Isolation and Characterization Chloroplast Marine Chromophyte

Plant Physiol. (1981) 68, 641-6470032-0889/81/68/0641/07/$00.50/0

Isolation and Characterization of Chloroplast DNA from theMarine Chromophyte, Olisthodiscus luteus: Electron MicroscopicVisualization of Isomeric Molecular Forms'

Received for publication August 26, 1980 and in revised form February 24, 1981

JANE ALDRICH2 AND ROSE ANN CATTOLICODepartment of Botany, AJ-JO, University of Washington, Seattle, Washington 98195

ABSTRACT

Chloroplast DNA (ctDNA) from the marine chromophytic alga, Olis-thodiscus luteus, has been isolated using a whole cel lysis method folowedby CsCI-Hoechst 33258 dye gradient centrifugation. This DNA, which hasa buoyant density of 1.691 grams per cubic centimeter was identified asplastidic in origin by enrichment experiments. Inclusion of the nucleaseinhibitor aurintricarboxylic acid in all lysis buffers was mandatory forisolation of high molecular weight DNA. Long linear molecules (40 to 48micrometers) with considerable internal organization comprised the major-ity of the ctDNA isolated, whereas supertwisted ctDNA and open circularmolecules averaging 46 micrometers were occasionally present. Also ob-served in this study were folded ctDNA molecules with electron densecenters ("rosettes") and plastid DNA molecules which have a tightly wound"key-ring" center. The ctDNA of Olisthodiscus has a contour length thatis median to the size range reported for chiorophytic plants.A minor component of the total celular DNA, which originates from a

DNase insensitive cellular structure, has a buoyant density of 1.694 gramsper cubic centimeter. This DNA consists predominantly oflinear molecules,but open circles 11.5 micrometers in length and rare 22-micrometer di-mers were also present.

This study represents the first analysis of the extranuclear DNA of achromophytic alga.

The Chromophyta represent a plant division whose memberspossess both Chl a and carotenoid pigments but lack Chl b. Areference (5) to the unpublished data of Hennig and Kowalliksuggests that chloroplasts of the Xanthophycean alga Vaucheriasessilis contain circular DNA molecules of 37 um contour length.This limited information exists as the only data on the chloroplastchromosome unit size for any member of this important evolu-tionary line of plants. In contrast to the paucity of information onctDNA3 structure among the Chromophyta, considerable data isavailable from a wide variety of Chlorophyta, those plants whichcontain both Chl a and b. Chloroplasts ofthe Chlorophyta containmultiple DNA copies (4, 18) whose unit chromosome is a circularmolecule (20, 26). Land plant representatives of this supertaxadisplay a ctDNA unit chromosome which ranges from 38.5 ,um in

'Supported by National Science Foundation Grant PCM7624440 toRAC and United States Public Health Grant HDO7183 from the NICHDto J. A.

2Present address: Standard Oil of Ohio, Cleveland, Ohio.3 Abbreviations: ctDNA, chloroplast DNA; Sarkosyl, sodium n-laurol

sarcosinate; ATA, aurintricarboxylic acid.

contour length (22) for liverwort, 43 to 45 ,Im for fern (22), and 38and 46 ,im for angiosperm species (20, 26) such as corn, oats, pea,snapdragon, and evening primrose. Chloroplast DNA contourlengths of four chlorophytic algal representatives have been suc-cessfully analyzed. A mol wt of 1.5 x IO' daltons has been reported(16, 33) for Acetabularia. Although linear molecules with contourlengths as high as 200 ,um have been recovered from Acetabularia,intact ctDNA molecules have not been recovered from this organ-ism. Among the less structurally complex unicellular Chlorophyta,the chloroplasts of Chlamydomonas reinhardtii have been shown(5) to contain a circular 62 ,um ctDNA molecule whereas Euglenagracilis which has green algal-like chloroplasts (15) containsctDNA whose unit chromosome is 40 um in size (30). Finally, themulti-cellular coenocyte, Codium fragile (Hedberg and Hammer-sand, personal communication) has a unit chloroplast chromo-some of approximately 28 ,um in length. This size represents thesmallest ctDNA chromosome among those studied.

In all Chlorophyta studied to date, the ctDNA is arranged (15,21) in nucleoidal packets dispersed throughout the organelle. Eachnucleoidal packet contains many unit chromosome sets (21). Al-though some members (15) of the Chromophyta also have thisnucleoidal arrangement, those Chromophyta in which girdle la-mella are present within the plastid have been shown (15) tocontain a single ring-shaped nucleoid. In this report, the firstdetailed study on the isolation and characterization of the chlo-roplast unit chromosome from a chromophytic alga, Olisthodiscusluteus4 is reported.

MATERIALS AND METHODS

Sources of Chemicals. BSA, Sarkosyl, mannitol, ATA and Cytc were purchased from Sigma. PEG was purchased as Carbowax6000 from Union Carbide Corp. (New York, NY) and PharmaciaFine Chemicals (Piscataway, NJ) was the source of Ficoll. Reno-grafm was purchased from Squibb. DNase I that was free ofRNase was purchased from Worthington Biochemical Corp.(Freehold, NJ). Calbiochem-Behring Corp. (La Jolla, CA) sup-plied the Hoechst 33258 dye, and optical grade CsCl was pur-chased from Harshaw Chemical Co. (Solon, OH). Baker ChemicalCo. (Phillipsburg, NJ) provided crystallized phenol and 2-mercap-toethanol. The phenol was redistilled before use and was stored ina light-protected container at -20 C. Collodion was purchasedfrom Ladd Laboratories, Burlington, VT.

Cell Maintenance and Harvest. Olisthodiscus luteus (Carter) wasgrown in 800 ml of artificial sea water medium contained in 3-liter Fernbach flasks. Cultures were maintained on a 12 h light/12

4 The taxonomic affmity of 0. luteus is disputed. The organism has beendescribed as a Xanthophyte (7) a Chrysophyte (14) and a Chloromonad(28).

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ALDRICH AND CATTOLICO

h dark regime as previously described (8). Cells were harvested inthe linear phase of growth (5 x 105 cells/mi) by centrifugationusing a GSA rotor in a Sorvall RC-5 centrifuge for 15 min at3,000 rpm at 4 C. Large culture volumes were harvested using anSS-34 rotor with a Szent-Gyorgi-Blum continuous flow attach-ment. Routine analysis for fungal and bacterial contaminationwas stringently maintained.

Buffers. Buffer A contains 0.23 M mannitol, 20 mm Tris, 0.1%w/v BSA, and 0.01% v/v 2-mercaptoethanol (pH 7.6), whereasbuffer P is composed of buffer A to which 10%o w/v PEG and 1%w/v Ficoll were added. These two buffers were used in cellulardisruption steps. Buffer R contains 10 mM Tris, 70 mm sucrose(pH 7.0) and was used in sucrose-Renografm gradient fractiona-tion of chloroplasts. Buffer D contains 5% w/v Sarkosyl, 0.15 MNa3C6H5O7.2H20, 50 mm EDTA, and 2 mM ATA (pH 7.0) andis used in DNA extraction.

Chloroplast Isolation.Protocol I. A total of 1 to 2 x 109 cells were collected by

centrifugation. Pellets, resuspended in 80 ml buffer A, were dis-rupted at 750 psi using an automatic French Press. The cellhomogenate was centrifuged at 2,700 rpm for 15 min at 5 C usinga Sorvall RC-5 centrifuge and an HB-4 rotor. Alternatively, aDNase treatment (see below) occurred before this centrifugationstep. The chloroplast pellet was resuspended in buffer R whichcontained 50%o w/v Renografm. This suspension became the mostdense solution in a 0, 10, 20, 25, and 50% sucrose-Renografmgradient (6). The gradient was centrifuged at 10,000 rpm for 1 hat 5 C using a Beckman L5-50 centrifuge and SW 27 rotor.Chloroplasts which banded at the 10 to 20%/o interface were col-lected by use of a pasteur pipette and diluted by adding 100volumes of half strength buffer R. The diluted suspension ofplastids was centrifuged at 10,000 rpm for 15 min using an SS-34rotor. The pellets were stored at -20 C or were used immediatelyfor ctDNA isolation.

Protocol II. Depending on the experiment, a total of 1 to 20 x109 cells were collected by centrifugation. Pellets, resuspended in100 ml buffer P were disrupted in a French Press at 1,000 psi.DNase treatment followed and was identical to that describedbelow except that the final chloroplast washes were with buffer Prather than buffer A. The homogenate was centrifuged at 2,700rpm at S C for 15 min in an SS-34 rotor and the pellet was

resuspended in buffer P. The DNase-treated, washed pellet was

placed on a sucrose pad (0.8 M sucrose, 0.15 M NaCl, 0.2 M EDTA,50 mM Tris, 1 mm ATA, pH 8.0) and centrifuged at 5,000 rpm for1 h at 5 C. The pellet was used immediately for ctDNA isolation.DNase Treatment. Following cellular disruption, both DNase

(100 ,ug/ml) and MgCl2.6H20 (10 mM final concentration) were

added to the homogenate. This solution was then incubated at 0 Cfor 1 h. After enzymic treatment, the chloroplasts were washed 5times using buffer A which contained 50 mM EDTA and 1 mmATA. Centrifugation was at 2,700 rpm at 5 C in an SS-34 rotorfor each wash step.DNA Extraction.Whole Cell. Chilled pellets of approximately 4 x 108 cells were

lysed in 6 ml of buffer D. After 5 mm, when lysis was complete,solid CsCl was added and the lysate was then prepared for CsCldensity gradient centrifugation in the presence of Hoechst 33258dye (see below).

Chloroplast. Chloroplasts prepared by Protocol II were lysed inan equal volume of buffer D at 5 C for 20 to 40 min. Upon lysiscompletion, which was monitored by light microscropy, solid CsClwas added and Hoechst 33258 eye-density centrifugation followed.Alternatively, chloroplasts prepared by Protocol I were lysed as

described above and the DNA was purified by the phenol extrac-tion technique of Cattolico (9). Occasionally, in this phenol ex-traction technique, a pronase digestion step was included beforecentrifugation. DNA species ofmuch higher mol wt were obtained

when 1 mm ATA (17) was included in all buffer systems.Preparative CsCI Hoechst 33258 Dye Density Centrifugation.

Five-ml gradients contained CsCl at a density of 1.69, 3% w/vSarkosyl, a maximum of 200 ,ug DNA and 1 mg Hoechst dye(stock solution was 10 mg/ml distilled H20). Although increasedamounts ofDNA precludedDNA species separation, dye amountscould vary from 1 to 20 times the amount of DNA present.Gradients were centrifuged at 40,000 rpm for 24 to 36 h using aBeckman centrifuge and Ti 65 rotor. An easily removable floatinggreen mat was present on the gradient after the first centrifugation.These initial gradients were fractionated manually to remove bulknuclear DNA. Extranuclear DNA species were centrifuged tohomogeneity and fractionated using an ISCO fractionator (Instru-mentation Specialties Co., Lincoln, Nebr.) at a flow rate of 0.375ml/min. No additional dye was required for these subsequentpurification steps. Dye removal was effected by eight extractionsof the recovered DNA with CsCl-saturated isopropanol. To re-move CsCl, the DNA solution was dialyzed against two literchanges of a pH 8.0 buffer containing 100 mm NaCl, 50 mm Trisand 10 mm EDTA. This treatment was followed by dialysis withtwo 1-liter changes using a pH 8.0 buffer which contained 0.1 mmTris, 1 mm EDTA. Buffer changes were made every 6 or 12 hafter which the DNA was either used immediately for electronmicroscopy, analytical CsCl gradient pycnographic analysis ac-cording to the method of Cattolico (9), or was stored at -20 C.

Electron Microscopy. DNA obtained from CsCl-Hoechst dyegradients was prepared for electron microscopy by the Kleinsch-midt (25) method. DNA spread on a Cyt c monolayer was pickedup using collodion coated grids. The grids were stained withuranyl acetate, rotary shadowed with platinum/paladium (80/20)and examined using a JEOL 100 B electron Microscope. Negativesof electron micrographs ofDNA molecules were enlarged and themolecules were traced using an Electronic Graphics calculator(Numonics Corp., Lansdale, Pa.). A grating replication calibrationgrid (E. Fullam, Inc., Schenectady, N. Y.) of 2157 lines/mmprovided a magnification reference. The molecule linear density,2.08 x 106 daltons/,um, was used (40) to calculate the mol wt fromthe contour length. In later experiments, 4X174 was included asan internal reference DNA as Stuber and Bujard (40) have shownthat this DNA is adequate as an internal reference for higher molwt DNA contour length determinations.

RESULTSChloroplast DNA Enichment. Two DNA species are visible

when whole cell DNA is analyzed by isopycnic centrifugationusing Model E ultracentrifugation. The nuclear species has abuoyant density of 1.702 ± 0.001 g/cm3, whereas a maior satellitespecies has a buoyant density of 1.691 ± 0.001 g/cm (Fig. IA).DNA extracted from isolated chloroplasts recovered from a su-crose-Renografm gradient displays a Model E ?rofile in which 25to 30%o of the nucleic acid is of the 1.691 g/cm species (Fig. 1B).

1711.731 1.61 13 .

FIG. 1. Buoyant density distribution of Olisthodiscus DNA species. A,whole cell DNA; B, DNA from isolated chloroplasts; C, DNA fromisolated chloroplasts which have been DNase-treated. 1.691 = chloroplastDNA; 1.702 = nuclear DNA; 1.731 = Micrococcus luteus marker DNA.

642 Plant Physiol. Vol. 68, 1981

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ctDNA IN A CHROMOPHYTIC ALGA

If chloroplasts are DNase-treated before the Renografm sucrosegradient step to remove contaminating nuclear DNA, 75 to 80%oof the DNA in the resulting Model E profile is represented by the1.691 g/cm3 buoyant density component (Fig. IC). These enrich-ment experiments provide strong evidence that the 1.691 g/cm3component is chloroplast in origin.

Extranuclear DNA: the Presence of Two Satellites. Highlyrefractile chloroplasts give indication that the membrane systemof the organelle is intact (39). This factor is critical in reducingplastid DNA loss resulting from nuclease penetration of theorganelle. Although chloroplasts of excellent quality (Fig. 2) wereisolated from Olisthodiscus, recovery of only 1% of the theoreticalyield of ctDNA was obtained. This low yield was not a result ofDNase penetration, for high mol wt ctDNA was isolated (seebelow) from these DNase-treated organelle preparations. The lowctDNA recovery values obtained in this experimental approachwere probably due to the presence of PEG in the chloroplastisolation buffer. This reagent, although critical to chloroplastmaintenance in early DNase experiments caused extensive chlo-roplast clumping (Fig. 2B) when the DNase cofactor Mg2+ wasadded. Thus, PEG was virtually impossible to remove and tena-ciously protected the chloroplasts from disruption. For this reason,a whole cell lysis technique (see "Materials and Methods") wasdeveloped. This method represented a significant advance in theisolation of plastid DNA from Olisthodiscus, for ctDNA recoveriesnear 75% of that theoretically present within the cell were ob-tained.DNA isolated from these whole cell lysates was fractionated on

CsCl gradients. The gradients contained the dye, Hoechst 33258,which preferentially binds to AT-rich regions of the DNA (31).Three DNA species were separable using this dye-gradient com-bination (Fig. 3). The buoyant density of these DNA species byModel E analysis were 1.702 ± 0.001 g/cm3 (nuclear), 1.691 +0.001 g/cm3 (chloroplast), and 1.694 ± 0.001 g/cm3 (origin un-known). When DNA was isolated from chloroplast preparationsand analyzed by the Hoechst dye-CsCl method, the 1.691 g/cm3and 1.694 g/cm3 DNA species were present in highly enrichedquantities relative to 1.702 g/cm3 nuclear DNA. It should benoted that the origin of the cryptic 1.694 g/cm3 DNA species isunknown. Inasmuch as this satellite is seen in DNase-treatedchloroplast preparations, it must originate from a nonnuclear,DNase-insensitive organelle. Although not directly measured, thisDNA species represents approximately 0.05 to 0.1% of the totalDNA (2.2 x 10-12 g) within the cell; hence, isolation of thissatellite required enormous quantities of cells.

A b

FIG. 2. Isolated chloroplasts from Olisthodiscus luteus. A, chloroplastsare discrete refractile bodies following cellular disruption. B, clumping ofchloroplasts results from addition of Mg2" required for DNase treatment,but refractility is maintained.

FIG. 3. Separation of whole cell DNA into component species using a

Hoechst dye-CsCl gradient. Model E ultracentrifugation of recoveredbands (lower scans) revealed that band A is the nuclear 1.702 buoyantdensity component, band B is a satellite ofunknown origin having buoyantdensity of 1.694 and band C is chloroplast DNA which has a buoyantdensity of 1.691. Micrococcus luteus marker DNA has a buoyant densityof 1.731.

Size Analysis: 1.694 g/cm3 Satellite DNA. Six centrifugationcycles were required to obtain a homogeneous 1.694 g/cm3 satellitespecies before it was spread for electron microscopic analysis. Thepredominant proportion of this DNA species was present in linearform. However, 5 to 15% of the molecules were observed to occur(Fig. 4) in both relaxed and highly twisted configurations. Thepresence of the nuclease inhibitor ATA greatly increased thenumber of intact circular molecules recovered. The relaxed mol-ecules of the 1.694 g/cm3 DNA species had a contour length of 11

± 0.7 ,um (23.0 ± 1.5 x 106 daltons). A total of 60 moleculesobtained from both cell lysates and isolated chloroplast prepara-tions were measured. Occasionally, larger relaxed and highlytwisted molecules (Fig. 4-2) were observed (less than 0.1% of thepopulation). The relaxed molecules (12 measured) had a contourlength of 21.9 ± 1.2 tim (45.5 ± 2.5 x 106 daltons) and probablyrepresent a dimer of the smaller size class.

Size Analysis: 1.691 g/cm3 Chloroplast DNA. The 1.691 g/cm3

buoyant density component was centrifuged to homogeneity (fourrebandings) and spread for electron microscopic analysis. Al-though the majority of molecules observed were linear with a molwt of 80 to 100 x 106 daltons, a very low proportion of themolecules (0.5% or less) were either in supertwisted (Fig. SA) orrelaxed (Fig. 5B) form. A total of 15 circular molecules weremeasured and a contour length of 45.7 ± 2.5 ,um (94.9 ± 5.2 x 106daltons) was obtained. Five of these molecules were from DNase-treated chloroplasts (94.5 ± 4.7 x 106 daltons) and 10 were fromwhole cell lysates (95.1 ± 5.5 x 106 daltons).

qpC

A B C

1731 1702 1731 1.694 1691 1.731 1691

643Plant Physiol. Vol. 68, 1981

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.1

V~~~

FIG. 4. Circular DNA molecules from 0. luteus. These were obtained

from the 1.694 g/cm3DNA satellite. (1), indicates supertwisted and relaxed

23 x 106 daltons and (2), indicates supertwisted and relaxed 46 x 106daltons DNA.

0.5

FIG. 5. Circular DNA molecules from 0. luteus. These molecules were

from the 1.691 g/cm3 (chloroplast) DNA satellite. A, supertwisted, and B,

relaxed form.

Chloroplast DNA: Internal Organization. Although fully su-pertwisted or relaxed molecules were observed in the electronmicroscopic analysis of ctDNA, other topological forms in whicha high degree of internal structural organization occurred werealso evident. The linear molecule of Figure 6C, which representsthe major size class of ctDNA isolated displays distinct "organi-zational centers" from which loops of twisted or untwisted DNAextend. Organizational centers connected by several strands ofuntwisted DNA from which supertwisted or relaxed DNA loopsemanate are also present within the relaxed ctDNA dimer ofFigure 6E. An electron opaque core (Fig. 6, A and D) wasoccasionally located at the organization center. These rosetteforms (core + looped or linear DNA) were often clustered (Fig.6D) and frequently joined one another via strands of DNA.Rosettes of different sizes were observed to occur. For example,the two rosette structures of Fig. 6D were estimated to be 22 and39-x 106 daltons, whereas the molecule displayed in Figure 6Ahas a mol wt of 80 x 106 daltons, which approaches unit chro-mosome length. A most interesting topological form (Fig. 6B) wasseen only twice in all samples prepared for electron microscopicanalysis. Although this structure (approximately 80 x 106 daltons)lacked a core region, it did contain a "key-ring" arrangement (27)of tightly wound DNA (see Fig. 6B inset) from which loops ofDNA extend.To eliminate the possibility that rosette-like structures were an

artifact of spreading, ammonium acetate concentration in thehypophase and hyperphase was varied in DNA spreads preparedfor electron microscopy. Variations were also made in Cyt c ageand concentration. Although these factors were found (11) toaffect the quality of DNA spreading, rosette molecules remainedunder all conditions tested. Several additional observations suggestthat rosette structures were not artifactual. (a) It is known (3) thatintercalation of ethidium bromide into DNA decreases the buoy-ant density of the molecule. When Olisthodiscus ctDNA wasfractionated on CsCl-ethidium bromide gradients, rosette mole-cules were present only within the most dense portion of thebanded DNA. The gradient fraction in which the rosettes ap-peared (1) never contained supertwisted DNA molecules andmigrated lower than those fractions which contained either longlinear or relaxed circular DNA. This observation is consistent withlimited ethidium bromide uptake imposed (3) by a conformation-ally constrained molecule. (b) Enhanced recovery of rosette mol-ecules was always obtained when ctDNA was minimally cycledthrough CsCl-Hoechst dye gradients, suggesting that unfolding ofthe ctDNA organization centers and DNA purification were si-multaneous events. (c) Although very long, linear molecules ofnuclear DNA (1.702 g/cm3 buoyant density) with nucleosomalstructures were obtained when minimal CsCl gradient centrifu-gation was performed, no rosette structures were seen in this DNAspecies. Further cycling of the nuclear DNA through CsCl gra-dients produced only linear DNA which was devoid of nucleoso-mal particles. (d) As a control, 4X174 DNA was co-spread withctDNA. No rosette structures were induced by the experimentalconditions of DNA spreading in this exogenous DNA source.

DISCUSSION

The highly fluorescent dye, Hoechst 33258, which binds pref-erentially to AT-rich DNA sequences, has been employed suc-cessfully in CsCl gradients to accentuate the buoyant densitydifferences of cellular DNA (12, 31, 38). Excellent resolution ofthe three 0. luteus cellular DNA species present in crude celllysates is obtained in a one-step purification/separation procedure.A significant variation in DNA to dye ratio (1- to 10-fold) is easilytolerated, thus this preparative technique offers a valuable methodfor recovery ofDNA from crude cell lysates where DNA quantitymay only be estimated. The fact that the minor 1.694 g/cm3satellite was revealed and successfully purified from the chloro-


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FIG. 6. Highly organized molecules present in ctDNA preparations. A, folded molecule of near unit length (80 x 106 daltons) with core bodyattached at organization center. B, folded molecule of near unit length (80 x 106 daltons) lacking core body. Inset shows compact coiling of center key-ring. C, linear molecule of approximately 100 x 10 daltons; arrows indicate centers of organization. D, smaller folded rosette-like molecules joined attheir centers by DNA strands. The single center part of this joint structure is 39 x 106 daltons and the double center part is 22 x 106 daltons. E, dimerof approximately 200 x 106 daltons with super-twisted regions (long arrow), and organization centers (2 small arrows) from which large twisted anduntwisted loops extend.

plast and nuclear DNA species, demonstrates the power of the measurements of circular molecules in the 1.694 g/cm3 crypticmethod. satellite DNA is in agreement with values obtained (Aldrich et al,A mol wt of 23 x 106 daltons obtained from contour length manuscript in preparation) by restriction enzyme analysis of this


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DNA. Although the origin of this DNA is presently unknown, itis probably not a nuclear component.Enhanced recovery of thissatellite is routinely obtained after the DNase treatment step ofthe isolation procedure. Only a DNA present within a DNaseinsensitive structure wouldshow this refractility to destruction.Since the chloroplast preparations are contaminated by mitochon-dria as evidenced from electron microscopy, it is possible that the1.694 g/cm3 species originates from this extranuclear source. Al-though experiments are in progress, the isolation and characteri-zation of mitochondrial DNA from Olisthodiscus has been ex-tremely difficult.

Contour length measurements of the open circular molecules ofthe 1.691 g/cm3 ct DNA of 0. luteus provide a mol wt of anaverage of 95x 106 daltons. Restriction enzyme analysis indicatesthat this DNA is a homogeneous population of molecules of anaverage of 99 x 106 daltons (Aldrich et al., manuscript in prepa-ration). In addition, reassociation kinetic analysis (Ersland et al.,manuscript in preparation) using phage X and T4 DNA as internalstandards gives an Olisthodiscus ctDNA genome size of 91 x 106daltons and 105 x 106 daltons, respectively. Therefore, by threeindependent measurements, an average value of 97 x 10W daltonsmay be assigned to the ctDNA. Whether this unit chromosome iscircular orlinear remains to be confirmed by restriction enzymemapping.

It has been reported (9) that a change in the growth conditionsunder which an 0. luteus culture is maintained will induce theorganism to alter its chloroplast complement from 38 to 14 plas-tids. Both chloroplast and nuclear DNA amount per cell remainconstant during this transition. As these earlier studies demon-strated, a reciprocal relationship exists between chloroplast num-ber and DNA content per plastid. Knowledge of the size of the 0.luteus chloroplast chromosome permits assignment of a copynumber to these data. A cell which has a 14 plastid complementpotentially contains 34 ctDNA molecules per chloroplast whereasa cell with 38 plastids would contain 12. The lower limit of DNAneeded to maintain a fully functional chloroplast (one whichincreases mass and replicates) is still an unanswered question.The isolation of structural isomers of ctDNA may provide

insight into the possible organization and localization of the unitchromosome within the chloroplast nucleoid. Highly organizedinternal molecular arrangement of the ctDNA is consistentlyobserved. Frequently, molecules containing a double-strandedbreak unwind in only a restricted area, while maintaining otherlocal domains in a supertwisted configuration. Organization cen-ters or areas from which loops of DNA emanate in both asupertwisted or untwisted form were also frequently noted. Theorganization centers seen in the 0. luteus ctDNA molecules (Fig.6, C and E) strikingly resemble the complexes formed by catena-tion of the plasmid HM 456 and SV 40 viral DNA by topo-isomerase activity of Xenopus germinal vesicle extracts (2). Therare "key-ring" arrangement seen in Figure 6B is similar to thekey-ring structure observed (27) in the enormous Col El plasmidcatenates generated by Escherichia coli gyrase. These observationsimply that topoisomerase activity may exist within the chloroplast.DNA supercoiling which results from the double stranded DNAbreak-and-reunion activity of these enzymes is suggested to beimportant to the process of DNA replication, recombination andtranscription (10). The fact that ctDNA replication in Euglena isinhibited by naladixic acid (29), an inhibitor which specificallyinterferes (13) with subunit A activity (nicking-closing) of topo-isomerase, supports this hypothesis.

Folded chromosomes with twisted or relaxed loops attached toa core structure seen in 0. luteus have also been reported forctDNA of Spinacia (19, 41), Antirrhinum (19), and Sphaerocarpos(22). These molecules shows a remarkable similarity in appearanceto both the looped folded E. coli chromosome (24) and to thehistone-depleted HeLa cell metaphase chromosomes (35) wherein

concentric DNA loops are attached to scaffold proteins.In 0. luteus folded ctDNA chromosomes with core structures

are recovered most frequently in first run CsCl-Hoechst dyegradients. In these gradients, virtually no supercoiled or relaxedctDNA molecules are seen. Subsequent centrifugation results inloss of the folded ctDNA form and a low yield recovery ofsupertwisted or relaxed ctDNA molecules. This low recovery ofcircular DNA forms might result from a transition from the foldedmolecule directly to a nicked linear form which is induced by highsalt core stripping. The fact (20) that 80%o of the total plastid DNAof spinach is in the open circular form following repeated CsClgradient centrifugation suggests that the affinity of the core bodyto DNA varies among plant types. The molecular composition ofa"Gcore"l structure is not known. Work with spinach (41) hasdemonstrated that prolonged proteinase K treatment is necessaryto remove core structures from ctDNA indicating the presence ofa "masked" or of a "heterogeneous" protein component. Thisenzymic treatment causes conversion of the folded spinach ctDNAto the linear form. Spinach ctDNA cores are refractile to diethyl-pyrocarbonate, SDS, chloroform, and RNase treatment. Similarrefractility is seen in 0. luteus ctDNA, where core bodies survivemild pronase treatment, phenol extraction and RNase digestion.

Folding of ctDNA may not be entirely dependent upon thepresence of a core structure. When membrane-free E. coli chro-mosomes are isolated, an RNase sensitive site is apparent (36).Pettijohn and Hecht (36) have suggested that DNA-RNA inter-action is involved in the maintenance of folded chromosomeintegrity. Whether this mode of chromosome folding occurs withinthe chloroplast is unknown.The replicon model of Jacob et al. (23) postulates that DNA

membrane attachment sites are involved in DNA replication. Thefact that (a) ctDNA is closely associated with thylakoid mem-branes in higher plant (14, 37) and algal (14) cells, and (b)[3HJthymidine incorporation into ctDNA is closely associated withgranal thylakoids, suggests that membrane ctDNA interactionmight also be of importance in plastid DNA replication. Recently,Pardoll et al. (34) have presented a model of DNA replication inwhich DNA is attached to a nuclear matrix at numerous fixedreplication complexes. Looped DNA is reeled through these sitesand replicated. Isolation ofjoined rosette structures from 0. luteus(Fig. SD) supports this model. The presence of one or moreattachment sites per chloroplast chromosome of 0. luteus may belogistically advantageous in segregation of ctDNA within thechloroplast which divides (Cattolico, unpublished) its ring-shapednucleoid by central fission. In summary, the maintenance, repli-cation, and segregation of ctDNA may be dependent upon inter-action of the DNA with thylakoid membranes, intermolecularcatenation/decatenation promoted by topoisomerase(s) and pos-sible DNA-RNA interactions.The 0. luteus chloroplast chromosome (98 x 106 daltons) is

mid-range in size when compared to the chloroplast chromosomeof the Chlorophyta. It is interesting to note that the organellarDNA of these two plant supertaxa are similar (32) in size to thesymbiotic, nonfreeliving cyanelles of Cyanophoraparadoxa. Thesecyanelles, which have a genome size of 115 x 106 daltons, havebeen postulated to represent an intermediate between blue greenalgae and chloroplasts. The mechanism and significance of main-taining the remarkable size constancy among ctDNA populationsof different plant types remains an intriguing question.

Acknowledgment-We would like to thank D. McCabe and J. Voss for theirexcellent technical support and H. Lyman and L. McIntosh for helpful discussions.

LITERATURE CITED

1. ALDRICH J 1980 Extranuclear DNA from the marine chromophyte, Olisthodiscusluteus. PhD thesis, University of Washington, Seattle, Washington

2. BALDI MI, P BENEDETTI, E MATTOCCIA, GP TocCHINI-VALENTINI 1980 In vitrocatenation and decatenation of DNA and a novel eukaryotic ATP-dependent


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Isolation and Characterization Chloroplast Marine Chromophyte