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by Copyright Jay Randall Reichman 2002 The Dissertation Committee for Jay Randall Reichman Certifies that this is the approved version of the following dissertation: Characterization and Evolution of Peridinin-Chlorophyll a Binding Protein Gene Families in Symbiotic Dinoflagellates Committee: David M. Hillis, Supervisor Peter D. Vize, Co-Supervisor Robert K. Jansen Bassett Maguire Jr. Thomas P. Wilcox Characterization and Evolution of Peridinin-Chlorophyll a Binding Protein Gene Families in Symbiotic Dinoflagellates by Jay Randall Reichman, B.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin December, 2002 Dedication To all my family members present and past who encouraged and supported me emotionally, helped me financially and kept doing so through all seasons long after they ceased to understand what I was working on. And especially to the three women who have had the largest positive impacts on my life, my mother Ann, my wife Melada and my daughter Lauren. Acknowledgements Substantial help with this work in the form of molecular training and critical feedback came from Dr. Peter Vize of the University of Calgary and Dr. Thomas Wilcox of the University of Texas at Austin. Thanks to Dr. Robert Trench formerly of UC Santa Barbara for donation of Symbiodinium cultures and exchange of ideas. Dr. Eric Lader at Ambion, Inc. provided access to an ABI 7700 and provided valuable assistance with the quantitative real-time PCR experiments. Thanks to Drs. Bassett Maguire and Judy Lang for supervision in the early days when I was trying to figure out what I wanted to work on, and for getting me down on the reefs in the Bahamas where I could really clear my head and think. Paul Thompson and Walter Hokanson from the UT Austin found and/or derived the appropriate statistical equations to use for error propagation. Derrick Zwickl from UT Austin was instrumental in setting up and evaluating phylogenetic analyses. Thanks to lab mates Dr. Thomas Carroll for helping me get that first clone and Dr. Derek Hagman for getting me involved in many underwater science projects at the Flower Garden Banks National Marine Sanctuary and for playing a mean game of rum pong. Undergraduate lab assistants Maria Polycarpo and Tasmin Smith gave considerable help in carrying v the large number of PCR and sequencing reactions were conducted. Thanks to my fellow scuba instructors at the University of Texas Underwater Science and Scuba Diving Program who kept me laughing and took much of the load off of me while I was trying to finish this project. And special thanks Erik and Madonna Reichman for replacing my crashed computer at their own expense and who often baby sat in my hour of need. Funding was made available through the Caribbean Marine Research Center Perry Foundation, the U.S National Oceanic and Atmospheric Administration, Oryx, Inc. and The University of Texas at Austin. vi Characterization and Evolution of Peridinin-Chlorophyll a Binding Protein Gene Families in Symbiotic Dinoflagellates Publication No._____________ Jay Randall Reichman, Ph.D. The University of Texas at Austin, 2002 Supervisors: David M. Hillis and Peter D. Vize Abstract: This dissertation consists of three integrated chapters. Chapter One presents the first genomic characterization of the PCP gene family from a symbiotic dinoflagellate, Symbiodinium sp. from Hippopus hippopus (Symbiodinium 203). Symbiodinium 203 has long PCP genes organized like those of Amphidinium carterae and Lingulodinium polyedra, but with a putative promoter that is different from L. polyedra. There are at least 14 distinct coding regions out of 36 12 PCP genes in this family. Diversity of Symbiodinium 203's PCP gene family appears to be consequence of low levels of concerted evolution and acts as a primary source of variability in PCP isoforms. Predicted amino acid substitutions in Symbiodinium 203's PCP apoproteins result in shifts of isoelectric points, and protein modeling suggests that polymorphic sites may influence light vii harvesting of holoproteins. In Chapter Two, the first PCP coding sequences from S. pilosum, Symbiodinium sp. from Dichocoenia stokesii, S. pulchrorum and S. kawagutii were presented. Diverse PCP gene families occur in all major clades of Symbiodinium and in both size classes of the gene. As with Symbiodinium 203 in Chapter One, these PCP gene families do not appear to have been homogenized through mechanisms leading to concerted evolution. The predicted PCP apoproteins from S. pilosum and S. kawagutii have calculated isoelectric focusing points that generally match values previously measured for these species, which supports the hypothesis that genetic polymorphism is the primary source generating differences in PCP isoforms. Protein modeling produced a putative tertiary structure for S. pilosum apoproteins and was used to identify polymorphic sites in S. pilosum and S. kawagutii PCPs that could affect spectral tuning of peridinins. And Chapter Three contains the first phylogenetic analyses of the evolution of dinoflagellate PCP gene families. The objective of this section is to estimate the selective pressure at the codon level within PCP genes. PCP polymorphism is ancient, however, the polymorphism is not maintained by positive selection. Codon sites within PCP genes are evolving under purifying selection and are subjected to net reduced levels of concerted evolution. Isoform diversity is probably selected for within a functional range. viii Table of Contents List of Tables ....................................................................................................... xiii List of Figures........................................................................................................xv CHAPTER ONE 1 Characterization and organization of the peridinin-chlorophyll a binding protein (PCP) gene family in Symbiodinium sp. from Hippopus hippopus: low levels of concerted evolution among PCP genes contribute to PCP isoform diversity ...............................................................1 Abstract...........................................................................................................1 Introduction ....................................................................................................2 Background on Dinoflagellates and PCPs....................................2 Materials & Methods ....................................................................................13 Algal Cultures.............................................................................13 Nucleic Acid Extractions............................................................13 Primer Design .............................................................................14 PCR Conditions, Identification and Purification of Products.....15 Reverse Transcription PCR (RT-PCR).......................................17 Cloning & Screening Plasmid Libraries .....................................17 Sequencing & Sequence Analysis ..............................................18 Nucleotide Sequence Divergence ...............................................19 PCR Recombination and Fidelity Controls ................................20 PCP Gene Copy Number and Genome Size Estimation ............21 Amino Acid Substitution Modeling ...........................................26 Results ..........................................................................................................26 Organization and Diversity of Genomic Coding and Spacer Regions ..............................................................................26 Low PCR Recombination Frequency and High PCR Fidelity ...31 Diversity Also Expressed at mRNA Level.................................33 ix Symbiodinium 203 Gene Family Size.........................................35 Predicted Proteins, Isoelectric Points and Amino Acid Substitutions ......................................................................38 Discussion.....................................................................................................44 PCP gene organization, diversity and family size ......................44 Low levels of concerted evolution in PCP gene family .............48 Affects of genetic diversity on predicted PCP apoproteins ........49 Conclusion ....................................................................................................51 CHAPTER TWO 52 Analyses of PCP genes and predicted proteins from S. pilosum, Symbiodinium sp. from Dichocoenia stokesii, S. pulchrorum, and S. kawagutii: Diverse PCP gene families occur in all major clades of Symbiodinium and in both size classes of the gene .........................................................................52 Abstract.........................................................................................................52 Introduction ..................................................................................................52 Materials and Methods .................................................................................54 Algal Cultures.............................................................................54 DNA Extractions ........................................................................56 PCR Primers and Conditions ......................................................56 Cloning & Screening Plasmid Libraries .....................................57 Sequencing & Sequence Analysis ..............................................59 Amino Acid Substitution Modeling ...........................................59 Results ..........................................................................................................61 Multiple PCP coding regions cloned from each Symbiodinium species .......................................................61 Predicted amino acid sequences, isoelectric focusing points and protein modeling for S. pilosum PCPs ........................64 Predicated amino acid sequences, isoelectric focusing points and protein modeling for S. kawagutii PCPs .....................70 Discussion.....................................................................................................78 x Diverse PCP gene families found in Symbiodinium sp. from each major rDNA clade .....................................................78 Both small and large PCP genes from Symbiodinium occur in diversified families ............................................................79 Calculated pIs from predicted PCP apoproteins match measured values.................................................................80 Polarity polymorphic sites of PCP apoproteins likely to influence tuning of peridinins............................................81 Conclusion ....................................................................................................83 CHAPTER THREE 84 Phylogenetic analyses and evolution of dinoflagellate PCP gene families: diversity within families is consistent across lineages, but individual sites are most likely to be under purifying rather than positive selection. ...84 Abstract.........................................................................................................84 Introduction ..................................................................................................85 Materials and methods..................................................................................87 DNA sequences and alignments .................................................87 Nucleotide substitution model testing ........................................88 Bayesian inference of PCP gene phylogeny...............................89 Testing PAML CODONML models and detecting selection.....90 Results ..........................................................................................................91 Bayesian phylogenetic analysis of PCP genes with GTR+I+G..91 PAML CODONML analyses .....................................................96 Discussion...................................................................................................100 PCP gene diversity hypotheses.................................................100 Heterogeneity among PCP genes is ancient .............................101 PCP size class not correlated to ribosomal clade. ....................102 PCP gene codons under purifying selection rather than positive selection .............................................................103 Suggested experiments to test for adaptation through purifying selection ...........................................................104 xi Conclusion and Future Directions ..............................................................105 Appendices ..........................................................................................................107 Appendix 1.1 Symbiodinium 203 clone 41 tandem repeat. Upstream and downstream coding sequences are underlined. The putative promoter within the spacer is in bold. ...............................................107 Appendix 1.2 Alignment of cloned Symbiodinium 203 pcp gene nucleotide sequences (start and stop codons are underlined). ...........108 Appendix 1.3 Alignment of predicted amino acid sequences from Symbiodinium 203 clones. transit peptide is underlined. Numbers in square parenthases indicate locations within apoproteins .............115 Appendix 2.1 Alignment of cloned S. pilosum pcp gene nucleotide sequences (start and stop codons are underlined)..............................118 Appendix 2.2 Alignment of cloned Symbiodinium sp. from D. stokesii and S. pulchrorum pcp gene nucleotide sequences ...........................121 Appendix 2.3 Alignment of cloned S. kawagutii pcp gene nucleotide sequences (start and stop codons are underlined)..............................126 Appendix 2.4 Alignment of predicted amino acid sequences from S. pilosum clones. transit peptide is underlined. Numbers in square parenthases indicate locations within apoproteins.............................131 Appendix 2.5 Alignment of predicted amino acid sequences from S. kawagutii clones. transit peptide is underlined. Numbers in square parenthases indicate locations within apoproteins.............................132 Appendix 3.1 Alignment of PCP nucleotide sequences colored by amino acid translation...................................................................................134 References ...........................................................................................................144 Vita 157 xii List of Tables Table 1.1 Primers used for amplification and sequencing. ...................................15 Table 1.2 Primer Sets & Thermoprofiles..............................................................16 Table 1.3 Symbiodinium 203 PCP clone accession numbers................................19 Table 1.4 Ka/Ks values for paired comparisons of Symbiodinium 203 1095 bp coding regions...................................................................................30 Table 1.5 PCR Recombination Control ................................................................32 Table1.6 Relative fluorescence of DNA stained with PicoGreen.........................37 Table 1.7 Calculated mass and isoelectric points from Symbiodinium 203 apoproteins........................................................................................39 Table 1.8 Substitutions within polymorphic sites of predicted Symbiodinium 203 PCP apoproteins compared to 1PPR amino acids. Sites in bold accommodate the presence or absence of amino acids with polar side chains. ..............................................................................40 Table 2.1 Algal cultures ........................................................................................55 Table 2.2 PCR and sequencing primers ................................................................57 Table 2.3 PCP clone accession numbers for S. pilosum, Symbiodinium sp. from D. stokesii, S. pulchrorum and S. kawagutii. ...........................60 Table 2.4 Calculated mass and isoelectric points from S. pilosum apoproteins...65 Table 2.5 Substitutions within polymorphic sites of predicted S. pilosum PCP apoproteins. Bold signifies polarity polymorphic sites. ...................67 Table 2.6 Calculated mass and isoelectric points for S. kawagutii apoproteins ...71 xiii Table 2.7 Substitutions within polymorphic sites of predicted S. kawagutii PCP apoproteins. Sites in bold accommodate the presence or absence of amino acids with polar side chains. ................................74 Table 3.1 Accession numbers of PCP gene sequences used for phylogenetic analyses.............................................................................................88 Table 3.2 Substitution model likelihood ratio test results for PCP genes.............91 Table 3.3 PAML CONDONML model likelihood ratio test results.....................99 Table 3.4 PAML CODONML estimated parameters for PCP genes ...................99 xiv List of Figures Figure 1.1 General phylogenetic relationship between free-living dinoflagellates and clades of Symbiodinium based on large subunit ribosomal sequence (redrawn from Wilcox, 1998)................4 Figure 1.2 A. carterae trimer of 35 kDa PCPs. (NCBI PDB ID 1PPR; Hofmann et al. 1996). .........................................................................6 Figure 1.3 Chromophores and lipids within 35 kDa PCPs; (a) 2 chlorophyll-a. (b) 8 peridinin; (c) 2 digactosyl diacyl glycerol; (d) combined chromophores complex.......................................................................7 Figure 1.4 Regions of interaction between peridinin and amino acids with polar side chains within PCPs; (a) polyene chain and (b) furanic ring of peridinin. .................................................................................7 Figure 1.5 Isoelectric focusing patterns from (a) Symbiodinium microadriaticum; (b) S. goreauii; (c) S. kawagutii; and (d) S. pilosum. Reproduced with permission of the Journal of Phycology from Trench and Blank, 1987. ........................................10 Figure 1.6 Superimposed confocal images of Symbiodinium 203 (left) and L. polyedra (right) cells at their proportional sizes showing fluorescence of PicoGreen stained nuclear DNA and red autofluorescence of chloroplasts.......................................................24 xv Figure 1.7 Cloning strategies. Horizontal bars represent coding regions. Horizontal lines represent spacer. Smaller black vertical bars represent hypothetical locations of substitutions, Open vertical bars represent putative promoter; (a) original clone; (b) clones between adjacent coding regions; (c) clones of complete coding regions; and (d) cDNA clone............................................................28 Figure 1.8 Alignment of conserved upstream region of Symbiodinium 203 spacers vs. the L. polyedra promoter (both in bold). ........................29 Figure 1.9 Neighboring Joining tree of Symbiodinium 203 genomic and cDNA nucleotide sequences. ............................................................34 Figure 1.10 Quantitative Real-Time PCR amplification curves for Symbiodinium 203 genomic DNA vs. dilution series of cloned PCP cds.............................................................................................36 Figure 1.11 Standard curve comparing PCP gene copies in Symbiodinium 203 genomic DNA vs. dilution series of clones PCP cds........................36 Figure 1.12 Substitutions of amino acids with polar side chains occurring at polymorphic sites within Symbiodinium 203 predicted apoproteins show in relation to peridinins........................................41 Figure 1.13 Distance (E= ) between the polar side chain of threonine 118 and the furanic ring of peridinin 612. ...............................................42 Figure 1.14 Distance (E= ) between the polar side chain of serine 253 and the polyene chain of peridinin 624. ..................................................42 xvi Figure 1.15 Distances (E= ) between the polar side chain of serine 287 and the furanic rings of peridinins 621 and 622. .....................................43 Figure 2.1 Primer map; (a) S. pilosum amplifications sequentially consisted of a 3' segment (sPCP-F1/sPCP-R3), the 3' and 5' region from 2 adjacent coding genes (U(112)/L(36)) and the entire cds (U(758)/L(l89); (b) For Symbiodinium sp. from D. stokesii and S. pulchrorum, outward facing primers (U(448)/L(423)) amplified between adjacent genes that lead to inward facing primers (U(182)/L(1315)) producing the majority of the cds; (c) Complete S. kawagutii cds were amplified with Symbiodinium 203 primers (U(-28)/L(1180)). .........................................................58 Figure 2.2 Isoelectric focusing patterns from S. pilosum. The red double line indicates the range of calculated pIs for S. pilosum apoproteins, and the red dots show matches between the calculated and actual pIs (After figure 1d, Trench and Blank, 1987). ................................66 Figure 2.3 S. pilosum 16 kDa PCP monomer; (a) first approximation apoprotein; (b) refined holoprotein. .................................................68 Figure 2.4 Substitutions of amino acids with polar side chains occurring at polymorphic sites within S. pilosum predicted PCP apoproteins shown in relation to peridinins. ........................................................69 Figure 2.5 Distances (E= ) from the polar side chains of threonine 33 and asparagine 133 to the furanic ring and polyene chain of peridinin 622. ...................................................................................................70 xvii Figure 2.6 Isoelectric focusing patterns from S. kawagutii. The red double line indicates the range of calculated pIs for S. kawagutii apoproteins, and the red dots show matches between the calculated and actual pIs (After figure 1c, Trench and Blank, 1987). ................................................................................................72 Figure 2.7 Twenty two polar amino acids occurring in polymorphic sites of S. kawagutii predicted PCP apoproteins located relative to eight peridinin chromophores. ...................................................................75 Figure 2.8 Distances (E= ) from the polar side chains of threonine 70, asparagine 89 and glutamine 150 to the furanic ring and polyene chain of peridinin 614.......................................................................76 Figure 2.9 Distances (E= ) from the polar side chains of serine 169 and tyrosine 178 to the furanic ring and polyene chain of peridinin 624. ...................................................................................................76 Figure 2.10 Distances (E= ) from the polar side chains of threonine 280, serine 287 and asparagine 288 to the furanic ring and polyene chain of peridinins 621 and 622. ......................................................77 Figure 3.1 Consensus (50% majority rule) of 36,000 trees for complete PCP gene data set, colored by species. Numbers are posterior probability support............................................................................93 Figure 3.2 Highest posterior probability tree for the complete PCP gene data set, colored by species. .....................................................................94 xviii Figure 3.3 Highest posterior probability tree for the complete PCP gene set colored by large subunit ribosomal clade of the species. .................95 Figure 3.4 Cladograms comparing the evolution of PCP and large subunit ribosomal genes. Clade B 35 kDa PCPs and clade B small PCP are from different species. (Ribosomal tree was redrawn from Wilcox, 1998). ..................................................................................97 Figure 3.5 Highest posterior probability tree for PCP gene subset colored by species...............................................................................................98 xix CHAPTER ONE Characterization and organization of the peridinin-chlorophyll a binding protein (PCP) gene family in Symbiodinium sp. from Hippopus hippopus: low levels of concerted evolution among PCP genes contribute to PCP isoform diversity ABSTRACT Photosynthetic dinoflagellates have evolved unique light harvesting complexes that combine the accessory pigment peridinin and chlorophyll a within the hydrophobic center of water-soluble peridinin-chlorophyll a binding proteins (PCPs). Most species of dinoflagellates express either 14-17 & 32-35 kDa mature PCP apoproteins and do so in stable combinations of isoforms that differ in isoelectric point (pI). The source (post-translational modification, protein degradation or genetic) and functional significance of PCP isoform variation has remained unclear. PCPs are coded for by multi-gene families. However, previous reports on free-living dinoflagellates conflict over the diversity of PCP genes within gene arrays. I present the first genomic characterization of the PCP gene family from a symbiotic dinoflagellate. Symbiodinium sp. from the Pacific bivalve Hippopus hippopus (RK Trench culture collection # 203) contains genes for 33 kDa PCP apoproteins that are organized in tandem arrays like those of free-living dinoflagellates Lingulodinium (Gonyaulax) polyedra and Heterocapsa pygmaea. The Symbiodinium 203 PCP cassette consists of 1098 bp coding regions separated 1 by spacers of variable length that are approximately 900 bp. The spacers contain a conserved upstream sequence similar to the promoter in L. polyedra. There is surprising diversity between cloned genomic coding regions. Ka/Ks divergence analyses between paired coding regions generally had values < 1. These differences are not accounted for by recombinations or errors introduced during PCR. Reverse Transcription PCR cDNA clones also show the same pattern of diversity. Quantitative Real-Time PCR and flow cytometry indicate that the Symbiodinium 203 genome has 36 12 PCP genes per genome. Thus the PCP gene family of Symbiodinium 203 appears to be evolving under little or no of concerted evolution. The predicted proteins have pIs that are within the range of those published for other species of Symbiodinium. Post-translational modifications are not necessary to explain the multiple PCP isoforms. Amino acid substitutions were mapped onto the Amphidinium carterae PCP crystal structure and identified several polymorphic sites that may influence spectral absorption tuning of chromophores. INTRODUCTION Background on Dinoflagellates and PCPs Dinoflagellates are an ancient and diversified group of organisms that diverged from other eukaryotic lineages at or before the Cambrian (Moldowan and Talyzina, 1998) and underwent extensive radiation to become dominant members of phytoplankton communities by the Jurassic (Loeblich, 1976, 1984). There are roughly 130 extant genera in the division Pyrrophyta with more than 2 1800 marine and 200 freshwater species of described dinoflagellates (Spector, 1984; Falkowski and Raven, 1997). Among these species, there has been considerable niche diversification. Dinoflagellates have become adapted to several life styles, and there are species that fall into one or more of the following categories: bioluminescent, toxic, free-living, parasitic, symbiotic, nonphotosynthetic and photosynthetic. There are at least seven dinoflagellate genera that form endosymbioses with a wide variety of marine invertebrates (Banaszak et al. 1993; Trench 1993). The best known of these mutualism occur between photosynthetic dinoflagellates in the genus Symbiodinium and corals, anemones, jellyfish or bivalves. Biogeographic and phylogentic studies have identified at least 3 major clades (A, B and C; Figure 1.1) of Symbiodinium based on RFLPs and sequences of ss and ls RNA genes (Rowan and Powers, 1991a & b; 1992, Rowan and Knowlton, 1995; Rowan et al. 1997; Wilcox, 1998). An additional Symbiodinium clade E has been justified by comparison of ITS sequences (LaJeunesse and Trench 2000; LaJeunesse, 2001). Photosynthetic dinoflagellates have evolved unique light-harvesting pigment-proteins that bind the carotenoid peridinin in close proximity to chlorophyll a, most often in a 4:1(8:2) molar ratio. These complexes are known as PCPs, and they are unique in terms of their combination of pigments, water solubility, nucleotide sequence and occurrence solely in dinoflagellates. PCPs have been isolated and analyzed for several dinoflagellate species. Examples include PCPs from free-living A. carterae (Haxo et al. 1976; Siegelman et al. 1977; Sharples et al. 1996), Alexandrium cohorticula (Ogata et al. 1994) 3 Glenodinium (Heterocapsa) sp. (Roman et al., 1988; Prezelin and Haxo 1976) L. polyedra (Prezelin and Haxo 1976), and endosymbionts S. microadriaticum (Chang and Trench 1982; 1984; Trench and Blank, 1987; Iglesias-Prieto et al. 1991), S. goreauii (Trench and Blank, 1987) S. pilosum and S. kawagutii (Trench and Blank, 1987; Iglesias-Prieto et al. 1991). Figure 1.1 General phylogenetic relationship between free-living dinoflagellates and clades of Symbiodinium based on large subunit ribosomal sequence (redrawn from Wilcox, 1998). PCPs also do not show significant structural homology to other chromophore binding proteins (Triplett et al. 1993; Norris and Miller, 1994) including phycobiliproteins or membrane-bound light harvesting complexes. It 4 should be noted that discovery of dinoflagellate membrane-bound antennae which also utilize peridinin and chlorophyll a has prompted the development of more accurate but somewhat inconsistent naming of these complexes. Extrinsic watersoluble and intrinsic membrane-bound PCPs have been respectively referred to as PCP1 and PCP2 (Grossman et al. 1995), sPCP and acpPC (Iglesias-Preito et al, 1991; Hiller et al. 1993; Iglesias-Prieto et al. 1993) and also sPCP and iPCP (Durnford et al. 1999). For simplicity within this work, PCP is used to indicate the water-soluble form. The properties of PCPs and their chromophores have been extensively characterized. As mentioned above, PCPs are water-soluble light harvesting complexes (Haxo et al. 1976; Prezelin and Haxo, 1976). PCP preproteins contain transit peptides that presumably help translocate the mature polypeptides to the aqueous thylakoid lumen of dinoflagellate chloroplasts. One way that dinoflagellates photo-acclimate is by changing cellular concentration of PCPs (Prezelin, 1976; Roman et al., 1988; Iglesias-Prieto and Trench, 1997), and this occurs through light regulated transcription (ten Lohuis and Miller; 1998). Under low light conditions, PCPs can constitute a major percentage of the protein in a dinoflagellate cell (Jovine et al. 1993). The peridinin within PCPs harvests blue green 435-550 nm light where chlorophyll a does not (Seigelman et al. 1977; Larkum, 1996; Moffat, 1996). Peridinin also aids in photo-protection by dissipating excess light energy as heat and by limiting the production of singlet oxygen free-radicals that can disrupt photosynthesis (Larkum, 1996; Lakum and Howe, 1997; Pinto et al. 2000). There is near 100% efficiency of energy transfer 5 between peridinin and chlorophyll a in PCPs (Song, 1976; Damjanovic et al., 2000). Tuning of this transfer is achieved by the specific physical arrangement of the chromophore complex and the surrounding amino acids. A high resolution crystal structure of the PCP trimer from A. carterae (Figure 1.2) has indicated that peridinins are held as close as 3.3-3.8 of the tetrapyrole rings of the chlorophylls (Figure 1.3). Furthermore, changes in the polarity of the PCP protein environment neighboring the polyene chains and furanic rings of peridinins can modify the spectroscopic properties of these accessory pigments (Figure 1.4). Figure 1.2 A. carterae trimer of 35 kDa PCPs. (NCBI PDB ID 1PPR; Hofmann et al. 1996). 6 Figure 1.3 Chromophores and lipids within 35 kDa PCPs; (a) 2 chlorophyll-a. (b) 8 peridinin; (c) 2 digactosyl diacyl glycerol; (d) combined chromophores complex. Figure 1.4 Regions of interaction between peridinin and amino acids with polar side chains within PCPs; (a) polyene chain and (b) furanic ring of peridinin. 7 Interactions between chromophores and surrounding amino acids are also stabilized by the lipid DGDG which maintains hydrophobicity of the interior of the PCP monomers (Hofmann et al. 1996). Once the light energy is transferred from peridinin and chlorophyll-a, it is then passed to membrane-bound light harvesting complexes and then to photosystem II (Damjanovic et al. 2000). There are two general size classes of PCP apoproteins, 14-17 kDa monomers or homodimers and 31-35 kDa monomers. These are commonly called 15 kDa (short) and 35 kDa (long) PCPs. The axis of symmetry in the amino acid sequence of long PCPs strongly suggests that original gene for long PCPs arose from a duplication and fusion event between genes for short PCPs (Le et al. 1997). This is also supported by the similarity of short PCP gene nucleotide sequence to regions of long PCP genes (Hiller et al. 2001). It is unclear if long PCP genes have evolved more than once. Many dinoflagellate species express one size class of PCP or the other (Prezelin and Haxo, 1976), however, some express both (Govind et al. 1990). There is evidence that the 35 kDa PCPs of certain dinoflagellates do not contain a single long polypeptide, but rather are constructed with two dimeric short polypeptides (Prezelin and Haxo, 1976). S. microadriaticum is an interesting example of a dinoflagellate that has both sizes of PCPs, and its 35 kDa PCP is dimeric (Chang and Trench, 1984). It is possible that the same short polypeptides that form the 15 kDa PCPs in S. microadriaticum can also be used, perhaps after post-translational modification, to build the 35 kDa version (personal communication, RK Trench, UC Santa Barbara). It is not yet known if all 8 dinoflagellates that express both sizes have dimeric 35 kDa PCPs. Whether assembled with short or long polypeptides, PCP holoproteins probably interact with each other while in the thylacoid lumen. The crystal structure of the A. carterae PCP showed that its functional holoproteins exists as trimers of long PCPs (Hofmann et al. 1996). Furthermore, it has been suggested that the small PCPs of H. pygmaea may also aggregate to form similarly constructed homohexamer (Hiller et al. 2001). It has often been demonstrated that PCPs occur in multiple isoforms that have distinct isoelectric points (pI) that range from pH 4.5-9.0 (Haxo et al. 1976; Prezelin and Haxo, 1976; Seigelman et al. 1977; Chang and Trench, 1984). Stable combinations of isoforms are expressed in a manner that is inversely proportional to light intensity and these combinations are species specific (Chang and Trench, 1982; 1984; Trench and Blank, 1987; see Figure 1.5). The source of PCP isoform variation, be it post-translational modification (Haxo et al. 1976; Seigelman et al. 1977), protein degradation (Ogata et al 1994) or genetic (Chang and Trench, 1984; Triplett et al. 1993; Sharples et al. 1996; Hiller et al. 2001) is unclear. Individual species may contain PCPs that have unique spectroscopic absorbance maxima (Prezelin and Haxo, 1976; Song et al. 1976). While the spectral properties of different PCP isoforms from A. carterae were found to be identical (Haxo et al. 1975), subtle differences were detected in both absorption and fluorescence maxima between individual isoforms from S. microadriaticum (Iglesias-Prieto et al. 1991). However, the functional significance is unknown. 9 Figure 1.5 Isoelectric focusing patterns from (a) Symbiodinium microadriaticum; (b) S. goreauii; (c) S. kawagutii; and (d) S. pilosum. Reproduced with permission of the Journal of Phycology from Trench and Blank, 1987. PCPs are coded for by nuclear genes. Just as there are short and long PCPs, two sizes of PCP genes have also been identified. To date, complete short gene coding sequences have come from H. pygmaea and S. muscatinei, and long gene coding sequences have come from A. carterae, L. polyedra and Symbiodinium from the Pacific coral Acropora formosa (Triplett et al. 1993; 10 Norris and Miller, 1994, Sharples et al. 1996; Hiller et al. 2001, Weis et al. 2002). PCP genes from free-living dinoflagellates have been shown to be intron-less and to exist in tandem arrays (Sharples, et al. 1996; Le et al. 1997; Hiller et al. 2001). Characterization of PCP genes from L. polyedra indicates that they occur in approximately 5000 highly conserved copies (Le et al. 1997). By contrast, H. pygmaea is estimated to have about 50 PCP gene copies and at least five different versions of these genes (the most diversity described prior to results presented in this paper). Evidence from A. carterae or H. pygmaea has mounted to suggest that PCP gene families may not be conserved in general and that the expression of different PCP genes is a primary source distinct PCP isoforms (Triplett et al. 1993; Sharples et al. 1996; Hiller et al. 2001). Diversity has also been a common feature reported in other dinoflagellate multigene families including those for luciferin-binding protein (Lee et al. 1993; Machabee' et al. 1994) and Rubisco (Rowan et al. 1996). The organization and evolution of PCP genes is probably a reflection of the very unusual nature of dinoflagellate genomes. DNA content per dinoflagellate nucleus has been shown to range from 3.2 pg for A. carterae to 200 pg in L. polyedra (Holm-Hansen, 1969). Depending on species, dinoflagellate nuclei contain as few as 4 or many as 325 chromosomes (Spector, 1984). Changes in ploidy may have contributed to genome variability, but this has not been conclusively demonstrated. Furthermore, the distribution of PCP genes between chromosomes is completely unknown. Dinoflagellate chromosomes stay condensed and associated with nuclear membrane throughout most of the cell 11 cycle. Their DNA is coiled around non-histone proteins and the DNA contains the modified base 5-hydroxymethyluracil (Spector, 1984 and Rizzo, 1991). The expectation that genes in tandem arrays would tend to become homogenized through gene conversion and then evolve in concert may not be the case for PCP genes. Detailed information is needed about PCP genes from symbiotic dinoflagellates to lay the groundwork for broader comparative analyses. Several basic questions about PCP genes remain unanswered. Apart from size, which characteristics distinguish PCP genes from different dinoflagellate species? To what degree are PCP gene families composed of divergent coding regions? Do different dinoflagellates species allocated the same relative proportion of their genomes to the expansion of PCP gene families? Does genetic diversity translate to functional differences of PCP isoforms? This chapter presents the first extensive characterization of the PCP gene family from a symbiotic dinoflagellate, Symbiodinium 203 (ls rDNA clade C). Particular emphasis is placed on demonstrating the level PCP gene diversity in Symbiodinium 203 and how it acts as a primary source of variability in PCP isoforms. Symbiodinium 203 PCP gene structure, organization and copy number are compared to genes from L. polyedra. Predicted polypeptide pIs are calculated and compared to published pIs. Amino acid substitutions are mapped onto the A. carterae PCP crystal structure to consider functional significance especially with regard to how variation at polymorphic sites may influence the spectral tuning of peridinins. 12 MATERIALS & METHODS Algal Cultures Robert K. Trench donated Symbiodinium 203 from his culture collection at UC Santa Barbara. Uni-algal subcultures cultures of were maintained in 10 ml of 0.45 m filtered Instant Ocean plus either Provasoli s Enriched Seawater (PES) or Guillard s F/2 (Sigma, Inc.). The cultures were grown under full spectrum fluorescent light banks (Sylvania 40W 4100K Designer) at 80 mole quanta/m /sec. on a 12:12 light to dark cycle at 27 C. Cultures were serially transferred every 3-4 weeks. L. polyedra (CCMP no.1738) was obtained from the Provasoli - Guillard National Center for Culture of Marine Phytoplankton. One liter subcultures of L. polyedra were grown in 2.5 l Fernbach flasks under conditions similar to Symbiodinium 203 except that L. polyedra was only grown in F/2 and the temperature was maintained at 21 C. Nucleic Acid Extractions Log-phase culture cells were harvested by centrifugation at 10,000g for 1 minute in 1.5 ml microcentrifuge tubes. Total genomic DNA was extracted either by a method previously developed for symbiotic dinoflagellates (Rowan and Powers, 1991b) or by a modification of the DNAeasy tissue extraction kit (Qiagen, Inc.), 1999 protocol. Steps 1 and 2 of the DNAeasy protocol were replaced by the following: The algal pellet was re-suspended in 500 l of 2X CTAB buffer (100 mM Tris pH 8.0, 1.4 M NaCl, 25 mM EDTA, 2% (w/v) 13 2 CTAB, 0.2% v/v 2-mercaptoethanol). The re-suspended cells were ground within the microcentrifuge tubes with plastic pestles. 4 l of 10 mg/ml Proteinase K was added to each tube and mix by inversion. The tubes were incubated at 65 C for 2 hours and were inverted every 30 minutes. The extractions then carried forward from step 3 of the DNAeasy protocol. DNA yield was verified on 1% UltraPure (Gibco BRL, Inc.) agarose TAE gels stained with ethidium bromide. Nucleic acid extractions done by the Rowan and Powers method also contained substantial amounts of usable RNA. To purify the RNA for Reverse Transcription PCR (described below), 5 l of the total nucleic acid preps were diluted into 95 l of 1X DNAse Buffer and incubated with DNAse I (Ambion, Inc.) at 37 C for 1 hour. The RNA was phenol/chloroform extracted, ethanol precipitated, desalted, air dried and re-suspended in RNAse-free H2O. RNA yield from this purification was checked on 1% agarose TBE gels stained with ethidium bromide. Primer Design Primer used for standard PCR, Reverse Transcription PCR, Quantitative Real Time PCR and for sequencing are listed in Table 1.1. Published PCP gene sequences Symbiodinium from Acropora formosa (Norris and Miller 1994, GenBank accession no. L13613), A. carterae (Sharples et al. 1996, Z50792, and Z50793) and L. polyedra (Le et al. 1997,U93077) were aligned with Clustal X (Thompson et al. 1997) to identify conserved regions and for initial design of primer set U325/L537. Thereafter, sequence from derived clones was used to design further primers. Primer selection for standard and reverse transcription 14 PCR was optimized with Oligo 4.0 (National Biosciences, Inc.). Primers for quantitative real-time PCR were designed with Primer Express (Applied Biosystems, Inc.). Table 1.1 Primers used for amplification and sequencing. Numbers indicate location of 5' end of primer relative to the Symbiodinium 203 coding region. Those in parentheses are outside of the coding and variable due to indels in the spacers. Upper Primers U325 U448 L423 U935 L(-67) U(1540) L24 U3 L913 U(-28) L(1180) SYBRf938 SYBRr1006 Lower Primers L537 Primer Sequence (5' - 3') AAGAATTCGAAGGACGCAGCAGAAGC CAGAATTCCTTCATGTACGCTGGCAC TCGGTCCCCAAAGCAAAGGTCA CATTCACGGCATCCCAGTCAGC CCGTGCCCAAGTCCACTGTCA GCAGGATGATTGGGATGAGT GAGCCGAACACATCCAGCAG AGCTTTCCTTGCTCCACGCAC GGTGCGTGGAGCAAGGAAAG GCATTCACAGCTTCGTAGT TCCGGCCCACTTTTAGTTTT TTTTCCCATTTGTTTCAGAG TGCCCAAGTCCACTGTCATG TGTTGGTCACGGTGGAATCA PCR Conditions, Identification and Purification of Products Standard PCR conditions (Palumbi, 1996) were generally used with modification to the 10X PCR buffer (200 mM Tris-HCl pH 8.8, 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100 and 1 mg/ml BSA). 50 l reactions were carried out in MJ Research, Inc PTC-100 and Mini-Cycler thermocyclers. Thermal cycle profiles were adjusted to accommodate the 15 annealing temperature of the primer sets and the length of the expected PCR product (Table 1.2). Each round of PCR included a negative control to check for contamination of reagents. PCR reactions were generally run out on 2% agarose TAE gels stained with ethidium bromide. When multiple bands were present in a given reaction, Southern hybridization (Sambrook, 1989) was used to identify PCP gene amplification products. PCR products were transferred to Hybond-N+ nylon membranes (Amersham, Inc.). Probes were created with DECAprime II random priming DNA labeling kits (Ambion, Inc.). Gel excised PCR products of interest were purified with QIAEX II gel extraction kits (Qiagen, Inc.). Table 1.2 Primer Sets & Thermoprofiles. Steps are expressed as time in minutes at a temperature in degrees C or as cycles to repeat. Reaction PCR PCR PCR RT cDNA SYNTH CYCLE SEQ AUTO SEQ Q RT PCR Primer(s) U325/L537 U448/L423 U(-28)/L(1180) OLIGOdT U3/L913 VARIOUS VARIOUS SYBRf938/SYBRr1006 step 1 3:00/94C 3:00/94C 3:00/94C 5:00/65C :30/94C 2:00/95C :10/96C 5:00/94C step 2 1:00/50C 1:00/55C :45/50C 1:00/30C :30/55C :30/95C :05/50C :20/60C step 3 :45/72C 2:15/72C 1:15/72C 30:00/30C to 65C 1:00/72C :30/42C 4:00/60C :20/72C step 4 :45/94C 1:00/94C 1:00/94C 5:00/98C GO TO 2 30X 1:00/70C GO TO 1 24X :20/94C GO TO 2 40X :20/60C :20/72C step 5 GO TO 2 30X GO TO 2 30X GO TO 2 30X 5:00/ 5C 4:00/72C GO TO 2 30X 5:00/4C step 6 :45/60C 1:00/55C :45/ 50C step 7 :45/72C 3:00/72C 3:00/72C 16 Reverse Transcription PCR (RT-PCR) A BcaBEST RNA PCR Kit Ver. 1.1 (TaKaRa, Inc.) was used for Reverse Transcription and amplification of cDNA. The oligo dT primer included in the kit was used for synthesis of single stranded cDNA following a thermoprofile (including a 30-minute gradual rise in temperature from 30 C to 65 C) suggested by the manufacturer (Table 1.2). Subsequent amplification of double stranded cDNA was done with the U3/L913 primer set using A Method from the kit manual. Each round of RT-PCR included controls to check for contamination. Cloning & Screening Plasmid Libraries The PCR product from U325/L537 was blunt cloned into the pBluescript II SK vector (Stratagene, Inc.), and the ligation was used to transform chemically competent E. coli. All other PCR products were cloned into pCR 2.1 vectors with TA Cloning kits (Invitrogen, Inc.) and were used to transform INV F cells included in the kits according to the manufacturers protocol. White transformants growing on L-broth agar plates containing ampicillin and X-Gal were picked for replica plating and PCR screening of the plasmid libraries. To screen the libraries, colony bits were re-suspended into 50 l ddH2O in 1.5 ml tubes by vortexing for 30 seconds. Tubes were heated to 99 C for 5 minutes to lyse the cells and to denature DNAses and then were placed on ice. Lysates were spun at 10,000 g for 2 minutes to pellet bacterial debris. 10.825 l of lysate from each tube was combined with 14.125 l of PCR cocktail consisting of 6.25 l ddH2O, 5 l of 5 mM dNTPs, 2.5 l of standard 10X PCR buffer, 1.5 l 25 mM MgCl2, 17 0.625 l of each 20 M primer and 0.125 l of Taq. Amplifications were done with themoprofiles listed above (Table 1.2). Cells from positive clones were picked from replica plates grown overnight in 3 ml cultures of L-broth containing ampicillin. Clones were purified with QIAamp DNA Mini kits (Qiagen, Inc.). PCR products from these mini preps were gel excised, purified as described above and eluted in ddH2O for sequencing. Sequencing & Sequence Analysis All published sequences for this project were sequenced at least twice and most were done in both the forward and reverse directions. The sequencing thermoprofiles used are described in Table 1.2. The clone from the U325/L537 PCR product was sequenced with a fmol DNA Cycle Sequencing System kit (Promega, Inc.), electrophoresed on conventional 6% polyacrylamide gels and visualized on autoradiographs. Automated sequencing was done for all other clones using BigDyeV2 Terminator kits (ABI, Inc.). Half-reaction mixes consisted of a maximum of 3 l of template (adjusted to include 20-40 ng), 1 l of 10 M primer, 1 l 5X Sequencing Buffer, 2 l BigDyeV2 Terminator mix and the balance with ddH2O. Automated sequencing reactions were purified through Sephadex G-50 Medium (Sigma, Inc.) columns and vacuum dried before resuspensions in loading dye. Samples were run out in 6% ThermoPage acrylamide gels (Amerisco, Inc), and data was collected on a Perkin Elmer ABI PRISM 377 DNA Sequencer. Sequence contigs were assembled with Seqman (DNA Star, Inc). Translation of predicted proteins and pI estimation with done with Gene Runner v3.04 (Hastings Software Inc.) and with Edit Seq (DNA Star Inc.). 18 GenBank database searches for similar nucleotide and amino acid sequences were done using the BLAST algorithm (Altschul et al. 1990). Nucleotide sequences of clones used in the analyses within this chapter were submitted to GenBank and assigned the accession numbers listed in Table 1.3. Table 1.3 Symbiodinium 203 PCP clone accession numbers Species Symbiodinium 203 " " " " " " " " " " " " " " " " Clone Name Accession Number 203clone41 AY149122 203clone79 AY149123 203clone80 AY149124 203clone81 AY149125 203clone82 AY149126 203clone83 AY149127 203clone84 AY149128 203clone85 AY149129 203clone87 AY149130 203clone89 AY149131 203clone90 AY149132 203cDNAclone34 AY149133 203cDNAclone39 AY149134 203cDNAclone42 AY149135 203cDNAclone50 AY149136 203cDNAclone51 AY149137 203cDNAclone55 AY149138 AY149139 203cDNAclone59 Nucleotide Sequence Divergence K-Estimator 5.5 (Cameron, 1995; 1999) was used to calculate of the number of nonsynonymous (Ka) and synonymous (Ks) nucleotide substitutions per site for all paired comparison of complete coding regions. No comparison restrictions were introduced. 1095 bp sites within 365 codons were analyzed for 19 each comparison. The Kimura-2p method was used to correct the number of substitution hit per site. Ka and Ks values were generated from separate analyses, and then Ka/Ks was calculated for each pair of compared sequences. DNA coding regions were aligned with Clustal X (Thompson et al, 1997), MegaAlign (DNA Star, Inc.). Nexus files were generated with MegaAlign and with MacClade v4.0 (Maddison and Maddison, 2000). Execution of the phylogenetic analysis was done with PAUP* 4.0 (Swofford, 1998). Neighbor Joining analysis settings used were with DNA distances corrected by the Kimura-2p model. A Neighbor Joining tree was generated to visually depict the nucleotide sequence divergence. PCR Recombination and Fidelity Controls 2 l of each mini-prep from three unique clones were diluted 1:100 with Tris HCl buffer pH 8.3. Pair-wise combinations of the diluted clones (1 l/ea) were used as templates for 50 l PCR reactions that were run under conditions described above. Amplification products from each reaction were TA cloned. Three sets of 16 sub-clones were isolated, sequenced with U448 and with L423. Each was then identified as being either one of the original templates or a recombinant. The location of the recombination was roughly determined and the observable recombination frequency was calculated as: Number of recombinant clones total number of clones 100% = observable recombination frequency (1.1) 20 In addition, 1 l of 1:100 diluted complete cds clone was amplified in 5 separate PCR reactions under the same conditions as above. A single TA clone was isolated from each reaction and sequenced with U(-28), U448, L913 and L(1180) to check for nucleotide substitutions introduced by the PCR process. PCP Gene Copy Number and Genome Size Estimation The number of PCP genes/ genome for Symbiodinium 203 was calculated by the overall equation: PCP genes genes pg genomic DNA pg genomic DNA pg genome = PCP genes genes genome (1.2) where equals the standard deviation. PCP genes genes pg genomic DNA The term in equation 1.2 was determined by quantitative real-time PCR with a Perkin Elmer ABI 7700. Data was analyzed with ABI Sequence Detection Systems 1.7 software. Amplifications of a 69 bp PCP gene segment were compared between known amounts of Symbiodinium 203 genomic DNA and a linearized PCP clone. Concentrations of genomic and plasmid DNA were quantified in triplicate on a Beckman Coulter DU640 spectrophotometer. 1.492 g of the clone was digested with 2 units Not I (New England Biolabs, Inc.) at 37 C for 1 hour to cut the pUC19 vector at a single site but not the insert. The reaction was terminated by heat inactivation at 65 C for 20 minutes. The linearized plasmid DNA was used to create a dilution series that 21 provided the templates for the amplification standard curve. Replica 50 l reactions were set up for each of the standards and for negative controls. Triplicate 50 l reactions were set up for the Symbiodinium 203 genomic samples. Reagents were assembled in master mix and were distributed so that each reaction contained 4 l of 5 mM dNTPs, 5 l of 10X PCR buffer (100 mM Tris HCl pH 8.3, 500 mM KCl, 30 mM MgCl2, 10% glycerol, 0.1% Tween 20 and 1:4000 dilution of SYBR Green I (Molecular Probes, Inc.), 6 l of 25 mM MgCl2, 0.5 l (2 units) of SuperTaq (Ambion, Inc.), 1 l of 5 M SYBRf938 primer, 1 l of 5 M SYBRr1006 primer, 27.5 l ddH2O and 5 l DNA template (= 10,000 pg of genomic DNA, and 1492000 to 14.92 fg in 10-fold dilutions of plasmid DNA). In order to compare PCP copy number to ng of genomic DNA, PCP gene copies/ fg of PCP clone were converted as follows: (plasmid + insert ) 1 mole bp 6.02 10 5108 bp 660 g 23 mole bp 1g 179(plasmids + inserts) = 15 fg 10 fg (1.3) The pg genomic DNA pg genome term in equation 1.2 was estimated by comparing the average genome sizes of Symbiodinium 203 and L. polyedra by flow cytometry using a Beckton Dickenson FACS Calibur. The instrument was equipped with a 15 mW Argon laser producing excitation at 488 nm. A modified version of the Veldhuis et al. 1997 protocol for fixing and staining the cells was 22 used. An equal volume of algal cells suspended in F/2 was combined with 4% paraformaldehyde and let stand at room temperature for 15 minutes with 0.33 l of 1% Triton X-100 and 1 l of 1 g/ml RNAse A were added per ml of fixed cells. The solutions were mixed by gentle inversion several times and kept at room temperature for 20 minutes. Cells were pelleted by certifugation at 3000 rpm at 4 C in a Sorvall Ultrafuge. After the liquid was carefully decanted, the fixed cells were re-suspended in 500 l of TE. 1 l 1% Triton X-100 and 50 l of 20X PicoGreen (Molecular Probes, Inc.) were added and mixed by inversion. The cells were allowed to stain at room for at least one hour prior to flow cytometry (Figure 1.6). Fluorescence of PicoGreen stained DNA was measured through the FACS FL1 filter at 530 30 nm. To calculate pg genomic DNA for Symbiodinium 203: mean fluorescence fluorescence for Symbiodinium 203 200 pg DNA for L. polyedra mean fluorescence fluorescence for L. polyedra genome (1.4) 23 Figure 1.6 Superimposed confocal images of Symbiodinium 203 (left) and L. polyedra (right) cells at their proportional sizes showing fluorescence of PicoGreen stained nuclear DNA and red autofluorescence of chloroplasts. Within the simple mathematic operations above, the following equations were used to accurately account for the propagation of indeterminate errors (Melissinos, 1966). Let A through D stand for means of independent data sets or resultants from previous calculations. A A B B For the division : + B B 2 A B A B = ( ) ( ) A A 2 24 A B = A B ( ) ( ) A A 2 + B B 2 The resulting quotient will be: A A B B ( ) ( ) A A 2 + B B 2 (1.5) And for the multiplication (C C ) (D D ) : CD CD = ( ) ( ) C C 2 + D D C C 2 2 CD = CD ( ) ( ) + D D 2 The resulting product will be: CD CD ( ) ( ) C C 2 + D D 2 (1.6) 25 Amino Acid Substitution Modeling Predicted amino acid substitutions were mapped onto the crystal structure for the A. carterae PCP trimer 1PPR using Swiss-PDB Viewer v3.7(b2) (Glaxo Wellcome, Inc.) as follows: Pair wise amino acid alignments between Symbiodinium 203 and A. carterae PCPs were created to identify conserved and variable sites. Mutations were individually introduced in the 1PPR structure at each of the fixed and polymorphic sites. As substitutions were made, rotomer conformations were optimized by the software. Distances were then calculated between polymorphic sites and either molecules in neighboring monomers or chromophores within the same monomer. reflecting all changes. A single layer pdb was rendered 3D protein structure images were rendered using POV- Ray v3.1 (POV-Ray Team) and could only be created from single layer pdb files. RESULTS Organization and Diversity of Genomic Coding and Spacer Regions The cloning strategy for this project is summarized in Figure 1.7. The long PCP gene sequences Symbiodinium from Acropora formosa (GenBank accession no. L13613), A. carterae (Z50792, and Z50793) and L. polyedra (U93077) are highly divergent and consequently did not produce large, conserved blocks that could be easily targeted for amplification. The alignment suggested that an approximately 200 bp region in the 5 half of the long PCP genes was moderately 26 conserved. Primers U325 and L537 based on sequence L13613 successfully amplified a 212 bp fragment from Symbiodinium 203 (Figure 1.7a). The sequence of small cloned fragment was positively identified by a BLAST search as partial PCP gene. When this research began, only long PCP genes from the free-living dinoflagellates A. carterae and L. polyedra were shown to exist in tandem arrays (Sharples et al. 1996; Le et al. 1997). Although it was not known at the time whether Symbiodinium 203 had long or short PCP genes, it was reasonable to assume that either variety would be arranged in the same manner. Sequence data from the 212 bp fragment was used for generation of outward facing primers U448 and L423 to amplify between adjacent gene copies. Amplification products of various sizes were present in the reaction. A Southern Blot was created and probed with the small fragment. There was substantial hybridization to a 1.9 kb band which was then excised and purified from another gel. Direct sequencing of the 1.9 kb PCR product effectively failed, because the reads contained many unresolved bases. This was an important clue that multiple templates were present in the sequencing reaction. The 1.9 kb fragments were TA cloned, and a single clone was sequenced completely by primer walking (see Appendix 1.1). This sequence confirmed that the PCP genes from Symbiodinium 203 are arranged in tandem arrays like A. carterae and L. polyedra. Open reading frames at the 5' and 3' ends of this clone both translate to PCP and flank a 903 bp spacer region. 27 Figure 1.7 Cloning strategies. Horizontal bars represent coding regions. Horizontal lines represent spacer. Smaller black vertical bars represent hypothetical locations of substitutions, Open vertical bars represent putative promoter; (a) original clone; (b) clones between adjacent coding regions; (c) clones of complete coding regions; and (d) cDNA clone 28 Sequencing additional 1.9 kb clones revealed that there were numerous substitutions distributed throughout the coding and spacer regions (complete nucleotide sequence data for all tandem array clones not shown). As expected, the spacers contained the majority of the substitutions including many insertions and deletions. Despite the highly variable nature of the spacers, each was found to contain a conserved 13 bp sequence CTTGAATGCAGAA approximately -201 to -188 bp upstream of the start codon. Figure 1.8 shows that 9 of the 13 bases in this sequence are identical to and in the same relative location as the promoter previously identified from the L. polyedra luciferase and PCP spacers (Li and Hastings. 1998). This conserved sequence is very likely to be the Symbiodinium 203 promoter. (-228) . L poly promoter 203_41 203_43 203_44 203_46 203_49 203_50 203_52 203_54 203_55 (-218) . (-208) . (-198) . (-188) . (-178) . CGTGAACGCAGTG GTGCTTGTCC----------GTTTGCAATCTG---TCTTGAATGCAGAATCGAGACAGCA GGGCTTGTCC----------GTTTGCAATCTGTCTTCTTGAATGCAGAATCGAGAAAGCA GTGCTTGTCC----------GTTTGCAATCTGTCTTCTTGAATGCAGAATCCAGACAGCA GTGCTTGTCC----------GTTTGCAATCTG---TCTTGAATGCAGAATCGAGACAGCA GTGCTTGTCC----------GTTTGCGATCTGTCTTCTTGAATGCAGAATCGAGACAGCA GTGCTTGTCC----------GTTGGCAATCTGTCTTCTTGAATGCAGAACCGAGACAGCA GTGTTTGTCC----------GTTTGCAATCTGTCTTCTTGAATGCAGAATCCAGACAGCA GTGCTTGTCCGTTTATGTCCGTTTGCAATCTGTCTTCTTGAATGCAGAATCGAGACAGCA GTGCTTGTCC----------GTTTGCAATCTGTCTTCTTGAATGCAGAATCGAGACAGCA * * ****** ************ ** **** **** ** *** **** Figure 1.8 Alignment of conserved upstream region of Symbiodinium 203 spacers vs. the L. polyedra promoter (both in bold). Amplification of Symbiodinium 203 genomic DNA with inward facing primers U(-28) and L(1180) produced a 1.1 kb band. After gel extraction and 29 purification, direct sequencing resulted in the same pattern of numerous unresolved bases seen with the 1.9 kb PCR products and again suggested that multiple templates were present. TA cloning produced clones 79-90. Clones 79 and 81-90 contained a 1098 bp complete long PCP coding sequence (Figure 1.7c). The open reading frame in clone 80 had a stop 27 bases earlier than other. All coding sequences except those within clones 86 and 88 were distinct from each other in terms of their nucleotide substitutions (97.8-99.5% identical). Of the published PCP gene sequences in GenBank, the Symbiodinium 203 coding sequences were most similar to the coding sequence of Symbiodinium sp. L13613 (92.6-94.5% identical). An alignment of genomic and partial cDNA clones (described below) is presented in Appendix 1.2. The majority of nucleotide substitutions between the complete 1098 bp coding regions were in synonymous codon positions resulting in Ka/Ks <1 (Table 1.4). Only the comparison of clones 79 and 82 had a Ka/Ks >1. Table 1.4 Ka/Ks values for paired comparisons of Symbiodinium 203 1095 bp coding regions clone 79 clone 81 clone 82 clone 83 clone 84 clone 85 clone 87 clone 89 0.385 1.115 0.373 0.326 0.526 0.375 0.294 0.0222 0.26 0.203 0.276 0.208 0.233 0.178 0.276 0.363 0.248 0.399 0.213 0.0113 0.038 0.324 0.274 0.266 0.218 0.333 0.474 0.111 0.306 0.404 0.399 0.335 0.186 0.162 0.22 0.196 clone 81 clone 82 clone 83 clone 84 clone 85 clone 87 clone 89 clone 90 30 Low PCR Recombination Frequency and High PCR Fidelity To control for possible generation of recombinant DNA sequences by amplifying genes in tandem (Bradley and Hillis, 1997), a re-amplification and sub-cloning experiment was conducted using pairs of distinctive clones as the templates. All other PCR conditions were the same as when genomic Symbiodinium 203 was amplified. The results are presented in Table 1.5. Sixteen sub-clones from each of the amplifications were partially sequenced to identify them as either one of the original templates within the PCR reaction or as recombinant. The first 500 bp of sequence from both the U448 (5') and L423 (3') ends were used for the identification. Four of the 48 sub-clones had different U448 and L423 identities. For each of these recombinants, the flanking regions sequenced completely matched one or other of the templates suggesting that recombination had likely occurred in the spacer. This may be attributable to secondary structure formation within the spacer regions of the clones during the PCR reactions. One of the sub-clones was excluded as a contaminant. The observable recombination frequency was estimated from equation 1 as: 4 recombinants 47 total sub-clones 100% = 8.5% 31 Table 1.5 PCR Recombination Control Identity of 1st 500bp from U448 Sub-clone A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 Sub-clone B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 Sub-clone C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 CLONE 41 * * * * * * * * * * * * * * CLONE 41 * * * * * * * * * * * * * * * CLONE 52 * * * CLONE 52 * * * * * * * * * * * CLONE 54 * * * * * * * * CLONE 41 * * * * * * CLONE 54 * RECOMBINANT * CLONE 52 * Identity of 1st 500bp from L423 CLONE 41 * * * * * * * CLONE 52 * RECOMBINANT * CLONE 54 * * * CLONE 54 * * * RECOMBINANT * * * * * * * not PCP * not PCP * * * * * * * * * * not PCP * * not PCP * * * 32 The rate at which Taq error introduced substitutions in our PCR reactions was also estimated. A single complete cds clone was re-amplified in five separate PCR reactions and a single sub-clone from each reaction was completely sequenced in both directions. There were no changes introduced in to any of the 1198 base sub-clones. Diversity Also Expressed at mRNA Level The surprising diversity found in the genomic clones was also present in partial cDNA clones. Poly-A mRNA from Symbiodinium 203 was reverse transcribed with oligo dT primers and then double-stranded cDNA was amplified with the U3/L913 primer set (Figure 1.7d). A single band with the expected size was seen after gel electrophoresis, and this was excised and purified. As with the previous genomic amplifications, directed sequencing failed because of apparent multiple temples and individual cloned RT-PCR products had diversified nucleotide sequences. Eight positive cDNA clones were sequenced and none were identical to the overlapping regions in the complete coding sequence clones. Five of these cDNAs were unique (Appendix 1.2). The absence of any insertions in the complete coding sequences compared to the cDNAs is evidence that Symbiodinium 203 PCP genes, like L. polyedra (Le et al. 1997) and H. pygmaea (Hiller et al. 2001) are intron-less. The overall diversity of genomic and cDNA nucleotide sequences is graphically depicted in the Neighbor Joining tree in Figure 1.9. 33 Figure 1.9 Neighboring Joining tree of Symbiodinium 203 genomic and cDNA nucleotide sequences. 34 Symbiodinium 203 Gene Family Size Quantitative Real-Time PCR and flow cytometry were used to estimate the number of PCP genes in the Symbiodinium 203 genome. The curves in Figure 1.10 represent the changing quantities of 68 bp SYBRf938/SYBRr1006 PCR products as amplified from 10 ng of Symbiodinium 203 genomic DNA verses the dilution series of a single compete cds clone. Figure 1.11 is the standard curve generated by comparing the quantity of amplicons in each reaction at the threshold value in the log phase of amplification suggested by the ABI 377. There was a 0.996 correlation coefficient between all points (unknowns and standards) on the curve. The mean amplification value for three replicates of 10 ng of genomic DNA was 667.60 with a standard deviation of 51.89. This indicates that 10 ng of Symbiodinium 203 genomic DNA contains about the same number of PCP gene copies as 667.60 fg of clone 79. From equation 3, each fg of clone 79 has 179 PCP gene copies. Therefore 10 ng of genomic DNA contains 119500 9288 PCP genes. In other words, there are roughly 12 0.9 PCP genes per pg of genomic DNA. The genome size of Symbiodinium 203 was compared to L. polyedra by flow cytometry to estimate the DNA content per nucleus. Table 1.6 shows the comparison of relative mean fluorescence of PicoGreen strained DNA from at least 3000 cells of each species. 35 Figure 1.10 Quantitative Real-Time PCR amplification curves for Symbiodinium 203 genomic DNA vs. dilution series of cloned PCP cds. Figure 1.11 Standard curve comparing PCP gene copies in Symbiodinium 203 genomic DNA vs. dilution series of clones PCP cds. 36 Table1.6 Relative fluorescence of DNA stained with PicoGreen Sample name Symbiodinium 203 L. polyedra Parameter FL1-H FL1-H Mean 1.92 152.09 CV (%) Std. Dev 16.73 0.32 26.47 40.26 Using equations 1.4 and 1.5: 1.92 .32 for Symbiodinium 203 for L. polyedra 152.09 40.26 for L. polyedra 1.92 1.92 = 152.09 152.09 ( ) ( ) 40.26 2 200 pg DNA .32 2 + genome 152.09 1.92 = 3 1 pg DNA for Symbiodinium 203 genome Combining the quantitative real-time PCR and flow cytometry results within equations 1.2 and 1.7: 12 0.9 PCP genes 3 1 pg genomic DNA for Symbiodinium 203 pg genomic DNA genome = 12 3 12 3 ( ) () 37 0.9 2 1 2 + 3 12 = 36 12 PCP genes for Symbiodinium 203 genome Predicted Proteins, Isoelectric Points and Amino Acid Substitutions Despite Ka/Ks values <1 for most of the compared Symbiodinium 203 coding regions, there was still considerable amino acid sequence variation. An alignment of predicted proteins from cloned complete coding regions is presented in Appendix 1.3. All clones but clone 80 (which had a stop codon 27 bases upstream of others) coded for 365 aa PCP pre-proteins with 52 aa transit peptides and 313 aa apoproteins. Pre-proteins ranged from 96.2-99.7% identical when compared to each other and 93.3-94.9% when compared to Symbiodinium sp. L13613. Within the Symbiodinium 203 transit peptides and apoproteins there were 9 and 20 polymorphic sites respectively. The 313 aa apoproteins had an average mass of 33 kDa and were the same length as those predicted from the Symbiodinium sp. L13613 and A. carterae Z50792 and Z50793 sequences. By contrast, the L. polyedra PCP gene sequence U93077 codes for a 375 aa preprotein with a 59 aa transit peptide and a 316 aa apoproteins. Variability in long PCP mass is attributable to amino acids composition and also to polypeptide length. Most of the predicted apoproteins from the clones had subtle differences in their mass, but they also varied in terms of their isoelectric points (Table 1.7). The pIs for each of the clones fell within a range of pH 5.73-6.78. The PCP isoforms reported from other dinoflagellates are not acidic for all species. Most isoforms from A. carterae (Haxo et al. 1976) and Glenodinium (Heterocapsa) sp. (Prezelin and Haxo 1976) are basic. However, there are several examples of 38 symbiotic dinoflagellates that produce predominantly acidic PCP isoforms with pI ranges similar to those predicted here including S. microadriaticum, S. goreauii (Trench and Blank, 1987) Symbiodinium from Montastrea annularis and Symbiodinium from M. cavernosa (Chang and Trench, 1982). In addition, the calculated apoprotein pI of the Symbiodinium from A. formosa sequence is 5.28. Table 1.7 Calculated mass and isoelectric points from Symbiodinium 203 apoproteins 203 clone 79 80 81 82 83 84 85 87 89 90 Apoprotein mass (kDa) 33.38 31.956 33.01 32.965 33.115 33.068 32.983 32.983 33.012 32.926 Isoelectric point (pI) 6.03 6.37 6.78 6.78 5.74 6.37 6.03 6.37 5.73 5.73 The variability of calculated Symbiodinium 203 PCP pIs is a direct result of predicted amino acid substitutions. To investigate possible functional significance, a composite of these changes was mapped onto the A. carterae PCP apoproteins crystal structure 1PPR. There were 70 sites at which Symbiodinium 203 and A. carterae 1PPR sequences differed. Among these sites, 43 were fixed substitutions between Symbiodinium 203 and A. carterae 1PPR apoproteins, and 27 were polymorphic between individual Symbiodinium 203 clones. Table 1.8 39 lists the specific amino acids and types of side chains substituted at each polymorphic site. Ten of the polymorphic sites (positions 6, 24,134,137,182,222, 239,244, 253 and 287) are predicted to accommodate the presence or absence of amino acids with polar side chains. Table 1.8 Substitutions within polymorphic sites of predicted Symbiodinium 203 PCP apoproteins compared to 1PPR amino acids. Sites in bold accommodate the presence or absence of amino acids with polar side chains. Site # 6 24 39 51 96 101 103 105 107 118 Symbiodinium 203 Substitutions Ala Thr Asn Asp Glu Lys Gly Glu Ile Val Glu Lys Lys Arg Met Val Val Ala Lys Ile Ala Ala Asp Val Lys Asn Gln Lys Ala Arg Side Chain nonpolar uncharged polar uncharged polar acidic acidic basic nonpolar acidic nonpolar nonpolar acidic basic basic basic nonpolar nonpolar nonpolar nonpolar basic nonpolar nonpolar nonpolar acidic nonpolar basic uncharged polar uncharged polar basic nonpolar basic A. carterae 1PPR Substitutions Ala Asn Glu Gly Ile Glu Met Met Val Lys Side Chain nonpolar uncharged polar acidic nonpolar nonpolar 239 acidic 244 nonpolar nonpolar nonpolar 251 basic 253 Ala nonpolar 275 245 Site # 172 182 217 222 Symbiodinium 203 A. carterae 1PPR Substitutions Side Chain Substitutions Ala nonpolar Gln Val nonpolar Lys basic Lys Gln uncharged polar Met nonpolar Ala Val nonpolar Ala nonpolar Leu Lys basic Thr uncharged polar Lys basic Thr Gln uncharged polar Ala nonpolar Ala Lys basic Thr uncharged polar Ala nonpolar Ala Gly nonpolar Asp acidic Asn uncharged polar Asn Tyr uncharged polar Ala nonpolar Ala Ser uncharged polar Gly nonpolar Gly Gln nonpolar Lys basic Val nonpolar Val Ile nonpolar Thr uncharged polar Thr Ser uncharged polar Ser uncharged polar Ser Pro nonpolar Side Chain uncharged polar basic nonpolar nonpolar uncharged polar nonpolar nonpolar uncharged polar nonpolar nonpolar 121 134 137 154 Lys Glu Ala basic acidic nonpolar 276 279 287 nonpolar uncharged polar uncharged polar Figure 1.12 is a rendering of all 10 polar substitutions showing their spatial orientations in relation to the 8 peridinins within 1PPR. Thr118, Ser253 and Ser287 are of particular interest because of their proximities to peridinin polyene chains and furanic rings and possible influence on spectral tuning of these chromophores. The polar side chain of Thr118 is 7.19 from PID612 (Figure 40 1.13). Ser253 is 10.06 from PID624 (Figure 1.14). Ser287 is from 8.99 of PID622 and 9.15 from PID621 (Figure 1.15). Figure 1.12 Substitutions of amino acids with polar side chains occurring at polymorphic sites within Symbiodinium 203 predicted apoproteins show in relation to peridinins. 41 Figure 1.13 Distance (E= ) between the polar side chain of threonine 118 and the furanic ring of peridinin 612. Figure 1.14 Distance (E= ) between the polar side chain of serine 253 and the polyene chain of peridinin 624. 42 Figure 1.15 Distances (E= ) between the polar side chain of serine 287 and the furanic rings of peridinins 621 and 622. 43 DISCUSSION PCP gene organization, diversity and family size This is the first characterization of a PCP gene family from any dinoflagellate in the genus Symbiodinium. Earlier descriptions of the organization of PCP genes came from free-living species A. carterae (Sharples et al. 1996), L. polyedra (Le et al. 1997) and H. pygmaea (Hiller et al. 2001). There are only 2 previous reports of Symbiodinium PCP nucleotide sequences and both were single cDNAs (Norris and Miller, 1994; Weis et al. 2002). The results of these molecular experiments demonstrate that the PCP genes of Symbiodinium 203 are organized essentially like those of A. carterae and L. polyedra in that the genes are arranged in tandem arrays, the coding regions code for long PCP polypeptides and the coding regions are intronless. The coding regions of Symbiodinium 203 are very similar in size to those from A. carterae and Symbiodinium from A. formosa yet shorter than L. polyedra. The Symbiodinium 203 PCP cassette has untranscribed spacers of variable sizes that are smaller than those from L. polyedra, but longer than H. pygmaea. Li and Hastings (1998) identified a likely promoter sequence from L. polyedra luciferase and PCP spacers. The putative promoter sequence that we found in the same relative location upstream of Symbiodinium 203 coding regions was 69% identical to the L. polyedra promoter. Dinoflagellate promoters do not fit into the common motifs used by other eukaryotes, and dinoflagellate promoters may be genus or species specific. 44 There is far greater diversity in the coding regions of Symbiodinium 203 PCP genes than previously described from any other dinoflagellate species. I have identified ten unique complete coding sequences and an additional five unique partial cDNA sequences. The majority of positive clones screened from genomic and cDNA PCR libraries were distinct however, redundant sequences were also found. For reasons of practicality, the searches were not exhaustive, so the limit on the number of unique PCP genes in Symbiodinium 203 may not been reached. The low Ka/Ks ratios for paired comparison between coding sequences from ten clones indicate that the majority of nucleotide substitutions occur at synonymous codon positions and that there is not a clear signal of entire coding regions being selected for within the Symbiodinium 203 PCP gene family. Although not shown here, the same is true when these sequences are compared to A. carterae or Symbiodinium from A. formosa coding sequences. This does not exclude the possibility that specific sites within PCP genes could be under selection. When PCP gene sequences are described for more species, Bayesian and likelihood analyses of PCP gene phylogenies may detect such sites. As it became apparent that very few of coding sequences were identical, there was concern that at least part of the variation that was being observed was the result of recombination within the PCR reactions (Bradley and Hillis, 1997). When amplifying genes from within tandem arrays it is possible to generate incomplete extensions products that can act as primers and anneal to various locations in the array resulting in PCR fragments whose sequences can be different from those actually present in the genomic DNA. The results of the 45 recombination experiment showed that 8.5% of subclones had different 5' and 3' identities with all recombinations apparently occurring in spacer regions. Early termination of PCR extensions in the spacers may be attributable to formation of secondary structure during the annealing phase of the reactions. Conclusions to be drawn from this type of control to detect recombination are limited in a few regards. Reamplification of cloned DNA can be different than amplifying genomic regions. An increase in the number of unique templates available within each reaction could increase recombination frequency. And this type of analysis only detects recombinants that are observable by comparing coding sequence in subclones to coding sequence in the original templates. Assuming that under our PCR conditions early extension termination is an artifact of amplifying across spacers, it is possible that clone 41 is a chimera, but it is less likely that the complete coding sequence clones of 79-90 or cDNA clones 34-59 are recombinants. Another source or artificial substitution is through Taq polymerase errors. The Taq polymerase fidelity experiment did not detect any introduced changes in subclones of reamplified clone 79 further suggesting that the coding sequence differences reported here are reliable. Le et al. (1997) reported that L. polyedra had a PCP gene family of roughly 5000 copies per 200 pg genome, which is one of the largest gene families for any organism. It should be noted that the L. polyedra genome is also unusually large compared to many other dinoflagellates and other eukaryotes. For perspective, human haploid cells typically have 3.2 pg of DNA per nucleus (Holm-Hanson, 1969). The Le et al. copy number was based on intensity of 46 hybridization signals of slot blots. I sought to further refine the methods of estimating PCP gene family size in this investigation. Quantitative Real-Time PCR is an extremely accurate and reliable way of determining the amount of starting template within amplification reactions and was used here to give a mean the number of PCP genes per pg of genomic DNA for Symbiodinium 203. Flow cytometry is now routinely used to measure various cellular parameters including DNA content and offers the advantage of being able to quickly collect data on large numbers of cells. Fluorescence is typically standardized to chick red blood cells (CRBCs), chick erythrocyte nuclei (CENs) or synthetic beads. When Veldhuis et al. (1997) quantified the DNA content of 121 strains of marine phytoplankton by flow cytometry, they pointed out that the staining of nuclei in whole cells with cell walls was not directly comparable to CRBCs. I chose not to standardize to CRBCs or CENs, but rather to make a relative comparison of the mean genome sizes of the Symbiodinium 203 and L. polyedra. L. polyedra cells are less than perfect standards for this purpose because they have armored theca while Symbiodinium 203 cells do not. In addition, the genome size of L. polyedra is substantially bigger than Symbiodinium 203. Nevertheless, comparing fluorescence of stained DNA between these two species is more valid than comparing either to the commercially available standards which stain much more readily than intact algal cells. The combined results of our quantitative real-time PCR and flow cytometry experiments show that Symbiodinium 203 has 36 12 PCP genes per 3 1 pg genome. This gene family size is much closer to the 50 PCP genes per genome for H. pygmaea suggested by Hiller et al. (2001) than to 47 L. polyedra. The PCP gene copy number estimates for all three species rely in one way or the other to the Holm-Hansen (1969) algal DNA content values based on amount of nuclear organic carbon as standards. As the DNA content of dinoflagellates becomes more accurately measured with newer techniques, the current PCP gene family sizes may be revised. Beyond the absolute differences in PCP gene family sizes, Symbiodinium 203 has proportionally fewer PCP genes per pg of genomic DNA than L. polyedra. There may be selection in symbiotic dinoflagellates toward smaller allocation of genomes to PCP gene families compared to free-living species. The test of this hypothesis awaits the characterization of PCP gene families from several more species of each type. Low levels of concerted evolution in PCP gene family Symbiodinium 203 has a relatively small PCP gene family that contains numerous divergent coding sequences. Despite the fact that these sequences bear greater resemblance to each other than they do to other published long PCP genes, I found little evidence that Symbiodinium 203 PCP genes are evolving in concert. Concerted evolution of tandemly repeated sequences such as the genes for ribosomal RNA, transfer RNA and histones is usually explained in terms of two mechanisms; continual expansion and contraction due to unequal crossing over and biased gene conversion. The relative importance of one or the other mechanism varies depending on which organisms are studied (see Hillis et al. 1991). Both rely on duplex formation between homologous loci as most often occurs in meiosis. While sexual reproduction has been reported in other dinoflagellate genera (Pfiester, 1984), there is an apparent absence of a haploid 48 sexual phase in Symbiodinium (Schoenberg and Trench, 1980a, Trench, 1993). However, Baillie et al. (2000) reported that RAPD variation in Symbiodinium from giant clams was comparable to patterns seen in dinoflagellates undergoing sexual recombination. Lack of heterogeneity and ease of direct sequencing of amplified large subunit ribosomal genes from various Symbiodinium species suggest that concerted evolution does occur within Symbiodinium genomes (see Wilcox, 1998). It is unclear if this is accomplished in the formation of cryptic haploid sexual cells, through mitotic crossing over or other as of yet described mechanisms particular to dinoflagellate genomes. Nevertheless, the current results strongly suggest that PCP genes are handled differently than ribosomal genes. PCP genes may contain variable sites into which mutations are introduced faster than they can be removed by homogenizing mechanisms. Another possibility is that PCP genes may reside in regions of dinoflagellate genomes where such mechanisms occur less often. The net effect is that PCP genes are evolving under little or no concerted evolution. PCP isoform diversity may be important in broadening the range of light wavelength that can be harvested by dinoflagellates for photosynthesis. Affects of genetic diversity on predicted PCP apoproteins While there are no reports of empirically determined PCP isoforms for Symbiodinium 203, translations of coding sequences cloned in this project demonstrate that there is sufficient genetic diversity to account for a suite of PCP apoprotein pIs comparable to those found in several other Symbiodinium species. This does not rule out the possibility that post-translational modification of PCP 49 polypeptides could still occur. The results suggest that post-translational modifications are not necessary to explain the multiple PCP isoforms. This point could be tested by additional PCR based characterizing PCP coding sequences of species for which PCP pIs are known. The potential for examining functionally significant differences between predicted PCP apoproteins was greatly enhanced by mapping amino acid substitutions on to the A. carterae 1PPR crystal structure. Collectively, the Symbiodinium 203 polypeptides differed from A. carterae 1PPR at 70 out of 312 sites within each monomer. The 43 fixed substitutions were distributed through all domains of 1PPR including regions near adjacent monomers, the hydrophilic exterior and chromophores. The majority of these changes that faced hydrophobic interior of monomers did not affect the polarity of this environment. However, they could be important in giving Symbiodinium 203 PCP holoproteins a different conformation than 1PPR. Ten of the 27 polymorphic sites hold amino acids with or without polar side chains. The apoproteins from clones 79 and 83 contain polar substitutions with OH groups in positions that could influence the spectral tuning of nearby peridinins. Predicting the direction and magnitude of overall changes to the spectroscopic properties of holoproteins is beyond the scope of this investigation. Distances calculated by Swiss-PDB Viewer v3.7(b2) between polar side chains and peridinins could shift for any individual apoprotein if only substitutions from a single clone were introduced. Rendering of new tertiary structures using more sophisticated software could also improve accuracy of their physical arrangement. 50 CONCLUSION Several points regarding the PCP gene family of Symbiodinium 203 have been established in this chapter. Symbiodinium 203 has long PCP genes organized like those of A. carterae and L. polyedra, but with a putative promoter that is different from L. polyedra. There are at least 14 distinct coding regions out of 36 12 PCP genes in this family. Diversity of Symbiodinium 203's PCP gene family appears to be consequence of low levels of concerted evolution and acts as a primary source of variability in PCP isoforms. Amino acid substitutions in Symbiodinium 203's PCP apoproteins result in shifts of isoelectric points and probably influence light harvesting of holoproteins. Heterogeneity in dinoflagellate PCP gene families may provide a selective advantage as means of broadening the range of wavelengths of light that can be captured for photosynthesis. 51 CHAPTER TWO Analyses of PCP genes and predicted proteins from S. pilosum, Symbiodinium sp. from Dichocoenia stokesii, S. pulchrorum, and S. kawagutii: Diverse PCP gene families occur in all major clades of Symbiodinium and in both size classes of the gene ABSTRACT Multiple PCP coding regions were cloned and sequenced from S. pilosum (ls rDNA clade A), Symbiodinium sp. from D. stokesii (clade B), S. pulchrorum (clade B) and S. kawagutii (clade C). S. pilosum has small PCP genes while the other species in this study have the large variety. In each case, the pattern of PCP gene diversity was similar to that reported for Symbiodinium 203 in Chapter One. These results demonstrate that diverse PCP gene families are a common feature of Symbiodinium species dinoflagellates, and that both size classes of PCP genes appear to be evolving under low levels of concerted evolution. PCP pIs predicted from S. pilosum and S. kawagutii complete coding sequences match data previously published from analyses of their apoproteins. This reinforces a conclusion from Chapter One that genetic diversity is the primary source of PCP isoform variation. Both S. pilosum and S. kawagutii PCP apoproteins contain polymorphic sites that are likely to influence the spectral tuning of peridinins. INTRODUCTION Chapter One established the structure, organization and size of the PCP gene family of Symbiodinium 203. This gene family was shown to be highly 52 diversified and capable of expressing distinct 33 kDa apoproteins with a range of pIs comparable to those previously reported for several other species of Symbiodinium. These results agree with previous reports that PCP gene families are probably diversified (Chang and Trench, 1984; Triplett et al. 1993; Sharples et al. 1996; Hiller et al. 2001) rather than highly conserved (Le et al. 1997). Furthermore, they support the hypothesis that genetic diversity, rather than posttranslational modification, is the primary source of PCP isoform variation. The results from Chapter One also revealed possible functional significance of isoform variation by demonstrating that substitutions of amino acids with polar side chain may effect spectral tuning of peridinins within PCP holoproteins (see Hofmann et al. 1996). By expressing multiple isoforms each absorbing light at subtly different maxima, dinoflagellates effectively harvest light from a broader range of light wavelengths than they would otherwise do with only a single PCP isoform. It is unclear if the diversity at the protein level is adaptive. It is also unknown if it being maintained by positive selection or if it is simply a tolerable byproduct of genetic polymorphism. Groundwork on Symbiodinium 203 provided the basis for the study of PCP genes and their predicted proteins in other Symbiodinium species. PCP gene sequences described here for four additional species will then be combined with Symbiodinium 203 sequences and previously published data for a comparative analysis of the evolution of PCP genes in Chapter Three. The current investigation builds on the detailed characterization of Symbiodinium 203 PCP genes to specifically address the following questions about PCP genes in other 53 symbiotic dinoflagellates. Do Symbiodinium species from each of the major ribosomal clades have diversified PCP gene families? Does this diversification extend to small and large PCP genes? Do predicted PCP pIs for Symbiodinium species match results of isoelectric focusing experiments on apoproteins from those same organisms (see Trench and Blank, 1987)? Do the predicted PCP apoproteins of other Symbiodinium species besides 203 also contain polymorphic sites that could effect the spectral tuning of peridinins? To answer these questions, analyses of PCP genes and predicted proteins from S. pilosum, Symbiodinium sp. from D. stokesii, S. pulchrorum, and S. kawagutii are presented here. This group of symbiotic dinoflagellates was selected because it contains representatives from ls rDNA clades A, B, and C with either large or small PCP genes, and species for which the actual isoelectric forms were already known. MATERIALS AND METHODS Algal Cultures The four species of Symbiodinium used in this investigation are described in Table 2.1. Robert K. Trench (University of California, Santa Barbara) donated three starter cultures of S. pilosum, S. pulchrorum and S. kawagutii. These are endosymbionts from a Hawaiian stony coral, a Hawaiian anemone, and a Jamaican zoanthid respectively. I isolated Symbiodinium sp. from the colonies of the stony coral Dichocoenia stokesii collected at Lee Stocking Island, Bahamas. My cultures 28, 54 32 and 35 were established from separate D. stokesii colonies located at depths of 2 m, 15 m and 34 m. Algal cells were extracted by modification of standard methods. A WaterPic was used to remove tissue from coral skeletons. Slurries were filtered through sterile gauze to remove large debris and algal cells were pelleted by centrifugation. The cells were resuspended in distilled water to lyse ciliate contaminates, pelleted again and resuspended in 10 ml of Provasoli s Enriched Seawater plus antibiotics. Cultures were grown for two days under broad-spectrum fluorescent lights on a 12:12 light to dark cycle at 27 C. Motile cells from these cultures were plated on to agar plates containing PES and antibiotics and were kept at the same lighting and temperature conditions. Algal colonies were picked from these plates after two weeks and used to establish unialgal cultures in 10 ml of 0.45 m filtered Gulliard s F/2. Isolates 28, 32 and 35 were indistinguishable from each other in terms of behavior in culture, external morphology and ls rDNA sequence (data not shown). They are presumably the same dinoflagellate species. All Symbiodinium cultures were subsequently maintained under conditions described above and were serially transferred every 3-4 weeks in 10 ml volumes of filtered Gulliard s F/2. Table 2.1 Algal cultures Symbiodinium Culture S. pilosum Symbiodinium sp. S. pulchrorum S. kawgutii Host Zoanthus sociatus (Zoantharia) Dichocoenia stokesii (Scleractinaria) Aiptasia pulchella (Actinaria) Montipora verrucosa (Scleractinaria) Isolate ls rDNA clade PCP apoprotein size RK Trench #185 A 17kDa JR Reichman #28, 32& 35 B undescribed RK Trench #8 B 35 kDa RK Trench #135 C 35 kDa 55 DNA Extractions Genomic DNA was extracted from pelleted log growth phase algal cells using the modification to the Qiagen DNAeasy kit protocol described in Chapter One in which steps one and two were replaced with CTAB resuspension and grinding followed by proteinase K digestion. Extraction yields were verified on ethidium bromide stained agarose gels. Genomic DNA was resuspended in Qiagen EB (0.01 M Tris buffer without EDTA). PCR Primers and Conditions The primers used to amplify and sequence PCP genes from each of the four Symbiodinium species are listed in Table 2.2. The sPCP-F1/sPCP-R3 primer sequences were provided by V.M. Weis (Weis et al. 2002). The U(448)/L(423) and U(-28)/L(1180) primers set were used in Chapter One on Symbiodinium 203. PCR reactions mixes contained the same components in the same concentrations used to amplify PCP genes from Symbiodinium 203 in Chapter One. Other primers were designed with Oligo 4.0 (National Biosciences, Inc.) based preliminary PCP gene sequences of the species in this study. Figure 2.1 shows relative primer locations. 56 Table 2.2 PCR and sequencing primers Upper/forward and Lower/reverse primers are listed sequentially in the pairs that were most often used for amplifications. Numbers in parentheses represent arbitriary location within contigs. sPCP-F1 and sPCP-R3 primers were originally designed for S. muscatinei (Weis et al.,2002). Upper Primers Lower Primers sPCP-F1 sPCP-R3 U(112) L(36) U(758) L(189) U(448) L(423) U(182) L(1315) U(-28) L(1180) Primer Sequence (5' - 3') CGCTTCAAAGACCATGGATG TAACGCTGGGATGCTTTGAC AAGGTTAAACTTGGCGAACG AGCTTACAAGGCACTCATGG GGCTTGAGGTCAGACACATCCAGGC GCTTGCCGCACACGTTTAGAAAATG TCGGTCCCCAAAGCAAAGGTCA CATTCACGGCATCCCAGTCAGC GGCATGATGTGGCTAGACGA TGTATGCAATTAAAAAAGTATG TCCGGCCCACTTTTAGTTTT TTTTCCCATTTGTTTCAGAG Genomic DNA Applied to S. pilosum " " " " " S. sp. & S. pulchrorum " " " S. kawagutii " Cloning & Screening Plasmid Libraries PCR products of interest were gel excised, purified with QIAEX II kits (Qiagen, Inc.) and cloned into pCR2.1 vectors with TA Cloning kits (Invitrogen, Inc.). Blue/white screens were performed on transformant INV F bacterial colonies. PCR screens of all plasmid libraries were done using the same protocol described on Chapter 1. Positive PCP gene clones were verified by separate amplification and sequencing of inserts. 57 Figure 2.1 Primer map; (a) S. pilosum amplifications sequentially consisted of a 3' segment (sPCP-F1/sPCP-R3), the 3' and 5' region from 2 adjacent coding genes (U(112)/L(36)) and the entire cds (U(758)/L(l89); (b) For Symbiodinium sp. from D. stokesii and S. pulchrorum, outward facing primers (U(448)/L(423)) amplified between adjacent genes that lead to inward facing primers (U(182)/L(1315)) producing the majority of the cds; (c) Complete S. kawagutii cds were amplified with Symbiodinium 203 primers (U(-28)/L(1180)). 58 Sequencing & Sequence Analysis The nucleotide sequences for all clones were determined by automated sequencing with BigDye V2 Terminator mix (Applera, Inc.) using the same half reaction volumes described in Chapter One. Sequence data were collected on either Perkin Elmer ABI PRISM 377 or 3100 DNA Sequencers. Sequence contigs were assembled with Seqman (DNA Star, Inc). All sequences were verified by sequencing twice and in most cases by sequencing in both directions. Sequences of clones were submitted to GenBank (accession numbers listed in Table 2.3). Translation of predicted proteins and pI estimation with done with Edit Seq (DNA Star Inc.). Amino Acid Substitution Modeling The predicted 16 kDa PCP apoprotein structure for S. pilosum was rendered by submitting the mature polypeptide sequence for a single clone to the Swiss Model server (Peitsch and Guex, 1997) with coordinates of the A. carterae 1PPR (monomer M) C-terminate domain as the template. The structure was imported into Swiss-PDB Viewer v3.7(b2) (Glaxo Wellcome, Inc.) where chromophores were added and merged into a single pdb layer. Polymorphic sites were mapped and distances from polar side chain of amino acids in polymorphic sites were measured in relation to furanic rings and polyene chains of the nearest peridinin molecules as was previously done with Symbiodinium 203. 59 The S. kawagutii 35 kDa predicted apoprotein was rendered using the methods applied to Symbiodinium 203 in Chapter One. Likewise, mapped polymorphic sites of S. kawagutii PCPs were converted to a single layer pdb file containing the substitutions prior to rendering graphics. For both S. pilosum and S. kawagutii 3D protein structure graphics were rendered using POV- Ray v3.1 (POV-Ray Team). Table 2.3 PCP clone accession numbers for S. pilosum, Symbiodinium sp. from D. stokesii, S. pulchrorum and S. kawagutii. Species Clone Name S. pilosum 185clone23 " 185clone25 " 185clone28 " 185clone30 " 185clone32 " 185clone33 " 185clone34 " 185clone35 Symbiodinium sp . from D. stokesii 28clone1 " 28clone2 " 28clone5 " 32clone1 " 35clone3 " 35clone42 " 35clone43 " 35clone44 " 35clone46 " 35clone47 " 35clone49 S. pulchrorum 8clone1 " 8clone2 " 8clone3 " 8clone4 S. kawgutii 135clone37 " 135clone38 " 135clone41 " 135clone42 " 135clone43 " 135clone49 " 135clone50 " 135clone52 " 135clone54 " 135clone60 Accession Number AY149140 AY149141 AY149142 AY149143 AY149144 AY149145 AY149146 AY149147 AY149148 AY149149 AY149150 AY149151 AY149152 AY149153 AY149154 AY149155 AY149156 AY149157 AY149158 AY149159 AY149160 AY149161 AY149162 AY149163 AY149164 AY149165 AY149166 AY149167 AY149168 AY149169 AY149170 AY149171 AY149172 60 RESULTS Multiple PCP coding regions cloned from each Symbiodinium species Primers that were previously designed for Symbiodinium 203 did not amplify PCP genes from S. pilosum. This is not surprising and is attributable to two things. First, S. pilosum has small PCP genes unlike Symbiodinium 203. Hiller et al. (2001) and Weis et al. (2002) demonstrated that small PCPs were most homologous to the C-terminal end of the large PCPs from A. carterae, L. polyedra and Symbiodinium sp. from A. formosa. The same homology applies to small and large PCP genes nucleotide sequences. The outward facing U(448)/L(423) primer set is nested in the N-terminal end of the Symbiodinium 203 coding region, and is not compatible with S. pilosum. Second, the Symbiodinium 203 U(-28)/L(1180) primers were designed to flank the coding region in positions just upstream and downstream in untranscribed spacers. Spacers evolve more rapidly than coding regions and may be highly divergent from species to species. While these primers were not useful on S. pilosum, the general cloning strategy used for Symbiodinium 203 was successfully applied here again. S. pilosum PCP coding regions were cloned by initially amplifying a small 3 segment using the sPCP-F1/sPCP-R3 primer set designed by VM Weis. The sequences from two clones of this region (data not shown) were used to design outward facing primers U(112)/L(36). This set amplified fragments approximately 1.2 kb in length between adjacent PCP coding regions containing roughly 500 bp spacers sequences and demonstrated the size of the S. pilosum 61 small PCP gene cassette. Sequences from four of these clones (data not shown) were used to design inward facing primers U(758)/L(189) that amplified entire coding regions. As with Symbiodinium 203, multiple coding regions were cloned and sequenced. After screening 20 complete cds clones, 8 unique sequences (clones 23,25,28,30 and 32-35) were found. The nucleotide sequences for these clones are aligned in Appendix 2.1. Comparison of the unique S. pilosum PCP gene clones to other sequences in GenBank via BLAST search indicated that they were most similar to the S. muscatinei PCP gene (AF425735). While S. pilosum small PCP genes are roughly half the size of the 1098 bp versions found in Symbiodinium 203, S. pilosum PCP genes contain proportionally fewer polymorphic sites. Across the 615 bp cds there were 36 polymorphic sites compared to 83 sites in Symbiodinium 203. The nucleotide sequences of 8 unique S. pilosum coding regions also shared from 96.6% to 99.7% identity with each other. The U(448)/L(423) primers did amplify 2.3 kb fragments between adjacent PCP coding regions from both Symbiodinium sp. from D. stokesii and S. pulchrorum, compared to 1.9 kb in Symbiodinium 203. Govind et al. (1990) reported that S. pulchrorum expressed 35 kDa PCP apoproteins, so it is reasonable to assume that that it's coding regions are approximately the same size (1.1 kb) as other large PCP genes including those from Symbiodinium 203. Both clade B species in this study had larger untranscribed spacers (1.2kb) than either Symbiodinium 203 or S. pilosum. As before, the objective of beginning with outward facing primers was to identify the 5 and 3 ends of the genes and to 62 design flanking inward facing primers that would amplify entire coding regions. Although a functional primer downstream of the stop codon was designed, L(1315), none of the primers intended to anneal upstream of the start codon worked reliably for amplifying and sequencing. These difficulties appear to be the result of false priming sites and formation of primer dimers. The U(182)/L(1315) combination amplified the majority of the coding regions from Symbiodinium sp. from D. stokesii and was the source of 28clone5 through 35clone49. Symbiodinium sp. 28clones1&2 and all S. pulchrorum clones were amplified with U(448)/L(1315). Eleven unique partial PCP gene fragments were cloned from Symbiodinium sp. from D. stokesii and four from S. pulchrorum. An alignment of these nucleotide sequences is presented in Appendix 2.2. Because each of the clones from clade B species contained partial cds, the total number of polymorphic sites could not be determined. Nevertheless, the same pattern of genetic diversity found in the PCP gene families of Symbiodinium 203 and S. pilosum was clearly present in Symbiodinium sp. from D. stokesii and S. pulchrorum with eleven and four distinct sequences respectively. Complete coding regions from S. kawagutii were amplified with little difficulty using the Symbiodinium 203 flanking primers U(-28)/L(1180). S. kawagutii is a large subunit rDNA clade C species and is more closely related to Symbiodinium 203 than the other species in this report. Of the 20 clones screened, 10 unique sequences were detected, 135clones 37, 38, 41, 42, 43, 49, 50, 52, 54 and 60. An alignment of these nucleotide sequences is present in Appendix 2.3. Nine coding regions were 1098 bp long as were those from Symbiodinium 203 63 and Symbiodinium sp from A. formosa (Norris and Miller, 1994; accession L13615). The coding region in 135clone42 was 6 bp longer. Minor differences in the lengths of PCP coding regions within the same species have been previously been noted for A. carterae (Sharples et al. 1996) and for Symbiodinium 203 in Chapter One. Although generally the same size as Symbiodinium 203 PCP genes, the S. kawagutii genes were substantially more polymorphic with 138 variable nucleotide sites. The coding regions shared from 95.7% to 98.4% identity with each other. Predicted amino acid sequences, isoelectric focusing points and protein modeling for S. pilosum PCPs The predicted PCP amino acid sequences for S. pilosum are shown in as an alignment in Appendix 2.4. The small PCPs from S. pilosum are structured essentially like those of S. muscatinei and H. pygmaea. Each S. pilosum clone codes for a 21 kDa PCP preprotein consisting of a 54 aa transit peptide plus a 150 aa 16 kDa mature apoprotein. The S. pilosum transit peptides are 6 aa longer than those identified for the S. muscatinei PCP (Weis et al. 2002) and 3 aa longer than those from H. pygmaea (Hiller et al., 2001), yet the transit peptides from all three species share common elements. Each contains an N-terminal positively charged sequence followed by two hydrophobic domains that are separated by a another positively charged residue containing the general motif (F/H)V(+/-P)GP(+/-G)R. In S. pilosum, this region is conserved as FVPGPGR. The transit peptides all end at a predicted cleavage point with the motif A(Y/H)A. muscatinei end with AYA while H. pygmaea ends with AHA. S. pilosum and S. 64 S. pilosum, like S. muscatinei and H. pygmaea, produces 150 aa apoproteins that show sequence homology with the C-terminal domains of the large PCP apoproteins in GenBank. All apoproteins had isoelectric focusing points in the basic range from pH 7.28 to 8.77 (see Table 2.4). The range and specific pIs values calculated here correspond with data previously published by Trench and Blank (1987) on S. pilosum PCPs (Figure 2.2). This strongly supports the hypothesis that genetic diversity among PCP genes contributes to the generation of PCP isoforms. Table 2.4 Calculated mass and isoelectric points from S. pilosum apoproteins S. pilosum clone 23 25 28 30 31 33 34 35 Apoprotein mass (kDa) 16.056 16.01 16.056 16.075 16.056 16.066 16.07 16 Isoelectric point (pI) 7.28 8.77 7.28 8.27 7.28 7.28 7.28 7.28 65 Figure 2.2 Isoelectric focusing patterns from S. pilosum. The red double line indicates the range of calculated pIs for S. pilosum apoproteins, and the red dots show matches between the calculated and actual pIs (After figure 1d, Trench and Blank, 1987). 66 As with the apoproteins of Symbiodinium 203 in Chapter One, the differences in pI for S. pilosum PCPs are the direct result of amino acid substitution at polymorphic sites. The predicted apoproteins from S. pilosum are polymorphic at nine sites. Three of these sites (positions 33, 100, and 113) accommodate the presence or absence of amino acids with polar side chains (Table 2.5). As with the nucleotide sequences, amino acid polymorphism among predicted S. pilosum PCPs was proportionally less than in Symbiodinium 203. Table 2.5 Substitutions within polymorphic sites of predicted S. pilosum PCP apoproteins. Bold signifies polarity polymorphic sites. Site # 11 23 29 33 58 Substitutions Lys Pro Trp Arg Ala Gly Thr Ala Leu Pro Side Chain basic nonpolar nonpolar basic nonpolar nonpolar uncharged polar nonpolar nonpolar nonpolar Site # 92 100 113 145 Substitutions Side Chain His basic Arg basic Ser uncharged polar Pro basic Asn uncharged polar Lys basic Val nonpolar Ile nonpolar In Chapter One, substitutions at polarity polymorphic sites of Symbiodinium 203 PCPs were shown to potentially influence the spectral tuning of peridinins. This was accomplished by mapping the amino acids substitutions into the A. carterae 1PPR crystal structure and measuring the distance between polar side chains and specific regions of peridinins. However, mapping S. pilosum PCP substitutions is more complicated because no crystal structure for a 67 small PCP is currently available. To get a reasonable approximation of S. pilosum PCP conformation, the C-terminal half of 1PPR was used as a template and a structure was generated through the Swiss-Prot server as described by Hiller et al. (2001). Previous characterization of the 16 kDa PCPs from S. pilosum by Govind et al. (1990) indicated that they existed only as monomers and not as hexadimers suggested by Hiller et al. (2001) for H. pygmaea PCPs. The first approximation monomer is shown in Figure 2.3a. After refining the structure by the addition of chromophores and rotomer optimization (Figure 2.3b), amino acid substitutions were mapped and the locations of the 3 polarity polymorphic sites were determined in relation to four peridinins (Figure 2.4). Figure 2.3 S. pilosum 16 kDa PCP monomer; (a) first approximation apoprotein; (b) refined holoprotein. 68 Figure 2.4 Substitutions of amino acids with polar side chains occurring at polymorphic sites within S. pilosum predicted PCP apoproteins shown in relation to peridinins. 69 Figure 2.5 Distances (E= ) from the polar side chains of threonine 33 and asparagine 133 to the furanic ring and polyene chain of peridinin 622. Predicated amino acid sequences, isoelectric focusing points and protein modeling for S. kawagutii PCPs Ten unique S. kawagutii PCP preproteins sequences are aligned in Appendix 2.5. All polypeptides but that predicted from a single clone are 365 aa long and like the majority from Symbiodinium 203 contain a 52 aa 12 kDa transit peptide followed by a 313 aa 33 kDa mature apoprotein. The apoprotein from 135clone42 differs slightly in that it is 2 aa longer on the C-terminal end. 70 The transit polypeptides from S. kawagutii PCPs are arranged like those from Symbiodinium 203 and Symbiodinium sp. from A. formosa (Norris and Miller, 1994). The positively charged central domain has a F(V/A)PGPR motif while the cleavage site is denoted by an AFA sequence. The apoproteins from S. kawagutii were most similar in terms of amino acid sequence to those described in Chapter One for Symbiodinium 203, but the calculated isoelectric focusing points for S. kawagutii PCPs were not restricted to solely acid or basic forms. To the contrary, they span from pH 5.5 to 8.38 (see Table 2.6). Table 2.6 Calculated mass and isoelectric points for S. kawagutii apoproteins 135 clone 37 38 41 42 43 49 50 52 54 60 Apoprotein mass (kDa) 32.9378 33.012 32.8408 33.057 32.1997 33.0351 32.9777 32.994 33.0941 33.0158 Isoelectric point (pI) 6.04 7.41 8.1 7.46 5.73 7.97 5.5 8.38 8.11 6.51 Just as there is correlation between predicted and previously measured pIs for S. pilosum apoproteins, the same is true for S. kawagutii. Figure 2.6 shows that the range of S. kawagutii PCP pIs encompasses many of the isoforms reported by Trench and Blank (1987), and that there are six matches between the data sets. 71 This relationship reinforces the idea that genetic diversity of PCP gene families is the underlying source of isoform generation. Figure 2.6 Isoelectric focusing patterns from S. kawagutii. The red double line indicates the range of calculated pIs for S. kawagutii apoproteins, and the red dots show matches between the calculated and actual pIs (After figure 1c, Trench and Blank, 1987). 72 The S. kawagutii PCPs are significantly more diversified than the PCPs from either Symbiodinium 203 or S. pilosum. Among the S. kawagutii clones, there are 9 polymorphic sites in the transit peptides and 55 in the apoproteins. Of the differences in the apoproteins, 22 sites (positions 15, 16, 24, 36, 70, 89, 130, 150, 154, 155, 162, 169, 178, 199, 215, 235, 238, 239, 280, 287 and 288) are predicted to accommodate either the presence or absence of amino acids with polar side chains. There are an additional four polymorphic sites that hold various polar amino acids. Specific amino acid substitutions and types of side chains are listed in Table 2.7. Mapping substitutions into the A. carterae 1PPR crystal structure revealed that there were 94 out of 313 sites at which the S. kawagutii and A. carterae PCPs differed. Beyond the 55 polymorphic sites, there were 39 fixed changes present in each S. kawagutii apoprotein. A composite structure containing all of the polar substitutions within polymorphic sites was used to show the positions of these amino acids in relation to the 8 peridinins present in large PCP (see Figure 2.7). As with protein modeling presented for Symbiodinium 203 and S. pilosum, polar side chains within 10 of furanic ring and polyene chains of peridinins are considered likely to affect the specific wavelength of blue-green light harvested by the accessory pigment (see Hofmann et al. 1996). Eight amino acids predicted from S. kawagutii PCP apoproteins (positions 70, 89, 150, 169, 178, 280, 287 and 288) meet this proximity criterion and are rendered in Figures 2.8-10. 73 Table 2.7 Substitutions within polymorphic sites of predicted S. kawagutii PCP apoproteins. Sites in bold accommodate the presence or absence of amino acids with polar side chains. Site # 9 10 15 16 24 Substitutions Lys Arg Lys Pro Tyr Phe Ser Pro Asn Asp Tyr Phe Leu Gln His Glu Lys Val Met Lys Met Lys Arg Val Ala Gly Thr Ile Val Phe Ser Thr Trp Cys Asn Asp Ala Glu Val Ala Side Chain basic basic basic nonpolar uncharged polar nonpolar uncharged polar nonpolar uncharged polar acidic uncharged polar nonpolar nonpolar uncharged polar basic acidic basic nonpolar nonpolar basic basic basic basic nonpolar nonpolar nonpolar uncharged polar nonpolar nonpolar nonpolar uncharged polar uncharged polar nonpolar nonpolar uncharged polar acidic nonpolar acidic nonpolar nonpolar Site # 118 130 138 142 146 150 152 153 154 155 160 162 166 168 Substitutions Ala Lys Pro Ser Ala Gly Phe Ile Val Asp Gln His Thr Ser Thr Ser Ala Thr Arg Ser Val Ala Ser Phe Ile Val Glu Gly Val Ala Ser Ala Val Tyr Phe Asp His Thr Pro Side Chain nonpolar basic nonpolar uncharged polar nonpolar nonpolar nonpolar nonpolar nonpolar acidic uncharged polar basic uncharged polar uncharged polar uncharged polar uncharged polar nonpolar uncharged polar basic uncharged polar nonpolar nonpolar uncharged polar nonpolar nonpolar nonpolar acidic nonpolar nonpolar nonpolar uncharged polar nonpolar nonpolar uncharged polar nonpolar acidic basic uncharged polar nonpolar Site # 202 Substitutions Glu Gln Ala Ile Val Ala Ser Val Ala Phe Ile Ser Cys Ala Thr Lys Asn Asp His Gly Lys Ile Val Thr Pro Ser Pro Lys Lys Asn Ser Asn Met Ile Val Lys Arg Ala Gly Side Chain acidic uncharged polar nonpolar nonpolar nonpolar nonpolar uncharged polar nonpolar nonpolar nonpolar nonpolar uncharged polar nonpolar nonpolar uncharged polar basic uncharged polar acidic basic nonpolar basic nonpolar nonpolar uncharged polar nonpolar uncharged polar nonpolar basic basic uncharged polar uncharged polar uncharged polar nonpolar nonpolar nonpolar basic basic nonpolar nonpolar 211 215 226 233 235 238 239 246 275 276 280 287 28 36 39 50 57 61 64 70 288 290 295 169 170 178 185 199 81 85 89 102 111 305 310 74 Figure 2.7 Twenty two polar amino acids occurring in polymorphic sites of S. kawagutii predicted PCP apoproteins located relative to eight peridinin chromophores. 75 Figure 2.8 Distances (E= ) from the polar side chains of threonine 70, asparagine 89 and glutamine 150 to the furanic ring and polyene chain of peridinin 614. Figure 2.9 Distances (E= ) from the polar side chains of serine 169 and tyrosine 178 to the furanic ring and polyene chain of peridinin 624. 76 Figure 2.10 Distances (E= ) from the polar side chains of threonine 280, serine 287 and asparagine 288 to the furanic ring and polyene chain of peridinins 621 and 622. 77 DISCUSSION Diverse PCP gene families found in Symbiodinium sp. from each major rDNA clade Evidence of genetic variability within individual dinoflagellate PCP gene families began to emerge in 1993 when Triplett et al. cloned 2 distinct small PCP transcripts from H. pygmaea. Despite reading frame problems with their data that were noticed after publication, their general multi-gene premise was correct. Sharples et al. (1996) reported two versions of large PCPs in A. carterae after cloning transcripts and genomic coding regions. Le et al. (1997) concluded that the large PCP gene family of L. (Gonyaulax) polyedra was highly conserved as judged by Southern hybridization, yet they acknowledged the possibility that differences could exist between individual gene copies. More recently, Hiller et al. (2001) indicated that up to five different PCP genes were found in H. pygmaea. The only previous data on PCP genes from Symbiodinium prior to Chapter One were single sequence reports (see Norris and Miller, 1994; Weis et al. 2002). The results from Chapter One demonstrated several important points regarding PCP genes. This was the first detailed characterization of a PCP gene family from a Symbiodinium species. The results showed that the PCP gene family in Symbiodinium 203 is highly diversified, is apparently subjected to little or no homogenization by concerted evolution, contains sufficient nonsynonymous substitutions to code for multiple PCP isoforms, and that its predicted apoproteins have polymorphic sites that could affect the tuning of peridinins. 78 One of the objectives of the present report is to determine if the diversity of Symbiodinium 203 PCP gene family is common to other Symbiodinium species or if Symbiodinium 203 is anomalous. Multiple PCP coding regions were found for each of the additional species tested here (S. pilosum, Symbiodinium sp. from D. stokesii, S. pulchrorum, and S. kawagutii). Together with Symbiodinium 203 there are now examples of diversified PCP gene families from species in each major Symbiodinium rDNA clade; 1 from A, 2 from B and 2 from C. This set of taxa includes endosymbionts from various Pacific and Caribbean invertebrate hosts, and it is reasonable to assume that the pattern of genetic diversity may extend to all Symbiodinium species. Both small and large PCP genes from Symbiodinium occur in diversified families Beyond showing that diversified PCP gene families are found in species from Symbiodinium clades A, B, and C, the present results also demonstrate that both size classes of PCP genes are diversified as well. This agrees with previous reports of multiple small and large PCP coding regions from H. pygmaea and A. carterae respectively (Triplett et al, 1993; Sharples et al., 1996; and Hiller et al., 2001). This widespread diversity should be considered within the context of dinoflagellate and PCP gene evolution. Free-living dinoflagellates are generally considered to be ancestral to symbiotic species. In addition, the axis of symmetry in large PCP genes and the nucleotide sequence homology between small and large PCP genes (Norris and Miller, 1994; Hiller et al., 2001) support the 79 hypothesis that large PCP genes evolved from the duplication and fusion of small PCP genes. An implication of these points taken together is that PCP gene polymorphism is probably ancient. This characteristic of PCP genes may predate the divergence of Symbiodinium from free-living dinoflagellates and the evolution of species with large genes from those with small genes. However, it is also possible that evolutionary history of PCP genes is more complicated. Large PCP genes and endosymbioses may both have evolved more than once. It is also possible that certain small PCP genes have been formed via truncation of large genes. The observable differences in level of diversification between species suggest that various factors affecting PCP gene diversification (selection, drift, etc.) are still at work. Although polymorphism among PCP genes has probably existed for millions of years, it is not clear if it offers a selective advantage and is being maintained. In general, PCP gene families appear to have evolved under little to no concerted evolution, unlike dinoflagellates ribosomal gene families that are more uniform and easily sequenced directly from PCR amplifications (see Rowan and Powers, 1991a & b; 1992, Rowan and Knowlton, 1995; Rowan et al. 1997; Wilcox, 1998). Calculated pIs from predicted PCP apoproteins match measured values From some of the earliest characterizations of PCPs, it was noted that these dinoflagellate light harvesting complexes were normally expressed in multiple isoforms distinguished by their isoelectric focusing points. While it was recognized that genetic diversity might be the source of the isoforms, preliminary 80 experimental results pointed to post-translational modification instead (see Haxo et al. 1975; Haxo et al. 1976; Seigelman et al. 1977). Trench and Chang (1984) suggested to the contrary, that the genetics were likely responsible. It was not until the tools of molecular biology were applied to the cloning and sequencing of PCP transcripts and genomic coding regions that evidence supporting the genetic hypothesis was produced from experiments on free-living dinoflagellates (Triplett et al. 1993; Sharples et al. 1996; Hiller et al. 2001). The predicted isoelectric focusing points for S. pilosum and S. kawagutii presented in this chapter reasonably match the Trench and Blank (1987) data on the actual apoproteins for these species. For both species, the range of predicted pIs overlap with the majority of dominant PCP isoforms expressed. However, the fit is not perfect in either case. This may be attributable to the fact that the searches for unique coding regions were not exhaustive. Each species may contain many more versions of PCP genes than were detected here. While results from this and the previous chapter substantiate that genetic diversity is common to PCP gene families in Symbiodinium, post-translational modification may also be contributing to isoform diversity. It now seems very unlikely that dinoflagellates only express a single PCP preprotein that is then converted to multiple isoforms. Polarity polymorphic sites of PCP apoproteins likely to influence tuning of peridinins Not all PCPs capture exactly the same wavelengths of light, but they generally overlap. Different dinoflagellates species can contain PCPs that have unique spectroscopic absorbance maxima (Prezelin and Haxo, 1976; Song et al. 81 1976), and individual PCP isoforms within a single species can have different absorption and fluorescence maxima (Iglesias-Prieto et al. 1991). The protein modeling results from Symbiodinium 203 (Chapter One), S. pilosum and S. kawagutii provide the likely structural basis for these observations. The predicted PCP apoproteins for all three species contain polarity polymorphic sites that may house amino acids with or without polar side chains. In each case there is a subset of these polymorphic sites that have side chains within 10 of furanic rings and polyene chains of peridinins potentially affecting spectral tuning. Although, a consistent signature of polarity polymorphic sites is present in each group of predicted PCP apoproteins, there is variation between species in terms of the relative proportion of these sites (Symbiodinium 203, 3%; S. pilosum, 2%; and S. kawagutii, 7%) and their specific locations. Symbiodinium 203 and S. kawagutii have three polarity polymorphic sites in common and only one (position 287) is close to a peridinin in both cases. Neither species has sites of this nature that correspond to those in S. pilosum. Despite the apparent functional significance of presence or absence of polar residues in areas that differentially tune the peridinins of PCPs, it is entirely possible that the substitutions within PCP genes occur at random locations and are only weakly filtered out by gene conversion. Depending on their host association and dominant habitat, different species of dinoflagellates may be able to tolerate more or less variability of PCP light harvesting characteristics that are simply the by products of genetic polymorphism. On the other hand there may be regions of PCPs that are under selection that are not yet revealed by the methods applied here or there may be 82 purifying selection for a range spectral absorption maxima. Phylogenetic analyses of the now expanded PCP gene sequences available will determine if the nucleotide substitutions are non-random and if they are, then sites under selection could also potentially be detected. CONCLUSION The first PCP coding sequences from S. pilosum, Symbiodinium sp. from D. stokesii, S. pulchrorum and S. kawagutii were presented in this chapter. Multiple coding sequences were cloned from each species. I established that diverse PCP gene families occur in all major clades of Symbiodinium and in both size classes of the gene. As with Symbiodinium 203 in Chapter One, these PCP gene families do not appear to have been homogenized through concerted evolution. The predicted PCP apoproteins from S. pilosum and S. kawagutii have calculated isoelectric focusing points that generally match values previously measured for these species, which supports the hypothesis that genetic polymorphism is the primary source generating differences in PCP isoforms, however, post-translational modification cannot be ruled out as another possible mechanism. Protein modeling based on A. carterae PCP produced a putative tertiary structure for S. pilosum apoproteins and was used to identify polymorphic sites in S. pilosum and S. kawagutii PCPs that could affect spectral tuning of peridinins. Additional phylogenetic work is needed to determine if amino acids sites within PCP are under selection. 83 CHAPTER THREE Phylogenetic analyses and evolution of dinoflagellate PCP gene families: diversity families within is consistent across lineages, but individual sites are most likely to be under purifying rather than positive selection. ABSTRACT Photosynthetic dinoflagellates use multiple PCP isoforms to harvest bluegreen light from aquatic habitats. Genetic differences among copies in PCP gene families account for expression of many of these isoforms, but it is unclear if selective pressures are maintaining the polymorphism. This is the first phylogenetic study of the evolution of PCP gene families with the aim of measuring selection at the codon level. Nucleotide substitution model likelihood ratio tests (LRTs) on PCP coding sequences from seven symbiotic and three free living dinoflagellate species suggests that PCP gene sequence changes are most closely fit by the GTR+I+G model. Consensus and highest posterior probability trees from Bayesian analyses show that topologies of PCP gene and large subunit (ls) rDNA trees are generally similar. As expected, PCP sequence data indicate that symbiotic species evolved from free-living ancestors and that the small PCP gene state consistently appears to be ancestral to large PCP gene state. There is also not a clear correlation between PCP gene size class and ribosomal clade. However, there are inter-digitations between large PCP sequences from clade B 84 and C species. These results suggest that PCP gene polymorphism is ancient and preceded the evolution of large PCP genes by fusion of small genes, the divergence of the genus Symbiodinium from free-living dinoflagellates and speciation within Symbiodinium. They also suggest that small and large PCP genes may have previously been present or may still be present in species from each Symbiodinium clade. Several PAML CODONML models were tested on a single 17 sequence tree with the highest posterior probability. LRTs indicate that that data is best fit by Model M3 (discrete s). There was no indication of codons under positive selection. To the contrary, purifying selection is affecting PCP genes. It is likely that PCP polymorphism is adaptive and that there is purifying selection for isoforms within a range of spectral absorption maxima. INTRODUCTION The evolution of PCPs has been a prominent adaptation for photosynthetic dinoflagellates. Free-living and symbiotic dinoflagellates utilize their unique PCP light harvesting complexes to capture blue-green light with remarkable efficiency from aquatic habitats. Regardless of whether it has small or large PCP genes, an individual species will express a stable combination of apoprotein isoforms in an inversely proportional response to light intensity (Chang and Trench, 1982; 1984; Trench and Blank, 1987). The source of PCP isoform diversity has been a matter of speculation that was addressed in the previously sections of this dissertation. In Chapters One and Two, I established that diverse PCP genes are common to several Symbiodinium species and that genetic polymorphism can account for the 85 majority of isoform pI variation through amino acid substitutions. Some of these changes can potentially affect absorption maxima of chromophores within PCP complexes and provide a structural mechanism for subtle absorption maxima differences reported for isoforms from S. microadriaticum (Iglesias-Prieto et al. 1991). Despite the genetic explanation for these PCP properties, it is not clear that PCP isoform polymorphisms are adaptive to the present environments that dinoflagellates live in. If they are adaptive, then positive selection of sites within PCP genes may be maintaining the polymorphism, or there may be purifying selection for ranges of isoforms. Sequence data on PCP genes have been slowly accumulating for almost a decade. While one phylogenetic study has shown how soluble and membranebound PCPs are related to light harvesting complexes of other algae and higher plants (Durnford et al. 1999), no investigation to date has concentrated at the evolution of PCP genes between dinoflagellates species or among gene families. There are now examples of multi-version PCP gene families from two free-living species, H. pygmaea (Triplett et al. 1993; Hiller et al. 2001), and A. carterae (Sharples et al. 1996) and five symbiotic species, Symbiodinium 203, S. pilosum, Symbiodinium sp. from D. stokesii, S. pulchrorum and S. kawagutii (Chapter One and Two). There are additional single sequence characterizations from free- living L. polyedra and endosymbionts Symbiodinium from A. formosa and S. muscatinei. The total number of PCP nucleotide sequences available for phylogenetic analyses was increased over six fold by the results from Chapters One & Two. 86 Comparative methods need to be applied to this collection of sequence to answer the following basic questions regarding PCP evolution. Does the evolution of PCP genes between species simply reflect the divergence of species as indicated by ribosomal sequences or does the phylogenetic reconstruction of PCP gene evolution show a more complex pattern? Are there codon sites that are under positive selection ( (= dN/dS = Ka/Ks) >1)? If so, are they associated with spectral tuning of chromophores or some other domains? Are PCP genes subject to purifying selection ( < 1)? This chapter seeks to answer these questions through the use of combined Bayesian analysis and maximum likelihood (ML) approaches. MATERIALS AND METHODS DNA sequences and alignments Fifty eight PCP coding sequences from Genbank were used in this project, and their accession numbers are shown in Table 3.1. Sequences were aligned with MegaAlign (DNA Star, Inc.) and imported into Se-Al Sequence Alignment Editor v2.0 (Copyright 1996-2002, Andrew Rambaut). Within Se-Al, the nucleotide sequences were translated and manual adjustments were made to the amino acid alignment. The final alignment was back-translated (see Appendix 3.1) and exported as a Nexus file. 87 Table 3.1 Accession numbers of PCP gene sequences used for phylogenetic analyses Species A. carterae H. pygmaea L. polyedra Symbiodinium sp . from A. formosa Symbiodinium sp . from H. hippopus S. pilosum Symbiodinium sp . from D. stokesii S. pulchrorum S. kawgutii Accession Numbers AF298220, Z50792 & Z50793 AJ298192 & AJ298193 U93077 L13613 AY149123 - AY149139 AY149140 - AY 149147 AY149148 - AY149158 AY149159 - AY149162 AY149163 - AY149172 Nucleotide substitution model testing The Nexus file described above was executed in PAUP* 4.0 (Swofford, 1998). With the optimality criterion set to maximum parsimony, a heuristic search of 10,000 trees was conducted and the best tree was retained. Likelihood scores for this tree were successively generated under the JC69, K2P, HKY85, GTR, GTR+I and GTR+I+G series of nucleotide substitution models. The PAUP* setting were as follows: JC69 (one substitution type, nucleotide frequencies equal, no invariable sites and equal rates at variable sites), K2P (two substitution types, transition / transversion ratio estimated by ML, nucleotide frequencies equal, no invariable sites and equal rates at variable sites), KHY85 (two HKY85 variant substitution types, transition / transversion ratio estimated by ML, nucleotide frequencies estimated by ML, no invariable sites and equal rates at variable sites), 88 GTR (six substitution types, rate matrix estimated by ML, nucleotide frequencies estimated by ML, no invariable sites and equal rates at variable sites), GTR+I (six substitution types, rate matrix estimated by ML, nucleotide frequencies estimated by ML, proportion of invariable sites estimated and equal rates at variable sites) and GTR+I+G (six substitution types, rate matrix estimated by ML, nucleotide frequencies estimated by ML, proportion of invariable sites estimated, gamma distribution of variable sites with shape parameter estimated and four rate classes). Pairs of nested models (a model in the series vs. the next more general model) were compared using a standard LRT in which twice the difference of negative log likelihood was compared to a 2 distribution at the degrees of freedom resulting from the difference in the number of free parameters of the models under comparison. In each comparison, the more specific model represents the null hypothesis and the next more general model is the alternative. The "p" statistic reported is probability of observing the LRT result if the null hypothesis was true. Bayesian inference of PCP gene phylogeny The same Nexus file was executed in MrBayes 2.01 and 3.0 (Huelsenbeck and Ronquist, in press) with the parameters of the likelihood model setting (lset) corresponding to GTR+G+I. The prior probabilities settings (prset) for the rate maxima were adjusted to give a negative exponential curve (exp (.2)). Using a negative exponential curve more accurately parameterizes the substitution rate matrix (personal communication, Derrick Zwickl, University of Texas at Austin). 89 In two MrBayes runs, the Markov Chain Monte Carlo (MCMC) settings included 500,000 generations with trees sampled every 100 generations with 4 MCMC chains. The temperature of the three "heated chains" was left at the default (.2). To check for convergence an additional 4,000,000 generation run was executed with the same settings. For all runs, -ln likelihood scores from the output files were graphically post "burnin" to determine which trees should be excluded from the calculation of consensus trees. Trees that were retained were sorted by in order to identify the tree with highest posterior probability. An additional 4,000,000 generation MrBayes run (same settings and postrun analyses) was executed on a 17 PCP gene sequence subset of the original nexus. The subset nexus file was generated by removing sequences from the alignment within PAUP*. The subset contained a single sequence from each freeliving species, the two Symbiodinium sequences published prior to Chapter One and pairs of sequences from each of the species in Chapters One and Two that were most divergent in terms of their "p" distance as identified by PAUP*. This subset was generated to reduce to occurrence of near zero branch lengths in the highest posterior probability tree. Testing PAML CODONML models and detecting selection. The 17 sequence Nexus file and the corresponding tree with the highest posterior probability were loaded in to PAML 3.13 (Yang, 1997). The data was analyzed with six codon substitution models from CODONML; M0 (single estimated for all sites), M1 ( 0 = 0 and 1 = 1), M2 ( 0 = 0, 1 = 1 and 2 90 estimated from the data), M3 (three discrete s all of which could be >1), M7 (ten discrete s picked from gamma distribution) and M8 (ten discrete s picked from gamma distribution plus one estimated from the data that can be >1). LRTs were performed on the likelihood scores from nested pairs of models to determine which models best fit the data. The values and probabilities generated by the best fitting models were compared for indications of selective pressure on PCP genes and to check for sites under positive selection (see Yang et al. 2000; Yang and Bielawski, 2000). RESULTS Bayesian phylogenetic analysis of PCP genes with GTR+I+G Based on the likelihood ratios of six standards nucleotide substitution models against a parsimony tree of PCP gene sequences, each more general set of parameters had a significantly better fit than the previous model (Table 3.2). Consequently, GTR+I+G was specified as the likelihood model for all MrBayes runs. Table 3.2 Substitution model likelihood ratio test results for PCP genes Model JC69 K2P HKY85 GTR GTR+I GTR+I+G - 9526.02663 9417.92171 9392.31734 9375.89712 9224.37258 9144.89927 LRT JC69 vs K2P K2P vs HKY85 HKY85 vs GRT GTR vs GTR+I GTR+I vs GTR+I+G -2 216.20984 51.20874 32.84044 303.04908 158.94662 df 1 3 5 1 1 p 6.06941E-49 4.41592E-11 4.04818E-06 7.13659E-68 1.92222E-36 91 Post-run analyses indicated that there was overall convergence between the likelihood scores and consensus tree from the 500,000 and 4,000,000 generation MrBayes runs (data not shown). The longer run produced over 36,000 trees with approximately the same posterior probabilities. The consensus (Figure 3.1 colored by species) and highest posterior probability trees (Figure 3.2 colored by species and 3.3 colored by ribosomal clade) retained from this group both are shown below. Some broad aspects of the topology of the PCP trees were present as expected. The tree structures are in agreement with the widely held view that species with small PCP genes gave rise to those with large PCP genes. PCP sequence data also support the hypothesis that Symbiodinium species have evolved from free-living ancestors. H. pygmaea small PCP genes are more basal than either S. muscatinei or S. pilosum. A. carterae and L. polyedra PCP genes are more basal than all other large PCP genes from Symbiodinium species. In addition, high levels of similarity between individual copies of the same PCP gene family (see Chapters One and Two) resulted in lack of resolution and polytomies in the consensus tree. This is also reflected in the short branch lengths near terminal nodes of the highest posterior probability tree. Finally, PCP size classes are not strictly correlated with ribosomal clades as demonstrated by the PCP genes from clade B species S. muscatinei, Symbiodinium from D. stokesii and S. pulrchrorum. 92 Figure 3.1 Consensus (50% majority rule) of 36,000 trees for complete PCP gene data set, colored by species. Numbers are posterior probability support. 93 Figure 3.2 Highest posterior probability tree for the complete PCP gene data set, colored by species. 94 Figure 3.3 Highest posterior probability tree for the complete PCP gene set colored by large subunit ribosomal clade of the species. 95 By contrast, there were also unanticipated aspects to these trees. Figures 3.1 and 3.2 suggest that there were interdigitations of branches between the large PCP genes from different Symbiodinium species. Furthermore, Figure 3.3 shows that large PCP genes from a clade C species (S. kawagutii) could be more basal to those from clade B species (Symbiodinium from D. stokesii and S. pulrchrorum). A summary of the results presented in the cladograms in Figure 3.4. The highest posterior probability tree from Bayesian analysis on the subset of most divergent PCP sequences is shown (Figure 3.5). This tree retains most of the same characteristics of the larger tree in Figures 3.2 and 3.3. This tree and its corresponding Nexus files were analyzed with PAML CODONML substitution models. PAML CODONML analyses The output files from PAML CODONML include likelihood scores, values (nonsynonymous to synonymous substitution ratios) and p scores (probability of sites having a corresponding value). As with the nucleotide substitution models in PAUP*, it was necessary to conduct LRTs on PAML codon substitution models to determine which model best fits the data. This was done prior to interpretation of estimated parameters. Table 3.3 show the comparisons made in these tests and probabilities where significant differences between models exited. M3 had the best fit. M2 and M3 fit the data better than either M0 or M1. M0 had a smaller negative log-likelihood score than M1, so an LRT was not appropriate. Neither M7 nor M8 fit significantly better than M3, 96 and there was also no significant difference between the likelihoods of M7 and M8. Figure 3.4 Cladograms comparing the evolution of PCP and large subunit ribosomal genes. Clade B 35 kDa PCPs and clade B small PCP are from different species. (Ribosomal tree was redrawn from Wilcox, 1998). 97 Figure 3.5 Highest posterior probability tree for PCP gene subset colored by species. 98 Table 3.3 PAML CONDONML model likelihood ratio test results Model Code M0 (one-ratio) M1 (neutral) M2 (selection) M3 (discrete) M7 (beta) M8 (beta& ) - 6328.168135 6493.227752 6234.371789 6222.006042 6224.635905 6224.066493 LRT M0 vs M2 M0 vs M3 M1 vs M2 M1 vs M3 M2 vs M3 -2 187.592692 212.324186 517.711926 542.44342 24.731494 df 2 4 2 4 2 p 1.83977E-41 8.40292E-45 3.8044E-113 4.414E-116 4.26211E-06 The estimated parameters for all six codon substitution models are listed in Table 3.4. PAML CODONML can conduct paired comparisons between models based on the estimated parameters in order to identify the presence of any codons under positive selection (M2 vs. M3) and to more accurately estimate the strength of selection (M7 vs. M8). No sites were identified as being under positive selection. Probabilities reported for M2 and M3 suggest that most codons have s < 0.2 and are under purifying selection instead. Table 3.4 PAML CODONML estimated parameters for PCP genes Model Code M0 M1 M2 M3 M7 M8 d N /d S for all branches 0.1025 0.5875 0.1467 0.1303 0.1238 0.1244 Parameters = 0.1025 p0 = 0.41252 0 = 0.00 p0 = 0.28871 0 = 0.00 p0 = 0.54959 0 = 0.02218 p = 0.53740 p = 0.72473 p1 = 0.58748 1 = 1.00 p1 = 0.06443 1 = 1.00 p1 = 0.40577 1 = 0.19529 q = 3.68372 q = 3.84597 p2 = 0.64685 2 = 0.12718 p2 = 0.04464 2 = 0.87118 p1 = 0.22793 1 = 0.01947 99 DISCUSSION PCP gene diversity hypotheses The novel evolution of PCP light harvesting complexes was a substantial adaptive success for photosynthetic dinoflagellates. The vast majority of extant photosynthetic dinoflagellate species express PCPs. It is now well established that small and large PCP genes from free-living and symbiotic dinoflagellates occur in diversified families and that this genetic polymorphism is the primary basis for expression of multiple PCP isoforms (Triplett et al. 1993; Sharples et al. 1996; Hiller et al. 2001; Chapters One and Two). Given the prevalence of PCP gene heterogeneity among divergent lineages of photosynthetic dinoflagellates, it is easy to assume that this characteristic has been maintained over evolutionary time because it too is adaptive, perhaps in terms of broadening light harvesting range. Selective pressures could lead to adaptive polymorphism in at least two scenarios. First, there is positive selection for individual PCP isoforms with distinct spectral properties, and consequently corresponding codon sites within PCP genes have > 1. Second, there is purifying selection for isoforms that have spectral properties within a specific range of absorption maxima (all codons with < 1). While it seems unlikely that PCP gene polymorphism would be so wide spread without some adaptive functional significance, it is possible that there is purifying selection for isoforms with a specific absorption maximum, and that minor variants of that isoform are tolerated (also codons with < 1). 100 Heterogeneity among PCP genes is ancient The main objective of the present research was to use Bayesian phylogenetic reconstruction and maximum likelihood methods to determine the selective pressures that exist within PCP genes at the codon level and to thereby narrow the explanation of PCP polymorphism. The consensus tree, phylograms and cladograms presented in this report (see Figures 3.1 3.5) revealed that the evolution of PCP genes within families and between species is not simply a reflection of divergence between taxa, but rather that the history has been more complex and interesting. For example, PCP polymorphism appears to be ancient. Its presence in lineages of small and large PCP genes and in both free-living and Symbiodinium species suggests that the polymorphism predates the beginning of these divergences. This is also in agreement with the inter-digitations of PCP gene families among more closely related Symbiodinium species. If differences in PCP genes were not instrumental in speciation and the polymorphism was present as speciation events occurred, then overlap between gene families of recently divergent species would be expected. In addition functional constraints on PCP polypeptides may contribute to keeping sequences between species similar as in major histocompatibility complex genes. The nesting of large PCP genes from clade B species within those from clade C species is curious. More accurate placement of clade B large PCP genes may come by obtaining complete coding region sequences instead of partials. 101 PCP size class not correlated to ribosomal clade. The lack of strict correlation between PCP gene size and ribosomal clade (see Figure 3.3 and 3.4) agrees with a similar observation about PCPs size classes and ITS clades (LaJeunesse, 2001). These results suggest that first fusions of small PCP genes to produce the large variety probably preceded the initial split of Symbiodinium from free-living species. Figure 3.4 demonstrates that different clade B species have either small or large PCP genes. It is very likely that all Symbiodinium clades once contained both size classes of PCP genes. In fact, C is the only major clade in which all characterized apoproteins are of one size class; 35 kDa (Govind et al. 1990; LaJeunesse, 2001). Clade C and clade A species may have lost their small and large PCP genes, respectively. On the other hand, it is also possible that these genes are still present yet were not detected by methods used in Chapter Two. In most instances, species specific primers had to be designed for each new PCP gene family. Characterization of PCP gene sequences from additional clade A and C species may demonstrate that both size classes of genes are still present. It would be interesting to learn if any single species possesses both small and large PCP genes as this could represent an intermediate state in the evolution of PCP gene size classes. There are examples of clade A species such as S. microadriaticum that express small and large apoproteins. However, the large polypeptides from these species are homodimers and may come from the same small PCP genes that code for their small apoproteins. Some of the small 102 apoproteins may then undergo post-translational modification to allow dimerization (personal communication, Robert Trench, UC Santa Barbara). PCP gene codons under purifying selection rather than positive selection The results from the two PAML CODONML models which best fit the data, M2 and M3, were in agreement and were conclusive. They both indicate that it is most likely for codons to be conserved and have ratios estimated from the data at < 0.2. Neither model assigned codons to any class with a value higher than 1. Furthermore, M3 estimated s and probabilities suggest that codons from the PCP gene compared here have 55% probability being highly conserved with a < 0.02, 41% probability of have < 0.2 and only 4% probability of experiencing the highest ratio of 0.87. The consensus after comparison of the results from both models is that no PCP codon sites are under positive selection. Instead of having sites under positive selection, codons within PCP genes are subject to purifying selection like most other duplicated genes (Wagner, 2002). Although nonsynonymous substitutions exist between copies, the locations are not consistent within families or across species. This may be an important reflection of selective pressures in different habitats occupied by different species. Because the tertiary structure of PCP polypeptides has a direct effect on the physical arrangement of chromophores and the efficiency of energy transfer from peridinin to chlorophyll a, there would clearly be constraints on the type and amount of amino acid substitutions that could occur without disrupting the light 103 harvesting function of the holoproteins. Purifying selection has the affect of removing deleterious mutations while leaving behind both nonsynonymous and synonymous substitutions of marginal to zero functional consequence. If PCP genes families were also subjected to highly efficient homogenization, then differences between copies would fade rapidly. However, there can be numerous differences at the nucleotide level between copies, and the distinctive pI properties of expressed isoforms are stable. This suggests that PCP genes are not being thoroughly homogenized by biased gene conversion or other mechanisms. Why PCP genes should be homogenized to a lesser degree than ribosomal genes (or any other gene family) is still unclear. However, it may be related to where PCP genes reside within dinoflagellate chromosomes. Their locations have yet to be mapped. Suggested experiments to test for adaptation through purifying selection If PCPs are adapted to harvest specific light wavelengths, then the PCPs from dinoflagellate species living under different light environments should also have absorption maxima that correspond to available light in their specific habitat. For instance, the expectation would be that dinoflagellates living in clear seawater dominated by blue light would have PCP isoforms with different absorption maxima than those from dinoflagellates living in turbid freshwater in which most of the ambient light is green. It should also be possible to determine if PCPs are under purifying selection to harvesting a range of wavelengths rather than a single wavelength. 104 The 3.6 nm range of absorption maxima reported by Iglesias-Prieto et al. (1991) for S. microadriaticum may be well suited to the fluctuations in the light field most often experienced by this organism. Endosymbiotic dinoflagellates of hosts that live in the shallow water with highly variable light environment should have PCP isoforms with a relatively broad range of absorption maxima, while those from deep water with generally uniform lighting should have a more narrow range of PCP variability. Furthermore, if PCP isoform polymorphism is adapted to variable light, then a physiological experiment designed to measure the rate of saturation of photosystem II should demonstrate more rapid saturation under fluctuating light wavelengths than when exposed to a single wavelength. CONCLUSION AND FUTURE DIRECTIONS This chapter has presented the first phylogenetic analyses of the evolution of dinoflagellate PCP gene families. There has been special emphasis on incorporating new sequence data generated for five Symbiodinium species in Chapters One and Two of this dissertation. Together, the results of this dissertation have demonstrated several main points. PCP genes from Symbiodinium species occur in diversified families. Genetic heterogeneity is the primary source of PCP isoform variation. Amino acid substitutions within isoforms may affect spectral tuning of peridinins. PCP polymorphism is ancient and present in major lineages of photosynthetic dinoflagellates. Codon sites within PCP genes are evolving under purifying selection and are subjected to net 105 reduced levels of concerted evolution. The polymorphism likely to be adaptive, but additional experiments are required to substantiate this hypothesis. It is possible that PCP genes contain sites that are under positive selection that were not detected here. Supplementary work is needed to characterize the PCP genes families from other species. The inclusion PCP gene sequences from additional dinoflagellate species in future phylogenetic analyses will provide a clearer picture of PCP gene evolution. Where possible the use of complete coding sequences will also improve the accuracy and resolution of PCP gene phylogenies and determination of selective pressures affecting PCP evolution. In situ hybridization experiments could be used to determine the distribution of PCP genes among dinoflagellate chromosomes. PCP genes could potentially be used as molecular markers to identify certain chromosomes or to gauge changes in ploidy level between species. The location of PCP genes on dinoflagellate chromosomes may also be relevant to the levels of concerted evolution that the genes experience. Finally, studies on the functional significance of amino acid substitution in PCPs will be enhanced by the determination of PCP crystal structures from many more species. The protein modeling presented here was dependent on the single structure that is currently available. Combined comparisons of nucleotide sequences, amino acid sequences and crystal structures from various dinoflagellate species may demonstrate if and how PCPs have been specialized for light harvesting in particular habitats. 106 Appendices APPENDIX 1.1 SYMBIODINIUM 203 CLONE 41 TANDEM REPEAT. UPSTREAM AND DOWNSTREAM CODING SEQUENCES ARE UNDERLINED. THE PUTATIVE PROMOTER WITHIN THE SPACER IS IN BOLD. TGGCCGTTTACGATTCAGTGAAAGGCATCACGGACCCCAAAGTGCCAGCTTACATGAAGTCCTTGG TGAACGGGCCCGATGCTGAGAACGCCTACAAAGCGTTCCTGGAATTCAAGGATGTTGTTGCAAAGA ACCAGGTGACCACCGCCAGTGCCCCTGCAGCTGTGCCTTCTGGGGACAAGATTGGTGTAGCTGCAA AAGCGTTGTCCGATGCATCCTATCCTTTCATCAAGGACATCGATTGGCTGTCCGACATTTACCTGA AGCCGCTGCCCGGCAAGACTGCCCCGGAGACCCTGAAAGCCATTGACAAGATGATCGTGATGGGTG CCAAGATGGATGGGAACCTCTTGAAGGCAGCAGCAGAGGCACACCACAAGGCCATTGGCAGCATTG ATGCCAAGGGTGTGACGTCCGCGGCCGACTACGAAGCTGTGAATGCAGCATTGGGGCGCTTGGTGG CATTCCGTGCCCAAGTCCACTGTCATTGGACGTGTACAATTCCATGGCCAAAGTCGTTGGTTCCAC CGTGACCAACAACATGTTCTCGAAGGTGAATCCATTGGATGCAGTGGCTGCCGCCAAGGGTTTCTA CACCTTCAAGGATGTTGTGGAGGCTTCCCAGCGCTGAAGGTGAAGACCATCGGCATGAAGTTTCGA CATTGCTTTGATGTGCGTTGACCCTTTTTTTAATCTTGTATAGCACGAGCCTCTGAAAATGGAAAA AGAAGCTCACAATTTGCTTTGTCACCAATGTATTGCCCGCTGCAGCTCCACTGTTTTCCAGATCTT TCGATATCTTTCAAGACATTTTAAAAGTCATAGCCATGCCTCGATTCAACTAGAGGCGCCAAAGCC TTGCTCCAACTCCCAACTCACCGACTTGGAGCCGAACACATCCAGCAGTTGAAGCCTCAGCATGAC ACGATATGACATGATATGACACGATTCCTCCATGTTCTCGCTTTGTTGCTCGTGTACCAATCGAAC ACCACAGAAGCATCATGAGATGGTGATGAAATGGAAGTGGCCGGTGGCGAGAAACCACCCTGCGTC ACGTTGAATCTGATCAGTGATTGCATTGTTTTGTTTTGATATGGTAAGCACCCCTTGATAGCTCTT AGGACTAGATGGACTAGGATCTCACATAGATGTCCAGTGACATAGATTCTGTGTTGATTTCTTGCT ACAAGATACAGTACGTCTCTGGCAGTGCTTGTCCGTTTGCAATCTGTCTTGAATGCAGAATCGAGA CAGCAAGAGGTAGGATTTTCGTGGATTTTGGTAACTCAATGATGATAGCGATATGGCCCGGGCCCG GCCCTGCTTATTTGGTTTATCTGTACTCATCCCAATCGTCCTGCGGTACTTGGTTCAAGTTAGTTT CAGAGGCTATTTTTTGAGCAACTCCGGCCCACTTTTAGTTTTTGGTGGCCATGGTGCGTGGAGCAA GGAAAGCTGTTGCTGTCGGAGTTGCGGTGGCTGTTGCCTGCAGTGTCCAGCAGCATTTGAATTTTG TGCCTGGGCCTCGGCATGCTGCTCCAGTGGCCGCAGCAGCAGCCAGCATGATGATGGCTCCCGCGG CTTTTGCTGATGAGATCGGCGATGCTGCAAAGAAGCTTGGAGATGCTTCCTACTCTTTTGCCAAGG AAGTGGATTGGAGCAATGGAATTTTCCTCCAGGCCCCTGGCAAGTTTCAGCCCTTGAAGGCGTTGA AAGCGATTGACAAGATGATCGAAATGGGGGCAGCTGCCGATCCCAAGCTTCTCAAAGAGGCAGCAG AAGCACATCACAAGGCCATCGGGAGCATCAGCGGGCCAAATGGTGTGACTTCGCGT 107 APPENDIX 1.2 ALIGNMENT OF CLONED SYMBIODINIUM 203 PCP GENE NUCLEOTIDE SEQUENCES (START AND STOP CODONS ARE UNDERLINED). 10 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 20 . 30 . 40 . 50 . 60 . 60 60 60 57 60 60 60 60 60 60 30 30 30 30 30 GTGGCCATGGTGCGTGGAGCAAGGAAAGCTGTTGTTGTCGGAGTTGCGGTGGCTGTTGCC ..................................C......................... ..................................C......................... ---...............................C......................... ..................................C......................... ..................................C....A.................... ..................................C......................... ..................................C......................... .C................................C......................... ..................................C......................... ------------------------------....C............A............ ------------------------------....C............A............ ------------------------------....C......................... ------------------------------....C......................... ------------------------------...TC............A............ 70 . 80 . 90 . 100 . 110 . 120 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 TGCAGTGTCCAGCAGCATTTGAATTTTGTGCCTGGGCCTCGGCATGCTGCTCCAGTGGCC ....T.T...G................................................. .................CC......................................... ...................C....C................................... .................CC........................................T ...........................................................T ...........................................................T ............................................................ ...........................................................T ............................................................ ..............A............................................. ..............A............................................. ............................................................ ........A........CC......................................... ..............A............................................. 130 . 140 . 150 . 160 . 170 . 180 . 120 120 120 117 120 120 120 120 120 120 90 90 90 90 90 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 GCAGCAACAGCCAGCATGATGATGGCTCCTGCGGCTTTTGCTGATGAGATTGGCGATACT .........................................................G.. ......G.........................A.................C......G.. ......G..................................................G.. ......G..A...................C....................C......G.. ......G...........................................C......G.. ......G...........................................C......G.. ......G..................................................G.. ......G...........................................C......G.. ......G..................................................G.. ......G..................................................G.. ......G..................................................G.. .........................................................G.. ......G..............................................T...G.. ......G..................................................G.. 180 180 180 177 180 180 180 180 180 180 150 150 150 150 150 108 190 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 200 . 210 . 220 . 230 . 240 . 240 240 240 237 240 240 240 240 240 240 210 210 210 210 210 GCAAAGAAGCTTGGAGATGCTTCCTACTCTTTTGCCAAGGAAGTGGATTGGAACAATGGA ............................................................ ............................................................ ............................................................ ............................................................ ..............G............................................. ............................................................ ............................................................ ...................................................G........ ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ 250 . 260 . 270 . 280 . 290 . 300 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 ATTTTCCTCCAGGCCCCTGGCAAGTTTCAGCCCTTGGAGGCGTTGAAAGCAATTGACAAG ............................................................ ....................................A.............G......... ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ..................................................G......... ............................................................ ............................................................ ............................................................ ....................................A....................... ............................................................ 310 . 320 . 330 . 340 . 350 . 360 . 300 300 300 297 300 300 300 300 300 300 270 270 270 270 270 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 ATGATCGAAATGGGGGCAGCCGCCGATCCCAAGCTTCTCAAAGAGGCAGCAGAAGCACAT ............................................................ ............................................................ ............................................................ .............A.............................................. ............................................................ ............................................................ ............................................................ ............................................................ ....................T....................................... ..........................C................................. ..........................C................................. ............................................................ ............................................................ ............................................................ 360 360 360 357 360 360 360 360 360 360 330 330 330 330 330 109 370 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 380 . 390 . 400 . 410 . 420 . 420 420 420 417 420 420 420 420 420 420 390 390 390 390 390 CACAAGGCCATCGGGAGCATCAGCGGACCAAATGGTGTGACTTCGCGTGCTGACTGGGAT ..........................G................................. ...........T..............G................................. ..........................G................................. ...........T..............G................................. ............................................................ ............................................................ ..........................G................................. ............................................................ ...........T..............G................................. ...........T..............G................................. ...........T..............G................................. ...........T..............G................................. ..........................G................................. ...........T..............G................................. 430 . 440 . 450 . 460 . 470 . 480 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 GCCGTGAATGCAGCCCTGGGCCGCGTAGTCGCTTCGGTCCCCAAAGCAAAGGTCATGGCC .................T.......................................... .................T........................G................. .................T.......................................... .................T.......................................... .................T.......................................... .................T.......................................... .................T.......................................... .................T.......................................... .................T.......................................... .................T.............................G.G.......... .................T.............................G.G.......... ..............T..T.........A..........................G..... .................T.......................................... .................T.............................G............ 490 . 500 . 510 . 520 . 530 . 540 . 480 480 480 477 480 480 480 480 480 480 450 450 450 450 450 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 GCTTACGATTCAGTGAAAGACATCACGGACCCCACAGTGCCAGCTTACATGAAGTCCTTG .T.......................................................... .T................................A......................... ............................................................ .T.........................................A................ .T.......................................................... .T.......................................................... .T.........G................................................ .T.......................................................... .T.......................................................... .T.........................................T................ .T.........................................T................ .T...............................G.......................... .T................................A......................... .T.........................................T................ 540 540 540 537 540 540 540 540 540 540 510 510 510 510 510 110 550 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 560 . 570 . 580 . 590 . 600 . 600 600 600 597 600 600 600 600 600 600 570 570 570 570 570 GTGAACGGGCCCGATGCTGAGAAGGCCTACCAAGCATTCCTGGAATTCAAGGATGTTGTT .......................C......A.G.................A......... .......................C......A............................C ..............................A............................. ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ..............................A............................. ...........T................................................ ............................................................ 610 . 620 . 630 . 640 . 650 . 660 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 GCAAAGAACCAGGTGACCACCGCCAGTGCTCCTGCAGTTGTGCCTTCTGGGGACAAGATT ............................................................ ............................................................ ............................................................ ............................................................ .....................CG..................................... ............................................................ ............................................................ ..G......................................................... ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ 670 . 680 . 690 . 700 . 710 . 720 . 660 660 660 657 660 660 660 660 660 660 630 630 630 630 630 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 GGTGTAGCTGCAAAAGCTTTGTCCGATGCATCCTATCCTTTCATCAAGGACATCGATTGG .................G.....T.................................... .................G.......................................... ............................................................ .................G.......................................... .................G.............................A............ .................G.......................................... .................G.......................................... ................TG.......................................... .................G...........................C.............. .................G.............................A............ .................G.............................A............ ............................................................ .................G.......................................... .................G.............................A............ 720 720 720 717 720 720 720 720 720 720 690 690 690 690 690 111 730 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 740 . 750 . 760 . 770 . 780 . 780 780 780 777 780 780 780 780 780 780 750 750 750 750 750 CTGTCCGACATTTATCTGAAGCCGCTGCCCGGCAAGACTGCCCCAGAGACCCTGAAAGCC ............................................................ ..............C............................................. ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ..............C..............T.............................. ............................................................ ............................................................ ............................................................ ............................................................ ..............C............................................. ............................................................ 790 . 800 . 810 . 820 . 830 . 840 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 ATTGACAAGATGATCGTGATGGGCGCCAAGATGGATGGAAACCTCTTGAAGGCAGCAGCA ............................................................ ............................................................ ............................................................ ............................................................ .......................T.................................... .......................T.................................... ............................................................ .......................T.................................... ............................................................ ............................................................ ............................................................ ..............................G............................. ............................................................ ............................................................ 850 . 860 . 870 . 880 . 890 . 900 . 840 840 840 837 840 840 840 840 840 840 810 810 810 810 810 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 GAGGCACACCACAAGGCCATTGGCAGCATTGATGCCAAGGGTGTGACGTCCGCGGCCGAC ............................................................ ............................................................ ............................................................ ............................................................ ....................................C.......C.....T..A...... ....................................C.......C.....T......... ............................................................ ....................................C.......C.....T......... ............................................................ ..........................................................-...................................................AA..G..-..........................................................-...................................................A......-...................................................A...A..-- 900 900 900 897 900 900 900 900 900 900 868 868 868 868 868 112 910 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 920 . 930 . 940 . 950 . 960 . 960 960 960 957 960 960 960 960 960 960 868 868 868 868 868 TACGAAGCTGTGTATGCAGCATTGGGGCGCTTGGTGGCATCCGTGCCCAAGTCCACTGTC ............A............................................... ............A............................................... ............A............................................... ............A.....T......................................... ............A............................................... ............A............................................... ............A............................................... ............A............................................... ............A..............................................G -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------970 . 980 . 990 . 1000 . 1010 . 1020 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 ATGGACGTGTACAATTCCATGGCCCAAGTCGTTGATTCCACCGTGACCAACAACATGTTC ........................A................................... ........................A...........A....................... ........................A................................... ........................A................................... ........................A................................... ........................A................................... ........................A................................... ........................A................................... .......................TGGCA................................ -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1030 . 1040 . 1050 . 1060 . 1070 . 1020 1020 1020 1017 1020 1020 1020 1020 1020 1020 868 868 868 868 868 1080 . 1080 1080 1080 1077 1080 1080 1080 1080 1080 1080 868 868 868 868 868 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 TCGAAGGTGAATCCATTGGATGCGATGGCTGCCGCCAAGGGTTTCTACACCTTCAAGGAT .......................A..............................T..... .......................A....................T............... C..............C............................................ .......................A..............A...........T......... .......................T.................................... .......................A.................................... .......................A....................T.....T......... .......................A....................T............... .......................A..............A..................... -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 113 1090 . 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 1100 . 1110 . 1120 . 1130 . 1140 . 1140 1140 1140 1137 1140 1140 1140 1140 1140 1140 868 868 868 868 868 GTTGTGGAGGCTTCCCAGCGCTGAAGGTGAAGACCATCAGCATGAAGTTTCGACCTTGCT ............................................................ .................................................C....A..... ............................................................ ............................................................ ......................................................A..... ......................................................A..... ............................................................ ......................................................A..... ......................................................A..... -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1150 . 1160 . 1170 . 1180 . 1190 . 1200 . 1196 1196 1190 1196 1193 1193 1192 1199 1189 1191 868 868 868 868 868 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 TTGACATGCTTTGACACTTTTTTA-AATCTTGTATCTTGTATAGCAGTATGTCACGG--........................-.........................A...G.A--....T....G.....C.......TT.....................------.GA.---........................-.......C.................TCAGGA.TT........................-.........................-...-----C ........................-.......CG.............------GA.CTTT ..........C.............-.......CG.............------GA-CTTT ............A...........-.........................A...GACTT........................-.......CG............C------GA-C--........................-.........................--------TC -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 203_79 203_80 203_81 203_82 203_83 203_84 203_85 203_87 203_89 203_90 203cDNA34 203cDNA39 203cDNA42 203cDNA55 203cDNA59 GC .. -. AA .AA AA A-A------ 1198 1198 1191 1198 1194 1195 1194 1200 1189 1192 868 868 868 868 868 114 APPENDIX 1.3 ALIGNMENT OF PREDICTED AMINO ACID SEQUENCES FROM SYMBIODINIUM 203 CLONES. TRANSIT PEPTIDE IS UNDERLINED. NUMBERS IN SQUARE PARENTHASES INDICATE LOCATIONS WITHIN APOPROTEINS 10 . 203 79aa 203 80aa 203 81aa 203 82aa 203 83aa 203 84aa 203 85aa 203 87aa 203 89aa 203 90aa 203cDNA 34aa 203cDNA 39aa 203cDNA 42aa 203cDNA 55aa 203cDNA 59aa 20 . 30 . 40 . 50 . 60[8] . 60 60 60 60 60 60 60 60 60 60 52 52 52 52 52 MVRGARKAVVVGVAVAVACSVQQHLNFVPGPRHAAPVAAATASMMMAPAAFADEIGDTAK .........A.........IFR...................................A.. .........A..............................A................A.. .........A..............S.L.............A................A.. .........A..............................AT...............A.. .........A.R............................A................A.. .........A..............................A................A.. .........A..............................A................A.. .........A..............................A................A.. .........A..............................A................A.. --------.A..............................A................A.. --------.A..............................A................A.. --------.A...............................................A.. --------.A..............................A................A.. --------.S..............................A................A.. 70[18] . 80[28] . 90[38] . 100[48] . 110[58] . 120[68] . 120 120 120 120 120 120 120 120 120 120 112 112 112 112 112 203 79aa 203 80aa 203 81aa 203 82aa 203 83aa 203 84aa 203 85aa 203 87aa 203 89aa 203 90aa 203cDNA 34aa 203cDNA 39aa 203cDNA 42aa 203cDNA 55aa 203cDNA 59aa KLGDASYSFAKEVDWNNGIFLQAPGKFQPLEALKAIDKMIEMGAAADPKLLKEAAEAHHK ............................................................ ..............................K............................. ............................................................ ..........................................E................. ............................................................ ............................................................ ............................................................ ...............D............................................ ............................................................ ............................................................ ............................................................ ............................................................ ..............................K............................. ............................................................ 130[78] . 140[88] . 150[98] . 160[108] . 170[118] . 180[128] . 180 180 180 180 180 180 180 180 180 180 172 172 172 172 172 203 79aa 203 80aa 203 81aa 203 82aa 203 83aa 203 84aa 203 85aa 203 87aa 203 89aa 203 90aa 203cDNA 34aa 203cDNA 39aa 203cDNA 42aa 203cDNA 55aa 203cDNA 59aa AIGSISGPNGVTSRADWDAVNAALGRVVASVPKAKVMAAYDSVKDITDPTVPAYMKSLVN ......................................V..................... ................................E.....V..........K.......... ............................................................ ......................................V.............D....... ......................................V..................... ......................................V..................... ......................................V..................... ......................................V..................... ......................................V..................... ..................................R...V.............V....... ..................................R...V.............V....... ...........................I........V.V..........A.......... ......................................V..........K.......... ......................................V.............V....... 115 190[138] . 203 79aa 203 80aa 203 81aa 203 82aa 203 83aa 203 84aa 203 85aa 203 87aa 203 89aa 203 90aa 203cDNA 34aa 203cDNA 39aa 203cDNA 42aa 203cDNA 55aa 203cDNA 59aa 200[148] . 210[158] . 220[168] . 230[178] . 240[188] . 240 240 240 240 240 240 240 240 240 240 232 232 232 232 232 GPDAEKAYQAFLEFKDVVAKNQVTTASAPAVVPSGDKIGVAAKALSDASYPFIKDIDWLS .....N..K................................................... .....N..K................................................... ........K................................................... ............................................................ .........................R.................................. ............................................................ ............................................................ ...........................................V................ .....................................................Q...... ............................................................ ............................................................ ........K................................................... ............................................................ ............................................................ 250[198] . 260[208] . 270[218] . 280[228] . 290[238] . 300[248] . 300 300 300 300 300 300 300 300 300 300 289 289 289 289 289 203 79aa 203 80aa 203 81aa 203 82aa 203 83aa 203 84aa 203 85aa 203 87aa 203 89aa 203 90aa 203cDNA 34aa 203cDNA 39aa 203cDNA 42aa 203cDNA 55aa 203cDNA 59aa DIYLKPLPGKTAPETLKAIDKMIVMGAKMDGNLLKAAAEAHHKAIGSIDAKGVTSAADYE ............................................................ ............................................................ ............................................................ ............................................................ ..................................................Q......... ..................................................Q......... ............................................................ ..................................................Q......... ............................................................ .........................................................--.......................................................KG--............................V............................--.......................................................T.--.......................................................TD--310[258] . 320[268] . 330[278] . 340[288] . 350[298] . 360[308] . 360 356 360 360 360 360 360 360 360 360 289 289 289 289 289 203 79aa 203 80aa 203 81aa 203 82aa 203 83aa 203 84aa 203 85aa 203 87aa 203 89aa 203 90aa 203cDNA 34aa 203cDNA 39aa 203cDNA 42aa 203cDNA 55aa 203cDNA 59aa AVYAALGRLVASVPKSTVMDVYNSMAQVVDSTVTNNMFSKVNPLDAMAAAKGFYTFKDVV ..N.......................K.............................---..N.......................K...T............................. ..N.......................K...........P..................... ..N.S.....................K................................. ..N.......................K................................. ..N.......................K................................. ..N.......................K................................. ..N.......................K................................. ..N.......................GI................................ -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 116 203 79aa 203 80aa 203 81aa 203 82aa 203 83aa 203 84aa 203 85aa 203 87aa 203 89aa 203 90aa 203cDNA 34aa 203cDNA 39aa 203cDNA 42aa 203cDNA 55aa 203cDNA 59aa EASQR ----..... ..... ..... ..... ..... ..... ..... ..... --------------------- 365 356 365 365 365 365 365 365 365 365 289 289 289 289 289 117 APPENDIX 2.1 ALIGNMENT OF CLONED S. PILOSUM PCP GENE NUCLEOTIDE SEQUENCES (START AND STOP CODONS ARE UNDERLINED). 10 . 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 20 . 30 . 40 . 50 . 60 . 58 58 59 58 59 58 57 57 GTGGGTCCCAG-TGCATTTTT-AGCCATGGCAAAGACGGCTCGCGCGGTTTCAGTTCTGC ......T....-.......G.-...................................... ...........G-........T..................................T... ...........-.........-...................................... ...........G.........-....................A................. ...........-.........-...................................... ........--AG.......-.T...................................... C.......--.G.......-.T...................................... 70 . 80 . 90 . 100 . 110 . 120 . 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 TTGCGGGAGTTTGCCTCTGCTGCCTCCCGGCCTTCGTGCCGGGGCCTGGTCGCTTTGCTC ..............................................C..........T.. ..............................................C............. ............................................................ ..............................................C............. ............................................................ ..................................T...........C............. ..............................................C............. 130 . 140 . 150 . 160 . 170 . 180 . 118 118 119 118 119 118 117 117 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 GCAGTGTGGCCCCTGCAGCTGTTGGGGCTGGGGCGCTGGGCATGCTTGGCGCAGCTCCTG ............................................................ ....C.....A................................................. ............................................................ ....C.....A................................................. ............................................................ ....C.....A................................................. ....C..A..A................................................. 190 . 200 . 210 . 220 . 230 . 240 . 178 178 179 178 179 178 177 177 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 CCTACGCCGACAAGATCGATGATGCCGCCAAAGTCCTCTCAGAGAAGTCCTATCCTTTCC ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ....................................C.....................T. 250 . 260 . 270 . 280 . 290 . 300 . 238 238 239 238 239 238 237 237 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 TGAAGGAGATCGATTGGACCTCGGACGTCTATGCCAAGCTCCCCACGCAGCCTCCTCTGA ....A.........C..................G.......................... ....A....................................................... ............................................................ ....A....................................................... ............................................................ ............................................................ ....A..A.........................G..........G............... 298 298 299 298 299 298 297 297 118 310 . 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 320 . 330 . 340 . 350 . 360 . 358 358 359 358 359 358 357 357 AGGTGATGACAGCCATTGACACAATGCTGAAGATGGGTGCTGCCATGGATCCAGCTGCTC ................C........................................... ................C........................................... ............................................................ ................C........................................... ............................................................ ................C........................................... .............A..C........................................... 370 . 380 . 390 . 400 . 410 . 420 . 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 TTAAGACGGGTGTTCTGGCCCACAGCCAGGCGATTGCCAACATGGATTCCAAAGGCGTGG C...............T......................................T.... .......................................................T.... ............................................................ .......................................................T.... .......C.................................................... .......................................................T.... .......C.......................C.......................T.... 430 . 440 . 450 . 460 . 470 . 480 . 418 418 419 418 419 418 417 417 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 CGACGCTGGCCGATTACACCGCCATCAACTCAGCGATTGGTCACATGATCTCTTCAGTGC ....C...........T..........................T................ ....C..............G.......................T................ ..........................................G................. ....C...........T..........................T................ ............................................................ ....C...........T.................A........T................ ................T.................A......................... 490 . 500 . 510 . 520 . 530 . 540 . 478 478 479 478 479 478 477 477 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 CCGCCTCGAAGACCATGGATGTTTACAATGCGTTCGCCAAGTTTAACCTTGGCTCAGACG ..............................................G........G.... .......................................................G.... ............................................................ .......................................................G.... .....C.................................................G.... .......................................................G.... ..............................................T........G.... 550 . 560 . 570 . 580 . 590 . 600 . 538 538 539 538 539 538 537 537 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 TTGGCCCCTACATGATGAGCAAGGTGAATGCCGCAGATGCCCAGGCAGCTTACAAGGCAC .......T.......................T............................ .......T.......................T............................ ...............................T............................ .......T.......................T............................ .......T.......................T............................ .......T.......................T............................ .......T.......................T............................ 598 598 599 598 599 598 597 597 119 610 . 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 620 . 630 . 640 . 650 . 660 . 658 658 659 658 659 658 657 657 TCATGGACTTCAAGGATGTTGTCAAAGCCTCCCAGCGCTGAGCCCCAAACTCAGAAACCT .............................................A...........T.. .................................................G.......TAG ............................................................ .................................................G.......TAG ............................................................ ....................A........................A...........T.. .........................................................T.. 670 . 185clone23 185clone25 185clone28 185clone30 185clone32 185clone33 185clone34 185clone35 TCTTGAAACAA ........... .....C......................... ........... ...-................- 669 669 669 668 670 669 666 667 120 APPENDIX 2.2 ALIGNMENT OF CLONED SYMBIODINIUM SP. FROM D. STOKESII AND S. PULCHRORUM PCP GENE NUCLEOTIDE SEQUENCES 10 . 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 20 . 30 . 40 . 50 . 60 . 60 0 8 60 60 60 60 60 60 60 60 0 0 0 0 ATGATCCAGATGGGAGCCGCCGCTGATCCTGAATTGTTGAAGAAGGCAGCTGATGCTCAC ---------------------------------------------------------------------------------------------------------------........ ......G..................................................... ......G..................................................... ......G..................................................... ......G..................................................... ......G..................................................... ......G.................................................G... ......G..................................................... ......G..................................................... --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------70 . 80 . 90 . 100 . 110 . 120 . 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 CACAAAGCCATCGGCAGCATTAGTGGACCAAATGGAGTAACTTCTCGTGCTGACTGGGAT -----------------------------------------------------------............................................................ ............................................................ ............................................................ ............................................................ ...........................................T................ ...........................................T................ ............................................................ ............................................................ ............................................................ --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------130 . 140 . 150 . 160 . 170 . 180 . 120 0 68 120 120 120 120 120 120 120 120 0 0 0 0 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 GCAGTGAATGCGGCCCTTGGCCGTGTGGTCGCTTCTGTCCCAAAGCAAAAGGTCATGGAC -------------------------------------------------------..... ............................................................ ............................................................ ............................................................ ...A........................................................ ...AA..................A..A................................. ...CA......A...........A......T............................. ...............T............................................ ...........A................................................ ...........A................................................ ---------------------------------------------------------... ---------------------------------------------------------... ---------------------------------------------------------... ------------------------------------------------------TGCC.. 180 5 128 180 180 180 180 180 180 180 180 3 3 3 6 121 190 . 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 200 . 210 . 220 . 230 . 240 . 240 65 188 240 240 240 240 240 240 240 240 63 63 63 66 GTCTATAATGCAGTCAAAGACATCACCGACCCCAAGGTGCCAGCTTACATGAAGTCCTTG ......G..................................................... ......G..................................................... ......G..................................................... ......G..................................................... ......G..............G...................................... ......G..................................................... ......G.....T.............................T................. ......G..................................................... ......G..................................................... ......G..................................................... ......G..................................................... ......G..............................C...................... ......G..................................................... ......G...............................C..................... 250 . 260 . 270 . 280 . 290 . 300 . 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 GTGAACGGTGCAGATGCGGAGAAAGCCTACCAAGGTTTCTTGGAGTTCAAGGATGCTGCG .......................................................T..T. ....G..............G...................................T..T. .......................................................T..T. .......................................................T..T. ..A....................................................T..T. .......................................................T..T. .......................................................T..T. .......................................................T..G. .......................................................T..T. .......................................................T..T. .......................................................T..T. ...........G...........................................T..T. .......................................................T..T. .................A.....................................T..TC 310 . 320 . 330 . 340 . 350 . 360 . 300 125 248 300 300 300 300 300 300 300 300 123 123 123 126 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 GCCGCAAACCAGGTGACCACCGCTAGCGCTGCTGCCACGGTGCCTACTGGAGACAAGATT ..........T.................................C............... ............................................................ ............................................C............... ............................................C............... ............................................C....A.......... ............................................................ ...........T..................T............................. ......................A..................................... ............................................................ ............................................................ ......................................T........A............ ......................................T........A............ ......................................T........A............ .....G..............T.................A........A............ 360 185 308 360 360 360 360 360 360 360 360 183 183 183 186 122 370 . 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 380 . 390 . 400 . 410 . 420 . 420 245 368 420 420 420 420 420 420 420 420 243 243 243 246 GGCACAGCTGCTAAAGCTTTGTCCGATGCATCATATCCATTCATCAAGGACATCGATTGG ............................................................ ............................................................ ............................................................ ............................................................ ........................................A................... ....................................T..........A.....A...... ....................A....................................... ....................A....................................... ....................A....................................... ....................A....................................... .....G...................................................... ...G.G...................................................... .....G...................................................... .....G........................C..C.......................... 430 . 440 . 450 . 460 . 470 . 480 . 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 CTGTCAGACATTTACCTGAAGCCATTGCCTGGCAAGACTGCCCCAGAGACCTTGACAGCC ..................................G......................... .........G.............................................A.... ............................................................ ............................................................ ..............................................G............. .........G.............................................A.... .........G.............................................A.... .........G.............................................A.... .........G.............................................A.... .........G.............................................A.... .......................................................A.... ..............................................G....C...A...T .......................................................A.... ......................................................CA.... 490 . 500 . 510 . 520 . 530 . 540 . 480 305 428 480 480 480 480 480 480 480 480 303 303 303 306 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 ATCGACAAGATGATCGTCATGGGAAGCAAAATGGATGGCAACCTCTTGAAGGCAGCCGCT .......................................G.................... ...................................C........................ ............................................................ ............................................................ ...............................C............................ ...................................C........................ ............................................................ ............................................................ ............................................................ ............................................................ .................G.......................................... .................G.......................................... .................G.......................................... ............................................................ 540 365 488 540 540 540 540 540 540 540 540 363 363 363 366 123 550 . 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 560 . 570 . 580 . 590 . 600 . 600 425 548 600 600 600 600 600 600 600 600 423 423 423 426 GAGGCACACCACAAGGCCATTGGCAGCATTGATGCCAAGGGTGTCACATCTCCAGCAGAC ............................C............................... ............................................................ ............................................................ ............................................................ ..............T.............................G...........G... ............................................................ .......................................................A.... ............................................................ .......................................................A.... .......................................................A.... ........................T..............................A.... .......................................................A.... .......................................................A..G. ............................................T..........A.... 610 . 620 . 630 . 640 . 650 . 660 . 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 TACGAAGCGGTGAACGCAGCTTTGGGCCGTATTGTGGCGTCGGTGCCAAAACAAACCGTC ............................................................ ..........................................A................. ............................................................ ............................................................ ............................................................ ............................................................ ........A.............................A..................... ............................................................ ............................................................ ............................................................ .................................................G.......... ............................................................ ............................................................ ............................................................ 670 . 680 . 690 . 700 . 710 . 720 . 660 485 608 660 660 660 660 660 660 660 660 483 483 483 486 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 ATGGATGTGTACAACTCAATGGCAAAGATTGTGGACCCAAGCGTGACCAACAACATGTTC ............................................................ ............................................................ ............................................................ ............................................................ ..................................T......................... ...........................G................................ ....................................T....................... .C.........................G................................ ...........................G................................ ...........................G................................ ............G.......................T....................... ............G.......................T....................... ............G.......................T....................... .........................................T.................. 720 545 668 720 720 720 720 720 720 720 720 543 543 543 546 124 730 . 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 740 . 750 . 760 . 770 . 780 . 780 579 728 780 780 780 780 780 780 780 780 586 599 599 584 TCTAAGGTCAACCCCTTGGATGCATTGTCTGCTGCCAAGGGCTTTTACACCTTCAAGGAT ..................................-------------------------............................................................ ............................................................ ..................................................T......... ............................................................ .............................C.............................. .............................G....................T......... .............................C.............................. ..C..................................G............T......... ..C..................................G............T......... ..G.C...................C.............A....----------------..G.C...................C.............A...........A.....---..G.C...................C.............A...........A.....---..G.C.........T.......................---------------------790 . 800 . 804 579 752 804 804 804 804 804 804 804 804 586 599 599 584 28clone5 28clone1 28clone2 32clone1 35clone3 35clone42 35clone43 35clone44 35clone46 35clone47 35clone49 8clone1 8clone2 8clone3 8clone4 GTTGTGGAAGCTGTCCAGCGTTAA -----------------------........................ .................A..C... ....................C..G ....................C... ....................C... ....................C..G ....................C... ....................C..G ....................C..G --------------------------------------------------------------------------------------------- 125 APPENDIX 2.3 ALIGNMENT OF CLONED S. KAWAGUTII PCP GENE NUCLEOTIDE SEQUENCES (START AND STOP CODONS ARE UNDERLINED). 10 . 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 20 . 30 . 40 . 50 . 60 . 59 59 59 60 58 59 58 59 59 51 -CGGTGGCCATGGTGCGTGGAGCAAGGAAAGCTGTTGCTGTCGGAGTTGCGGTGGCTGTT -T......................................................C... -T.......................................................... G........................................................... -T......-................................................... -........................................................... -T.-...............................................T........ -T...............................C.......................... -T.......................................................... ---------................................................... 70 . 80 . 90 . 100 . 110 . 120 . 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GACTGCAGTGTCCAGCAGCACTTGAATTTTGTGCCTGGGCCTCGGCGTGCTGCTCCAGTG .C...............A..............T.............A......C...... .C............T...............................A............. .C....................................A.......A............. .C...............A............................A............. .C.............................C......A.......A............. .C............................................A............. .C............................................A............. .C...............A..............T.............A......C...... .C....................................A.......A............. 130 . 140 . 150 . 160 . 170 . 180 . 119 119 119 120 118 119 118 119 119 111 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GCCGCAGCAGCAGCCAGCATGATGATGGCTCCTGCGGCTTTTGCTGATGAGATCGGCGAT ............................................................ .....................................................T..T... .....................................................T...... ..........................A................................. .....................................................T...... .....................G...............................T...... .....................................................T...... ....................A....................................... .....................................................T...... 190 . 200 . 210 . 220 . 230 . 240 . 179 179 179 180 178 179 178 179 179 171 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GCTGCAAAGAAGCTTGGAGATGCTTCCTACTCTTTTGCCAAGGAAGTGGATTGGAACAAT ............................................................ ............................................................ ............................T.........................G..... ............................................................ ......................................T...............T..... ............................................................ ..........G................................................. .............C.............................................. .....G...........G............C............................. 239 239 239 240 238 239 238 239 239 231 126 250 . 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 260 . 270 . 280 . 290 . 300 . 299 299 299 300 298 299 298 299 299 291 GGAATTTTCCTCCAGGCCCCTGGCAAGTTTCAGCCCTTGGAGGCGTTGAAAGCAATTGAC .......................................A.................... .......................................A.................... ................................C..G...A.................... ......C..........T.....................A.................... .................T.....................A.................... ......C..................................................... .......................................A.................... .......................................A.................... .........................................A.................. 310 . 320 . 330 . 340 . 350 . 360 . 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 AAGATGATCGAAGTGGGGGCAGCCGCCGATCCCAAGCTTCTCAAAGAGGCAGCAGAAGCA ............A.....................T......................... ............A............................................... ............A..............................................G ............A............................................... ............A............................................... ............A.......................................G....... ............A............................................... ............A..............................G................ ............A............................................... 370 . 380 . 390 . 400 . 410 . 420 . 359 359 359 360 358 359 358 359 359 351 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 CATCACAAGGCCACTGGGAGCATCAGCGGGCCAAATGGTGTGACTACGCGTGCTGACTGG .............................................T.............. .............T.....................C.........T.............. ............GT...............................T.T............ ............TTC..............................T.............. .............TC..............................T.............T ............TTC....................C.........T.A............ .............TC....................C.........T....C......... .............TC..A...........................T.............. ...........T.T...............................T.............. 430 . 440 . 450 . 460 . 470 . 480 . 419 419 419 420 418 419 418 419 419 411 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GATGCCGTGAATGCAGCCCTTGGCCGCGTAGTCGCTTCGGTCCCCAAAGCAAAGGTCATG ............................................................ ............................................................ ............................................................ ........AG............................A..................... ......................................A..................... .................................................A.......... ............................................................ ............................................................ ............................................................ 490 . 500 . 510 . 520 . 530 . 540 . 479 479 479 480 478 479 478 479 479 471 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GCCGTTTACGATTCAGTGAAAGACATCACGGACCCCGCAGTGCCAGCTTACATGAAGTCT ....................................AA.....................C ....................................AA.....................C ....................................AA.....................C ...........................................................C ................C...................AA.....................C ....................................AA.....................C ....................................AA.....................C ....................................AA.....................C ....................................AA.....................C 539 539 539 540 538 539 538 539 539 531 127 550 . 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 560 . 570 . 580 . 590 . 600 . 599 599 599 600 598 599 598 599 599 591 TTGGTGAACGGGCCCGATGCTGAGAAGGCCTACCAAGCATTCCTGGAATTCAAGGATGTT ..........................................T................. ...........T..T......................G...................... ...........T..T......................G...................... ..........................................T..........A...... ............................................................ ............T...................................A........... .....................................G...................... ..........................................T................. ............................................................ 610 . 620 . 630 . 640 . 650 . 660 . 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GTTGCAAAGAACCAGGTGACCACCGCCAGGGCTCCTGCAGTTGTGCCTTCTGGAGACAAG .............................T.......................G...... ...................G..G......T.............C.........G...... ......................G......T.......................G...... .............................T...........C...........G...... .....T..................A....T.......................G...... .......................T.....T.......................G...... ..C...................G......T.......................G...... ..............C..............T...................T...G...... .A...........................T.......................G...... 670 . 680 . 690 . 700 . 710 . 720 . 659 659 659 660 658 659 658 659 659 651 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 ATTGGTGAAGCTGCAAAAGCGTTGTCCGATGCATCCTATCCTTTCATCAAGGACATCGAT G......T.................................A.................. .......T................................................T... .......T................................................T... .................G.......................................... .......T.................................A.................. .....................................T...A........A......... .......T.....T.............................................. G....A.TGT...............................A.................. .......G.................................A...............C.. 730 . 740 . 750 . 760 . 770 . 780 . 719 719 719 720 718 719 718 719 719 711 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 TGGCTGTCCGACATCTACCTGAAGCCGCTGCCCGGCAAGACTGCCCCAGAGACCCTGAAA ............................................G............... ........G..............................C........C........... ........G.......................................C........... ........G.....T............................................. ........G.................A.................G............... ........G................................................... ........G........................................C.......... ............................................G............... ........G.....T........A.................................... 790 . 800 . 810 . 820 . 830 . 840 . 779 779 779 780 778 779 778 779 779 771 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GCCATTGACAAGATGATCGTGATGGGCGCCAAGATGGATGGTAACCTCTTGAAGGCAGCA ..........................TT.............A.................. .........................................A.................. .........................................A.................. .............................A...........A.................. .........................................A.................. .........................................A.................. .....C...................................A.................. ...............G..........TT.............A..T............... .........................................A.................. 839 839 839 840 838 839 838 839 839 831 128 850 . 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 860 . 870 . 880 . 890 . 900 . 899 899 899 900 898 899 898 899 899 891 GCAGAGGCACACCACAAGGCCATTGGCAGCATTGATGCCAAGGGTGTGACGTCCGCGGCC .......................C..........................A......... .......................C.................................... .......................C.................................... .......................C.................C..............C... .T.....................C............A...................A... .........................................C.................. .....................T.....T................................ .......................C..........................A......... .........................................C.................. 910 . 920 . 930 . 940 . 950 . 960 . 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GACTACGAAGCTGTGAATGCAGCATTGGGGCGCTTGGTGGCATCCGTGCCCAAGTCCACT ............................................................ ...........................................................A ...........................................................A ........................................................A... ............................................................ ............................................................ C........................................................... ............................................................ ............................................................ 970 . 980 . 990 . 1000 . 1010 . 1020 . 959 959 959 960 958 959 958 959 959 951 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GTCATGGACGTGTACAATTCCATGGCTGGCATCGTTGATTCCACCGTGACCAACAACATG ..........................GAAAG............................. ..............................G...........C................. ............................................................ ............................................T............... ..........................CAAAG............................. ..........................CAAAG............................. ...........T................................G...........T... ..........................GAAAG............................. ..........................CAAAG............................. 1030 . 1040 . 1050 . 1060 . 1070 . 1080 . 1019 1019 1019 1020 1018 1019 1018 1019 1019 1011 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 TTTTCGAAGGTGAGCCCATTGGATGCAATGGCTGCCGCCAAGGGTTTTTACACCTTCAAG ..CC.........AT............................................. ..C..........AT..............T.............................. ..C..........AT............G................................ ..C..........AT............................................. ..C..........AT....................T........................ ..C.....T....AT..............T..C..........................A ..C..........AT............................................. ..C..........AT............................................. ..C.T........AT...C............................C..........G. 1090 . 1100 . 1110 . 1120 . 1130 . 1140 . 1079 1079 1079 1080 1078 1079 1078 1079 1079 1071 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GATGTTGTGGAGGCTTCCCAGCGCTGAAGGTGAAGACCATCAGCAGGAAGTTTCGACATT .............................................T.............. .............................................T.............A ...........A..............G..................T.............. .................T...........................C.............. .............................................T.............. .............................................T.............. .............................................T.............. .............................................T.............. .............G...T...........................T.............. 1139 1139 1139 1140 1138 1139 1138 1139 1139 1131 129 1150 . 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 1160 . 1170 . 1180 . 1190 . 1200 . 1198 1178 1171 1174 1171 1164 1176 1173 1173 1170 GCTTT-GACATGCTTTAACAGAATTTTAAATCTTGCATCTGCCTTTGCAATTGCATTGCC .....-...........CA.CTT....G.......T....-------------------.....-..............ATT....-----.--A..AA-------------------.....-..........G..CCTT....-----..AA..A.-------------------.....-..............CTT...A....G..-------------------------.....-..............CTT...---------------------------------.....-...........G..CTT...-........T....-------------------.....T..............-CT....-----..AA....-------------------.....-..............CTT............------------------------.....-A.............CTT............T....-------------------1210 . 1220 . 1230 . 1240 . 1250 . 1260 . 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 ATCTTTGTAATTGCATTGTCATGCAGCGTCGCAGAGTGAGTGAGATTTGTTTAAGATATT -------....-------------------------------------------------------.----------------------------------------------------------.--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.----------------------------------------------------------.---------------------------------------------------------------------------------------------------------------------.---------------------------------------------------1270 . 1280 . 1278 1202 1192 1195 1191 1179 1185 1194 1185 1175 1258 1182 1172 1175 1171 1164 1177 1174 1173 1171 135clone37 135clone38 135clone41 135clone42 135clone43 135clone49 135clone50 135clone52 135clone54 135clone60 GTATCTTGTATAGCAAAAGC .................... .................... .................... ...A................ -A..............----------........----.................... -............-------------...C--------- 130 APPENDIX 2.4 ALIGNMENT OF PREDICTED AMINO ACID SEQUENCES FROM S. PILOSUM CLONES. TRANSIT PEPTIDE IS UNDERLINED. NUMBERS IN SQUARE PARENTHASES INDICATE LOCATIONS WITHIN APOPROTEINS 10 . 185clone23aa 185clone25aa 185clone28aa 185clone30aa 185clone31aa 185clone33aa 185clone34aa 185clone35aa 20 . 30 . 40 . 50 . 60[6] . 60 60 60 60 60 60 60 60 MAKTARAVSVLLAGVCLCCLPAFVPGPGRFARSVAPAAVGAGALGMLGAAPAYADKIDDA ..............................V............................. ............................................................ ............................................................ .....H...................................................... ............................................................ ............................................................ ............................................................ 70[16] . 80[26] . 90[36] . 100[46] . 110[56] . 120[66] . 120 120 120 120 120 120 120 120 185clone23aa 185clone25aa 185clone28aa 185clone30aa 185clone31aa 185clone33aa 185clone34aa 185clone35aa AKVLSEKSYPFLKEIDWTSDVYAKLPTQPPLKVMTAIDTMLKMGAAMDPAALKTGVLAHS ................R.....G............................P........ ............................................................ ............................................................ ............................................................ ............................................................ ............................................................ ...P..................G...A................................. 130[76] . 140[86] . 150[96] . 160[106] . 170[116] . 180[126] . 180 180 180 180 180 180 180 180 185clone23aa 185clone25aa 185clone28aa 185clone30aa 185clone31aa 185clone33aa 185clone34aa 185clone35aa QAIANMDSKGVATLADYTAINSAIGHMISSVPASKTMDVYNAFAKFNLGSDVGPYMMSKV ..............................................K............. ............................................................ .........................R.................................. ............................................................ .................................P.......................... ............................................................ ............................................................ 190[136] . 200[146] . 205 205 205 205 205 205 205 205 185clone23aa 185clone25aa 185clone28aa 185clone30aa 185clone31aa 185clone33aa 185clone34aa 185clone35aa NAADAQAAYKALMDFKDVVKASQR. ......................... ......................... ......................... ......................... ......................... ..................I...... ......................... 131 APPENDIX 2.5 ALIGNMENT OF PREDICTED AMINO ACID SEQUENCES FROM S. KAWAGUTII CLONES. TRANSIT PEPTIDE IS UNDERLINED. NUMBERS IN SQUARE PARENTHASES INDICATE LOCATIONS WITHIN APOPROTEINS 10 . 135clone37aa 135clone38aa 135clone41aa 135clone42aa 135clone43aa 135clone49aa 135clone50aa 135clone52aa 135clone54aa 135clone60aa 20 . 30 . 40 . 50 . 60 . 60 60 60 60 60 60 60 60 60 60 MVRGARKAVAVGVAVAVDCSVQQHLNFVPGPRRAAPVAAAAASMMMAPAAFADEIGDAAK .................A..............H........................... .................A...H..........H........................... .................A..............H........................... .................A..............H............I.............. .................A.........A....H........................... ..............L..A..............H...........V............... ........L........A..............H........................... .................A..............H..........I................ .................A..............H........................... 70 . 80 . 90 . 100 . 110 . 120 . 135clone37aa 135clone38aa 135clone41aa 135clone42aa 135clone43aa 135clone49aa 135clone50aa 135clone52aa 135clone54aa 135clone60aa KLGDASYSFAKEVDWNNGIFLQAPGKFQPLEALKAIDKMIEVGAAADPKLLKEAAEAHHK ..............................K..........M......M........... ..............................K..........M.................. ......F........D...........H..K..........M.................. ...................L..........K..........M.................. ...............Y..............K..........M.................. ...................L.....................M............G..... R.............................K..........M.................. .P............................K..........M.........R........ .......P.................................M.................. 130 . 140 . 150 . 160 . 170 . 180 . 120 120 120 120 120 120 120 120 120 120 135clone37aa 135clone38aa 135clone41aa 135clone42aa 135clone43aa 135clone49aa 135clone50aa 135clone52aa 135clone54aa 135clone60aa ATGSISGPNGVTTRADWDAVNAALGRVVASVPKAKVMAVYDSVKDITDPAVPAYMKSLVN ............S....................................K.......... .I..........S....................................K.......... .V..........S....................................K.......... .F..........S.......D....................................... .I..........S...C.........................A......K.......... .F..........S....................E...............K.......... .I..........S....................................K.......... .I..........S....................................K.......... .I..........S....................................K.......... 190 . 200 . 210 . 220 . 230 . 240 . 180 180 180 180 180 180 180 180 180 180 135clone37aa 135clone38aa 135clone41aa 135clone42aa 135clone43aa 135clone49aa 135clone50aa 135clone52aa 135clone54aa 135clone60aa GPDAEKAYQAFLEFKDVVAKNQVTTARAPAVVPSGDKIGEAAKALSDASYPFIKDIDWLS ..........................S..........V.V.................... .........G.............SS.S....A.......V.................... .........G..............S.S............V.................... ..........................S................................. .........................TS............V.................... .S...........I............S......................F.......... .........G..............S.S............V.V.................. .....................H....S......F...V.VS................... .................D........S............G................H... 240 240 240 240 240 240 240 240 240 240 132 250 . 135clone37aa 135clone38aa 135clone41aa 135clone42aa 135clone43aa 135clone49aa 135clone50aa 135clone52aa 135clone54aa 135clone60aa 260 . 270 . 280 . 290 . 300 . 300 300 300 300 300 300 300 300 300 300 DIYLKPLPGKTAPETLKAIDKMIVMGAKMDGNLLKAAAEAHHKAIGSIDAKGVTSAADYE ..........................S................................. ..........P..Q.............................................. .............Q.............................................. ..................................................N......... .....................................V...........T.......... ..................................................N......... .............A..............................F.C..........H.. ......................V...S................................. ..................................................N......... 310 . 320 . 330 . 340 . 350 . 360 . 135clone37aa 135clone38aa 135clone41aa 135clone42aa 135clone43aa 135clone49aa 135clone50aa 135clone52aa 135clone54aa 135clone60aa AVNAALGRLVASVPKSTVMDVYNSMAGIVDSTVTNNMFSKVSPLDAMAAAKGFYTFKDVV ..........................KV..........P..N.................. ...........................V...P.........N....I............. .........................................N....V............. .........................................N.................. ..........................KV.............N.................. ..........................KV...........N.N....I............. .........................................N.................. ..........................KV.............N.................. ..........................KV..........L..N..............R... 360 360 360 360 360 360 360 360 360 360 135clone37aa 135clone38aa 135clone41aa 135clone42aa 135clone43aa 135clone49aa 135clone50aa 135clone52aa 135clone54aa 135clone60aa EASQR.-......-......-.....WR. ......-......-......-......-......-.G....-- 366 366 366 368 366 366 366 366 366 366 133 APPENDIX 3.1 ALIGNMENT OF PCP NUCLEOTIDE SEQUENCES COLORED BY AMINO ACID TRANSLATION 134 135 136 137 138 139 140 141 142 143 References Altschul SF, Gish W, Miller W, Myers EW and Lipman DJ (1990). 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Changes in Cytosine Methylation Accompany Photoadaptation. Plant Physiol 117: 189-196. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F and Higgins DG (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24):4876-82. Trench RK (1981). Cellular and molecular interactions in symbioses between dinoflagellates and marine invertebrates. Pure and Appl Chem, 53:819835. 154 Trench RK (1993). Microalgal-invertebrate symbioses: a review. Ectocytobiosis & Cell Res, 9:135-175. Trench RK and Blank RJ (1987). Symbiodinium microadriaticum Freudenthal, S. goreauii sp. nov., S. kawagutii sp. nov. and S. pilosum sp. nov.: Gymnodinioid dinflagellates of marine invertebrates. J Phycol 23:469481. Triplett EL, Jovine RVM, Govind NS, Roman SJ, Chang SS, & Prezelin BB (1993). Characterization of two full-length cDNA sequences encoding for apoproteins of peridinin-chlorophyll a-proptein (PCP) complexes. Mol Mar Biol Biotechnol. 2(4):246-254. Wagner, A (2002). Selection and gene duplication: a view from the genome. Genome Biology 2002 3(5): reviews1012.1-1012.3 Weis VM, Verde EA, and Reynolds WS (2002). Characterization of a short form peridinin-chlorophyll-protein (PCP) cDNA and protein from the symbiotic dinoflagellate Symbiodinium muscatinei (Dinophyceae) from the sea anemone Anthopleura elegantissima (Cnidaria). J Phycol 38 (1), 157-163. Wilcox TP (1998). Large-subunit ribosomal RNA systematics of symbiotic dinoflagellates: Morphology does not recapitulate phylogeny. Mol Phylogenet Evol 10 (3): 436-448. Yang Z (1997). PAML: a program package for phylogenetic analysis by maximum likelihood Cabios 13:555-556. 155 Yang Z and Bielawski JP (2000). Statistical methods for detecting molecular adaptation. Tree 15(12): 496-503. Yang Z, Nielsen R, Goldman N, and Pedersen AK (2000). Codon-Substitution Models for Heterogeneous Selection Pressure at Amino Acid Sites. Genetics 155: 431-499. Veldhuis MJ, Cucci TL and Sieracki ME (1997). Cellular DNA content of marine phytoplankton using two new fluorochromes: taxonomic and ecological implications. J Phycol 33: 527-541. 156 VITA Jay R. Reichman was born on January 20, 1960 in Odessa, Texas to parents Ann and Roy. Jay was raised in Dallas, Texas where he attended Lamplighter School, St. Marks School of Texas and Jesuit College Preparatory School. He received a B.S. from Texas A & M in 1982. Jay met his future wife Melada in 1983. Jay worked as a purchasing agent and as an operations manager in the private sector in Austin, Texas prior to entering graduate school. While in graduate school at UT Austin, Jay served as a teaching assistant in the Department of Zoology for courses in genetics, oceanography and limnology, ecology, and human physiology. Jay and Melada were married in 1994. In 1998, he was promoted to Lecturer in the Department of Kinesiology and Health Education where he supervised the University of Texas Underwater Science and Scuba Diving Program. Jay and Melada's daughter Lauren was born was born in 2001. Jay will be doing postdoctoral research with the US Environmental Protection Agency's Western Ecology Division in Corvallis, Oregon. Permanent address: Unknown at this time. This dissertation was typed by the author. 157

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