Expression of the mitochondrial genome in HeLa cells

J. Mol. Biol. (1971) 55,251-270

Expression of the Mitochondrial II. Evidence for Complete Transcription

Genome in HeLa Cells of Mitochondrial

DNA

YOSEF ALONI AND GIUSEPPE ATJXIZDI

Division of Biolog?l, California Institute of Tehobgy Pamdena, Calif. 91109, U.&A. (Received26 May 1970, and in revised form 27 Aup.& 1970) The fraction of mitochondrial DNA which is complementary to mitochondriaassociated RNA in exponentially growing HeLe, cells has been investigated by RNA-DNA hybridization experiments using mitochondrial DNA strands separated by alkaline CsCl density-gradient centrifugcttion and RNA from cells uniformly labeled with [5-3H]uridine. It has been found that mitochondrial RNA hybridizes almost exclusively with the heavy strand, in agreement with the complementarity of their base compositions. Less than 2% of the light strand, after puriiioation by two cycles of alkaline C&l gradient centrifugation, was found to form hybrids with mitochondrial RNA. In hybridization saturation experiments, the RNA sedimenting slower than 22 s or faster than 30 s gave about 100% saturation level, whereas the components in the intermediate range of sedimentation coefficients gave about 86% saturation level. An analysis in CsaS04 gradients of the density of the RNA-DNA hybrids formed at saturation showed that the hybrids formed with the RNA of less than 22 s and greater than 30 s banded at a density of I.491 g/cm3, i.e. the density expected for fully base-paired hybrids; whereas the hybrids formed with the components of intermediate sedimentation coefficients banded at a slightly lower density (I.485 g/cm3), in agreement with the lower saturation level obtained with these components. An analysis by electron microscopy of the hybrids sepamted in a Cs&lO~ density-gmdient showed that essentially all the material from the 1.491 g/cm3 band. consists of nucleic acid duplexes. In view of the available evidence for the absence of major internal repetitions in mammalian mitochondrial DNA, the results obtained by the three approaches described here indicate that the whole or a;lmost whole mitochondrial genome is transcribed in HeLa cells. The possibility that this transcription occurs in the form of a continuous RNA ohain is discussed.

1. Introduction The occurrence of both heterogeneous and discrete RNA components in mitochondria from animal cells has recently been shown in several l&or&oriea (Attardi & Attardi, 1967,1969; Attardi et ccl., 1969b; Dubin, 1967; Dubin & Montenecourt, 1970; Vesoo BE Penman, 1969 ; Zylber & Penman, 1969 ; Dawid, 1969a). Evidence haa been presented for the synthesis of these components on a mit-DNAt template (Attardi & Attardi, 1968,1969; David, 1969b; Naas & Buck, 1969,197O). In HeLa oells, the occurrence in pulse-labeled RNA associated with mitochondris of fast sedimenting components (up t Abbreviations

used: mit-DNA,

mitochondrial DNA; dodecyl SO1, sodium dodecyl sulf..te. 261

252

Y.

ALONI

AND

G. ATTARDI

to 60 s and more) which are homologous to n&-DNA and the kinetics of labeling of different size molecules suggest the possibility that mit-DNA is transcribed in the form of long RNA chains, possibly as long aa the whole mit-DNA molecule, destined to be processed to molecules of smaller size. In order to obtain information on the fraction of m&DNA being transcribed in exponentially growing HeLa cells, RNA-DNA hybridization experiments have been carried out between separated strands of m&DNA and mitochondria-associated RNA from cells uniformly labeled with [S3H]uridine. In view of the absence of major internal repetitions in mammalian n&DNA (Borst, 1969), the fraction of this DNA hybridizable at saturation with mitochondrial RNA was expected to reflect the extent of i?z vivo transcription. It has been found that mitochondrial RNA is synthesized in HeLa cells almost exclusively on the heavy strand of n&-DNA and, moreover, that all or almost all the sequences of this strand are represented in mitochondria-associated RNA.

2. Materials and Methods (a) Cells and method of growth The method of growth of HeLa cells in suspension (53 clonal strain) has already been described (Amaldi & Attardi, 1968). The cultures were free of any detectable contamination by pleuropneumonia-like organism (Mycoplasma). (b) Buffers The buffer designations are as follows. (1) T: 0.01 M-Tri8 buffer (pH 6.7, 25°C) ; (2) TrisK-EDTA: 0.05 M-Tris buffer (pH 6*7), 0.025 mKC1, 0.001 M-EDTA; (3) low ionic strength Trk-K-EDTA: 0.01 M-Tris buffer (pH 6.7), 0.01 M-KCl, 0.001 M-EDTA; (4) dodecyl SO4 buffer (Gilbert, 1963): 0.01 M-Tris buffer (pH 7*0), 0.1 M-NaCl, 0.001 M-EDTA, 0.5% dodecyl SO,; (5) acetate-NaCl buffer: 0.01 M-acetate buffer (pH 5-O), 0.1 M-NaCI; (6) SSC (standard saline citrate): 0.15 M-NaCI, 0.015 M-sodium citrate; (7) Tris-K-Mg: 0.05 M-Tris buffer (pH 6.7), 0.025 M-KCl, 0.0025 M-MgCI,. (c) Labeling conditions Long-term labeling of RNA was carried out by growing HeLa cells (initial concentration lo5 cells/ml.) for 46 hr in modified Eagle’8 medium (Levintow & Darnell, 1960) with 5% dialyzed calf serum in the presence of [5JH]uridine (22.8 mc/pmole) according to the following schedule, designed to label to the same average specific activity the stable and unstable RNA specie8 : 1.25 pc of the labeled precursor/ml. was added to the medium at the beginning of the incubation, 1.25 rc/ml. after 25 hr of growth and 0.87 PC/ml. after 35.5 hr of growth; In order to estimate the amount of [3H]uridine incorporated into the cells, at various intervals 2-ml. portions of the cell suspension were removed, the cells centrifuged at 900 g,, for 5 min, and 5-~1. samples of the supernatant were plated in duplicate on nitrocellulose membranes and counted in the scintillation spectrometer. Mitochondrial DNA was labeled by growing HeLa cells for 60 hr in the presence of [2-14C]thymidine (53 pc/~mole, 0.05 PC/ml.). (d) Preparakn of subcellular fractions, extraction and pur&a.tion of RNA The preparation by differential centrifugation of the mitochondrial fraction from 3.1 x log cells long-term labeled with [5-3H]uridine and the extraction of the RNA wa carried out as described previously (Attardi & Attardi, 1971). The tial RNA preparation was run through a 5 to 20% sucrose gradient. in acetate-N&l buffer in the Spinco SW25.2 rotor under the conditions specified below. The analysis of radioactivity and the isotopecounting procedure have been described elsewhere (Attardi, Pernas, Hwang & Attardi, 1966). In order to purify the RNA fractions to be used in the hybridization experiments from any DNA contaminant, they were precipitated with 2 vol. of ethanol, redissolved in 3 ml. Tris-K-Mg and incubated with electrophoretically purified DNase (Worthington) (100 pg /ml.) for 60 min at room temperature. (Control experiments showed that digestion under

TRANSCRIPTION

OF MITOCHONDRIAL

DNA

268

these conditions with 20 s DNase rendered 10 ccg[“C]DNA acid-soluble up to 679&) The RNA W&Bthen extracted with an equal volume of phenol in the cold in the presence of O+% dodecyl S04, precipitated with 2 vol. of ethanol, redissolved in 2 ml. of 2 x SSC and run through a Sephadex Cl00 column (0.9 cm x 55 cm equilibrated with 2 x SSC) at room temperature. For de&mm&ion of the specific activity of the final RNA preparations, a portion was plated directly on a nitrocellulose membrane of the same type used in the RNA-DNA hybridization experiments (see section (f)), and counted in the scintillation counter. The concentrations of RNA solutions-were determined from 0.D.2eo measurements by using a value of 214 for E:Trn0 at 260 nm in O-1 M-N&I (Hall & Doty, 1969). (e) Iaoldion

of W-i&&d

closed cir&r mitochondrial

mitochondti DNA &an&

DNA

and separation of the

The closed circular mit-DNA wae isolated from HeLa cells labeled for 60 hr with [2-14C]thymidine as deecribed by Hudson & Vinogmd (1907). The specific activity of the preparation wsa determined by measuring its optical density at 260 nm (by using the equiunit, derived from a value of E(P), molar extinction valence 48 pg/ml. = 1.0 o.D.~~~ coefficient with respect to phosphorus, of 6500, as reported for native calf thymus DNA at 22°C in O*Ol M-N&I (Shack, 1968)), and by counting in the scintillation counter a portion directly plated on a nitrocellulose membrane. The specific activity thus obtained was 8020 cts/min/r*g. For the separation of the complementary strands of HeLa mit-DNA in an alkaline UsCl gradient, a solution containing closed circular mit-DNA in 4.0 ml. 0.065 M-K~PO~0.01 y’ dodecyl SO4 was brought to a refraotive index (n,a60c) of about 1.405 with solid CsCl and to pH 12.4 with KOH. The mixture was centrifuged in a Polyallomer tube in the Spinco 66 angle rotor at 42,000 rev./min for 42 hr at 20°C. Eleven-drop fractions were collected from the bottom of the tube and aesayed for radioactivity. The fractions corresponding to the peaks of the heavy and light strands were separately pooled and brought to pH 8.0 with 1 M-Tris buffer. The specific activities of the heavy and light strands were determined ae explained above, by using a correction fsator of 40% for hyperchromicity (Nass, 1969). The specific activity of the heavy strand thus obtained was 9380 cts/min/pg and that of the light strand 7300 cts/min/pg. Mitochondrial DNA from unlabeled cells wss purif%d and fractionated into light and heavy ‘strands in the same way as described above. Methylated albumin-kieselguhr purified SV40 DNA (component I) wss prepared as described elsewhere (Aloni, Winocour, Sachs & Torten, 1969).

(i) Hybridization

experhents

(f) RNA-DNA hybridizdion ?&&zing DNA &nmobik?d on nitroce&dose memb?wnea

The technique used wss similar to that described by Gillespie & Spiegelman (1966). Samples of single-stranded DNA (0.0125 to 0.025 M) in 6 to 10 ~1. CsCl-Tris (pH 8.0) were diluted to 2 ml. with 6 x SSC and loaded under low suction onto nitrocellulose membranes (Bat-T-Flex type B6, Schleicher & Schuell), which had been soaked in 6 x SSC for 10 min and washed with 25 ml. of the same buffer. The DNA-carrying filters were then washed with 50 ml. of 6 x SSC, dried at room temperature overnight and then in an oven at 80°C for 3 hr. 90 to 100% of the 14C-lebeled heavy or light mit-DNA strands deposited on the filters wae retained, and not more than 10% of the DNA initially retained was lost in the subsequent incubation and washings. The filters were incubated in scintillation vials with various concentrations of [3H]RNA in 2.0 ml. of 4 x SSC containing 0.01 r+Tris buffer, pH 7.8, at 68°C for 24 hr. (In preliminary experiments these conditions were found to give the highest efficiency of hybridization and the lowest loss of RNA-DNA hybrids during the incubation and washing steps.) After incubation, the titers were removed from the vials, washed by suction filtration on each side with 100 ml. of 2 x SSC, and incubated with 6 ml. of 2 x SSC containing 20 ~/ml. of pancreatic RNsee (pre-heated at 8O“C for 10 min) for 1 hr at room temper&ire. The filters were then rewashed on each side with 100 ml. of 2 x SSC, and assayed for radioactivity in a scintillation spectrometer.

Y. ALONI

2.54

AND

G. ATTARDI

(ii) Hybridizdm experimeata in liqwid medium ad a&y& of RNA-DNA hybrid by ceeium &fate density-gradient centtifugat&3n For the preparation of RNA-DNA hybrids to be anaLyzed in CszSOc density-gradients, samples of mit-DNA heavy strands (0.08 to 0.2 pg) were incubated with saturating amounts of [3H]RNA in 0.5 to I-5 ml. of 4 x SSC containing 0.1 m-Tris buffer, pH 8.0, at 70°C for 5 hr. After incubation, the mixtures were brought to room temperature and diluted with an equal volume of water, and the pH was then brought to 7.2 with 1 M-Tris buffer, pH 6.7. Pancreatic RNase (200 r*glml.) and T, RNase (100 units/ml.) were added, and the mixtures incubated for 1 hr at room temperature. After the enzymic digestion, each sample was run through a 0.9 cm x 55 cm Sephadex GlOO column equilibrated with 2 x SSC at room temperature. 1-ml. fractions were collected and assayed for radioactivity. The fractions of the peak corresponding to RNA-DNA hybrids were pooled, and incubated with pancreatic RNase (5 pg/ml.) and T, RNase (5 units/ml.) for 45 min at room temperature. Dodecyl SO1 was then added to 0.5%, and the mixtures were extracted with an equal volume of phenol in the cold. Traces of phenol were removed from the aqueous phase by ether, and the ether was then evaporated by bubbling nitrogen through the solution. The samples were then dialyzed for 12 hr at 4°C against 2 x SSC. The final recovery of the i*C-labeled mit-DNA heavy strand used in the hybridization mixture was about 80%. The samples, in 2 x SSC, were adjusted to 1.530 g/ cm3 with Cs,SO, in a total volume of 5.0 ml. and centrifuged at 32,000 rev./min for 5 days in a Spinco SW39 rotor at 20°C using nitrocellulose tubes. A serious problem in these experiments was found to be the almost complete loss of the small amounts of RNA-DNA hybrids (and likewise that of mit-DNA heavy-strand or of mitochondria-associated RNA in the control experiments) during the Cs,S04 densitygradient centrifugation, presumably due to adsorption to the walls of the nitrocellulose or Polyallomer tubes. It was found that addition to the Cs,S04 solution of sonicated and denatured DNA (HeLa DNA sonicated in a Branson sonifier (model S-125) at 20 kc./sec for 90 see was used for this purpose) at a concentration of 1 rg/ml. allowed a recovery of about 40% of the sample analyzed. The recovery was further increased to about 65% by presoaking the nitrocellulose tubes (previously washed for 24 hr in 5 x 10V3 M-EDTA containing 1% dodecyl SO,) in a solution of 25 pg denatured HeLa DNA/ml. of 6 x SSC for 24 hr at room temperature, and then drying them at 37°C for 12 hr. After centrifugation, 50 fractions were collected from the bottom of each gradient into glass tubes which were immediately stoppered and kept at 2°C for the measurement of the refractive index. The density gradient was determined from the refractive index measurements by using an experimentally established relationship between refractive indexes of CszSOI solutions in 2 x SSC and their densities, as measured by weighing 25-~1. samples in calibrated micropipettes. In the case of analysis of RNA-DNA hybrids, individual fractions of the Cs2S04 density-gradient or portions of them were diluted with 1 ml. of 6 x SSC and filtered slowly through nitrocellulose membranes (pre-soaked in 6 x SSC for 10 mm) ; the filters were then washed with 50 ml. of 2 x SSC, dried, and counted in the scintillation spectrometer. (It was found in preliminary experiments that this procedure allowed the removal of acid-precipitable RNA fragments spread throughout the gradient, without affecting the recovery of RNA-DNA hybrids.) In the case of analysis of either mit-DNA, heavy-strand or mitochondria-associated RNA, the fractions of the gradient were precipitated with 10% trichloroacetic acid and the precipitates collected on Millipore membranes.

3. Results (a) Preparation and fractionation of RNA

of unifomz specific activity

(i) Labeling conditions In order to label HeLa cells in such a way that both the metabolically unstable and stable RNA species present in the mitochondrial fraction had a similar specific activity, the level of r3H]uridine in the medium was adjusted at various intervals over a period of 46 hours. On the basis of preliminary experiments, the schedule indicated in

TRANSCRIPTION

OF MITOCHONDRIAL

256

DNA

TABLET Uniform labeling of RNA from the n&!odmdd [W’H]Uridine added per 800 ml.

Inoubation time w

Cts/min/ml. in the medium (x10-6)

Cts/min/ml. incorporated (x10-5)

frmtion of He&z cella

IllCreaSe

in no. of Odl+l.

Aversge Ct+ill

incorporated per ce11t

1 mc

0 24

6.4 2.0

4.4

1V

2.2

1 mc

26 34.5

8.4 4.0

4.4

105

2.9

@7 mc

36.6 46

8-O 4.8

3.2

106

3.0

[$aH]Uridine (22.8 mc/Fole) wan added to a HeLe cell euspeneion ( 10s cells/ml.) in modified Eagle’s medium with 6% oalf eerum in the amounts and at the times indieatad. The amount of radioactive material incorporated into the cells at various times was estimated from the radioactivity diaappewing from the medium, determined aa detailed in Materials and Methods, seotion (0). t The lo6 cells/ml. present at time 0 are inoluded in the caloulation.

Table 1 was adopted. As one can see from the Table, the average cts/min incorpomted per cell during the total incubation period (3.0) (i.e. the total number of cts/min incorporated by the cell suspension divided by the total number of cells) is about equal to that incorporated during the last IO-5 hr incubation (3.2). From the amount of rtulioactive material in the supernatant fluid at the end of incubation, it can be expected that even fast turning-over RNA species would have a specific activity similar to the average specific activity of the stable RNA species, in particular of rRNA. (In fact, the average concentration of [3H]uridine in the medium during the 48-hr growth period corresponded to 56 x IO6 cts/min/ml., which for the purpose of estimating the specific activity of stable RNA species should be decreased by 25% to take into account the initial cell concentration.) (ii) Fractionation gradient

of the unifor&y

labelso?RNA by sedimentation velocity in 8ucro8e

In order to verify the assumption, implicit in the results of the previous section, that the fast turning-over non-ribosomal RNA components present in the mitochondrial fraction, under the labeling conditions used in the present experiments, had the same average specific activity as the stable rRNA species, the extracted RNA was fractionated by repeated sucrose-gradient centrifugations. Figure l(a) shows the sedimentation profle of the RNA extracted from the EDTAtreated mitochondrial fraction. One recognizes a prominent 28 s peak, pertaining to the EDTA-resistant membrane-stuck 50 s subunits of the rough endoplasmic reticulum (Attardi et al., 1969u), and a smaller broad peak in the 18 s region, containing the discrete RNA species discussed in the preceding paper (Attardi & Attardi, 1971). The sedimentation cattern was divided into three cuts, as indicated by arrows, corresponding to approximate S values greater than 40, between 22 and 40 and less than 22 S. The RNA from the cut corresponding to S values greater than 40 was precipit&ed with ethanol, centrifuged down, dissolved in ctcetate-N&l buffer and run through

266

Y. ALONI

AND

U. ATTARDI

4

6-O-

3

4.0 -

2 b\ -b

dd

2,0dd % % 0 cii d

IO (cl

I 20 I

i0

40

I.0 1

0

I

f-------3

c,28s

4

* 2 \E

v)

0

IO

20 Fraction

30

40

no.

Fm. 1. Sedimentation profile of RNA extracted from the EDTA-treated mitochondrial fraction of HeLa cells labeled for 46 hr with [5-sH]uridine. (a) RNA was extracted with dodecyl SO,-phenol from the EDTA-treated mitochondrial fraction of 3.1 x 10s HeLa cells labeled with [S-sH]uridine under the conditions described in Materials and Methods, section (0). The labeled RNA was run through a 5 to 20% (w/w) sucrose gradient in acetate-NaCl buffer over a cushion of 6 ml. 64% sucrose in the same buffer in the Spinco SW25.2 rotor at 21,000 rev./min for 10.5 hr at 3°C. The sedimentation profile was divided, as indicated by arrows, into three cuts corresponding to8 values greater than 40, between 22 and 40 and less than 22 S, and the material present in each out was collected by ethanol precipitation and centrifugation. --O--O--, 0.D.2Bo nm. (b) The components in the profile of (a) corresponding to S values greater than 40s were re-run through a 5 to 20% sucrose gradient in acetate-NaCl buffer in the Spinco SW25.2 rotor at 19,000 rev./min for 10.5 hr at 3°C. --O--O--, O.D.Zeo nm; --m-e-, sH radioactivity. (c) The components corresponding tosvalues greater than 30 8 in pattern (b) (indicated by arrows) wem collected by ethanol precipitation and centrifugation, redissolved in acetate-NaCl buffer and, after addition of 30 pg of i4C-labeled 28 s RNA from large ribosomal subunits of free polysomes, passed through a third 5 to 20% sucrose gradient in acetate-NaCl buffer in the Spinco SW25.2 rotor at 19,000 rev./min for 6 hr 15 min at 3°C. The insert shows the 3H radioactivity profile pertaining to non-ribosomal RNA components, determined by subtracting from the total profile the contribution due to 28 s RNA, as reconstructed from the r*C-profile. -- 0 -- 0 --, 0.D.260 ,,,,, ; - e-e-, aH radioactivity;-a -- A --, “C radioactivity.

TRANSCRIPTION

OF MITOCHONDRIAL

DNA

267

another sucrose gradient (b). After the rerun, one can recognize a main 28 s peak with a pronounced leading edge of heavier material spread down to the bottom of the tube and some lighter material. The components sedimenting faster than 32 s (indicated by arrows) were precipitated with ethanol, redissolved in acetate-NaCl buffer and run through a third sucrose gradient together with 14C-labeled 28 8 RNA from large ribosomal subunits of free polysomes (Fig. l(c)). A comparison of the sedimentation pro6le of the 3H and 14Cradioactivity shows a considerable enrichment of 8H cts/min over 14C cts/min in the leading edge of the 28 s peak, indicating the presence of 3Hlabeled non-ribosomal RNA. By subtracting the contribution to the 3H profile of the 28 s RNA, as estimated from the ‘“C pro6le, the pattern shown in the insert of Figure l(c) is obtained. A prominent pes$ at about 33 s is apparent. This peak is skewed toward the fast-sedimenting side. The existence of this RNA component was also observed in the sedimentation profile of pulse-labeled mitochondrial RNA (Attardi et al., 19693). The sedimentation distribution shown in Figure l(c) was divided into three cuts (as indicated by the arrows) corresponding toS values less than 30, between 30 and 70 and greater than 70 s. The RNA in each cut was precipitated with ethanol and redissolved in Tris-K-l@. The RNA components from the fractions corresponding to S values between 22 and 49 and less than 22 s in the first sedimentation run (Fig. l(a)) were also precipitated with ethanol and redissolved in Tris-K-I@. Table 2 shows the TABLE

SpeciJic activities of RNA

2

fractionafrom different

reqiu~~ in the .sedi?nentuticn

POJile

suorose grsdiellt oentrifugtstion

RNA fraotion (S value)

Speo*o aativity WbW43)

“J’.aeo O.D.sso

First

<22 2240

31,000 31,000

2.13 2.11

Third

<30 30-70 >70

30,000 28,000 18,600

2.12 2.06 1432

The RNA components in the regions corresponding to S values less than 22 and between 22 and 32 s in the sedimentation profile of Fig. l(e) and in the regions oorresponding to S values less than 30, 30 to 70, and greater than 70 8 in the sedimentation pro& of Fig. l(o) were preoipitated with ethanol and dissolved in Trk-K-Mg. After DNase treatment and dodeoyl SO*--phenol extraction, the samples were run through Sephadex alO0 columns, snd their speoifio aotivitiee determined es detailed in Materials and Methods, section (d).

specific activity and the o.D.,~,, to o.D.~*~ ratio of these five RNA fractions, after DNase treatment and Sephadex chromatography (Materials and Methods, section (d)). It appears that, with the exception of the material with S values greater than 70 s (Fig. l(c)), all RNA fractions have about the same specific activity and an o.D.,,, to O.D.,,, ratio of about 2-l. The lower specific activity and the abnormally low 0.D.26,-, ratio of the material sediment&g fester than 70 s suggests that the RNA t0 O.D.,,, present in this cut is contaminated by, and presumably attached to, other components from the ED!L’A-treated mitochondrial fraction, which have survived the extraction

268

Y.

ALONI

AND

G. ATTARDI

procedure. The results shown in Table 2 indicate that the RNA components corresponding to the S range between 22 and 40 s in the first sucrose gradient centrifugation, the great majority of which was represented by rRNA, and the components sedimenting between 30 and 70 s in the third sucrose gradient centrifugation, consisting of about 50% of non-ribosomal RNA, which is fast turning-over (as shown by the observation that this RNA is very rapidly labeled in pulse experiments and does not accumulate (Attardi & Attardi, 1967 ; Attardi et al., 1969b)), had about the same specific activity. In the hybridization experiments to be described below, the components corresponding to S values less than 22 s and between 22 and 40 in the first sucrose gradient centrifugation (Fig. l(a)), and the components from the middle cut (30 to 70 s) in the third run (Fig. I(c) and insert) were used. (b) Sqnaration of the naitochundrial DNA strands In order to avoid the complications deriving from possible renaturation of n&DNA prior to fixation to the nitrocellulose filters or during the hybridization in solution, and also to obtain information concerning the strands being transcribed in HeLa cells, all the hybridization experiments described in this work were performed with separated mit-DNA strands. For this, advantage was taken of the fact that the two complementary strands of human mit-DNA can be separated in alkaline CsCl gradients (Corneo, Zardi & Polli, 1968; Borst & Ruttenberg, 1969; Clayton, Davis & Vinograd, 1970) due to their different G+ T/C+ A ratio (Vinograd, Morris, Davidson & Dove, 1963). Figure 2 shows the result of such a separation. The ratio of radioactivities associated with the two bands in this experiment was l-4. The same ratio was found in several other experiments in the present work and also by Hallberg & Vinograd (personal communication). In an experiment using unlabeled mit-DNA, the ratio of O.D.zso associated with the two bands was close to unity, suggesting that no preferential loss of the light strands occurred during the fractionation; therefore, the difference in [14C]thymidine radioactivity associated with the two strands presumably reflects solely the difference in thymine content. From a value of 1.4 for the ratio of thymine 400

/

I

I

I

I

Hey y

Fraction no.

Fm. 2. Separation of the complementary strands of i4C-labeled HeLa mit-DNA in an alkaline CsCl density-gradient. A solution containing 13 pg closed circular mit-DNA in 4.0 ml. 0.066 w-KsP04, 0.01% dodecyl SO*, was brought to a refractive index of about 1.406 with solid CsCl and to pH 12.4 with KOH. The mixture was oentrifuged in a Polyallomer tube in the Spinco 06 angle rotor at 42,000 rev./min for 42 hr at 20°C. Il-drop fractions were collected from the bottom of the tube and assayed for radioactivity.

TRANSCRIPTION

OF MITOCHONDRIAL TABLE

DNA

269

3

Bme compdtb of separatedstrands of m&DNA and of J2Pp&e-labeled mitochondk-associated RNA

Component A

Heavy strand m&DNA Light strand m&DNA =P puke,-labeled mitoohondria-esswietedRNAt +X174 DNAQ

22.2 31.8 33.9 31.4 24.7

9-26 s 26-48 8

C

U(T)

24.6 23.9 18.6

31.8 22.2 22-6 26.3 32.6

G

G+C%

18-9 19.4 24.2

46$ 46$ 43.4 43-3 42.7

The A and T contents of the heavy and light mit-DNA strands were determined from the ratio of [2-W]thymidine incorporated into the separated strands (see Results, seotion (b)). t Attardi & Attardi, 1967. $ Clayton & Vinograd, 1967. 5 Sinaheimer, 1969. in the two strands and from the G+ C content of human mit-DNA (46%, Clayton $ Vinogrsd, 1967) the proportion of A and T in the two strands can be calculated (Table 3). The specific activities determined for the separated m&DNA strands in the experiment recorded in Figure 2, calculated by assuming a hyperchromic effect of 40yo (Nass, 19691, were 7300 cts/min/~g for the light strand and 9380 cts/min/pg for the heavy strand: these values are fairly close to those expected from the specific activity of the native mit-DNA (8020 cts/min/& and from a ratio of 1.4 for the thymine content in the two strands. content

(c) The fraction of mitochondrid

DNA which is homologcn~ to mitochondriaa.awoc&edRNA

In order to obtain direct evidence as to the fraction of the m&DNA which has base sequence homology to mitochondrie-sssociated RNA in HeLa cells, RNA-DNA hybridization experiments were performed between [6-3H]uridine-labeled RNA from the EDTA-treated and isopycnically separated mitochondrial fraction, and [2-14C]thymidine-labeled separated strands of n&DNA, prepared as described in the previous sections. The fraction of mit-DNA complementary to mitochondria-assoc. i&ed RNA was determined from the maximum amount of RNA which could be specifically hybridized to n&DNA, and, independently, from the density of the RNA-DNA hybrids formed with satursting amounts of RNA and from their appesrance in the electron microscope (see Appendix). (i) Saturation experiments with separatedmitoc~rial

DNA strands

Samples of ~4C-labeled heavy or light mit-DNA strands immobilized on Alters were incubated, in three series of experiments, with increasing amounts of [3H]RNA components of different X range, as specified in Results, section (a) (ii). As appears from Figure 3, HeLa mitochondrial RNA hybridized almost exclusively with the heavy strand of n&-DNA. This result is in agreement with the observation that the base composition of the 3aPpulse-labeled mitochondria-associated heterogene-

Y. ALONI

260

AND

G. ATTARDI

Heavy strand ./-------

20 i Light strand ,--+----t---: b-

40 20

li --

-*-

Light strand ---.A----,-----j====b

+t Cc) 2oc

I---Heavy

strgind

/.~‘--+-‘-:

200

400 600 800 Input RNA/DNA

IO00

1500

FIQ. 3. Hybridization of separated strands of i4C-labeled HeLa mit-DNA with increasing amounts of RNA from EDTA-treated and isopycnicJly separated mitochondrial fraction of HeLa aells labeled for 46 hr with [5-sH]uridine. The RNA components from the EDTA-treated mitochondrial fraction corresponding to s values between 30 and 70 (Fig. l(c)), between 22 and 40 and less than 22 (Fig. l(a)) were collected by ethanol precipitation and centrifugation, subjected to DNase digestion-Sephadex chromatogmphy as desaribed in Materials and Methods, section (d), and used for hybridization tests by the Gillespie & Spiegelman (1965) procedure. Samples of 0.0125 pg (a), or 0.025 pg ((b) and (c)), of [14C]thymidine-labeled heavy or light strand of mit-DNA were immobilized on nitrocellulose membrane and inaubated with the RNA samples ((a) 30 to 70 s; (b) 22 to 40 s; (c) less than 22 s) in 2 ml. of 4 x SSC containing 0.01 rd-Tris buffer (pH 7.8) for 24 hr at 68°C. The fraction of mit-DNA strands which was involved in hybrid formation was determined from the amount of the DNA remaining on the filter and its specific activity (see text), and from the 3H cts/min bound and the specific activity of the RNA preparations used (Table 2). The data are corrected for nonspecific background (less than 10% of the cts/min hybridized with the heavy mit-DNA strand), as determined without DNA or with purified SV 40 DNA( component I) (Aloni, Winooour, Sachs & Torten, 1969). -a-@--, Heavy strand; --O----O--, light strand; -O-O--, light strand re-run.

ous RNA, which is mit-DNA coded (Attardi & Attitrdi, 1971), is complementary, as concerns the A and U content, to that of the heavy strand of mit-DNA (Table 3). The low level of hybridization observed with the light strand (about 6% of that obtained with the heavy strand) is presumably due in part to a small amount of contaminating heavy strand; in fact, this level was considerably reduced (to about 2%) by using light strand run twice through an alkaline CsCl density-gradient (Fig. 3(c)).

TRANSCRIPTION

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As shown in Figure 3, the RNA components sedimenting between 39 and 70 s (Fig. l(c)) or slower than 22 s (Fig. l(a)) saturated the heavy strand of n&DNA at a level corresponding to about 199%. The RNA components corresponding to 8 values between 22 and 46 s (Pig. I(a)) gave a slightly lower saturation value (about 86%). These results indicated that all or almost all the sequences of the heavy strand of m&DNA are represented in mitochondria-associated RNA. This conclusion was corroborated by the analysis of the density of the RNA-DNA hybrids. (ii) Buoyant den&y in cesium sulfate of RNA-DNA

hybrid8

It has been shown that complete RNA-DNA hybrids between #X174 DNA and complementary RNA synthesized in vitro have a buoyant density in Cs,SOI of 1.491 g/cm3 (Sinsheimer & Lawrence, 1964; Chamberlin & Berg, 1964). The base composition of the heavy strand of m&DNA is roughly similar to that of #X DNA, as judged from its mole percentage of A and T and from the nucleotide composition of 3aP pulselabeled heterogeneous mitochondrial RNA (Table 3). Therefore, one would expect that 1: 1 hybrids between heavy strand of mit-DNA and mitochondrial RNA would have a density in Cs,SOc of 1,491 g/cm3. Hybrids between heavy strand of m&-DNA and saturating amounts of mitochondria-associated RNA of different sedimentation range, formed in liquid medium, were isolated by RNase digestion and Sephadex chromatogmphy and analyzed in CasElO density-gradients. As a control for density measurements in these experiments, #Xl’74 DNA was run in a Cs,SO, density-gradient under the same conditions: as shown in Figure 4(f), this DNA banded at a density of 1452 g/cm3, in agreement with published data, (Sinsheimer & Lawrence, 1964). Figure 4(a) shows the density distribution of the [14C]DNA and [3H]RNA present in the hybrids formed with the RNA components sediment@ slowerthan 22 s (Fig. 1 (a)). It appears that the [14C]DNA forms a sharp band at the density of l-491 g/cm3 expected for 1 :l RNA-DNA hybrids. No [14C]DNA is found at the density of unhybridised heavy strand (l-452 g/cm 3, Fig. 4(b)) nor at intermediab densities. Furthermore, the great majority of the r3H]RNA bands in correspondence with the [14C]DNA peak. The RNA to DNA ratio in the peek was estimated from the specific activities of RNA and DNA to be about 1-O. The small amount of 3H ctslmin on the heavy side of the [14C]DNA peak (about 5%) may reflect the presence of hybrids with incompletely digested RNA tails. The small 3H peak at the density of 145 g/cm3 is probably due to trapping of [3H]RNA by the sonic&ted denatured HeLa DNA added as a carrier, which banded at approximately that position (shown only in Fig. 4(c)). The recovery after Cs,SO, density-gradient centrifugation of the [14C]DNA originally present in the hybrid mixture was about 60%. That this incomplete recovery w&s not due to lack of retention of the hybrids by the nitrocellulose membranes is indicated by control experiments, which showed a very similar recovery of RNA-DNA hybrids by trichloroacetic acid precipitation and by filtration through nitrocellulose membranes. Also the non-hybridized heavy n&-DNA strand was recovered after Cs,SO, densitygradient centrifugation in the proportion of about SO%, as determined by trichloroacetic acid precipitation. The majority of the [3H]RNA present in the hybrids formed with the fast-sedimenting components (30 to 70 s in Fig. I(c)) banded also at a density of l-491 g/cm” (Fig. 4(d)). However, about 25% of the 3H cts/min were found in the density region l-5 to l-6 g/cm3. The significanoe of this material is not clear.

Y.

262

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G. ATTARDI

The hybrid mixture using RNA components of intermediate sedimentation range (22 to 40 s in Fig. 1(a)) gave in a C&SO, density-gradient a sharp band of 14Cand 3H radioactivity at 1.485 g/cm3 (Fig. 4(e)). Figure 4( 0) shows the banding position for the input RNA (22 to 40 s). The extreme sharpness of the band suggests aggregation of RNA due to the excess of rRNA, previously described by others (Spiegelman & Doi, 1963).

I

1

I

(a)

I

I

(d)

“‘Y

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I

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- IC

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- IC 1.

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- 0413 ’ -1.6

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-I,4

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0, I0

nA4 Frcct~on no

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FIG. 4.

Q.

TRANSCRIPTION

OF MITOCHONDRIAL

DNA

263

4. Discussion Our purpose in this paper was to obtain information relevant to the question of what fraction of mit-DNA is transcribed in exponentially growing HeLa cells. Since the available evidence indicates that there are no major internal repetitions in mammalian mit-DNA (Borst, 1969), it was reasoned that measurements of the fraction of mit-DNA hybridizable at saturation with mitochondria-associated RNA would provide an estimate of the extent of in vivo transcription. Three approaches were followed in this work. The first was to determine the maximum amount of mitochondria-associated RNA which can be hybridized to a known amount of mit-DNA in hybridization-saturation experiments; the second approach used centrifugation in C&SO, density-gradients to analyze the density of RNA-DNA hybrids formed at saturation; the third approach involved analysis of RNA-DNA hybrids by electron microscopy (see Appendix). While the results of the first approach depended heavily on the accuracy of determination of the specific activity of the RNA and DNA involved in hybrid formation, the second and third approach were completely independent of these parameters. (a) Preparation of RNA of unijorm specific activity An essential requirement for experiments aimed at a quantitative analysis of RNADNA hybrids is the accurate determination of the specific activity of both the DNA and the RNA involved in hybrid formation. As concerns the RNA, this can pose a problem if the RNA preparations used consist of metabolically stable and unstable components, and if the labeled precursor is present in low molar concentration and can, therefore, be exhausted from the medium during incubation. Mitochondrial preparations from HeLa cells, as well as from other animal cells, are contaminated by elements of rough endoplasmic reticulum, in which the majority of RNA consists of stable rRNA species (Attardi et aE., 1969a). Even after EDTA treatment, extramitochondrial rRNA represents the major RNA component in crude mitochondrial preparations. It is also known that most of the mitochondrial RNA in HeLa cells has a fairly short half-life (Attardi & Attardi, 1967; Attardi et al., 1969b). In the present work, the labeling conditions were chosen in such a way that even fast turning-over RNA would have a specific activity similar to the average specific activity of stable RNA species. Furthermore, fractionation in a sucrose gradient of the labeled RNA extracted from _. FIG. 4. Analysis of RNA-DNA hybrids in Cs$O, density-gradients. In the experiment recorded in (a), 0.20 pg of 14C-labelod mit-DNA heavy strand mixed with cold mit-DNA heavy strand to give a specific activity of 3800 cts/min/pg was incubated, under the conditions described in Materials and Methods, section (f) (ii), with 200 pg of the [3H]RNA components lighter than 22 s (Fig. l(a) ), purified by DNase digestion-Sephadex chromatography as explained in Materials and Methods, section (d); in the experiment recorded in (d), 0.08 pg of unlabeled mit-DNA heavy strand was incubated with 72 pg of [3H]RNA of the 30 to 70 s sedimentation range (Fig. l(c) ) ; in the experiment shown in (e), 0.20 pg of unlabeled mit-DNA heavy strand was incubated with 360 pg of [3H]RNA from the 22 to 40 s sedimentation region (Fig. l(a) ). After pancreatic and T, RNase digestion, Sephadex chromatography and second enzymic digestion, the hybrids were isolated in a Cs,SOd density-gradient as detailed in Materials and Methods. section (f) (ii). In (b), 0.025 pg of 14C-labeled mit-DNA heavy strand (spec. act. 9380 cts/min/pg), in (c) 5 pg of [3H]RNA (22 to 40 s), and in (f) 40 pg 4X174 DNA were run in CszS04 densitygradients. Centrifugation was in all cases at 32,000 rev./min for 5 days in a Spinco SW39 rotor at 2O’C. Fractions were collected from the bottom of the tube and the refractive index was determined on selected fractions. The whole fractions ((d) and (e)) or 50-~1. samples (a) were diluted with 1 ml. of 6 x SSC and filtered through nitrocellulose membrane filters. In (b) and (c), the fractions were precipitated with 10% trichloroacetic acid as described in Materials and Methods, section (f) (ii). 18

Y.

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the EDTA-treated mitochondrial fraction showed that the non-ribosomal RNA components sedimenting faster than 30 s had about the same specific activity as the rRNA species. Since the RNA components heavier than 30 s are labeled very rapidly in pulse experiments and do not accumulate (Attardi et al., 19693), it is reasonable to think that most of this material is fast turning-over. Therefore, these observations strongly suggest that, under the labeling conditions used in the present experiments, the unstable and stable RNA species had about the same specific activity. The nature of the RNA components heavier than 30 s is at present being investigated as regards their physical properties and their significance in relation to the synthesis and processing of mitochondrial RNA. (b) DNA

saturation experiments

The results obtained in the present work indicate that HeLa mitochondrial RNA hybridizes almost exclusively with the heavy strand of mit-DNA. That the low level of hybridization observed with the light mit-DNA strand purified through two cycles of alkaline CsCl density-gradient centrifugation (about 2% of that obtained with the heavy strand) may be significant is suggested by recent experiments by Nass & Buck (1970), which show hybridization of two specific mitochondrial tRNA species to the light strand of rat liver mit-DNA. The exclusive transcription of the heavy strand of mit-DNA had been previously reported for rat liver cells (Borst & A&j, 1969). The saturation levels for the heavy mit-DNA strand obtained with the fast-sedimenting RNA components (30 to 70 S) and with the slow-sedimenting components (less than 22 s) approached lOO%, whereas only about 85% of the heavy strand appeared to be saturated with the RNA components in the intermediate range of sedimentation coefficients (22 to 40 s). The error in the determination of the specific activity of the separated mit-DNA strands or in that of the RNA was probably less than 5%, thus giving a maximum uncertainty of 10% in the saturation values observed. Evidence supporting the validity of these saturation values has been derived from an analysis of the density in Cs,SO, gradients of RNA-DNA hybrids formed at saturation, as discussed below. (c) Analysis

of RNA-DNA

hybrids in cesium sulfate density-gradients

The alkali treatment used for the separation of mit-DNA strands and the thermal treatment during the hybridization reaction were found to break the mit-DNA strands to sizes considerably smaller than the original one (see Appendix). Therefore, it was anticipated that an analysis of the hybridization mixtures, after RNase digestion and Sephadex chromatography, in Cs,SO, density-gradients would provide information on the fraction of the heavy strand of mit-DNA complementary to mitochondria-associated RNA, without any indication of the distribution of the complementary regions in the intact molecules. In the present experiments, all the mit-DNA (heavy strand) which was annealed with saturating amounts of mitochondria-associated RNA components with S values less than 22 and between 30 and 70 s was recovered in the form of a sharp band at the density of 1.491 g/cm3, corresponding to a [3H]RNA peak. The RNA to DNA ratio in the peak was estimated from the specific activities of RNA and DNA to be about 1.0. Electron microscopy of samples from this peak con6rmed the duplex nature of this material (Appendix). No mit-DNA was found in the density position of non-hybridized DNA or at intermediate densities. Although only about 60% of the mit-DNA originally

TRANSCRIYTION

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present was recovered after Cs,SOc density-gradient centrifugation, this was not due to preferential losses of non-hybridized or partially hybridized mit-DNA, since a similar recovery was obtained with samples of non-hybridized mit-DNA heavy strand. The retention of RNA-DNA hybrids which were fully or almost fully basepaired (as judged from the density) on nitrocellulose membranes was surprising, since it is known that double-stranded DNA segments are not retained (Nygaard & Hall, 1963) : it is likely that the retention is due to small imperfections in these hybrids, like short non-base-paired regions in the DNA chains, or RNA tails. The banding at the position expected for 1: 1 RNA-DNA hybrids of the heavy strand of m&-DNA amealed with the 30 to 70 s RNA components or with components of less than 22 s confirmed the results of the DNA saturation experiments, suggesting full-length base-sequence complementarity of this strand with mitochondria-associated RNA. The hybrids formed with the 22 to 40 s components banded at a slightly lower density (about l-485 g/cm3), again in agreement with the results of the DNA saturation experiments. (d) Conclusions Previous work from this laboratory (Attardi & Attardi, 1967,1968,1969 ; Attardi et al., 1969 ; Attardi t Attardi; 1971) has shown that mit-DNA is transcribed very adtively in exponentially growing HeLa cells. The results discussed above of the DNA saturation experiments and of the Cs,SO, density-gradient analysis of RNA-DNA hybrids have clearly indicated that almost all the sequences of the heavy strand of mit-DNA in these cells are represented in mitochondria-associated RNA. The electron microscopic examination of the RNA-DNA hybrids (Appendix) has corroborated. this conclusion. Since the evidence derived from measurements of DNA renaturation kinetics argues against the presence of major internal repetitions in mammalian mit-DNA (Borst, 1969), the present results can be interpreted to indicate that the mitochondrial genome in HeLa cells is completely or almost completely transcribed. This conclusion and the observation that transcription of mit-DNA occurs almost exclusively from the heavy strand are in agreement with the idea that reading of the mit-DNA template takes place in the form of a continuous long RNA chain. This idea had been suggested by the occurrence of fast sedimenting RNA molecules, ethidium bromide-sensitive and homologous to mit-DNA (Attardi et al., 19693), in the sedimentation profile of pulse-labeled mitochondrial RNA. Implicit in this idea is that the slower sedimenting discrete species coded by m&-DNA described in the preceding paper (Attardi St Attardi, 1971) derive from processing of the large precursors. The observed kinetics of labeling of molecules of different size in pulse experiments is indeed consistent with a precursor-to-product relationship between the faster and the slower sedimenting molecules (Attardi et al., 19696). The 33 s peak skewed to the heavy side observed both in pulse-labeled and long term-labeled RNA preparations may represent one of the 6rst intermediates in the processing of mitochondrial RNA. RNA-DNA hybridization experiments (Attardi & Attardi, 1969), kinetics and inhibition studies (Vesco & Penman, 1969 ; Zylber & Penman, 1969; Attardi & Attardi, 1971) have shown that the bulk RNA synthesized on mit-DNA in HeLa cells is represented by discrete species with sedimentation coeflicients of 4 s, 12 s and 16 s; the heterogeneous RNA components spread in the 4 s to 70 s region of the gradient and the discrete 33 s component represent a relatively minor part of the total

266

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mit-DNA coded RNA. However, these heterogeneous RNA components and the 33 s discrete species are presumably those involved in hybrid formation in the DNA saturation experiments carried out with the Rh’A heavier than 22 S. $lthough the 4 s, 12 s and 16 s RNA components certainly participated in hybrid formation in the experiments carried out with the RNA sedimcnting slower than 22 s, the high RN14 to DNA input ratio needed to reach DNA saturation with this RNA fraction suggests the contribution to hybrid formation of more rare RXA sequences not represented in the above mentioned discrete species. It seems unlikely that the complete saturation of the heavy strand of mit-DNA by this RNA cut is due to its contamination by components heavier than 30 s, although a contribution to the hybridization of components in the intermediate sedimentation range cannot be excluded. If the above discussed model of continuous transcription of the heavy strand of mit-DKA is correct, the presence of all transcribed sequences of mit-DNA in the region of the gradient of less than 22 s could result either from physiological processing of the large precursors, or from artificial degradation during extraction, or from the presence of incomplete nascent RNA chains starting on the template from multiple initiation points, or a combination of these processes. The possibility that saturation of the heavy strand of mit-DNA with the components slower than 22 s is due to the fact that the genes corresponding to the discrete species 16 s, 12 s and 4 s account for all its length is excluded by the results of RNA-DNA hybridization with these individual components (Aloni & Attarcli, 1971). The saturation level appreciably lower than 100% observed with the RNA components of intermediate sedimentation coefficient (22 to 40 s in Fig. l(a)), both in the saturation experiments and in the Cs,SO, density-gradient analysis, may indicate that some sequences are absent in these components: a possible candidate for these missing sequences is 4 s RNA. The shape of the saturation curve obtained with these components would tend to exclude interference by the large excess of rRNA as an alternative explanation for the incomplete saturation. This investigation was supported by a grant from the National Institutes of Health (GM-11726). The work reported in this paper was undertaken during the tenure of a research training fellowship awarded by the International Agency for Research on Cancer to one of us (Y. A., on leave from the Weizmann Institute of Science, Rehovot, Israel). $X DNA was the generous gift of Dr A. Razin and J. Sedat. The excellent technical assistance

of Mrs LaVerne

Wenzel and Es

Benneta Keeley is gratefully

acknowledged.

REFERENCES Aloni,

Y. & Attardi,

G. (1971) J. Mol. Biol. 55, 255. Aloni, Y., Winocour, E., Sachs, L. & Torten, J. (1969). J. Mot. Biol. 44, 333. Amaldi, F. & Attardi, G. (1968). J. Mol. Biol. 33, 737. Attardi, B. & Attardi, G. (1967). Proc. Nat. Acad. Sci., Wash. 58, 1051. Attardi, B. & Attardi, G. (1969). Nature, 224, 1079. Attardi, B. & Attardi, G. (1971). J. Mol. Biol. 55, 215. Attardi, B., Cravioto, B. & Attardi, G. (1969a). J. Mol. Bzol. 44, 47. Attardi, G., Aloni, Y., Attardi, B., Lederman, M., Ojala, D., Pica-Mattoccia, L. 65 Storrie, B. (1969b). In Int. Syrnp. on Autonomy and Biogenesis of Mitochondria u.rz,d Chloropkzsts. Canberra. Amsterdam: North-Holland Publ. Co., in the press. Attardi, G. & Attardi, B. (1968). Proc. Nat. Acad. SC%., Wash. 61, 261. Attardi, G., Parnas, H., Hwang, M-I. H. & Attardi, B. (1966). J. Mol. Biol. 20, 146. Borst, P. (1969). In Int. Symp. on AzLtonomy and Biogenesis of Mitochondtia and Chloropkwts, Canberra. Amsterdam : North-Holland Publ. Co., in the press. Borst, P. & Aaij, C. (1969). Biochem. Biophys. Res. Comm. 34, 205.

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DNA

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Borst, P. & Ruttenberg, 5. c1. M. (1969). Biochim. biophys. Acta, 190, 391. Chamberlin, M. & Berg, I’. (1964). J. XoZ. Biol. 8, 297. Clayton, D. A., Davis, 12. W. & Vinograd, J. (1970). J. Mol. Biol. 47, 137. J. (1967). Nature, 216, 652. + Clayton, D. A. & Vinograd, Corneo, G., Zardi, L. 85 Polli, E. (1968). J. Mol. BioZ. 36, 419. Dawid, I. B. (1969a). Fed. Proc. 28, 349. Dawid, I. B. (19695). Symp. Sot. Exp. BioZ. 24, 227. Dubin, D. T. (1967). Biochem. Biophys. Res. Comm. 29, 655. Dubin, D. T. & Montenecourt, -B. S. (1970). J. Mol. BioZ. 48, 279. Gilbert, W. (1963). J. Mol. Biol. 6, 389. Gillespie, D. & Spiegelman, S. (1965). J. Mol. BioZ. 12, 829. Hall, B. D. & Doty, P. (1959). J. Mol. Biol. 1, 111. Hudson, B. & Vinograd, J. (1965). Nature, 216, 647. Levintow, L. & Darnell, J. E. (1960). J. Biol. Chem. 235, 70. Nass, M. M. K. (1969). J. Mol. BioZ. 42, 521. Nass, M. M. K. & Buck, C. A. (1969). Proc. ivat. Acad. Xci., Wash. 62, 506. Nass, M. M. K. & Buck, C. A. (1970). J. Mol. BioZ. 54, 187. Nygaard, A. P. & Hall, B. D. (1963). Biochem. Biophys. Res. Corm. 12, 98. Radloff, R., Bauer, W. & Vinograd, 5. (1967). Proc. Nat. Acad. Sci., Wash. 57, 1514. Shack, 5. (1958). J. BioZ. Chem. 233, 677. Sinsheimer, R. L. (1959). J. Mol. BioZ. 1, 43. Sinsheimer, R. L. & Lawrence, M. (1964). J. Mol. BioZ. 8, 289. Spiegelman, S. & Doi, R. H. (1963). Cold Spr. Hark Sywvp. f&ant. BioZ. 28, 109. Vesco, C. & Penman, S. (1969). Proc. Nat. Acad. Sci., Wash. 62, 218. N. & Dove, W. F., Jr. (1963). Proc. Nat. Acad. Sci., Vinograd, J., Morris, J., Davidson, Wash. 49, 13. Zylber, E. & Penman, S. (1969). J. Mol. BioZ. 46, 201.

APPENDIX

Electron Microscopic

Visualization

D. ROBBERSON,Y. Division

of Mitochondrial ALONIAND

RNA-DNA

Hybrids

G.ATTARDI

of Biology, California Institute of Technology Pasadena, C&f. 91109, U.S.A.

Hybrids between mitochondrial RNA (<22 s components) and mit-DNA banded in a Cs,SOa gradient, as described in Results section (c)(ii), were examined by electron microscopy for their content of duplex structure. The basic protein film technique (Kleinschmidt & Zahn, 1959) was used, with a spreading solution of 05 M-ammonium acetate, 0.1 M-Tris, 0.005 M-EDTA at pH 8,05 pgnucleic acid/ml., O-2 mg cytochrome c/ml., and a hypophase of O-2m-ammonium acetate, pH 8. Films were picked up on 3.57; Parlodion-coated 200-mesh screens and stained with uranyl acetate (Wetmur, Davidson & Scaletti, 1966). Staining was used in order to allow detection of small regions of non-hybridized single-stranded DNA. The grids were examined by dark-field electron microscopy on a Philips EM300 electron microscope utilizing the electronic beam tilt and a 40 p objective aperture. The dark-field technique enhanced the contrast of the stained structures. Several of the hybrid molecules mounted by the above technique and photographed in dark-field are shown in Plate AI(b). For comparison, the heavy strand of n&-DNA and double-stranded +X RF DNA mounted under the same conditions are presented in Plate AI(a) and (c), respectively. Single-stranded DNA appears collapsed in