Table 1.
SL RNA-related oligonucleotides used in this study.
Table 2.
Mitochondrial gene primers designed in this study.
Table 3.
Best substitution models selected by ProtTest for phylogenetic analyses.
Figure 1.
Alignments of spliced leader (SL) RNAs of Perkinsus marinus and dinoflagellates.
A) Representative genomic sequences of two types of P. marinus SL RNA. B) P. marinus SL RNAs with the reported representatives of dinoflagellate SL RNA genomic sequences (modified according to [9]; the number of identical clones retrieved for each type is indicated by “@number” following the species abbreviation and type number). The SL region (boxed) is shown in uppercase letter, intron and the flanking regions are shown in lowercase letters, * indicates the conserved nucleotide (nt). The first ‘A’ of SL is numbered as nt 1. SL RNA transcripts mapped by 3′ RACE analyses are denoted by arrows to indicate the terminal positions, thickness with darkness of the arrows denote relative frequency of clones that ends where it is indicated. Note that the PCR-amplified Amoebophrya sp. genomic sequences contain only one unit of SL RNA gene, the partial SL sequence is of the primer used. Per, P. marinus, Amo, Amoebophrya sp.; Har, Heterocapsa arctica; Kbr, Karenia brevis; Kve, Karlodinium veneficum; Ppi, Pfiesteria piscicida; Pgl, Polarella glacialis; Pmi, Prorocentrum minimum. SL refers to SL RNA sequences obtained from SL-only repeats; SL-5S indicates SL RNA sequences from genes associated with 5S rRNA genes. *: sequences from [8]; **: sequence from [46]; #: sequences from [9], $1-4: GQ178071-GQ178074; •: sequences missing in the original reports. Shaded are conserved positions defined as identical in over six sequences in at least three species. A non-canonical C in the splice donor site of KbrSL-3 is boxed. Gaps introduced in the sequence alignment are shown as ‘–’.
Table 4.
Genomic sequences containing SL RNA genes identified from P. marinus genome data.
Figure 2.
Perkinsus spp. substrate SL RNA larger than typical of dinoflagellates.
A) Denaturing 8% polyacrylamide/8 M urea gel of total cell RNA from Perkinsus spp. and other organisms. Lane 1, Leishmania tarentolae; 2, P. chesapeaki; 3, P. marinus; 4, Prorocentrum minimum; 5, Polarella glacialis; 6, Karenia brevis and 7, Karlodinium veneficum. B–F) Probing of the blot shown in A) using oligonucleotides DinoSLa/s, PmaSL-La/s, PmaSL-Sa/s, and designed from intron regions in the PmaSL-L and PmaSL-S genotypes, respectively. Arrows highlight the SL RNA transcripts. G) and H) Dot blots of the PmaSLRNA-L (G) and PmaSLRNA-S (H) cDNA clones using the same probes as in (E) and (F), respectively.
Figure 3.
Perkinsus marinus SL RNA secondary structure similar to DinoSL RNA.
Predicted structures of SL RNA for P. marinus L-type (A) and S-type (B) based on the most abundant cDNAs obtained. Model simulation was run using MFOLD: Prediction of RNA secondary structure modeling program (http://bioweb.pasteur.fr/seqanal/interfaces/mfold-simple.html) under default settings except that the folding temperature was set at 27°C, the culture temperature.
Figure 4.
Perkinsus spp. cob and the predicted aa sequences.
Sequences of P. marinus, and for P. chesapeaki only the sites with different nt/aa sequences, are shown. Four invariant His residues that are ligands for heme β are highlighted in blue. ‘-’ indicates missing sequence; the potential quadruplet codons ‘aggy’ for glycine are marked in grey, and quintuplet codons ‘uaggc’ and ‘ucggu’ for glycine are boxed.
Figure 5.
Perkinsus spp. cox1 and the predicted aa sequences.
Sequences of P. marinus, and for P. chesapeaki only the sites with different nt/aa sequences, are shown. Six invariant His residues that are ligands for heme α, CuB and heme α3 are highlighted in blue. ‘-’ indicates missing sequence; the potential quadruplet codons ‘aggy’ for glycine and ‘ccccu’ quintuplet codons for proline are marked in grey, and a standard ‘ggu’ codon for glycine in P. chesapeaki cox1 is marked in red.
Figure 6.
Phylogenetic affiliation of P. marinus with apicomplexans based on mitochondrial COB and COX1.
The consensus trees with support from NJ (bootstrap, only >84% are shown), ML (aLRT), and MB (posterior probability). Brackets indicate clades of apicomplexans (AP), dinoflagellates (DI) and ciliates (CI).
Figure 7.
Phylogenetic affiliation of P. marinus with dinoflagellates and apicomplexans based on 30 conserved protein sequences.
The consensus trees of concatenated genes of 19 ribosomal proteins (RPs) for 17 taxa (A), 8 RPs for 18 taxa including Oxyrrhis (B) and 11 non-RP proteins (C). Supports of nodes are from NJ (bootstrap), ML (aLRT), and MB (posterior probability). Brackets indicate clades of apicomplexans (AP), dinoflagellates (DI) and ciliates (CI).
Figure 8.
Phylogenetic affiliation of P. marinus with apicomplexans based on histone H2A.
The canonical H2A and the isoform H2A.X consensus tree with support from NJ (bootstrap), ML (aLRT), and MB (posterior probability). Brackets indicate clades of apicomplexans (AP), dinoflagellates (DI) and ciliates (CI).
Figure 9.
Phylogenetic affiliation of Perkinsus marinus with apicomplexan based on three histone proteins.
Histone H2B(A), H3 (B), and H4 (C) consensus trees with support from NJ (bootstrap, only >70% are shown), ML (aLRT), and MB (posterior probability). Brackets indicate clades of apicomplexans (AP), dinoflagellates (DI) and ciliates (CI).