The dinoflagellates are an interesting model for eukaryotic evolutionary studies, due to their extraordinary genomic characteristics. Dinoflagellate chromosomes remain permanently condensed during the entire cell life cycle, their nuclear membranes remain intact during mitosis, and they lack nucleosomes and typical histones(Dodge 1966, Hackett et al. 2004, Moreno Diaz de la Espina et al. 2005, Lin et al. 2010). Moreover, dinoflagellates contain modified nuclear DNA; for example, 5-hydroxymethyluraci replaces 12-70% of the nuclear DNA’s thymine, while 5-methylcytosine replaces some cytosine(Lin 2011). Dinoflagellates possess a sizable quantity of DNA, ranging from 1.5 to 225 pg per cell (LaJeunesse et al.2005). In addition, dinoflagellates’ gene regulation mechanisms,such as alternative splicing and post-transcriptional regulation, differ substantially from those of typical eukaryotes (Brunelle and Van Dolah 2011, Zhang et al.2011). In particular, studies have shown spliced leader(SL)
Recently, Lin and colleagues (Zhang et al. 2007, Zhang and Lin 2009) have studied dinoflagellate SL (dinoSL)
In the present study, we investigated the dinoSL sequence using our EST databases that comprised a naked dinoflagellate,
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Cochlodinium polykrikoides and Prorocentrum minimum cultures
We obtained the two dinoflagellate strains,
To isolate the total RNA from these harvested cells, we used the TRIzol method (Invitrogen), according to the manufacturer’s instructions. After physically breaking the cells via freeze-thawing in liquid nitrogen, we homogenized them using zirconium beads (diameter 0.1 mm) and a Mini-Beadbeater (BioSpec Products Inc., Bartlesville, OK, USA). We measured each RNA sample’s concentration and purity using a DU730 life science UV-Vis spectrophotometer (Beckman Coulter, Fullerton, CA, USA) and verified the RNA’s integrity via electrophoresis on agarose gels.
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EST sequencing and annotations
First, we pooled a variety of total RNAs from various conditions (e.g., heat shock, cold shock, toxic chemical exposure, and different life stages) into a single tube, which we then subjected to EST sequencing via a GS-FLX Titanium instrument (454 Life Sciences, Roche), assembling the EST sequences having 95% similarity levels with one another. Next, we separately characterized contigs and singletons of each EST data set by means of BLAST-X comparisons, using public domain databases. This process allowed us to treat EST sequences with E-values over 1.0E-05 as “No Hit,” as they probably belonged to UTRs.
DinoSL sequence searches
Finally, we investigated EST sequences having more than 100 bp of 5′-UTR for the SL sequence. In addition, we constructed local nucleotide databases of our
[Table 1.] Summary of EST data constructed from Cochlodinium polykrikoides and Prorocentrum minimum
Summary of EST data constructed from Cochlodinium polykrikoides and Prorocentrum minimum
version 5.0.6 (Hall 1999), and used them for local BLAST searches. To analyze nuclear encoded transcripts (or EST sequences) that had the dinoSL sequence, we used Genetyx version 7.0 software (Genetyx Corp., Tokyo, Japan).
In the present study, we determined the large-scale EST sequences of two dinoflagellates,
In addition, we used BLAST searches to investigate dinoSL-containing transcripts in our local nucleotide database. Through this analysis, we detected 55 dinoSL sequence-containing ESTs (17 contigs, 38 singletons) from the
To our knowledge, the dinoSL sequence is added to the 5′-end of dinoflagellate gene transcripts. For investigating whether all or parts of dinoflagellate nuclear gene transcripts contain dinoSL sequence, researchers should retain intact 5′-ends of the genes. In addition, to detect the dinoSL-containing transcripts, studies should amplify transcripts entirely by means of reverse transcriptase.
Cochlodinium and Prorocentrum ESTs containing dinoSL RNA sequences at the 5′-end and their closest matches from GenBank data
However, many dinoflagellates contain inhibitors that will strongly inhibit either reverse transcriptase or Taq DNA polymerase activity (Zhang and Lin 2009). Problems such as these may explain why few nuclear gene transcripts contain the dinoSL sequence, in both the previous data (Zhang et al. 2007, Bachvaroff and Place 2008) and in our EST data. On the other hand, Bachvaroff and Place (2008) showed that the SL
This study investigated the dinoSL sequence location by surveying reported dinoSL-containing gene transcripts and our EST data (Table 3). Having detected the dinoSL sequence in
Genes GenBank accession numbers and locations of the dinoSL sequence upstream of ATG summarized from available dinoflagellates’ trans-spliced genes
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the start codon (ATG). Moreover, additional summarized data (Table 3) showed that the dinoSL sequence’s major locations ranged from 40 to 160 bp upstream of ATG. Only in a few genes, and particularly in unknown function genes, did the dinoSL sequence occur more than 170 bp upstream of ATG. Perhaps the SL