.0 Copy Number 47 3.5.0 Discussion 47 IV CHAPTER 4: EVOLUTION OF THE APICOMPLEXAN PLASTID GENOME 4.1.0 Introduction 51 4.2.0 Cross Hybridisation 51 4.3.0 The Inverted Repeat and Downstream Region 52 4.4.0 The tuf Region 52 4.5.0 Phylogenetic Analysis of the tuf Gene 53 4.6.0 Discussion 55 CHAPTER 5: PROTEIN ENCODING GENES 5.1.0 Introduction 60 5.2.0 ORF470 60 5.3.0 The tuf Gene 61 5.4.0 ORF78 62 5.5.0 The Ribosomal Protein Genes 62 5.6.0 Protein Analysis 63 5.7.0 Discussion 64 CHAPTER 6: RIBOSOMAL RNA GENES 6.1.0 Introduction 74 6.2.0 Conservation and Transcription of the Ribosomal RNA Genes 74 6.3.0 Antibiotic Susceptibility 75 6.4.0 Discussion 77 CHAPTER 7: CODON USAGE AND TRANSFER RNAs 7.1.0 Introduction 83 7.2.0 Plastid Encoded Transfer RNAs 83 7.3.0 Amino Acid Frequencies and Codon-Usage 84 7.4.0 Known Anti-Codon Frequency 85 7.5.0 Discussion 85 CHAPTER 8: CONCLUSION 96 CHAPTER 9: REFERENCES 101 APPENDIX: PUBLICATIONS 130 VI FIGURES AND TABLES Figure 1.1: Apicomplexan Merozoite 23 Figure 1.2: Toxoplasma Life-Cycle 24 Figure 1.3: Complete Map of the Malarial Plastid Genome 25 Figure 1.4: Cruciforms 26 Table 2.1: Oligonucleotide Primers for Amplification of the P30 Nuclear Gene 39 Table 2.2: Oligonucleotide Primers for Amplification of Isolated Toxoplasma Plastid DNA 40 Table 2.3: Oligonucleotide Primers for Amplification of Eimeria Plastid DNA 41 Table 2.4: Oligonucleotide Primers for Amplification of Theileria Plastid DNA 42 Table 2.5: Oligonucleotide Primers for RT-PCR 43 Table 2.6: Oligonucleotide Primers for Amplification of Toxoplasma Plastid Genes for Fusion Protein Production 44 Figure 3.1: Caesium Cbloride/DAPI Gradients of Toxoplasma DNA 48 Figure 3.2: Identification of Gradient Fractions 49 Figure 3.3: Determination of the Plastid Copy Number 50 Figure 4.1: Preliminary Identification of Malarial Plastid Gene Homologues 57 Figure 4.2: Conservation of Gene Order in Apicomplexan Plastid Genomes 58 Figure 4.3: Phylogenetic Tree Based on Plastid Sequence Data 59 Figure 5.1: Analysis of ORF470 Homologues 66 Figure 5.2: Alignment of EF-Tu Proteins 67 Figure 5.3: Analysis of ORF78 Homologues 68 Figure 5.4: Analysis of Predicted S7 Proteins 69 Figure 5.5: Alignment of Predicted S12 Proteins 70 Figure 5.6: Analysis of Putative Lll Proteins 71 Vll Figure 5.7: RT-PCR Analyses in Toxoplasma 72 Figure 5.8: ORF470 Protein Analysis 73 Figure 6.1: SSU rRNA Sequence Alignment 79 Figure 6.2: LSU rRNA Sequence Alignment 80 Figure 6.3: Northern Analysis of rRNA 81 Figure 6.4: Susceptibility of Apicomplexan GTPase Centres to Thiostrepton 82 Figure 7.1: Toxoplasma Transfer RNAs 90 Figure 7.2: Eimeria Transfer RNAs 91 Table 7.1: Toxoplasma Plastid Codon Usage 92 Table 7.2: Eimeria Plastid Codon Usage 93 Table 7.3: Toxoplasma Plastid Anti-Codon Frequency 94 Table 7.4: Eimeria Plastid Anti-Codon Frequency 95 V lll ABBREVIATIONS A2 6 0 absorbance at 260 nm aa amino acid AIDS acquired immunodeficiency syndrome bp base pairs DAPI 4',6-diamidino-2-phenylindole DNA deoxyribonucleic acid DNase deoxyribonuclease dNTP deoxyribonucleotide triphosphate DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EF-Tu elongation factor Tu Et Eimeria tenella EtBr ethidium bromide PCS foetal calf serum g gram GDP guanosine diphosphate GST glutathione S-transferase GTP guanosine triphosphate HEPES N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid HRPL horseradish peroxidase luciferase HPLC high performance liquid chromatrography I inosine Ig immunoglobulin IPTG isopropyl |3-D-thiogalactopyranoside IR inverted repeat IX kb kilobase kD kilodalton 1 litre L-agar luria agar L-broth luria broth LSC large single copy region LSU large subunit M molar m milli mA müliamp |i micro MDCK Madin-Darby canine kidney mol mole MOPS 3-(N-morphohno) propanesulfonic acid MRC Medical Research Council mRNA messenger RNA n nano NTMR National Institute for Medical Research nt nucleotide nucL nuclear CD optical density ORF open reading frame p pico PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction Pf Plasmodium falciparum PK proteinase K plas.