The isolation and characterization of DNA associated with chloroplast preparations

The isolation and characterization of DNA associated with chloroplast preparations

J. Mol. Biol. (1963) 7, 130-141 The Isolation and Characterization of DNA Associated with Chloroplast Preparations EDWARD H. L. CHUN, MAURICE H. ...

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J. Mol. Biol. (1963) 7, 130-141

The Isolation and Characterization of DNA Associated with Chloroplast Preparations EDWARD

H. L.

CHUN, MAURICE

H.

VAUGHAN, JR. AND

ALEXANDER RICH

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. (Received 3 March 1963) DNA has been isolated from spinach and beet leaves, and then subjected to density gradient ultracentrifugation. In addition to the major component of density 1·695 gJcm 3 , there are two minor components of density 1·705 and 1·719 gJcm3 • The leaves have been separated into a nuclear fraction and a chloroplast fraction. The nuclear fraction contains only the major DNA component, but the chloroplast fraction shows a considerable enrichment (ten- to thirtyfold) of the minor DNA components. It is known that chloroplasts have non-chromosomal inheritance, and it is suggested that the minor DNA components may provide the physical basis for this inheritance. Minor DNA components have also been isolated from the photosynthetic green algae Chlamydomonas reinhardi and Chlorella ellipsoidea. By denaturation studies, it is concluded that the minor as well as the major DNA components are all two-stranded.

1. Introduction Chloroplasts have been intensively studied in relation to their central role in photosynthesis. However, at the present time there is no clear understanding of how these organized cellular structures are produced. It is believed that they arise from pre-existing structures which enlarge and divide in the cytoplasm of the growing plant cell. For many years it has been known that certain genetic factors governing the production and function of chloroplasts are inherited in an extra-nuclear fashion; the transmission is maternal and is not linked to any chromosomal genes. Since DNA is the major repository for genetic information, it is of interest to determine whether there is DNA in chloroplasts. Previous studies have suggested that DNA is present in chloroplasts. Stocking & Gifford (1959) have shown that tritiated thymidine is incorporated into the chloroplasts of the green alga Spirogyra. Experiments with staining reactions, as well as electron microscope studies (Ris & Plaut, 1962), have also suggested that DNA is present in chloroplasts of Chlamydomonas. Furthermore, u.v, irradiation of the one-celled green alga Euglena can prevent the formation of chloroplasts in the progeny and its action spectrum suggests that nucleoprotein is the probable site of action (Lyman, Epstein & Schiff, 1961). However, many of these experiments are indirect and we have been stimulated to use a more direct approach, namely the isolation of DNA from chloroplast preparations and its characterization by density gradient ultracentrifugation. This method (Meselson, Stahl & Vinograd, 1957) is capable of detecting a microgram of DNA and, in addition, the position of the band may be used to obtain some information about 130

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the composition of the DNA. This method has been utilized here to determine whether plant plastids might contain small amounts of DNA and whether it is the same as the majority of the cellular DNA found in the nucleus. The higher plants beet (Beta vulgaris) and spinach (Spinacia oleracea) were studied, since chloroplasts can be concentrated from them. In addition, two single-celled green algae, Chlamydomonas reinhardl, and Ohlorella ellipsoidea, were studied.

2. Materials and Methods Green succulent spinach and beet leaves obtained from commercial sources were washed thoroughly with distilled water, stripped of stalks and weighed. Chloroplasts were concentrated according to a modification of the method of Jagendorf & Avron (1958).100 g of leaves was ground in a large mortar with sea sand and 140 ml. of buffered sucrose solution (0'4 M-sucrose, 0·02 M-tris, 0·01 M-NaCl and 0·005 M-ethylenediamine tetraacetic acid (EDTA, pH 8)). The suspension was filtered through four thicknesses of cheesecloth and centrifuged for 10 min at 400 g to obtain a white pellet, the nuclear fraction P I. The supernatant solution was recentrifuged for 20 min at 1000 g to obtain a green pellet, the chloroplast fraction P II. P I and P II were separately resuspended in 0·15 M-NaCI-0·I0 M-EDTA, pH 7'4, and treated with 1% sodium dodecyl sulfate to lyse the nuclei and the chloroplasts. The procedure of Marmur (1961) for the isolation of DNA was followed, except that the alcohol precipitation steps in the procedure were omitted. This is especially important in the case of P II where the concentration of DNA is very low. Instead, the high perchlorate concentration was reduced by dialysis against 0·15 M-NaCI-0'015 M-Na citrate buffer. After treatment with ribonuclease for the digestion of the RNA, the solution was dialysed against the same citrate buffer to remove u.v. light-absorbing material. In some cases, a series of centrifugal fractions was obtained between the precipitates P I and P II by centrifuging for various time periods at different speeds. This divided P II into three or four fractions. Several strains of Ohlamydomonas reinhardi (wild, mutants Yl, Y2) were grown in a standard medium (Levine & Ebersold, 1959) supplemented with yeast extract and sodium acetate. The algae were harvested after 2 weeks of growth at room temperature under room illumination. In addition, several cultures were obtained through the kind cooperation of Professor R. P. Levine. Chlorella ellipsoidea was grown at room temperature under room illumination on a photosynthetic agar. The medium for the agar was basically the same as that used for Ohlamydomonas except that no acetate or yeast extract was added. No bacterial contamination was observed in these algal cultures. The DNA isolated was analysed by CsCl density-gradient centrifugation in the Spinco model E analytical ultracentrifuge. The CsCI solution was at a mean density of 1·70 g/cm 3 and contained 2/Lg of plant DNA and l/Lg Micrococcus lysodeikticUB DNA used as a density reference (p = 1·731 g/cm 3 ) . Substantially higher concentrations of the sample DNA were used in some experiments to permit detection of the minor components; however, the density of the DNA components was obtained only from centrifuge runs containing 2/Lg. After 20 hr centrifugation at 44,770 rev.jrnin at 25°C, u.v. absorption photographs of the cell were taken. Tracings of these photographs were made with a Joyce-Loebl double-beam recording microdensitometer. Denaturation temperatures were measured in a Beckman DU spectrophotometer equipped with thermal spacers and a thermometer for measuring temperature in the cell compartment. In order to isolate or enrich for the minor DNA components, samples were centrifuged in 56% CsCI solution in the SW 39 rotor at 35,000 rev.huu» in the Spinco model L ultracentrifuge for 65 hr at room temperature. The fractions were collected dropwise after puncturing the bottom of the centrifuge tube. They were diluted with 1 ml. of buffer, and their absorbances at 260 miL measured in a Cary model 14 spectrophotometer. Selected fractions were then analysed by CsCl density-gradient centrifugation in the analytical ultracentrifuge.

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E. H. L. CHUN, M. H. VAUGHAN, JR. AND A. RICH

3. Results (a) Experiments with spinach. and beet chloroplast preparations

The first experiments were carried out with spinach preparations. Figure 1 shows densitometer tracings of the u.v, absorption photographs of DNA obtained from a whole cell preparation and the DNA obtained from the nuclear fraction and the chloroplast fraction of the same sample. The band at the right in the Figure (M) is the reference marker band of added Micrococcu8 lY8odeikticu8 DNA. In this whole ex

t

1'65

M '

1'70 1'75 Density (g/cm3)

FIG. 1. Densitometer tracing of u.v. absorption photographs of spinach DNA in the analytical ultracentrifuge. The photographs were made after 20 hr of density-gradient centrifugation at 44,770 rev.ymin and 25°C. M represents the marker band of added Micrococcu8lY8odeikticua DNA which has a density of 1·731 g/cm 3 • The whole cell preparation shows a major component (e<) and a minor component (y). The nuclear fraction contains only e< while the chloroplast fraction shows a ten-to twentyfold enrichment of y.

cell preparation it can be seen that the major band (lX) has a density of 1·695 g/cm 3 and a very small subsidiary trace band (y) can be seen at slightly higher density between the main component lX and the marker band (M). The minor component, y, is not observed in the nuclear fraction while, in the chloroplast fraction, this minor constituent has been considerably concentrated. The component with a density 1·719 g/cm3 (y) is always concentrated in the chloroplast preparations from spinach, although there is some variability in the appearance of another trace constituent as shown in Fig. 2. In Fig. 2(a) the whole cell preparation shows only the nuclear DNA fraction lX and very little, if any, of the additional minor components. However, the chloroplast preparation (Fig. 2(b)) shows a considerable enrichment of two minor components, f3 and y, to the nuclear fraction lX. The f3 band appears on these tracings more as a shoulder of the lX band than as a discrete band, even though it can be seen rather clearly by eye when looking at the photographs themselves. However, the independent existence of the f3 peak is shown in Fig. 2(c). The DNA for this was

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obtained by banding the DNA from the chloroplast preparation (Fig. 2(b)) in the preparative ultracentrifuge and collecting fractions as described in Materials and Methods. A subfraction was then taken in the region of the f3 band and this fraction was run in the analytical ultracentrifuge. The results of the banding (Fig. 2(c)) show clearly an enrichment of the f3 band relative to the ex band with very little of the y band showing up. In this spinach preparation we estimate that the y band at density 1·719 g/cm3 is about 2 to 3 times more plentiful than the f3 band which has a density of 1·705 g/cm3 . ex

+ (0)

;3

ex

t-

t

I

i

:

t« rA

/

-: {)vi~

FIG. 2. Densitometer tracing of spinach DNA as in Fig. 1. (a) contains DNA from the whole cell; (b) shows the chloroplast fraction with an enrichment of fJ and y bands; (c) a fraction was collected from the fJ region of chloroplast preparation B and then centrifuged again in the CsCl density gradient. The separate fJ peak is now visible. M is added marker DNA.

Eight different preparations of chloroplast fraction DNA from different spinach samples have been investigated. The results have yielded varying amounts of enrichment of the f3 and y bands relative to the ex band in the chloroplast fraction. In Fig. 1 the enrichment of the y band relative to the whole cell preparation is approximately 10 to 20 times. It represents about 45% of the total DNA in the chloroplast fraction. In the preparation shown in Fig. 2 the f3 and y bands cannot be seen in the whole cell preparation, therefore we estimate that their concentration is less than 1 to 2%. In the chloroplast preparation (Fig. 2(b)), f3 and y account for about 20% of the total DNA. Thus, this represents a concentration of at least ten- to twentyfold in this particular spinach preparation. We have observed that different spinach preparations have varying amounts of f3 and y bands in the whole spinach preparation covering the range shown in Figs. 1 & 2. These results are tabulated in Table 1. It should be pointed out that the inability to see a minor band in the whole cell preparation means that there is not enough to form a discrete band when the ultracentrifuge cell is loaded with a small amount of DNA. However, trace bands

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E. H. L. CHUN, M. H. VAUGHAN, JR. AND A. RICH

of this type can be seen readily if the cell is heavily loaded with DNA. In this case the main component forms a very broad band and the minor components become visible. The y band is characteristically concentrated in the chloroplast preparations (ten- to thirtyfold), while the f1 band lies so close to the main 0: peak that it is difficult to estimate its concentration. TABLE

1

Densities in CsCl solution and abundances of DNA components in plants Species

% Guanine + cytosine t

% in whole leaves or cell

%inPI

%inPII

1·695 1·705 1·719

36 46 60

90 to 100 < 4 < 8

95 to 100 < 3 < 3

50 to 80 5 to 15 10 to 40

1·695 1·705 1·719

36 46 60

90 to 100 < 10

1·723 1·695

64 36

95 to 99 1 to 5

1·716 1·695

57 36

99 1

Component Density

Spinacia oleracea (8 samples)

f3

Beta vulgaris (5 samples)

f3

Chlamydomonas reinhardi (6 samples)t

ex

Chlorella elli psoidea (3 samples)

f3

ex y

ex y

f3 ex

100

75 to 90 5 to 15 5 to 10

t Composition is obtained from the density, assuming no unusual bases. t Includes "wild" Yl, Y2, Y2 grown in the dark. Our initial interpretation of these results was that the chloroplast fraction had predominantly the f1 and y components, since they were both concentrated, whereas the 0: component diminished considerably relative to the whole cell preparation. The nuclear fraction itself had only the 0: component and this suggested the possibility that the 0: component of the chloroplast fraction arose from an incomplete separation of the nuclei or fragments of disrupted nuclei from the chloroplasts. Additional experiments were carried out to examine the effectiveness of differential centrifugation in separating chloroplasts from nuclei. The standard preparative method uses a 20 minute centrifugation at 1000 g to pull down a green pellet of the chloroplasts (P II). Experiments were carried out in which intermediate levels of centrifugation were employed in order to examine the relative concentration of the 0: versus the f1 plus y components in the supernatant solution. In addition, other experiments were carried out in which the green chloroplast pellet (P, II) was resuspended and then spun again at 400 g. When this was done, a pellet was obtained which was colored white on the bottom and green on top. This showed that the preparations which we have been using to prepare chloroplasts still had residual nuclei in them. When a chloroplast pellet was made from the supernatant solution of this material, there was a further relative enhancement of the f1 and y bands compared to the nuclear band (0:). This clearly suggested that the 0: component of the chloroplast fraction was due to contaminating nuclei which had not yet been pelleted in the initial nuclear fraction. Unfortunately, it was not possible to use a continual recycling

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of chloroplast pellets to effect a clean separation of the nuclei from the chloroplasts. The chloroplasts clump together in this process and cannot be resu spended. in addition, there appears to be a heterogeneity in the sedimentation coefficients of the nuclei or their fragments. A vigorous mechanical disruption is used to break open the leaf cells and liberate the chloroplasts and it is quite likely that this is accompanied by a certain amount of nuclear breakdown in the process . This would produce a heterogeneity of sedimentation coefficients from these nuclear fragments and is not surprising that they would be found along with the chloroplasts. Even though we were unable to get a chloroplast fraction containing only fJ and y bands,

II

M

~

FIG. 3. Dens itometer tracing of DNA from beet under conditions as in Fig. 1. (a) DNA from whole leaf showing major (X com pone nt ; (b) DNA from chloroplast preparation showing additional f3 and y bands; (c) DNA isolated from the f3 region of chloroplast preparation B and then centrifuged again in the density gradient. The f3 peak is clearly visible, but the (X peak is still present. J\.I is the added marker DNA.

these experiments nonetheless suggested that the fJ and y components were associated with the chloroplast fraction rather than the ex band which is found associated with the nuclei. The chloroplast preparations routinely had an enrichment of from ten- to thirtyfold of the fJ and y bands as compared to the ex band in the original whole cell DNA preparation. After observing the presence of minor DNA components obtained from the chloroplast fraction of spinach, we then carried out experiments with beet leaves to see whether the results were comparable. For this purpose the same preparative technique was carried out and the result of density gradient centrifugation is shown in Fig. 3. Figure 3(a) shows a tracing of a whole beet leaf DNA preparation. It can be seen that the main («) peak appears at a density of 1·695 g/cm3 , but no trace components are observed. However, the chloroplast preparation (Fig. 3(b)) now shows a considerable enhancement of two components which are not seen in the original

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E. H. L. CHUN, M. H. VAUGHAN, JR. AND A. RICH

whole cell preparation. These are labeled f3 and 'Y as with spinach and have the same densities, namely 1·705 and 1·719 gjcm 3 respectively. However, in this case there is relatively more of the f3 peak than of the 'Y in contrast to the spinach. The f3 peak of beet can be seen more clearly by fractionating the DNA in a preparative ultracentrifuge and recentrifuging a fraction obtained in the f3 regioh, This produces the tracing shown in Fig. 3(c) where the
The results described above suggested that minor DNA components with a different base composition from that which exists in the nucleus are associated with the chloroplast fractions in two higher plants. This suggested to us the possibility that chloroplasts in general may have associated with them DNA which has a base

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137

composition which differs from that of the nucleus. Unfortunately, very few experimental procedures have been developed for isolating chloroplasts from other species in general and in particular from lower organisms. However, we decided to explore the total DNA obtained from two lower organisms, namely the green algae Chlamydomonas reinhardi and Ohlorella ellipsoidea, to look for minor DNA components. Finding these components would not, of course, prove that they are associated with the chloroplasts but leaves this open as a possibility. The DNA preparations obtained from Chlamydomonas were mixed with the Micrococcus lysodeikticus marker DNA and run in the ultracentrifuge. The densitometer tracing of this mixture is seen in Fig. 4(a). It can be seen that the major ex

(b)

(el

1·65

FIG. 4. Densitometer tracing of DNA isolated from Ohlamydomonas and then subjected to density gradient centrifugation as in Fig. 1. (a) Normal loading of centrifuge cell shows only main '" peak; (b) heavy loading of cen trifuge cell shows minor peak (Il); (c) {3 peak isolated as shown in Fig. 5 and then centrifuged in density.gradient. The (3 peak is now completely isolated. M is added marker DNA.

peak has a density of 1·723 gJcm3 in agreement with the value reported by Sueoka (1960) and, with normal loading, no other bands can be seen. If the centrifuge cell is overloaded with several times the normal amount of DNA, it results in a very dense ex band as shown in Fig. 4(b) in which the optical density is greater than 1·0. Accordingly, there is a non-linear response in the photographic film in that region. However, this overloading makes it possible to visualize a minor DNA component, fJ, which is found at a density of 1·695 gJcm 3 • The minor component (fJ) of Chlamydomonas can be separated completely from the major ex component in the preparative ultracentrifuge by using a CsCl solution in the swinging bucket rotor. Figure 5 shows the optical density of the fractions obtained in this way. It can be seen that fJ corresponds to about 5% of the total DNA in this Chlamydomonas preparation. By selecting a fraction from the peak of the fJ component and mixing it

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E. H. L. CHUN, M. H. VAUGHAN, JR. AND A. RICH

with marker DNA for centrifugation, we obtain the densitometer tracing shown in Fig. 4(c), in which the f3 peak is free of any ex component. Six samples of Chlamydomonas DNA were prepared, including three wild-type preparations and the mutants Yl, Y2, and Y2 grown in the dark. A variable amount of the f3 band is found ranging from approximately I to 5%i of the total DNA (Table 1).

0·3

0·2

J6 0·1

15

20

25

30

Fraction number

FIG. 5. Optical density readings at 260 m,.. of the diluted fractions of DNA from Chlamydomonas reinhardi Y2 after preparative density gradient ultracentrifugation. The ex component has a density of 1·723 g/cm'; the fJ component has a density of 1·695 g/cm",

I

f

M

t

FIG. 6. Densitometer tracing showing IX and fJ components of a Chlorella DNA fraction enriched for fJ by preparative density gradient ultracentrifugation. The conditions are those listed in Fig. 1; M is added marker DNA.

A similar investigation was carried out on the DNA obtained from Chlorella. By heavily loading the ultracentrifuge cell, a f3 peak was visualized which was close to the ex peak (density 1·716 g/cm3 ) . In this case, however, the f3 peak represents approximately I to 2% of the total DNA in the Chlorella preparation. Density gradient preparative centrifugation was carried out and fractions were collected. DNA which was isolated from the f3 region was then centrifuged in a CsCIgradient in the analytical ultracentrifuge. The densitometer tracing of this preparation is shown in Fig. 6. The large f3 peak can be seen with a density of 1·695 g/cm 3 • However, the

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f3 component was not isolated completely from the 0: peak, and a small amount of the latter appears in the final preparation. The 0: and f3 peaks in both Chlamydomonas and Chlorella were shown to be normal two-stranded DNA by two experiments. They are both completely digestible by deoxyribonuclease and, in addition, heat denaturation and subsequent banding produce an increase in density of 0·015 g/cm3 • Thus, these organisms both have minor DNA components.

4. Discussion In the experiments described above we have shown that minor DNA components are found in the green leaves of two higher plants and in two photosynthetic green algae. Chloroplast preparations from the two higher plants have been accompanied by a concentration of the two minor DNA components, ranging from ten- to thirtyfold. However, despite careful differential centrifugation, it was impossible to remove the nuclear DNA component from the chloroplast preparation. It is possible that the concentration of the minor DNA components (f3 and y) in the chloroplast fraction may occur because they are found within the chloroplasts, and the nuclear component may arise from the presence of contaminating nuclear fragments. This explanation is suggested by the experiments in which a series of differential centrifuge fractions were isolated. Alternatively, we cannot rule out the possibility that some of the major component, 0: , is also found in the chloroplasts. One interesting feature of these preparations has been the fact that the DNA isolated from the whole cell often shows a somewhat variable amount of the minor components, f3 and y , compared to ex. This variability is indicated in Table 1 for the eight samples of spinach and five samples of beet. Accompanying this variability in the amount of f3 and y in the whole cell is a comparable variability in the amount of f3 and y in the chloroplast fraction. Thus, if a whole cell sample contains an amount of f3 .and y which is visible directly when the DNA of the whole cell is centrifuged, then f3 and y will appear in very large proportion in the chloroplast fraction. It is possible that this variability represents uncontrolled differences in the way in which the extraction procedure was carried out, since it is very difficult to quantitate the results of the initial grinding of the leaves with sand in the mortar. Alternatively, however, it may represent an inherent heterogeneity in these plants. The variability may be due to seasonal factors, as the specimens were collected over an 8-month period. Different physiological states may be reflected in differences in the turnover rates of DNA components as has been suggested by Iwamura (1960). The inheritance of chloroplasts is one of the outstanding examples of nonchromosomal heredity. These organelles increase in size during the growth of the cell and in young plants are believed to divide by fission. Many features of chloroplast function can be influenced by chromosomal mutations. Nonetheless, it has been clearly demonstrated in a large number of plants that the inheritance of the chloroplast is largely non-chromosomal. The results of these studies in higher plants have been reinforced by studies carried out in photosynthetic algae where it is possible to obtain chloroplast mutants. For example, streptomycin treatment of Euglenaleads to an irreversible loss of chlorophyll-forming ability, and the same result can be obtained with u.v, radiation (Lyman et al., 1961). It is not clear from these experiments that the site of the altered or lost genetic determinant is the chloroplast itself, but these

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E. H. L. CHUN, M. H. VAUGHAN, JR. AND A. RICH

experiments point out that the ability to form chloroplasts can be separated from the other genetic activities of the cell. In view of the fact that the minor DNA components which we observe in the chloroplast fraction have a substantially different base composition from that found in the nucleus, we are inclined to relate this to the non-chroniosomal heredity of the chloroplast. Thus, it is reasonable to make a hypothesis to the effect that the minor component DNA contains the genetic information necessary for replicating the cWoroplast and that this DNA is not integrated into the DNA of the nucleus. This hypothesis will have to be tested by further investigations in a variety of other photosynthetic organisms. In particular, our experiments on two photosynthetic green algae lend only minimal support to such an interpretation, since we have not shown that the minor DNA component in the green algae is associated with its cWoroplasts. It is of interest, however, that another example of a non-chromosomal genetic factor has been found to be associated with DNA which has a base composition different from that of the rest of the organism. Kappa particles can infect Paramecium aurelia and are known to be inherited through the cytoplasm. Recently Smith-Sonneborn, Green & Marmur (1963) have shown that this agent contains DNA with a base composition substantially different from that of the host nucleus. As with cWoroplasts, reproduction of kappa particles depends upon their living in the cytoplasm of the cell, since they are unable to function otherwise. We have wondered why there are two minor components (f3 and y) in the cWoroplast preparations of both spinach and beet. It may be that these are both associated with the chloroplasts. Alternatively, it is also possible that one of these may be associated with another organelle which is concentrated together with the chloroplasts. In particular, it may be that one of the minor DNA constituents is associated with the mitochondria rather than with the chloroplasts, since they would also accumulate in these centrifuge fractions. The fact that the ratio of f3 and y varies somewhat in different preparations suggests that they may not be both associated with the same structure. Further experiments are under way to investigate the possibility of a DNA fraction associated with mitochondria. The minor DNA component of the green algae has a density similar to that of the major nuclear component of both the higher plants; on the other hand, the major DNA component of the green algae has a density close to that of the y component of the two higher plants. This may be entirely fortuitous, or it may represent an interesting evolutionary pattern. Further experiments of this type will have to be carried out on a variety of organisms before any conclusions can be drawn. We gratefully acknowledge discussions during this work with Dr. V. M. Ingram and Dr. R. P. Levine. This research was supported by grants from the U.S. Public Health Service and the National Science Foundation, including a predoctoral fellowship (number 16,281) from the Division of General Medical Sciences, U.S. Public Health Service, awarded to M. H. V. REFERENCES Iwamura, T. (1960). Biochim. biophys. Acta, 42, 161. Jagendorf, A. T. & Avron, M. (1958). J. Biol. Chem. 231, 277. Levine, R. P. & Ebersold, W. T. (1959). Z. Vereb.-Lehre, 90,74. Lyman, H., Epstein, H. T. & Schiff, J. A. (1961). Biochim. biophys. Acta, 50,301. Marmur, J. (1961). J. Mol. Biol. 3, 208.

DNA ASSOCIATED WITH CHLOROPLAST PREPARATIONS

Mannur, J. & Doty, P. M. (1962). J. Mol. Biol. 5, 109. Meselson, M., Stahl, F. W. & Vinograd, J. (1957). Proc. Nat. Acad. Sei., Wa8h. 43, 581. Ris, H. & Plaut, W. (~962). J. Cell. Biol. 13, 383. Rolfe, R. & Meselson, M. (1959). Proc. Nat. Acad. Sci., Wa8h. 45, 1039. Schildkraut, C. L., Marmur, J. & Doty, P. (1962). J. Mol. Biol. 4, 430. Smith-Sonneborn, J., Green, L. & Marmur, J. (1963). Nature, 197, 385. Stocking, C.11. & Gifford, E. M. (1959). Biophq«. Biochem, Res. Comm. 1, 159. Sueoka, N. (1960). Proc. Nat. Acad. Sci., Wash. 46, 83. Sueoka, N., Marmur, J. & Doty, P. (1959). Nature, 183, 1427.

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