Hybridization studies with yeast mitochondrial RNAs

396

BIOCHIMICA ET BIOPHYSICA ACTA

8BA 97295

H Y B R I D I Z A T I O N S T U D I E S W I T H YEAST M I T O C H O N D R I A L RNAs L. R E I J N D E R S , C. M. K L E I S E N , L. A. G R I V E L L AND P. B O R S T

Department of Medical En~ymology', Laboratory of Biochemistry, University of Amsterdam, Amsterdam (The Netherlands) {Received F e b r u a r y I4th, 1972)

SUMMARY

I. RNA from highly purified mitochondrial ribosomes of Saccharomvces carlsbergensis was hybridized with homologous mtDNA, purified by a procedure that avoids equilibrium centrifugation in CsC1. Maximally 2.4 % of the DNA could be converted into hybrid, showing that not more than one gene is present for each of the rRNAs. 2. Separate plateaus were observed for the rRNAs from purified ribosomal subunits, showing that substantial base sequence homologies between these RNAs are absent. 3. Co-sedimentation of mitochondrial rRNA with Escherichia coli rRNA through gradients containing 99 % dimethyl sulphoxide, shows that both mitochondrial rRNAs are slightly larger than the corresponding E. coli rRNAs. 4. The base composition of mitochondrial rRNA in mole percent is 39 % A, 3 9 ° o U, 14 % G a n d 9 % C. 5. Total m t R N A hybridized to a large extent with nuclear DNA of S. carlsbergensis, but the hybridization was suppressed to near background (0.07 % of DNA in hybrid) by excess cold cell-sap rRNA. This indicates that yeast mitochondria do not contain substantial amounts of imported nuclear transcripts.

INTRODUCTION

Hybridization experiments have shown that the mtDNAs of Xenopus laevis L~ and of H e L a cells 3'4 contain one cistron for each of the two m t R N A components. Sinfilar experiments with the ascomycetes Saccharornyces and Neurospora have led to controversial results. From 2.3 to 14 OJjoof the m t D N A was found to be complementary to rRNA in different studies (refs 5-8), whereas de Kloet et al. 9 have even expressed doubt whether yeast m t D N A codes for mitochondrial rRNA at all. Accurate knowledge of the number of cistrons for rRNA on yeast m t D N A is not only of intrinsic interest, it is also a prerequisite for the interpretation of data on antibiotic-resistant yeast mutants in which resistance is due to a change in the mitochondrial ribosome (see ref. IO). In this paper we present the results of a reAbbreviation: I x SSC, o.15 M NaC1 O.Ol5 IV[ s o d i u m citrate ( p i t 7.0). * Postal address: J a n S w a m m e r d a m i n s t i t u t e , Eerste Constantijn H u y g e n s s t r a a t 20, A m s t e r d a m , The Netherlands.

Biochim. Biophys. Acta, 272 (1972) 396-4o 7

HYBRIDIZATIONSTUDIES WITH MITOCHONDRIALRNAs

397

investigation of this problem. We show that only one cistron of each of the mitochondrial rRNAs is present on yeast mtDNA; we provide an explanation for the higher values obtained b y some other groups; and we present additional data on the base composition and molecular weights of yeast mitochondrial rRNAs and on the possible import of nuclear m R N A into mitochondria. METHODS

Preparation o / m t D N A ]rom S. carlsbergensis S. carlsbergensis NCYC 74 was grown to late-logarithmic phase in a minimal medium containing 0.08/~Ci ~2,4-3H]adenine per ml. Mitochondria were then prepared essentially according to the procedure of Ohnishi et al. ~1. The yeast lysate was centrifuged for 5 rain at 3coo×g, the mitochondria were pelleted from the resulting supernatant by a spin at IO ooo × g for IO rain, and taken up in 0.6 M mannitol- 5 mM MgC12-IO mM Tris-HC1 (pH 7.6) and incubated for 28 rain at o°C with 15o #g pancreatic deoxyribonuclease per nil. After this treatment the mitochondria were spun down and washed 4 times with 0.6 M mannitol--2 mM EDTA (pH 7.5). The washed mitochondria were lysed in a medium containing 2 % sodium dodecyl sulphate, I o/ triisopropyl naphthalene disulphonate, 4 % sodium aminosalicylate, 6 o/ iO , 0 sec-butanol and 50 mM Tris-HC1 (pH 7-5)- The lysate was extracted twice with an equal volume phenol: cresol TM and the RNA and DNA were precipitated from the aqueous layer with ethanol at --2o°C. The precipitate was dissolved in o.i ×SSC and incubated for I h at 25°C with 50 fig bovine pancreas ribonuclease and 25 units T 1 ribonuclease per ml both pre-heated for io min at 8o°C, followed by a 2-h incubation with 5o/2g pronase per ml (pre-heated for IO rain at 8o°C) at 25°C. DNA was recovered by chromatography on a column of methylated albumin on Kieselguhr la. The DNA solution was dialysed, adjusted to a suitable volume by rotary evaporation, and centrifuged through a Spinco SW-4I isokinetic sucrose gradient, containing IO mM Tris-HC1 (pH 7.5) and IOO mM NaC1, made according to the method of Noll 1~, assuming a particle density of 1.65. DNA fragments sedimenting faster than the open circular duplex from phage PM2 (s20,w == 21.2) (ref. 15) were recovered and used for hybridization experiments. In analytical CsC1 equilibrium runs of this DNA, no material could be seen banding at the nuclear density, indicating that nuclear contamination is probably less than 3 %. Preparation o / m t R N A and mitochondrial ribosomes/tom S. carlsbergensis 32P-labelled total m t R N A was prepared as described b y Cohen et al. TM. For the preparation of mitochondrial ribosomes and the mitochondrial ribosomal subunits of S. carlsbergensis the procedure of Grivell et al. 18 was followed. RNA from ribosomes or ribosomal subunits was obtained by suspending ribosomes in 50 mM Tris-HC1 (pH 7.5). In the case of pelleted ribosomes MgCle was added to a final concentration of 5 mM and the ribosomes were incubated at 25°C for 45 rain with 2o/,g deoxyribonuclease I (ribonuclease free), followed by the addition of 0.5 M E D T A (pH 7.5) to a final concentration of 7 raM. The suspension of ribosomes or ribosomal subunits was made 2 % in sodium dodecyl sulphate and 4 % in sodium aminosalicylate and extracted twice with an equal volume of phenol: cresol 1~. The water phase was dialysed for 48 h against 3 changes of 3 × SSC at 4°C. Biochim. Biophys. Acta, 272 (1972) 396-4o 7

398

L. REIJNDERS et al.

Preparation o~ D N A , R N A and ribosomes/rom other sources ~*C-labelled nuclear DNA from S. carlsbergensis was prepared according to Cohen et al. TM. 14C-labelled E. coli RNA was prepared as described elsewhere iv. Cell-sap ribosomes of S. carlsbergensis were obtained by layering a clarified post-mitochondrial supernatant over o.4vol. I M sucrose, containing 5oomM NH,C1, IO mM magnesium acetate, io mM Tris-HC1 (pH 7.5) and spinning for io h in the Spinco-4o rotor at 4 ° ooo rev./min and 5°C.

H),bridizaliol¢ Hybridization was carried out according to Gillespie and Spiegelman TM. Filters (o.I/~m pore size) were pre-soaked in 2 × SSC-o.1% sodium dodecyl sulphate before loading with DNA. DNA was denatured at pH 13, neutralized and brought onto the filter in a concentration of o.I/~g/ml at 5°C. Three filters were always incubated together in 4 ml of 3 × S S C - o . 1 % sodium dodecyl sulphate; one containing 3Hlabelled mtDNA or 14C-labelled nuclear DNA, one containing a corresponding amount of E. coli DNA and one without DNA. The incubation was carried out in a shaking water bath for the period and at the temperature indicated in the text. Acid-precipitable material at the end of the incubation was never less than 70 °'0 of the a2P-labelled input. The filters recovered from the vessels (containing crude hybrids) were treated as follows: a wash at 63°C for 15 rain in 3 ×SSC (5 ml), a wash at 25°C for 15 rain in 5 ml 3 × SSC, an incubation in 4 ml of 2 × SSC, containing io/~g ribonuclease A and 25 units T 1 ribonuclease (pre-heated for IO rain at 8o°C) per ml for the time and at the temperature indicated in the text, a wash at 25°C for 15 rain in 5 ml of 3 × SSC-o. I °/o sodium dodecyl sulphate, a wash at 63°C for 15 rain in 5 ml of 3 ×SSC-o.I °/o sodium dodecyl sulphate and a wash at 25°C for 15 rain in 5 ml of 3 × SSC. The filters were dried and counted in P P O - P O P O P toluene solution in the Nuclear Chicago Mark-I liquid scintillation system. Values for hybridization with mtDNA were corrected for hybridization with E. coli DNA. Blank filters (containing no DNA) did not bind more than o.oi °/o of the input a2p radioactivity. The concentration of DNA was determined by the method of Burton TM using calf-thymus DNA as standard. The concentration of RNA was determined spectrophotometrically, assuming that I mg RNA has an absorbance at 260 nm of 24/cm. Specific activities of RNA and DNA samples used in hybridization were determined by spotting a small volume RNA or DNA solution of known concentration on the membrane filters used for hybridization experiments, drying and counting in P P O - P O P O P - t o l u e n e solution in the Mark-i Nuclear Chicago liquid scintillation system.

Measurement o/ribonuclease resistance Duplicate samples containing O.l-O.3 #g RNA were incubated at the temperature indicated in the text for different periods in o.25-ml volumes, containing SSC and ribonucleases as indicated in the text, at 25 or 80°C. At the end of the incubation i o o / , g bovine serum albumin was added, followed by an equal volume of IO °/o trichloroacetic acid. The turbid suspension was filtered through Millipore MF-45 filters and the filters were washed with 25 ml 2 °/o trichloroacetic acid. Ribonuclease

Biochim. Biophys. Acta, 272 (1972) 396-407

HYBRIDIZATIONSTUDIES WITH MITOCHONDRIALRNAs

399

resistance is expressed as percentage of the initial amount of trichloroacetic acidprecipitable radioactivity, corrected for the material that remained acid precipitable after an incubation with 0.3 M K O H for 36 h at 37°C.

Dimethyl sulphoxide-suerose gradient centri]ugation The method used was essentially that of Strauss et al. 2°. RNA samples were dissolved in 99 % dimethyl sulphoxide-I mM E D T A (pH 7.o), layered on a o - I o % sucrose gradient in 99 % dimethyl sulphoxide-I mM E D T A (pH 7.o) and centrifuged for 22 h at 25°C at 4 ° ooo rev./min in the Spinco SW-41 rotor. Our experience showed that centrifugation for a longer time led to a diminished resolution. Fractions were collected manually from a hole in the bottom of the tube and RNA was precipitated with 4 ml 5 % trichloroacetic acid in the presence of IOO #g bovine serum albumin as carrier. The precipitates were filtered onto glass fibre discs, dried and counted in P P O - P O P O P - t o l u e n e in the Nuclear Chicago Mark-I liquid scintillation system. Base analysis oI R N A RNA was hydrolysed in o.3 M K O H for 36 h at 37°C. The solution was neutralised with perchloric acid and base analysis was performed following the method of Markham and Smith 21, using 2o mM sodium citrate (pH 3.5) as electrophoresis buffer. Materials Saccharose (reinst), NaC1 and NH4C1 (suprapur) and magnesium acetate (pro analysi) were obtained from Merck; sodium dodecyl sulphate from Serva; bovine serum albumin (Fraction V), Trizma base and pancreatic deoxyribonuclease from Sigma; ribonucleases A and T 1 and deoxyribonuclease I (ribonuclease-free) from Worthington; triisopropyl naphthalene disulphonate from Eastman. 14C- and 3H-labelled chemicals were obtained from the Radiochemical Centre, Amersham; [~2plphosphate from Philips Duphar, Petten. All other chemicals were obtained from British Drug Houses, Ltd. o.I # m pore size membrane filters were obtained from Sartorius Membranfilter G m b H ; MF-45 membrane filters from Millipore Filter Corporation. RESULTS

Hybridization o] mitochondrial r R N A with mtDNA Two pitfalls complicate the interpretation of published experiments with yeast and Neurospora: (I) In most experiments whole m t R N A was fraetionated on sucrose gradients and the RNA in the r R N A peaks was used for hybridization. If m R N A s with the same sedimentation coefficients as the rRNAs are present, this approach could lead to gross overestimation of the number of rRNA cistrons on mtDNA. We have avoided this complication b y using RNA from highly purified mitochondrial ribosomes and from subunits separated b y sucrose gradient centrifugation (Fig. I). Since these ribosomes are almost as active as E. coli ribosomes in amino acid incorporation directed b y poly(U) (ref. 17) or phage MS2 RNA (L. Reijnders, unpublished observations), and subunits can be recombined to give full protein synthetic activity, they must contain a full complement of rRNA. Biochim. Biophys. Acta, 272 (1972) 396-407

L. REIJNDERS el al.

400

2000

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Fig. I. P r e p a r a t i o n of s u b u n i t s of m i t o c h o n d r i a l r i b o s o m e s from S. carlsbergensis. 32p-labelled r i b o s o m e s p r e p a r e d as described in M e t h o d s , were s p u n in t h e Spinco SW-41 r o t o r for 5 h a t 39 ooo r e v . / m i n (5°C) t h r o u g h a n isokinetic sucrose g r a d i e n t (see ref. 16) c o n t a i n i n g 500 m M NH4C1, io m M m a g n e s i u m acetate, IO m M Tris-HC1 (pH 7.6). F r a c t i o n s were m o n i t o r e d for 32p r a d i o a c t i v i t y b y C e r e n k o v c o u n t i n g . T h e c r o s s - h a t c h e d regions indicate t h e fractions pooled for R N A e x t r a c t i o n . Fig. 2. H y b r i d i z a t i o n of S. carlsbergensis m t R N A w i t h m t D N A . R N A was e x t r a c t e d f r o m w h o l e (74 S) r i b o s o m e s ( + - + ) , f r o m 5o-S s u b u n i t s ( O - C ) ) , a n d from 37-S s u b u n i t s ( × - x ) . H y b r i d i z a t i o n p r o c e d u r e as described in M e t h o d s ; h y b r i d i z a t i o n for 2o h a t 63°C a n d digestion of t h e c r u d e h y b r i d w i t h r i b o n u c l e a s e s for 2 h a t Io°C. T h e specific a c t i v i t y of t h e 32P-labelled r R N A w a s 74 i o o c p m per ktg. T h e 3H-labelled D N A h a d a specific a c t i v i t y of 857 c p m p e r / ~ g ; m i t o c h o n d r i a l a n d E. coli filters c o n t a i n e d 0.6/~g D N A . All v a l u e s are corrected for R N A b o u n d to E. coli filters (5-2o c p m ) .

(2) In all previous experiments with yeast, mtDNA was purified by repeated CsC1 equilibrium gradient centrifugation. The mole percent G + C of yeast rRNA has been reported to be 26 (ref. 22), i.e. 9 % higher than that of yeast mtDNA 23. Since isolated yeast mtDNA is heavily fragmented, purification by density gradients may remove rDNA fragments and, therefore, lead to an underestimation of the number of rRNA cistrons. We have avoided this second complication by only using mtDNA that had not been fractionated by CsC1 gradient centrifugation. The necessary purification was obtained by extracting mtDNA from isolated mitochondria, pretreated with deoxyribonuclease, and removing the remaining nuclear DNA fragments by sucrose gradient centrifugation. Hybridization experiments with these DNA and RNA preparations are presented in Fig. 2 and Table I. Fig. 2 shows that maximal hybridization is reached under our conditions at an input of 3-4/tg RNA. Further increase of the input by a factor three had no effect on the hybridization level. This shows that the rRNA preparations were not contaminated to a significant extent with mRNA and that experiments at a single RNA input of > 5/~g could be used under these conditions to get information about hybridization plateaus. The hybridization plateau obtained in Fig. 2 was about 30 °,/olower than the plateau expected if mtDNA contains one cistron for each of the Biochim. Biophys. Acta, 272 (1972) 396-4o7

HYBRIDIZATION STUDIES WITH MITOCHONDRIAL TABLE

RNAs

4Ol

I

INFLUENCE OF DIFFERENT PROCEDURES FOR HYBRIDIZATION AND TREATMENT OF CRUDE HYBRID ON PLATEAU VALUES FOUND FOR THE HYBRIDIZATION OF MITOCHONDRIAL r R N A AND m t D N A Hybridization of szP-labelled mitochondrial rRNA with 81q-labelled mtDNA was performed as described in Methods, with conditions of hybridization and of ribonuclease digestion of the c r u d e h y b r i d a s i n d i c a t e d i n c o l u m n s I a n d 2. H y b r i d i z a t i o n w a s m e a s u r e d i n d u p l i c a t e i n c u b a t i o n s of" 5 . 3 5 leg s 2 p - l a b e l l e d m i t o c h o n d r i a l r R N A (32 6 o 0 c p m p e r /~g) w i t h 0. 3 /2g S H - l a b e l l e d m t D N A ( 8 4 o c p m p e r / ~ g ) a n d c o r r e c t e d f o r h y b r i d i z a t i o n o f m i t o c h o n d r i a l r R N A w i t h o.3 /*g E. coli D N A ( l O - 2 O c p m ) .

Hybridi,mtion Temp.

Time

CC)

(h)

Ribonuclease treatment of crude hybrid Temp.

DE,4 converted into hybrid (% of D N A on filter) Time

(oc)

(h)

63

20 40 20 20

25 25 IO IO

I I 2 3

2. 4 2.45 2.45 2.5

58

2o 20 4°

25 IO 25

I 3 I

2. 4 2.35 2.45

rRNAs (see Discussion). Slightly lower values (2.15-2. 4 %) were obtained with three other r R N A preparations in initial experiments in which the hybrid was exposed to more drastic conditions of ribonuclease treatment (I h at 25°C). We, therefore, analysed the hybridization conditions in more detail. The results, presented in Table I show that the hybridization level was not significantly increased b y increasing the time of hybridization, b y lowering the hybridization temperature or b y using milder conditions for the ribonuclease treatment of the hybrid. In contrast to recent observations on yeast cell-sap rRNA 24, we do not find extensive cross-hybridization between the RNAs of the two mitochondrial ribosomal subunits. Fig. 2 shows that the RNAs isolated from subunits separated as in Fig. I, hybridize to different extents with mtDNA. The fact that an increase of the input above about 5 #g RNA has no effect on the hybridization level, shows that crosscontamination between the subunits is negligible and that the rRNAs of the two subunits do not have a substantial degree of base sequence homology. The plateau observed for the RNA from the 5o-S subunit in this experiment is lower than expected. We have not determined the reason for this anomaly. Ribonuclease resistance o / r R N A

A possible explanation for the low plateau value found for rRNA in the experiments presented in the previous section, could be a partial self-complementarity of mitochondrial rRNA. The corresponding regions in the rDNA genes will be in a duplex form during the hybridization and they will, therefore, be unable to participate in hybrid formation leading to a systematic underestimation of the fraction of the m t D N A complementary to rRNA. This possibility was suggested b y a recent report t h a t 5 ° % of the mitochondrial rRNA of N. crassa is resistant to degradation b y ribonuclease 2s. The result in Table I I shows, however, that only 6 % of this yeast r R N A was resistant; only about half of this resistance was lost when the ribonuclease Biochim. Biophys. Acta, 2 7 2 ( 1 9 7 2 ) 3 9 6 - 4 o 7

402 TABLE

L. REIJNDERS el a,/. II

RIBONUCLI~ASE RESISTANCE OF R N A s EXTRACTED FROM 5 o - S AND 3 7 - S MITOCHONDRIAL RIBOSOMAL SUBUNITS OF S. carlsbergensis Ribonuclease resistance was measured as described in Methods under the conditions indicated i n c o l u m n I. V a l u e s a r e m e a n s o f p l a t e a u s f o u n d in t w o t i m e c u r v e d e t e r m i n a t i o n s o n t w o d i f f e r e n t p r e p a r a t i o n s o f 8 ~ P - l a b e l l e d r R N A , h a v i n g s p e c i f i c a c t i v i t i e s of a b o u t 25 8 o o a n d 4 4 o o o c p m p e r p g , r e s p e c t i v e l y . A l k a l i - s t a b l e m a t e r i a l w a s less t h a n o . i % o f t h e i n p u t .

Conditions of ribonuclease digestion

Ribonuclease resistance in % of input 37-S RNA

5o-S RNA

25°C, 2 x SSC, 25 u n i t s T 1 r i b o n u c l e a s e / m l , io p g r i b o n u c l e a s e A / m l

6.2

5.9

8o°C, o.i × SSC, 25 u n i t s T1 r i b o n u c l e a s e / m l , 50 ktg r i b o n u c l e a s e A / m l

3.0

2.0

treatment was carried out at 8o°C in low salt, conditions that should allow degradation of any duplex RNA present. We conclude that self-complementarity of the mitochondrial rRNA cannot explain the low hybridization plateaus observed. It is of interest that the ribonuclease resistance of RNA extracted from pelleted mitochondrial 74-S ribosomes, rather than from purified subunits, was much higher, i.e. lO-25 % of the alkali-labile, acid-insoluble radioactivity. This suggests that nfitochondria from ascomycetes contain an alkali-labile, non-RNA contaminant that is removed during extensive purification of ribosomes. The presence of high concentrations of this contaminant could explain the very high ribonuclease resistance reported ~5 for rRNA preparations from Neurospora mitochondria.

Sedimentation o/mitochondrial r R N A through dimethyl sulphoxide-sucrose gradients The unusual structure of yeast mitochondrial rRNA makes it hazardous to infer its molecular weight from sedimentation through standard sucrose gradients or electrophoretic mobility in acrylamide gels even after treatment with formaldehyde (see ref. 17). To exclude that tile low hybridization plateau found was due to an unusually small size of yeast mitochondrial rRNA we have, therefore, compared the sedimentation of mitochondrial and E. coli rRNA through dimethyl sulphoxidesucrose gradients. Under tile conditions used (see Methods), both RNAs are completely denatured random coils, because increase in temperature did not lead to an increase in A260 nm (experiments not shown). Fig. 3 shows that the mitochondrial rRNAs sediment slightly ahead of E. coli rRNAs through the dimethyl sulphoxide sucrose. It is clear, however, that the relative sedimentation rates of the two subunit RNAs are anomalous, the large subunit RNA sedimenting more slowly than would be expected. This is due to the fact that the sedimentation rate through these gradients decreases in the lower part of the tube, leading to loss of resolution. Although this effect precludes the assignment of a precise molecular weight to the yeast rRNAs, the results exclude the possibility that these RNAs are smaller "~han tile rRNAs of

E. coli. Hybridization o~ z6-S and 23-S m t R N A The hybridization plateaus found by us for yeast mitochondrial rRNA are much lower than those obtained by others, who used I6-S and 23-S RNA from sucrose Biochim. Biophys. Acta, 272 (1972) 396-407

RNAs

HYBRIDIZATION STUDIES WITH MITOCHONDRIAL

403

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Fig. 3. S e d i m e n t a t i o n of l~C-labelled E. colt R N A a n d 32P-labelled m t R • A f r o m S. cavlsbergensis in d i m e t h y l s u l p h o x i d e - s u c r o s e gradients. P r o c e d u r e as described in M e t h o d s . Specific a c t i v i t y o f t h e 14C-labelled E. colt R N A , 358 c p m p e r t,g; 32P-labelled m t R N A , 345 ° c p m per/2g. Fig. 4. P r e p a r a t i o n of a2P-labelled 23-S a n d I6-S m t R N A . T o t a l 32P-labelled m t R N A (specific a c t i v i t y a b o u t i i ooo c p m p e r / ~ g ) w a s s p u n for 14 h at 33 ooo r e v . / m i n in t h e Spinco S W - 4 I r o t o r into an isokinetic 5 25 % sucrose g r a d i e n t (cf. ref. I7), c o n t a i n i n g i o o m M NaC1, io m M T r i s - H C 1 (pH 7.6). F r a c t i o n s were collected a n d m o n i t o r e d for 3,p r a d i o a c t i v i t y b y C e r e n k o v c o u n t i n g . T h e c r o s s - h a t c h e d regions indicate t h e fractions pooled to o b t a i n 23-S a n d I6-S R N A , respectively. Before h y b r i d i z a t i o n t h e pooled fractions were dialysed a g a i n s t 3 × SSC.

Q

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Fig. 5. H y b r i d i z a t i o n of 23-S a n d I6-S m t R b l A w i t h m t D N A . I n c r e a s i n g a m o u n t s of 23-S (a) a n d I6-S a2P-labelled R N A (b) were h y b r i d i s e d to filters c o n t a i n i n g 0.6/~g mtDl~'A, as in M e t h o d s for 20 h a t 63°C. T h e c r u d e h y b r i d w a s t r e a t e d as described in M e t h o d s , u s i n g a r i b o n u c l e a s e t r e a t m e n t of 2 h a t Io°C. Specific a c t i v i t y of t h e R N A w a s 9060 c p m p e r /~g for b o t h R N A s ; .specific a c t i v i t y of 3H-labelled m t D N A w a s 820 c p m per/~g,

Biochim. Biophys. Acta, 272 (1972) 3 9 6 - 4 o 7

404

L. REIJNDERS et

al.

gradients of total mtRNA. An explanation for this discrepancy is provided by the following results. Such I6-S and 23-S RNA fractions, taken from the gradient in Fig. 4, form hybrids with more than 6 °/o of the m t D N A (Fig. 5)- Comparison of Figs 2 and 5 shows, however, that hybridization continues to increase in the latter beyond an input of 4 #g RNA and that no plateau is reached even at 15/~g RNA. This indicates that the I6-S and 23-S regions of the gradients are heavily contaminated by non-ribosomal RNAs.

Base composition o/mitochondrial r R N A The base composition of yeast mitochondrial rRNA has been determined by Fauman et al. ~2, using I6-S and 23-S RNA fractions from sucrose gradients of total mtRNA. Our finding that such fractions are contaminated with non-ribosomal RNA made it desirable to repeat the analysis with an alkaline digest of RNA from purified ribosomes. Fig. 6 shows that all radioactivity migrated with the four standard nucleotides and that the peak migrating between AMP and GMP found by Ktintzel and Nol126 in Neurospora m t R N A and considered by Noll ~7 to be 2'-O-methyldinucleotide, is completely missing in veast mitochondrial rRNA. The average mole o/ G + C found by us is only 22. 7 o/o, . . . .3. % lower than that reported by Fauman et al. 22. This would mean that the non-ribosomal RNA contaminants in I6-S and 23-S RNA have a surprisingly high G + C content. :O

12 C

c~

A

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U

x >,

T.2

4

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0 )

5

10 15 Distance Migrated (cm)

20

Fig. 6. P a p e r electrophoresis of an alkaline h y d r o l y s a t e of mitochondrial r R N A . The R N A was extracted from ribosomes isolated from S. carlsbergensis mitochondria, t h a t showed less t h a n 5 % c o n t a m i n a t i o n with cell-sap ribosomes, as judged from gel electropherograms of total n l t R N A in low salt (see ref. 17). Unlabelled m a r k e r nucleotides were co-electrophoresed. Specific activity r R N A was a b o u t 25 ooo epm per ]*g. @-(2), 32p radioactivity; c~7~, unlabelled nucleotides.

Hybridization o / m t R N A with nuclear DNA Previous experiments have shown that RNA complementary to yeast m t D N A does not hybridize to a significant extent with yeast nuclear DNA 13. Nevertheless, mitochondrial preparations contain a substantial amount of RNA that hybridizes with nuclear DNA and the question arises if this RNA fraction represents contaminating cell-sap RNA or RNA that is synthesized in the nucleus and specifically taken up by the mitochondria. To test these alternatives total mtRNA, obtained from cells labelled for 24 h (several generations) with E32Plphosphate as described in Methods, Biochim. Biophys. Mcta, 272 (1972) 396-407

HYBRIDIZATION STUDIES WITH MITOCHONDRIAL R N A s i

i

405

i

x

g

/

+-

÷7

o

'u

~

-e

0

IL

ii0

o

115

t

2O

pg[~P]mtR NA added

b

t

x

j e~

pgp2P]mt RNA added Fig. 7- H y b r i d i z a t i o n of m t R N A w i t h nuclear DNA. a~P-labelled total m t R N A w a s hybridised with x4C-labelled nuclear D N A (a) and all-labelled m t D N A (b) as described in Methods for 4 ° h at 64°C in 2 × SSC with or w i t h o u t a Ioo-fold excess of cold cell-sap r R N A . Nuclear D N A filters contained 1.2 /~g D N A ; m t D N A filters, 0. 3 ~tg DNA. The crude h y b r i d w a s t r e a t e d as described in Methods, using a ribonuclease t r e a t m e n t of i h at 25°C. + - + , h y b r i d i z a t i o n in t h e absence of cold cell-sap RNA; O - Q , h y b r i d i z a t i o n in the presence of cold cell-sap RNA. Specific activities were: m t R N A , 34 2o0 c p m per #g; nuclear DNA, 760 c p m per/~g; m t D N A , 830 c p m per ~tg.

was hybridised with nuclear DNA with and without a ioo-fold excess of cold cell-sap rRNA. During the hybridization time used (4 ° h) the hybridization reached a plateau (data not shown). Fig. 7 a shows that the competing RNA reduces the hybridization level of m t R N A to less than 24 cpm, even at the highest radioactive RNA input. This is not due to gross contamination of the cold cell-sap r R N A with m t R N A , because this RNA preparation had no effect on the hybridization of m t R N A with m t D N A (Fig. 7b). The remaining hybridization corresponds to 0.07 ~o of the nuclear DNA in hybrid (Fig. 7a). Biochim. Biophys. Acta, 272 (1972) 396--407

406

L. REIJNDERS et al.

DISCUSSION From the molecular weight of yeast m t D N A of 49" lO6 (ref. '28) and the approximate total molecular weights of both mitochondrial rRNAs of 1. 7 • lO 6, one would expect a hybridization plateau of 3.5 % if each m t D N A molecule contained one cistron for each rRNA. We find only 2.4 % and our results suggest that this discrepancy is not due to an overestimation of the molecular weight of mitochondrial r R N A (see Fig. 3), inadequate saturation of the DNA with RNA (see Fig. 2 and Table f), or self-complementarity of the mitochondrial rRNA (see Table II). We cannot exclude, however, that some loss of hybrid is responsible for the low plateaus observed, for instance because post-transcriptional modification leads to poorly base-paired hybrid regions, or because fully base-paired hybrids without ss-DNA ends are pulled from the filter. It seems more likely, however, that the error is in the assumed value for the molecular weight of the DNA. The molecular weight of yeast m t D N A has been determined by measuring the contour length of twisted circular DNA molecules in electron mierographs of mitochondria isolated by osmotic shock, using a mass/length ratio of 1.96 • lO 6 per #m. Since the circles were highly twisted and since there is uncertainty about the correct value for the mass/length ratio ~'9,the calculated value for the molecular weight could well be underestimated. In conclusion, we consider our results fully compatible with the idea that yeast m t D N A consists of a homogeneous population of DNA molecules, each containing one cistron for each of the rRNAs. We cannot exclude the unlikely alternative, however, that the DNA is heterogeneous and that 30 % of the molecules lack rRNA cistrons. Our experiments show that the higher levels of hybridization for yeast mitochondrial rRNA reported b y others, result from the presence of non-ribosomal transcripts of m t D N A sedimenting at 16 and 23 S. A similar conclusion has been reached independently for Neurospora by Sch~ifer and Ktintzel 3~. The good agreement between the value found by us and that reported by Morimoto et al. 6, who used m t D N A fractionated in CsCl gradients, was unexpected. It could be due to the fact that the difference in G + C content between m t D N A (17 °/o ) and mitochondrial rRNA is not 9 % but only 6 °/o (Table I I I ) . Hence loss of rDNA by CsC1 gradient centrifugation could be less than expected. TABLE IH NUCLEOTIDE

C O M P O S I T I O N OF M I T O C H O N D R I A L A N D C E L L - S A P R I B O S O M & L R~x~A OF

Source of r R N A

Mitochondria Cell-sap

S. carlsbergensis

Nucleotide composition (mole %) ;4

U

G

C

38.7 28.9

38.6 23.5

14.° 29.o

8.7 18.6

Finally, our results show that the stable RNA in mitochondrial preparations that hybridizes with nuclear DNA can be competed out with cold cell-sap rRNA. The remaining hybridization (0.07 °/o ) is barely above background and similar to Biochim. Biophys. Acta, 272 (1972) 396-407

HYBRIDIZATION STUDIES WITH MITOCHONDRIAL

RNAs

407

the level observed with mtRNA, purified b y a hybridization-dehybridization cycle with m t D N A 13. The experiment does not exclude the presence in initochondria either of small amounts of nuclear RNA with a short half-life or of RNA complementary to a small section of the nuclear genome 3000-4000 nucleotides in length. The results are compatible, however, with previous conclusions that mitochondria make up for the lack of genetic information primarily by import of proteins, rather than by import of mRNAs.

ACKNOWLEDGEMENTS

We thank Dr J. W. Th. Coolsma for help and advice and Miss G. T. Noordhoek for technical assistance. This work was in part supported by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). REFERENCES i I. 13. Dawid, J. Mol. Biol., 63 (I972) 2Ol. 2 I. B. Dawid, in P. L. Miller, Control of Organelle Development, C a m b r i d g e U n i v e r s i t y Press, C a m b r i d g e , 197 o, p. 227. 3 Y. Aloni a n d G. A t t a r d i , J. Mol. Biol., 55 (1971) 271. 4 G. A t t a r d i , Y. Aloni, B. A t t a r d i , D. Ojala, L. Pica-lV[attoccia, D. L. R o b b e r s o n a n d ]3. Storrie, Cold Spring Harbor Symp. Quant. Biol., 35 (197 o) 599. 5 E. W i n t e r s b e r g e r a n d G. V i e h h a u s e r , Nature, 220 (1968) 699. 6 I-L Morimoto, A. t-L Scragg, J. N e k h o r o c h e f f , V. Villa a n d I-L O. I-~alvorson, in N. K. B o a r d m a n , A. W. L i n n a n e a n d R. 1V[. Smillie, Autonomy and Biogenesis of Mitochondria and Chloroplasts, North-I-Iolland, A m s t e r d a m , 1971, p. 282. 7 J. Casey, M. Cohen, M. R a b i n o w i t z , t t . F u k u h a r a a n d G. S. Getz, J. Mol. Biol., 63 (1972) 431. 8 D. D. W o o d a n d D. J. L. Luck, J. Mol. Biol., 41 (1969) 211. 9 S. R. De Ktoet, B. A. G. A n d r e a n a n d V. S. Mayo, Arch. Biochem. Biophys., 143 (1971) 175. IO L. A. Grivell, L. R e i j n d e r s a n d I-L de Vries, F E B S Lett., 16 (1971) 159. I i T. Ohnishi, K. K a w a g u c h i a n d ]3. I{agihara, J. Biol. Chem., 241 (1966) 1797. 12 J. i-L P a r i s h a n d K. S. K i r b y , Biochim. Biophys. Acta, 129 (1966) 554. 13 L. H. Cohen, C. P. H o l l e n b e r g a n d P. ]3orst, Biochim. Biophys. Acta, 224 (197 o) 61o. 14 I-I. Noll, Nature, 215 (1967) 360. 15 R. M. Espejo, E. S. Canelo a n d R. L. Sinsheimer, Proc. Natl. Acad. Sci. U.S., 63 (1969) 1164. 16 L. A. Grivell, L. R e i j n d e r s a n d P. Borst, Biochim. Biophys. Acta, 247 (1971) 91. 17 L. A. Grivell, L. R e i j n d e r s a n d P. Borst, Eur. J. Biochem., 19 (1971) 64. 18 D. G. Gillespie a n d S. Spiegelman, J. Mol. Biol., 12 (1965) 829. 19 K. B u r t o n , Biochem. J., 62 (1956) 315 • 20 J. I-L Strauss, Jr, R. ]3. K e l l y a n d R. L. Sinsheimer, Biopolymers, 6 (1968) 793. 21 R. M a r k h a m a n d J. D. S m i t h , Bioehem. J., 52 (1952) 522. 22 M. F a u m a n , M. R a b i n o w i t z a n d G. S. Getz, Biochim. Biophys. Acta, 182 (1969) 355. 23 G. ]3ernardi, M. F a u r e s , G. P i p e r n o a n d P. P. Slonimski, J. Mol. Biol., 48 (197 o) 23. 24 J. Retbl a n d R. J. P l a n t a , Biochim. Biophys. Acta, 169 (1968) 416. 25 K. P. Sch~kfer, G. B u g g e , M. G r a n d i a n d I-L Kfintzel, Eur. J. Biochem., 21 (1971) 478. 26 I-L Kfintzel a n d ]-L Noll, Nature, 215 (1967) 134o. 27 H. Noll, in P. L. Miller, Control of Organelle Development, C a m b r i d g e U n i v e r s i t y Press, C a m bridge, 197 o, p. 419. 28 C. P. H o l l e n b e r g , P. 13orst a n d E. F. J. v a n B r u g g e n , Biochim. Biophys. Aeta, 209 (197 o) I. 29 D. Freifelder, J. Mol. Biol., 54 (197 o) 567 . 3 ° K. P. Schi~fer a n d t{. Ktintzel, Biochem. Biophys. Res. Commun., 46 (1972) 1312.

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