PCP Gene Family in Symbiodinium from Hippopus hippopus: Low Levels of Concerted Evolution, Isoform Diversity, and Spectral Tuning of Chromophores

Jay R. Reichman*,, Thomas P. Wilcox* and Peter D. Vize{dagger}

* School of Biological Sciences, University of Texas at Austin
{dagger} Department of Biological Science, University of Calgary, Calgary, Canada

Correspondence: E-mail: reichman.jay{at}epamail.epa.gov.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 Literature Cited
 
Photosynthetic dinoflagellates have evolved unique water-soluble light harvesting complexes known as peridinin-chlorophyll a–binding proteins (PCPs). Most species of dinoflagellates express either 14 to 17 kDa or 32 to 35 kDa mature PCP apoproteins and do so in stable combinations of isoforms that differ in isoelectric point (pI). The source (posttranslational modification, protein degradation, or genetic) and functional significance of PCP isoform variation have remained unclear. PCPs are encoded by multigene families. However, previous reports conflict over the diversity of PCP genes within gene arrays. We present the first genomic characterization of the PCP gene family from a symbiotic dinoflagellate. Symbiodinium from the Pacific bivalve Hippopus hippopus (203) contains genes for 33 kDa PCP apoproteins that are organized in tandem arrays like those of free-living dinoflagellates Amphidinium carterae, Lingulodinium (Gonyaulax) polyedra, and Heterocapsa pygmaea. The Symbiodinium 203 PCP cassette consists of 1,098-bp coding regions separated by approximately 900-bp spacers. The spacers contain a conserved upstream sequence similar to the promoter in L. polyedra. Surprisingly, sequences of cloned coding regions are not identical, and can differ at up to 2.2% of the nucleotide sites. Sequence variation is found at both silent and nonsilent sites, and analysis of cDNA clones indicate that the variation is present in the mRNA pool. We propose that this variation represents nucleotide diversity among PCP gene copies that are evolving under low-level concerted evolution. Interestingly, the predicted proteins have pIs that are within the range of those published for other species of Symbiodinium. Thus, posttranslational modifications are not necessary to explain the multiple PCP isoforms. We have also identified several polymorphic sites that may influence spectral absorption tuning of chromophores.

Key Words: peridinin • PCP • gene family • dinoflagellate • Symbiodinium • concerted evolution


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 Literature Cited
 
Photosynthetic dinoflagellates have evolved unique extrinsic light-harvesting complexes known as peridinin-chlorophyll a–binding proteins (PCPs). PCPs apparently function within the aqueous lumen of dinoflagellate chloroplasts and are distinct in terms of their combination of pigments and water solubility (Haxo et al. 1976; Prezelin and Haxo 1976). Additionally, they occur solely in dinoflagellates and do not share significant amino acid sequence homology with other chromophore-binding proteins (Triplett et al. 1993; Norris and Miller 1994), including phycobiliproteins or membrane-bound, light-harvesting complexes.

Within chloroplasts, PCPs allow harvesting of blue-green 435 to 550 nm light by peridinins and efficient transfer of energy to chlorophyll a (Song et al. 1976; Siegelman, Kycia, and Haxo 1977; Larkum 1996; Moffat 1996; Damjanovic, Ritz, and Schulten 2000). The first high-resolution crystal structure of PCP from the free-living dinoflagellate Amphidinium carterae (Hofmann et al.1996) indicates that tuning of this transfer is achieved by the specific physical arrangement of the chromophore complex and the surrounding amino acids. In addition, changes in the polarity of the PCP protein environment neighboring the furanic rings and polyene chains of peridinins could modify the spectroscopic properties of these accessory pigments (fig. 1).



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FIG. 1. (a) A. carterae trimer of 35-kDa PCPs (NCBI PDB ID 1PPR [Hofmann et al. 1996]). These monomers typically contain (b) eight peridinin, (c) two chlorophyll a, (d) two digactosyl diacyl glycerol, and (e) combined chromophores complex. Regions of interaction between peridinin and amino acids with polar side chains within PCPs include (f) furanic ring and (g) polyene chain of peridinin

 
There are two general size classes of PCP apoproteins, commonly called short and long PCPs:14 to 17 kDa monomers/homodimers and 31 to 35 kDa monomers. Most dinoflagellate species express only one size class of apoproteins, although a few express both (Prezelin and Haxo 1976; Govind et al. 1990). The axis of symmetry in the amino acid sequence of long PCPs strongly suggests that the original gene for long PCPs arose from a duplication and fusion event between genes for short PCPs (Le et al. 1997), and analysis of nucleotide sequences supports this hypothesis (Hiller et al. 2001).

Regardless of which size PCP an individual dinoflagellate species expresses, it does so in multiple isoforms with distinct isoelectric points (pIs). Stable combinations of isoforms are species specific (Haxo et al. 1976; Prezelin and Haxo 1976; Siegelman, Kycia, and Haxo 1977; Chang and Trench 1982, 1984; Trench and Blank 1987). There are differences in spectroscopic properties of isoforms between species and among isoforms within species (Haxo et al. 1976; Prezelin and Haxo 1976; Song et al. 1976; Iglesias-Prieto, Govind, and Trench 1991). The source of PCP isoform variation, be it posttranslational modification, protein degradation, or genetic, has remained unclear (Haxo et al. 1976; Siegelman, Kycia, and Haxo 1977; Chang and Trench 1984; Triplett et al. 1993; Ogata et al. 1994; Sharples et al. 1996; Hiller et al. 2001). Likewise, the functional significance of isoform diversity has yet to be resolved.

With the exception of descriptions of cDNAs from two Symbiodinium species, all other details on PCP gene structure and organization are based on three free-living dinoflagellates: A. carterae, Lingulodinium (Gonyaulax) polyedra, and Heterocapsa pygmaea (Triplett et al. 1993; Norris and Miller 1994; Sharples et al. 1996; Le et al. 1997; Hiller et al. 2001; Weis, Verde, and Reynolds 2002). The nuclear genes that encode PCPs are intronless and exist as multigene families set in tandem arrays. However, previous reports conflict over the diversity of genes within these arrays. Evidence has mounted to suggest that PCP gene families may not be highly conserved in general and that the expression of distinct PCP isoforms is primarily due to genetic diversity among gene copies in PCP arrays (Triplett et al. 1993; Sharples et al. 1996; Le et al. 1997; Hiller et al. 2001). If so, the expectation that genes in tandem arrays evolve in concert may not strictly be true for PCP genes. Interestingly, nucleotide diversity has also been reported in other dinoflagellate multigene families, including those for luciferin-binding protein and Rubisco (Lee et al. 1993; Machabee', Wall, and Morse 1994; Rowan et al. 1996).

Detailed information about PCP gene families from Symbiodinium is needed to lay the groundwork for evolutionary analyses of PCP genes across dinoflagellate genera and species. Furthermore, the issue of genetic diversity contributing to expression of multiple isoforms in a functional manner also needs to be addressed. We present the first genomic characterization of the PCP gene family from a symbiotic dinoflagellate, Symbiodinium from the Pacific bivalve Hippopus hippopus (RK Trench culture collection number 203; clade C, sensu Rowan and Powers 1991a, 1991b, 1992; Wilcox 1998; LaJeunesse and Trench 2000; LaJeunesse 2001). Symbiodinium 203 PCP gene structure, organization, and copy number are compared with genes from L. polyedra. The presented data uncover considerable PCP gene diversity in Symbiodinium 203 and demonstrate how this diversity acts as a primary source of variability in PCP isoforms. Amino acid substitutions are mapped onto the A. carterae PCP crystal structure to consider potential functional significance of variable residues, especially with regard to how polymorphic sites may influence the spectral tuning of peridinins.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 Literature Cited
 
Algal Cultures
Dr. Robert K. Trench kindly donated Symbiodinium 203 from his culture collection at University of California Santa Barbara. Uni-algal subcultures were maintained in 0.45 mm filtered 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 µ mole quanta/m2/s on a 12:12 light-to-dark cycle at 27°C. Cultures were serially transferred every 3 to 4 weeks. L. polyedra (CCMP number 1738) was obtained from the Provasoli – Guillard National Center for Culture of Marine Phytoplankton. Subcultures of L. polyedra were grown 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
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 one and two of the Qiagen protocol were replaced by the following: Algal pellets were resuspended in 500 ml of standard 2X CTAB buffer (Coffroth et al. 1992). Resuspended cells were ground within microcentrifuge tubes and then 4 ml of 10 mg/ml Proteinase K was added to each tube. The tubes were incubated at 65°C for 2 h and were inverted every 30 min. The extractions then carried forward from step three of the Qiagen protocol.

Nucleic acid extractions done by the Rowan and Powers (1991b) method also contained substantial amounts of usable RNA. To purify the RNA for reverse transcription PCR (described below), total nucleic acid preps were diluted 1:20 in 1X DNase buffer and incubated with DNase I (Ambion, Inc.) at 37°C for 1 h. The RNA was phenol/chloroform extracted, ethanol precipitated, desalted, air-dried, and resuspended in RNase-free ddH2O.

Primer Design
Primer used for standard PCR, reverse transcription PCR, and quantitative real-time PCR and for sequencing are listed in table 1. Published PCP gene sequences Symbiodinium from Acropora formosa, A. carterae and L. polyedra were aligned with ClustalX to identify conserved regions and initial design of primer set U325/L537 (Norris and Miller 1994; Sharples et al. 1996; Le et al. 1997; Thompson et al. 1997). Thereafter, sequences from derived clones were used to design primers. Primer selection for standard and reverse transcription PCR was optimized with Oligo 4.0 (National Biosciences, Inc.). Primers for quantitative real-time PCR were designed with Primer Express (Applied Biosystems, Inc.).


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Table 1 Primers Used for Amplification and Sequencing.

 
PCR Conditions, Identification, and Purification of Products
Standard PCR conditions (Palumbi 1996) were 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). Thermal cycle profiles were adjusted to accommodate the annealing temperature of the primer sets and the length of the expected PCR product (table 2). Each round of PCR included a negative control to check for contamination of reagents. When multiple bands were present in a given reaction, Southern hybridization (Sambrook 1989) identified 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.)


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Table 2 Primer Sets, Annealing, and Extension Parameters.

 
Reverse Transcription PCR
A BcaBEST RNA PCR kit version 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 suggested by the manufacturer (table 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 reverse transcription PCR included controls to check for contamination.

Cloning and Screening Plasmid Libraries
The PCR product from U325/L537 was blunt cloned into the pBluescript II SK vector (Stratagene, Inc.). All other PCR products were cloned into pCR2.1 vectors with TA cloning kits (Invitrogen, Inc.) according to the manufacturer's protocol. Cells from transformed colonies were lysed by boiling in ddH2O and were screened by PCR to detect inserts. Standard minipreps were created for each positive clone. Plasmid DNA was extracted and purified with QIAamp DNA Mini kits (Qiagen, Inc.). PCR products from these clones were gel excised, purified as described above, and eluted in ddH2O for sequencing.

Sequencing and Sequence Analysis
All published sequences for this project were sequenced at least twice and were done in both the forward and the reverse directions. The sequencing annealing and extension parameters are described in table 2. The clone from the U325/L537 PCR product was sequenced with an fmol DNA Cycle Sequencing System kit (Promega, Inc.). Automated sequencing was done for all other clones using BigDye (version 2) Terminator kits (ABI, Inc.), and data was collected on a PerkinElmer ABI PRISM 377 DNA Sequencer. Sequence contigs were assembled with Seqman (DNASTAR, Inc). Translation of predicted proteins and pI estimation with done with Gene Runner version 3.04 (Hastings Software Inc.) and with Edit Seq (DNASTAR, Inc.). GenBank database searches for similar nucleotide and amino acid sequences were done using the Blast algorithm (Altschul et al. 1990). Nucleotide sequences of the clones and an alignment (described below) were submitted to GeneBank under accession numbers AY149122 to AY149139.

PCR Recombination and Fidelity Controls
Three unique clones, amplified from tandem coding regions, were combined in equal quantities and used as templates for PCR reactions that were run under conditions described above. Amplification products from each reaction were TA cloned. Three sets of 16 subclones were isolated and then sequenced with U448 and L423. Each was then identified as being either one of the original templates or a recombinant. The location of the recombination was determined to be in either the coding region or the spacer, and then the observable recombination frequency was calculated as a percentage.

In addition, a complete coding sequence (cds) clone was amplified in five separate PCR reactions under the same conditions as above. A subclone 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. The maximum Taq error rate that would produce the observed number of mutations was estimated. A standard likelihood analysis assuming a binomial distribution was conducted to determine the 95% confidence intervals for the Taq error rate. A comparison was then made to the substitution rate observed within the region common to all genomic complete cds and cDNA clones.

Nucleotide Sequence Divergence
K-Estimator version 5.5 (Comeron 1995, 1999) was used to calculate the number of nonsynonymous (Ka) and synonymous (Ks) nucleotide substitutions per site for all paired comparisons of complete coding regions. No comparison restrictions were introduced, and all base pair sites within 365 codons were analyzed for each comparison. The Kimura two-parameter method was used to correct the number of substitution hits 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 ClustalX (Thompson et al. 1997) and MegaAlign (DNA Star, Inc.). To visually depict the nucleotide sequence divergence, we used PAUP* version 4.0b10 (Swofford 1998) to generate a Neighbor-Joining tree from Kimura two-parameter DNA distances.

PCP Gene Copy Number and Genome Size Estimation
The number of PCP genes per genome for Symbiodinium 203 was calculated by the overall equation:


where {sigma} equals the standard deviation.

The (PCP genes ± {sigma}genes)/(pg genomic DNA) term in equation 1 was determined by quantitative real-time PCR with a PerkinElmer ABI 7700. Data were analyzed with ABI Sequence Detection Systems software version 1.7. Amplifications of a 69-bp PCP gene segment were compared between known amounts of Symbiodinium 203 genomic DNA (10,000 pg) and a dilution series of linearized PCP clone (1,492,000 fg to 14.92 fg in 10-fold dilutions). Concentrations of genomic and plasmid DNA were quantified in triplicate on a Beckman Coulter DU640 spectrophotometer. A 1.492 µg sample of the clone was digested with two units NotI (New England Biolabs, Inc.) at 37°C for 1 h to cut the pUC19 vector at a single site outside the insert. Duplicate reactions were set up for each of the standards and for negative controls. Triplicate reactions were set up for the Symbiodinium 203 genomic samples. Reagents were assembled in master mix and were distributed so that each reaction contained 0.4 mM dNTPs, 10.0 mM Tris-HCl pH 8.3, 50.0 mM KCl, 6.0 mM MgCl2, 1.0% glycerol, 0.01% Tween 20, and 1:4000 dilution of SYBR Green I (Molecular Probes, Inc.), two units of SuperTaq (Ambion, Inc.), 0.1 µM SYBRf938 primer, 0.1 µM SYBRr1006 primer, and the DNA template.To compare PCP copy number to ng of genomic DNA, PCP gene copies/fg of PCP clone were converted as follows:


The (pg genomic DNA ± {sigma}pg)/(genome) term in equation 1 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, Cucci, and Sieracki (1997) protocol for fixing and staining the cells was used. Five hundred microliters of algal cells suspended in F/2 was combined with an equal volume of 4% paraformaldehyde and incubated at 22°C for 15 min, after which Triton X-100 and RNase-A were added to concentrations of 0.0033 % (v/v) and 1 ng/ml, respectively. The solutions were mixed by inversion and kept at room temperature for 20 min. Cells were pelleted by centrifugation at 3000 rpm at 4°C. After decanting the liquid, the fixed cells were resuspended in 500 µl of TE. Triton X-100 concentration was adjusted to 0.002 % (v/v) and 20X PicoGreen (Molecular Probes, Inc.) was added to achieve a final 2X staining concentration. Cells and reagents were mixed by inversion. Cells were stained at 22°C for 1 to 2 h before flow cytometry. PicoGreen Fluorescence of DNA was measured through the FACS FL1 filter at 530 ± 30 nm. The DNA content of Symbiodinium 203 nuclei was then estimated as the ratio of fluorescence from Symbiodinium 203 and L. polyedra times the estimated DNA content of 200 pg/cell for L. polyedra (Holm-Hansen 1969; Spector 1984). The methods from Melissinos (1966) were used to accurately account for the propagation of indeterminate errors.

Amino Acid Substitution Modeling
Predicted amino acid substitutions were mapped onto the crystal structure for the A. carterae PCP trimer 1PPR (Hofmann et al. 1996) using Swiss-PDB Viewer version 3.7(b2) (Glaxo Wellcome, Inc.) as follows: Pairwise 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 polar side-chain residues in polymorphic sites and furanic rings and/or polyene chains of peridinins within the same monomer. A single-layer pdb was rendered, reflecting all changes. Three-dinemsional(??) structure images of selected molecules from the holoprotein were rendered using POV-Ray version 3.1 and version 3.5 (POV-Ray Team). Residue numbers within renderings were based on the 1PPR structure.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 Literature Cited
 
Organization and Diversity of Genomic Coding and Spacer Regions
The cloning strategy for this project is summarized in figure 2. Long PCP coding regions of Symbiodinium from Acropora formosa (L13613), A. carterae (Z50792, and Z50793), and L. polyedra (U93077) shared 70.4% to 90.8% identity and consequently when aligned did not produce large, conserved blocks that could be easily targeted for amplification. However, primers U325 and L537 based on sequence from the 5' half of L13613 successfully amplified a 212-bp fragment from Symbiodinium 203 (fig. 2a). The sequence of the small cloned fragment was identified by a Blast search as partial PCP gene.



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FIG. 2. 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 clones

 
PCP genes from the free-living dinoflagellates A. carterae, L. polyedra, and H. pygmaea were previously shown to exist in tandem arrays (Sharples et al. 1996; Le et al. 1997; Hiller et al. 2001). Although it was not known whether Symbiodinium 203 had long or short PCP genes, we assumed that either variety would also be arranged in tandem. Sequence from the 212-bp fragment was used to design outward facing primers U448 and L423 to amplify between adjacent gene copies. Amplification products of multiple sizes were present in the reaction. A genomic Southern blot probed with the small PCP fragment produced hybridization to a 1.9-kb band (not shown) that was subsequently excised and purified. Direct sequencing of the 1.9-kb PCR product contained many unresolved bases, suggesting that multiple templates were present in the reaction. The 1.9-kb fragments were TA cloned, and a single clone was completely sequenced by primer walking. Open reading frames at the 5' and 3' ends of this clone both translated to PCP and flanked a 903-bp spacer region. This sequence confirmed that the PCP genes from Symbiodinium 203 are arranged in tandem arrays like those from free-living dinoflagellates.

Sequencing additional 1.9-kb clones revealed substitutions among gene copies distributed throughout the coding and spacer regions (fig. 2b). As expected, the spacers contained the majority of the substitutions, including insertions and deletions. Despite the highly variable nature of the spacers, each contained a conserved 13-bp sequence CTTGAATGCAGAA, approximately –201 to –188 bp upstream of the start codon. Nine of the 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 PCP gene promoter.

Spacer sequence was also used to design primers that flank coding regions. Amplification of Symbiodinium 203 genomic DNA with inward-facing primers U(–28) and L(1180) produced a 1.1-kb band. Because direct sequencing of the PCR product indicated that multiple templates were again present, these products were cloned and individually sequenced. Of the 11 clones sequenced, 10 contained a 1,098-bp complete, long PCP coding sequence (fig. 2c). One clone contained a truncated coding region with a stop codon occurring 27 bases earlier than others. All but two of the complete coding regions were distinct from each other in terms of their nucleotide substitutions (97.8% to 99.5% identical). The majority of nucleotide substitutions between Symbiodinium 203 complete coding regions were in synonymous codon positions resulting in Ka/Ks < 1 (mean 0.29 ± 0.18). Only a single pairwise comparison had a Ka/Ks > 1 (= 1.11).

Low PCR Recombination Frequency and High PCR Fidelity
To control for possible generation of recombinant DNA sequences in amplifying genes arrayed in tandem (Bradley and Hillis 1997), a reamplification and subcloning experiment was conducted using pairs of distinctive 1.9-kb clones as the templates. All other PCR conditions were the same as used on Symbiodinium 203 genomic DNA. Sixteen subclones 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 5' and the 3' end were used for the identification. Four of the 48 subclones had different 5' and 3' identities. For each of these recombinants, the 5' and 3' coding regions sequenced completely matched one or the other template, suggesting that recombination had likely occurred in the spacer. One of the subclones was excluded as a contaminant. The observable recombination frequency was 8.5%.

The maximum rate at which Taq error introduced substitutions in the PCR reactions was also estimated. There were no changes introduced into any of the five 1,198-bp subclones amplified from the same template in separate reactions. For the 5,990 bp sequenced, the maximum error rate that would produce zero substitutions is approximately 5 x 10–4 errors/bp with a 95% likelihood confidence interval (0, 5 x 10–4). If our PCR conditions caused Taq errors to occur at the estimated maximum rate, this would only account for seven of the 58 mutations that were present within the 13,685 bp of sequence from the region common to all genomic cds and cDNA clones. Furthermore, if all 58 substitutions were the result of Taq error, then the error rate would have been 4.24 x 10–3 errors/bp, considerably higher than previous estimates of Taq error rates (First 2003).

Diversity Also Expressed at mRNA Level
The surprising diversity found in the genomic clones was also present in cDNA clones. Poly-A mRNA from Symbiodinium 203 was reverse transcribed with an oligo dT primer and then cDNA amplified with the U3/L913 primer set (fig. 2d). A single band with the expected size was observed after gel electrophoresis, and this was excised, purified, and cloned. As with the previous genomic amplifications, individual cDNA clones had diversified nucleotide sequences. Eight cDNA clones were sequenced and none were identical to the corresponding regions in the complete cds clones. Seven of these cDNAs were unique. The absence of any insertions in the complete coding sequences compared with the cDNAs is evidence that Symbiodinium 203 PCP genes, like L. polyedra and H. pygmaea, are intronless (Le et al. 1997; Hiller et al. 2001). The overall diversity of genomic and cDNA nucleotide sequences is graphically depicted in the Neighbor-Joining tree in figure 3.



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FIG. 3. Neighboring-Joining tree of Symbiodinium 203 genomic and cDNA nucleotide sequences

 
Symbiodinium 203 Gene Family Size
Quantitative real-time PCR and flow cytometry were used to estimate the number of PCP genes per Symbiodinium 203 genome. There was a 0.996 correlation coefficient between all points (unknowns and standards) on the standard curve generated by quantitative real-time PCR (curve not shown). The mean amplification value for three replicates of 10 ng of genomic DNA was 667.60 ± 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 complete cds clone, which converts (with equation 2) to 12 ± 0.9 PCP genes per pg of genomic DNA.

The genome size of Symbiodinium 203 was compared with the 200-pg genome of L. polyedra (Holm-Hansen 1969; Spector 1984) by flow cytometry to estimate the DNA content per nucleus. Table 3 shows the comparison of relative mean fluorescence of PicoGreen-stained DNA from at least 3,000 cells of each species. The flow cytometry results indicate that Symbiodinium 203 has 3 ± 1 pg DNA per genome. Although Blank and Huss (1989) determined that the genome size of S. microadriaticum was larger than 2 x 108 bp (0.2 pg), no other estimates for additional species in this genus were found. By combining the quantitative real-time PCR and flow cytometry results within equation 1, we calculated that the Symbiodinium 203 genome contains 36 ± 12 PCP gene copies.


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Table 3 Relative Fluorescence of DNA Stained with PicoGreen Based on Flow Cytometry from at Least 3,000 Cells per Species.

 
Predicted Proteins, Isoelectric Points, and Amino Acid Substitutions
There was considerable variation between amino acid sequences predicted from complete coding regions. All but one clone (which had a stop codon 27 bp upstream of others) coded for 365-aa PCP preproteins with 52-aa transit peptides and 313-aa apoproteins. Preproteins ranged from 96.2% to 99.7% identical when compared with each other and 93.3% to 94.9% when compared with Symbiodinium sp.L13613. Within the Symbiodinium 203 transit peptides and apoproteins, there were nine 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 encodes a 375-aa preprotein with a 59-aa transit peptide and a 316-aa apoprotein. Variability in long PCP mass is attributable to amino acid composition and also to polypeptide length.

Most of the predicted apoproteins from our clones had subtle differences in their mass, but they also varied in terms of their calculated isoelectric points (table 4). The pIs for each of the clones fell within a range of pH 5.73 to 6.78. Most isoforms from A. carterae and Glenodinium (Heterocapsa) sp. have a basic pI (Haxo et al. 1976; Prezelin and Haxo 1976). However, there are several examples of symbiotic dinoflagellates that produce predominantly acidic PCP isoforms with pI ranges similar to those predicted here, including 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.


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Table 4 Calculated Mass and Isoelectric Points from Symbiodinium 203 Apoproteins.

 
The variability of calculated Symbiodinium 203 PCP pIs is a direct result of predicted amino acid substitutions. To investigate possible significance of these substitutions to PCP function, a composite of these changes was mapped onto the A. carterae PCP crystal structure 1PPR. There were 70 sites at which Symbiodinium 203 cds and A. carterae 1PPR sequences differed (excluding 5' and 3' gaps). Among these sites, 44 were fixed substitutions between Symbiodinium 203 and A. carterae 1PPR, and 26 were polymorphic between individual Symbiodinium 203 clones. Table 5 lists the specific amino acids and types of side chains substituted at each polymorphic site. Eleven of the polymorphic sites (positions 6, 24,118,134,137,182, 239,244, 253, 275, and 287) were predicted to accommodate the presence or absence of amino acids with polar side chains. Figure 4 is a rendering of all 11 polar substitutions showing their spatial orientations in relation to the eight peridinins within 1PPR. Thr118, Ser253, and Ser287 are of particular interest because of their proximities to peridinin furanic rings and polyene chains and possible influence on spectral tuning of these chromophores (fig. 5). The polar side chain of Thr118 is 7.19 from PID612. Ser253 is 10.06 from PID624. Ser287 is 8.99 from PID622 and 9.15 from PID621.


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Table 5 Substitutions Within Polymorphic Sites of Predicted Symbiodinium 203 PCP Apoproteins Compared with 1PPR Amino Acids.

 


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FIG. 4. Substitutions of amino acids with polar side chains occurring at polymorphic sites within Symbiodinium 203 predicted apoproteins shown in relation to peridinins. Numbering is based in PPR structure

 


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FIG. 5. Distances (E = ) between the polar side chains of (a) threonine 118, (b) serine 253, and (c) serine 287 and the furanic ring and polyene of nearest peridinins

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 Literature Cited
 
PCP Gene family Organization and Diversity
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, L. polyedra, and H. pygmaea (Sharples et al. 1996; Le et al. 1997; Hiller et al. 2001). There are only two previous reports of Symbiodinium PCP nucleotide sequences and both were single cDNAs (Norris and Miller 1994; Weis, Verde, and Reynolds 2002). The results of our molecular experiments demonstrate that the PCP genes of Symbiodinium 203 are organized essentially like those of A. carterae, L. polyedra, and H. pygmaea in that the genes are arranged in tandem arrays. The coding regions specify long PCP polypeptides and 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 those of H. pygmaea. The putative promoter sequence that was identified in the same relative location upstream of Symbiodinium 203 coding regions was 69% identical to the L. polyedra promoter described by Li and Hastings (1998). Dinoflagellate promoters do not fit into the common motifs used by other eukaryotes and may be genus or species specific.

There is far greater nucleotide diversity in the coding regions of Symbiodinium 203 PCP genes than previously described from any other dinoflagellate species. Eighty nine percent of positive clones screened from genomic and cDNA PCR libraries were distinct at the nucleotide level. The heterogeneity of Symbiodinium 203 PCP genes appears to be inconsistent with a pattern of concerted evolution. Concerted evolution of tandem repeated sequences such as the coding regions for ribosomal RNA is usually explained in terms of two mechanisms: continual expansion and contraction due to unequal crossing over and biased gene conversion (see Hillis et al. 1991). Both mechanisms rely on duplex formation between homologous loci as most often occurs in meiosis. Despite reported sexual reproduction in some dinoflagellate genera and RAPDs suggesting sexual recombination in Symbiodinium, there is an apparent absence of a haploid sexual phase in this genus, even though microsatellite evidence indicates that Symbiodinium vegetative cells are haploid (Schoenberg and Trench 1980; Pfiester 1984; Trench 1993; Baillie et al. 2000; Santos and Coffroth 2003). Homogeneity of ribosomal coding regions within individual species suggests that concerted evolution does occur within Symbiodinium genomes (see Rowan and Powers 1991a,1992; Wilcox 1998). Nevertheless, the diversity of Symbiodinium 203 PCP genes is more comparable to that found in dinoflagellate luciferin-binding protein and rubisco gene families (Lee et al. 1993; Machabee', Wall, and Morse 1994; Rowan et al. 1996). Perhaps mutations are introduced into PCP coding regions 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 are less efficient. The net effect is that PCP genes are evolving under reduced concerted evolution.

The low Ka/Ks ratios for paired comparison between complete coding sequences indicate that the majority of nucleotide substitutions occur at synonymous codon positions and that there is not a clear signal of positive selection across entire coding regions within the Symbiodinium 203 PCP gene family. Rather, these coding regions appear to be under purifying selection. The same is true when the Symbiodinium 203 sequences are compared with A. carterae or Symbiodinium from A. formosa coding sequences (data not shown). This does not exclude the possibility that specific codon sites within PCP genes could be under positive selection. Analyses of PCP gene phylogenies containing sequences from multiple species may detect such sites.

We were concerned that at least part of the PCP gene variation 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 extension 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 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 recombination control are limited in a few regards. Reamplification of small number of cloned DNA templates can be different from amplifying genomic regions. An increase in the number of unique templates available within each reaction could increase recombination frequency. Additionally, 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 our PCR conditions occasionally produced early extension terminations as an artifact of amplifying across spacers, it is possible that AY149122 is a chimera, but it is less likely that the complete cds clones (AY149123 to AY149132) or cDNA clones (AY149133 to AY149139) are recombinants.

Another potential source of artificial substitution is through Taq polymerase errors. Our estimated maximum Taq error rate is 4.5 to 25 times higher than previous reports for Taq (see references within First [2003]), yet our maximum is still about eight times lower than the substitution rate within the region common to genomic cds and cDNA clones. These results strongly suggest that the Symbiodinium 203 PCP coding sequence differences presented here are reliable and were not caused by Taq error.

PCP Gene Family Size
Le et al. (1997) reported that L. polyedra had a PCP gene family of roughly 5,000 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 with many other dinoflagellates and other eukaryotes (Holm-Hansen 1969; Spector 1984). For perspective, human haploid cells typically have 3.5 pg per nucleus (Gregory 2001). The L. polyedra copy number was based on intensity of hybridization signals of slot blots. We sought to further refine the methods of estimating PCP gene family size in this investigation through a combination of quantitative real-time PCR and flow cytometry. Quantitative real-time PCR is an extremely accurate and reliable way of determining the amount of starting template within amplification reactions when compared with known standards and was used here to give a mean 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, Cucci, and Sieracki (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. We 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 an armored theca, whereas Symbiodinium 203 cells do not, potentially resulting in underestimation the genome size of Symbiodinium 203. In addition, the wide range between the genome sizes of these species makes calibration difficult. Nevertheless, comparing fluorescence of stained DNA between these two species is likely to be more relevant than comparing either with commercially available standards. 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 L. polyedra, and the Symbiodinium 203 genome size is comparable to A. carterae (Holm-Hansen 1969; Spector 1984). The PCP gene copy number estimates for L. polyedra, H. pygmaea, and Symbiodinium 203 all rely on the accuracy of the Holm-Hansen (1969) algal DNA content values based on amount of nuclear organic carbon. As the DNA content of dinoflagellates is measured with additional techniques, the current PCP gene family sizes may be revised. If the Symbiodinium 203 PCP gene family size is accurate, then the unique coding sequences that we identified represent 35% to 71% of the genes. Furthermore, if the diversity between our clones reflects the overall diversity within the gene family, then there may be as many as 42 unique PCP coding regions present in this species.

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 with free-living species. The test of this hypothesis awaits the characterization of PCP gene families from several more species of each type.

Affects of Genetic Diversity on Predicted PCP Apoproteins
Although there are no reports of empirically determined pIs for Symbiodinium 203 PCP isoforms, translation of coding sequences cloned in this project demonstrate that there is sufficient genetic diversity to account for a suite of pIs comparable with those found in several other Symbiodinium species through protein analysis. This does not rule out the possibility that posttranslational modification of PCP polypeptides could still occur. Our results suggest that posttranslational modifications are not necessary to explain the multiple PCP isoforms. Isoelectric focusing of PCPs from Symbiodinium 203 could test this point, as could additional PCR-based characterizations of PCP coding sequences from species for which PCP pIs are already known.

The potential for examining functionally significant differences between predicted PCP apoproteins was greatly enhanced by mapping amino acid substitutions onto 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 44 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 different conformations than 1PPR. Eleven of the 26 polymorphic sites hold amino acids with or without polar side chains. The apoproteins from two clones contain polar substitutions with side chain groups in positions that could influence the spectral tuning of nearby peridinins. There may still be additional members of the Symbiodinium 203 PCP gene family that produce isoforms with similar effects that went undetected. Predicting the direction and magnitude of overall changes to the spectroscopic properties of holoproteins is beyond the scope of this investigation. Distances calculated by between polar side chains and peridinins could shift for individual apoproteins if substitutions from single clones were introduced separately within renderings.


    Conclusion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 Literature Cited
 
Symbiodinium 203 has long PCP genes that are intronless and arranged in tandem like those of A. carterae and L. polyedra but with a putative promoter that is different from L. polyedra. There are at least 17 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 provides the primary source of variability in PCP isoforms. Amino acid substitutions in Symbiodinium 203's PCP apoproteins result in shifts of isoelectric points and potentially 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.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 Literature Cited
 
We would like to thank Professor Robert K. Trench formerly of UC Santa Barbara for donation of Symbiodinium cultures and exchange of ideas. Dr. Eric Lader provided valuable assistance with the quantitative real-time PCR experiments. Paul Thompson, Walter Hokanson, and Dr. E. Henry Lee assisted with statistics. Undergraduate laboratory assistants Maria Polycarpo and Tasmin Smith gave considerable help with PCR and sequencing routines. The Caribbean Marine Research Center Perry Foundation, the U.S National Oceanic and Atmospheric Administration, Oryx, Inc. and The University of Texas at Austin provided funding for this project.


    Footnotes
 
Claudia Kappen, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
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Accepted for publication July 30, 2003.





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