Properties of the satellite DNA associated with the chloroplasts of Euglena gracilis

J. Mol. Biol. (1964) 9, 812-824

Properties of the Satellite DNA Associated with the Chloroplasts of Euglena gracilis DAN

S.

RAY AND

P. O. HANAWALT

Biophysics Laboratory, Stanford University, Stanford, Oalifornia, U.S.A. (Received 4 May 1964, and in revised form 1 June 1964) Chloroplasts of the algal flagellate Euglena gracilis were isolated by sucrose gradient sedimentation of crude lysates and were shown to be enriched with a satellite DNA component. The satellite and principal DNA components were separated in a cesium chloride density-gradient and compared with respect to buoyant density, thermal denaturation, base composition and molecular weight distribution. The satellite DNA (p = 1·685 g/cc) had a lower melting temperature than the principal DNA (p = 1·707 g/cc). Consistent with this was a high adeninethymine content; 76% as compared to 47% in the principal DNA. However, the principal DNA contained 2·3 mole per cent of the rare base 5-methylcytosine, whereas none could be detected in the satellite DNA. The molecular weight range of the principal DNA was 20 to 40 million. More than half of the satellite DNA appeared in a comparable molecular weight range; but the remainder was less heterogeneous, with lower molecular weight of 2·6 million. The component of small molecular weight is probably not a breakage product of the larger one.

1. Introduction The recent discovery of DNA in association with chloroplasts (Ris & Plaut, 1962; Leff, Mandel, Epstein & Schiff, 1963; Sager & Ishida, 1963; Ohun,Vaughan & Rich, 1963; Kirk, 1963; Baltus & Brachet, 1963; Gibor & Izawa, 1963) is of considerable interest in view of the autonomous replication of these organelles (Granick, 1963). Growth on tritiated thymidine and subsequent autoradiography of labeled cells had previously led to the suggestion that DNA was present in the cytoplasm of algae (Stocking & Gifford, 1959; Sagan & Scher, 1961) and higher plants (Wollgiehn & Mothes, 1963), although sensitivity of the label to deoxyribonuclease was demonstrated only in the latter two papers. The DNA of wild type Euglena gracilis was observed by Leff et al. (1963) to have a small satellite band at a density lighter than the main DNA band in a cesium chloride density-gradient, whereas a mutant without plastids did not have a detectable satellite band. These workers concluded that the satellite DNA was associated with the capacity to form chloroplasts. It is tempting to identify chloroplast-associated DNA as the chemical species responsible for the extra-nuclear genetic control of chloroplast development and replication. However, the evidence that DNA components in higher plants (Sampson, Katoh, Hotta & Stern, 1963) and chloroplast-associated DNA in algae (Iwamura, 1962) might be metabolically labile tends to make this identification uncertain. Thus, further studies on the properties of chloroplast-associated DNA are required in order to assign to it an important biological role. The present report concerns the characterization of the satellite DNA associated with the chloroplasts in Euglena gracilis 812

SATELLITE DNA IN E UGLENA

813

and a comparison with corresponding properties of the major DNA component in th is system .

2. Materials and Methods (a) Organism and growth conditions The al gal flag ellate Euglena graci lis var, bacillar is was obt ained fr om the University of Indiana cult u re collection and grown asep tically on Hutner 's m edium (Greenblatt & Schiff, 1959) at pH 3·5 with glut amic and malic a cids as carbo n sources; 15-ml. cultu res were gro wn in I -in . di amet er t ubes at 27°C with occas ion al shak in g. Continuous illuminat ion at 400 ft. candles was pr ovided by a bank of fluorescen t tubes . The m ean gen erati on t ime was approxi mately 15 hr as determined by cell cou n ts with a hem ocytometer.

(b) Isolation of DNA A modification of the Marmur (1961) procedure was u sed for the isolation of DNA from wh ole cells. The cells wer e harvest ed by low-speed centrifugation at 4°C and washed twice at 4°C with buffer, pH 8, cons ist in g of 0·1 M-NaCl, 0·001 M-EDTA and 0·001 M-tris (Sigma121). This buffer is hencefor th abbreviated 0,1-0,001-0,001 NET and the molarities of the differ ent components are indicated in respective order as their conc ent rat ions are altered (e.g. 0,1-0,01-0,01 NET cited below). The green pellet was then resuspended in 95 % ethanol and allowed to st and at 4°C for 10 min. The mater ial was ag ain sedimented by low-speed cent r ifu gat ion and the et han ol ext ract ion was rep eat ed. F ollowing another cent rifugation, 1% sodiu m dod ecyl sulfate in 0·1-0·01-0'01 NET at pH 8 was added t o t he pellet at room t emperature and t he prep arati on allowed to st and for 30 m in; 3 M-sodium perchl orate (i vol.) was added a nd the mixture was sub jected t o vortex mixing (Cyclomixer, Clay-Ad ams, N.Y.) wit h an equal vol. of 9 : 1 (v/v) chloro form-oct ano l for t h ree 5 to 10-sec periods. The aqueo us pha se was decanted aft er a low- sp eed centrifugation and di alysed overnigh t at 4°C against 0,1-0,01-0,01 NET, pH 8. A sim ilar procedure was u sed for isolat ing DNA fr om purified chloro plast preparations (see below) ex cep t that t he ethanol extrac t ion was omi t ted and the sodium dodecyl sulfate was a dde d directly to the chloroplast-enriched fractions from the suc ros e gradient. (c) I solation of chloroplasts

Cells wer e disrupted at 2000 p.s.i, in a French pressure cell and the lysate was sed imented through a d iscontinuous suc rose gradient as descri bed by Sager & I shida (1963) . After sedimentation at 2°0 for 90 min at 25,000 r ev Jtuu: in the SW25·1 ro tor of a Spinco m od el L ultracentrifuge, the fractions were collect ed through a pinhole in t he bottom of the cellulose tube. (d) Radioactive labeling and assay Labeling of Euglena DNA with 32p was carried out in a m odified Hutner's medium in volving a 15-fold reduction in phosphate concentration, a change which had no observable effect on growth rate or appearance of cells. [32P]orthophosphate was obtained from Nuclear Chicago Corp., and 250 fLO (carrier-free) was us ed in a 15-ml. culture. In extensive preliminary st udies involving attempts to label the DNA highly with radioactive thymine or thymidine, m ost of t h e label was incorporated into non-nucleic acid fractions. Exogenously supplied deoxyadenosine, d eoxycytidine, deoxyuridine, and brom od eox yuridine were als o po orly in corporated into DNA (Ray, 1964). Incorporated radioac ti ve material was ass aye d b y adding a sm all sam ple to 5 ml. ice cold 5 % T CA,t collect ing by suction on type HA Millip ore filt ers (Bedford, Mass .), and r ins ing with t wo 5-ml. p ortions of d istilled water. Aft er drying under a heat lamp, t he filt ers wer e added to counting vial s con t a ining 5 ml. toluene, 18 mg PPO and 0·45 mg POPOP and then assayed in a P ackard Tri-Carb scintillation sp ectrometer. Alkalinere sistant radioactive materi al wa s de te rmin ed by in cubating the sample overnight at room temperature in 1 M·KOH prior to the addition of an ex cess of cold 5 % TCA.

t

Abbreviations us ed: TeA, trichloroacetic acid; 5BU. 5-bromouracil.

814

D. S. RAY AND P. C. HANAWALT (e) OsOl density-gradient analysis

The method of Meselson & Stahl (1958) was used to determine the buoyant density of DNA. DNA (1 to 4 p.g), along with a similar amount of marker DNA of known density, was added to CsCI (Harshaw, optical grade) and the density of the solution was adjusted to 1·71 gje«, The solution was centrifuged in the Spinco model E ultracentrifuge for 20 hr at 44,770 rev./min in an aluminium cell with a - 10 window and a Kel-F centerpiece. Ultraviolet absorption photographs were taken at 20 hr and scanned with the J oyce--Loebl recording microdensitometer. The density of the DNA was determined by its position in the gradient relative to the marker DNA (Sueoka, 1961). The buoyant density of native E. coli DNA was assumed to be 1·710 g/cc (Schildkraut, Marmur & Doty, 1962). For preparative density.gradient centrifugation, 3 ml. of the DNA preparation was added to 3'85 g of CsCI, poured into cellulose tubes, overlaid with mineral oil and centrifuged at 37,000 rev.jmin for 48 hr at 20°C in the SW39 rotor of a Spinco model L ultracentrifuge. The density fractions from the tube were collected and diluted with 0·5 ml. of 0,1-0'001-0,001 NET at pH 8 before taking samples for radioactive assay. To purify fractions further in a particular density range, the fractions of interest were pooled and subjected to a second preparative centrifugation in CsCI.

(f) Molecular weight determination

To determine the molecular weight and the heterogeneity of DNA, 0·15 ml. of 32p -labeled DNA was mixed with 0·05 ml. of tritium-labeled DNA from A bacteriophage (mol. wt 31 X 10 6 , kindly supplied by Dr. V. Bode) and layered on top of a linear 5 to 20% sucrose gradient in a cellulose tube. The final concentration of the [32P]DNA was always less than 1 p.g/ml. and that of the 1I[3H]DNA equal to 12 p.g/ml. A small correction was made for the dependence on concentration ofthe sedimentation of the ADNA. The tube was centrifuged for 4 to 5 hr at 28,000 rev.jmin in the Spinco model L ultracentrifuge and fractionated as in (e) above. Unlabeled cytosine was added to the sucrose solutions so that the size of each drop could be determined by the absorbance of each fraction at 260 xxu». Samples were assayed for 32p and tritium and the molecular weight of the 32P-Iabeled DNA was calculated from the distance sedimented relative to the distance sedimented by the II DNA (Burgi & Hershey, 1963).

(g) Base composition of [32 P]DNA fractions

Pooled fractions along with 100 p.g of sahnon sperm DNA (Cal. Biochem.) were precipitated with an equal volume of 7% HClO. at O°C and allowed to stand for 10 min. The precipitate was collected by centrifugation at 4°C for 10 min at 8,000 g, washed twice with distilled water at 4°C and once with ether at 4°C. The precipitate was resuspended in 0·2 ml. of 0·01 M:-tris (Sigma-Lz l} pH 7·6 and 0·005 M-MgCl 2. Enzymic degradation to mononucleotides was accomplished by incubation for 2·5 hr at 37°C with 0·02 ml. of pancreatic deoxyribonuclease (Cal. Biochem.) at 1 rng/ml. (in 0·001 M-NaCI and 1 mg/ml. bovine serum albumin) followed by incubation at pH 8·5 for 2·5 hr at 37°C with 0·01 ml. of venom phosphodiesterase (Worthington) at 4 mg/ml. (in 0·001 M-NaCI and 4 mgjml. bovine serum albumin). The resulting digest was evaporated to dryness under vacuum at 20°C, redissolved in 0·050 ml. of distilled water, and applied in a narrow line to a strip of What-. man no. 1 filter paper. The nucleotides were separated by descending paper chromatography in the isobutyric acid-ammonium isobutyrate solvent system (Wyatt, 1955). Under our chromatographic conditions, dMCMP consistently appeared between dAMP and dCMP rather than ahead of dAMP as observed by Tamm, Shapiro, Lipshitz & Chargaff (1953). This difference may be due to our use of Whatman no. 1 filter paper rather than Schleicher & Schull no. 597 filter paper. Chromatograms were scanned with a Vanguard automatic chromatogram scanner to determine the relative amounts of each of the nucleotides. Ultraviolet-absorbing marker nucleotides (Cal. Biochem.) were used to identify the peaks of radioactivity.

SATELLITE DNA IN EUGLENA

815

3. Results (a) Physical association of the satellite DNA with chloroplasts

To investigate the possibility that the satellite DNA might be physically associated with the chloroplasts, we have isolated chloroplasts by sucrose density-gradient sedimentation of celllysates (see Materials and Methods). The cells were labeled with 32p for approximately five generations so that small amounts of DNA might be detected. Sedimentation of the lysate in the discontinuous sucrose density-gradient for 90 minutes yielded a green pellet at the bottom of the tube and two chloroplast-containing bands. As shown in Fig. 1, both chloroplast 30 25

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240 280 320 360 Drop no.

FIG. 1. Distribution of 32p following sucrose density-gradient sedimentation of a. 32P-la.beled lysate of Euglena gracilis. The two major bands of radioactive material (I and II) correspond to the two chloroplast-containing bands. - . - . - , TCA-insoluble radioactivity; - ... - ... - , KOH.resistant, TCA·insoluble radioactive material.

bands contained alkaline-resistant 32p as well as acid-precipitable radioactive material. The amount of material in the pellet was variable, amounting to 40 to 60% of the total alkaline-resistant 32p radioactive material. The pellet remained in the tube during drop collecting and did not appear to contaminate the fractions collected. The relative amounts of activity in bands I and II were also variable, with band II generally being in considerable excess. However, a second sedimentation of band I gave only the band I component, whereas a second sedimentation of band II yielded both components in approximately the same proportions as in the first sedimentation. The density distribution of the DNA obtained from the chloroplast-containing bands was examined in the cesium chloride density-gradient as shown in Fig. 2. For comparison, a DNA preparation from whole cells was also examined, and it showed a principal band and a lighter satellite band, in agreement with the results of Leff et al. (1963). The DNA from band I of the sucrose density-gradient was similar to the whole cell DNA preparation and little, if any, enrichment for the satellite DNA was apparent. In contrast, the DNA from band II gave approximately a fourfold enrichment for the satellite component, consistent with a physical association of the

D. S . RAY AND P. C. HANAWALT

816

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3000 2000

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FIG. 2. CsCI density-gradient distributions of alkaline-resistant, TCA-insoluble 32p isolated from: (a ) whole cells , (b) band I from Fig. I and (c) band II from Fig. 1.

satellite DNA with the chloroplasts. Further enrichment for the satellite could not be obtained by a second sucrose density-gradient sedim entation of ba nd II. (b) Purification by rebanding in the cesium chloride gradient

I solat ion of the principal DNA component was achieved in a single banding of t he whole cell DNA in the cesium chloride gradient. No satellite DNA could be det ect ed in a rebanding of peak fractions from this principal band. The pooled satellite DNA fractions, however, required a second banding in CsCI to obtain a preparation relatively free of the principal band, as shown in Fig. 3, where the principal DNA now appears as a small shoulder on the heavy side of the satellite. Not shown in Fig . 3 is an optical density marker of added unlabeled E. coli DNA at a density slightly greater than that of the principal DNA component. Fractions from the light side ofthe satellite band through the first fraction on the heavy side were pooled for further cha racterization of the satellite DNA. Less than 2% of the DNA in t hese pooled fractions came from the original principal DNA component. Figure 4(a) shows the banding of principal DNA in the analytical ultracentrifuge to yield a calculated density of 1·707 glee and Fig . 4(b) gives a similar banding of the

SATELLITE DNA IN EUGLENA

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FIG. 3. CsCl density-gradient distribution of TCA-insoluble 32p following rebanding of satellite DNA fractions from a whole-cell DNA preparation. The original banding in CsCl was similar to that shown in Fig. 2(a).

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FIG. 4. Densitometer tracings of the ultraviolet absorption photographs taken after equilibrium density-gradient sedimentation of (a) the purified principal DNA and (b) the purified satellite band DNA. The band at 1·756 is a marker of known density, E. coli 5BU hybrid DNA.

purified satellite DNA to yield a buoyant density of 1·685 gJcc. E. coli 5BU hybrid DNA was used as a density marker at 1·756 gJee. (c) Selective denaturation of the satellite DNA

Since both the denaturation temperature and buoyant density of DNA are presumably linearly related to the adenine-thymine content (Marmur & Doty, 1962; Schildkraut et al., 1962) it might be expected that the denaturation temperature of the satellit e DNA would be considerably lower than that of the principal DNA (if its

818

D. S. RAY AND P. C. HANAWALT

lighter density were indeed due to a higher adenine-thymine content). To verify this hypothesis, we have selectively melted the satellite DNA in a mixture of the two components at a temperature just below the onset of thermal denaturation of the principal component. Preliminary studies on the principal component indicated that thermal denaturation in 0·15 M-NaOI does not occur until temperatures above 90°0 are reached (Ray, 1964). The expected effect of selective denaturation of the satellite DNA is demonstrated in Fig. 5 by a selective shift in the buoyant density of the

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FIG. 5. Selective melting of the satellite DNA. (a) Rebanding satellite (32P]DNA fractions and purified principal DNA in a CsCI density-gradient along with E. coli (3H]DNA as a density marker. (b) Rebanding ofidentical fractions after heating for 10 min at 90°C and rapidly cooling, along with E. coli (3H]DNA. - . - . - , optical density 260 mu (unlabeled principal DNA); (32P]DNA fractions (satellite DNA and some principal DNA); - .. - .. - , E. coli (3H]DNA.

-e--e-,

satellite DNA while not affecting the density ofthe principal DNA. Unlabeled principal DNA was combined with [32P]DNA from the satellite region of a preparative OsC] banding of whole-cell DNA in a final buffer consisting of 0·15--0·005--0·005 NET at pH 8. Half of this mixture was heated to 90°0 for 10 minutes and was rapidly chilled. Both fractions then received E. coli [3H]DNA as a density marker prior to densitygradient centrifugation. The final mixtures were added to solid OsOI and buffered at pH 10·2 with 0·03 M-K2HP0 4 • Only the unlabeled principal DNA component was present in large enough quantity to be detectable by ultraviolet absorbance in the experiment illustrated in Fig. 5. The position of the principal band relative to the density marker was unchanged following the 90°0 treatment, whereas the position of the satellite band was shifted into the vicinity of the principal band. This corresponds to a buoyant density shift of approximately 0·02 glee, which is consistent with the observed density of the denatured satellite DNA (Leff et al., 1963). Thus, this treatment has selectively denatured the satellite DNA while leaving the principal DNA in native form.

SATELLITE DNA IN EUGLENA

819

(d) Base composition

The composition of both principal and satellite DNA's was determined by enzymic degradation and paper chromatography of the 32P-Iabeled material from respective bands in the CsCI gradient. Some of the preparations were observed to contain small amounts of [32P ]R NA which were removed by incubation for 30 minutes at 37°C with 15 fLgfml. ribonuclease, dialysis overni ght in 0·1-0·001-0·001 NET at pH 8, and treatment with active charcoal prior to precipitation of the DNA. Typical chromatedAMP

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FIG. 6. Radiochromatograms of (a) purified principal DNA and (b) purified satellite DNA. Arrows indicate the position of ultraviolet-absorbing marker nucleotides.

grams are shown in Fig. 6 and a quantitative summary of a number of independent preparations and determinations is given by Table 1. TABLE

1

Base composition of the principal and sateUite DNA's

Component

dAMP dTMP dGMP dCMP dMCMP

Mole percentage of DNA componentst Principal Satellite

22·6 24·4 27 ·7 23·2 2·3

38·2 38·1 12·3 11·3 <0·3

t Each value represents an average for three preparations. Two striking differences between principal and satellite DNA components are immediately obvious in Fig . 6 and Table 1. First, the satellite DNA has an extremely high adenine-thymine content relative to that of the principal DNA . Second, the satellite DNA has little, if any, 5-methyleytosine, whereas the principal DNA has a small but detectable amount of this rare base. Chromatograms of the satellite DNA

D. S. RAY AND P. C. HANAWALT

820

hydrolysate were also scanned at a threefold lower range of count-rate than that shown in Fig. 6 in order to set an upper limit on the amount of 5-methylcytosine which might be present. No peak was observed at the position of 5-methylcytosine. If the satellite DNA contained this base in the same proportion to cytosine as in the principal DNA, we would have expected more than one mole per cent of 5-methylcytosine in the satellite DNA, whereas our results indicate that there is less than 0·3 mole per cent. The presence of 5-methylcytosine in whole cell DNA preparations from Euglena has been reported by Brawerman, Hufnagel & Chargaff (1962), who found a base composition very close to that reported here for the principal DNA component. This seems reasonable in view of the small amount of the satellite DNA present in whole-cell preparations. (e) Molecular weight distribution

Sedimentation of DNA in a linear sucrose density-gradient yields information both about the molecular weight and about heterogeneity of molecular weight. In Fig. 7

200

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FIG. 7. Sucrose density-gradient sedimentation of purified principal [32P]DNA and [H3]DNA of molecular weight 31 X 106 from the ,\ bacteriophage. - . - . - , 32p radioactive material; - • - • - , 3H radioactive material.

the sedimentation of purified principal DNA is compared to that of a 3H-Iabeled DNA from Abacteriophage. The ADNA served as a homogeneous marker with established molecular weight of 31 million (Burgi & Hershey, 1963). The distance along the abscissa represents the approximate distance sedimented. Accurate calculations of this distance were made only for the fractions containing the marker and the "unknown" DNA. The purified principal DNA sediments in a broad band which appears to consist of two components, both evident in a number of independent preparations. The calculated molecular weights for the components are 20 million and 40 million, and the former could reasonably be a breakage product of the latter since it has been demonstrated that DNA molecules tend to shear near the middle (cf. Kaiser, 1962). A similar sedimentation of purified satellite DNA yields a profile which is qualitatively quite different from that of the principal DNA. As shown in Fig. 8, in addition to a high molecular weight DNA in the 20 to 40 million molecular weight range, there is a also a slowly sedimenting component of molecular weight 2·6 million. We have observed variable amounts of this low molecular weight component in purified

SATELLITE DNA IN EUGLENA

821

c:

c:

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j

j

E

u

u

400

200

3

4

Distance from meniscus (em)

FIG. 8. Sucrose density-gradient sedimentation of (a) purified satellite [32P]DNA and A[3H]DNA, and (b) an identical sample of satellite [32P]DNA, which was sheared by vortex mixing, and A[3H]DNA. 32p radioactive material; - . - . - , 3H radioactive material.

-e-e-,

satellite DNA preparations, but always within 35 to 50% of the total. Since there are few, if any, components between 20 million and 2·6 million, it seems unlikely that the low molecular weight component is a breakage product of the larger DNA. To test this hypothesis we have subjected an identical portion of the satellite DNA (compare Fig. 8(a) and (b)) to shearing for two minutes in a vortex mixer prior to sedimentation in the sucrose gradient. Although the large component now sediments at a rate corresponding to a molecular weight of 17 million, both the molecular weight and the relative amount of the small component are unchanged (Table 2). TABLE

2

Effect of shearing on the molecular weight distribution of satellite

32 P-labeled

DNA

Distribution (%) in tube Treatment

None Sheared

20 to 40 X 10 mol.wt

6

17 X 10 6 mol.wt

2·6 X 10 6 mol. wt

Bottom and meniscus

54

37 37

16 9

47

822

D. S. RAY AND P. C. HANAWALT

4. Discussion We have shown that enrichment for chloroplasts of Euglena gracili.
SATELLITE DNA IN EUGLENA

823

pared to a buoyant density of 1·707 glcc for the principal band. These values are in reasonably good agreement with those reported by Leff et al. (1963), and the higher density (1,688 g/cc) reported by those workers for the satellite may have resulted from the contribution from the tail of the principal band. Our observed selective denaturation of the satellite DNA in the presence of the principal DNA is consistent with the lighter density and the high adenine-thymine content of the former. In agreement with the melting of both DNA components (Leff et al., 1963), the satellite and the principal DNA components were shown to be double stranded on the basis of complementary base ratios obtained in the chromatographic experiments. In view of the small number of DNA species having greater than 70% adeninethymine, it is of interest to see how the Euglena satellite DNA, with 76% adeninethymine, fits the linear relationship between buoyant density and base composition (cf. Schildkraut et al., 1962). This relationship predicts an adenine-thymine content of 74·5% for DNA having a buoyant density of 1·685 g/cc. This slight difference is within the error of our experiments. Of particular interest, in addition to its high adenine-thymine content of 76%, is the apparent absence of 5-methylcytosine in the satellite DNA. In contrast, the principal DNA has an adenine-thymine content of 47% and contains 2·3% of 5-methylcytosine. This rare base appears to be distributed among all the principal DNA molecules of Euglena. Otherwise a heavy satellite DNA would be expected to result from the replacement of cytosine by 5-methylcytosine in a special class of DNA molecules (Schildkraut et al., 1962). In experiments to detect such a heavy satellite, we have re-banded DNA molecules from the heavy side of the principal DNA band; however, these fractions re-banded at the position of the principal DNA, with no evidence of a heavy satellite (Ray, 1964). The existence of DNA in physical association with chloroplasts lends support to the existing evidence that chloroplasts may be autonomously replicating organelles. It has recently been shown that a specific species of ribosomes is also associated with the chloroplasts of Euglena (Brawerman, 1963); and that these ribosomes have a different base composition from the microsomal particles and exhibit a different sedimentation behaviour in a sucrose gradient. Both the isolated chloroplasts and the chloroplast ribosomes have been shown to be active in protein synthesis in vitro (Eisenstadt & Brawerman, 1963). The apparent and striking differences betweenthe nucleic acids associated with Euglena chloroplasts and those of the whole cell are not surprising if the chloroplast is an evolved endosymbiont, as Ris & Plaut (1962) have suggested. These authors have pointed out a similarity in ultrastructural organization of chloroplasts and blue-green algae. Studies on the DNA of blue-green algae might substantiate this hypothesis. We wish to thank Dr A. D. Kaiser for discussions on a portion of this work and Dr W. R. Briggs for frequent discussions and reading of the manuscript. This research was supported by the U.S. Public Health Service (GM09901) and the Atomic Energy Commission (AT(04-3)326-7). REFERENCES Baltus, E. & Brachet, J. (1963). Biochim. biophys. Acta, 76, 490. Brawerman, G. (1963). Biochim. biophys. Acta, 72, 317. Brawerman, G., Hufnagel, D. A. & Chargaff, E. (1962). Biochim. biophys. Acta, 61, 340. Burgi, E. & Hershey, A. D. (1963). Biophys. J. 3, 309.

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