Genes Genet. Syst. (2007) 82, p. 161–166 Cryptic long internal repeat sequences in the ribosomal DNA ITS1 gene of the dinoflagellate Cochlodinium polykrikoides (dinophyceae): a 101 nucleotide six-repeat track with a palindrome-like structure

Jang-Seu Ki1* and Myung-Soo Han2 1Department of Biology, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China 2Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, Korea

(Received 27 December 2006, accepted 16 February 2007)

Extremely long PCR fragments were generated by PCR amplification of ITS and 5.8S rDNA from Cochlodinium polykrikoides against other dinoflagellates. These patterns were consistent among geographically different isolates of C. polykrikoies. DNA sequencing reactions revealed that the PCR products were 1,166 bp in length and consisted of 813 bp of ITS1, 160 bp of 5.8S rDNA and 193 bp of ITS2. Thus, the long length was caused mainly by the long ITS1 sequence. Cryptically, the ITS1 contained a tract of 101 bp that occurs six times in tandem. The six repeated elements had identical nucleotide sequences. ITS1, therefore, separated three distinct regions: the 5’ end (122 bp), the six parallel repeats (606 bp), and the 3’ region (85 bp). Interestingly, both the single and six-repeat sequences should be palindrome-like sequences. In inferred secondary structures, both repeat sequences formed a long helical structure. This is the first reported dis- covery of comparatively long internal repeats in the ITS1 of dinoflagellates.

Key words: dinoflagellate, Cochlodinium polykeikoides, internal transcribed spacer (ITS), long repeat sequence, palindrome

Ribosomal DNA (rDNA) genes, including the internal polykrikoides in Korean coastal waters have significantly transcribed spacer (ITS) regions, are the most frequently increased in frequency, severity, and duration (Kim et al., used target for phylogenetic studies, because not only do 1999). These dinoflagellates closely resemble the related they occur in all living organisms, but they typically are dinoflagellates Akashiwo sanguinea, Gymnodinium cate- present in several copies which are distributed through- natum and G. impudicum. Because of this close resem- out the (Harmsen and Karch, 2003). Particu- blance, Cho et al. (2001) tried to discriminate C. larly, eukaryotic nuclear rDNA is tandemly organized, polykrikoides from others using DNA sequences. They with copy numbers up to the order of 10,000 (Schlötterer, suggested that the ITS1 region was valuable for this tax- 1998). Because of their microscopic sizes and fragile onomic discrimination. Independently, we have ana- bodies, species of microalgae, like , are commonly lyzed the sequence of C. polykrikoides to identify nuclear identified using these gene sequences (Ki et al., 2004; Ki DNA markers that will facilitate the taxonomic identifi- and Han, 2006). At present, more than 20,000 strands of cation of this harmful microalgae. Recently, we reported rDNA sequences from dinoflagellates have been identified that the partial 18S rDNA was sufficient to discriminate and deposited into public databases (i.e. EMBL/DDBJ/ C. polykrikoides from other related dinoflagellates (Ki et GenBank). al., 2004). In the course of analyzing dinoflagellate The ichthyotoxic Cochlodinium polykrikoides Margalef rDNA sequences, we found consistent patterns of extre- is a harmful dinoflagellate that is one of the most mely long PCR fragments in the ITS and 5.8S rDNA frequent causes of fish kills (Kim et al., 1999; Ahn et al., sequences of C. polykrikoides compared to other 2006). Over the last two decades, outbreaks of C. dinoflagellates. This study reports the complete nucle- otide sequences of the ITS and 5.8S rRNA genes from C. Edited by Akio Toh-e polykrikoides and its close relatives. In addition, we * Corresponding author. E-mail: [email protected] report the characterization of the long ITS in C. 162 J.-S. KI and M.-S. HAN polykrikoides and our prediction of the secondary struc- 2004). In brief, DNA sequencing was performed using ture of ITS1. PCR products and near infrared dye (IRD)-labeled Clonal cultures of Cochlodinium polykrikoides (CcPk02, primers. Identical PCR and nested primers were used 03, 05, 06), including Akashiwo sanguinea (GnSg03) were for sequencing reactions on each amplicon containing kindly provided by Dr. M. Chang of the Korean Ocean entire ITS, 5.8S and partial LSU rDNAs. PCR products Research and Development Institute. An additional (3–6 μL) were subjected to DNA cycle sequencing using a strain (CCMP414) of Gymnodinium catenatum was obtai- ThermoSequenaseTM version 2.0 Cycle Sequencing Kit ned from a commercial source (the Provasoli-Guillard (USB, Cleveland, OH, USA) in the presence of 1.5 pico- National Center for Culture of Marine Phytoplankton, moles of sequencing primers. The four base-specific CCMP). All cultures were grown in f/2 medium pH 8.2 reactions were initially subjected to 94°C for 1 minute, at 15°C, with a 12:12-h light:dark cycle and a photon flux followed by 40 cycles consisting of 94°C for 20 s, 50°C for density of ca. 65 μmol photons/m2/s. Mid-exponential 30 s, and 72°C for 60 s in the UNO-II Thermoblock. batch cultures were harvested by centrifugation at 3,000g When complete, reactions were stopped by adding 4 μL of for 10 min. The concentrated cells were mixed with 100 IR2 stop/loading buffer (Licor®, NE, USA), and the prod- μL of 1X TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH ucts were heat-denatured and analyzed on a Model 4200 8.0), and stored at –20°C until DNA extraction. Genomic Dual Dye Automated Sequencer (Licor), according to the DNA was isolated from the stored cells using the DNeasy manufacturer’s instructions. All of the sequences obta- Plant mini kit (Qiagen, Valencia, CA) according to the ined here were completely assembled with the Sequ- manufacture’s instructions. encher3.0 software (Gene Codes, MI, USA). All DNA PCR was used to amplify complete ITS regions includ- sequences reported here have been deposited in GenBank ing 5.8S rDNA with a set of PCR primers (18F1641, database as accession numbers AY347309, AY831411, 28R01) as shown in Fig. 1. In addition, three Cochlodin- DQ779984-6 and DQ779990. ium polykrikoides-specific PCR primers (Fig. 1) were A secondary structure of ITS1 was estimated using the designed based on the 18S and 28S rDNA sequences program Mfold, version 3.2 (http://www.bioinfo.rpi.edu/ determined previously (Ki et al., 2004), by comparing applications/mfold/old/rna/), according to Zuker and other dinoflagellate sequences available in GenBank. Turner (Zuker, 2003). With the default option (e.g. tem- Overall specificities were tested by phylogenetic analyses perature setting, T = 37°C), Mfold predicted 7 secondary of most dinoflagellate sequences retrieved from the structures from ITS1 sequence including a single repeat database. Additional tests were performed on BLAST unit and 27 secondary structures from the entire ITS1 search by comparing these sequences against all publicly sequence, respectively. Using different parameter set- available data. Fifty-microliter PCR reactions were car- tings of, for example, temperature (T = 10°C, T = 20°C, T ried out in 1X PCR buffer (10 mM Tris-HCl, 50 mM KCl, = 37°C), did not affect the general architecture, but did

1.5 mM MgCl2, 0.001% gelatin; pH 8.3) with < 0.1 μg result in different energy levels for the secondary struc- genomic DNA template, 200 μM each of the four dNTPs, tures. The secondary structure models inferred here 0.5 μM of each primer and 1 Unit Taq polymerase were redrawn with the software RNAViz (De Rijk and De (Promega, Madison, WI, UAS). Using a UNO-II Ther- Wachter, 1997), a versatile program developed to draw moblock (Biometra, Göttingen, Germany), PCR thermocy- secondary structures of molecules in a fast and user- cling parameters were as follows: 95°C for 5 min; 35 friendly way. Among them, we finally selected a general cycles of denaturation at 95°C for 20 s, annealing at 55°C model of secondary structure for C. polykrikoides ITS1. for 30 s and extension at 72°C for 60 s and a final exten- PCR amplification following the typical PCR protocol sion at 72°C for 5 min. The PCR products (2 μL) were generated relatively long PCR fragments (approximately analyzed by 1.5% agarose gel electrophoresis according to 1,200 bp) from entire ITS rDNA regions of Cochlodinium standard methods. polykrikoides. DNA sequencing revealed the PCR prod- ITS1 in Cochlodinium polykrikoides contained long ucts to be 1,166 nucleotides in length. Cryptically, this repetitive sequences, which apparently formed strong contained six long internal repeat sequences (Fig. 1A). hairpin structure in sequencing reactions. Consequently, To find general ITS characteristics and to determine the the helix structure might hinder elongation of the enzyme relationship between the ITSs of C. polykrikoides and its at the base of the hairpin and cause the enzymatic reac- relatives, we determined the complete sequences of ITS1, tion to stop there or render unreadable, weakened sig- 2, and 5.8S rDNA from Gymnodinium catenatum and nals. Hence, we could not analyze the repetitive regions Akashiwo sanguinea, which are considered phylogenti- with the dye terminator sequencing method. In the cally as members of the closest Cochlodinium relative present study, using the dye-labeled primer sequencing (Cho et al., 2007). With these sequences and available method, we were able to determine the complete sequ- GenBank data (e.g. A. sanguinea, AY831412; G. corii, ences of the ITS regions in C. polykrikoides. The AF318226; G. maguelonnense, AF318225; G. mikimotoi, sequencing protocol has been described in detail (Ki et al., AF318224), we found that the armoured dinoflagellate Cryptic long internal repeats in the rDNA ITS1 of dinoflagellate C. polykrikoides 163

Fig. 1. A map of Cochlodinium polykrikoides ITS and 5.8S rDNA and PCR products. (A) diagrammatic presentation of C. polykrikoides ITS and 5.8S rDNA, indicating the long internal repeats in ITS1. Solid boxes refer to rDNA genes and open boxes are represented repeat units, respectively. The R1 to 6 in ITS1 refer to six internal repeats, to which individual sequences are aligned and represented below. Arrows indicate the position of primers used in this study. Forward primers, Cp18-3F3 (5’-TTAGATGTTC TGGGCTGCACG-3’, positioned at 1446-1466), 18F1641 (5’-GAGGAAGGAG AAGTCGTAACAAGG-3’, 1622-1641), ITSF01 (5’-TCCCT- GCCCTTTGTACACAC-3’, 1747-1770), are shown above 18S rDNA, near the priming site. Reverse primers, ITSR01 (5’-TCCGCTTAC- TTATATGCTTAAATTCAGC-3’, 29-56), Cp28R2 (5’-GTTGGCGT TGCATTTCGAGAC-3’, 148-168), Cp28-3R2 (5’-ACGGAGGGCTGCA- GATTGAC-3’, 227-246) and 28R01 (5’-AAACTTCGGAGGGAACCAGCTAC-3’, 1019-1041), are shown below 28S rDNA. Each primer position is based on the complete 18S and 28S rDNA sequence of C. polykrikoides (Accession no. AY347309). (B) PCR products am- plified with a C. polykrikoides genomic DNA (CcPk06) and four different primer sets. Each PCR product is expected to be, in order, 1579 bp, 1691 bp, 1769 bp and 2564 bp in length, respectively. (C) PCR products amplified with various combinations of both different templates and two primer sets (ITSF01 + ITSR01, Cp18-3F3 + ITSR01). Genomic used here are from each strain of C. polykrikoides (CcPk05: Lane 1, 5; CcPk06: lane 2, 6), Akashiwo sanguinea (GnSg03: lane 3, 8) and Gymnodinium catenatum (CCMP 414: lane 4, 9), respectively. M1 and M2 indicate 100-bp and lamda hind III DNA markers. species have around 200 bp in each ITS1 and ITS2, and DDBJ/GenBank) revealed that the ITS1 sequence of C. all species had either 160 bp or 161 bp of 5.8S rDNA. The polykrikoides was the longest among dinoflagellate complete sequence length of the ITS region was, there- sequences recorded to date. The majority of dinoflagel- fore, less than 560 bp in most dinoflagellates. In con- late ITS1 sequences deposited in the database were less trast, the ITS regions of C. polykrikoides was considerably than 300 bp in length, and many of them were shorter longer. The ITS of C. polykrikoides consisted of 813 bp than 250 bp. of ITS1, 160 bp of 5.8S rDNA, and 193 bp of ITS2. The extreme length of the PCR products was confirmed Among these regions, the ITS1 was extremely long and through the application of C. polykrikoides specific-prim- thus contributed substantially to the length of the PCR ers (Fig. 1A) to be PCR-amplified the complete ITSs fragments. Additional database searches (e.g. EMBL/ regions. PCRs, employing combinations of the primers, 164 J.-S. KI and M.-S. HAN apparently generated bands of each expected size (Fig. lack of such constraints in the middle of the spacer might 1B). In addition, when a universal primer set (ITSF01 + have favored the rise of repetition in this region. From ITSR01) was applied to the closely related dinoflagellates a structural perspective, Bakker et al. (1995) reported A. sanguinea and G. catenatum, the PCR product pat- that the ITS is believed to have few evolutionary con- terns differed comparatively in size (Fig. 1C). However, straints and might be expected to evolve at or near the PCR, employing C. polykrikoides specific primers, did not neutral level. generate any fragments from the relatives (Fig. 1C). A predicted model of the secondary structure of the For intraspecific variation of ITS1, we investigated four ITS1 pre-RNA transcripts conforms well to the hairpin Cochlodinium polykrikoides stains isolated from geo- structure present in C. polykrikoides (Fig. 2). In addi- graphically different coastal waters in Korea (CcPk02, tion, the predicted secondary structures clearly indicated Tongyoung; CcPk03, Narodo; CcPk05, Hakdong; CcPk06, that there are three distinct regions within ITS1. Each Sarangdo). Upon comparison, the sequences from these single unit of each repeat sequence represents a palindro- strains were 100% identical to one another. This sug- mic DNA sequence (see DeBoer and Ripley, 1984), as gested that the long sequences in ITS1 might be a com- shown in the dotted box of Fig. 2. Thus these sequences mon feature of populations of C. polykrikoides rather than can form hairpins as a double helix (Fig. 2) when pre- a mutation of particular strains. Previously, however, rRNA genes express these regions. In addition, the long Cho et al. (2001) reported 243 bp of full ITS1 sequences ITS1 containing the entire repeats should form a com- (GenBank access. no. AF208248; isolate, CP-1) from C. pletely helical structure as a long chain (Fig. 2). In fact, polykrikoides. By comparison, this sequence was com- these hairpin structures are believed to play a role in pro- pletely identical to the sequences reported here, indicat- cessing signals. Recently, Campbell et al. (2005) pointed ing that all strains were apparently the same species of out that small hairpins, such as those formed by these C. polykrikoies. Although the reasons for the disparity regions, are functional and that natural selection would with our results are unclear, one possible reason is that favor GC-rich sequences to stabilize the stems. Actually, the previous sequence represented the incomplete sequ- C. polykrikoides has considerably high G/C content ence of ITS1. Indeed, the GenBank record for the sequ- (61.4%) in ITS1, particularly in repeat units (63.9%) when ence shows that it is not full-length sequences of the ITS compared to other dinoflagellates: A. anguinea, 56.2%; G. regions. Another possible explanation might be intrage- catenatum, 50.0; G. impudicum, 53.7%. These findings nomic rDNA diversity in ITS1 of C. polykrikoides. That is, suggest that the hairpin in C. polykrikoides might be the 18S–26S rDNA as a multigene family is subject to structurally stable and play an important role in ribo- (Elder and Turner, 1995), and few some biogenesis through the processing of the pre-RNA. variations have been reported. These variations are To date, many studies have investigated ITS1 varia- thought to stem from an individual unequal crossing over tions (van Herwerden et al., 1999; Harris and Crandall and subsequent (Hugall et al., 1999). 2000; von der Schulenberg et al., 2001; Campbell et al., Although the chances of gene conversion are low and few 2005). Commonly, short tandem repeats (STR) are con- cases have been reported (Hugall et al., 1999), the finding sidered to be responsible for the length variation in that the PCR of the ITS from C. polykrikoides does not ITS1. The presence of STR in fungi is well documented amplify two different-sized bands (see Fig. 1B) unsup- (Platas, 2001). In addition, these STR motifs have been ports this possibility. found in various arthropods (Kumar, 1999; Harris and The ITS1 of Cochlodinium polykrikoides contains a Crandall, 2000), and flowering plants (Liston et al., 1996). tract of 101 nucleotides, which occurs six times in tan- Previously, Levinson and Gutman (1987) reviewed the dem, as noted previously. The six repeated elements repetitive DNA sequences in light of STRs. However, to were exactly identical, except for two nucleotides, in com- the best of our knowledge, no studies have identified plete sequences (Fig. 1A). The ITS1 of C. polykrikoides STRs in ITS1 of dinoflagellates. Several mechanisms can therefore be separated into three distinct regions: the have been proposed to generate length variation. These 5’ end (122 nucleotides), the six parallel repeats, and the include intra- and inter-strand recombinational effects, 3’ region (85 nucleotides). In addition, the tandem repe- such as unequal crossing over; other mechanisms involve ats in C. polykrikoides were present in the middle of the failures in the replication of DNA such as slipped-strand spacer positioned between the 3’ and 5’ ends of ITS1. mispairing (Levinson and Gutman, 1987) or replication This is consistent with the previous studies (van Herwer- slippage (Pinder et al., 1998). In contrast to STR, long den et al., 1999; von der Schulenburg et al., 2001). von internal repeats are not frequent in ITS1 regions. Long der Schulenburg et al., (2001) reported that long repeti- internal repeats include repetitive elements with compar- tive elements were always confined to the middle of the atively long repeat units, and they have only been spacer in coccinellid species. They suggested that the reported in few taxonomic groups such as the trematode reason for this might be that the 39- and 59-end regions Paragonimus westermani (van Herwerden et al., 1999) of ITS1 might have functional importance, whereas the and the dipteran Simulium damnosum (Tang et al., Cryptic long internal repeats in the rDNA ITS1 of dinoflagellate C. polykrikoides 165

1996). Previously, von der Schulenburg et al. (2001) found long tandemly motifs of greater than 60 bp in several insects Adalia, Harmonia and Psyllobora. More recently, Warberg et al. (2005) repor- ted that the repetitive sequences (130 bp) in the ITS1 region of rDNA was consistently present in congeneric microphallid species (Trematoda: Digenea). In these cases, the generation of the repetitive sequences has been proposed to occur through the same mechanisms as STR: replication slippage, unequal crossing over, and biased gene conversion (e.g. Levinson and Gutman, 1987; Elder and Turner, 1995). However, these studies did not find any relationship between the long repeats and taxonomi- cal groups. Furthermore, it is not known whether the long repeat units within the ITS1 region have any func- tional importance, nor is it known how they might have arisen and how they are maintained. Additionally, we found that C. polykrikoides has comparatively long inter- nal repeat sequences in the ITS1, which is the first such repeats to be discovered in alveolates. At the present stage, the function and biogenesis of repeat sequences are not clear, and later more detailed studies are needed to improve understanding of both evolutionary variation and the molecular processes that lead to the generation of repetitive sequences.

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