Plant Physiol. (1982) 70, 92-960032-0889/82/70/0092/05/$00.50/0

Isolation and Characterization of Chloroplast DNA from Chlorellaellipsoidea

TAKASHI YAMADAMitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan

ABSTRACT

A circular DNA molecule was isolated from chloroplasts of ChorellaeHlipsoidea. The DNA had a buoyant density of 1.695 grams per cubiccentimeter (36% GC) and a contour length of 56 micrometers (175 kilobasepairs). The restriction endonuclease analysis gave the same size. Agarosegel electrophoretic patterns of chloroplast DNA digested by several restric-tion endonucleases were also presented. The digestion by the restrictionenzymes, Hpall, Mspl, SmaI, and XmaI revealed no appreciable methyl-ation at CG sites in chloroplast DNA.

The green alga represented by the genus Chlorella includesspecies that differ considerably in biochemical and physiologicalcharacteristics (11). The GC content of the DNAs from differentspecies ranges from 43 to 79% (7). Hybridization of DNAs fromdifferent species of Chlorella to one another indicate that thereare several distinctively different groups of species within thisgenus (10). In contrast, studies on cp DNAs' from different strainsof Chlorella by means of thermal denaturation and renaturationkinetics showed that this genome was quite homogeneous withinthis genus (2) and similar to those of other algae such as Chla-mydomonas and Euglena (1). Recently, the cp DNAs of Chiamy-domonas reinhardii (14, 15) and of Euglena gracilis bacillaris (5)have been characterized. Both cp DNAs were physically mappedwith restriction endonucleases and some specific genes (e.g.rRNAs, etc.) were located on these maps. The cp DNA of Chla-mydomonas was also shown to be methylated during certain stagesof the life cycle of this alga (16). The cp DNAs of these two algaeare physically organized quite differently. This raises the questionas to the organization of other algal cp DNAs such as those fromthe genus Chlorella.

Recently, Yamada and Sakaguchi (25) induced protoplast for-mation in Chlorella species and discussed the possibility of isolat-ing intact chloroplasts and cp DNA from the protoplasts. Thepresent study includes the isolation of an intact circular cp DNAof C. ellipsoidea C-87, the measurement of its buoyant density(GC content) and contour length (mol wt), and some restrictionendonuclease fragment patterns. This is the first report of theintact isolation of cp DNA from Chlorella.

MATERIALS AND METHODS

Plant Material. Chlorella ellipsoidea Gerneck (IAM C-87) wasobtained from the algal culture collection of the Institute ofApplied Microbiology, University of Tokyo. The cells were cul-tured in a modified Bristol medium (24) supplemented with 0.1%(w/v) proteose peptone. The cells were grown in flasks on a

'Abbreviation: cp DNA, chloroplast DNA.

reciprocal shaker with a 16 h light (3,000 lux) and 8 h dark cyclefor 4 to 5 d.

Preparation of Protoplasts and Chloroplasts. Protoplasts wereprepared as previously described from freshly harvested cells (25).For the isolation of chloroplasts, the prepared protoplasts werewashed and resuspended in the homogenization medium A (13)with a modification (0.3 M mannitol was replaced by 0.3 Msorbitol). The protoplast suspension (109 cells ml-1) was homoge-nized at 0°C by a glass homogenizer with a motor-driven Teflonpestle. The homogenate was centrifuged at 1,000 rpm for 5 min toremove unbroken cells and autospores. The supernatant (about 5ml) was then layered onto a discontinuous density gradient con-taining 1.0, 1.5, 2.0, and 2.5 M sucrose (17). The gradients werecentrifuged at 4°C in an RPS 25 rotor at 25,000 rpm for 90 minin a Hitachi 65P ultracentrifuge. The green bands correspondingto chloroplasts (17) were harvested.

Isolation ofcp DNA. Chloroplast DNA for electron microscopicobservations was prepared according to Kolodner and Tewari(13). Chloroplast DNA for restriction endonuclease analyses wasprepared as described by Kolodner and Tewari (12) and purifiedtwice by ultracentrifugation in a neutral CsCl gradient (p = 1.700g cm-3). Analytical CsCl density equilibrium ultracentrifugationin neutral CsCl solution was performed at 40,000 rpm for 30 h at25°C in a Hitachi analytical ultracentrifuge Model 282, withphage SPO 1 DNA (p = 1.741 g cm-3), Escherichia coli DNA (p= 1.710 g cm-3), and Bacillus subtilis DNA (p = 1.703 g cm-3) asdensity markers.

Restriction Endonuclease Analyses. Restriction endonucleasesBamHI, BclI, BglI, HaeII, SstI, SstII, and XhoI were purchasedfrom Bethesda Research Labs (USA); BglII, EcoRI, PstI, andSmaI from Takara Shuzo Co. Ltd. (Japan); HpaII from Boehrin-ger, Mannheim (Germany); and MspI and PvuI from New Eng-land Biolabs (USA). XmaI was purified according to Endow andRoberts (6). Chlorella cp DNA was incubated at 37°C for 1 h witha sufficient amount of restriction endonucleases to effect completedigestion in the buffers recommended by the suppliers. Doubledigestion with KpnI and SstI or XhoI was carried out in the bufferfor KpnI. The buffer for SstI was used for the double digestionwith SstI and XhoI. Agarose gel electrophoresis ofDNA fragmentswas carried out according to Rochaix (14). EcoRI and Hindlllfragments of ADNA were used as mol wt markers.

Electron Microscopy of Chlorella cp DNA. Chloroplast DNAin 10 mm Tris-HCl (pH 8.5) containing 1 mm EDTA, 0.03% Cytc, and 50% formamide was spread onto the hypophase consistingof 1 mm Tris-HCl (pH 8.5), 0.1 mm EDTA, and 20% formamideaccording to Davis et al. (3). The spreads were examined in a JEM100B (JEOL) electron microscope after rotary shadowing with Pt-Cd. Contour length of circular cp DNA molecules was determinedby comparison with plasmid pBR322 DNA (22).

RESULTS

Isolation of cp DNA. DNA prepared from protoplasts of C.ellipsoidea showed two UV absorbing bands in neutral CsCldensity equilibrium gradients (Fig. la); the minor band at density

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CHLOROPLAST DNA FROM CHLORELLA

FIG. 1. Absorbance profile of DNAs from C. ellipsoidea, separated bypreparative CsCl gradient centrifugation. a, DNAs from protoplasts; b,from isolated chloroplasts. Chloroplast DNA was considerably enriched(arrow).

less than 1.700 g cm-3 accounted for about 8% of total DNA.When DNA was prepared from isolated chloroplasts, the band atthe lower density accounted for more than 85% of total DNA (Fig.lb) suggesting that it be identified as cp DNA band. Although theband of cp DNA was greatly enriched (about 10 times), the DNAof the higher density could not be completely eliminated even bythe treatment of chloroplast preparations with DNase (13). Forrestriction endonuclease analyses, the cp DNA fractions wererepurified by ultracentrifugation in CsCl gradient.

Characterization of cp DNA. The buoyant densities of the cpDNA and nuclear DNA in CsCl equilibrium density gradientswere found to be 1.695 ± 0.002 g cm3 and 1.717 ± 0.002 g cm3,respectively (Fig. 2). The density of cp DNA corresponds to GCcontent of 35.7% (in molar percent) in the DNA (21) which iscomparable to that of cp DNAs from higher plants and severalalgae (8). Electron micrographs of the cp DNA isolated by CsCl-ethidium bromide ultracentrifugation (13) revealed circular mol-ecules and linear fragments (Fig. 3a); most circles were in the openconfiguration and some were supercoiled. In Figure 3b, the con-tour lengths of several circular cp DNA molecules from C. ellip-soidea are given in pBR322 units (4,362 base pairs) (22). Fromthese determinations, the population of the cp DNA circles ap-peared to be uniform in size. The mean length was calculated as40 pBR322 units which corresponds to 56 ,um, 175 kilobase pairs,or 115 x 106 daltons. The size was also determined by restrictionendonuclease analyses where the cp DNA was digested with KpnI,SstI, XhoI, KpnI + SstI, KpnI + XhoI, and SstI + XhoI (Fig. 4).A list ofDNA fragments including their sizes and stoichiometriesis given in Table I. The largest size (about 170 Kbp) obtained withSstI agrees with the size of cp DNA determined by electronmicroscopy.

Restriction Endonuclease Patterns of cp DNA. Since the intra-molecular heterogeneity of cp DNA from Chlorella strains wasknown (1, 2), the fragment patterns of cp DNA from C. ellipsoideaproduced by the digestion with several restriction endonucleaseswere studied. Figure 5 shows the restriction patterns of the cp

FIG. 2. Photoelectric scans from analytical ultracentrifugations of C.ellipsoidea DNAs. The buoyant densities of cp DNA and nuclear DNAwere determined as 1.695 and 1.717 (g cm-3), respectively. Density markersused were as follows: DNA of B. subtilis (1.703 g cm-3), of E. coli (1.710g cm-3), and of phage SPOI (1.741 g cm-3).

DNA digested with EcoRI, BglI, BglII, Hae II, KpnI, PstI, SalI,SstI, BamHI, and XhoI. From the size and the base compositionof the DNA, and from the specificity of each restriction endonu-clease, the number of cleavage sites per cp DNA is expected asfollows: 57.8 for EcoRI and BglII; 18.2 for BamHI, KpnI, PstI,SalI, SstI, and XhoI; 24.0 for HaeII; 5.8 for BglI. In most cases,the observed number of sites agree with the predicted values: 23for EcoRI; 11, BglI; 22, BglII; 30, HaeII; 14, KpnI; 19, PstI; 20,SstI; 18, XhoI. BamHI and SalI, apparently, cut the cp DNAsignificantly less.

Recently, DNA modification in eukaryotes, such as the meth-ylation of C residues, has been demonstrated and discussed withrespect to its significance (4). For the green alga Chiamydomonasreinhardi, the methylation of cp DNA is known to play an impor-tant role in the maternal inheritance ofchloroplast (18). Therefore,the cp DNA of Chlorella was tested for the methylation of Cresidues. Figure 6 shows a comparison of the restriction fragmentpatterns with SmaI, XmaI, HpaII, and MspI, which were used toprobe for methylation of DNA (16, 26). If the methylation at theinternal CG sites of cp DNA occurs, SmaI which recognizes thesequence ofCCCGGG and HpaII (CCGG) would not cleave theDNA in contradistinction to their isoschizomers XmaI and MspI,respectively. Inasmuch as most band patterns were the samebetween the isoschizomers (Fig. 6), methylation at the CG siteswas very rare if they occurred. On the other hand, the isoschizo-mers gave different band patterns, when the nuclear DNAs of C.ellipsoidea were digested. This indicated a low level of methylationat the CG sites.

DISCUSSIONThe cp DNA from C. ellipsoidea is a large circular molecule

with a buoyant density of 1.695 g cm-3 (36% GC). In this respect,

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FIG. 3. Electron micrograph of an open circular cp DNA molecule of C. ellipsoidea (a). Bar marker represents 1 Jtm. b, Length frequency distribution:)f the cp DNA. Size was presented in pBR322 units (4,362bp).

_..11I111..111..1.................. . .

FIG. 4. Agarose gel electrophoresis for size determination of cp DNA from C. ellipsoidea after cleavage with several restriction endonucleases: (1),A DNA digested with Hind111; (2), Kpnl; (3), Sstl; (4), KpnI + SstI; (5), Kpnl; (6), Xhol; (7), Kpnl + XhoI; (8), Sstl; (9), Xhol; (10), SstI + Xholcleavage pattern of cp DNA; and (i1), A DNA digested with EcoRI (all on the same 0.5% agarose gel).

it is similar to those reported for several higher plant and algae(8). However, the size of the cp DNA of C. ellipsoidea (175kilobase pairs, 115 x 106 daltons) is considerably larger than thoseof higher plant whose mol wt are reportedly, at most, 100 x 106daltons (8); one exception is the cp DNA of duckweed Spirodelaoligorrhiza which was reported to have a size of 115 to 120 x 106daltons (23). In algae, Euglena gracilis (Euglenophyceae) andVaucheria sessilis (Xanthophyceae) possess a very small cp DNA(8). Chlamydomonas reinhardii as well as C. ellipsoidea (Chloro-phyceae) seems to have larger cp DNAs; the size of C. reinhardiicp DNA is 126 x 106 daltons (14). The size variation of cp DNAsfrom various origins may have some evolutionary significance. In

this connection, DNA from cyanelles of Cyanophoraparadoxa hasa size of 110 x 106 daltons, though its buoyant density is veryhigh (1.716) (9).

Restriction endonuclease analyses of the Chlorella cp DNArevealed that the sites for some enzymes such as BamHI(GGATCC) and SailI (GTCGAC) were significantly fewer thanexpected (Fig. 5). PvuI (CGATCG) and BclI (TGATCA) did notdigest the cp DNA (data not shown). This may be due to modi-fications at these sites or to a nonrandom base arrangement of thecp DNA. The methylation of the C residue of cp DNA such asreported for C. reinhardii (18) might occur in C. ellipsoidea, thoughmethylation of the internal CG sites of the sequence CCGG or

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CHLOROPLAST DNA FROMCHLORELLA9

Table I. Sizes ofDNA Fragments Resultingfrom Digestion with Several Restriction Endonucleases of cp DNA from C. ellipsoidea (in Base Pairs)

The sizes were determined from the electrophoretic mobility in 0.5% agarose gel.

RestrictionEnzymes KpnI SstI XhoI KpnI/SstI KpnI/XhoI SstI/XhoI

(Fragment No.)

I >30a 23.5 21.7 23.5 20.0 15.02 16.3 17.0 20.0 17.0 12.6 12.13 13.2 15.0 17.5 12.7 11.8 10.1(2)4 10.5 12.9 10.7 12.1 10.8 9.005 9.61 12.1 9.76 9.40 10.7 7.656 9.40 10.8 8.49 9.00 9.40 6.807 8.49 10.4 7.01 8.08 8.49(2) 6.558 8.08 9.15(2)b 6.35 6.82(2) 6.95 6.159 6.95 6.80 6.00(2) 6.25 6.35 5.9510 5.25 6.25 5.27 5.78 6.15 5.301 1 4.87 5.78 4.85 5.50 6.00 5.25(2)12 4.52 4.80 4.65 4.95 5.27 4.8513 4.08 4.40(2) 3.72(2) 4.40 4.80 4.4014 3.85 4.25 3.30 4.25 4.65 4.20(2)15 1.80 4.00 2.85 4.00 3,72(2) 3.9516 3.64 1.95 3.64 3.30 3.6017 3.22 3.22 2,85 3.26(2)18 1.80 2.86 1.95 3.5519 2.45(2) 2.3020 2.16 2.1221 1.77(2) 1.8722 1.53 1.6923 1.19 1.2924 1.19

Sum >136.9 169.34 143.82 163.59 147.99 150.93

a Large smear bands; the sizes could not be determined.b The numbers in parentheses indicate a probable stoichiometry of 2.

JlFBEEC At SW AA~~~~~~~~~~~~~~~~~~~~~~~~~~~~~......-Aaoegl1 2 3 4 5 6 7 8 9 1011 1 ?2 5 i /2 d^

FIG. 5. Aaoeglelectrophoresis of cp DNA after cleavage with various restriction endonucleases: (1), EcoRI; (2), BglI; (3), BglII; (4), HaeII; (5),KpnI; (6), PstI; (7), Sa~l; (9), SstI; (10), BamHI; (11), XhoI fragments of cp DNA; and (12), A DNA digested with HmndIII (0.5% agarose gel).

CCCGGG was absent in the Chiorella cp DNA. Methylation at The cp DNA used mn this study was isolated from nonsynchron-other DNA sites might have taken place. Indeed, methylation ously and photosynthetically growing cells of C. ellipsoidea; it willenzymes were isolated and characterized in C. reinhardli (19, 20), be interesting to compare the present results with those ofcp DNAone of which existed in vegetative cells and was specific to the from cells at various growth phases in synchronous culture orbase sequences TCG, TCC, and TCA. from cells growing under different conditions (heterotrophically

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FIG. 6. Agarose gel electrophoresis of cp DNA (a) and nuclear DNA(b) after cleavage with restriction enzymes SmaI, Xmal, HpaII, and MspI.a, Cp DNA digested with SmaI (1), XmaI (2), HpaII (3), and MspI (4). X

DNA digested with HindIl was used as a size marker (5). b, NuclearDNA digested with SmaI (2), XmaI (3), HpaII (4), and Mspl (5). A DNA-HindIII marker was in (1). Electrophoreses were carried out on 0.7%agarose gel.

or mixtotrophically).Based on the restriction endonuclease patterns obtained in this

work, a physical map of the cp DNA from C. ellipsoidea is beingconstructed. Specific genes for the rRNAs and RuBPCase will belocated on this map.

Acknowledgments-The author is very grateful to Dr. Shinkichi Irie for analyticalultracentrifugation and to Drs. Kenji Sakaguchi and Hiroshi Mitzutani for helpfuldiscussions.

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