Proc. Nat. Acad. Sci. USAVol. 69, No. 7, pp. 1830-1834, July 1972

Physicochemical Characterization of Mitochondrial DNA from Pea Leaves(circular conformation/molecular size/renaturation kinetics)

RICHARD KOLODNER AND K. K. TEWARI

Department of Molecular Biology and Biochemistry, University of California, Irvine, Calif. 92664

Communicated by Martin D. Kamen, March 31, 1972

ABSTRACT The mitochondrial DNA from pea leavesexists in a circular conformation. 25% of the circular mole-cules exist as supercoils, and 10% of the molecules are di-mers. The molecular weight of mitochondrial DNA isabout 66 to 70 X 106 by electron microscopy, and 74 X 106from its renaturation kinetics. No evidence for inter- andintramolecular heterogeneity is found.

The mitochondrial (mt-) DNA from animal tissues exists inthe form of closed circular duplex molecules of 10 X 106molecular weight (1). Luck and Reich (2, 3) reported theisolation of linear molecules of molecular weight 13 X 106from Neurospora mitochondria, and calculated that the molec-ular weight of mt-DNA might be about 66 X 106 from itsrenaturation kinetics. Recent studies (4) have shown thatmt-DNA from Neurospora can be obtained in a linear struc-ture 26 jum in length. Similarly, mt-DNA from slime moldPhysarium has been isolated in linear forms, with molecularweights ranging from 20 to 30 X 106 (5). Extensive studies inosmotically shocked preparations of yeast have demon-strated the existence of mt-DNA in circular form (6). How-ever, all attempts to isolate these circular molecules -wereunsuccessful. The molecular weight of yeast mt-DNA wascalculated to be 50 X 106 (6). Mt-DNA from protozoa,Tetrahymena, and Paramecium, are linear structures of 17.6jim in length (7). Mt-DNA from higher plants has alwaysbeen isolated in linear molecules with mean lengths rangingfrom 10 to 20 ,um (7, 8). We have studied mt-DNA from pealeaves; it has a molecular weight of 74 X 106 from its renatura-tion rate. The DNA released from osmotically shocked mito-chondria, as well as that isolated by deproteinization, has acircular form with an average contour length of 30 jum underexperimental conditions in which replicative form (RF) IIDNA from bacteriophage OX-174 has a length of 1.45 Mum.This is the largest circular mt-DNA isolated to date.

MATERIALS AND METHODS

I8olation of Mitochondrial DNA. 12- to 15-day-old pealeaves were homogenized with 4 volumes of buffer containing0.3 M mannitol-0.05 M Tris HCl (pH 8.0)-3 mM EDTA-0.1% bovine-serum albumin-1 mM 2-mercaptoethanol.Homogenates were filtered through cheese cloth and centri-fuged at 1020 X g for 15 min; the supernatant was thencentrifuged at 12,000 X g for 20 min. The mitochondrialfraction (contaminated with broken nuclei and chloroplasts)was suspended in the buffer (200 ml/kg of leaves) and centri-fuged twice at 1020 X g for 15 min; the final mitochondrial

pellet was obtained after centrifugation at 12,000 X g for 20min. This pellet was suspended in the buffer (40 ml/kg ofleaves) and treated with 50 MLg/ml of DNase for 1 hr at 4°.All subsequent operations to obtain DNA free from RNA andprotein were those reported elsewhere (9).

Isolation of Mitochondrial DNA for Electron Microscopy.100 g of leaves were chopped with razor blades in 250 ml ofbuffer as reported (10). The mitochondrial pellet obtained asdescribed above was suspended in 10 ml of buffer, treatedwith DNase, and washed -three times by suspension in 3volumes of washing medium containing 0.35 M sucrose and0.1 M EDTA (pH 8.0). The final pellet was taken up in 1.0ml of washing medium. For treatment of osmotic shock 0.02ml of this suspension was added to 1.0 ml of 4 M ammoniumacetate-0.1 M EDTA (pH 8.0), and after 15 min the solutionwas spread on a hypophase of ice-cold glass-distilled water.After 15 min the monolayer was picked up on carbon-Formvarcoated mesh (200) grids and prepared as described (9). Forisolations of mt-DNA, 1.0 ml of 0.4% sodium dodecyl sulfatein 20 mM EDTA and 30 NaCl was added to 1.0 ml of themitochondrial fraction obtained after DNase treatment asdescribed above. The mixture was allowed to stand at 250for 30 min; then 0.1 ml of Pronase (25 mg/ml) was added,and the mixture was incubated at 370 for 2 hr. Further de-proteinization was done with chloroform-isoamyl alcohol95:5. The aqueous phase was dialyzed overnight against asolution of 0.15M NaCl-20 mM EDTA (pH 8.0). The isolatedDNA was prepared for electron microscopy as described (9).

Electron Microscopy. Preparations were examined at amagnification of X8250, and tracings were made at a finalmagnification of X250,000. A Zeiss EM9A with a 30-jumobjective aperture was used and calibrated with a diffractiongrating (Ladd 28,200 lines/inch).

RESULTS

Purity of mitochondrial DNA

The DNA isolated from crude mitochondrial fractions withoutDNase treatment showed the CsCl banding pattern pre-sented in Fig. 1A. There are two kinds of DNA, one bandingat a density of 1.698 g/cm3 and the other at a density of1.706 g/cm3. DNA banding at the density of 1.698 g/cm3could be a mixture of nuclear and chloroplast DNA (10).The DNA obtained from mitochondria treated with DNaseshowed only one band at a density of 1.706 g/cm3 (Fig. 1B).The localization of this DNA in the mitochondria of higherplants has been established in a recent review (10). The mt-DNA of pea leaves increased in density by 0.013 g/cm3

1830

Abbreviations: mt-DNA, mitochondrial DNA; RF II DNA,replicative form II DNA.

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Mitochondrial DNA in Pea Leaves 1831

FIG. 1. Photoelectric scans of DNA after analytical density-gradient centrifugation in CsCl. The centrifugation was per-formed in an initial density of 1.710 g/cm3 at 44,700 rpm for 18hr at 20° with Micrococcus lysodeikticus DNA of density 1.731g/cm3 as a marker. (A) DNA isolated from mitochondrial frac-tion, (B) DNA isolated from mitochondria treated with DNase.

after denaturation (Fig. 2C). Incubation of denatured DNAat 60° for 3 hr resulted in lowering the buoyant density by0.011 g/cm3. Thus, the mt-DNA obtained here shows thecharacteristic properties of organelle DNA.

Homogeneity of mitochondrial DNA

Mt-DNA was thermally denatured in 0.15 M NaCl-0.015 MNa citrate. A melting profile of such an experiment is pre-sented in Fig. 3. The mt-DNA melted with a sharp transitionmidpoint, with a Tm of 88 0.5°, maximal hyperchromicity(hmax) of 0.34, and dispersion (a- 2/3) of 7°. The absorbancetemperature profile of Fig. 3 does not show any intramolecularheterogeneity of the kind reported for Chlamydomonaschloroplast-DNA (11), which melted over a broad range oftemperatures. Since there is more than one molecule of mt-DNA in each mitochondrion (unpublished results), a hetero-geneous melting pattern of the isolated DNA would indicatethat these molecules have different genetic information orthat the base sequences in a given molecule are heterogeneous.The native mt-DNA gave a symmetrical peak in equilibriumcentrifugation in CsCl (Fig. 1B), indicating that significantintermolecular heterogeneity does not exist. The meltingpattern obtained further demonstrates that it is not possibleto detect any inter- and intramolecular heterogeneity withinthe limits of the sensitivity of the method. We determinedthe Tm of mt-DNA in 0.01 NaCl-Na citrate in order to furtherinvestigate the possibility of heterogeneity. The mt-DNAshowed a Tm of 680 but still melted sharply with a a 2/3 of70 and hmax of 0.32. On the other hand, nuclear DNA, whichshowed a 2/3 of 70 NaCl-Na citrate, a a 2/3 of 100 in 0.01NaCl-Na citrate.The mt-DNA was denatured and brought to 650. The

absorbance was continuously recorded and is presented in

FIG. 2. Same as in Fig. 1. (A) Neutral mt-DNA, (B) rena-tured, mt-DNA, (C) alkali-denatured mt-DNA.

FIG. 3. The relative absorbance at 260 nm of mt-DNA as afunction of time and temperature.

Fig. 3. The denatured mt-DNA returned to its native ab-sorbance in 2.5 hr. That the hypochromicity observed wasthe result of actual formation of native base pairs is seen fromthe data when renatured DNA is again heated. A sharpmelting of DNA at Tm of 88.50 is observed instead of meltingover a broad range of temperatures characteristic of un-paired structures.

Molecular size of mitochondrial DNA fromrenaturation rate-constant

Mt-DNA was broken into fragments of 17-18 S, denaturedby alkali, neutralized to pH 7.0, and added to a cuvetteequilibrated at 650. The rates of renaturation are plotted inFig. 4 for three different concentrations of mt-DNA. Thestraight-line plot shows that up to about 75% renaturation,the reaction obeys second-order kinetics (Fig. 4). No evidencefor any rapidly renaturing component is seen. Our resultsare in complete contrast to those reported by Wells and

3.2

3.0-

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2.6 ,c /

~2.4-,

B

2.2- A/

4( 2.0 0

~ie6

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1.2-

1.0

20 40 60 80 100 120 140 160Minutes

FIG. 4. Second-order rate plots of mt-DNA. The concen-trations of A, B, and C were 8, 11, and 16,.g/ml, respectively.DNA was fragmented by passage through a No. 27 gauge needle.It was made alkaline with 1 N NaOH and neutralized. The con-centration of salt was adjusted to (0.15 NaCl-0.015 M Na-citrate. Renaturation was done at 250 below the Tm.

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1832 Biochemistry: Kolodner and Tewari

TABLE 1. Molecular weight of mitochondrial DNA

Mol. wt. of mt-DNA (106)Renaturation rate- calculated from T4 DNA

Prep- constant (mol-i sec-') assuming mol. wt ofaration

no. T4 DNA mt-DNA 130 X 106 106 X 106

1 12.2 17.8 89.0 72.72 12.1 17.4 90.4 73.73 12.1 17.1 92.0 75.04 12.2 16.9 93.8 76.55 12.0 17.9 97.2 71.5

Mean 90.48 73.8

Birnstiel (12), where the rate-constant plot of lettuce mt-DNA suggested considerable base-sequence heterogeneity.It is difficult to explain these differences in results betweenmt-DNA of peas and lettuce, since we have found no evidencefor such heterogeneity in mt-DNA isolated from beans,lettuce, spinach, or tobacco (13).The renaturation rate-constant of mt-DNA was calculated

from the slope of the curve by the method of Wetmur andDavidson (14). The values for the renaturation rate constant(k2) for five different preparations of mt-DNA ranged from16.9 to 17.9 mol-1 sec-' (Table 1). Under the same experi-mental conditions, DNA from bacteriophage T4 gave valuesof k2 ranging from 12.0 to 12.2 mol-' sec-'. The molecularweight of mt-DNA was calculated from the analyticallydetermined values for the molecular weight of T4 DNA of130 X 101 and 106 X 106 (15) as a standard. The mean molec-ular weight of mt-DNA was calculated to be 90.5 X 101 and73.8 X 106, respectively, using the two different values for T4DNA. A molecular weight of 106 X 106 for T4 DNA is moreaccurate (15) and is also correct when calculated against peachloroplast-DNA as a standard (9). Thus, the molecularweight of mt-DNA from pea leaves was 73.8 X 106 by re-naturation kinetics.The second-order rate-constants calculated for mt-DNA

were independent of the initial reactant concentration (Table2). The renaturation rate-constants were dependent on themolecular weight of DNA and exhibited the square rootrelationship between the second-order rate-constant and themolecular weight (Table 3).

Electron microscopy of mitochondrial DNA

Osmotically shocked mitochondria were prepared as describedin Methods and examined in the electron microscope. The

TABLE 2. Renaturation rates at different concentrations ofmitochondrial DNA

Mol. wt. of mt-DNA (106)calculated from T4 DNA

Concentration Rate- assuming mol. wt. ofof mt-DNA constant

(Mg/Ml) k2 130 X 106 106 X 106

10 18.1 86.0 70.315 17.2 90.6 74.020 16.6 93.4 76.625 16.3 95.7 78.030 17.7 88.1 71.840 18.6 83.8 68.4

Mean 89.6 73.2

TABLE 3. Renaturation rate-constants of mitochondrial DNAat different molecular weights

Molecular Rate-weight* constant

82l0o X106 k21 11 1.14 12.7

(obtained by sonication ofmt-DNA)

2 16 3.37 17.4(obtained by passage of DNAthrough No. 27 gauge needle)

3 26 13.72 30.04 32 24.98 39.95 36 35.12 45.8

(preparations 3, 4, and 5 wereisolated DNA without further

fragmentation)

* Calculated by Studier's method (16).

preparation contained circular forms of DNA. The frequencydistribution of the lengths of DNA molecules is presented inFig. 5. 55% of the DNA released was in the circular conforma-tion of monomer length 30.3 Mm (SD ± 1.0 Am). 1% of theDNA was in the form of circles, ranging in contour lengthsfrom 3 to 5 Mm. 10% of the circular molecules were present indimer-length circles. The remaining DNA was linear, and themolecules ranged in length from 2 to 27 Mm. The circularmolecules found in these preparations are of mitochondrialorgin because no such molecules were seen in the nuclear orchloroplast fractions. Furthermore, chloroplast-DNA existsin circular molecules of 39 Am in length (9). The deproteinizedmt-DNA was prepared as described in Methods. 25% of theisolated mt-DNA was in the form of circles of contour length29.9 ,m (SD + 1.0 Mm). The supercoiled molecules constituted30% of the circular molecules. A typical picture of an opencircle is presented in Fig. 6.The necessity of including an internal standard when

making molecular weight determinations from contour-lengthmeasurements of DNA molecules has been discussed (9).Therefore, 4X174 RF II DNA was spread along with isolated

0

C

5 10 15 20 25 30Length (jim)

50 55 60 65

FIG. 5. Frequency distribution of the DNA released fromosmotically shocked mitochondria. The values expressed are thepercentage of the total DNA lengths measured, which was 800,um. Three of the molecules shown are of dimer lengths. Only oneof these could be distinguished as a circular dimer. The others areeither catenated or tangled circles.

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Mitochondrial DNA in Pea Leaves 1833

DNA (Fig. 7). In 20 molecules measured, contour lengthsof 4X174 RF II DNA ranged from 1.30 to 1.45,um (SD ±t0.08 Mm). Sixteen molecules of mt-DNA ranged from 28.4to 31.8 ,um, with an average contour length of 29.9 Am (SD i=1.0 Mm). The ratio of lengths between mt-DNA and 4X174RF II DNA is 20.6. From this ratio, and assuming a molecularweight of 3.2 X 101 for OX174 RF II DNA (Strider andWarner, personal communication), the molecular weight ofmt-DNA is 66 X 106.

DISCUSSION

The isolation of mt-DNA from sources other than animaltissues has always resulted in linear molecules (1-8). In

Euglena gracilis, the longest linear molecule found was 19Mm, even though circular molecules of 40 Am could be isolatedfrom chloroplasts (17). Recent studies in pea mitochondria byMikulska et al. (18) showed that only linear molecules of DNAwere present in the lysed mitochondria, and the size of mt-DNA in the isolated DNA was only 10 Mm. Our success inisolating intact mt-DNA in circular forms of contour lengthsof 30 Mm depends on gentle conditions of lysis. These circularmolecules could not be due to non-DNA links, since suchmolecules amounted to 25% of the DNA isolated after ex-traction with detergent, Pronase, and chloroform-isoamylalcohol. Wong and Wildman (19) have reported the presenceof circular molecules with contour lengths of 2 Mm in the

eor~,,--Aa r - I. ," --.1

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FIG. 6. Electron micrograph of a typical DNA molecule isolated from mitochondria. The contour length of the molecule is28.6,m. Bar length equals 1 gm.

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1834 Biochemistry: Kolodner and Tewari

'5

`4

0

0

z

1.0 1.2 1.4 1.6 1.8 26 28 30 32 34Length (pm)

FIG. 7. Length distributions of OX174 RF II DNA andisolated mt-DNA spread at the same time. The average length ofisolated mt-DNA is 29.9 Am (SD 4 1.0 ,m) Qompared to theaverage length of 4X174 RF II of 1.45,Mm (SD 4 0.08,m). Theratio between the lengths of the two DNAs is 20.6

mitochondrial fraction of tobacco leaves. We have observedsuch molecules in our preparations (Fig. 5), but these con-stituted an insignificant amount of the total DNA. Smallmolecules are also present in yeast mitochondria (6), buttheir significance has yet to be understood.The molecular weight of mt-DNA from pea leaves has been

calculated to be 70 X 106 and 66 X 106, assuming corre-sponding values of 3.4 X 106 and 3.2 X 108 for 4X174 RFDNA (9). The calculation of the molecular weight of mt-DNAfrom renaturation rates yields a value of 73.8 X 106. Thus,the mean value for the molecular weight of pea mt-DNA is70 X 108.The banding in CsCl density gradients and the melting

characteristics of mt-DNA have failed to show any inter-or intramolecular heterogeneity in the base compositions ofdifferent molecules. Also, there is no evidence of repeatingsequences in the mt-DNA. The excellent agreement observedbetween the molecular weight of 70 X 106 obtained by elec-tron microscopy and 73.8 X 106 obtained by kinetics analysisprecludes the presence of mt-DNA molecules containing dif-ferent base sequences.

Mt-DNA of pea leaves of molecular weight 70 X 108 is thelargest DNA molecule reported in mitochondria. The ques-tion arises whether mt-DNA from sources other than animalmitochondria are of similar complexity or differ in theirmolecular sizes. Our experiments with spinach, lettuce, andbeans have shown that the molecular weight of mt-DNA inthese plants is also about 70 X 106 (13).

This work was supported by the National Science FoundationGrant GB-20674. R. K. is a predoctoral trainee of NIGMS(grant no. GM-02063-01). We thank Dr. R. C. Warner for pro-viding helpful discussions and liberal use of his research facilities.We are especially thankful to Dr. C. N. Gordon for teaching usthe protein-monolayer technique. The excellent technical assist-ance of Mr. K. Evans and Juta Keithe is gratefully acknowledged.

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224.4. Schafer, K. P., Bugge, G., Grandi, M. & Kuntzel, H. (1971)

Eur. J. Biochem. 21, 478-488.5. Evans, T. E. & Suskind, D. S. (1971) Biochim. Biophys. Acta

228, 350-364.6. Hollenberg, C. P., Borst, P. & Van Bruggen, E. F. J. (1970)

Biochim. Biophys. Acta 209, 1-15.7. Suyama, Y. & Mivra, K. (1968) Proc. Nat. Acad. Sci. USA

60, 235-242.8. Wolstenholme, D. R. & Gross, N. J. (1968) Proc. Nat. Acad.

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Meeting Electron Microscopy Society of America, ed. Arcen-eaux, C. J. (Clator's Publ.), in press.

14. Wetmur, J. G. & Davidson, J. (1968) J. Mol. Biol. 31, 349-370.

15. Friefelder, F. (1970) J. Mol. Biol. 54, 567-577.16. Studier, F. W. (1965) J. Mol. Biol. 11, 373-390.17. Manning, J. E., Wolstenholme, D. R., Ryan, R. S., Hunter,

J. A. & Richards, 0. C. (1971) Proc. Nat. Acad. Sci. USA 68,1169-1173.

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