Dinoflagellate Genes Research Articles

Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic

Dinoflagellates are an important component of the marine biota, but a large genome with high-copy number (up to 5,000) tandem gene arrays has made genomic sequencing problematic. More importantly, little is known about the expression and conservation of these unusual gene arrays. We assembled de novo a gene catalog of 74,655 contigs for the dinoflagellate Lingulodinium polyedrum from RNA-Seq (Illumina) reads. The catalog contains 93% of a Lingulodinium EST dataset deposited in GenBank and 94% of the enzymes in 16 primary metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, indicating it is a good representation of the transcriptome. Analysis of the catalog shows a marked underrepresentation of DNA-binding proteins and DNA-binding domains compared with other algae. Despite this, we found no evidence to support the proposal of polycistronic transcription, including a marked underrepresentation of sequences corresponding to the intergenic spacers of two tandem array genes. We also have used RNA-Seq to assess the degree of sequence conservation in tandem array genes and found their transcripts to be highly conserved. Interestingly, some of the sequences in the catalog have only bacterial homologs and are potential candidates for horizontal gene transfer. These presumably were transferred as single-copy genes, and because they are now all GC-rich, any derived from AT-rich contexts must have experienced extensive mutation. Our study not only has provided the most complete dinoflagellate gene catalog known to date, it has also exploited RNA-Seq to address fundamental issues in basic transcription mechanisms and sequence conservation in these algae.

Transcriptomic response of the red tide dinoflagellate, Karenia brevis, to nitrogen and phosphorus depletion and addition.

BackgroundThe role of coastal nutrient sources in the persistence of Karenia brevis red tides in coastal waters of Florida is a contentious issue that warrants investigation into the regulation of nutrient responses in this dinoflagellate. In other phytoplankton studied, nutrient status is reflected by the expression levels of N- and P-responsive gene transcripts. In dinoflagellates, however, many processes are regulated post-transcriptionally. All nuclear encoded gene transcripts studied to date possess a 5' trans-spliced leader (SL) sequence suggestive, based on the trypanosome model, of post-transcriptional regulation. The current study therefore sought to determine if the transcriptome of K. brevis is responsive to nitrogen and phosphorus and is informative of nutrient status.ResultsMicroarray analysis of N-depleted K. brevis cultures revealed an increase in the expression of transcripts involved in N-assimilation (nitrate and ammonium transporters, glutamine synthetases) relative to nutrient replete cells. In contrast, a transcriptional signal of P-starvation was not apparent despite evidence of P-starvation based on their rapid growth response to P-addition. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. Both N- and P-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p ≤ 10-4. By far, the earliest responding genes were dominated in both nutrient treatments by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 1 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes.ConclusionsMicroarray analysis provided transcriptomic evidence for N- but not P-limitation in K. brevis. Transcriptomic responses to the addition of either N or P suggest a concerted program leading to the reactivation of chloroplast functions. Even the earliest responding PPR protein transcripts possess a 5' SL sequence that suggests post-transcriptional control. Given the current state of knowledge of dinoflagellate gene regulation, it is currently unclear how these rapid changes in such transcript levels are achieved.

Post-transcriptional control of circadian rhythms

Circadian rhythms exist in most living organisms. The general molecular mechanisms that are used to generate 24-hour rhythms are conserved among organisms, although the details vary. These core clocks consist of multiple regulatory feedback loops, and must be coordinated and orchestrated appropriately for the fine-tuning of the 24-hour period. Many levels of regulation are important for the proper functioning of the circadian clock, including transcriptional, post-transcriptional and post-translational mechanisms. In recent years, new information about post-transcriptional regulation in the circadian system has been discovered. Such regulation has been shown to alter the phase and amplitude of rhythmic mRNA and protein expression in many organisms. Therefore, this Commentary will provide an overview of current knowledge of post-transcriptional regulation of the clock genes and clock-controlled genes in dinoflagellates, plants, fungi and animals. This article will also highlight how circadian gene expression is modulated by post-transcriptional mechanisms and how this is crucial for robust circadian rhythmicity.

Comparative gene expression in toxic versus non-toxic strains of the marine dinoflagellate Alexandrium minutum

BackgroundThe dinoflagellate Alexandrium minutum typically produces paralytic shellfish poisoning (PSP) toxins, which are known only from cyanobacteria and dinoflagellates. While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production in dinoflagellates remains elusive.ResultsWe constructed and analysed an expressed sequence tag (EST) library of A. minutum, which contained 15,703 read sequences yielding a total of 4,320 unique expressed clusters. Of these clusters, 72% combined the forward-and reverse reads of at least one bacterial clone. This sequence resource was then used to construct an oligonucleotide microarray. We analysed the expression of all clusters in three different strains. While the cyanobacterial PSP toxin genes were not found among the A. minutum sequences, 192 genes were differentially expressed between toxic and non-toxic strains.ConclusionsBased on this study and on the lack of identified PSP synthesis genes in the two existent Alexandrium tamarense EST libraries, we propose that the PSP toxin genes in dinoflagellates might be more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer. As a starting point to identify possible PSP toxin-associated genes in dinoflagellates without relying on a priori sequence information, the sequences only present in mRNA pools of the toxic strain can be seen as putative candidates involved in toxin synthesis and regulation, or acclimation to intracellular PSP toxins.

From Stop to Start: Tandem Gene Arrangement, Copy Number and Trans-Splicing Sites in the Dinoflagellate Amphidinium carterae

Dinoflagellate genomes present unique challenges including large size, modified DNA bases, lack of nucleosomes, and condensed chromosomes. EST sequencing has shown that many genes are found as many slightly different variants implying that many copies are present in the genome. As a preliminary survey of the genome our goal was to obtain genomic sequences for 47 genes from the dinoflagellate Amphidinium carterae. A PCR approach was used to avoid problems with large insert libraries. One primer set was oriented inward to amplify the genomic complement of the cDNA and a second primer set would amplify outward between tandem repeats of the same gene. Each gene was also tested for a spliced leader using cDNA as template. Almost all (14/15) of the highly expressed genes (i.e. those with high representation in the cDNA pool) were shown to be in tandem arrays with short intergenic spacers, and most were trans-spliced. Only two moderately expressed genes were found in tandem arrays. A polyadenylation signal was found in genomic copies containing the sequence AAAAG/C at the exact polyadenylation site and was conserved between species. Four genes were found to have a high intron density (>5 introns) while most either lacked introns, or had only one to three. Actin was selected for deeper sequencing of both genomic and cDNA copies. Two clusters of actin copies were found, separated from each other by many non-coding features such as intron size and sequence. One intron-rich gene was selected for genomic walking using inverse PCR, and was not shown to be in a tandem repeat. The first glimpse of dinoflagellate genome indicates two general categories of genes in dinoflagellates, a highly expressed tandem repeat class and an intron rich less expressed class. This combination of features appears to be unique among eukaryotes.

Molecular Characterization of Iron-Containing Superoxide Dismutases in the Heterotrophic Dinoflagellate Crypthecodinium cohnii

Superoxide dismutases (SODs) are a family of antioxidant enzymes that catalyse the degradation of toxic superoxide radicals in obligate and facultative aerobic organisms. Here, we report the presence of a multi-copy gene family encoding SODs in the heterotrophic dinoflagellate Crypthecodinium cohnii. All the genes identified (sod1 to sod17) have been cloned and sequenced, and shown to encode potentially functional dimeric iron-containing SOD isozymes. Our data revealed a considerable molecular heterogeneity of this enzyme in C. cohnii at both genomic and transcriptional levels. The C. cohnii SOD1, overexpressed in Escherichia coli, was active and its structure obtained by homology modeling using X-ray crystal structures of homologues exhibited the typical fold of dimeric FeSODs. Phylogenetic studies including 110 other dimeric FeSODs and closely related cambialistic dimeric SOD sequences showed that the C. cohnii SODs form a monophyletic group and have all been acquired by the same event of horizontal gene transfer. It also revealed a dichotomy within the C. cohnii SOD sequences that could be explained by an ancestral sod gene duplication followed by subsequent gene duplications within each of the two groups. Enzyme assays of SOD activity indicated the presence of two FeSOD activities in C. cohnii cell lysate whereas MnSOD and Cu/ZnSOD were not detected. These activities contrasted with the SOD repertoire previously characterized in photosynthetic dinoflagellates. To explain these differences, a hypothetical evolutionary scenario is proposed that suggests gains and losses of sod genes in dinoflagellates.

MICROARRAY ANALYSIS OF DIURNAL‐ AND CIRCADIAN‐REGULATED GENES IN THE FLORIDA RED‐TIDE DINOFLAGELLATE KARENIA BREVIS (DINOPHYCEAE)1

The photoperiod plays a central role in regulating the physiology and behavior of photosynthetic phytoplankton, and many of these processes are controlled by an underlying circadian rhythm. In dinoflagellates, circadian rhythms have been shown to depend largely on posttranscriptional regulation. However, the extent to which dinoflagellates modulate transcript levels to regulate gene expression in response to light and dark has not been addressed. Here we utilized an oligonucleotide microarray containing probes to 4629 unique genes from the Florida red‐tide dinoflagellate Karenia brevis (C. C. Davis) G. Hansen et Moestrup to characterize global gene expression patterns over a 24 h day in cells exposed to a 16:8 light:dark cycle (LD treatment) and cells under constant light (LL treatment). We determined that 9.8% of the queried genes were differentially expressed during LD, while 3% of genes were differentially expressed over the 24 h day when exposed to LL. Most genes exhibited either peak or minimum expression in early dark phase. Of the 453 differentially expressed genes in LD, 104 were assigned predicted annotations based on BLASTX searches. Few of the identifiable genes that were differentially expressed in LD appear to be under circadian control, as their cyclical expression did not persist in LL. Analysis of coordinately expressed genes revealed several novel insights into dinoflagellate gene expression, including an unusually high representation of genes involved in post‐transcriptional processing of RNA and protein turnover, the regulation of PSII genes in response to light and dark, and the absence of transcriptional regulation of cell‐cycle genes.

S-adenosylmethionine synthetase genes from eleven marine dinoflagellates

L.D. Harlow, A. Koutoulis and G.M. Hallegraeff. 2007. S-adenosylmethionine synthetase genes from eleven marine dinoflagellates. Phycologia 46: 46–53. DOI: 10.2216/06-28.1Paralytic shellfish poisoning toxins (PSTs) such as saxitoxin are believed to be synthesised from precursor molecules including arginine, S-adenosylmethionine (SAM) and acetate. The enzyme SAM synthetase is a good candidate as an early acting enzyme in the PST biosynthetic pathway. We have used degenerate PCR to identify the gene encoding SAM synthetase, Sam, in dinoflagellates. Several different PCR products were cloned and sequenced from PST-producing strains of the dinoflagellates Alexandrium minutum, A. catenella and Gymnodinium catenatum and primers specific to dinoflagellate Sam used for further sequence analysis in 11 species of toxic and nontoxic dinoflagellate genera, including: Alexandrium, Gymnodinium, Karenia, Karlodinium, Noctiluca, Prorocentrum and Takayama. At the nucleotide level, Sam clones were unique to dinoflagellates and the most commonly identified Sam was highly conserved between dinoflagellate species. Dinoflagellate Sam G + C content was both typical (60.8%) or lower (40.7%) compared to A. tamarense. The derived protein sequences showed a low and equal level of similarity to Sam from other species representing a range of Kingdoms. DNA sequence information for dinoflagellate Sam provides important insights for dinoflagellate codon usage, which will facilitate primer design to target novel dinoflagellate genes.

Topology of splicing and snRNP biogenesis in dinoflagellate nuclei

Dinoflagellates are protists that are hypothesized to have experienced a secondary loss of histones. Amongst eukaryotes, they are unique in lacking these proteins. To date, information on the mechanisms involving remodelling, transcription and splicing of their chromatin is limited. Dinoflagellate genes lack TATA boxes and downstream polyadenylation sites and particular linear arrangements. They have an alpha-amanitin-sensitive RNA polymerase, specific transcription factors and regulators, and both transcriptional and post-transcriptional regulation of gene expression. Dinoflagellates produce either polycistronic or discrete mRNAs, and have conserved snRNAs (small nuclear RNAs), indicating that their genes are spliced. Five representative dinoflagellate species (Amphidinium carterae, Akashiwo sanguinea, Alexandrium lusitanicum, Alexandrium fundyense and Prorocentrum micans), which show diversity in their DNA content, nuclear organization and taxonomic position, were investigated. The nuclear distribution and ultrastructural organization of splicing and snRNP (small nuclear ribonucleoprotein) biogenesis were determined by fluorescent and electron microscopy immunolabelling with Y12 sera [recognizing the sDMA (symmetrical dimethylarginine) domain of Sm and other nuclear proteins], anti-p105-PANA [proliferation-associated nuclear antigen; a marker of IGs (interchromatin granules)] and anti-DNA antibodies. In parallel, ultrastructural analysis, including cytochemical staining for RNA, phosphorylated proteins and DNA, was carried out. Splicing factors were distributed in a diffuse perichromosomal layer containing perichromatin granules and fibrils that co-localized with the decondensed peripheral DNA loops, but not with the main chromosome body. Interchromosomal domains with IGs and Cajal-like bodies were also detected. Dinoflagellates are rather dissimilar to other eukaryotes in their genomes, their mechanisms of gene expression and their chromosome ultrastructure. However, they share common splicing nuclear domains and snRNP biogenesis with that of other eukaryotes.

EFL GTPase in Cryptomonads and the Distribution of EFL and EF-1α in Chromalveolates

EFL (EF-like protein) is a member of the GTPase superfamily that includes several translation factors. Because it has only been found in a few eukaryotic lineages and its presence correlates with the absence of the related core translation factor EF-1alpha, its distribution is hypothesized to be the result of lateral gene transfer and replacement of EF-1alpha. In one supergroup of eukaryotes, the chromalveolates, two major lineages were found to contain EFL (dinoflagellates and haptophytes), while the others encode EF-1alpha (apicomplexans, ciliates, heterokonts and cryptomonads). For each of these groups, this distribution was deduced from whole genome sequence or expressed sequence tag (EST) data from several species, with the exception of cryptomonads from which only a single EF-1alpha PCR product from one species was known. By sequencing ESTs from two cryptomonads, Guillardia theta and Rhodomonas salina, and searching for all GTPase translation factors, we revealed that EFL is present in both species, but, contrary to expectations, we found EF-1alpha in neither. On balance, we suggest the previously reported EF-1alpha from Rhodomonas salina is likely an artefact of contamination. We also identified EFL in EST data from two members of the dinoflagellate lineage, Karlodinium micrum and Oxyrrhis marina, and from an ongoing genomic sequence project from a third, Perkinsus marinus. Karlodinium micrum is a symbiotic pairing of two lineages that would have both had EFL (a dinoflagellate and a haptophyte), but only the dinoflagellate gene remains. Oxyrrhis marina and Perkinsus marinus are early diverging sister-groups to dinoflagellates, and together show that EFL originated early in this lineage. Phylogenetic analysis confirmed that these genes are all EFL homologues, and showed that cryptomonad genes are not detectably related to EFL from other chromalveolates, which collectively form several distinct groups. The known distribution of EFL now includes a third group of chromalveolates, cryptomonads. Of the six major subgroups of chromalveolates, EFL is found in half and EF-1alpha in the other half, and none as yet unambiguously possess both genes. Phylogenetic analysis indicates EFL likely arose early within each subgroup where it is found, but suggests it may have originated multiple times within chromalveolates as a whole.

Global transcriptional profiling of the toxic dinoflagellate Alexandrium fundyense using Massively Parallel Signature Sequencing

BackgroundDinoflagellates are one of the most important classes of marine and freshwater algae, notable both for their functional diversity and ecological significance. They occur naturally as free-living cells, as endosymbionts of marine invertebrates and are well known for their involvement in "red tides". Dinoflagellates are also notable for their unusual genome content and structure, which suggests that the organization and regulation of dinoflagellate genes may be very different from that of most eukaryotes. To investigate the content and regulation of the dinoflagellate genome, we performed a global analysis of the transcriptome of the toxic dinoflagellate Alexandrium fundyense under nitrate- and phosphate-limited conditions using Massively Parallel Signature Sequencing (MPSS).ResultsData from the two MPSS libraries showed that the number of unique signatures found in A. fundyense cells is similar to that of humans and Arabidopsis thaliana, two eukaryotes that have been extensively analyzed using this method. The general distribution, abundance and expression patterns of the A. fundyense signatures were also quite similar to other eukaryotes, and at least 10% of the A. fundyense signatures were differentially expressed between the two conditions. RACE amplification and sequencing of a subset of signatures showed that multiple signatures arose from sequence variants of a single gene. Single signatures also mapped to different sequence variants of the same gene.ConclusionThe MPSS data presented here provide a quantitative view of the transcriptome and its regulation in these unusual single-celled eukaryotes. The observed signature abundance and distribution in Alexandrium is similar to that of other eukaryotes that have been analyzed using MPSS. Results of signature mapping via RACE indicate that many signatures result from sequence variants of individual genes. These data add to the growing body of evidence for widespread gene duplication in dinoflagellates, which would contribute to the transcriptional complexity of these organisms. The MPSS data also demonstrate that a significant number of dinoflagellate mRNAs are transcriptionally regulated, indicating that dinoflagellates commonly employ transcriptional gene regulation along with the post-transcriptional regulation that has been well documented in these organisms.

NOVEL AND RAPIDLY DIVERGING INTERGENIC SEQUENCES BETWEEN TANDEM REPEATS OF THE LUCIFERASE GENES IN SEVEN DINOFLAGELLATE SPECIES1

Tandemly arranged luciferase genes were previously reported in two dinoflagellates species, but their intergenic regions were strikingly different and no canonical promoter sequences were found. Here, we examined the intergenic regions of the luciferase genes of five other dinoflagellate species along with those of the earlier two. In all cases, the genes exist in multiple copies and are arranged tandemly, coding for proteins of similar sizes and sequences. However, the 5′ untranslated region, 3′ untranslated region, and intergenic regions of the seven genes differ greatly in length and sequence, except for two stretches that are conserved in the intergenic regions of two pairs of phylogenetically close species. Microsatellites and minisatellites were detected in the intergenic sequences of four species, Alexandrium affine (H. Inoue & Y. Fukuyo) E. Balech, A. tamarense (Lebour) E. Balech, Protoceratium reticulatum (Claparède & Lachmann) Butschli, and Pyrocystis lunula (Schütt) Schütt, the first three of which have unusually high percentages of particular sets of dinucleotides. Most remarkably, the P. reticulatum intergenic region is almost exclusively made up of 19 nearly identical repeats of an 11‐nucleotide sequence. Dinoflagellate luciferase intergenic regions bear similarities to ribosomal genes and to some protein‐encoding genes in trypanosomes, both of which are transcribed by RNA polymerase I. It is possible that the transcription of the dinoflagellate genes are catalyzed by an RNA polymerase with novel properties.

Rate Variation as a Function of Gene Origin in Plastid-Derived Genes of Peridinin-Containing Dinoflagellates

Peridinin-pigmented dinoflagellates contain secondary plastids that seem to have undergone more nearly complete plastid genome reduction than other eukaryotes. Many typically plastid-encoded genes appear to have been transferred to the nucleus, with a few remaining genes found on minicircles. To understand better the evolution of the dinoflagellate plastid, four categories of plastid-associated genes in dinoflagellates were defined based on their history of transfer and evaluated for rate of sequence evolution, including minicircle genes (presumably plastid-encoded), genes probably transferred from the plastid to the nucleus (plastid-transferred), and genes that were likely acquired directly from the nucleus of the previous plastid host (nuclear-transferred). The fourth category, lateral-transferred genes, are plastid-associated genes that do not appear to have a cyanobacterial origin. The evolutionary rates of these gene categories were compared using relative rate tests and likelihood ratio tests. For comparison with other secondary plastid-containing organisms, rates were calculated for the homologous sequences from the haptophyte Emiliania huxleyi. The evolutionary rate of minicircle and plastid-transferred genes in the dinoflagellate was strikingly higher than that of nuclear-transferred and lateral-transferred genes and, also, substantially higher than that of all plastid-associated genes in the haptophyte. Plastid-transferred genes in the dinoflagellate had an accelerated rate of evolution that was variable but, in most cases, not as extreme as the minicircle genes. Furthermore, the nuclear-transferred and lateral-transferred genes showed rates of evolution that are similar to those of other taxa. Thus, nucleus-to-nucleus transferred genes have a more typical rate of sequence evolution, while those whose history was wholly or partially within the dinoflagellate plastid genome have a markedly accelerated rate of evolution.

A Transcriptional Fusion of Genes Encoding Glyceraldehyde‐3‐Phosphate Dehydrogenase (GAPDH) and Enolase in Dinoflagellates

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase are enzymes essential for glycolysis and gluconeogenesis. Dinoflagellates possess several types of both GAPDH and enolase genes. Here, we identify a novel cytosolic GAPDH-enolase fusion protein in several dinoflagellate species. Phylogenetic analyses revealed that the GAPDH moiety of this fusion is weakly related to a cytosolic GAPDH previously reported in dinoflagellates, ciliates, and an apicomplexan. The enolase moiety has phylogenetic affinity with sequences from ciliates and apicomplexans, as expected for dinoflagellate genes. Furthermore, the enolase moiety has two insertions in a highly conserved region of the gene that are shared with ciliate and apicomplexan homologues, as well as with land plants, stramenopiles, haptophytes, and a chlorarachniophyte. Another glycolytic gene fusion in eukaryotes is the mitochondrion-targeted triose-phosphate isomerase (TPI) and GAPDH fusion in stramenopiles (i.e. diatoms and oomycetes). However, unlike the mitochondrial TPI-GAPDH fusion, the GAPDH-enolase fusion protein appears to exist in the same compartment as stand-alone homologues of each protein, and the metabolic reactions they catalyze in glycolysis and gluconeogenesis are not directly sequential. It is possible that the fusion is post-translationally processed to give separate GAPDH and enolase products, or that the fusion protein may function as a single bifunctional polypeptide in glycolysis, gluconeogenesis, or perhaps more likely in some previously unrecognized metabolic capacity.

Insights into a dinoflagellate genome through expressed sequence tag analysis

BackgroundDinoflagellates are important marine primary producers and grazers and cause toxic "red tides". These taxa are characterized by many unique features such as immense genomes, the absence of nucleosomes, and photosynthetic organelles (plastids) that have been gained and lost multiple times. We generated EST sequences from non-normalized and normalized cDNA libraries from a culture of the toxic species Alexandrium tamarense to elucidate dinoflagellate evolution. Previous analyses of these data have clarified plastid origin and here we study the gene content, annotate the ESTs, and analyze the genes that are putatively involved in DNA packaging.ResultsApproximately 20% of the 6,723 unique (11,171 total 3'-reads) ESTs data could be annotated using Blast searches against GenBank. Several putative dinoflagellate-specific mRNAs were identified, including one novel plastid protein. Dinoflagellate genes, similar to other eukaryotes, have a high GC-content that is reflected in the amino acid codon usage. Highly represented transcripts include histone-like (HLP) and luciferin binding proteins and several genes occur in families that encode nearly identical proteins. We also identified rare transcripts encoding a predicted protein highly similar to histone H2A.X. We speculate this histone may be retained for its role in DNA double-strand break repair.ConclusionThis is the most extensive collection to date of ESTs from a toxic dinoflagellate. These data will be instrumental to future research to understand the unique and complex cell biology of these organisms and for potentially identifying the genes involved in toxin production.

Isolation of a dinoflagellate mitotic cyclin by functional complementation in yeast

Dinoflagellates are protists with permanently condensed chromosomes that lack histones and whose nuclear membrane remains intact during mitosis. These unusual nuclear characters have suggested that the typical cell cycle regulators might be slightly different than those in more typical eukaryotes. To test this, a cyclin has been isolated from the dinoflagellate Gonyaulax polyedra by functional complementation in cln123 mutant yeast. This GpCyc1 sequence contains two cyclin domains in its C-terminal region and a degradation box typical of mitotic cyclins. Similar to other dinoflagellate genes, GpCyc1 has a high copy number, with ∼5000 copies found in the Gonyaulax genome. An antibody raised against the N-terminal region of the GpCYC1 reacts with a 68 kDa protein on Western blots that is more abundant in cell cultures enriched for G2-phase cells than in those containing primarily G1-phase cells, indicating its cellular level follows a pattern expected for a mitotic cyclin. This is the first report of a cell cycle regulator cloned and sequenced from a dinoflagellate, and our results suggest control of the dinoflagellate cell cycle will be very similar to that of other organisms.

COMPLEX GENE STRUCTURE OF THE FORM II RUBISCO IN THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

We analyzed the unusually complex organization of the nuclear‐encoded (form II) RUBISCO gene in the dinoflagellate Prorocentrum minimum (Parvillard) Schiller by intensive genomic DNA and cDNA sequencing and Western blotting. Over 10 transcribed units (TUs) were detected, which varied dramatically in their 3′ untranslated region. Each TU appeared to contain four tandem copies of the RUBISCO coding region (1.46 kb each; coding unit, or CU) interspersed by a 63‐bp spacer; the four CUs in each TU were cotranscribed and apparently cotranslated to a tetrameric polyprotein that may undergo successive cleavage steps to yield mature RUBISCO. By means of real‐time PCR analysis, it was estimated that each of the P. minimum genome harbored 148±16 CUs. Although nucleotide sequences varied by 1%–9% among the detected CUs, their inferred amino acid sequences were essentially identical. Our results suggest that the complex structure of Pmrbc has been derived from extensive and repeated gene duplications, an evolutionary process that has also been observed for other dinoflagellate genes.

184 Expressed Gene Arrays in Three Marine Dinoflagellates with Contrasting Trophic Modes Analyzed Via a Small‐Scale Est Sequencing

In an attempt to determine the common and unique the genes functioning for different trophic modes in dinoflagellates, cDNA libraries were constructed for Prorocentrum minimum (photoautotrophic), Karlodinium micrum (mixotrophic), and Pfiesteria piscicida (kleptoplastic heterotrophic). Randomly selected 100–350 clones were sequenced for each species. Results revealed a small number of genes transcribed in all three species (basic nuclear proteins, ribosomal proteins). There appeared to be a suite of unique genes in each species. In P. piscicida that was fed Rhodomonas sp., the array of expressed genes differed clearly among different growth conditions. The cDNA library prepared for samples collected 12 h after feeding contained more photosynthetic genes (light harvesting complex proteins) and ribosomal proteins, while growth‐related genes were expressed more abundantly at 24 hours after feeding. At 48 hours when growth rate was decreased dramatically, the most abundant gene transcripts found were those involved in metabolism (mt cob, cox1 and cox3). In addition, several genes (ras, rac, chaperonin) appeared to be identical at amino acid level among the two heterotrophic or mixotrophic dinoflagellates (P. piscicida and K. micrum) and a prey alga (Rhodomonas sp.), although the likelihood of contamination was small. These results suggest that some of the dinoflagellate genes may be originated through horizontal transfer from historical prey species and RT‐PCR measurements indicate that these genes maybe highly expressed in dinoflagellates.

A New Class of Transcription Initiation Factors, Intermediate between TATA Box-binding Proteins (TBPs) and TBP-like Factors (TLFs), Is Present in the Marine Unicellular Organism, the Dinoflagellate Crypthecodinium cohnii

Dinoflagellates are marine unicellular eukaryotes that exhibit unique features including a very low level of basic proteins bound to the chromatin and the complete absence of histones and nucleosomal structure. A cDNA encoding a protein with a strong homology to the TATA box-binding proteins (TBP) has been isolated from an expressed sequence tag library of the dinoflagellate Crypthecodinium cohnii. The typical TBP repeat signature and the amino acid motives involved in TFIIA and TFIIB interactions were conserved in this new TBP-like protein. However, the four phenylalanines known to interact with the TATA box were substituted with hydrophilic residues (His(77), Arg(94), Tyr(171), Thr(188)) as has been described for TBP-like factors (TLF)/TBP-related proteins (TRP). A phylogenetic analysis showed that cTBP is intermediate between TBP and TLF/TRP protein families, and the structural similarity of cTBP with TLF was confirmed by low affinity binding to a consensus' TATA box in an equivalent manner to that usually observed for TLFs. Six 5'-upstream gene regions of dinoflagellate genes have been analyzed and neither a TATA box nor a consensus-promoting element could be found within these different sequences. Our results showed that cTBP could bind stronger to a TTTT box sequence than to the canonical TATA box, especially at high salt concentration. Same binding results were obtained with a mutated cTBP (mcTBP), in which the four phenylalanines were restored. To our knowledge, this is the first description of a TBP-like protein in a unicellular organism, which also appears as the major form of TBP present in C. cohnii.

Primer Premier 5

Primer Premier 5