Through the analysis of hundreds of full-length cDNAs from fifteen species representing all major orders of dinoflagellates, we demonstrate that nuclear-encoded mRNAs in all species, from ancestral to derived lineages, are trans-spliced with the addition of the 22-nt conserved spliced leader (SL), DCCGUAGCCAUUUUGGCUCAAG (D ‫؍‬ U, A, or G), to the 5 end. SL trans-splicing has been documented in a limited but diverse number of eukaryotes, in which this process makes it possible to translate polycistronically transcribed nuclear genes. In SL trans-splicing, SL-donor transcripts (SL RNAs) contain two functional domains: an exon that provides the SL for mRNA and an intron that contains a spliceosomal (Sm) binding site. In dinoflagellates, SL RNAs are unusually short at 50 -60 nt, with a conserved Sm binding motif (AUUUUGG) located in the SL (exon) rather than the intron. The initiation nucleotide is predominantly U or A, an unusual feature that may affect capping, and hence the translation and stability of the recipient mRNA. The core SL element was found in mRNAs coding for a diverse array of proteins. Among the transcripts characterized were three homologs of Sm-complex subunits, indicating that the role of the Sm binding site is conserved, even if the location on the SL is not. Because association with an Sm-complex often signals nuclear import for U-rich small nuclear RNAs, it is unclear how this Sm binding site remains on mature mRNAs without impeding cytosolic localization or translation of the latter.inoflagellates are unicellular eukaryotes that contribute significantly to marine primary production, coral reef growth, and marine toxins. They are members of the Alveolata, which also include ciliates and apicomplexa (1). Dinoflagellate genomes are enormous (3-200 pg of DNA per cell) and lack typical histones, with chromosomes permanently condensed, nuclear membranes remaining intact in mitosis, and mitotic spindle being extranuclear (for review see ref.2). The mechanism of gene regulation is largely unknown. Sporadic investigations have shown that a relatively small fraction of genes are under transcriptional control (3-6) and that introns are not common (7,8). The few comprehensive studies of dinoflagellate gene structures reveal genes with high copy number and arrangement in polycistronic or otherwise tandem arrays (e.g., refs. 7-10).Spliced leader (SL) trans-splicing has been found in a disjointed group of eukaryotes, in which a short RNA fragment (i.e., SL, Ϸ15-50 nt) from a small noncoding RNA (SL RNA) is transplanted to the 5Ј end of independently transcribed pre-mRNAs to yield mature mRNAs. This process converts a polycistronic transcript into translatable monocistronic mRNAs. SL trans-splicing has been well studied in Euglenozoa. It has been detected in nematodes, Platyhelminthes, cnidarians, rotifers, ascidians, and appendicularia (for review, see 11-13). SL RNA contains two functional domains: an exon (i.e., SL) that is spliced to an mRNA and an intron that contains a spliceosomal (Sm) binding site be...

Quantitative real-time PCR (qPCR) has become a gold standard for the quantification of nucleic acids and microorganism abundances, in which plasmid DNA carrying the target genes are most commonly used as the standard. A recent study showed that supercoiled circular confirmation of DNA appeared to suppress PCR amplification. However, to what extent to which different structural types of DNA (circular versus linear) used as the standard may affect the quantification accuracy has not been evaluated. In this study, we quantitatively compared qPCR accuracies based on circular plasmid (mostly in supercoiled form) and linear DNA standards (linearized plasmid DNA or PCR amplicons), using proliferating cell nuclear gene (pcna), the ubiquitous eukaryotic gene, in five marine microalgae as a model gene. We observed that PCR using circular plasmids as template gave 2.65-4.38 more of the threshold cycle number than did equimolar linear standards. While the documented genome sequence of the diatom Thalassiosira pseudonana shows a single copy of pcna, qPCR using the circular plasmid as standard yielded an estimate of 7.77 copies of pcna per genome whereas that using the linear standard gave 1.02 copies per genome. We conclude that circular plasmid DNA is unsuitable as a standard, and linear DNA should be used instead, in absolute qPCR. The serious overestimation by the circular plasmid standard is likely due to the undetected lower efficiency of its amplification in the early stage of PCR when the supercoiled plasmid is the dominant template.

The ability to predict gene content is highly desirable for characterization of not-yet sequenced genomes like those of dinoflagellates. Using data from completely sequenced and annotated genomes from phylogenetically diverse lineages, we investigated the relationship between gene content and genome size using regression analyses. Distinct relationships between log10-transformed protein-coding gene number (Y′) versus log10-transformed genome size (X′, genome size in kbp) were found for eukaryotes and non-eukaryotes. Eukaryotes best fit a logarithmic model, Y′ = ln(-46.200+22.678X′, whereas non-eukaryotes a linear model, Y′ = 0.045+0.977X′, both with high significance (p<0.001, R2>0.91). Total gene number shows similar trends in both groups to their respective protein coding regressions. The distinct correlations reflect lower and decreasing gene-coding percentages as genome size increases in eukaryotes (82%–1%) compared to higher and relatively stable percentages in prokaryotes and viruses (97%–47%). The eukaryotic regression models project that the smallest dinoflagellate genome (3×106 kbp) contains 38,188 protein-coding (40,086 total) genes and the largest (245×106 kbp) 87,688 protein-coding (92,013 total) genes, corresponding to 1.8% and 0.05% gene-coding percentages. These estimates do not likely represent extraordinarily high functional diversity of the encoded proteome but rather highly redundant genomes as evidenced by high gene copy numbers documented for various dinoflagellate species.

Proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerases delta and epsilon, is essential for DNA replication and repair. Understanding the structure and expression characteristics of this gene in dinoflagellates would enable us to gain insights into how the cell cycle in these enigmatic eukaryotes is regulated and whether this gene can be a growth marker of these ecologically important organisms. We analyzed pcna and its encoded protein from Pfiesteria piscicida (Ppi_PCNA). Using reverse transcription-polymerase chain reaction (RT-PCR) and RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE) methods, Ppi_pcna cDNA was isolated; it contained a coding region for 258 amino acid residues (aa) preceded by various 5'- and 3'-untranslated ends. The deduced protein length was similar to that of typical vertebrate and plant PCNA. PCR using genomic DNA as the template yielded multiple products whose sequences revealed multiple copies of pcna in tandem repeats separated by an unknown sequence. Using real-time PCR, we estimated 41+/-7 copies of this gene in each P. piscicida cell. Reverse transcription real-time PCR indicated a similar pcna mRNA level between the exponential and the stationary growth phases. Western blot analysis revealed a slightly higher PCNA level (<2-fold) in the exponential than in the stationary growth phases. We conclude that (1) P. piscicida possesses a typical eukaryote PCNA; (2) unlike in other eukaryotes, pcna in P. piscicida occurs in multiple copies arranged in tandem; and (3) regulation of P. piscicida PCNA probably lies in post-translational modification.

DNA barcoding is a diagnostic technique for species identification using a short, standardized DNA. An effective DNA barcoding marker would be very helpful for unraveling the poorly understood species diversity of dinoflagellates in the natural environment. In this study, the potential utility for DNA barcoding of mitochondrial cytochrome c oxidase 1 (cox1) and cytochrome b (cob) was assessed. Among several primer sets examined, the one amplifying a 385-bp cob fragment was most effective for dinoflagellates. This short cob fragment is easy to sequence and yet possess reasonable taxon resolution. While the lack of a uniform gap between interspecific and intraspecific distances poses difficulties in establishing a phylum-wide speciesdiscriminating distance threshold, the variability of cob allows recognition of species within particular lineages. The potential of this cob fragment as a dinoflagellate species marker was further tested by applying it to an analysis of the dinoflagellate assemblages in Long Island Sound (LIS) and Mirror Lake in Connecticut. In LIS, a highly diverse assemblage of dinoflagellates was detected. Some taxa can be identified to the species and some to the genus level, including a taxon distinctly related to the bipolar species Polarella glacialis, and the large number of others cannot be clearly identified, due to the inadequate database. In Mirror Lake, a Ceratium species and an unresolved taxon were detected, exhibiting a temporal transition from one to the other. We demonstrate that this 385-bp cob fragment is promising for lineage-wise dinoflagellate species identification, given an adequate database.DNA barcoding is a diagnostic technique for species identification using a short, standardized DNA (i.e., DNA barcode) (15). For microbial organisms, this PCR-based technique is useful not only for identifying cultured species but also for rapid retrieval and species identification for uncultured taxa from natural environments. A good DNA barcoding marker should be simple (easy to PCR amplify and sequence) and universal (effective for a wide range of lineages), with a high resolving power (high interspecific and low intraspecific variations). Therefore, an ideal DNA barcoding marker is a relatively short and reasonably variable gene fragment (for species discrimination) flanked by highly conserved sequences (for primer design). The pioneering DNA barcoding work used mitochondrial cytochrome c oxidase 1 (cox1) to identify animal species (9, 10). Mitochondrial genes are a good barcode choice for animals, because they are markedly more variable than nuclear genes (3, 32) and contain conserved regions for primer design. Among other organisms, cox1 has also been shown to be useful for barcoding other organisms, such as fungi (35). Initial attempts at cox1 barcoding for macroalgae (rhodophyte and phaeophyte) also showed good potential (21,29,34). In land plants, the mitochondrial genome evolves substantially more slowly than the nuclear genome (26, 27), rendering its genes less useful than genes...

We developed dinoflagellate-specific 18S rRNA gene primers. PCR amplification using these oligonucleotides for a picoplanktonic DNA sample from Long Island Sound yielded 24 clones, and all but one of these clones were dinoflagellates primarily belonging to undescribed and Amoebophrya-like lineages. These results highlight the need for a systematic investigation of picodinoflagellate diversity in both coastal and oceanic ecosystems.Dinoflagellates have received considerable attention due to their ecological and economical significance and their remarkable cytological and genetic features (6,7,20). However, our knowledge of the species diversity of these organisms remains limited even though novel living (2,3,10,14) and fossil lineages (4, 19) have been discovered. Knowledge of the diversity of "small" dinoflagellates is particularly deficient. The recent discovery of ultraplanktonic (Ͻ5-m) and picoplanktonic (Ͻ3-m) dinoflagellates in Antarctica and the Pacific Ocean (13, 15), respectively, is the first demonstration of a rich biodiversity of small dinoflagellates that have escaped routine microscopic detection. A better understanding of dinoflagellate biodiversity requires targeted approaches, particularly for picoplanktonic species.Development of dinoflagellate-oriented primers. Based on a large database of nuclear small-subunit (18S) rRNA genes, we designed PCR primers that target dinoflagellates. A total of 140 18S rRNA gene sequences, including sequences from dinoflagellates, diatoms, chlorophytes, haptophytes, cryptophytes, and other algae, were obtained from GenBank and were aligned using ClustalW1.8; 11 of the dinoflagellate species were sequenced in this study (GenBank accession no. DQ388456 to DQ388466). Regions unique to dinoflagellates were used to design three forward and two reverse PCR primers (Table 1), which were paired with previously described eukaryotic 18S rRNA gene universal primers (22) for DNA amplification.The primers were tested with 33 genera of cultured dinoflagellates (35 species, including Oxyrrhis marina), as well as nine other taxa (Table 2). Algal cultures were grown in f/2 medium (28‰ or 15‰ salinity), cells were harvested, and DNA was purified as previously described (23). Briefly, after cell lysis in 1 ml DNA buffer (100 mM EDTA [pH 8.0], 0.5% sodium dodecyl sulfate, 200 g ml Ϫ1 proteinase K), DNA was purified using DNA Clean and Concentrator columns (Zymo Research, Orange, CA). With these DNA samples as templates, PCR was performed using five combinations of the primers, as follows: primers 18ScomF1 and Dino18SR1 (expected product size, 0.65 kb), primers 18ScomF1 and Dino18SR2 (0.92 kb), primers Dino18F1 and 18Scom R1 (1.60 kb), primers Dino18F2 and 18Scom R1 (0.92 kb), and primers Dino18F3 and 18S com R1 (0.90 kb). All primer sets except the Dino18SF2-18ScomR1 set exhibited specificity for dinoflagellate 18S rRNA genes, which allowed amplification from most taxa examined (Table 2). The only exceptions were O. marina (often referred to as an ancestral dinoflagellate [17] or a p...

TABLE 4. Distances between species in the same genus and between strains within species derived from cob, based on the TVMϩG model Group Range Mean SD No. of sequences No. of taxa

BackgroundForaminiferan protists, which are significant players in most marine ecosystems, are also genetic innovators, harboring unique modifications to proteins that make up the basic eukaryotic cell machinery. Despite their ecological and evolutionary importance, foraminiferan genomes are poorly understood due to the extreme sequence divergence of many genes and the difficulty of obtaining pure samples: exogenous DNA from ingested food or ecto/endo symbionts often vastly exceed the amount of "native" DNA, and foraminiferans cannot be cultured axenically. Few foraminiferal genes have been sequenced from genomic material, although partial sequences of coding regions have been determined by EST studies and mass spectroscopy. The lack of genomic data has impeded evolutionary and cell-biology studies and has also hindered our ability to test ecological hypotheses using genetic tools.Results454 sequence analysis was performed on a library derived from whole genome amplification of microdissected nuclei of the Antarctic foraminiferan Astrammina rara. Xenogenomic sequence, which was shown not to be of eukaryotic origin, represented only 12% of the sample. The first foraminiferal examples of important classes of genes, such as tRNA genes, are reported, and we present evidence that sequences of mitochondrial origin have been translocated to the nucleus. The recovery of a 3' UTR and downstream sequence from an actin gene suggests that foraminiferal mRNA processing may have some unusual features. Finally, the presence of a co-purified bacterial genome in the library also permitted the first calculation of the size of a foraminiferal genome by molecular methods, and statistical analysis of sequence from different genomic sources indicates that low-complexity tracts of the genome may be endoreplicated in some stages of the foraminiferal life cycle.ConclusionsThese data provide the first window into genomic organization and genetic control in these organisms, and also complement and expands upon information about foraminiferal genes based on EST projects. The genomic data obtained are informative for environmental and cell-biological studies, and will also be useful for efforts to understand relationships between foraminiferans and other protists.