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Origin of mitochondrial ribosomal RNA in Candida utilis. Hybridization studies
BIOCH1MIE, 1974, 56, 269-274.
Origin of mitochondrial ribosomal RNA in Candida utilis. Hybridization studies. Angeline REBOUL and Pierre VmNAIS ('). D~partement de R e c h e r c h e Fondamentale (Biochimie), Centre d'Etudes Nucl~aires et Unioersit~ Scienti[ique et M~dicale de Grenoble, BP n ° 85 .-- 38041 Grenoble - - C~dex (France). Summary. - - 1) This paper reports studies of the hybridization between RNAs isolated from purified mitoribosomes and mitochondrial DNA of Candida utilis. 2) Mitoehondrial rRNA's appeared to have sequences complementary to mitochondrial DNA. The average value for the hybridization plateau was 4.0 ± 0.2 p. cent, a result which points to the presence in mitochondrial DNA of only one cistron for each mitochondrial rRNA. 3) The hybridization between mitochondrial rRNA and mitoehondrial DNA was not reduced by competition with cytoplasmic rRNA. 4) Mitoehondrial rRNA hybridized with nuclear DNA to a level of 0.9 p. cent. Competition with cytoplasmic rRNA reduced this hybridization to 0.2 p. cent.
INTRODUCTION. Mitochondrial ribosomes (mitoribosomes) have a n u m b e r of distin,ctive features, such as size, RNA a n d p r o t e i n composition, a n d sensitivity to a n t i b i o t i c i n h i b i t o r s , w h i c h clearly d i s t i n g u i s h them from the c y t o p l a s m i c ribosomes (cytoribosornes) of the same cell [1]. It is n o w r e a s o n a b l y certain that the majority, if not all, of the proteins of the mitochon;drial r i b o s o m e are coded for a n d synthesized by the n u c l e o c y t o p l a s m i c system [2, 3], w h i l e the p r i n c i p a l r i b o s o m a l RNA (rRNA) species are t r a n s c r i b e d from m i t o c h o n d r i a l DNA [2, 4]. Several areas of u n c e r t a i n t y still r e m a i n : one c o n c e r n s the n u m b e r of cistrons in m i t o c h o n drial DNA w h i c h code for m i t o c h o n d r i a l rRNA ; a n o t h e r c o n c e r n s the possible d u p l i c a t i o n of these genes in n u c l e a r DNA. Both possibilities have been raised on the basis of e x p e r i m e n t a l data o b t a i n e d by h y b r i d i z a t i o n of m i t o c h o n d r i a l rRNA w i t h m i t o c h o n d r i a l a n d n u c l e a r DNA. Yeast is a suitable material for the isolation in good yield of m i t o c h o n d r i a l rRNA because of the relatively large n u m b e r of ribosomes in yeast m i t o c h o n d r i a . I n this laboratory, purified mitoribosomes have been o b t a i n e d from Candida utilis a n d t h e i r m o n o m e r i c form and s u b u n i t structure (*) With the technical assistance of R. Cesarini. Abbreoiations : SDS : Sodium dodeeyl sulfate, I × SSC : 150 mM NaCI, 15 mM sodium citrate pH 7.0, EtBr: Ethidium bromide. Abbreviations for nucleic acids follow CBN rules, Eur. J. Biochem., 15, (1970) 203.
have been characterized on the basis of m o r p h o ligical, p h y s i c a l a n n chemical criteria [5]. The results r e p o r t e d in this paper, u s i n g rRNA's prep a r e d from mitoribosomes of C. utilis, show that the m i t o c h o n d r i a l DNA of this o r g a n i s m c o n t a i n s only one cistron for each type of rRNA molecule, (10S a n d 21S). EXPERIMENTAL PROCEDURE. Materials.
DNAase (I) was obtained from Sigma, RNAase A (Bovine Pancreas) from P.L. Biochemieals, a n d P r o n a s e from Calbioehem. 2-04C)uracil, 6-(all)thymine, w e r e o b t a i n e d from the Commissariat h l ' E n e r g i e Atomique Saclay. Preparation of mitochondria. Candida utilis (strain CBS 1516) was g r o w n for 18hr at 280C u n d e r forced aeration i n f e r m e n t o r jars c o n t a i n i n g 10 liters of a m e d i u m made of 1 p. cent yeast extract, 2 p. cent bacto-peplone a n d 1.5 p. cent glucose (standard m e d i u m ) . W h e n m i t o c h o n d r i a w e r e used for p r e p a r i n g t h y m i n e labelled DNA, the c o n c e n t r a t i o n of the yeast extract was decreased to 0.05 p. cent. After m e c h a n i c a l d i s r u p t i o n of cells, m i t o c h o n d r i a were isolated a c c o r d i n g to Mattoon a n d Sherm a n [6]. Preparation o[ DNA.
M i t o c h o n d r i a l DNA was p r e p a r e d by a procedure slightly modified from that of Sehiifer et al. [7]. M i t o c h o n d r i a were s u s p e n d e d i n 0.44 M
270
A. Reboul and P. Vignais.
sucrose, 5 mM MgCl_o, 20 mM Tris HC1, pH 7.9, (15 mg p r o t e i n per ml) and i n c u b a t e d with DNAase I (0.1 m g / m l ) for 30 m i n at 4°C. The r e a c t i o n was stopped b y a d d i n g 0.2 volume of 0.5 M EDTA, pH 7.0. The s u s p e n s i o n was then diluted 3 times w i t h 0.44 M sucrose, 100 mM EDTA, 10 mM Tris-HC1, pH 8.0 a n d centrifuged for 20 rain. at 15 000 g. The m i t o c h o n d r i a l pellet was s u s p e n d e d in the same m e d i u m to about 100 m g / m l , a n d m i x e d w i t h 2 volumes 2.2 M sucrose, 100 mM EDTA, 10 mM Tris-HC1, pH 8.0. T h e n 0.05 volume RNase A (2 m g / m l ) in 0.15 M NaCI pH 5.1, p r e v i o u s l y heated for 10 m i n at 90°C a n d 0.25 volume P r o n a s e (20 m g / m l ) p r e v i o u s l y self digested for 2 h r at 37°C were added to this suspension. The m i x t u r e 'was dialyzed for 17 h r at 37°C against about 50 volumes 40 p. cent sucrose, 10 mM Tris-HC1, 100 mM EDTA, 1 p. cent SDS, pH 8.0 at 4°C w i t h several changes of the dialysis solution. The dialyzate was c o n c e n t r a t e d 2 to 3 fold p u t t i n g the dialysis bag on a bed of d r y Sephadex G200, a n d then centrifuged for 15 rain at 75,000 g in a Spinco model 30 rotor. The s u p e r n a t a n t was m i x e d w i t h 1.3 g CsC1 per ml to give a d e n s i t y of 1.7 m g / m l (n20 = 1.403), a n d centrifuged i n a Spinco model 40 rotor at 90,000 g for 45 h r at 20°C. After centrifugation, the contents of the tubes were p u s h e d w i l h m e r c u r y through a flow cell of a LKB U v i c o r d for analysis of a b s o r b a n c e at 254 nm, a n d collected into fractions of 0.15 ml. The p o r t i o n s of the g r a d i e n t c o r r e s p o n d i n g to m i t o c h o n d r i a l DNA were pooled, dialyzed extensively against 1 × SSC, and precip i t a t e d by 2 volumes ethanol. The p r e c i p i t a t e was collected by centrifugation, d r i e d u n d e r v a c u u m , a n d stored at 0°C. N u c l e a r DNA was p r e p a r e d from w h o l e ceils essentially as described by Schfifer et al. ET], and purified by CsC1 gradient centrifugation. W h e n l a b e l l i n g of DNA was necessary, the culture m e d i u m was s u p p l e m e n t e d w i t h 25 mCi p e r liter of [aH] labelled t h y m i n e .
Preparation of mitoribosomes. Mitoribosomes w e r e p r e p a r e d as described by Vignais et al. [5]. Mitochon,dria w e r e s u s p e n d e d i n 10 mM Tris-HC1, 10 mM MgCle, pH 7.5 at the c o n c e n t r a t i o n of 10 mg per m l a n d an equal volume of 0.5 p. cent DOC, 1 mM Tris-HC1, pH 7.5 was added d r o p w i s e (ratio of DOC to m i t o c h o n drial p r o t e i n s equal to 0.5). After s t a n d i n v for 10 minutes, the m i x t u r e was centrifuged at 17,500 r p m for 20 m i n in a Spinco model 30 rotor. The r e s u l t i n g s u p e r n a t a n t was s p u n at 29,000 r p m for 2 hours in the same rotor. The recovered ribos o m a l pellet was w a s h e d twice w i t h 10 mM Tris-
BIOCHIMIE, 1974, 56, n ° 2.
HC1, 100 mM NH~C1, 5 mM MgCle, p t l 7.5, suspended in the same m e d i u m (1 ml for 100 mg mitoc h o n d r i a l protein) a n d clarified bv a spin at 2 000 g for 5 minutes.
Preparation of rRNA. rRNA was always p r e p a r e d from isolated ribosomes. I n the first stage of this w o r k p h e n o l i c extraction was used [8] ; but in most of the later e x p e r i m e n t s we a p p l i e d the method described by F o r e s t e r et aI. EOJ, after slight modifications, w h i c h yielded a more stable RNA p r e p a r a t i o n . According to this last p r o c e d u r e m i t o r i b o s o m e s p r e p a r e d as described above a n d s u s p e n d e d in 10 mM Tris-HC1, 100 mM NH4C1, 5 mM MgC1z, pH 7.5 (1 ml for 100 mg m i t o c h o n d r i a l protein) were stirred w i t h 0.1 volume ethyl pyrocarbona,~e for 10 m i n at 4°C. A solution of 10 p. cent SDS in 10 mM Tris-HC1, 100 mM NH4C1, 5 mM MgCI.,, pH 7.5 was added to give a final c o n c e n t r a t i o n of 2.5 p. cent SDS, a n d the m i x t u r e , was centrifuged for 10 m i n at 5,000 g. The s u p e r n a t a n t was made 1 M NaC1 w i t h solid NaC1 a n d centrifuged for 30 m i n at 15,0.00 g. After centrifugation, 0,1 volume ethyl p y r o c a r b o n a t e was added to the supern a t a n t , a n d RNA was p r e c i p i t a t e d by a d d i t i o n of 2 volumes cold ethanol a n d s t a n d i n g at least 10 h r at - - 2 ' 0 o C. RNA from cytoribosomes w a s p r e p a r e d accord i n g to the same procedure. [14C]RNA was p r e p a r e d as described above from cells cultivated in p r e s e n c e of ~14C]uraeil, (0.2 mCi per liter).
Subfractionation of mitochondrial rRNA. Mitochondrial rRNA was diluted in 10 m M TrisHC1, 100 mM NH~C1, ~ mM EDTA, pH 7.5, a n d l a y e r e d on a l i n e a r g r a d i e n t of 5-20 p. cent sucrose in the same buffer. After c e n t r i f u g a t i o n at 4°C for 4hr at 45,000 r p m in a SWS0 rotor, the tube was p u n c t u r e d a n d the elution was performed from top to bottom b y p u s h i n g a solution of 40 p. cent sucrose into the bottom of the tube w i t h a s y r i n g e p u m p . E l u t i o n of the fractions was m o n i t o r e d w i t h an U v i c o r d cell at 2.54 nm. F r a c tions c o r r e s p o n d i n g to the 16S a n d 21S peaks were pooled, a n d p r e c i p i t a t e d b y 2 volumes ethanol at 20°C pooled d u r i n g one night.
Hybridization. H y b r i d i z a t i o n was c a r r i e d out a c c o r d i n g to Gillepsie a n d Spiegelman [10]. Schleicher and Schuell B6 filters were soaked and w a s h e d with 2~5 ml 2 × SSC before l o a d i n g of DNA. DNA in 8 volumes 0.01 × SSC was alkali d e n a t u r e d by 1 volume 1 M NaOH for 15 m i n at room tempe-
Origin of mitochondrial rRNA. rature, n e u t r a l i z e d by 1 v o l u m e 2 M NaHePO 4, q u i c k l y cooled to 2-4°C a n d a l l o w e d to pass t h r o u g h each filter, in 2.5 ml 2 X SSC, w i t h o u t suction. T h e filters w e r e w a s h e d w i t h 10 ml 2 × SSC and d r i e d o v e r n i g h t u n d e r v a c u u m . H y b r i d i z a t i o n w a s e a r r i e d out at 65°C-67°C in s c i n t i l l a t i o n vials. T h e filters (one p e r vial) w e r e c o v e r e d by 5 ml 2 X SSC c o n t a i n i n g the rRNA to be h y b r i d i z e d . After a 20 h r i n c u b a t i o n , the vials w e r e cooled to r o o m t e m p e r a t u r e and the filters w e r e r e m o v e d , w a s h e d w i t h several changes of 2 X SSC, and i n c u b a t e d for 1 h r at r o o m t e m p e r a t u r e w i t h 5 ml 2 X SSC c o n t a i n i n g 100 :~g RNAase A. After RNAase treatment, the filters w e r e again w a s h e d w i t h 2 X SSC, a n d then dried. T h e h y b r i d i z e d DNA and RNA r e m a i n i n g on the filters 'were e s t i m a t e d f r o m aH and z4C eounts r e s p e c t i v e l y . T h e non specific b i n d i n g of RNA w a s estimated in the same c o n d i t i o n s by using filters c o n t a i n i n g no DNA. Before counting, the filters w e r e d i s s o l v e d in vials c o n t a i n i n g 100 g n a p h t a l e n e , 6 g of 2-5 d i p h e n y l o x a z o l e and 300 mg o.f p-bis [2-5(phenyl-oxazolyl)] b e n z e n e p e r liter of 1,4-dioxanne. H y b r i d i z a t i o n b e t w e e n n u c l e a r DNA and mitoe h o n d r i a l DNA w a s p e r f o r m e d using the DNADNA h y b r i d i z a t i o n m e t h o d d e s c r i b e d by Kourilsky et al. [11].
271
tion obtained w i t h the 16S and 21S m i t o c h o n d r i a l rRNA c o m p a r e d to the value o b t a i n e d for total m i t o c h o n d r i a l rRNA. The sum of the h y b r i d i z a -
ZlS 21S t MITO rRNA's 165 25S 17S "E
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Diredion of migr~ion ~ A 21S
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"6 RESULTS. O .Q
Purity of mitochondrial ribosomal RNA. M i t o c h o n d r i a i rRNA w a s a l w a y s p r e p a r e d f r o m isolated m i t o r i b o s o m e s . T h e p u r i t y of m i t o c h o n d r i a l rRNA p r e p a r a t i o n s was assessed by elect r o p h o r e s i s (fig. 1A) or c e n t r i f u g a t i o n (fig. /B). M i t o c h o n d r i a l rRNA w a s a p p a r e n t l y not d e g r a d e d and not significantly c o n t a m i n e d w i t h cytoplasm i c rRNA. E l e c t r o p h o r e s i s was m o r e c o n v e n i e n t in r o u t i n e assays since the 16S and 21S m i t o c h o n drial rRNA could be r e a d i l y s e p a r a t e d f r o m the 17S a n d 25S c y t o p l a s m i c rRNA.
Hybridization of mitochondrial DNA.
mitochondrial
rRNA
with
W h e n h y b r i d i z i n g rRNA f r o m m i t o r i b o s o m e s w i t h m i t o c h o n d r i a l DNA, a w e l l - d e f i n e d saturation p l a t e a u was o b t a i n e d w h i c h established that m i t o c h o n d r i a l rRNA w a s not c o n t a m i n a t e d w i t h o t h e r m o l e c u l a r w e i g h t s m i t o c h o n d r i a l RNA's. In the e x p e r i m e n t illustrated in figure 9, the p l a t e a u of h y b r i d i z a t i o n w a s 4 p. cent, a n d w a s r e a c h e d w h e n the a m o u n t of a d d e d RNA w a s about ten times the a m o u n t of DNA r e t a i n e d on the filter. T a b l e I gives the values of s a t u r a t i o n h y b r i d i z a -
BIOCHIMIE, 1974,
56, n ° 2.
.ta <
B
FIG. 1. - - A - - Electrophoresis pattern of mitochondrial rRNA from C. utilis. Electrophoresis was performed on 2.4 p. cent polyaerylamide gel with 50 u,g ]RNA. The buffer used was 30 mM Tris-HC1, 2 mM Mg Acetate, 0.1 mM EDTA, pH 8.1 (9). The gel was in the buffer containing 0.2 p. cent SDS. Electrophoresis was carried out for 3.5 hr at 5 m A p e r gel at room temperature. The gel yeas scanned in tlie ultraviolet with a Polyfrae UV apparatus. B - - Sedimentation pattern of mitochondrial rRNA [rom C. utilis. Conditions as described in Methods. The fractions corresponding to 21S and 16S RNA (hatched) were pooled and used for hybridization with mitochondrial DNA.
tion values for 16S and 21S RNA is h i g h e r than the value found for total m i t o r i b o s o m a l RNA. This m a y be related to the high lability of the 21S RNA, w h i c h breaks easily d u r i n g its isolation into smaller c o m p o n e n t s s e d i m e n t i n g at 15-16S. It is thus possible that the 16S f r a c t i o n w h i c h w a s r e c o v e r e d f r o m the g r a d i e n t c o n t a i n e d both the g e n u i n e 16S RNA a n d some 15-16S d e g r a d a t i o n p r o d u c t s f r o m 21S R N A , l e a d i n g to an overesti-
A. Reboul and P. Vignais.
272 m a t i o n of the 16S RNA.
hybridization
value
of l h e
true
As s h o w n i n t a b l e I I h y b r i d i z a t i o n of m i t o c h o n drial rRNA w i t h m i t o c h o n d r i a l DNA w a s not prevented by cold RNA prepared from cytoribo-
s o m e s . I n t h e s e c o m p e t i t i o n e x p e r i m e n t s , suffic i e n t m i t o e h o n , d r i a l RNA w a s a l w a y s t a k e n to s a t u r a t e t h e DNA.
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Fro. 3• - - Hybridization of nuclear DNA with mitochondrial of C. utilis RNA from mitoribosomes of C. utilis. Hybridization was p e r f o r m e d as described in Methods. The DNA i n p u t was 8 gg for each filter.
]Jg rnitochondrial rRNA
Fro. 2. --- Hybridization of mitochondrial DNA ,with RNA prepared from mitoribosomes. Increasing a m o u n t s of mit rRNA were incubated w i t h filters containing 4.5 ,~g mit DNA. Hybridization w a s p e r f o r m e d as described in Methods. TABLE I.
H y b r i d i z a t i o n o[ m i t o c h o n d r i a l r R N A ' s w i t h m i t o c h o n d r i a l DNA f r o m C a n d i d a utilis. Mitoehondrial rRNA
Hybridization with wit DNA
Total . . . . . . . . . . . . . . . . 16 S . . . . . . . . . . . . . . . . . 21S . . . . . . . . . . . . . . . . .
Hybridization nuclear DNA.
Hybridization as described in Methods. For h y b r i d i zation of total m i t o e h o n d r i a l rRNA see Fig. 2. 16S and 21S RNA were separated by sucrose density gradient and precipitated w i t h EtOH. Filters hybridized with 16S and 21S RNA contained 2 't~g mJt DNA. The a m o u n t of RNA ranged between 1.5 and 40 ~g. T a . B ~ II.
Competition b e t w e e n m i t o c h o n d r i a l r R N A and c y t o p l a s m i c r R N A for h y b r i d i z a t i o n w i t h mitochondrial DN A of C a n d i d a u t i l i s .
mitochondrial
rRNA
and
M i t o c h o n d r i a l r R N A a p p e a r e d to h y b r i d i z e w i t h n u c l e a r D N A (fig. 3), 0.9 p. c e n t h y b r i d i z a t i o n b e i n g r e a c h e d at t h e s a t u r a t i o n p l a t e a u . S u c h a h i g h p e r c e n t a g e o f h y b r i d i z a t i o n is p r o b a b l y d u e to a s l i g h t c o , n t a m i n a t i o n o f m i t o r i b o s o m a l RNA b y c y t o p l a s m i c RNA as i n d i c a t e d b y t h e f a c t that c y t o p l a s m i c rRNA r e d u c e s the level of h y b r i d i z a t i o n to 0.2 p. c e n t (fig. 4). S i n c e t h i s v a l u e
4.0 p. cent + 0 . 2 ( ' ) ( 4 ) ( " ) 2.4 p. cent 3.0 p. cent
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200 1oo pg cytoplasmic rRNA
Fro. 4. - - C o m p e t i t i o n bel,ween mitochondrial rRNA and cytoplasmic rRNA for hybridization ~with nuclear DNA. H y b r i d i z a t i o n as described in Methods. Filters
~tg of competing cytoribosomal RNA 1 in the hybridization medium . . . . . . 0 35 90 80 360
containing 20 Ilxg nuclear DNA w e r e incubated w i t h 45 ,t~g m i t o r i b o s o m a l 14C-RNA and increasing a m o u n t s of RNA prepared f r o m cytoribosomes.
Hybridization as described in Methods. Filters containing 6 1o.g mitoeho,ndrial DNA were incubated w i t h 70 ,Ixg 1~C ix/itochandrial rRNA and increasing a m o u n t s of cold RNA p r e p a r e d f r o m eytoribosomes.
w a s s i g n i f i c a n t l y a b o v e b a c k g r o u n d it s u g g e s t e d s o m e h o m o l o g y b e t w e e n t h e nt~clear D N A a n d t h e m i t o c h o n d r i a l r R N A . H o w e v e r , if s u c h w e r e
BIOCHIMIE, 1974, 56, n ° 2.
Origin of mitochondrial r R N A . the case, one between the DNA, a n d we hybridization
w o u l d also expect some homology n u c l e a r DNA a n d m i t o c h o n d r i a l were u n a b l e to detect measurabIe between these DNA's.
DISCUSSION. RNA purified from isolated m i t o r i b o s o m e s of
C. utilis has been s h o w n to give h y b r i d i z a t i o n s a t u r a t i o n values of 4.0 +_ 0.2 p. cent w i t h mitoc h o n d r i a l DNA. The m o l e c u l a r weights of mitoc h o n d r i a l rRNAs calculated e m p i r i c a l l y from s e d i m e n t a t i o n p a t t e r n s in E D T A - s u p p l e m e n t e d sucros~ gradients are respectively 1.2 X 106 for the 21S RNA a n d 0.7 X 106 for the 16S RNA [5_3. Assuming a m o l e c u l a r weight of 50 × 106 for yeast m i t o c h o n d r i a l DNA [12], a h y b r i d i z a t i o n plateau of 3.8 p. cent w o u l d be expected if each m i t o c h o n d r i a l DNA molecule c o n t a i n e d one cistron for each m i t o c h o n d r i a l rRNA. Even taking into a c c o u n t some i n a c c u r a c y in the r e p o r t e d molecular weights of m i t o c h o n d r i a l DNA and RNA, our results i n d i c a t e that r e p e t i t i o n of rRNA genes i n the m i t o c h o n d r i a l DNA of C. uliIis is unlikely. The same c o n c l u s i o n has been r e a c h e d by Reijnders et al. E13] a n d Morimoto et al. [14] who studied h y b r i d i z a t i o n of rRNA from isolated m i t o r i b o s o m e s of Saccharomyces cerevisiae a n d Saccharomyces carlbergensis a n d f o u n d a plateau of 2.3-2.4 p. cent, a value w h i c h would exclude multiple copies coding for m i t o r i b o s o m a l RNA i n this m i t o c h o n d r i a l DNA. However, W i n t e r s b e r g e r et al. [1:5] using rRNA purified by sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of total m i t o c h o n d r i a l RNA from S. cerevisiae o b t a i n e d s u b s t a n t i a l l y higher values of 3.92 p. cent h y b r i d i z a t i o n w i t h 23S RNA and 4.73 p. cent w i t h 16S RNA w h i l e De Klo6t et al. [16] have c o n c l u d e d tbat only a high m o l e c u l a r weight fraction of m i t o c h o n d r i a l RNA, p r o b a b l y mRNA, h y b r i d i z e s w i t h m i t o c h o n d r i a l DNA. Similarly divergent results have been obtained w i t h Neurospora crassa. I n a p r e l i m i n a r y r e p o r t [17] Wood a n d Luck f o u n d that the 25S a n d 19S m i t o c h o n d r i a l RNA's were c o m p l e m e n t a r y to 6.1 p. cent a n d 2.8 p. cent of the m i t o c h o n d r i a l DNA, values w h i c h w o u l d c o r r e s p o n d to at least four cistrons on m i t o c h o n d r i a l DNA coding for m i t o c h o n d r i a l r R N A ; these results have been r e c e n t l y reassessed by K u r i y a m a a n d Luck [18] who f o u n d a s a t u r a t i o n value of only 3.3 p. cent for the h y b r i d i z a t i o n of m i t o c h o n d r i a l DNA w i t h m i t o c h o n d r i a l rRNA's, in agreement w i t h Sch~ifer a n d Kiintzel [19] ; these authors have noted that rRNA isolated from whole m i t o c h o n d r i a is cont a m i n a t e d by m i n o r RNA species w h i c h saturate 10-11 p. cent of m i t o c h o n d r i a l DNA. Chi a n d
BIOCHIMIE, 1 9 7 4 ,
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Suyama [20] have reported that the 21S a n d 14S m i t o c h o n d r i a l RNA's of Tetrahymena pyriformis are c o m p l e m e n t a r y to 3.8 a n d 1.9 p. cent of the m i t o c h o n d r i a l DNA respectively. Based on a value of 34 × 106 for the m o l e c u l a r weight of the mitoc h o n d r i a l DNA in Tetrahymena a n d on values of 0.927 X 106 a n d 0.431 X 106 for the m i t o c h o n drial rRNAs [21] one m a y calculate a theoretical h y b r i d i z a t i o n close to 4 p. cent, a s s u m i n g no r e d u n d a n c y of rRNA genes, w h i c h is i n the range of the e x p e r i m e n t a l value (5.7 p. cent). The mitoc h o n d r i a l DNA's of Xenopus laevis [22] and of HeLa cells [23] have also been s h o w n to c o n t a i n only one cistron for each rRNA. I n the case of HeLa cells, the genes c o d i n g for m i t o e h o n d r i a l rRNA are located on ne heavy s t r a n d of mitoc h o n d r i a l DNA. All these a p p a r e n t d i s c r e p a n c i e s b e t w e e n the results obtained w i t h rHNA p r e p a r e d from nlitoribosomes or purified from total mitoc h o n d r i a l RNA emphasize the i m p o r t a n c e of the methods used for p r e p a r i n g rRNA. I n general, higher values of h y b r i d i z a t i o n have been o b t a i n e d w i t h rRNA purified from total m i t o c h o n d r i a l RNA, suggesting that this rRNA may be contam i n e d by m i t o c h o n d r i a l mRNA. W h e n the rRNA was p r e p a r e d from mitoribosomes, h y b r i d i z a t i o n curves with a well defined s a t u r a l i o n plateau were obtained, p o i n t i n g to the absence of contam i n a n t s . A n o t h e r factor of d i s c r e p a n c y m a y be the use of 32p instead of 14C for labeling the rRNA. Since polyphosphate-like c o n t a m i n a n t s have been found in RNA p r e p a r a t i o n s [17, 24-3, the use of 32p l e a d i n g to a labeling of both RNA a n d polyphosphate could explain some low h y b r i d i z a t i o n values reported in literature. Good i n t e g r i t y of m i t o c h o n d r i a l DNA is also necessary for accurate h y b r i d i z a t i o n estimation. If m i t o c h o n d r i a l DNA is highly fragmented, p u r i f i c a t i o n by CsC1 g r a d i e n t c e n t r i f u g a t i o n m a y remove the DNA fragments c o d i n g for rRNA. The method we used for prep a r i n g DNA is s i m i l a r to the one used for prep a r i n g high m o l e c u l a r weight DNA [25]. According to the results of Schhfer a n d K/intzel [19!, it yielded intact DNA molecules w h i c h did not c o n t a i n a p p r e c i a b l e a m o u n t s of b o u n d RNA. To conclude, it a p p e a r s that some of the r e p o r t e d discrepencies m a y be due to differences i n techniques. However in spite of the a p p a r e n t discrepencies, the bulk of the data reported up to n o w a p p e a r s to p o i n t to the absence of r e d u n d a n c y of rRNA genes i n m i t o c h o n d r i a l DNA. The ratio b e t w e e n the h y b r i d i z a t i o n values of 16S a n d 21S m i t o c h o n d r i a l rRNAs with m i t o c h o n drial DNA in C. utilis is 1.25 (cf table I) ; the ratio b e t w e e n their molecular weights calculated empirically is 1.7 [5]. This difference could signify a 18
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A. Reboul
and P. Vignais.
p a r t i a l h o m o l o g y of n u c l e o t i d e s e q u e n c e b e t w e e n the two RNA molecules ; this would also explain w h y t h e s a t u r a t i o n v a l u e f o r h y b r i d i z a t i o n of m i t o c h o n d r i a l r R N A s w i t h m i t o c h o n d r i a l D N A is lower when determined with mixed rRNA than when calculated by summation of t h e v a l u e s o b t a i n e d ~vith e a c h s e p a r a t e r R N A . H o w e v e r i t c a n n o t b e e x c l u d e d t h a t t h e 16S R N A f r a c t i o n contains some degradation products arising from t h e b r e a k a g e of 21S RNA. I n d e e d w e h a v e o b s e r v e d t h a t s e p a r a t i o n o n s u c r o s e g r a d i e n t of m i t o c h o n d r i a l 16S a n d 21S r R N A ' s m a k e s t h e 21S R N A very sensitive to degradation. A base sequence homology between mitochond r i a l D N A a n d n u c l e a r D N A i n S. c e r e v i s i a e h a s b e e n r e c e n t l y r e p o r t e d , o n t h e b a s i s of D N A - D N A and DNA-RNA hybridization assays [26_]. I n C. utilis, h y b r i d i z a t i o n of m i t o c h o n d r i a l r R N A w i t h n u c l e a r D N A is r e d u c e d b y c o m p e t i t i o n w i t h c y t o r i b o s o m a l RNA. T h i s r e s u l t is i n a g r e e m e n t w i t h t h a t r e p o r t e d b y R e i j n d e r s et al. [ t 3 ] . A l t h o u g h t h e r e m a i n i n g l e v e l of h y b r i d i z a t i o n w a s n o t n e g l i g i b l e i n o u r case, w e t h i n k t h a t t h i s is n o t s u f f i c i e n t p r o o f f o r t h e e x i s t e n c e of a <> of m i t o c h o n d r i a l D N A i n t h e nucleus for we have not been able to observe any hybridization between mitochondrial DNA and n u c l e a r DNA. T h e f a c t t h a t n u c l e a r D N A h y b r i dizes very poorly with mitochondrial rRNA purified b y p r e h y b r i d i z a t i o n w i t h m i t o c h o n d r i a l D N A [24, 27] d o e s n o t f a v o r t h e h y p o t h e s i s of a <) and the ¢ D616gation G6n6rale /t la Recherche Scientifique et Technique >>. R~suM~. 1 - Dans cette c o m m u n i c a t i o n , nous r a p p o r t o n s des r6sultats sur l ' h y b r i d a t i o n e n t r e les RNA isol~s h p a r t i r de m i t o r i b o s o m e s et le DNA mitochorLdrial de Candida utilis. 2 - - Les rRNA m i t o c h o n d r i a u x poss6dent des s6queuces c o m p l 6 m e n t a i r e s du DNA m i t o c h o n d r i a l . La v a l e u r m o y e n n e p o u r le p l a t e a u d ' h y b r i d a t i o n est de 4.0 _ 0.2 p. cent, r 6 s u l t a t qui indique la pr6sence dans le DNA m i t o c h o u d r i a l d ' u u seul cistron pour chaque rRNA m i t o c h o n d r i a l .
BIOCHIMIE, 1974, 56, n ° 2.
3 - - L ' h y b r i d a t i o n e n t r e le rRNA m i t o c h o n d r i a l et le DNA m i t o c h o n d r i a l n'est pas r6duite p a r competition aver le rRNA dytoplasmique. 4 - - Le p l a t e a u d ' h y b r i d a t i o n e n t r e le rRNA mitoc h o n d r i a l et le DNA uucl6aire est de 0.9 p. cent. La com~6titiou avec le rRNA c y t o p l a s m i q u e r6duit cette h y b r i d a t i o n h 0.2 p. cent. REFERENCES. 1. Borst, P. ~ Grivell, L. A. (1972) FEBS-Letters, 13, 73-88. 2. Mason, T., Ebner, E., Poyton, R. O., Saltzgaber, J., W h a r t o n , D. C , Menucci, L. ~ Schatz, G. (1972) in Mitochondria : Biogenesis and Bioenergetics (Vand der Bergh, S., Borst, P. a n d Slater, E. C. eds), 8th Meeting of Fed. of Enr. Biochem., (1972) p. 53-69, Elsevier, Amsterdam. 3. Tzagotoff, A., Rubin, M. S. a Sierra, M. F. (19731 Biochim. Biophys. Acta, 301, 71-104. 4. Borst, P. (1972) Ann. Bey. Biochem. 41, 333-376. 5. Vignais, P. V., Stevens, B. J., Huet, J. ~ Andre, J. (1972) J. Cell. Biol., 54, 468-492. 6. Mattoon, J. R. ~ S h e r m a n , F. (1966) J. Biol. Chem., 241, 4330-4338. 7. Schiller, K. P., Biigge, G., Grandi, M. • Kiintzel, H. (1971) Eur. J. Biochem., 21, 478-488. 8. Wilson, S. L. ~ Quincey, R. V. (1969) J. Biol. Chem., 244, 1092-1096. 9. Forester, I. T. Nagley, P. ~ IAnnane, A. W. (1970) FEBS-Letters, 11, 59-61. 10. Gillepsie, D. ~ Spiegelman, S. (1965) J. Mol. Biol., 12 829-842. 11. Kourilsky, Ph., Leidner, J. ~ Tremblay, G. Y. (1971) Biochtmie, 53, 1111-1114. 12. Hollenberg C. P., Borst, P. ,¢ Van Bruggen, E. F. J. (1970) Biochim. Biophys. Acta, 209, 1-15. 13. Reijnders, L., Kleisen, C. M., Grivell, L. A. & Borst, P. (1972) Biochim. Biophys. Acta, '272, 396-407. 14. Morimoto, H., Scragg, A. H., Nekho Rocheff, J., Villa, V. ~ Halvorson, H. O. in Boaz'dman, N. K., L i n n a n e A. W. a n d Smillie, R. M. (1971) A u t o n o m g and Biogenesis of Mitochondria and chloroplasts, p. 282-292, North-Holand, Amsterdam. 15. Wintersberger, E. ~ Viehhouser, G. (1968) Nature, 220, 699-702. 16. De Klo~t, S. R., Andrean, B. A. C. & Mayo, V. S. (1971) Arch. Biochem. Biophys., 143, 17"5-186. 17. Wood, D. D. ~ Luck, D. J. L. (1969)J. Mol. Biol., 41, 211-224 18. Kuriyama, Y. ,~ Luck, D. J. L. (1973) J. Mol. Biol., 73, 425-437. 19. Sch~ifer, K. P. a Kfintzel, H. (1972) Biochem. Biophys. Res. Commun., 46, 1312-1319. 20. Chi, S. C. H. & Suyama, Y. (1970) J. Mol. Biol., 53, 531-556. 21. Suyama, Y. ~ Miura, K. (1968) Proc. Nat. Acad. Sci. U.S., ¢JO, 235-242. 22. Dawid, I. B. (1972) J. Mol. Biol., 63, 201-2!16. 23. Aloni, "~. ,~ Attardi, G. (1971) J. Mol. Biol., 55, 201216. 23. Aloni, Y. ~ Attardi, G. (1971) J. Mol. Biol., 55, 271276. 24. F u k u h a r a , H., Faures, M. ~ Genin, C. (1969) Mol. Gen. Genet., 104, 264-281. 25. Bhargava, M. M., Cramer, J. H. & Halvorson, H. O. (1972) Analyt. Biochcm., 49, 276-284. 26. Storti, R. V. ~ Sinclair, J. H. (1972) J. Cell. Biol., 55, p. 252a, Abstr. n ° 503. 27. Cohen, L. H., Hollenberg, C. P. a Borst, P. (1970) Biochim. Biophys. Acta, 224, 610-613.
Origin of mitochondrial ribosomal RNA in Candida utilis. Hybridization studies. Angeline REBOUL and Pierre VmNAIS ('). D~partement de R e c h e r c h e Fondamentale (Biochimie), Centre d'Etudes Nucl~aires et Unioersit~ Scienti[ique et M~dicale de Grenoble, BP n ° 85 .-- 38041 Grenoble - - C~dex (France). Summary. - - 1) This paper reports studies of the hybridization between RNAs isolated from purified mitoribosomes and mitochondrial DNA of Candida utilis. 2) Mitoehondrial rRNA's appeared to have sequences complementary to mitochondrial DNA. The average value for the hybridization plateau was 4.0 ± 0.2 p. cent, a result which points to the presence in mitochondrial DNA of only one cistron for each mitochondrial rRNA. 3) The hybridization between mitochondrial rRNA and mitoehondrial DNA was not reduced by competition with cytoplasmic rRNA. 4) Mitoehondrial rRNA hybridized with nuclear DNA to a level of 0.9 p. cent. Competition with cytoplasmic rRNA reduced this hybridization to 0.2 p. cent.
INTRODUCTION. Mitochondrial ribosomes (mitoribosomes) have a n u m b e r of distin,ctive features, such as size, RNA a n d p r o t e i n composition, a n d sensitivity to a n t i b i o t i c i n h i b i t o r s , w h i c h clearly d i s t i n g u i s h them from the c y t o p l a s m i c ribosomes (cytoribosornes) of the same cell [1]. It is n o w r e a s o n a b l y certain that the majority, if not all, of the proteins of the mitochon;drial r i b o s o m e are coded for a n d synthesized by the n u c l e o c y t o p l a s m i c system [2, 3], w h i l e the p r i n c i p a l r i b o s o m a l RNA (rRNA) species are t r a n s c r i b e d from m i t o c h o n d r i a l DNA [2, 4]. Several areas of u n c e r t a i n t y still r e m a i n : one c o n c e r n s the n u m b e r of cistrons in m i t o c h o n drial DNA w h i c h code for m i t o c h o n d r i a l rRNA ; a n o t h e r c o n c e r n s the possible d u p l i c a t i o n of these genes in n u c l e a r DNA. Both possibilities have been raised on the basis of e x p e r i m e n t a l data o b t a i n e d by h y b r i d i z a t i o n of m i t o c h o n d r i a l rRNA w i t h m i t o c h o n d r i a l a n d n u c l e a r DNA. Yeast is a suitable material for the isolation in good yield of m i t o c h o n d r i a l rRNA because of the relatively large n u m b e r of ribosomes in yeast m i t o c h o n d r i a . I n this laboratory, purified mitoribosomes have been o b t a i n e d from Candida utilis a n d t h e i r m o n o m e r i c form and s u b u n i t structure (*) With the technical assistance of R. Cesarini. Abbreoiations : SDS : Sodium dodeeyl sulfate, I × SSC : 150 mM NaCI, 15 mM sodium citrate pH 7.0, EtBr: Ethidium bromide. Abbreviations for nucleic acids follow CBN rules, Eur. J. Biochem., 15, (1970) 203.
have been characterized on the basis of m o r p h o ligical, p h y s i c a l a n n chemical criteria [5]. The results r e p o r t e d in this paper, u s i n g rRNA's prep a r e d from mitoribosomes of C. utilis, show that the m i t o c h o n d r i a l DNA of this o r g a n i s m c o n t a i n s only one cistron for each type of rRNA molecule, (10S a n d 21S). EXPERIMENTAL PROCEDURE. Materials.
DNAase (I) was obtained from Sigma, RNAase A (Bovine Pancreas) from P.L. Biochemieals, a n d P r o n a s e from Calbioehem. 2-04C)uracil, 6-(all)thymine, w e r e o b t a i n e d from the Commissariat h l ' E n e r g i e Atomique Saclay. Preparation of mitochondria. Candida utilis (strain CBS 1516) was g r o w n for 18hr at 280C u n d e r forced aeration i n f e r m e n t o r jars c o n t a i n i n g 10 liters of a m e d i u m made of 1 p. cent yeast extract, 2 p. cent bacto-peplone a n d 1.5 p. cent glucose (standard m e d i u m ) . W h e n m i t o c h o n d r i a w e r e used for p r e p a r i n g t h y m i n e labelled DNA, the c o n c e n t r a t i o n of the yeast extract was decreased to 0.05 p. cent. After m e c h a n i c a l d i s r u p t i o n of cells, m i t o c h o n d r i a were isolated a c c o r d i n g to Mattoon a n d Sherm a n [6]. Preparation o[ DNA.
M i t o c h o n d r i a l DNA was p r e p a r e d by a procedure slightly modified from that of Sehiifer et al. [7]. M i t o c h o n d r i a were s u s p e n d e d i n 0.44 M
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sucrose, 5 mM MgCl_o, 20 mM Tris HC1, pH 7.9, (15 mg p r o t e i n per ml) and i n c u b a t e d with DNAase I (0.1 m g / m l ) for 30 m i n at 4°C. The r e a c t i o n was stopped b y a d d i n g 0.2 volume of 0.5 M EDTA, pH 7.0. The s u s p e n s i o n was then diluted 3 times w i t h 0.44 M sucrose, 100 mM EDTA, 10 mM Tris-HC1, pH 8.0 a n d centrifuged for 20 rain. at 15 000 g. The m i t o c h o n d r i a l pellet was s u s p e n d e d in the same m e d i u m to about 100 m g / m l , a n d m i x e d w i t h 2 volumes 2.2 M sucrose, 100 mM EDTA, 10 mM Tris-HC1, pH 8.0. T h e n 0.05 volume RNase A (2 m g / m l ) in 0.15 M NaCI pH 5.1, p r e v i o u s l y heated for 10 m i n at 90°C a n d 0.25 volume P r o n a s e (20 m g / m l ) p r e v i o u s l y self digested for 2 h r at 37°C were added to this suspension. The m i x t u r e 'was dialyzed for 17 h r at 37°C against about 50 volumes 40 p. cent sucrose, 10 mM Tris-HC1, 100 mM EDTA, 1 p. cent SDS, pH 8.0 at 4°C w i t h several changes of the dialysis solution. The dialyzate was c o n c e n t r a t e d 2 to 3 fold p u t t i n g the dialysis bag on a bed of d r y Sephadex G200, a n d then centrifuged for 15 rain at 75,000 g in a Spinco model 30 rotor. The s u p e r n a t a n t was m i x e d w i t h 1.3 g CsC1 per ml to give a d e n s i t y of 1.7 m g / m l (n20 = 1.403), a n d centrifuged i n a Spinco model 40 rotor at 90,000 g for 45 h r at 20°C. After centrifugation, the contents of the tubes were p u s h e d w i l h m e r c u r y through a flow cell of a LKB U v i c o r d for analysis of a b s o r b a n c e at 254 nm, a n d collected into fractions of 0.15 ml. The p o r t i o n s of the g r a d i e n t c o r r e s p o n d i n g to m i t o c h o n d r i a l DNA were pooled, dialyzed extensively against 1 × SSC, and precip i t a t e d by 2 volumes ethanol. The p r e c i p i t a t e was collected by centrifugation, d r i e d u n d e r v a c u u m , a n d stored at 0°C. N u c l e a r DNA was p r e p a r e d from w h o l e ceils essentially as described by Schfifer et al. ET], and purified by CsC1 gradient centrifugation. W h e n l a b e l l i n g of DNA was necessary, the culture m e d i u m was s u p p l e m e n t e d w i t h 25 mCi p e r liter of [aH] labelled t h y m i n e .
Preparation of mitoribosomes. Mitoribosomes w e r e p r e p a r e d as described by Vignais et al. [5]. Mitochon,dria w e r e s u s p e n d e d i n 10 mM Tris-HC1, 10 mM MgCle, pH 7.5 at the c o n c e n t r a t i o n of 10 mg per m l a n d an equal volume of 0.5 p. cent DOC, 1 mM Tris-HC1, pH 7.5 was added d r o p w i s e (ratio of DOC to m i t o c h o n drial p r o t e i n s equal to 0.5). After s t a n d i n v for 10 minutes, the m i x t u r e was centrifuged at 17,500 r p m for 20 m i n in a Spinco model 30 rotor. The r e s u l t i n g s u p e r n a t a n t was s p u n at 29,000 r p m for 2 hours in the same rotor. The recovered ribos o m a l pellet was w a s h e d twice w i t h 10 mM Tris-
BIOCHIMIE, 1974, 56, n ° 2.
HC1, 100 mM NH~C1, 5 mM MgCle, p t l 7.5, suspended in the same m e d i u m (1 ml for 100 mg mitoc h o n d r i a l protein) a n d clarified bv a spin at 2 000 g for 5 minutes.
Preparation of rRNA. rRNA was always p r e p a r e d from isolated ribosomes. I n the first stage of this w o r k p h e n o l i c extraction was used [8] ; but in most of the later e x p e r i m e n t s we a p p l i e d the method described by F o r e s t e r et aI. EOJ, after slight modifications, w h i c h yielded a more stable RNA p r e p a r a t i o n . According to this last p r o c e d u r e m i t o r i b o s o m e s p r e p a r e d as described above a n d s u s p e n d e d in 10 mM Tris-HC1, 100 mM NH4C1, 5 mM MgC1z, pH 7.5 (1 ml for 100 mg m i t o c h o n d r i a l protein) were stirred w i t h 0.1 volume ethyl pyrocarbona,~e for 10 m i n at 4°C. A solution of 10 p. cent SDS in 10 mM Tris-HC1, 100 mM NH4C1, 5 mM MgCI.,, pH 7.5 was added to give a final c o n c e n t r a t i o n of 2.5 p. cent SDS, a n d the m i x t u r e , was centrifuged for 10 m i n at 5,000 g. The s u p e r n a t a n t was made 1 M NaC1 w i t h solid NaC1 a n d centrifuged for 30 m i n at 15,0.00 g. After centrifugation, 0,1 volume ethyl p y r o c a r b o n a t e was added to the supern a t a n t , a n d RNA was p r e c i p i t a t e d by a d d i t i o n of 2 volumes cold ethanol a n d s t a n d i n g at least 10 h r at - - 2 ' 0 o C. RNA from cytoribosomes w a s p r e p a r e d accord i n g to the same procedure. [14C]RNA was p r e p a r e d as described above from cells cultivated in p r e s e n c e of ~14C]uraeil, (0.2 mCi per liter).
Subfractionation of mitochondrial rRNA. Mitochondrial rRNA was diluted in 10 m M TrisHC1, 100 mM NH~C1, ~ mM EDTA, pH 7.5, a n d l a y e r e d on a l i n e a r g r a d i e n t of 5-20 p. cent sucrose in the same buffer. After c e n t r i f u g a t i o n at 4°C for 4hr at 45,000 r p m in a SWS0 rotor, the tube was p u n c t u r e d a n d the elution was performed from top to bottom b y p u s h i n g a solution of 40 p. cent sucrose into the bottom of the tube w i t h a s y r i n g e p u m p . E l u t i o n of the fractions was m o n i t o r e d w i t h an U v i c o r d cell at 2.54 nm. F r a c tions c o r r e s p o n d i n g to the 16S a n d 21S peaks were pooled, a n d p r e c i p i t a t e d b y 2 volumes ethanol at 20°C pooled d u r i n g one night.
Hybridization. H y b r i d i z a t i o n was c a r r i e d out a c c o r d i n g to Gillepsie a n d Spiegelman [10]. Schleicher and Schuell B6 filters were soaked and w a s h e d with 2~5 ml 2 × SSC before l o a d i n g of DNA. DNA in 8 volumes 0.01 × SSC was alkali d e n a t u r e d by 1 volume 1 M NaOH for 15 m i n at room tempe-
Origin of mitochondrial rRNA. rature, n e u t r a l i z e d by 1 v o l u m e 2 M NaHePO 4, q u i c k l y cooled to 2-4°C a n d a l l o w e d to pass t h r o u g h each filter, in 2.5 ml 2 X SSC, w i t h o u t suction. T h e filters w e r e w a s h e d w i t h 10 ml 2 × SSC and d r i e d o v e r n i g h t u n d e r v a c u u m . H y b r i d i z a t i o n w a s e a r r i e d out at 65°C-67°C in s c i n t i l l a t i o n vials. T h e filters (one p e r vial) w e r e c o v e r e d by 5 ml 2 X SSC c o n t a i n i n g the rRNA to be h y b r i d i z e d . After a 20 h r i n c u b a t i o n , the vials w e r e cooled to r o o m t e m p e r a t u r e and the filters w e r e r e m o v e d , w a s h e d w i t h several changes of 2 X SSC, and i n c u b a t e d for 1 h r at r o o m t e m p e r a t u r e w i t h 5 ml 2 X SSC c o n t a i n i n g 100 :~g RNAase A. After RNAase treatment, the filters w e r e again w a s h e d w i t h 2 X SSC, a n d then dried. T h e h y b r i d i z e d DNA and RNA r e m a i n i n g on the filters 'were e s t i m a t e d f r o m aH and z4C eounts r e s p e c t i v e l y . T h e non specific b i n d i n g of RNA w a s estimated in the same c o n d i t i o n s by using filters c o n t a i n i n g no DNA. Before counting, the filters w e r e d i s s o l v e d in vials c o n t a i n i n g 100 g n a p h t a l e n e , 6 g of 2-5 d i p h e n y l o x a z o l e and 300 mg o.f p-bis [2-5(phenyl-oxazolyl)] b e n z e n e p e r liter of 1,4-dioxanne. H y b r i d i z a t i o n b e t w e e n n u c l e a r DNA and mitoe h o n d r i a l DNA w a s p e r f o r m e d using the DNADNA h y b r i d i z a t i o n m e t h o d d e s c r i b e d by Kourilsky et al. [11].
271
tion obtained w i t h the 16S and 21S m i t o c h o n d r i a l rRNA c o m p a r e d to the value o b t a i n e d for total m i t o c h o n d r i a l rRNA. The sum of the h y b r i d i z a -
ZlS 21S t MITO rRNA's 165 25S 17S "E
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cyto rRNA's
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Diredion of migr~ion ~ A 21S
1 E E
"6 RESULTS. O .Q
Purity of mitochondrial ribosomal RNA. M i t o c h o n d r i a i rRNA w a s a l w a y s p r e p a r e d f r o m isolated m i t o r i b o s o m e s . T h e p u r i t y of m i t o c h o n d r i a l rRNA p r e p a r a t i o n s was assessed by elect r o p h o r e s i s (fig. 1A) or c e n t r i f u g a t i o n (fig. /B). M i t o c h o n d r i a l rRNA w a s a p p a r e n t l y not d e g r a d e d and not significantly c o n t a m i n e d w i t h cytoplasm i c rRNA. E l e c t r o p h o r e s i s was m o r e c o n v e n i e n t in r o u t i n e assays since the 16S and 21S m i t o c h o n drial rRNA could be r e a d i l y s e p a r a t e d f r o m the 17S a n d 25S c y t o p l a s m i c rRNA.
Hybridization of mitochondrial DNA.
mitochondrial
rRNA
with
W h e n h y b r i d i z i n g rRNA f r o m m i t o r i b o s o m e s w i t h m i t o c h o n d r i a l DNA, a w e l l - d e f i n e d saturation p l a t e a u was o b t a i n e d w h i c h established that m i t o c h o n d r i a l rRNA w a s not c o n t a m i n a t e d w i t h o t h e r m o l e c u l a r w e i g h t s m i t o c h o n d r i a l RNA's. In the e x p e r i m e n t illustrated in figure 9, the p l a t e a u of h y b r i d i z a t i o n w a s 4 p. cent, a n d w a s r e a c h e d w h e n the a m o u n t of a d d e d RNA w a s about ten times the a m o u n t of DNA r e t a i n e d on the filter. T a b l e I gives the values of s a t u r a t i o n h y b r i d i z a -
BIOCHIMIE, 1974,
56, n ° 2.
.ta <
B
FIG. 1. - - A - - Electrophoresis pattern of mitochondrial rRNA from C. utilis. Electrophoresis was performed on 2.4 p. cent polyaerylamide gel with 50 u,g ]RNA. The buffer used was 30 mM Tris-HC1, 2 mM Mg Acetate, 0.1 mM EDTA, pH 8.1 (9). The gel was in the buffer containing 0.2 p. cent SDS. Electrophoresis was carried out for 3.5 hr at 5 m A p e r gel at room temperature. The gel yeas scanned in tlie ultraviolet with a Polyfrae UV apparatus. B - - Sedimentation pattern of mitochondrial rRNA [rom C. utilis. Conditions as described in Methods. The fractions corresponding to 21S and 16S RNA (hatched) were pooled and used for hybridization with mitochondrial DNA.
tion values for 16S and 21S RNA is h i g h e r than the value found for total m i t o r i b o s o m a l RNA. This m a y be related to the high lability of the 21S RNA, w h i c h breaks easily d u r i n g its isolation into smaller c o m p o n e n t s s e d i m e n t i n g at 15-16S. It is thus possible that the 16S f r a c t i o n w h i c h w a s r e c o v e r e d f r o m the g r a d i e n t c o n t a i n e d both the g e n u i n e 16S RNA a n d some 15-16S d e g r a d a t i o n p r o d u c t s f r o m 21S R N A , l e a d i n g to an overesti-
A. Reboul and P. Vignais.
272 m a t i o n of the 16S RNA.
hybridization
value
of l h e
true
As s h o w n i n t a b l e I I h y b r i d i z a t i o n of m i t o c h o n drial rRNA w i t h m i t o c h o n d r i a l DNA w a s not prevented by cold RNA prepared from cytoribo-
s o m e s . I n t h e s e c o m p e t i t i o n e x p e r i m e n t s , suffic i e n t m i t o e h o n , d r i a l RNA w a s a l w a y s t a k e n to s a t u r a t e t h e DNA.
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Fro. 3• - - Hybridization of nuclear DNA with mitochondrial of C. utilis RNA from mitoribosomes of C. utilis. Hybridization was p e r f o r m e d as described in Methods. The DNA i n p u t was 8 gg for each filter.
]Jg rnitochondrial rRNA
Fro. 2. --- Hybridization of mitochondrial DNA ,with RNA prepared from mitoribosomes. Increasing a m o u n t s of mit rRNA were incubated w i t h filters containing 4.5 ,~g mit DNA. Hybridization w a s p e r f o r m e d as described in Methods. TABLE I.
H y b r i d i z a t i o n o[ m i t o c h o n d r i a l r R N A ' s w i t h m i t o c h o n d r i a l DNA f r o m C a n d i d a utilis. Mitoehondrial rRNA
Hybridization with wit DNA
Total . . . . . . . . . . . . . . . . 16 S . . . . . . . . . . . . . . . . . 21S . . . . . . . . . . . . . . . . .
Hybridization nuclear DNA.
Hybridization as described in Methods. For h y b r i d i zation of total m i t o e h o n d r i a l rRNA see Fig. 2. 16S and 21S RNA were separated by sucrose density gradient and precipitated w i t h EtOH. Filters hybridized with 16S and 21S RNA contained 2 't~g mJt DNA. The a m o u n t of RNA ranged between 1.5 and 40 ~g. T a . B ~ II.
Competition b e t w e e n m i t o c h o n d r i a l r R N A and c y t o p l a s m i c r R N A for h y b r i d i z a t i o n w i t h mitochondrial DN A of C a n d i d a u t i l i s .
mitochondrial
rRNA
and
M i t o c h o n d r i a l r R N A a p p e a r e d to h y b r i d i z e w i t h n u c l e a r D N A (fig. 3), 0.9 p. c e n t h y b r i d i z a t i o n b e i n g r e a c h e d at t h e s a t u r a t i o n p l a t e a u . S u c h a h i g h p e r c e n t a g e o f h y b r i d i z a t i o n is p r o b a b l y d u e to a s l i g h t c o , n t a m i n a t i o n o f m i t o r i b o s o m a l RNA b y c y t o p l a s m i c RNA as i n d i c a t e d b y t h e f a c t that c y t o p l a s m i c rRNA r e d u c e s the level of h y b r i d i z a t i o n to 0.2 p. c e n t (fig. 4). S i n c e t h i s v a l u e
4.0 p. cent + 0 . 2 ( ' ) ( 4 ) ( " ) 2.4 p. cent 3.0 p. cent
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Fro. 4. - - C o m p e t i t i o n bel,ween mitochondrial rRNA and cytoplasmic rRNA for hybridization ~with nuclear DNA. H y b r i d i z a t i o n as described in Methods. Filters
~tg of competing cytoribosomal RNA 1 in the hybridization medium . . . . . . 0 35 90 80 360
containing 20 Ilxg nuclear DNA w e r e incubated w i t h 45 ,t~g m i t o r i b o s o m a l 14C-RNA and increasing a m o u n t s of RNA prepared f r o m cytoribosomes.
Hybridization as described in Methods. Filters containing 6 1o.g mitoeho,ndrial DNA were incubated w i t h 70 ,Ixg 1~C ix/itochandrial rRNA and increasing a m o u n t s of cold RNA p r e p a r e d f r o m eytoribosomes.
w a s s i g n i f i c a n t l y a b o v e b a c k g r o u n d it s u g g e s t e d s o m e h o m o l o g y b e t w e e n t h e nt~clear D N A a n d t h e m i t o c h o n d r i a l r R N A . H o w e v e r , if s u c h w e r e
BIOCHIMIE, 1974, 56, n ° 2.
Origin of mitochondrial r R N A . the case, one between the DNA, a n d we hybridization
w o u l d also expect some homology n u c l e a r DNA a n d m i t o c h o n d r i a l were u n a b l e to detect measurabIe between these DNA's.
DISCUSSION. RNA purified from isolated m i t o r i b o s o m e s of
C. utilis has been s h o w n to give h y b r i d i z a t i o n s a t u r a t i o n values of 4.0 +_ 0.2 p. cent w i t h mitoc h o n d r i a l DNA. The m o l e c u l a r weights of mitoc h o n d r i a l rRNAs calculated e m p i r i c a l l y from s e d i m e n t a t i o n p a t t e r n s in E D T A - s u p p l e m e n t e d sucros~ gradients are respectively 1.2 X 106 for the 21S RNA a n d 0.7 X 106 for the 16S RNA [5_3. Assuming a m o l e c u l a r weight of 50 × 106 for yeast m i t o c h o n d r i a l DNA [12], a h y b r i d i z a t i o n plateau of 3.8 p. cent w o u l d be expected if each m i t o c h o n d r i a l DNA molecule c o n t a i n e d one cistron for each m i t o c h o n d r i a l rRNA. Even taking into a c c o u n t some i n a c c u r a c y in the r e p o r t e d molecular weights of m i t o c h o n d r i a l DNA and RNA, our results i n d i c a t e that r e p e t i t i o n of rRNA genes i n the m i t o c h o n d r i a l DNA of C. uliIis is unlikely. The same c o n c l u s i o n has been r e a c h e d by Reijnders et al. E13] a n d Morimoto et al. [14] who studied h y b r i d i z a t i o n of rRNA from isolated m i t o r i b o s o m e s of Saccharomyces cerevisiae a n d Saccharomyces carlbergensis a n d f o u n d a plateau of 2.3-2.4 p. cent, a value w h i c h would exclude multiple copies coding for m i t o r i b o s o m a l RNA i n this m i t o c h o n d r i a l DNA. However, W i n t e r s b e r g e r et al. [1:5] using rRNA purified by sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of total m i t o c h o n d r i a l RNA from S. cerevisiae o b t a i n e d s u b s t a n t i a l l y higher values of 3.92 p. cent h y b r i d i z a t i o n w i t h 23S RNA and 4.73 p. cent w i t h 16S RNA w h i l e De Klo6t et al. [16] have c o n c l u d e d tbat only a high m o l e c u l a r weight fraction of m i t o c h o n d r i a l RNA, p r o b a b l y mRNA, h y b r i d i z e s w i t h m i t o c h o n d r i a l DNA. Similarly divergent results have been obtained w i t h Neurospora crassa. I n a p r e l i m i n a r y r e p o r t [17] Wood a n d Luck f o u n d that the 25S a n d 19S m i t o c h o n d r i a l RNA's were c o m p l e m e n t a r y to 6.1 p. cent a n d 2.8 p. cent of the m i t o c h o n d r i a l DNA, values w h i c h w o u l d c o r r e s p o n d to at least four cistrons on m i t o c h o n d r i a l DNA coding for m i t o c h o n d r i a l r R N A ; these results have been r e c e n t l y reassessed by K u r i y a m a a n d Luck [18] who f o u n d a s a t u r a t i o n value of only 3.3 p. cent for the h y b r i d i z a t i o n of m i t o c h o n d r i a l DNA w i t h m i t o c h o n d r i a l rRNA's, in agreement w i t h Sch~ifer a n d Kiintzel [19] ; these authors have noted that rRNA isolated from whole m i t o c h o n d r i a is cont a m i n a t e d by m i n o r RNA species w h i c h saturate 10-11 p. cent of m i t o c h o n d r i a l DNA. Chi a n d
BIOCHIMIE, 1 9 7 4 ,
56, n ° 2.
273
Suyama [20] have reported that the 21S a n d 14S m i t o c h o n d r i a l RNA's of Tetrahymena pyriformis are c o m p l e m e n t a r y to 3.8 a n d 1.9 p. cent of the m i t o c h o n d r i a l DNA respectively. Based on a value of 34 × 106 for the m o l e c u l a r weight of the mitoc h o n d r i a l DNA in Tetrahymena a n d on values of 0.927 X 106 a n d 0.431 X 106 for the m i t o c h o n drial rRNAs [21] one m a y calculate a theoretical h y b r i d i z a t i o n close to 4 p. cent, a s s u m i n g no r e d u n d a n c y of rRNA genes, w h i c h is i n the range of the e x p e r i m e n t a l value (5.7 p. cent). The mitoc h o n d r i a l DNA's of Xenopus laevis [22] and of HeLa cells [23] have also been s h o w n to c o n t a i n only one cistron for each rRNA. I n the case of HeLa cells, the genes c o d i n g for m i t o e h o n d r i a l rRNA are located on ne heavy s t r a n d of mitoc h o n d r i a l DNA. All these a p p a r e n t d i s c r e p a n c i e s b e t w e e n the results obtained w i t h rHNA p r e p a r e d from nlitoribosomes or purified from total mitoc h o n d r i a l RNA emphasize the i m p o r t a n c e of the methods used for p r e p a r i n g rRNA. I n general, higher values of h y b r i d i z a t i o n have been o b t a i n e d w i t h rRNA purified from total m i t o c h o n d r i a l RNA, suggesting that this rRNA may be contam i n e d by m i t o c h o n d r i a l mRNA. W h e n the rRNA was p r e p a r e d from mitoribosomes, h y b r i d i z a t i o n curves with a well defined s a t u r a l i o n plateau were obtained, p o i n t i n g to the absence of contam i n a n t s . A n o t h e r factor of d i s c r e p a n c y m a y be the use of 32p instead of 14C for labeling the rRNA. Since polyphosphate-like c o n t a m i n a n t s have been found in RNA p r e p a r a t i o n s [17, 24-3, the use of 32p l e a d i n g to a labeling of both RNA a n d polyphosphate could explain some low h y b r i d i z a t i o n values reported in literature. Good i n t e g r i t y of m i t o c h o n d r i a l DNA is also necessary for accurate h y b r i d i z a t i o n estimation. If m i t o c h o n d r i a l DNA is highly fragmented, p u r i f i c a t i o n by CsC1 g r a d i e n t c e n t r i f u g a t i o n m a y remove the DNA fragments c o d i n g for rRNA. The method we used for prep a r i n g DNA is s i m i l a r to the one used for prep a r i n g high m o l e c u l a r weight DNA [25]. According to the results of Schhfer a n d K/intzel [19!, it yielded intact DNA molecules w h i c h did not c o n t a i n a p p r e c i a b l e a m o u n t s of b o u n d RNA. To conclude, it a p p e a r s that some of the r e p o r t e d discrepencies m a y be due to differences i n techniques. However in spite of the a p p a r e n t discrepencies, the bulk of the data reported up to n o w a p p e a r s to p o i n t to the absence of r e d u n d a n c y of rRNA genes i n m i t o c h o n d r i a l DNA. The ratio b e t w e e n the h y b r i d i z a t i o n values of 16S a n d 21S m i t o c h o n d r i a l rRNAs with m i t o c h o n drial DNA in C. utilis is 1.25 (cf table I) ; the ratio b e t w e e n their molecular weights calculated empirically is 1.7 [5]. This difference could signify a 18
274
A. Reboul
and P. Vignais.
p a r t i a l h o m o l o g y of n u c l e o t i d e s e q u e n c e b e t w e e n the two RNA molecules ; this would also explain w h y t h e s a t u r a t i o n v a l u e f o r h y b r i d i z a t i o n of m i t o c h o n d r i a l r R N A s w i t h m i t o c h o n d r i a l D N A is lower when determined with mixed rRNA than when calculated by summation of t h e v a l u e s o b t a i n e d ~vith e a c h s e p a r a t e r R N A . H o w e v e r i t c a n n o t b e e x c l u d e d t h a t t h e 16S R N A f r a c t i o n contains some degradation products arising from t h e b r e a k a g e of 21S RNA. I n d e e d w e h a v e o b s e r v e d t h a t s e p a r a t i o n o n s u c r o s e g r a d i e n t of m i t o c h o n d r i a l 16S a n d 21S r R N A ' s m a k e s t h e 21S R N A very sensitive to degradation. A base sequence homology between mitochond r i a l D N A a n d n u c l e a r D N A i n S. c e r e v i s i a e h a s b e e n r e c e n t l y r e p o r t e d , o n t h e b a s i s of D N A - D N A and DNA-RNA hybridization assays [26_]. I n C. utilis, h y b r i d i z a t i o n of m i t o c h o n d r i a l r R N A w i t h n u c l e a r D N A is r e d u c e d b y c o m p e t i t i o n w i t h c y t o r i b o s o m a l RNA. T h i s r e s u l t is i n a g r e e m e n t w i t h t h a t r e p o r t e d b y R e i j n d e r s et al. [ t 3 ] . A l t h o u g h t h e r e m a i n i n g l e v e l of h y b r i d i z a t i o n w a s n o t n e g l i g i b l e i n o u r case, w e t h i n k t h a t t h i s is n o t s u f f i c i e n t p r o o f f o r t h e e x i s t e n c e of a <
BIOCHIMIE, 1974, 56, n ° 2.
3 - - L ' h y b r i d a t i o n e n t r e le rRNA m i t o c h o n d r i a l et le DNA m i t o c h o n d r i a l n'est pas r6duite p a r competition aver le rRNA dytoplasmique. 4 - - Le p l a t e a u d ' h y b r i d a t i o n e n t r e le rRNA mitoc h o n d r i a l et le DNA uucl6aire est de 0.9 p. cent. La com~6titiou avec le rRNA c y t o p l a s m i q u e r6duit cette h y b r i d a t i o n h 0.2 p. cent. REFERENCES. 1. Borst, P. ~ Grivell, L. A. (1972) FEBS-Letters, 13, 73-88. 2. Mason, T., Ebner, E., Poyton, R. O., Saltzgaber, J., W h a r t o n , D. C , Menucci, L. ~ Schatz, G. (1972) in Mitochondria : Biogenesis and Bioenergetics (Vand der Bergh, S., Borst, P. a n d Slater, E. C. eds), 8th Meeting of Fed. of Enr. Biochem., (1972) p. 53-69, Elsevier, Amsterdam. 3. Tzagotoff, A., Rubin, M. S. a Sierra, M. F. (19731 Biochim. Biophys. Acta, 301, 71-104. 4. Borst, P. (1972) Ann. Bey. Biochem. 41, 333-376. 5. Vignais, P. V., Stevens, B. J., Huet, J. ~ Andre, J. (1972) J. Cell. Biol., 54, 468-492. 6. Mattoon, J. R. ~ S h e r m a n , F. (1966) J. Biol. Chem., 241, 4330-4338. 7. Schiller, K. P., Biigge, G., Grandi, M. • Kiintzel, H. (1971) Eur. J. Biochem., 21, 478-488. 8. Wilson, S. L. ~ Quincey, R. V. (1969) J. Biol. Chem., 244, 1092-1096. 9. Forester, I. T. Nagley, P. ~ IAnnane, A. W. (1970) FEBS-Letters, 11, 59-61. 10. Gillepsie, D. ~ Spiegelman, S. (1965) J. Mol. Biol., 12 829-842. 11. Kourilsky, Ph., Leidner, J. ~ Tremblay, G. Y. (1971) Biochtmie, 53, 1111-1114. 12. Hollenberg C. P., Borst, P. ,¢ Van Bruggen, E. F. J. (1970) Biochim. Biophys. Acta, 209, 1-15. 13. Reijnders, L., Kleisen, C. M., Grivell, L. A. & Borst, P. (1972) Biochim. Biophys. Acta, '272, 396-407. 14. Morimoto, H., Scragg, A. H., Nekho Rocheff, J., Villa, V. ~ Halvorson, H. O. in Boaz'dman, N. K., L i n n a n e A. W. a n d Smillie, R. M. (1971) A u t o n o m g and Biogenesis of Mitochondria and chloroplasts, p. 282-292, North-Holand, Amsterdam. 15. Wintersberger, E. ~ Viehhouser, G. (1968) Nature, 220, 699-702. 16. De Klo~t, S. R., Andrean, B. A. C. & Mayo, V. S. (1971) Arch. Biochem. Biophys., 143, 17"5-186. 17. Wood, D. D. ~ Luck, D. J. L. (1969)J. Mol. Biol., 41, 211-224 18. Kuriyama, Y. ,~ Luck, D. J. L. (1973) J. Mol. Biol., 73, 425-437. 19. Sch~ifer, K. P. a Kfintzel, H. (1972) Biochem. Biophys. Res. Commun., 46, 1312-1319. 20. Chi, S. C. H. & Suyama, Y. (1970) J. Mol. Biol., 53, 531-556. 21. Suyama, Y. ~ Miura, K. (1968) Proc. Nat. Acad. Sci. U.S., ¢JO, 235-242. 22. Dawid, I. B. (1972) J. Mol. Biol., 63, 201-2!16. 23. Aloni, "~. ,~ Attardi, G. (1971) J. Mol. Biol., 55, 201216. 23. Aloni, Y. ~ Attardi, G. (1971) J. Mol. Biol., 55, 271276. 24. F u k u h a r a , H., Faures, M. ~ Genin, C. (1969) Mol. Gen. Genet., 104, 264-281. 25. Bhargava, M. M., Cramer, J. H. & Halvorson, H. O. (1972) Analyt. Biochcm., 49, 276-284. 26. Storti, R. V. ~ Sinclair, J. H. (1972) J. Cell. Biol., 55, p. 252a, Abstr. n ° 503. 27. Cohen, L. H., Hollenberg, C. P. a Borst, P. (1970) Biochim. Biophys. Acta, 224, 610-613.