J. Phycol. 42, 96–103 (2005) r 2005 Phycological Society of America DOI: 10.1111/j.1529-8817.2005.00165.x NOVEL AND RAPIDLY DIVERGING INTERGENIC SEQUENCES BETWEEN TANDEM REPEATS OF THE LUCIFERASE GENES IN SEVEN DINOFLAGELLATE SPECIES1 Liyun Liu and J. Woodland Hastings2 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138, USA Tandemly arranged luciferase genes were previ- Our previous studies of the structure of dinoflagel- ously reported in two dinoflagellates species, but lates genes and their circadian regulation revealed that their intergenic regions were strikingly different several occur in tandemly arranged copies (Le et al. and no canonical promoter sequences were found. 1997, Li and Hastings 1998, Okamoto et al. 2001). Here, we examined the intergenic regions of the Other than for ribosomal genes (Sollner-Webb and luciferase genes of five other dinoflagellate species Tower 1986) and a few protein-coding genes in two along with those of the earlier two. In all cases, the protozoa, Trypanosoma brucei and Babesia bovis (Lee and genes exist in multiple copies and are arranged Van der Ploeg 1997, Suarez et al. 1998), such an ar- tandemly, coding for proteins of similar sizes and rangement is not known in other eukaryotes. Indeed, sequences. However, the 50 untranslated region, 30 it is well known that the dinoflagellate nucleus is very untranslated region, and intergenic regions of the unusual; its envelope remains intact throughout the seven genes differ greatly in length and sequence, cell cycle, with the separation of the chromosomes in except for two stretches that are conserved in the mitosis being carried out by an external mitotic spindle intergenic regions of two pairs of phylogenetically (Taylor 1987). Microscopically, the chromosomes have close species. Microsatellites and minisatellites a ‘‘cholesteric-like liquid crystalline organization,’’ with were detected in the intergenic sequences of four a characteristic banding pattern in transmission elec- species, Alexandrium affine (H. Inoue & Y. Fukuyo) tron microscopy and a whorled appearance in the 3D E. Balech, A. tamarense (Lebour) E. Balech, Proto- models (Herzog and Soyer 1981, Spector 1984, Soyer- ceratium reticulatum (Clapare`de & Lachmann) Gobillard and Geraud 1992, Rizzo 2003). Dinoflagel- Butschli, and Pyrocystis lunula (Schu¨tt) Schu¨tt, the lates typically possess a large amount of DNA (up to 40 first three of which have unusually high percentages times that of the human cell) and an enormous of particular sets of dinucleotides. Most remarkably, number of chromosomes (4100), which lack typical the P.reticulatum intergenic region is almost exclu- eukaryotic histones and nucleosomes, and remain con- sively made up of 19 nearly identical repeats of an densed permanently. This last feature raises the ques- 11-nucleotide sequence. Dinoflagellate luciferase tion of how transcription factors and RNA polymerase intergenic regions bear similarities to ribosomal have access to the DNA, because in other eukaryotes genes and to some protein-encoding genes in try- transcription is temporarily repressed when the chro- panosomes, both of which are transcribed by RNA mosomes are condensed during mitosis (White et al. polymerase I. It is possible that the transcription of 1995, Gottesfeld and Forbes 1997). the dinoflagellate genes are catalyzed by an RNA To understand the molecular machinery for tran- polymerase with novel properties. scription and its regulation, workers have studied pro- Key index words: intergenic region; luciferase; mi- teins that interact with the chromosomal DNA. crosatellites; minisatellites; promoter; tandem genes Histone-like proteins have been isolated from a few dinoflagellate species (Rizzo 1981) and one from Ling- Abbreviations: Aa, Alexandrium affine;At,Ale- ulodinium polyedrum (Lp) binds weakly to DNA (Chud- xandrium tamarense; LCF and lcf, luciferase protein novsky et al. 2002). In addition, two transcription and gene; Lp, Lingulodinium polyedrum (F. Stein) factors, a WW domain protein and a TATA box bind- J.D. Dodge; Pf, Pyrocystis fusiformis (Wyville– ing protein (TBP), have been identified in the dino- Thomson ex Haeckel) Blackman; Pl, Pyrocystis flagellate Crypthecodinium cohnii (Guillebault et al. 2001, lunula; Pn, Pyrocystis noctiluca Murray ex Haec- 2002). At the sequence level, a C. cohnii TBP homolog kel; Pr, Protoceratium reticulatum; TEM, transmis- differs considerably from those of other eukaryotes, sion electron microscopy; 30 UTR, 30 untranslated and its expressed protein can bind weakly to a syn- region; 50 UTR, 50 untranslated region thetic TATA box and more strongly to a TTTT box. Sequences in the 50 upstream region (up to 500 nt from the translation start codon) have been obtained previously for seven dinoflagellate genes (Le et al. 1997, Li and Hastings 1998, Okamoto et al. 2001, 1Received 19 May 2005. Accepted 20 October 2005. Guillebault et al. 2002). None of them have canonical 2Author for correspondence: e-mail [email protected]. eukaryotic promoter sequences such as a TATA box, a 96 INTERGENIC SEQUENCES OF LUCIFERASES 97 CAAT box, or an initiator element. Furthermore, no pelleted at 10,000g for 30 min, washed once with 80% sequence similarities were found among these, except ethanol, and dissolved in TE (10 mM Tris, 1 mM EDTA, for a 13-nt sequence present in the 50 upstream region pH 8.0). One-third volume of 8M LiCl was then added; of both the peridinin binding protein and luciferase after 3h on ice, RNA was obtained by centrifugation at 10,000g for 30 min. The DNA was recovered from the supe- genes of Lp (Li and Hastings 1998), and GC-rich se- rnatant by adding an equal volume of isopropanol. Residual quences in both Pyrocystis lunula (Pl) luciferase and Peri- DNA in the RNA sample was digested with DNase I (Ambion dinium bipes ferredoxin genes(Yoshikawa et al. 1997, Inc., Austin, TX, USA) at 0.5 U per 10 mgRNAin10mL Okamoto et al. 2001). Neither of the above two se- at 371 C for 20 min. Similarly, trace RNA was removed from quences was demonstrated to mediate transcription. the genomic DNA using RNase A (Sigma Inc., St. Louis, MO, Dinoflagellate luciferase catalyzes a light-emitting USA). DNase I and RNase A were inactivated by phenol:chlo- roform (1:1). One-tenth volume of 3M sodium acetate reaction in which the luciferin (a tetrapyrrole) is oxi- (pH 5.2) and 2.5 volumes of ethanol were added to the aque- dized to give a product in an electronically excited state ous phase containing DNA or RNA. DNA or RNA was pel- (Wilson and Hastings 1998). In Lp, the protein itself leted, washed with 80% ethanol and resuspended in TE. was found to consist of three intramolecular repeats, After measurement of concentration by OD at 260 nm, the each of which can act as an active luciferase (Li et al. DNA or RNA was stored at À 801 Cuntiluse. 1997). The coding sequences of luciferase were later RNA electrophoresis and Northern blotting: Ten micrograms of total RNA were denatured at 751 C for 5 min, separated on isolated from six other photosynthetic dinoflagellate a 1.4% agarose gel by electrophoresis, and transferred onto a species; they share high sequence similarity to the Lp N þ nylon membrane (Amersham Biosciences Corp., Piscat- luciferase gene and possess a similar three-repeat con- away, NJ, USA) as described (Pelle and Murphy 1993). After figuration (Okamoto et al. 2001, Liu et al. 2004). Both the transfer, the blot was baked at 801 C for 1 h to fix the RNA Lp and Pl lcf genes were found to occur in tandem andprehybridizedfor1hat501 C in the hybridization buffer copies, separated by intergenic sequences with no simi- (20% formamide, 6ÂSSPE,0.5%SDS,5ÂDenhardt’s reagent, larity to each other or to others in the database (Li and and 20 mg/mL sonicated salmon sperm DNA). Afterwards, radioactive probes generated from the full-length Lp lcf DNA Hastings 1998, Okamoto et al. 2001). by random priming labeling (Amersham) were added to In the present report, the genomic structure and 106 cpm/mL. The hybridization lasted for 12 h and the blot organization of luciferase genes from five additional was washed with three consecutive washes (2ÂSSPE, 0.1% species were determined and analyzed along with SDS, 15 min, 201 C; 0.5ÂSSPE, 0.1% SDS, 15 min, 601 C; those of Lp and Pl. All were found to occur in tandem 0.2ÂSSPE,0.1%SDS,15min,601 C). The blot was detected copies and, except for closely related species, to have by a phosphorimaging machine. Protein extraction and Western blotting: To prepare the total very different intergenic sequences and no evident proteins, the cells were harvested and homogenized as for promoter elements in the region upstream from the the nucleic acids described above but resuspended in protein 0 0 5 untranslated region (5 UTR). The results suggest extraction buffer (100 mM Tris-HCl, 10 mM EDTA, and that the RNA polymerase responsible for transcription 5 mM 2-mecaptethanol, pH 8.5) (Johnson et al. 1984). After of the luciferase genes, and probably other dinoflagel- centrifugation, 10 mg of total protein from the supernatant lates genes, is likely to be very different from those in was adjusted to 1Â Laemmli gel loading buffer and sepa- rated on 10% SDS-polyacrylamide gel by electrophoresis most eukaryotes. at 200 V for 50 min (Laemmli 1970). The separated protein was then transferred onto nitrocelluose BA 85 (Schleicher & Schuell, Keene, NH, USA) and probed with an affinity- MATERIALS AND METHODS purified luciferase antibody (Knaust et al. 1998). The blot was detected by chemiluminescence (Amersham) following Algal cultures: Dinoflagellates were grown in F2 medium the manufacturer’s instructions. Protein concentration was under 12:12 LD cycles at 191 C and a light intensity of determined by the Bradford method (Bradford 1976). 150 mmol photons Á m À 2 Á s À 1 (Guillard and Ryther 1962, 30 RACE, and PCR amplification of the intergenic re- Liu et al.