Plant Physiol. (1991) 97, 61 3-6180032-0889/91/97/061 3/06/$01 .00/0

Received for publication March 7, 1990Accepted June 18, 1991

Characterization of Satellite DNA from Three MarineDinoflagellates (Dinophyceae): Glenodinium spi andTwo Members of the Toxic Genus, Protogonyaulax'

Barbara A. Boczar2, John Liston, and Rose Ann Cattolico*Department of Botany KB-15, University of Washington, Seattle, WA 98195 (B.A.B.), Departments of Botany andOceanography KB-15, University of Washington, Seattle, WA 98195 (R.A.C.), and Institute for Food Science and

Technology, University of Washington, Seattle, WA 98195 (J.L.)

ABSTRACT

Using CsCI-Hoechst dye or CsCI-ethidium bromide gradients,satellite and nuclear DNAs were separated and characterized inthree marine dinoflagellates: Glenodinium sp., and two toxic di-noflagellates, Protogonyaulax tamarensis and Protogonyaulax ca-tenella. In all three dinoflagellates, the lowest density fraction,satellite DNA1, hybridized to chloroplast genes derived from ter-restrial plants and/or other algae. Dinoflagellate chloroplast DNAsexhibited molecular sizes of 114 to 125 kilobase pairs, which isconsistent with plastid sizes determined for other chromophyticalgae (120-150 kilobase pairs). Mitochondrial DNA was not re-solved from nuclear DNA in this system. Two additional satelliteDNAs, satellite DNA2 and satellite DNA3, recovered from P. tamar-ensis and P. catenella were similar to one another, both withinand between species, when characterized by restriction enzymeanalysis. These satellites were 85 to 95 kilobase pairs in size,and exhibited restriction fragments that hybridized to yeast nu-clear ribosomal RNA genes. Restriction enzyme analyses andDNA hybridization studies of cpDNA document that the two Pro-togonyaulax isolates are not evolutionarily identical.

The Dinophyceae is a group of unicellular, eukaryoticorganisms that is composed of photosynthetic, heterotrophic,phagocytic, and saprophytic forms. These algae are secondonly to diatoms in their contribution to primary productionin marine and freshwater ecosystems. In addition, species ofphotosynthetic dinoflagellates have been identified as thedominant organisms comprising the toxic "red tides" thatperiodically close shellfish aquaculture facilities on both theEast and West Coasts.

Dinoflagellates possess unique DNA characteristics. Cellsof this taxonomic group frequently contain very high levels(32-200 pg/cell) of DNA packaged into chromosomes thatlack classic histone-like proteins. These chromosomes remaincondensed throughout cellular interphase (28) and contain an

' Supported by Washington Sea Grant Postdoctoral Award toB.A.B.; National Science Foundation PCM 8402322 and Universityof Washington Special Projects Award to R.A.C.; Washington SeaGrant and Egvet Food Research Fund to J.L.

2 Present address: Environmental Safety Department, Procter andGamble Co., Ivorydale Technical Center, 5299 Spring Grove Avenue,Cincinnati, OH 45217.

unusual pyrimidine, hydroxymethyl uracil, that replaces thy-mine to varying degrees (23). Historically, preparativeamounts of DNA have been difficult to obtain from manydinoflagellate species due to the presence ofboth thecal platesand a thick cell wall (pellicle) (26). Although several investi-gators have analyzed total cellular DNA and/or nuclear genes(e.g. ribosomal RNA) of dinoflagellates, no reports exist con-cerning the isolation and characterization of organellar (chlo-roplast/mitochondrial) DNA from this taxon.

In recent years, Hoechst dye 33528, which binds preferen-tially to A-T3 rich regions of DNA, has become increasinglyuseful in resolving cp- and mtDNA from nDNA in algae (1,14, 19) and terrestrial plants (25). Organelle DNA speciesfrequently migrate as low density satellite bands above themain nuclear DNA band in the Hoechst dye-CsCl gradients.These Hoechst dye-CsCl gradients have also been exploitedin the separation of terrestrial plant nuclear ribosomal DNAfrom total cellular DNA. Ribosomal DNA frequently appearsas a high density satellite band below the nDNA in thesegradients (25).The isolation and characterization of satellite DNA from

the dinoflagellates will be important to future studies concern-ing the organellar gene regulation, genomic organization, andmolecular evolution of this group. Earlier investigators haveobserved the presence of nonnuclear dinoflagellate DNA spe-cies in CsCl gradients (13, 26); however, the origin of theseDNA species was not determined.

In this study, the Hoechst dye CsCl method is applied tothree marine dinoflagellate representatives. Glenodinium sp.has been used as a representative species in numerous pho-tosynthetic studies (7, 22), whereas the isolates of Protogon-yaulax tamarensis and Protogonyaulax catenella are repre-sentative of those organisms responsible for the occurrence ofred tides in the northwestern and northeastern United States.

MATERIALS AND METHODS

Cultures

Cultures of Glenodinium sp. (University of California,Santa Barbara, No. 5M29 1), Protogonyaulax catenella (iso-

I Abbreviations: A-T, adenine-thymine; EtBr, ethidium bromide;sDNA, DNA satellite band; kb, kilobase pair; cpDNA, chloroplastDNA.

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lated by L. Nishitani from Whidbey Island, WA, 1980) andProtogonyaulax tamarensis (obtained from S. Hall, FDA, IDNo. P06) were cultured in 2.0 L off/2 medium (15) containedin 2.8 L Fernbach flasks. Glenodinium sp. cells were main-tained at 20°C on a 12 h light/12 hour dark regimen atapproximately 80 ,uEm -2 s-', whereas Protogonyaulax cellswere grown at 15oC on a 16 h light/8 hour dark cycle atapproximately 50,uE m-2 s-2.

DNA Isolation

Eight (Glenodinium sp.) to 16 (Protogonyaulax) L of midto late logarithmic cells (Glenodinium: 1 x 104 cells/mL;Protogonyaulax: 8 x 103 cells/mL) were harvested by centrif-ugation (5000g, 10 min, 5C) and resuspended in 5 mL of abuffer that contained 0.05 M Tris, 0.50 M EDTA (pH 8.0).Cells were disrupted by vortexing with 0.45 mm glass beads(4 x 50 s). Solutions were kept on ice. The cell homogenatewas centrifuged to remove cell debris (120g, 10 min at 5C)and then incubated with SDS (final concentration: 2%) andproteinase K (final concentration: 400 ,ug/mL) at 37°C for 30min.EtBr was added to Protogonyaulax lysates at a final concen-

tration of 100 ,ug/mL. Solid CsCl was added (1 g/mL), andthe CsCl lysate was centrifuged at 1.5 x 105g for 18 h at 20°Cusing a fixed-angle rotor. The fluorescent DNA bands fromeach isolate were removed from the gradient using a 16 gaugeneedle. EtBr was removed by eight extractions with isopro-panol saturated with TE buffer (10 mM Tris, 1 mm EDTA,pH 8.0). Hoechst dye was added to Glenodinium or Protogon-yaulax DNA-CsCl solutions at a final concentration of 400,ug/mL. The refractive index was adjusted to 1.3960 and thepreparation was centrifuged at 1 x 105g for 40 h in a verticalrotor. DNA bands were removed and each DNA species wasrecentrifuged without further Hoechst dye addition, using theconditions outlined above. Each DNA species was recoveredand recentrifuged at least one additional time. In the finalDNA purification procedure, EtBr was added to each DNAspecies (final concentration: 100 ,ug/mL), the refractive indexwas adjusted to 1.3860, and the solution centrifuged at 1 x105g for 24 h.After EtBr purification, dye was removed from the isolated

DNA species by eight extractions with isopropanol saturatedwith NaCl-saturated TE buffer. The recovered DNA wasdialyzed overnight against TE buffer at 2°C. Sodium acetatewas added to each DNA fraction (final concentration: 0.3 M)and DNA was precipitated with two volumes of 95% ethanolat -20C. Recovered DNA was resuspended in TE buffer andstored at -20°C.

DNA Analysis

DNA was digested with approximately 20 units of restric-tion enzyme per ,ug DNA using conditions recommended bythe manufacturer (Bethesda Research Laboratory). DNA frag-ments resulting from this treatment were separated on a 0.7%agarose slab gel in 1 x TBE buffer (90 mM Tris, 64.6 boricacid, 2.5 mm EDTA, pH 8.3) at 20°C. Negatives of Polaroid545 photographs of EtBr stained gels were scanned with aBio-Rad model 620 video densitometer for estimation of

fragment sizes. Transfer of DNA fragments to Gene ScreenPlus membrane followed the protocol described by DuPont.DNA fragments to be used for hybridization were nick-translated using [32P]ATP (Amersham kit).Gene Screen blots were hybridized with the following genes:

Olisthodiscus luteus chloroplast rbcL (24), mung bean psbA(provided by Drs. K. Ko and N. Strauss, University of To-ronto, Canada), 0. luteus chloroplast rDNA (10), spinachpsaA (29), maize mitochondrial Cox 11 (12), and a nuclearyeast ribosomal operon (4). Membranes were prehybridizedovernight at 60°C in 1.0 M NaCl and 1% SDS and hybridizedat 65°C (high stringency) or 42C (low stringency) for 16 to24 h in 1.0 M NaCl, 1% SDS and 32P-labeled DNA (boiled for5 min before use). After hybridization, blots were washed ateither 65 or 42°C in 2 x SSC (0.15 M NaCl, 0.0155 M Nacitrate, pH 7.5) and exposed to x-ray film using a Kodak X-omat AR intensity screen for 10 to 48 h at -70°C.

RESULTS

Using CsCl-Hoechst dye gradient separation procedures,sDNAs were separated from main band nDNA in Glenodi-nium sp. as well as the toxic dinoflagellates, P. tamarensisand P. catenella. Optimal separation of DNA species inProtogonyaula.x only occurred if a CsCl-EtBr gradient ultra-centrifugation was inserted prior to the CsCl-Hoechst 33258gradient separation. In contrast, an initial CsCl-EtBr gradientstep did not enhance DNA separation in Glenodinium sp.Two sDNAs were separated from nDNA in Glenodinium sp.preparations, whereas three sDNAs were separated in bothProtogonyaulax species. All sDNAs were obtained in lowyields (3-10 ,ug/preparation).

Restriction endonuclease digestion and heterologousctDNA hybridization analyses were used to characterize thethree DNA species isolated from Glenodinium sp. Theseanalyses demonstrated that sDNA,, the satellite of lowestbuoyant density (refractive index: approximately 1.404 ininitial buffer system), was of chloroplast origin, with a molec-ular size of 1 8.2 ± 3.4 kb (Table I, II). A mitochondrial gene(Cox II) did not hybridize to sDNA, or sDNA2 at either highor low stringencies (data not shown). Although sDNA2 had ahigher genomic complexity (> 180 kb) than sDNA,, it did nothybridize to selected chloroplast genes. The third Glenodi-nium sp. DNA band, which was ofhighest density and greatestabundance in CsCl gradients, only hybridized to a nuclearyeast ribosomal DNA probe, and not to any chloroplast geneprobe.

Similar to Glenodinium sp. (data not shown), P. tamarensisand P. nyaulax catenella satellite bands were present in lowabundance and, therefore, were recovered in minimal quan-tity. The sDNA, satellite that exhibited the lowest buoyantdensity (refractive index: approximately 1.401 in initial buffersystem) for both Protogonyaulax isolates was identified aschloroplast DNA (Table I) using heterologous hybridizationanalysis. Molecular size for this DNA was estimated fromrestriction fragment analyses to be approximately 119.3 kbfor P. tamarensis and 124.2 kb for P. catenella (Table II).The sDNA2 and sDNA3 satellites of both Protogonyaulax

isolates are cryptic in origin. When these DNAs were hybrid-ized to chloroplast psbA, psaA, rDNA, rbcL genes, or the

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SATELLITE DNA SPECIES IN DINOFLAGELLATES

Table I. Hybridization Analysis of Dinoflagellate sDNA1 toChloroplast Specific Probes

Gene Probe rRNAa pSaAb psbAc rbcLdkb

GlenodiniumEnzymePvull 5.5 0.6

3.5 *e * 0.42.5

Bstell 19.0 23.0 * 11.03.0

Xhol 12.0 12.0 *Haelll * * 2.4 *

1.9P. tamarensisEcoRl * 6.2 * *

P. catenellaPvuII * 16.0 * *

2.11.4

a rRNA, 0. luteus chloroplast ribosomal RNA operon (10).bpsaA, Spinach PSI (27). CpsbA, Spinach PSII 32 kb protein(provided by K. Ko and N. Strauss, University of Toronto). d rbcL,0. luteus large subunit of nubisco (22). e An asterisk (*) indicatesno hybridization data for these enzyme fragments.

mitochondrial Cox II genes, no hybridization signals were

observed. In addition, sDNA2 and sDNA3 were not cleavedby several restriction enzymes. Of the 14 enzymes tried, onlythose capable of cutting at methylated sites (MspI), a four-base recognition enzyme (HaeIII), and three six-base recog-

nition enzymes (PvuII, HpaI, BglII) would digest these DNAs.When sDNA2 and sDNA3 from both P. tamarensis and P.catenella were digested with these enzymes, restriction pat-terns looked remarkably similar to each other both within a

species (Fig. 1, lanes 2 and 4) and between species (Fig. 1,lanes 2 and 8).

Restriction endonucleases MspI and HpaII can be used todetermine the presence or absence of methylation within a

specific sequence. If methylation occurs at a site (usuallyCCGG), digestion with MspI but not HpaII will occur. Asshown in Figure 2, restriction analyses of sDNA2 and sDNA3from both P. tamarensis and P. catenella indicate that meth-ylation of internal cytosines occurs in these DNAs.To characterize sDNA2 and sDNA3 from Protogonyaulax,

these DNAs were hybridized to yeast nuclear ribosomal DNA(Fig. 3). Positive hybridization signals were seen. Althoughhybridization patterns were identical in sDNA2 and sDNA3within Protogonyaulax species, they differed when comparedbetween P. tamarensis and P. catenella. It should be notedthat the yeast ribosomal DNA used to probe these satelliteDNAs only hybridized to discrete restriction fragments, indi-cating that much sDNA2 and sDNA3 was not encoding ribo-somal DNA.

Additional studies with P. tamarensis sDNA2 indicated

Table II. Summary of Dinoflagellate cpDNA Sizes Obtained from Restriction Enzyme AnalysisaGlenodinium Protogonyaulax tamarensis Protogonyaulax catenella

FragmentBSTE 11 Yhol EcoRl PstI EcoRl Sst1 EcoRl Pvull

1 23.0 24.0 16.0 23.5 16.5 (2x) 14.4 13.0 18.02 11.0 12.0 14.0 12.5 13.0 13.6 12.0 (2x) 17.03 10.0 9.0 12.0 12.0 12.0 12.0 10.5 11.04 9.5 (2x) 7.0 10.5 10.0 11.0 10.6 10.0 10.05 7.0 6.5 9.0 9.0 10.0 8.8 9.5 9.06 6.5 6.0 8.0 7.0 7.0 8.3 9.3 8.07 5.5 5.8 7.0 6.5 6.0 7.8 8.0 8.08 5.0 5.0 (2x) 6.2 6.0 5.0 7.3 7.0 4.69 4.9 4.5 (2x) 5.7 5.0 3.7 6.9 6.0 4.010 3.5 4.2 5.0 4.5 3.5 6.1 5.0 3.511 3.2 4.0 4.6 4.2 3.0 5.1 4.0 3.212 3.0 3.8 4.2 4.0 2.8 3.7 3.0 2.8 (2x)13 2.5 3.7 4.0 3.5 (2x) 2.7 2.9 2.7 2.714 2.0 (2x) 3.2 3.5 3.2 2.0 2.3 2.6 1.915 1.8 3.1 3.2 3.0 1.6 1.9 2.5 1.5 (2x)16 1.4 3.0 3.0 2.8 1.4 1.7 (2x) 2.0 1.217 1.2 2.3 2.8 1.3 1.2 1.7 (2x) 1.0 (2x)18 0.9 2.2 0.7 1.0 1.3 0.919 0.7 2.1 0.6 0.5 1.0 0.320 0.6 0.4 0.521 0.55 0.3

Sum of fragment sizes 114.1 122.1 118.7 120.2 120.3 118.2 125.6 122.7Average genome size 118.8 119.3 124.2

a Data expressed in kb.

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Figure 1. EtBr stained sDNA2 and sDNA3 from P. tamarensis (TAM)and P. catenella (CAT) cut with restriction endonucleases. Lanes 1,7: X DNA digested with HindlIl; lanes 2, 3: TAM sDNA2 digested withHaelll and HindlIl, respectively; lanes 4, 5: TAM sDNA3 digested withHaelll and HindlIl, respectively; lanes 8, 9: CAT sDNA2 digested withHaelll and HindlIl, respectively; lanes 10, 11: CAT sDNA3 digestedwith Haelll and HindlIl, respectively; lanes 6, 12: Bethesda ResearchLaboratory 1 kb ladder.

that, when this DNA was digested with the restriction endo-nucleases HpaI or BglII, a "ladder" or repetitive DNA serieswas seen with a repeat unit length of 333 base pairs (Fig. 4).

DISCUSSION

Although satellite DNAs have been observed in DNA prep-arations from some dinoflagellate species (6, 26), to date therehas been no definitive characterization of the origin of thesesDNAs. In this study of three dinoflagellate species, totalDNA was extracted from algal cells and bands of satelliteDNA were resolved from the majority of nDNA in CsClgradients containing Hoechst dye. These DNA species werethen characterized using restriction endonucleases and heter-ologous gene hybridization.

Unlike terrestrial plants, molecular sizes of the plastid ge-nome of algae are variable, and range from the smallestgenome on record (Codium: 85kb) (16) to the genome ofAcetabularia (>200 kb) (21). Within taxonomic groups, chlo-rophytic algae have exhibited a wide range of plastid genomesizes (85-200 kb), whereas the plastid genomes of thoserhodophytes investigated to date exist in a narrow range of170 to 200 kb (14, 19, M. S. Shivji and R. A. Cattolico,unpublished observation). Plastid DNAs (sDNA,) isolatedhere from three marine dinoflagellates had average molecularsizes of 124 kb (P. catenella), 119 kb (Glenodinium sp.) and1 9 kb (P. tamarensis). When the dinoflagellate genome sizesare compared with other chromophytic algal plastid genomes,

Figure 2. EtBr stained sDNA2 and sDNA3 from P. tamarensis (lanes1-3) and P. catenella (lanes 4-6) digested with the restriction endo-nucleases Hpall and Mspl to determine the presence of methylatedresidues. Lanes 1, 4: undigested DNA; lanes 2, 5: digested withHpall; lanes 3, 6: digested with Mspl; lane 7: X DNA digested withHindlll.

Figure 3. Radioautogram of sDNA2 from P. tamarensis (lanes 1-3)and P. catenella (lanes 4-6) digested with the restriction endonucle-ases, Pvull (lanes 1 and 4), HindlIl (lanes 2 and 5), and Haelll (lanes3 and 6), transferred to Gene Screen Plus, and hybridized to yeastnuclear ribosomal DNA.

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SATELLITE DNA SPECIES IN DINOFLAGELLATES

1 2

Figure 4. EtBr stained gel with DNA from P.with restriction endonuclease, Hpal.

tamarensis digested

such as 0. luteus (150 kb), Vaucheria (121 kb), and Ochro-monas (121 kb), it appears that chromophytes have a rela-tively conserved range of chloroplast genome size.

Steele and Rae (27) have used gene hybridization methodsto determine the relatedness of geographical isolates of thenonphotosynthetic dinoflagellate, Crypthecodinium cohnii. Asimilar approach has been used in this study to determineProtogonyaulax isolate relatedness. Historically, the taxo-nomic position of the toxigenic P. tamarensis/catenella spe-cies complex remains controversial (9). The two generallyrecognized morphotypes within the complex, tamarensoidand catenelloid, cannot be highly correlated with toxin con-

centration, composite toxin profiles, or isozyme assessment(9). Results described here show that the restriction profilesof plastid DNAs from isolates of P. tamarensis and P. cate-nella are not the same, suggesting that these two isolates are

separate species. In addition, hybridization patterns ofsDNA,and sDNA2 to heterologous rDNA are also dissimilar betweenthese isolates. Data such as these may be extended to a largenumber of morphological and/or geographical isolates todetermine Protogonyaulax isolate boundaries.Some conservation of chloroplast gene sequence between

terrestrial plants and dinoflagellates can be inferred fromDNA hybridization analyses. Genes encoding photosystemreaction center proteins (psaA, psbA) derived from spinachhybridize to dinoflagellate ctDNA at relatively high stringen-cies, indicating that sequence homologies are maintainedamong these phylogenetically distinct organisms. In contrast,hybridization does not occur, even at relatively low stringen-cies, between DNA sequence coding for the terrestrial plantlarge protein subunit of rubisco and dinoflagellate cpDNA.However, a positive hybridization response is obtained when

the rubisco large subunit DNA derived from the chromo-phyte, 0. luteus, is used to probe dinoflagellate cpDNA. Apositive hybridization signal was also observed when the 0.luteus rubisco small protein subunit gene was hybridized todinoflagellate cpDNA (B. A. Boczar, J. M. Chesnick, and R.A. Cattolico, unpublished data). Recent investigations haveshown that the chloroplast-encoded 0. luteus rubisco small(8) and large (L. R. Hardison, B. A. Boczar, and R. A.Cattolico, unpublished data) subunit amino acid sequence issimilar to the rubisco small subunit of the chemolithotrophicbacterium, Alcaligenes eutrophus. 0. luteus rubisco large andsmall subunit amino acid sequences are similar to thosederived from Fucus (Phaeophyta) species and the red algaPorphyra yezoensis (J. Woolford and R. A. Cattolico, unpub-lished data). These data suggest that the dinoflagellate rubisco,like that of other chromophytes and rhodophytes, may havehad a purple bacterial origin.

Algal mtDNA has been identified as an adenine-thyminerich satellite DNA in some rhodophytes (14, 19) and chrom-ophytes (1). Genome sizes for these molecules have beendetermined to be approximately 35 to 70 kb in rhodophytes(19) and cryptomonads (J. M. Chesnick and R. A. Cattolico,unpublished data). However, it does not appear that dinofla-gellate mtDNA separates as a satellite in CsCl-Hoechst gra-dients. Yeast mtDNA (Cox II) did show some hybridizationto nDNA in all three dinoflagellates, suggesting a co-migrationof mt- and nDNA in these gradients.

Using renaturation kinetic analysis, the complexity of totalcellular dinoflagellate DNA has been studied by several inves-tigators (3, 23, 26). These studies indicated that 55 to 60% ofthe total DNA in C. cohnii and Prorocentrum cassubicumconsisted of repetitive sequences. Steele (26) observed a guan-osine-cytosin-rich satellite DNA species in CsCl density gra-dients containing DNA from P. cassubicum, and hypothe-sized that this satellite consisted of repetitive DNA sequences.However, this satellite DNA was not further characterized.Protogonyaulax sDNA2 and sDNA3 are relatively adenine-thymine rich DNAs that contain methylated cytosines. Suchmethylated cytosines are characteristics of nuclear derivedsatellite DNAs in both animals (5) and plants (1 1, 20). Diges-tion with HpaI and BglII indicated that sDNA2 and sDNA3also contain repetitive sequences, suggesting that dinoflagel-lates possess sDNAs similar to those of other organisms. Thefact that hybridization to ribosomal DNA genes was restrictedto a few DNA restriction fragments in the dinoflagellatesatellite DNAs suggests that additional coding potential ispresent. The occurrence of ribosomal DNA in A-T richsatellites contrasts with observations that nuclear ribosomalDNA in terrestrial plants has been localized to guanine-cytosine rich satellites, whose densities are heavier than thatof nDNA (17, 25). However, Chlamydomonas reinhardtiiribosomal DNA is found in Hoechst-CsCl gradients at arelatively A-T rich region (2).

Eukaryotic highly repeated DNA sequences are often local-ized in the heterochromatic regions of plant and animalchromosomes (1 1). Heterochromatin is thought to be tran-scriptionally inactive, and satellite DNA sequences often ap-pear to be located in the heterochromatin associated with thecentromeric regions ofchromosome. John and Miklos suggest(18) that repetitive satellite DNA may be important for chro-

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mosome function; however, no specific evidence has beenfound to support this hypothesis. Heterochromatin, as such,is not a structural feature of the highly condensed dinoflagel-late chromosome. Therefore, it would be interesting fromboth an evolutionary and structural view to determinewhether or not the satellite DNAs isolated from dinoflagellatesexhibit homology to those characterized from terrestrial plantDNAs.

ACKNOWLEDGMENTS

We would like to dedicate this paper in the memory of our

colleague, mentor, and friend, Beatrice M. Sweeney. We would alsolike to thank Linda Stolfi-Mecca for initial technical information andM. Kay Suiter for help in manuscript preparation.

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