Deoxyribonucleic Acid of the Blue-Green Algae (Cyanophyta)The Cyanophyta or blue-green algae share the kingdom Monera with the Schizomycophyta or bacteria, because both are distinguished

BACERIOLOGICAL REVEWS, Dec. 1967, p. 315-331 Vol. 31, No. 4Copyright © 1967 American Society for Mcrobiology Printed in U.S.A.

Deoxyribonucleic Acid of the Blue-GreenAlgae (Cyanophyta)

MARVIN EDELMAN,1 DAVID SWINTON,2 JEROME A. SCHIFF, H. T. EPSTEIN,AMD BERNICE ZELDIN

Department ofBiology, Brandeis University, Waltham, Massachusetts 02154

INTRODUCDONI................................................................MATERIALS AND METHODS.....................................................

Culture Conditions ........................................................Extraction of Blue-green Algal DNA ...........................................Fractionation of Cyanophora paradoxa........................................Density-gradient Centrifugation................................................

Chemical and Enzymatic Analyses............................................RESULTS AND DSCUSSION...................... ..............

Blue-green Algal DNA Patterns...............................................Question of Satellite Components..............................................Nature of the Satellite Component...........Distinguishing Between Light-absorbing and Light-scatterinlg Macromolecules ill the

Analytical Ultracentrifuge..................................................

DNA Density Shifts Due to Presence of Polysaccharides........................Taxonomic and Phylogenetic Significance of the DNA Base Compositions............Relationships between Blue-green Algal and Chloroplast DNA......................DNA of Cyanophora paradoxa ................................................

LITERATURE CITED....

315315315316316317317318318319319

320322322325327329

INTRODUCTrONThe Cyanophyta or blue-green algae share the

kingdom Monera with the Schizomycophyta orbacteria, because both are distinguished by aprocaryotic type of intracellular organization,where the protoplast of the cell does not con-tain organelles conspicuously delimited from thesurrounding cytoplasm. The blue-green algae,however, share the property of an oxygen-evolving type of photosynthesis with the eucary-otic algae and higher plants and produce phyco-erythrin and phycocyanin, two photosyntheticpigments characteristic of the Rhodophyta, orred algae, and one or two other smaller groups.It has been suggested that the organelles ofeucaryotic cells, such as the chloroplast, arosefrom a blue-green algal-like endosymbiontwithin an otherwise nonphotosynthetic cell.The phylogenetic affinities of the blue-greenalgae, therefore, are of considerable interest.With the finding (25) that deoxyribonucleic

acid (DNA) base compositions from relatedorganisms are similar and can be employed as anindex of phylogenetic relatedness (as judgedfrom more conventional taxonomic evidence),

I Present address: Department of Biochemistry,Weizmann Institute of Science, Rehovoth, Israel.

2Present address: Department of Biology, Stan-ford University, Stanford, Calif.

and with the accumulation of DNA base compo-sition data for certain bacteria and for chloro-plasts of several of the eucaryotic algae and higherplants, it became of interest to compare the DNAbase compositions of various blue-green algalspecies with each other, with those of othergroups of organisms, and with various chloro-plast DNA's.

This paper presents a characterization of theDNA of a number of blue-green algae with respectto several physical and chemical properties in-cluding their base-pair compositions. The DNAcharacteristics are reported and discussed withregard to their taxonomic and phylogeneticsignificance and their relation to hypotheses ofchloroplast origin. Several points of interestconcerning certain polysaccharides, which wereconsistently encountered in the DNA fraction ofthe Cyanophyta, are presented. A brief accountof this work has already appeared (M. Edelman,D. Swinton, J. A. Schiff, and H. T. Epstein,Plant Physiol. (Suppl.) p. x, 1966).

MATERIALS AND MErHODSCulture Conditions

Axenic cultures of blue-green algae, obtainedfrom several sources (acknowledged in Table 3),were usually grown at 25 to 30 C in 500-mlErlenmyer flasks containing 100 ml of liquid

315

on March 17, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

http://mmbr.asm.org/

EDELMAN ET AL.

media on a rotary shaker under an atmosphereof 1 to 5% CO2. Occasionally, when largeramounts were required, several liters of mediawere used. Intensity of illumination was from 50to 200 ft-c. Algae were harvested when growthappeared sufficient to yield a 2- to 5-ml packedvolume of cells. Stock cultures were maintainedon medium solidified with 1% agar withoutadded CO2 .

All cultures were adapted to, and grown onSoil Extract Medium (58) except the following:Nostoc species were maintained in a modifiedBristol's Nitrate Broth (24), marine species(Anacystis marina, Dermocarpa violacea, Lyngbyasp. 77, and Plectonema sp. 52) were grown inArtificial Seawater Medium (40) and Cyanophoraparadoxa was grown in Cyanophora ConservationMedium, II (Provasoli, personal communication).

Before a culture was used experimentally, itwas routinely checked for contamination. Inaddition to microscopic examination, a sampleof culture was added to 5 ml of sterile BrainHeart Infusion Medium (Difco) and aerated for24 hr at 30 C. When turbidity appeared at theend of this period, a large amount of the contami-nating cells was grown up, and the DNA wasextracted and subjected to analytical ultracen-trifugal analysis. The density pattern thus ob-tained was compared with a similar DNA densitypattern from the parent blue-green algal culture.In each instance of cell contamination encoun-tered by us (e.g., in cultures of Nostoc muscorumand Nostoc punctiforme, 384), a minor band inthe blue-green algal DNA pattern could bematched with the DNA profile of the freshlygrown contaminant and thereby identified andeliminated from consideration. The identityof each of the various blue-green algal speciesused in this study was accepted as indicated onthe label of the culture tube upon arrival. Otherthan microscopic examination to verify grossmorphological features, no attempt was made toconfirm the various assignments.

Extraction of Blue-Green Algal DNA

Cultures were harvested at 7,000 to 10,000 x gfor 5 min and suspended in 5 to 10 ml of salineethylenediaminetetraacetic acid (EDTA) [0.15M NaCl, 0.1 M EDTA, pH 8 (29)]. Filamentousblue-green algae were subjected, at this point, tohomogenization at 4 C for 30 sec with a Teflonhomogenizer to reduce the length of theirfilaments. Cell breakage was accomplishedwith a French pressure cell (4 C) applying from12,000 to 18,000 psi. Occasionally, breakage bylysozyme treatment (8) was also employed. TheFrench press effluent (or lysozyme extract) was

D(r0com

DENSITY (g/cm3)

FIG. 1. DNA density profiles from Plectonemaboryanum cells treated with lysozyme or pressure.Microdensitometer tracings from ultraviolet-absorp-tion photographs of Plectonema DNA centrifugedto equilibrium at 44,770 rev/min and 25 C in CsCldensity gradients. Photographs taken after 20 hrin the analytical ultracentrifuge. The peaks at 1.743g/cm3 are due to SP8 DNA, which was added as adensity standard to calibrate the gradients. Peaks at1.707 g/cm3 are due to Plectonema DNA.

directed into a chilled test tube containing 1 mlof a 10% sodium dodecyl sulfate solution (2 xrecrystallized) and was gently mixed with thissoap solution for 5 min to complete lysis.DNA was isolated by the method of Marmur

(29), with minor modifications (12, 26). Theisopropanol precipitation step was not omittedin this study.

Figure 1 compares ultracentrifugal DNA pat-terns from Plectonema boryanum cells broken bypressure and lysozyme treatment. As anticipated,pressure-treated samples yielded a larger variancein the DNA banding pattern than did lysozyme-treated samples, thus indicating the shearingeffects of the former treatment on the DNAmolecules. However, both techniques producedidentical buoyant density medians at 1.707g/cm3. The band of density 1.743 g/cm3 is adensity marker (SP8 phage DNA) introducedto calibrate the CsCl gradient. Since the averagebuoyant density was the datum sought in thisstudy, and cell breakage by lysozyme treatmentis not feasible for many blue-green algal species(8), pressure was employed routinely.

Fractionation of Cyanophora paradoxaCells were harvested at 500 x g for 10 min at

25 C and were resuspended in two times theirpacked volume of saline-EDTA with the aid ofa machine-driven Teflon homogenizer for 15

316 BACTERIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from


DNA OF BLUE-GREEN ALGAE

sec. From 60 to 80% of the host protozoan cellswere ruptured by this procedure, but few cyanelles(blue-green algal inclusions) were broken. Thehomogenate was divided into two parts. Onewas treated directly with 0.05% lysozyme in0.03 M sodium phosphate buffer, pH 6.8, for 4hr at 37 C, and then with 1.0% (final concentra-tion) sodium dodecyl sulfate (SDS) for 5 minat 20 C. DNA isolated from this fraction isdesignated as the whole cell extract. Incubationof a sample of this extract with deoxyribonucleaseproduced the deoxyribonuclease-treated wholecell extract.The second part of the homogenate was sub-

jected to 0.01% (final concentration) SDS for1 min at 20 C. This resulted in lysis of the re-maining whole protozoan cells, but not of thecyanelles. These were sedimented at 5,000 x gfor 5 min at 4 C and the supernatant fluid wasdecanted and extracted, yielding supernatantfraction DNA.The 5,000 X g pellet, containing cellular debris

and cyanelle particles, was further divided intotwo parts. The first part was resuspended directlyin 2 to 3 volumes of saline-EDTA, treated withlysozyme, and with 1.0% (final concentration)SDS as described above; then the lysate wasextracted to give the pellet fraction DNA. Asecond sample was taken up in 1 ml of 0.25 Msucrose and 0.005 M MgCl2 and was treated with

500 g10.0.... 251C

DC vol. 3.lneMMNa"u atsatoe

lwe_s S D S

I°°S%@ o.OI.o.o

8.05. 0.015hr... 37C 1.I, 2000

3 D 3 C.nt Lfuge

I.OS 5000 05 Eta., 200c l5 L., 4&

2rzt1OO15000 510S--ft0

000007008 0..o.pood~~DPA..t.etI hs1ed R-asndl ~~~~~~~~~~~~~~~Su-nt;t Practice,

1.e Call OoO0.tl 21 .1. 8.11. On 1.0 .1,0- 25 N .ueros

aliquot o0.0050 0C12

M00.. ly.OSS 200..(200 mg/e1. 0.055 ii.:It45 d.2, ;50 ( hr... 370 450L, 25&

"0 Tp.0.d SODS 0.0101100.|_1. 8.11 00Oo.oO

1.05 5000g5L.. 20°C 5.-L.. 00

OVA ..t..ctio PelletI Supn-tee,

|~ ~ ~~~~~~. pIn (Det.-L OD26)Pallet FraetLon

2X -I. SolLn_E

ly o...

10.05S. 4 hr... 37C

S D S1.0%, S5o.. 200&

OAl *.trectot.

Cymll, freettio

FIG. 2. Fractionation scheme for Cyanophoraparadoxa. Designations are explained in the text.

deoxyribonuclease. Just before the start of in-cubation, 50 ,ug of SP8 phage DNA (density1.743 g/cm3) was added to the reaction mixtureas a check on the effectiveness of the enzymatictreatment. At the termination of the incubationperiod (45 min), the mixture was sedimentedonce more at 5,000 X g for 5 min at 4 C, the su-pernatant fluid was saved for optical density meas-urements at 260 m,, and the pellet was resus-pended in 2 volumes of saline-EDTA. Lysozymetreatment, followed by the addition of 1.0%SDS (final concentration), brought about lysis ofthe isolated cyanelle particles, as confirmedby the release of the photosynthetic pigmentsinto the medium and by direct microscopicobservation. DNA was extracted from thislysate to yield the cyanelle fraction. An outlineof the fractionation procedure is shown in Fig.2.

Density-gradient CentrifugationCsCl (Harshaw, optical grade) density-

gradient centrifugation (35, 51) was carried outin a Spinco model E analytical ultracentrifuge,as previously described (12). A 0.5-,ug amountof SP8 or SPOI phage DNA was introducedinto the centrifuge cells in all experiments, toserve as a density standard. Experimental sam-ples were investigated at two different concen-trations: one, at 1 to 2 ,ug of DNA per centrifugecell, to determine mean buoyant densities, andthe other, at 25 to 50 ,ug of DNA per centrifugecell, to enable detection of minor DNA compo-nents, either endogenous or exogenous, downto the 1% level (M. Edelman, Ph.D. Thesis,Brandeis Univ., Waltham, Mass., 1965). Densi-tometer tracings were made with a Joyce-Loebldouble-beam recording microdensitometer.

Preparative gradient ultracentrifugation wascarried out in cellulose nitrate tubes in the SW39 rotor of a Spinco model L ultracentrifuge.A 4-ml sample in a CsCl solution was centri-fuged to equilibrium at 39,000 rev/min for 48hr at a chamber temperature of 4 C. Fractionswere collected dropwise through a puncture inthe bottom of the centrifuge tube and diluted to5 ml with SSC [0.15 M NaCl, 0.015 M trisodiumcitrate, pH 7.0 (29)] for optical density measure-ments (reported per milliliter) at 260 m,u.

Chemical and Enzymatic AnalysesDNA was determined by a modification of

the Dische diphenylamine procedure (4). RNAwas estimated as pentose-reacting material bythe Mejbaum modification of the Bial orcinolreaction (34) and the dichromatic procedureused by Dische (11). Total protein was assayedwith Folin reagent according to the method of

VOL. 31, 1967 317


http://mm

br.asm.org/

Dow

nloaded from


EDELMAN ET AL.

Lowry et al. (28). Carbohydrates were quali-tatively assayed by a modification of the orcinoltest described by Brown (3) and quantitativelydetermined by using Dreywood's anthronereagent as reported by Morris (36).

Deoxyribonuclease treatment was accom-plished with 200 jig of pancreatic deoxyribonu-clease (Worthington) in 1 ml of 0.25 M sucroseand 0.005 M MgC12. Incubation was carried outat 25 C for 45 min, and the reaction was termi-nated by the addition of 2 volumes of saline-EDTA. Ribonuclease treatment was carried out

1.706

NATIVE

1.743

1.721z

CID

DE Y (gDENATURED0

:j i.743

1.7 43

DNase TREATED

DENSITY (g/cm3)FIG. 3. DNA density profiles of Phormidium

luridum, a representative blue-green alga. Conditionsoj centrifugation are as in Fig. 1. Peaks at 1.743g/cm3 are ofdensity marker, SP8 DNA. Upper tracingshows the native Phormidium DNA profile peaking at1.706 g/cm3. Lower tracing reveals the sensitivity ofthe native DNA preparation (1.706 g/cm3) to de-oxyribonuclease treatment.

S ~~~~I'-,z 1.7234 ~~~~~I

W DENATURED

(10I

DNase TREATED

DENSITY (g/cm3)

FIG. 4. Satellite components in the DNA fractionof Plectonema boryanum. Conditions of centrifugationare as in Fig. 1. Peaks of density 1.743 g/cm3 showSP8 standard DNA. Upper tracing shows native DNA(35 sxg) with a main band at 1.707 g/cm3 and a satellitecomponent at 1.658 g/cm3. Middle tracing showsdenatured DNA (25 ,ug) with the main band shiftedto 1.723 g/cm3 and the satellite component remainingat 1.658 g/cm3. Lower tracing shows thze deoxyribo-nuclease-treated, native DNA profile. A 35-,ug amountof DNA was subjected to enzymatic treatment; asingle peak, at 1.658 g/cm3, remains after treatment.

as described by Marmur (29). Trypsin (100,ug/ml) digestion was performed at pH 7.5, 25 C,for 30 min.

RESULTS AND DISCUSSIONBlue-green Algal DNA Patterns

When DNA was extracted from a typical,axenically grown blue-green alga (in this case,Phormidium luridum, a representative memberof the Oscillatoriales) and subjected to analyticalultracentrifugation, the CsCl buoyant densityprofiles shown in Fig. 3 were obtained. The bandof density 1.743 g/cm3 is due to SP8 phage DNA.which was introduced as a density marker in allprofiles shown. Native DNA (upper tracing),after centrifugation to equilibrium, showed aunimodal density distribution at 1.706 g/cm3.Treatment to heat-denature the DNA (middletracing) resulted in an upward shift in buoyantdensity of 0.015 g/cm3 characteristic of double-stranded molecules (30), and produced an in-crease in band width in accordance with a re-duction in molecular weight (35). Treatment ofthe DNA sample with deoxyribonuclease (lower

707

NATIVE

318 BACTERIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



tracing), tollowed by dialysis against SSC,resulted in the failure of the treated sample toproduce a band in the CsCl gradient, due toenzymatic degradation of the DNA polymer.These characteristics were shared by all of theblue-green algal DNA samples tested by us.

Question of Satellite ComponentsIn an attempt to determine whether minor or

"satellite" components were present in anyof the DNA samples investigated, large amountsof DNA (from 25 to 50 ,ug) were introducedinto analytical ultracentrifuge cells and were

centrifuged to equilibrium. As is evident fromFig. 4, certain of the purified DNA samples did,indeed, show a satellite band. Plectonemaboryanum extracts are shown in which a mainband (1.707 g/cm3) and satellite band (1.658g/cm3) are evident (upper tracing). However,this satellite material neither increased in densityupon heat treatment (middle tracing) nor ex-

hibited a sensitivity to incubation with deoxyribo-nuclease (lower tracing), while the main bandDNA did exhibit such changes in each instance.Moreover, this satellite band exhibited an un-usually low CsCl buoyant density for a DNAspecies. [Crab testes poly-AT DNA and syntheticpoly-AT band at 1.680 g/cm3 and are the leastdense DNA species reported (51, 55).]

Nature of the Satellite ComponentThe satellite band material of Fig. 4 was sub-

jected to several preparative density gradientcentrifugations and collections to further purifyit for analysis. The final gradient pattern isshown in Fig. 5A, where a single peak at anaverage density of 1.658 g/cm3 accounts foressentially all of the material measurable at260 m,u.

Several tests were made on this purified ma-terial (Table 1). The enzymatic and chemical testsfor DNA, RNA, and protein clearly indicatedthat the satellite band was not composed of

FRACTION NUMBER24

WAVELENGTH (mph)

FIG. 5. Purification and spectrophotometric pro -

file of the Plectonema satellite band. (A) Preparat,vegradient ultracentrifugal pattern of the deoxyri bo-nuclease-treated DNA fraction from Fig. 3. Conditionsofcentrifugation as in Materials and Methods. Buoyantdensity was calculated from refractive index measure-ments (Si). (B) Spectrophotometric profile ofpurifiedsatellite band material. Fraction 32 of (A), dialyzedagainst distilled water, was scanned in the v:sible andultraviolet regions of the spectrum in a Cary model 14recording spectrophotometer. Insert shows a plot ofthe log of the optical density against the log of thewavelength for this data. Slope of the resulting straightline is -4.2.

TABLE 1. Analysis of blue-green algal satellite band materiala

Substance sought Treatmentb Result

Deoxyribonucleic acid .............. Deoxyribonuclease ResistantcDiphenyl amine <1 JAg

Ribonucleic acid................... Ribonuclease ResistantcOrcinol <5 ,sgd

Protein............................. Trypsin ResistantcFolin phenol <1 jAg

Carbohydrate ...................... Orcinol Positive for hexosedAnthrone 1,500 jug (equivalents of glucose)e

a From fraction 32, Fig. 5A.b See Materials and Methods.c Material having an optical density at 260 mju of 0.10 was treated. "Resistant" refers to the unaltered

density, concentration, and band width of the satellite component in CsCl equilibrium gradients aftertreatment.

d See Fig. 6 for relevant spectra.e Material having an optical density at 260 mnA of 0.50 was treated.

319VOL. 31, 1967


http://mm

br.asm.org/

Dow

nloaded from


EDELMAI

04

z

< 0 2

0-tL

400 450 500 550 600 650

WAVELENGTH (mHu)700

FIG. 6. Absorption spectra of Plectonema satelliteband material after treatment with modified orcinolreagent (3). Spectrochemical characteristics of similarconcentrations (approximately 100 ,ug) of RNA, glu-cose, and satellite material were determined with aCary model 14 recording spectrophotometer. Satellite-band material and glucose peak at 660, 530, and 435mA,u and RNA pentose peaks at 670 and 430 m,.

these molecular species. On the other hand,positive tests (qualitative and quantitative) forcarbohydrates were obtained and a value of 1,500-,ug equivalents of glucose per 0.5 optical den-sity units at 260 m,s of satellite band materialwas determined.

Figure 6 shows comparative data for the ab-sorption spectra of RNA (Worthington, commer-cial), glucose, and the purified satellite bandmaterial after treatment with orcinol reagent(34). The spectrum for glucose exhibited maximaat 660, 530, and 435 m, (3), as did the satelliteband material. The spectrum produced by RNApentose was qualitatively different, showing onlytwo maxima, at 670 and 430 m,u.The nondialysable nature of the satellite ma-

terial, together with the banding it exhibitedin equilibrium density gradiei,s (Fig. 5A),required it to be of a fairly high molecular weight(35). Coupled with the results of the analyticaltests in Table 1 and Fig. 6, a polysaccharide wasindicated. Further evidence for this identification(Fig. 5B) was in the spectrophotometric profileof the purified satellite material. It was apparentthat the carbohydrate-positive satellite materialwas scattering, rather than absorbing, UV light.The insert in Fig. SB shows a plot of the log ofoptical density against the log of the wave-length, in which the experimentally obtainedlinear slope is -4.2, closely approximating theexpectation of -4 from Rayleigh's law for lightscattering by ideal particles. The milky-whiteappearance of the concentrated, purified satelliteband in preparative CsCl gradient tubes and itsformation of a colloidal suspension in watersubstantiated this finding. Such light scattering

N ET AL. BACTERIOL. REV.

behavior is characteristic of large polysaccharides,such as starch or glycogen.

Distinguishing Between Light-absorbingLight-scattering Macromolecules in the

Analytical Ultracentrifuge

and

From a consideration of the spectrophotometricdata of Fig. SB and the quantitative determina-tion of carbohydrate (in glucose equivalentunits) by the anthrone test (Table 1), the specificextaiction coefficient (E260) of the satellite bandpolysaccharide material from Plectonemaboryanum was estimated to be 60-fold lower thanthe E260 for DNA. This difference between DNAand the polysaccharide material can be exploitedto provide a simple and rapid method for dis-tinguishing between these two macromolecularspecies in a single analytical ultracentrifugesample.

Figure 7 shows photographs of such a sampletaken with both the UV and Schlieren opticalsystems. In the first case, optical density, and inthe second, the rate of change in refractive index,was measured across the gradient. In the upperphotograph (UV optics), the band at 1.710g/cm3 represents 1 ,ug of Escherichia coli DNA,and that of density 1.658 g/cm3 represents 60ug of the purified Plectonema polysaccharidematerial based on the anthrone test shown inTable 1. These amounts were chosen to produceapproximately equal blackening of the UV-sensitive film by both macromolecular species.A comparison of the areas under the curvesobtained in microdensitometer tracings (notshown) of this film showed that this conditionwas approximately met. In the lower photograph,taken with the Schlieren optical system, the dif-ference between the optically similar bands in theUV-sensitive photograph is revealed. The poly-saccharide material at 1.658 g/cm3, due to itsgreater concentration, produced a much largerdisturbance in the refractive index gradient thandid the lesser amount of DNA at 1.710 g/cm3.Thus, the degree of disturbance of refractiveindex measured across the gradient, and the dif-ferent apparent absorbancies of the two sub-stances in UV light, permitted a clear distinctionto be made between light-absorbing and light-scattering macromolecules present at a givendensity in a sample.Based on this method of detection, a number of

polysaccharide species have been found in theDNA fractions from several blue-green algae.Some were undetectable using UV-absorptionphotographs but were clearly visible in Schlierenphotographs of the same sample. Some samples

- - - GLUCOSE- - RNA-SATELLITE BAND MATERIAL / t \

435 / 670

4~~~~~~~~~~~530 1 6

A"; 4 / \\430 \

\ ,I1

O I',

320


http://mm

br.asm.org/

Dow

nloaded from



contained two such bands. Table 2 gives thesources and buoyant densities in CsCl of thesepolysaccharides. All the blue-green algal speciesfound by us to contain a polysaccharide in theirisolated DNA fractions belong to the orderOscillatoriales, whose members possess extensivemucopolysaccharide sheaths surrounding theirfilaments. Possibly, therefore, these DNA-fraction contaminants were derived from thismaterial. However, no attempt was made byus to localize or further characterize these poly-saccharides.The buoyant densities of the polysaccharide

1.710

lI

1.658

II

I I

I I

I I

FIG. 7. Distinguishing between lighit-absorbing andlight-scattering macromolecules in the analyticalultracentrifuge. Conditions of centrifugation are as inFig. 1. Upper photograph made with the ultraviolet-absorbing optical system; lower photograph madewith the Schlieren-refractive index optical system.The band at 1.710 g/cm3 is from I ,ug of Escherichiacoli DNA, and that at 1.658 g/cm3 from 60 Ag ofPlectonema polysaccharide material.

TABLE 2. Buoyant densities of blue-green algalpolysaccharides in CsCI

Species Density (g/cm3)

Anabaena spiroides............... 1.654Tolypothrix sp ................... 1.655Lyngbya sp., 77 .................. 1.656Plectonema boryanum, 581 ........ 1. 658; 1.672Anabaenopsis sp.................. 1.659Anabaena variabilis............... 1.664Plectonema sp., 52 ............... 1.665Nostoc muscorum 12*3*2 .......... 1. 665; 1.672Nostoc punctiforme, 384 .......... 1.665; 1.675Nostoc muscorum................. 1.666Nostoc sp., 586................... 1.668Lyngbya sp., 487................. 1.684Nodularia sphaerocarpa, 11-1-1.... 1.704; 1.729Calothrix parietina............... 1.753

polymers shown in Table 2 varied to aslightly greater degree from one determinationto another than did the usual DNA densityvalues; they were reproducible to i 0.003 g/cm3.Although many of these polymers have densitieslower than those of known DNA species, themajor portion of the DNA density spectrum (51)is encompassed by these polysaccharide densitiesas well.

Observations concerning polysaccharide con-taminants in DNA fractions have recently beenmade by other investigators. The presence ofthese polymers in chloroplast (41), Drosophilia(47), and frog egg (10) DNA fractions has beennoted. Substances in E. coli lysates, which bandin the DNA buoyant density region in CsCland behave similarly to the polysaccharidesdescribed here with respect to their Schlierenoptical properties, have been described bySchumaker and Wagnild (52). In mammaliansystems, polysaccharides have been identifiedin HeLa cell DNA preparations (54) and charac-terized in mouse liver DNA fractions (7, 54).

Thus, in a wide spectrum of biological systems,the DNA fraction of the cell, extracted and puri-fied according to current methods (20, 29),appears to be contaminated with carbohydratepolymers. As it is now evident that these poly-saccharides can be responsible for the appearanceof minor components or "satellite bands" inUV-absorption photographs of purified DNAsolutions, it is suggested that a comparison ofUV-absorption and refractive-index patterns ofultracentrifuge samples be routinely made (asin Fig. 7) whenever multicomponent patternsare encountered and are to be interpreted. Thismethod should supplement, rather than replace,

321VOL. 31, 1967

.-; ,I s:

:.x:~


http://mm

br.asm.org/

Dow

nloaded from


EDELMAN ET AL.

identification of DNA by deoxyribonucleasesensitivity and other conventional assays whichare desirable.

DNA Density Shifts Due to Presence oJPolysaccharides

Two or more macromolecular components,present in small amounts and sufficiently dis-similar in buoyant density, will not measurablydistort each other's position in a gradient atequilibrium. The use of DNA species of dissimilarbut previously determined densities as standardsin CsCl gradients is based upon this fact (51).However, when the conditions mentioned abovedo not obtain, distorting effects may appear (M.Edelman, Ph.D. Thesis, Brandeis University,1965).

Figure 8 shows DNA extracts from Nodulariasphaerocarpa, which produced bimodal distribu-tions in the analytical ultracentrifuge. The uppertracing indicates the native, low concentration(6 j,g) pattern. Based upon heat denaturationtreatment, observation of the Schlieren refrac-tive index patterns, and deoxyribonuclease treat-ment (middle tracing), the component of density1.698 g/cm3 was DNA and that of 1.703 g/cm3a carbohydrate polymer. Upon centrifugation oflarger amounts of the native material (30 ,ug),the pattern shown in the lower tracing was pro-duced. The density differential between the medi-ans of the two components had now increasedconsiderably, giving the DNA an apparentdensity of 1.691 g/cm3 and the polysaccharide avalue of 1.712 g/cm3. The maintenance of sepa-rate modalities, and the shifts in density producedunder these conditions of centrifugation, raisedthe possibility that such carbohydrate polymersmay be useful in physically separating closelybanding DNA species in a gradient.The materials examined in the present investiga-

tion were studied under conditions which did notintroduce these distortions, and the DNA densityvalues reported throughout represent undistortedvalues.

Taxonomic and Phylogenetic Significance of theDNA Base Compositions

Lee et al. (25) were the first to recognize thepossibilities ofDNA base analysis as a taxonomicaid. Extensive analyses by several workers exam-ining bacteria showed that organisms which arevery closely related genetically have similar DNAbase compositions (31, 33). Table 3 summarizesthe DNA base-pair compositions of the variousspecies of the Cyanophyta investigated by us.The organisms are grouped according to a com-monly accepted classification for this phylum

LLI()

z

m

0

(I)

m

:j

DENSITY (g/cm3)FIG. 8. DNA fraction of Nodularia sphaerocarpa.

Conditions of centrifugation are as in Fig. 1. Peaksat 1.743 g/cm3 represent SP8-phage, standard DNA.The amount of material introduced into the centrifugecell in each case is indicated.

(56) based upon morphological and biochemicalcriteria other than DNA base composition. Allspecies were from fresh-water habitats, exceptAnacystis marina, Dermocarpa violacea, Lyngbyasp. 77, and Plectonema sp. 52, which were marine.In general, the CsCl buoyant density values andthe calculated base-pair compositions in terms ofper cent guanine plus cytosine (% GC; 51) were

based upon several separate DNA isolates, andthe values are significant to i0.001 g/cm3 or

1% GC. Single isolates, and determinationsmade on cell lysates, are noted in the footnotesto Table 3.The linear relationship between DNA buoyant

density and base-pair composition, upon whichthe % GC values of Table 3 were based, pre-

supposes the exclusive presence of the four usualDNA bases: adenine, guanine, thymine, andcytosine. Anomalous or "rare" bases presentin a sample might lead to a significant deviationfrom this correlation (51). In this regard, thethermal transition temperature (ti) of Anacystisnidulans DNA was determined to be 92 C in SSC(% GC = 56; 30), in agreement with the valuecalculated from buoyant density measurementsfor this strain. Chemical base analysis data are

also available for this species [B. B. Biswas,Plant Physiol. (Suppl.) 35: xxx, 1960] and are ingood agreement with the base-pair percentagesdetermined by the two indirect physical methodsmentioned above.

703

11.698

NATIVE(6EW9) 1.743(6 Ag)

1.703DNase

TREATED(4 g)

712169!

NATIVE(301s)g)

322 BACTERIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



The most conspicuous feature of Table 3 is thehomogeneity of base-pair compositions among

the members of the Oscillatoriales. The mean

% GC for the 20 different species of this group

examined by us is 45 i 6. The exhibitedhomogeneity is noteworthy, because it is clear

from other studies that a wide diversity in basecomposition is generally characteristic of groups

of unicellular organisms (33). This has beenshown to be the case for bacteria (31), protozoa(M. Mandel, Chem. Zool., in press), and algaeand fungi (50). The results obtained for the

TABLE 3. Densities and base compositions of blue-green algal DNA

Biological classification'

Order 1. ChroococcalesFamily 1. ChroococcaceaeAnacystis mariniaA. nidulansAnacystis sp.Coccochloris pentiocystisCoccochloris sp.Gleocapsa alpicola

Order 2. ChamaesiphonialesFamily 2. DermocarpaceaeDermocarpa violacea

Order 3. OscillatorialesSuborder 1. OscillatorineaeFamily 1. OscillatoriaceaeLyngbya sp.Lyngbya sp.Microcoleus sp.M. vaginatusPhorniidium luridum

var. olivaceaSuborder 2. NostochineaeFamily 1. NostocaceaeAnabaena spiroides

A. variabilisAnabaenopsis sp.Nodularia sphaerocarpaNostoc punctiformeN. muscorumN. muscorumNostoc sp.Nostoc sp.Nostoc sp.

Family 2. ScytonteniataceaeFremyella diplosiphonPlectonema boryanumP. boryanumP. calothricoidesPlectonema sp.Tolypothrix sp.

Family 4. RivulariaceaeCalothrix parietina

Strain

6311630766046308

47877

64016304

426

11.1.1384

12-3.212.4-1

5866305

481581

9-2-559852

Source

R. LewinM. M. Allen (from M. B. Allen)M. M. AllenM. M. AllenM. M. AllenM. M. Allen

R. Lewin

IUCCe

R. LewinM. M. AllenM. M. Allen

IUCC

M. M. Allen(from M. Shilo)

J. MeyersM. GibbsKFRIfIUCC

N. LazaroffKFRIKFRIIUCC

M. M. Allen

IUCC

IUCC

KFRIIUCC

R. LewinM. Gibbs

M. M. Allen (from M. B. Allen)

Density(g/cm3)

1.7281.7151.7151.7301.7221.694

1.703

1.7061.7101.7041.707

1.706

1.7051.7031.7011.6981.7031.7021.7021.7031.7031.703

1.7011.7071.7071.7071.7021.702

1.702

a After Smith (56).b Per cent guanine plus cytosine (% GC); determined from bouyant density (51).c Determined from single sample.d Determined from cell lysate (single sample); not examined for possible contamination.e IUCC, Indiana University Culture Collection (58).f KFRI, Kaiser Foundation Research Institute Collection, Richmond, Calif.

% GCb

69565671c63d

35c

44c

475145d48d

47

46444239444343444444

424848484343

43

VOL. 31, 1967 323


http://mm

br.asm.org/

Dow

nloaded from


TABLE 4. DNA of bacterial species thought to be related to the blue-green algae

Group Species Density % GC' Reference

Filamentous Flexibacter rubrum 1.696 37 Mandel (personal communtication)glidingbacteria

Saprospira thermalis 1.696 37 Mandel (personal communication)Flexothrix sp. 1.697 38 Mandel (personal communication)Vitreoscilla sp. 1.703 44 Mandel (personal communication)Vitreoscilla sp. 1.704 45 Mandel (personal communication)Saprospira grandis 1.706 47 Mandel (personal communication)Flexibacteria elegans 1.707 48 Mandel (personal communication)Leucothrix mucor (11 strains) 1 .708 49 (2)

Nonfilamentous Cytophaga johnsonii 33 (31)glidingbacteria

Sporocytophaga myxococcoides 35 (31)Cytophaga fermentans 39 (31)C. auranitiaca 39 (31)Myxococcus fulvus 67 (31)M. virescens 67 (31)M. xanthus 67 (31)Polyangium cellulosum 69 (31)

Per cent guanine plus cytosine; determined from buoyant density (51).

Oscillatoriales suggested that this group, as postu-lated from other evidence (14), may indeed be aconservative one which has undergone limitedgenetic diversification over the course of evolu-tion.

Morphological heterogeneity permits the divi-sion of this group into two suborders and severalfamilies. The sampling ofDNA base-pair compo-sitions permitted by the availability of axenic cul-tures may appear to reflect these divisions, but thedifferences are not statistically significant.Only one member of the Chaemosiphonales was

available, and the base-pair composition of thisspecies, Dermocarpa violacea, falls within therange of the base compositions of the Oscilla-toriales.The Chroococcales exhibit a much larger range

of buoyant densities in CsCl than do the Oscilla-toriales, their base compositions varying from 35to 71% GC for the six samples examined by us.In this respect, they resemble most of the otherunicellular groups investigated. Although the evi-dence presented for the Oscillatoriales is in agree-ment with a common evolutionary origin for thevarious species, the members of the Chroococcalesexhibit sufficiently diverse base-pair compositions(from this limited sample) to suggest polyphyleticorigins for this group. Indeed, the genera placedin this order are characterized by negative ratherthan positive characteristics, and form the resi-duum left after exclusion of the strictly filamen-

tous and regularly endospore-forming Cyanophyta(56).

In view of the data in Table 3, it is of interest toexamine the DNA base-pair compositions ofcertain other members of the Monera, e.g., the"gliding bacteria," which have been thought to berelated to the blue-green algae (17, 39, 57).The DNA base compositions of several mem-

bers of the filamentous gliding bacteria, generallyacknowledged to be closely related to the Oscilla-toriales (39), and the nonfilamentous gliding bac-teria, more distant, but possibly also related to theCyanophyta (39), have been characterized byMandel and co-workers and are listed in Table 4.Figure 9 compares these values with those of theblue-green algae. Note that the DNA base com-positions of the filamentous organisms overlapwith those of the Oscillatoriales. Thus, strains ofVitreoscilla, Saprospira, Leucothrix, and repre-sentatives of the Flexibacteriaceae (Soriano,Bergey's Manual of Determinative Bacteriology,6th ed., 1948) all fall within, or quite close to, thelimits of base composition exhibited by theOscillatoriales in the present study.The nonfilamentous unicellular gliding bacteria

generally fall outside the range exhibited by theOscillatoriales and appear similar to the non-filamentous, unicellular blue-green algae (Chro-ococcales) in that both show large differences inbase compositions among the various species in-vestigated. The unicellular gliding bacteria also

324 EDELMAN ET AL. BACTERIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



CH LOROPL AST DNA

ALGAEHlIGHE R PLANT S

3- nnnRw BLUE GREEN ALGAL DNA

w *CHROOCOCCALESm CHAMAESIPHONALES

o- 5- 0 OSCILLATORIALESiar 3- F nl

m~~~~~~~~Imz

SCHI ZOMYCOPHYTAflFILAMENTOUS GLIDING BACTERIA

INON FILAMENTOUS GLIDING BACTERIA

3 1,..,!1-, ,.. ....,

20 40 60 80% GUANINE + CYTOSINE

FIG. 9. DNA base composition relationships ofthe blue-green algae, the gliding bacteria, and eucaryoticchloroplasts. Blue-green algal, Schizomycophyta, andchloroplast DNA values are from Tables 3, 4, and 5,respectively.

appear to be a heterogeneous group based uponDNA homology studies (J. L. Johnson and E. J.Ordal, Bacteriol. Proc., p. 304, 1966).

It appears that, in several of the filamentousgliding bacteria, there are affinities indicated, onthe basis of DNA base composition, which sub-stantiate current phylogenetic notions based onother criteria.

Relationships Between Blue-green Algal andChloroplast DNA

Basically, the same two theories which havebeen suggested for the origins of viruses havebeen suggested to account for the origin of chloro-plasts in eucaryotic cells (50). In the first, a pieceof the central DNA of the cell is presumed tohave become detached from the main genome andestablished itself as a separate entity in the cyto-plasm. In time, this episomal DNA would or-ganize about itself a structure implicit in its owncode and might continue to evolve, dividing alongwith the rest of the cell. This would predict thatthe DNA of this episomally derived structure(which might evolve as far as a chloroplast orother organelle) should be related to the host'sDNA. It is possible, however, that, in terms ofbase composition, the host DNA could be hetero-geneous along its length, and the detached piecemight not be representative of the host DNA as awhole (e.g., see 43). It is also possible that, afterdetachment, the cytoplasmic fragment and the

major DNA of the cell diverged in base composi-tion as a result of subsequent evolution.A second theory supposes that a primitive,

procaryotic, photosynthetic cell, such as a primi-tive blue-green alga, invaded a nonphotosyntheticprocaryote and established itself as an endosym-biont. With time, interrelationships would de-velop which would further ensure that the endo--symbiont divided along with the host cell, andevolutionary pressure might lead to certain losses.in the invader which would no longer, normally,permit its existence outside the host. This theorydoes not require the invader and host necessarilyto have DNA's of related base composition. .iAlthough there is little direct evidence to sup-

port these two theories (or several others whichmight be proposed), bits of relevant informationexist from studies of the DNA of contemporaryorganisms.Table 5 shows the DNA base composition data

for several algal and higher plant species. A closerelationship between nuclear and chloroplastDNA base compositions in these organisms wouldbe viewed as supporting an episomal theory ofchloroplast origin. However, even from thelimited sampling available at present from theliterature, it is obvious that this condition doesnot obtain in the algae where chloroplast DNA isfrom 21 to 28% richer in adenosine plus thymi-dine content than the corresponding nuclearDNA. In the case of higher plants, the situation isless obvious, with the chloroplast-nuclear DNAdifferential ranging from 0 to 13% for the speciesinvestigated.

If chloroplasts originated by blue-green algalinvasion and endosymbiosis, a relationship at thelevel of DNA base-pair composition might existbetween the present-day representatives. A com-parison of the blue-green algal (Table 3) andchloroplast (Table 5) DNA base compositions isshown in Fig. 9. Significant correlation at thislevel between organism and organelle would bein agreement with the postulated relationshipbetween the two. However, too few algal species(which are of greater interest for this purpose)have been investigated to permit a meaningfulcomparison of their chloroplast DNA data withthe data from the Cyanophyta. For the higherplants, as well as the green algae (Chlorella andChlamydomonas), existing data indicate a partialmatching of chloroplast DNA with the DNA ofOscillatoriales.For the present, we will assess possible rela-

tionships between blue-green algal DNA andchloroplast DNA by comparing certain macro-molecular characteristics shared by procaryotic

325VOL. 31, 1967


http://mm

br.asm.org/

Dow

nloaded from


EDELMAN ET AL. BACTERIOL. REV.

TABLE 5. DNA of chloroplast-containing species

Euglena gracilis Z......................Euglenza gracillis v. bacillaris.....Chlamydomonas reinhardi, Y, ...........

Chlamydomonas reinhardi..............Chlorella ellipsoidea..................

Phaseolus vulgaris, v. Saxa (bean)..

Brassica rapa (turnip)Antirrhinum majus (snapdragon)....Viciafaba (broadbean).Beta vulgaris v. cicla (swiss chard) .....

Nicotiana tabacum (tobacco)..........Beta vulgaris (beet) ..................

Spinacia oleracea (spinach)Nicotiana rustica (turkish tobacco)...

Chloroplasts

Density %GCa(g/cm3)

1.685 25b1.686 26b1.694 351.695 361.695 36

1.694C 35

1.695

1.7001.7031.705e1 .705e

1 707C

3637b37d4244464648

Nucleus

.. IIDensity ,,GC(g/CM3)"

1 .7081 .7071.7211.7231.716

1 .694

1.692

1 .6891 .6901.6951.695

51b

52h

626457

35

3341 b40d30313636

References

(1)(12,42)(27,49)(6)(6,18)

(Beridze et al.Mol. Biologiya,in press, 1967)

(59)(48)(21)(22,61)(53)(6)(6)

(Green and Gor-don, FederationProc. 24:539,1965)

- Per cent guanine plus cytosine determined from bouyant density (51).bDetermined from chemical analyses.¢ Unidentified minor components are also present in the chloroplast fraction as isolated.d Chloroplast fraction, A:G = 1.67; nuclear fraction, A:G = 1.54.e The authors also report a satellite band of density 1.719 g/cm3 in the chloroplast fraction.

organisms and eucaryotic organelles. Chloro-plasts and mitochondria, viewed under the elec-tron microscope, contain clumped filamentouscomponents closely resembling structures in thebacterial nucleus (37, 44, 61). These clumps, pro-duced during specimen dehydration, are notcharacteristic of nuclear chromatin of higher or-

ganisms, presumably due to the presence ofDNA-histone complexes in the latter (61). Theobservation that exactly similar clumps occur inthe nuclear regions of blue-green algae (46, 62;L. V. Leek, J. Cell Biol. 31:67A, 1966) is ofspecific interest. In addition, after fixation withKellenberger's procedure, the nucleoplasm ofeucaryotic chloroplasts and mitochondria, alongwith that of the blue-green algae and bacteria,exhibit masses of ribonuclease-resistant, deoxy-ribonuclease-sensitive fibrils 25 A thick (37, 45,46).

Another similarity between blue-green algaland chloroplast DNA, and, in general, betweenDNA of procaryotes and eucaryotic organelles,is the apparent unimodality of both in CsCl equi-librium density gradients. The numerous bac-terial DNA samples investigated have consistentlybeen found to be unimodal, unless an episomalor similar type of particle is present (31, 50). The

organism investigated by Joshi, Guild, and Han-dler (19) is the only exception. DNA satellitebands are not found in our blue-green algalpreparations (Fig. 3). This characteristic, uniqueat the organismal level, to the DNA of procary-otic cells which lack organized intracellular com-partmentalization, appears to find a parallel inorganellular DNA from eucaryotes. Occasion-ally, in isolated chloroplast and mitochondrialfractions, several DNA species are encountered.On further purification or enzymatic treatment,however, a single DNA species, giving a uni-modal distribution in CsCl, often emerges freefrom cellular DNA contaminants (12, 23). Toestablish whether such a pattern is characteristicof all organelle samples will require furtherelucidation.

Other DNA characteristics in common be-tween cells of the Monera and organeile samples,but not reported present in eucaryotic chromatin,are the circular state of the bacterial (5) and mito-chondrial (23) DNA molecule and the similarityin the frequency of occurrence of the dinucleotideCpG (9). Equivalent studies with blue-greenalgal and chloroplast DNA have not yet beenreported.These similarities in DNA behavior at the

326

Group Species

Algae

Higher plants

-


http://mm

br.asm.org/

Dow

nloaded from



electron microscopic and ultracentrifugal levelbetween the blue-green algae and bacteria on theone hand and chloroplasts and mitochondria ofeucaryotes on the other, are consistent with thesuggestion that a genetic relationship may existbetween certain procaryotic cells and eucaryoticorganelles. Evidence supporting this hypothesisis presented in the next section.

DNA of Cyanophora paradoxaThe hypothesis of chloroplast origin by in-

vasion and endosymbiosis is attractive, becausesome instances of extensive symbiosis can befound in contemporary organisms. Examples ofblue-green algae, associated to various degreeswith a variety of other organisms, are to be foundin the literature (cf., 14, 56). In several of thesecases, cyanophytes coexist in a symbiotic rela-tionship with various fungi, protozoa, higheranimals, and plants. Often, the two partners canbe separated and grown independently of eachother, indicating that the relationship is not an

mX0

n

DENSITY (g/cm3)FIG. 10. DNA density profiles of Cyanophora

paradoxa. Conditions ofcentrifugation are as in Fig. 1.SP8 phage DNA (1.743 g/cm3) was added as a densitymarker in each case. Fractionation procedures anddesignations are explained in the text.

extremely close or reciprocal one. However, inat least two separate cases (15, 16, 27), a blue-green alga appears to have established a moreintimate relationship with a protistan cell. In thecase of Cyanophora paradoxa, a cryptomonad,a blue-green alga has become habituated in ananimal cell, has lost its cell wall, and dividesalong with its host (15). It might be but a smallseries of steps from here to becoming a chloro-plast. Our attempts to grow the "cyanelle," sodesignated by Pascher (38), outside of the crypto-monad have not been successful.The DNA of this organism was extracted and

examined in detail. Figure 10 shows the CsCiequilibrium density profiles for the various frac-tions of the cell prepared according to Fig. 2.The band at 1.743 g/cm3 in each case is a densitystandard introduced into the ultracentrifuge cellto calibrate the gradient. The whole cell extract,in which lysis of all ceUular components occurs,exhibits a trimodal DNA distribution with twocomponents of about equal concentrations at1.730 g/cm3 and 1.716 g/cm3 and a minor com-ponent at 1.697 g/cm3.The DNA banding profile of the pellet frac-

tion, containing broken nuclei, large cellulardebris, and unlysed cyanelles, is shown below(Fig. 10). A pattern similar to the whole cellextract is obtained, but the minor component isslightly diminished in concentration. The super-natant fraction (Fig. 10), which by microscopicexamination is completely free of cyanelle par-ticles as well as any large debris, contains theremainder of the minor DNA band free fromother components. The cellular localization ofthis DNA species is discussed below.

Since the protozoan nuclei do not remain in-tact during preparation of the pellet fraction,treatment of this fraction with deoxyribonucleasebefore DNA extraction was performed in anattempt to degrade selectively any susceptibleDNA fragments which might be adhering to themembranes of the intact cyanelle particles. A50-,ig amount of DNA of known density (SP8phage DNA) was added to the pellet beforeincubation, as a check on the effectiveness ofthe enzyme (13). At the end of the incubationperiod, the membrane-enclosed cyanelles wererecentrifuged, and an amount of low molecularweight, ultraviolet-absorbing material (opticaldensity maximal at 263 m,u) in excess of theadded SP8 DNA was recovered from the super-natant fraction. When the recentrifuged materialwas lysed and subjected to DNA extraction, theprofile designated "Cyanelle fraction" was ob-tained. Here, the DNA species of 1.730 g/cm3,present in the whole cell extract and in the pellet

1.716

1.730

WHOLE CELLEXTRACT 1.74I 1697

I.4 .697II

PELLET |FRACTION

SUPERNATANTFRACTION

CYANELLEFRACTION I

\IX

DNoseTREATED,

WHOLE CELLEXTRACT

VOL. 31,y 1967 327


http://mm

br.asm.org/

Dow

nloaded from


EDELMAN ET AL.

fraction in a partially degraded form (observeband widths), is totally absent. Bands of den-sity 1.716 g/cm3 and 1.697 g/cm3 are present,the former enriched about 50% over the latter.In other cyanelle fraction preparations, the DNAof density 1.716 g/cm3 constituted over 99% ofthe total. This DNA species, then, is identifiedwith the blue-green endosymbiont of Cyanophoraparadoxa.We also tested the possibility that the deoxy-

ribonuclease resistance exhibited by the bandspresent in the cyanelle fraction might be due topolysaccharide material or to some unusual prop-erty of the DNA. A comparison of refractive-index and UV-absorbance patterns in the ultra-centrifuge, however, revealed no evidence for thepresence of polysaccharides. Treatment of thewhole cell extract DNA with enzyme, as above,and dialyzing overnight against SSC, producedthe deoxyribonuclease-treated, whole cell ex-tract (bottom profile, Fig. 10) in which all bandsare shown to be sensitive to enzymatic degrada-tion under these non-membrane-protected con-ditions.The DNA profile of the protozoan partner can

now be assigned. Main band DNA of density1.730 g/cm3 is probably nuclear in origin, sinceit constitutes approximately 75% of the DNA ofthe whole cell extract minus the cyanelle-bandDNA, is absent from the 5,000 x g supernatantfraction, and is present in a degraded form in thepellet fraction in which ruptured nuclei are ob-served.

Satellite band DNA of density 1.697 g/cm3 ispresent in all fractions of the cell investigated. Itis not associated with the cyanelles, since it isprominently present in the 5,000 X g supernatantfraction, which is observed to be free of the blue-green particles. By its appearance in the cyanellefraction preincubated with deoxyribonuclease,it seems to be contained within a membrane-protected structure. These criteria suggest alocalization of this DNA species within an or-ganelle of the protozoan such as the mitochon-drion or nucleolus. Mitochondria are knownto protect their DNA from enzymatic digestion,under the conditions employed in this study (13,41), and both organelles would be, to some ex-tent, sedimented at 5,000 x g, the force necessaryto concentrate the cyanelles in the pellet fraction.The percentage of satellite DNA (about 25% ofthe DNA of the whole cell extract minus thecyanelle-band DNA) is appreciably greater thanhas been shown to be present in protozoan (60)and other (23, 59) mitochondrial fractions. [Thecase of frog egg DNA, where at least two-thirdsof the total egg DNA has been shown to bemitochondrial, is clearly different from the above.

Dawid (10) has shown the phenomenon of ex-cess cytoplasmic DNA in amphibian eggs to becaused by the large size of these cells and by thegreat number of mitochondria they contain.] Thehigh percentage of satellite DNA in Cyanophoramay be evidence in favor of a nucleolar locationfor this DNA species, since nuclear-localizedminor components constitute a greater percent-age of the total DNA than do mitochondrialcomponents and range as high as 30% of thetotal DNA (T. Beridze, M. Odintsova, and N.Sissakian, Mol. Biologiya, in press). Alternatively,the high percentages of both satellite and cyanelleDNA encountered in the whole cell extract couldresult from selective degradation of the DNA ofthe ruptured protozoan nuclei during the 4-hrperiod of lysozyme treatment at 37 C, whichoccurs before inactivation of endogenous nu-cleases with sodium dodecyl sulfate. From itsphylogenetic position in the evolutionary scale,one might expect the protozoan nucleus to con-tain appreciably more DNA than the two toeight blue-green endosymbionts within it (33).

Based upon the CsCl buoyant density values(51), the mole per cent of GC for the nuclear,cyanelle, and satellite DNA of Cyanophora para-doxa is 71, 57, and 38, respectively. The GCcontent of the cyanelle DNA (57%) places itoutside the DNA base-composition range of theOscillatoriales and very close to several speciesof Chroococcales (see Table 3). On morphologi-cal grounds, Hall and Claus (15) placed thecyanelle of Cyanophora paradoxa precisely inthis same taxonomic group, calling it CyanocytaKorschikoffiana in a new family Cyanocytaceae ofthe Chroococcales. Cyanocyta DNA is 14%richer in adenosine plus thymidine content thanis the nuclear DNA of its protozoan host. This isconsistent with the previous postulation that itis not necessary for invader and host to haveDNA of similar base composition. In this re-spect, CyanocytaDNA resembles the algal chloro-plast DNA's shown in Table 3.While the presence of organisms, such as

Cyanophora paradoxa, speaks in favor of thehypothesis of chloroplast origin through inva-sion, the heterogeneity in base composition ofchloroplast DNA among the algal genera studied,as well as among the higher plants (Table 5),suggests that, if its origin were through invasionand habituation, there may have been severaldifferent cases of invasion rendering the originsof chloroplasts in different species polyphyletic(50). In a similar case to that of Cyanophoraparadoxa, a blue-green alga lacking the typicalcyanophycean wall has been found inside anapochlorotic green alga, Glaucocystis nosto-chinearum (16, 27). This cyanophyte, on mor-

328 BACTERIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



phological grounds, is also classified in theChroococcales, but in a different genus thanCyanocyta. If a propensity had existed for differ-ent members of the early Chroococcales to becomeassociated in such a manner with early protistancells, then the present heterogeneity of this groupin DNA base composition (Table 3) would ac-count for the concomitant heterogeneity ob-served in chloroplast DNA's (Table 5).Although data for comparison are scanty, the

blue-green algal DNA parameters described anddiscussed in the preceding sections appear to bein agreement with an hypothesis of chloroplastorigin based on invasion and endosymbiosis.Further characterization of chloroplast DNA, interms of base composition and other physio-chemical parameters, is necessary if greatersignificance is to be given to the present compari-sons. Molecular hybridization studies, as a moredirect test of base sequence homology betweenrelated genomes (32), should be undertaken toaugment the existing data.

ACKNOWLEDGMENTs

We thank the following individuals who materiallyaided this research by providing or facilitating theacquisition of the various organisms listed in Table 3:Mary Belle Allen, (formerly at the Kaiser Founda-tion); Martin Gibbs, Dept. of Biology, BrandeisUniversity, Waltham, Mass. 02154; Norman Laza-roff, Microbiological and Biochemical Centers,Syracuse University, Research Corp., 1075, Com-stock, Syracuse, N.Y. 13210; Ralph A. Lewin, ScrippsInst. of Oceanography, La Jolla, Calif.; Jack Myers,Dept. of Zoology, University of Texas, Austin;M. M. Allen and R. Y. Stanier, Dept. of Micro-biology, University of California, Berkeley; andRichard C. Staff, Dept. of Botany, University ofIndiana, Bloomington (Director of the culture col-lection). We are also indebted to Luigi Provasoli,Haskins Labs., New York, for providing cultures ofCyanophora paradoxa, with instructions for theirmaintenance.We are especially grateful to Manley Mandel,

M.D. Anderson Hospital, Houston, Tex., for permis-sion to quote his unpublished data on the DNA of cer-tain gliding bacteria, and to Roger Stanier for his sus-tained interest and for valuable discussions. RobertHaselkorn, University of Chicago, kindly permittedone of us (D. S.) to use his facilities for a brief pe-riod.

This investigation was supported by grants fromthe National Institutes of Health, U.S. Public HealthService, and from the American Cancer SocietyGrant IN-29.

LITERATURE CITED

1. BRAWERMAN, G., AND J. M. EISENSTADT. 1964.DNA from the chloroplasts of Euglena gracilis.Biochim. Biophys. Acta 91:477-485.

2. BROCK, T. D., AND M. MANDEL. 1966. DNA base

composition of geographically diverse strains ofLeucothrix mucor. J. Bacteriol. 91:1659-1660.

3. BROWN, A. H. 1946. Determination of pentosein the presence of large quantities of glucose.Arch. Biochem. 11:269-278.

4. BURTON, K. 1956. A study of the conditions andmechanism of the diphenylamine reaction forthe colorimetric estimation of DNA. Biochem.J. 62:315-323.

5. CAIRNS, J. 1963. The bacterial chromosome andits manner of replication as seen by auto-radiography. J. Mol. Biol. 6:208-213.

6. CHUN, E. H. L., M. H. VAUGHAN, JR., AND A.RICH. 1963. The isolation and characteriza-tion of DNA associated with chloroplastpreparations. J. Mol. Biol. 7:130-141.

7. CouNTs, W. B., AMD W. G. FLAMM. 1966. Anartifact associated with the incorporation ofthymine into DNA preparations. Biochim.Biophys. Acta 114:628-630.

8. CREiSP, H. L., S. E. MANDEVILLE, AND J. J. KATZ.1962. The action of lysozyme on several blue-green algae. Biochem. Biophys. Res. Commun.9:569-573.

9. CUMMINS, J. E., H. P. RUSCH, AND T. E. EvANS.1967. Nearest neighbor frequencies and thephylogenetic origin of mitochondrial DNA inPhysarwn polycephalwn. J. Mol. Biol. 23:281-284.

10. DAWID, I. B. 1966. Evidence for the mitochondrialorigin of frog egg cytoplasmic DNA. Proc.Natl. Acad. Sci. U.S. 56:269-276.

11. DISCHE, Z. 1955. Color reactions of nucleic acidcomponents, p. 285-305. In E. Chargaff andJ. N. Davidson [ed.], The nucleic acids, vol. 1.Academic Press, Inc., New York.

12. EDELMAN, M., C. A. COWAN, H. T. EPSTEIN,AND J. A. SCHIFF. 1964. Studies of chloroplastdevelopment in Euglena. VIII. Chloroplast-associated DNA. Proc. Natl. Acad. Sci. U.S52:1214-1219.

13. EDELMAN, M., H. T. EPSTEIN, AND J. A. SCHIF.1966. Isolation and characterization of DNAfrom the mitochondrial fraction of Euglena.J. Mol. Biol. 17:463-469.

14. FRrrSCH, F. E. 1945. The structure and reproduc-tion of the algae, vol. 2. Cambridge Univ. Press,London.

15. HALL, W. T., AND G. CLAUs, 1963. Ultrastruc-tural studies on the blue-green algal symbiontin Cyanophora paradoxa, Korschikoff. J. CellBiol. 19:551-563.

16. HALL, W. T., AND G. CLAUs. 1967. Ultrastruc-tural studies on the cyanelles of Glaucocystisnostochinearum, Itzigsohn. J. Phycol. 3:37-51.

17. HAROLD, R., AND R. Y. STANIER. 1955. The generaLeucothrix and Thiothrix. Bacteriol. Rev.19:49-64.

18. IwAMURA, T., AND S. KUWASHNA. 1964. Furtherobservations on the DNA species in Chlorellashowing light-dependent metabolic turnover.Biochim. Biophys. Acta 82:678-679.

19. JOSHI, J., W. GUILD, AND P. HANDLER. 1963.

329VOL. 31, 1967


http://mm

br.asm.org/

Dow

nloaded from


330 EDELMAN ET AL.

The presence of two species of DNA in somehalobacteria. J. Mol. Biol. 6:34-38.

20. KiRBY, K. S. 1957. A new method for the isola-tion of DNAs: Evidence on the nature of bondsbetween DNA and protein. Biochem. J. 66:495-504.

21. KIRK, J. T. 0. 1963. The DNA of broad beanchloroplasts. Biochim. Biophys. Acta 76:417-424.

22. KLSLEV, N., H. SWIFT, AND L. BOGORAD. 1965.Nucleic acids of chloroplasts and mitochondriain Swiss chard. J. Cell Biol. 25:327-344.

23. KROON, A. M., P. BORST, E. F. J. VAN BRUGGEN,AND G. J. C. M. RuTrENBERG. 1966. Mito-chondrial DNA from sheep heart. Proc.Natl. Acad. Sci. U.S. 56:1836-1843.

24. LAZAROFF, N., AND W. VISHNIAC. 1961. The effectof light on the developmental cycle of Nostocmuscorum, a filamentous blue-green alga. J.Gen. Microbiol. 25:365-374.

25. LEE, K. Y., R. WAHL, AND E. BARBU. 1956.Contenu en bases puriques et pyrimidiques desacides desoxyribonucleiques des bacteries.Ann. Inst. Pasteur 91:212-224.

26. LEFF, J., M. MANDEL, H. T. EPSTEIN, AND J. ASCHIFF. 1963. DNA satellites from cells ofgreen and aplastidic algae. Biochem. Biophys.Res. Commun. 13:126-130.

27. LEFORT, M. 1965. Sur le chromatoplasma d'unecyanophycee endosymbiotique: Glaucocystisnostochinearum Itzigt. Compt. Rend. 261:233-236.

28. LOWRY, 0. H., N. J. ROSEBROUGH, L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

29. MARMUR, J. 1961. A procedure for the isolationof DNA from microorganisms. J. Mol. Biol. 3:208-218.

30. MARMUR, J., AND P. DOTY. 1962. Determinationof the base composition of DNA from itsthermal denaturation temperature. J. Mol.Biol. 5:109-118.

31. MARMUR, J., S. FALKOW, AND M. MANDEL. 1963.New approaches to bacterial taxonomy. Ann.Rev. Microbiol. 17:329-372.

32. MARMUR, J., C. L. SCHILDKRAUT, AND P. DOTY.1962. Biological and physical chemical aspectsof reversible denaturation of DNAs, p. 9-43.In The molecular basis of neoplasia. Universityof Texas Press, Austin.

33. MCCARTHY, B. J. 1965. The evolution of basesequences in polynucleotides. Progr. NucleicAcid Res. Mol. Biol. 4:129-160.

34. MEJBAUM, W. 1939. Uber die Bestimmung kleinerPentosemengen insbesondere in Derivaten derAdenylsaure. Z. Physiol. Chem. 258:117-120.

35. MESELSON, M., F. W. STAHL, AND J. VINOGRAD.1957. Equilibrium sedimentation of macro-molecules in density gradients. Proc. Natl.Acad. Sci. U.S. 43:581-583.

36. MORRIS, D. L. 1948. Quantitative determination

BACTERIOL. REV.

of carbohydrates with Dreywood's anthronereagent. Science 107:254-255.

37. NAss, M. M., S. NASS, AND B. A. AFZELIUS. 1965.The general occurrence of mitochondrial DNA.Exptl. Cell Res. 37:516-539.

38. PASCHER, A. 1929. Studien uber symbiosen. I.'Ober einige symbiosen von blaualgen ineingellern. Jahrb. Wiss. Bot. 71:386.

39. PRINGSHEIM, E. G. 1949. The relationship betweenbacteria and Myxophyceae. Bacteriol. Rev. 13:47-98.

40. PROVASOLI, L. 1964. Growing marine seaweeds,p. 9-17. In D. De Virville and J. Feldman [ed.],Proceedings of the 4th International Sea-weed Symposium, Macmillan Co., New York.

41. RABINOWITZ, M., J. SINCLAIR, L. DESALLE, R.HASELKORN, AND H. SWIFT. 1965. Isolation ofDNA from mitochondria of chick embryoheart and liver. Proc. Natl. Acad. Sci. U.S. 53:1126-1133.

42. RAY, D. S., AND P. C. HANAWALT. 1964. Prop-erties of the satellite DNA associated with thechloroplasts of Euglena gracilis. J. Mol. Biol.9:812-824.

43. RICHARDS, 0. C. 1967. Hybridization of Euglenagracilis chloroplast and nuclear DNA. Proc.Natl. Acad. Sci. U.S. 57:156-163.

44. RIs, H. 1961. Ultrastructure and molecular or-ganization of genetic systems. Can. J. Genet.Cytol. 3:95-120.

45. RLs, N., AND W. PLAUT. 1962. Ultrastructure ofDNA-containing areas in the chloroplast ofChlamydomonas. J. Cell Biol. 13:383-391.

46. Ris, H., AND R. N. SINGH. 1961. Electron micro-scope studies on blue-green algae. J. Biophys.Biochem. Cytol. 9:63-80.

47. RITOSSA, F. M., AND S. SPIEGELMAN. 1965. Local-ization of DNA complementary to ribosomalRNA in the nucleolus organizer region ofDrosophila melanogaster. Proc. Natl. Acad.Sci. U.S. 53:737-745.

48. RUPPEL, H. G., AND D. VAN WYK. 1965. Uberdie Deoxyribonucleinsaure in Chloroplastenvon Antirrhinum majus. Z. Pflanzenphysiol. 53:32-38.

49. SAGER, R., AND M. R. ISHIDA. 1963. ChloroplastDNA in Chlamydomonas. Proc. Natl. Acad.Sci. U.S. 50:725-730.

50. SCHWF, J. A., AND H. T. EPSTEIN. 1965. The con-tinuity of the chloroplast in Euglena, p. 131-189. In M. Locke [ed.], Reproduction: cellular,sub-cellular, and molecular. Academic Press,Inc., New York.

51. SCHILDKRAUT, C. L., J. MARMUR, AND P. DOTY.1962. Determination of the base compositionof DNA from its buoyant in CsCl. J. Mol.Biol. 4:430-443.

52. SCHUMAKER, V. N., AND J. WAGNILD. 1965. Zonecentrifugation in a CsCl density gradient causedby temperature change. Biophys. J. 5:947-964.

53. SHIPP, W. S., F. J. KIERAS, AND R. HASELKORN.1965. DNA associated with tobacco chloro-plasts. Proc. Natl. Acad. Sci. U.S. 54:207-213.


http://mm

br.asm.org/

Dow

nloaded from



54. SEGOVIA, Z. M. M., F. SOKOL. I. L. GRAvES, ANDW. W. ACKERMANN. 1964. Some properties ofnucleic acids extracted with phenol. Biochim.Biophys. Acta 95:329-340.

55. SmITH, M. 1964. DNAs of crustacea. J. Mol.Biol. 9:17-23.

56. SMrrH, G. 1950. Fresh water algae of the UnitedStates. McGraw-Hill Book Co., Inc., NewYork.

57. STANIER, R. Y., AND C. B. VAN NEL. 1941. Themain outlines of bacterial classification. J.Bacteriol. 42:437-466.

58. STARR, R. C. 1964. The culture collection of algae

at Indiana University. Am. J. Botany 51:1013-1044.

59. SUYAMA, Y., AND W. D. BONNER, JR. 1966. DNAfrom plant mitochondria. Plant Physiol. 41:383-388.

60. SUYAMA, Y., AND J. R. PREER, JR. 1965. Mito-chondrial DNA from protozoa. Genetics 52:1051-1058.

61. SwuFr, H. 1965. Nucleic acids of mitochondriaand chloroplasts. Am. Naturalist 99:201-227.

62. WILDON, D. C., AND F. V. MERCER. 1963. Theultrastructure of the vegetative cell of blue-green algae. Australian J. Biol. Sci. 16:585-596.

VOL. 31, 1967 331


http://mm

br.asm.org/

Dow

nloaded from


Documents

Deoxyribonucleic Acid of the Blue-Green Algae (Cyanophyta)The Cyanophyta or blue-green algae share the kingdom Monera with the Schizomycophyta or bacteria, because both are distinguished