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Mitochondrial DNA
J. Mol.
Biol.
(1969) 42, 521-528
Mitochondrial
DNA
I. Intramitochondrial Distribution and Structural Relations of Single- and Double-length Circular DNA MARGIT
Department
of Therapeutic
M. K. NASS
Researclt, Uniaersity of Pennsylvania Philadelphia, Pa. 19104, U.S.A.
(Received 25 April
1968, and in revised form 18 February
School of Medicine
1969)
The structure of the DNA of L-cell mitochondria was studied as it may relate to the organelle in wivo. Serial sections of mitochondria revealed two to three DNA-containing regions (nucleoids) per organelle; up to six regions were occasionally observed. Osmotic rupture of freshly isolated mitochondrie released 2 to 4% circular DNA dimers (lO*lp, mol. wt 20 x 106) in a population of circular monomers (5.2 p, mol. wt 10 x 106). The molecules were unbranched, usually folded in half, and either loosely coiled or extended. Most dimers were single circles, either lying free or attached at a polar region to another dimer or two monomers. It appears that each nucleoid may contain one to several monomers and dimers, to yield an average total of two to six molecules per mitochondrion. About 80% of the monomers were associated at polar regions with the mitochondrial membranes; experiments were performed that reduced the possibility of artifact formation. Osmotically shocked mitochondria of ascites tumor cells and adult rat and chicken liver were also compared. The observed membrane associations and the multiplicity of DNA molecules per mitochondrion or per nucleoid may be related to mitochondrial duplication; the high degree of variation in number of molecules per mitochondrion implies redundancy of informational content.
1. Introduction The presence of DNA and RNA in mitochondria and the capacity of these organelles to synthesize DNA, RNA and protein is now well documented (cf. reviews by Nass, 1967,1969a; Borst, Kroon & Ruttenberg, 1967). Mitochondrial DNA was originally identified in chick embryos by various electron cytochemical techniques (Nass & Nass, 1962,1963a,b) and was shown in mitochondria of all animal phyla and one plant species (Nass, Nass & Afzelius, 1965). The DNA released directly from isolated L-cell mitochondria, was found to be circular with a contour length of 5.2 TV(Nass, 1966). Estimates of DNA by chemical and electron microscopical methods yielded an amount equivalent to two to six DNA molecules per L-cell mitochondrion. Purified mitochondrial DNA of several vertebrates was found to consist of twisted and open circular molecules with an average perimeter of 5 CL, corresponding to a molecular weight of 9 to 10 x lo6 daltons (Nass, 1966; van Bruggen, Borst, Ruttenberg, Gruber & Kroon, 1966; Sinclair & Stevens, 1966). In addition, double- and multiple-size circular DNA molecules of yet unknown significance have been isolated 521
JI. M. Ii.
522
NASS
from mitochondria of HeLa cells (Radloff, Bauer & Vinograd, 1967; Hudson & Vinograd, 1967), leucocytes (Clayton & Vinograd, 1967), sea urchin (Pike, Blair, Tyler & Vinograd, 1968), and L-cells (Nass, 1969aJ). This report summarizes studies of mitochondria by serial sections and osmoticshock techniques to gain some insight into the structure, size and distribution of DNA molecules in the intact mitochondrion. The following paper (Nass, 19696) describes properties of purified mitochondrial DNA.
2. Materials and Methods (a) Isolation
of mitochondria
L-cells (mouse fibroblasts) were cultured in suspension in Eagle’s medium supplemented with 10% fetal calf serum. Aureomycin (50 pg/ml.) was added for 3 days/week to prevent infection by Mycoplasma. Cells were harvested in the logarithmic phase at a concentration of 5 to 7 x IO5 cells/ml. Approximately 1 x IO9 cells were washed twice with a solution of 0.16 M-NaCl, 0.01 M-phosphate buffer (pH 7.0). The cells were ruptured at 2°C in a hypotonic medium by homogenization with a Dounce homogenizer in 0.10 M-sucrose, 2 m&r-EDTA, 0.025 M-Tris-HCl buffer (pH 7.4). The homogenization was monitored by phase-contrast microscopy. Approximately 5% whole cells remained after homogenization. After rapid adjustment of the sucrose concentration to 0.30 M, the whole cells and nuclei were removed by centrifugation twice at 500 g. Mitochondria were sedimented at 10,000 g and washed twice with 0.30 M-sucrose, 2 m&r-EDTA, 0.025 M-Tris-HCl (pH 7.4). Residual nuclear DNA was removed by digestion with 10 pg DNase/ml. in 0.3 Msucrose, 5 m&r-MgCl, for 40 min at 2°C or for 30 mm at room temperature (Fig. 1). Mitochondria were purified further by centrifugation in a linear gradient of sucrose (1.03 to 1.91 M-sucrose, 2 mm-EDTA, 0.025 ivr-Tris-HCl, pH 7.4) for 90 min at 22,000 rev./min in a Spinco model L2 ultracentrifuge (SW25*1 rotor, 4°C). Figure 2 shows the distribution of radioactivity from [3H]thymidine, the content of DNA and protein, and
I I O 0
I
I
I
I
I
I
IO
20
30
40
50
60
Time (min) FIG. 1. Incubation of isolated L-cell mitochondria in the presence sucrose, 0.005 M-Mgcl,, 0.025 M-Tris-HCl, pH 7.3, at 2% (-O--O-,
fragments
added to one preparation
of mitochondria
(-m-m-).
of 10 pg DNase/ml., 0.3 M--(>--a--). Nuclear
I.
0
MITOCHONDRIAL
8
DNA
16
IN
24 Fraction
513
SITU
32
40
no.
FIG. 2. Sedimentation of isolated L-cell mitochondria through a linear gradient of sucrose, as described in Materials and Methods. Fractions of 1 ml. were collected and analyzed for radiocontent of DNA (-A-A--), protein (--O--O--), activity of [3H]thymidine (-O-O--), and cytochrome c oxidase activity (rel. act./ml.) (---x--x--). Molar sucrose (-A-n-).
TABLE
1
Content of DNA, RNA and protein in purified L-cell mitochondria Type of analysis (1) mg protein/mitoohondriont (lO)$ no. of mitochondria/mg proteint (2) pg DNA/mg mitochondrial protein (14)$ (3) g DNA/mitochondrion§ mol. wt of total DNA/mitochondrion mol. wt of single circular DNA molecule (4) pg RNA/mg mitochondrial protein (3)$ (5) pg mitochondrial protein/IO6 cells isolated (6) No. of mitochondria isolated/cell // Total no. of mitochondria/cell~ (7) g DNA/cell (8) ‘;/o mitochondrial DNA in cellular DNA
Amount 8.0 (hO.7) x 10-11 1.2 (&0.2) x 10’0 1.1 (kO.2) 8.8 (h2.4) x lo-i7 53.0 x 106 10.0 x 106 38.0 (h5.5) 14.8 (b4.1) 185*60 250&70 1.3 (10.2) x IO-” 0.15
i Preparations contained negligible amounts of electron microscopically detectable nonmitochondrial structures that would significantly contribute either to protein content or to mitochondrial particle counts. $ Number of determinations. $ Calculated from values (1) and (2) (Nass, 1966). I] Estimated by converting yield of mitochondrial protein (5) into numbers of mitochondria, using values (1). 7 Estimate includes the assessment of lost mitochondria in unbroken cells (5%) and during oentrifugation (15 to 35%), as judged by the analysis of cell fractions during the isolation procedure with respect to content of whole cells, electron microscopic composition and content of oytochrome c oxidase activity.
5’4
Xl. 11. I<. NAS‘iL L
the relative activity of cytochrome c oxidase after sedimentation of mitochondria in the sucrose gradient. Electron microscopical analysis of the middle zone showed mostly contracted and some slightly swollen mitochondria. The small ba,nd at the top of the gradient consisted of lysosome-like particles and membrane fragments. The small pe‘llet at the bottom contained traces of mitochondria and nuclear fragments. Table 1 s~nnmarizes the yield of mitochondria and the content of DNA, RNA and protein in the purified organelles. Mitochondria from Ehrlich ascites tumor cells and adult rat and chicken liver were prepared by the same procedure, except that liver mitochondria were homogenized in isotonic sucrose. (b) Chemical
analyses
DNA was measured by a microfluorometric method as previously described (Nass, 1966). RNA was analyzed by the orcinol reaction, and protein was determined by the Lowry method. Cytochrome c oxidase was measured as described by Cooperstein & Lazarow (1951); the activity was expressed relative to the absorbance of reduced and completely oxidized cytochrome c. Tritium-labeled mitochondria were obtained from cells incubat,ed for 17 hr with 1 PC [3H]thymidine/ml. (10 c/m-mole, Schwartz Bioresearch). Samples of mitochondria were extracted twice with cold 5% trichloroacetic acid, ethyl alcohol and ether and the pellets were suspended in 0.7 ml. NCS reagent (Nuclear Chicago) and 10 ml. toluene containing 4 g POP and 50 mg POPOP/l. The radioactivity was determined in a Packard Tricarb liquid-scintillation counter. Microscopic counting of isolated mitochondria was performed as reported previously (Nass, 1966).
(c) Electron microscopy L-cells were fixed, embedded and stained as previously described (Nass et ul., 1965). Serial sections were cut on a Porter-Blum ultramicrotome MT-2 and picked up on carboncoated Formvar films supported by copper grids with a large central opening. The spreading of DNA by the osmotic-shock method, shadowing, electron microscopy and measurement of the contour lengths of DNA molecules were performed as described previously (Nass, 1966).
3. Results (a) Serial sections of DNA
centers in mitochondria
of intact cells
Chemical and electron microscopical evidence, suggesting the presence of several DNA molecules per mitochondrion (Nass, 1966), raises the question whether DNA molecules are concentrated in one area of the mitochondrion or are distributed in several areas. Although the DNA of mitochondria from many cell types may be visualized by electron microscopy as fibrils of 20 A diameter in areas of low matrix density (Nass & Nass, 1963a,b; Nass et al., 1965), DNA was not observed in all electron-lucid areas of the mitochondrion in a given tissue section. Serial sections of mitochondria were therefore cut to assess the number of DNA centers in intact mitochondria
(Plate
I).
The
visibility
of the
mitochondrial
DNA
was
enhanced
by
omitting post-fixation of cells with uranyl acetate, thus preserving the DNA in a clumped rather than fine fibrillar state (see Nass & Nass, 1963aJ for details of the structure of DNA in ultrathin sections). The mitochondrion shown in the first series of serial sections contains in its matrix at least three spatially discrete DNA centers or nucleoids, and the two mitochondria in the second series have at least two and three DNA centers. Some highly filamentous organelles with four to six DNA centers were occasionally observed.
I.
(b) Structure
of circular
MITOCHONDRIAL
DNA
DNA
IN
52.j
SITU
monomers and dimers released by osmotic shock from isolated mitochondria
Mitochondris subjected to osmotic shock (Nass, 1966) released individual circular monomers and unresolved aggregates of DNA with a total mass equivalent to multiples of DNA monomers. To resolve these aggregates into individual units, larger have now been exnumbers of mitochondria (2000) and more dilute preparations amined. Mitochondria were homogenized in 4 M-ammonium acetate prior to spreading. It was ascertained by phase-contrast microscopy that aggregates of mitochondria were dispersed into single particles. Once a mitochondrion was ruptured, there was little evidence that DNA molecules remained inside undetected; judging from the low percentage of partially extruded molecules present, breaks in the mitochondrial membrane allowed the molecules to emerge at least partially. A clear area with a radius of at least 50 TV surrounding the structures to be described was considered sufficient to reduce strongly or eliminate t’he contribution of DNA molecules by other mitochondria. In mitochondria of L-cells and ascites tumor cells, circular DNA monomers (5.21 f. made up 80 to 90% of the extruded DNA (Plates II and 0.2 p in circumference) III(b)). Typically, the molecules were folded in half; both halves were coiled around each other or remained closely associated after unwinding (Plates II(b), III and VI(2)). Most coiled molecules contained up t’o 25 cross-overs ; 1% had up to 35 crossovers, similar to purified covalently closed DNA (Nass, 19698). The polar regions of these flattened circles appeared to be attached to membrane fragments. Approximately 10% free-lying molecules were observed. As compared with mitochondria of L-cells, mitochondria of rat and chicken liver appeared to release more free-lying molecules and more coiled molecules with 30 to 35 crossovers. Strikingly, 2 to 4% of the DNA seen in osmotically lysed L-cell mitochondria consisted of double-size circular molecules (Plate IV(a) and (b)). The contour length of eleven dimers was 10.1*0+4 p, corresponding to a molecular weight of 19.6 x lo6 daltons. This form of dimer was not apparent in mit’ochondria of other cell types. A dimer was sometimes attached to another dimer or two monomers by a common knob-like central point (Plate V). It is possible that the two monomers shown are actually cont,inuous at the central point to form another dimer. The fact that these DNA molecules are connected at a cent,ral point suggests that t’hey are derived from one mitochondrial nucleoid. Most mitochondria released two DNA molecules (Plates II and III). Higher aggregates of DNA associated with a ruptured mitochondrion could be partially or completely resolved into mixtures of monomers and dimers totalling up to six t’imes the unit size of 52 p (Plates VI and VII). (c) Relationship
of DNA
to the mitochondrial
membrane
Experiments were performed to see whet,her the apparent attachment of polar regions of many circular DNA molecules to the mitochondrial membrane may reflect the occurrence of attachments in viva or whether artifacts could arise due to nonspecific adsorption. First, purified circular DNA was mixed with mitochondrial suspensions and spread by the osmotic-shock method. Interfering endogenous DNA could be reduced to about 3% by self-digestion of the mitochondria for 20 minutes at 37°C prior to the addition of exogenous DNA. As shown in Table 2, 83% of the added
526
M. M. K.
NASS
TABLE 2 Relation
of surface-spread
Preparation?
Mitochondria ( +endogenous DNA), control preparation Mitochondria (+ endogenous DNA) + circular DNA added Mitochondria (pre-incubated, 37’C, 30 min) + circular DNA added5
DNA
to mitochondrial
(a) lying free
membranes
o/o DNA molecules :$ (b) membrane(c) membraneassociated associated at poles at random
7
14
12
44
49
I
83
10
7
acetate (pH 7.0) and 0.01 o/0 cytochrome c in 7 0.05 mg mitochondrial protein, 4 M-&%IIXnOniUXII 60 ~1. In experimental preparations, 0.07 pg purified circular DNA added (DNA isolated on CsClethidium bromide gradients and freed of the dye (Nass, 1969b)). $ 60 to 80 molecules counted in each preparation. 5 No DNA was seen in pre-incubated mitochondria without circular DNA added, apparently due to digestion by endogenous DNase.
DNA was not associated with membrane, whereas the reverse was true for endogenous DNA of control mitochondria, and an intermediate situation existed when the endogenous DNA was not removed. L-cell mitochondria were also incubated with 100 pg of either ribonuclease A, pronase, phospholipase C or phospholipase D (Calbiochem) per ml. for 30 minutes at room temperature prior to spreading. It was ascertained that the enzymes at least partially penetrated and were active (though not maximally) under the conditions used by chemically analyzing corresponding samples for RNA, protein content and dry weight after extraction with lipid solvents. The only obvious change was seen after treatment with pronase. The membrane fragments were somewhat smaller than in control preparations and points of association of polar regions of DNA with membrane fragments were rarely seen. These experiments reduce, but do not eliminate, the possibility of artifacts that may result from non-specific adsorption or unknown forces during osmotic shock and formation of the protein film.
4. Discussion The results described in this paper indicate that L-cell mitochondria contain mixtures of circular DNA monomers and dimers which are located in spatially distinct regions of the mitochondrial matrix. The presence of dimers in osmotically shocked mitochondria implies that these forms exist in the organelle in vivo. In previous studies dimers had been observed only in mitochondrial DNA isolated by centrifugation in cesium chloride gradients (see Introduction). No direct methods are presently available to analyze the structural arrangement of DNA molecules in an organelle. Each experimental approach has distinct limitations, and, taken by itself, would be insufficient to demonstrate unequivocally the multiplicity of DNA molecules in a mitochondrion. For this reason, the conclusions have been based on congruent results from several lines of investigation. First, the serial sections show that variable numbers of DNA-containing regions, usually two to three, are present per mitochondrion. They do not indicate how many DNA
243)
(W
(b)
(c) (4
(d)
(b)
(4
(4
(4
(b)
......_._____________....... -..-.
1. MITOCHONDRIAL
DNA
IL\: SITU
,527
molecules each region contains, nor whether any DNA exists between the regions. The latter possibility is unlikely, however, because the localization of DNA in electronlucid regions is typical for many biological systems, especially bacteria (cf. Nass et al., 1963u). Second, the joint attachment of some circular DNA molecules released by osmotic shock (Plate V) implies that more than one DNA molecule may be located in one nucleoid. If a mitochondrion possessestwo nucleoids, two to eight molecules may be included. In cases where liberated DNA molecules overlap (Plates VI and VII), it is obviously not possible to determine how many intramitochondrial nucleoids they represent. The high degree of dispersion and dilution of mitochondria suggests that the cluster of DNA represents the combined content of one mitochondrion (although it cannot be eliminated that some molecules may float off before they are adsorbed to the protein film). Thirdly, the results are compatible with the chemically determined DNA content per mitochondrion (Nass, 1966) ; 53 x lo8 daltons of DNA would allow for an average number of five DNA molecules of 10 x IO6 daltons per organelle. Whether the lack of supercoiling of many osmotically liberated DNA molecules reflects the structure in vivo is not known. The absence or presence of tertiary turns may be a function of the ionic and thermal environment in vivo (Wang, Baumgarten & Olivera, 1967) or upon spreading. Lack of supercoiling, noted especially in the fast-growing cells, may also be due to the introduction of single-strand scissions by nucleases prior to spreading or by an endonuclease that is preferentially liberated during osmotic shock. Lack of supercoiling may also be related to DNA replication or other functions (cf. Nass, 1969a). The apparent association of DNA with the membrane, observed also in ultrathin sections of mitochondria (cf. Nass et al., 1965) and of Bacillus subtilis (Ryter & Jacob, 1963), may form a physical basis for some of these functions. However, a clear definition of such connections will have to await analyses by other techniques. The biological significance of double-size circular DNA is not yet known. It has been suggested (Rush, Kleinschmidt, Hellmann & Warner, 1967) that t#he multiplelengt’h circular DNA molecules observed in preparations of the replicative form of +X174 represent intermediate forms in DNA replication or recombination. Although there is considerable evidence in support of mitochondrial division and DNA replication (cf. review by Nass, 1969a), it is premature to speculate about the nature of the DNA replication mechanism in mitochondria. It is conceivable, however, that the number and structural arrangement of the DNA-containing regions in filamentous mitochondria may represent products of duplication of pre-existing DNA cent,ers during growth and elongation of the organelles. Multiplicity and variation in number of DNA molecules per mitochondrion may imply redundancy of the informational content of a single DNA4 molecule. This investigation was supported by U.S. Public Health Service grant POl-A107005. I acknowledge the excellent technical assistance of Miss Anneke Theunissen in the biochemical work and Mr John R. W. Hobbs in the electron microscopic and photographic work. REFERENCES Rorst,
P., Kroon, A. M. & Ruttenberg, G. J. C. M. (1967). In Genetic Elements, Properties alad Function, p. 81. London & New York: Academic Press. Warsaw: Pn’N. v&n Hruggen, IS. F. J., Rorst, P., Rut)tenberg. G. .J. C. M., Gruber, M. & Kroon, A.M. (1966). Rioch,irn. hiophys. rlctm, 119, 437. 33
508
Al.
n1. ti.
L-ASS
Clayton, D. A. & Vinograd, J. (1967). Nature, 216, 652. Cooperstein, S. J. & Lazarow, A. (1951). J. Biol. Chem. 189, 665. Hudson, B. & Vinograd, J. (1967). Nature, 216, 647. Nass, M. M. K. (1966). Proc. Nut. Acad. Sci., Wash. 56, 1215. Nass, M. M. K. (1967). In Organizational Biosynthesis, ed. by H. J. Vogel, ,J. 0. Lampen and 1’. Bryson, p. 503. New York: Academic Press. Nass, M. M. K. (1969a). Science, in the press. Nass, M. M. K. (1969b). J. Mol. Biol. 42, 529. Nass, M. M. K. & Nass, S. (1962). Exp. Cell Ree. 26, 424. Nass, M. M. K. & Nass, S. (1963a,). J. Cell Biol. 19, 593. Nass, M. M. K., Nass, S. & Afzelius, B. A. (1965). Exp. Cell Res. 37, 516. Nass, S. & Nass, M. M. K. (1963b). J. Cell BioZ. 19, 613. Pik6, L., Blair, D. G., Tyler, A. & Vinograd, J. (1968). Proc. Nat. Acad. Sci., Wash. 59, 838. Radloff, R., Bauer, W. & Vinograd, J. (1967). Proc. Nat. Acad. Sci., Wash. 57, 1514. Rush, M. G., Kleinschmidt, A. T., Hellmann, W. & Warner, R. C. (1967). Proc. Nut. A end. Sk., Wash. 58, 1676. Ryter, A. & Jacob, F. (1963). G. R. Acad. Sci. Paris, 257, 3060. Sinclair, J. H. & Stevens, R. J. (1966). Proc. Nat. Acad. Sci., Wash. 56, 508. Wang, J. C.. Baumgarten, D. & Olivera, n. M. (1967). Proc. Nat. Acud. Sci., Wash. 58, 1852.
Biol.
(1969) 42, 521-528
Mitochondrial
DNA
I. Intramitochondrial Distribution and Structural Relations of Single- and Double-length Circular DNA MARGIT
Department
of Therapeutic
M. K. NASS
Researclt, Uniaersity of Pennsylvania Philadelphia, Pa. 19104, U.S.A.
(Received 25 April
1968, and in revised form 18 February
School of Medicine
1969)
The structure of the DNA of L-cell mitochondria was studied as it may relate to the organelle in wivo. Serial sections of mitochondria revealed two to three DNA-containing regions (nucleoids) per organelle; up to six regions were occasionally observed. Osmotic rupture of freshly isolated mitochondrie released 2 to 4% circular DNA dimers (lO*lp, mol. wt 20 x 106) in a population of circular monomers (5.2 p, mol. wt 10 x 106). The molecules were unbranched, usually folded in half, and either loosely coiled or extended. Most dimers were single circles, either lying free or attached at a polar region to another dimer or two monomers. It appears that each nucleoid may contain one to several monomers and dimers, to yield an average total of two to six molecules per mitochondrion. About 80% of the monomers were associated at polar regions with the mitochondrial membranes; experiments were performed that reduced the possibility of artifact formation. Osmotically shocked mitochondria of ascites tumor cells and adult rat and chicken liver were also compared. The observed membrane associations and the multiplicity of DNA molecules per mitochondrion or per nucleoid may be related to mitochondrial duplication; the high degree of variation in number of molecules per mitochondrion implies redundancy of informational content.
1. Introduction The presence of DNA and RNA in mitochondria and the capacity of these organelles to synthesize DNA, RNA and protein is now well documented (cf. reviews by Nass, 1967,1969a; Borst, Kroon & Ruttenberg, 1967). Mitochondrial DNA was originally identified in chick embryos by various electron cytochemical techniques (Nass & Nass, 1962,1963a,b) and was shown in mitochondria of all animal phyla and one plant species (Nass, Nass & Afzelius, 1965). The DNA released directly from isolated L-cell mitochondria, was found to be circular with a contour length of 5.2 TV(Nass, 1966). Estimates of DNA by chemical and electron microscopical methods yielded an amount equivalent to two to six DNA molecules per L-cell mitochondrion. Purified mitochondrial DNA of several vertebrates was found to consist of twisted and open circular molecules with an average perimeter of 5 CL, corresponding to a molecular weight of 9 to 10 x lo6 daltons (Nass, 1966; van Bruggen, Borst, Ruttenberg, Gruber & Kroon, 1966; Sinclair & Stevens, 1966). In addition, double- and multiple-size circular DNA molecules of yet unknown significance have been isolated 521
JI. M. Ii.
522
NASS
from mitochondria of HeLa cells (Radloff, Bauer & Vinograd, 1967; Hudson & Vinograd, 1967), leucocytes (Clayton & Vinograd, 1967), sea urchin (Pike, Blair, Tyler & Vinograd, 1968), and L-cells (Nass, 1969aJ). This report summarizes studies of mitochondria by serial sections and osmoticshock techniques to gain some insight into the structure, size and distribution of DNA molecules in the intact mitochondrion. The following paper (Nass, 19696) describes properties of purified mitochondrial DNA.
2. Materials and Methods (a) Isolation
of mitochondria
L-cells (mouse fibroblasts) were cultured in suspension in Eagle’s medium supplemented with 10% fetal calf serum. Aureomycin (50 pg/ml.) was added for 3 days/week to prevent infection by Mycoplasma. Cells were harvested in the logarithmic phase at a concentration of 5 to 7 x IO5 cells/ml. Approximately 1 x IO9 cells were washed twice with a solution of 0.16 M-NaCl, 0.01 M-phosphate buffer (pH 7.0). The cells were ruptured at 2°C in a hypotonic medium by homogenization with a Dounce homogenizer in 0.10 M-sucrose, 2 m&r-EDTA, 0.025 M-Tris-HCl buffer (pH 7.4). The homogenization was monitored by phase-contrast microscopy. Approximately 5% whole cells remained after homogenization. After rapid adjustment of the sucrose concentration to 0.30 M, the whole cells and nuclei were removed by centrifugation twice at 500 g. Mitochondria were sedimented at 10,000 g and washed twice with 0.30 M-sucrose, 2 m&r-EDTA, 0.025 M-Tris-HCl (pH 7.4). Residual nuclear DNA was removed by digestion with 10 pg DNase/ml. in 0.3 Msucrose, 5 m&r-MgCl, for 40 min at 2°C or for 30 mm at room temperature (Fig. 1). Mitochondria were purified further by centrifugation in a linear gradient of sucrose (1.03 to 1.91 M-sucrose, 2 mm-EDTA, 0.025 ivr-Tris-HCl, pH 7.4) for 90 min at 22,000 rev./min in a Spinco model L2 ultracentrifuge (SW25*1 rotor, 4°C). Figure 2 shows the distribution of radioactivity from [3H]thymidine, the content of DNA and protein, and
I I O 0
I
I
I
I
I
I
IO
20
30
40
50
60
Time (min) FIG. 1. Incubation of isolated L-cell mitochondria in the presence sucrose, 0.005 M-Mgcl,, 0.025 M-Tris-HCl, pH 7.3, at 2% (-O--O-,
fragments
added to one preparation
of mitochondria
(-m-m-).
of 10 pg DNase/ml., 0.3 M--(>--a--). Nuclear
I.
0
MITOCHONDRIAL
8
DNA
16
IN
24 Fraction
513
SITU
32
40
no.
FIG. 2. Sedimentation of isolated L-cell mitochondria through a linear gradient of sucrose, as described in Materials and Methods. Fractions of 1 ml. were collected and analyzed for radiocontent of DNA (-A-A--), protein (--O--O--), activity of [3H]thymidine (-O-O--), and cytochrome c oxidase activity (rel. act./ml.) (---x--x--). Molar sucrose (-A-n-).
TABLE
1
Content of DNA, RNA and protein in purified L-cell mitochondria Type of analysis (1) mg protein/mitoohondriont (lO)$ no. of mitochondria/mg proteint (2) pg DNA/mg mitochondrial protein (14)$ (3) g DNA/mitochondrion§ mol. wt of total DNA/mitochondrion mol. wt of single circular DNA molecule (4) pg RNA/mg mitochondrial protein (3)$ (5) pg mitochondrial protein/IO6 cells isolated (6) No. of mitochondria isolated/cell // Total no. of mitochondria/cell~ (7) g DNA/cell (8) ‘;/o mitochondrial DNA in cellular DNA
Amount 8.0 (hO.7) x 10-11 1.2 (&0.2) x 10’0 1.1 (kO.2) 8.8 (h2.4) x lo-i7 53.0 x 106 10.0 x 106 38.0 (h5.5) 14.8 (b4.1) 185*60 250&70 1.3 (10.2) x IO-” 0.15
i Preparations contained negligible amounts of electron microscopically detectable nonmitochondrial structures that would significantly contribute either to protein content or to mitochondrial particle counts. $ Number of determinations. $ Calculated from values (1) and (2) (Nass, 1966). I] Estimated by converting yield of mitochondrial protein (5) into numbers of mitochondria, using values (1). 7 Estimate includes the assessment of lost mitochondria in unbroken cells (5%) and during oentrifugation (15 to 35%), as judged by the analysis of cell fractions during the isolation procedure with respect to content of whole cells, electron microscopic composition and content of oytochrome c oxidase activity.
5’4
Xl. 11. I<. NAS‘iL L
the relative activity of cytochrome c oxidase after sedimentation of mitochondria in the sucrose gradient. Electron microscopical analysis of the middle zone showed mostly contracted and some slightly swollen mitochondria. The small ba,nd at the top of the gradient consisted of lysosome-like particles and membrane fragments. The small pe‘llet at the bottom contained traces of mitochondria and nuclear fragments. Table 1 s~nnmarizes the yield of mitochondria and the content of DNA, RNA and protein in the purified organelles. Mitochondria from Ehrlich ascites tumor cells and adult rat and chicken liver were prepared by the same procedure, except that liver mitochondria were homogenized in isotonic sucrose. (b) Chemical
analyses
DNA was measured by a microfluorometric method as previously described (Nass, 1966). RNA was analyzed by the orcinol reaction, and protein was determined by the Lowry method. Cytochrome c oxidase was measured as described by Cooperstein & Lazarow (1951); the activity was expressed relative to the absorbance of reduced and completely oxidized cytochrome c. Tritium-labeled mitochondria were obtained from cells incubat,ed for 17 hr with 1 PC [3H]thymidine/ml. (10 c/m-mole, Schwartz Bioresearch). Samples of mitochondria were extracted twice with cold 5% trichloroacetic acid, ethyl alcohol and ether and the pellets were suspended in 0.7 ml. NCS reagent (Nuclear Chicago) and 10 ml. toluene containing 4 g POP and 50 mg POPOP/l. The radioactivity was determined in a Packard Tricarb liquid-scintillation counter. Microscopic counting of isolated mitochondria was performed as reported previously (Nass, 1966).
(c) Electron microscopy L-cells were fixed, embedded and stained as previously described (Nass et ul., 1965). Serial sections were cut on a Porter-Blum ultramicrotome MT-2 and picked up on carboncoated Formvar films supported by copper grids with a large central opening. The spreading of DNA by the osmotic-shock method, shadowing, electron microscopy and measurement of the contour lengths of DNA molecules were performed as described previously (Nass, 1966).
3. Results (a) Serial sections of DNA
centers in mitochondria
of intact cells
Chemical and electron microscopical evidence, suggesting the presence of several DNA molecules per mitochondrion (Nass, 1966), raises the question whether DNA molecules are concentrated in one area of the mitochondrion or are distributed in several areas. Although the DNA of mitochondria from many cell types may be visualized by electron microscopy as fibrils of 20 A diameter in areas of low matrix density (Nass & Nass, 1963a,b; Nass et al., 1965), DNA was not observed in all electron-lucid areas of the mitochondrion in a given tissue section. Serial sections of mitochondria were therefore cut to assess the number of DNA centers in intact mitochondria
(Plate
I).
The
visibility
of the
mitochondrial
DNA
was
enhanced
by
omitting post-fixation of cells with uranyl acetate, thus preserving the DNA in a clumped rather than fine fibrillar state (see Nass & Nass, 1963aJ for details of the structure of DNA in ultrathin sections). The mitochondrion shown in the first series of serial sections contains in its matrix at least three spatially discrete DNA centers or nucleoids, and the two mitochondria in the second series have at least two and three DNA centers. Some highly filamentous organelles with four to six DNA centers were occasionally observed.
I.
(b) Structure
of circular
MITOCHONDRIAL
DNA
DNA
IN
52.j
SITU
monomers and dimers released by osmotic shock from isolated mitochondria
Mitochondris subjected to osmotic shock (Nass, 1966) released individual circular monomers and unresolved aggregates of DNA with a total mass equivalent to multiples of DNA monomers. To resolve these aggregates into individual units, larger have now been exnumbers of mitochondria (2000) and more dilute preparations amined. Mitochondria were homogenized in 4 M-ammonium acetate prior to spreading. It was ascertained by phase-contrast microscopy that aggregates of mitochondria were dispersed into single particles. Once a mitochondrion was ruptured, there was little evidence that DNA molecules remained inside undetected; judging from the low percentage of partially extruded molecules present, breaks in the mitochondrial membrane allowed the molecules to emerge at least partially. A clear area with a radius of at least 50 TV surrounding the structures to be described was considered sufficient to reduce strongly or eliminate t’he contribution of DNA molecules by other mitochondria. In mitochondria of L-cells and ascites tumor cells, circular DNA monomers (5.21 f. made up 80 to 90% of the extruded DNA (Plates II and 0.2 p in circumference) III(b)). Typically, the molecules were folded in half; both halves were coiled around each other or remained closely associated after unwinding (Plates II(b), III and VI(2)). Most coiled molecules contained up t’o 25 cross-overs ; 1% had up to 35 crossovers, similar to purified covalently closed DNA (Nass, 19698). The polar regions of these flattened circles appeared to be attached to membrane fragments. Approximately 10% free-lying molecules were observed. As compared with mitochondria of L-cells, mitochondria of rat and chicken liver appeared to release more free-lying molecules and more coiled molecules with 30 to 35 crossovers. Strikingly, 2 to 4% of the DNA seen in osmotically lysed L-cell mitochondria consisted of double-size circular molecules (Plate IV(a) and (b)). The contour length of eleven dimers was 10.1*0+4 p, corresponding to a molecular weight of 19.6 x lo6 daltons. This form of dimer was not apparent in mit’ochondria of other cell types. A dimer was sometimes attached to another dimer or two monomers by a common knob-like central point (Plate V). It is possible that the two monomers shown are actually cont,inuous at the central point to form another dimer. The fact that these DNA molecules are connected at a cent,ral point suggests that t’hey are derived from one mitochondrial nucleoid. Most mitochondria released two DNA molecules (Plates II and III). Higher aggregates of DNA associated with a ruptured mitochondrion could be partially or completely resolved into mixtures of monomers and dimers totalling up to six t’imes the unit size of 52 p (Plates VI and VII). (c) Relationship
of DNA
to the mitochondrial
membrane
Experiments were performed to see whet,her the apparent attachment of polar regions of many circular DNA molecules to the mitochondrial membrane may reflect the occurrence of attachments in viva or whether artifacts could arise due to nonspecific adsorption. First, purified circular DNA was mixed with mitochondrial suspensions and spread by the osmotic-shock method. Interfering endogenous DNA could be reduced to about 3% by self-digestion of the mitochondria for 20 minutes at 37°C prior to the addition of exogenous DNA. As shown in Table 2, 83% of the added
526
M. M. K.
NASS
TABLE 2 Relation
of surface-spread
Preparation?
Mitochondria ( +endogenous DNA), control preparation Mitochondria (+ endogenous DNA) + circular DNA added Mitochondria (pre-incubated, 37’C, 30 min) + circular DNA added5
DNA
to mitochondrial
(a) lying free
membranes
o/o DNA molecules :$ (b) membrane(c) membraneassociated associated at poles at random
7
14
12
44
49
I
83
10
7
acetate (pH 7.0) and 0.01 o/0 cytochrome c in 7 0.05 mg mitochondrial protein, 4 M-&%IIXnOniUXII 60 ~1. In experimental preparations, 0.07 pg purified circular DNA added (DNA isolated on CsClethidium bromide gradients and freed of the dye (Nass, 1969b)). $ 60 to 80 molecules counted in each preparation. 5 No DNA was seen in pre-incubated mitochondria without circular DNA added, apparently due to digestion by endogenous DNase.
DNA was not associated with membrane, whereas the reverse was true for endogenous DNA of control mitochondria, and an intermediate situation existed when the endogenous DNA was not removed. L-cell mitochondria were also incubated with 100 pg of either ribonuclease A, pronase, phospholipase C or phospholipase D (Calbiochem) per ml. for 30 minutes at room temperature prior to spreading. It was ascertained that the enzymes at least partially penetrated and were active (though not maximally) under the conditions used by chemically analyzing corresponding samples for RNA, protein content and dry weight after extraction with lipid solvents. The only obvious change was seen after treatment with pronase. The membrane fragments were somewhat smaller than in control preparations and points of association of polar regions of DNA with membrane fragments were rarely seen. These experiments reduce, but do not eliminate, the possibility of artifacts that may result from non-specific adsorption or unknown forces during osmotic shock and formation of the protein film.
4. Discussion The results described in this paper indicate that L-cell mitochondria contain mixtures of circular DNA monomers and dimers which are located in spatially distinct regions of the mitochondrial matrix. The presence of dimers in osmotically shocked mitochondria implies that these forms exist in the organelle in vivo. In previous studies dimers had been observed only in mitochondrial DNA isolated by centrifugation in cesium chloride gradients (see Introduction). No direct methods are presently available to analyze the structural arrangement of DNA molecules in an organelle. Each experimental approach has distinct limitations, and, taken by itself, would be insufficient to demonstrate unequivocally the multiplicity of DNA molecules in a mitochondrion. For this reason, the conclusions have been based on congruent results from several lines of investigation. First, the serial sections show that variable numbers of DNA-containing regions, usually two to three, are present per mitochondrion. They do not indicate how many DNA
243)
(W
(b)
(c) (4
(d)
(b)
(4
(4
(4
(b)
......_._____________....... -..-.
1. MITOCHONDRIAL
DNA
IL\: SITU
,527
molecules each region contains, nor whether any DNA exists between the regions. The latter possibility is unlikely, however, because the localization of DNA in electronlucid regions is typical for many biological systems, especially bacteria (cf. Nass et al., 1963u). Second, the joint attachment of some circular DNA molecules released by osmotic shock (Plate V) implies that more than one DNA molecule may be located in one nucleoid. If a mitochondrion possessestwo nucleoids, two to eight molecules may be included. In cases where liberated DNA molecules overlap (Plates VI and VII), it is obviously not possible to determine how many intramitochondrial nucleoids they represent. The high degree of dispersion and dilution of mitochondria suggests that the cluster of DNA represents the combined content of one mitochondrion (although it cannot be eliminated that some molecules may float off before they are adsorbed to the protein film). Thirdly, the results are compatible with the chemically determined DNA content per mitochondrion (Nass, 1966) ; 53 x lo8 daltons of DNA would allow for an average number of five DNA molecules of 10 x IO6 daltons per organelle. Whether the lack of supercoiling of many osmotically liberated DNA molecules reflects the structure in vivo is not known. The absence or presence of tertiary turns may be a function of the ionic and thermal environment in vivo (Wang, Baumgarten & Olivera, 1967) or upon spreading. Lack of supercoiling, noted especially in the fast-growing cells, may also be due to the introduction of single-strand scissions by nucleases prior to spreading or by an endonuclease that is preferentially liberated during osmotic shock. Lack of supercoiling may also be related to DNA replication or other functions (cf. Nass, 1969a). The apparent association of DNA with the membrane, observed also in ultrathin sections of mitochondria (cf. Nass et al., 1965) and of Bacillus subtilis (Ryter & Jacob, 1963), may form a physical basis for some of these functions. However, a clear definition of such connections will have to await analyses by other techniques. The biological significance of double-size circular DNA is not yet known. It has been suggested (Rush, Kleinschmidt, Hellmann & Warner, 1967) that t#he multiplelengt’h circular DNA molecules observed in preparations of the replicative form of +X174 represent intermediate forms in DNA replication or recombination. Although there is considerable evidence in support of mitochondrial division and DNA replication (cf. review by Nass, 1969a), it is premature to speculate about the nature of the DNA replication mechanism in mitochondria. It is conceivable, however, that the number and structural arrangement of the DNA-containing regions in filamentous mitochondria may represent products of duplication of pre-existing DNA cent,ers during growth and elongation of the organelles. Multiplicity and variation in number of DNA molecules per mitochondrion may imply redundancy of the informational content of a single DNA4 molecule. This investigation was supported by U.S. Public Health Service grant POl-A107005. I acknowledge the excellent technical assistance of Miss Anneke Theunissen in the biochemical work and Mr John R. W. Hobbs in the electron microscopic and photographic work. REFERENCES Rorst,
P., Kroon, A. M. & Ruttenberg, G. J. C. M. (1967). In Genetic Elements, Properties alad Function, p. 81. London & New York: Academic Press. Warsaw: Pn’N. v&n Hruggen, IS. F. J., Rorst, P., Rut)tenberg. G. .J. C. M., Gruber, M. & Kroon, A.M. (1966). Rioch,irn. hiophys. rlctm, 119, 437. 33
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n1. ti.
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Clayton, D. A. & Vinograd, J. (1967). Nature, 216, 652. Cooperstein, S. J. & Lazarow, A. (1951). J. Biol. Chem. 189, 665. Hudson, B. & Vinograd, J. (1967). Nature, 216, 647. Nass, M. M. K. (1966). Proc. Nut. Acad. Sci., Wash. 56, 1215. Nass, M. M. K. (1967). In Organizational Biosynthesis, ed. by H. J. Vogel, ,J. 0. Lampen and 1’. Bryson, p. 503. New York: Academic Press. Nass, M. M. K. (1969a). Science, in the press. Nass, M. M. K. (1969b). J. Mol. Biol. 42, 529. Nass, M. M. K. & Nass, S. (1962). Exp. Cell Ree. 26, 424. Nass, M. M. K. & Nass, S. (1963a,). J. Cell Biol. 19, 593. Nass, M. M. K., Nass, S. & Afzelius, B. A. (1965). Exp. Cell Res. 37, 516. Nass, S. & Nass, M. M. K. (1963b). J. Cell BioZ. 19, 613. Pik6, L., Blair, D. G., Tyler, A. & Vinograd, J. (1968). Proc. Nat. Acad. Sci., Wash. 59, 838. Radloff, R., Bauer, W. & Vinograd, J. (1967). Proc. Nat. Acad. Sci., Wash. 57, 1514. Rush, M. G., Kleinschmidt, A. T., Hellmann, W. & Warner, R. C. (1967). Proc. Nut. A end. Sk., Wash. 58, 1676. Ryter, A. & Jacob, F. (1963). G. R. Acad. Sci. Paris, 257, 3060. Sinclair, J. H. & Stevens, R. J. (1966). Proc. Nat. Acad. Sci., Wash. 56, 508. Wang, J. C.. Baumgarten, D. & Olivera, n. M. (1967). Proc. Nat. Acud. Sci., Wash. 58, 1852.