Molec. gen. Genet. 168, 101 -109 (1979) © by Springer-Verlag 1979

Inserted Sequence in the Mitochondrial 23S Ribosomal RNA Gene of the Yeast Saccharomyces cerevisiae G6rard Faye, Nicole Dennebouy 1, Chantal Kujawa 2, and Claude Jacq 2 Institut Curie*, Biologic, Centre Universitaire, Brit. 110, F-91405 Orsay, 1 Laboratoire de BiologicG6n6rale, Universit6 de Paris Sud, Brit. 400, F-91405 Orsay, 2 Centre de G+n~tique Mol6culaire du CNRS F-91190Gif sur Yvette, France.

Summary. The sequence organization of the yeast mitD N A region carrying the large ribosomal R N A gene and the polar locus co was examined. Hybridization studies using r h o - deletion mutants and electron microscopy of the heteroduplexes formed between 23S r R N A and the appropriate restriction fragments, lead to the conclusion that the 23S r R N A t gene of the co + strains is split by an insertion sequence of 1,000-1,100 bp. In contrast, no detactable insertion was found in the 23S r R N A gene of the co- strains. The size and the location of the insert found in the 23S r R N A gene of the co+ strains appear to be identical to those of the sequence A which had previously been found to characterize the difference (at the co locus) between the m i t D N A of the wild type strains carrying the co+ or co- alleles (Jacq et al., 1977).

Introduction The circular mitochondrial D N A of S a c c h a r o m y c e s c e r e v i s i a e contains about 75,000 base pairs (Hollenberg et al., 1970). It carries two ribosomal R N A genes: one for the large 23S ribosomal RNA, the other for the small 16S rRNA. These two r R N A genes are separated by a segment of about 30 kb long (Faye et al., 1975; Sanders et al., 1975) which contains at least eighteen t R N A genes (Martin et al., 1977) and the genes for two subunits of the cytochrome c-oxidase (Cabral et al., 1978). Three loci Rib I, Rib II and Rib III (also called R I, R II and R III) (Plischke et al., 1976) have been localized in the region of the * Formerly, Fondation Curie - Institut du Radium, Section de Biologic For offprints contact: C. Jacq a Abret'iations: rRNA: ribosomal RNA; mitDNA: mitochondrial DNA; r-protein: ribosomal protein; bp: base pair; S.D.: standard deviation

23S r R N A gene. Mutations at these loci confer mitochondrial resistance to chloramphenicol for R I, to erythromycin or/and spiramycin for R II and R I I I (Netter et al., 1974). The mitoribosomes of R I or R I I I mutants have been shown to be resistant to chloramphenicol or erythromycin respectively by in vitro functional test (Grivell et al., 1973). In the vicinity of the 23S r R N A gene, another locus called co has been genetically characterized and biochemically localized. Two allelic forms of co have been described: co+ and co-. Both forms give identical respiratory competent phenotypes. Crosses between co+ and co- strain exhibit a significant polarity of recombination in the R I, R II, R I I I region (polar region), this phenomenon is thought to be initiated at the co locus (Bolotin et al., 1971; Netter et al., 1974). The biochemical characterization and localization of the co alleles have been recently published (Dujon and Michel, 1976; Jacq et al., 1977; Borst et al., 1977; Morimoto et al., 1978). The loci R II, R I, co, R I I I have been mapped in this order, R I being very close to co. Biochemically, the difference between the two alleles co+ and co appeared to be a D N A sequence called A (Jacq et al., 1977). This sequence, 1,000 1,100 base pairs long, is present in all co+ strains examined, whereas it is absent in costrains; the sequences on both sides of co showed no difference between co+ and co mitDNAs. The detailed genetic implications of these results will be presented elsewhere. A set of r h o - mitochondrial DNAs carrying the R I or/and R I I I loci have been studied by hybridization with 23S r R N A (Faye et al., 1974; Faye et al., 1975). Also fine dissection by restriction enzymes of wild type m i t D N A and of several r h o - mitDNAs has established the physical map of this region (Jacq et al., 1977; Borst et al., 1977; Morimoto et al., 1978). These results have led to the conclusion that the R I I I locus is in the 23S r R N A coding sequence.

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G. Faye et al. : Mitochondrial 23S Ribosomal RNA Gene of the Yeast Saccharomyces cerevisiae

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The question whether R I and R II are in the 2 3 S r R N A c o d i n g s e q u e n c e is still c o n t r o v e r t e d . A t first, w e s u p p o s e d t h a t t h e R I l o c u s w a s o u t s i d e t h e 23S r R N A g e n e b u t v e r y c l o s e t o o n e o f its e x t r e m i t i e s ( F a y e et al., 1974). O n e p o s s i b i l i t y t h e r e f o r e e x i s t e d that the R I locus might affect one mitoribosomal p r o t e i n , a l t h o u g h o n l y a few, i f a n y , m i t o r i b o s o m a l proteins seemed to be translated on the mitoribosomes. However, analysis of mitoribosomal proteins of the wild type and R I mutants by two-dimensional p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s d i d n o t s h o w a n y differences in the migration of the r-proteins of the 37S a n d 50S r i b o s o m a l s u b u n i t s ( F a y e , 1977). I f t h e R I I a n d R I l o c i a r e r e a l l y l o c a l i z e d i n t h e 23S r R N A sequence, then a possibility arises that the sequence A is i n s e r t e d i n t h e 2 3 S r R N A g e n e . T o v e r i f y t h i s hypothesis we have carried out hybridization experiments and electron microscopy analyses of rRNADNA duplexes.

Materials and Methods Yeast strains Table 1 is a list of the strains used and their genetic markers in the Rib region.

Preparation of Yeast mitDNAs All mitDNAs were isolated from purified mitochondria prepared from protoplasts according to Petzuch (Petzuch, 1971 ; Faye et al., 1974). DNAs were then extracted from sodium dodecylsulphate lysates of mitochondria followed by several phenol-chloroformisoamylalcohol extractions and one precipitation with ethanol. The crude DNA preparations were then chromatographed on a small hydroxyapatite column; the bulk of RNA was removed by an extensive washing of the column with 0.2 M phosphate buffer, pH 6.8 and DNAs were eluted by 0.4 M phosphate buffer pH

Table 1. Strains used and their genotypes Name

Mitochondrial loci

IL8-8C ILS-8C/R53 KL479-2B/C721 KL263-2B/CO KL263-2B/B8 IL8-8C/D41 IL8-8C/D61 IL8-8C/C42

rho + rho rhorhorho rho rho rho-

b

Fukuhara et al. (1974) Jacq et al. (1977)

co+ co+ co 0 co+ (D+ co+ co+

cR21 C~21 C~24 cR24 cR21 cR21 C~21

+ + ERs3 ER53 ERs3 @ + +

ERI~ ER14 S~52 0 0 0 0 0

co

R I

R II

R III

cR24

a a b u b a a a

6.8. The yields of mtDNA were generally between 100 to 200 ~tg for 6 L cultures, harvested in the late exponential phase. The mitDNA preparations were contaminated by less than 5% of nuclear DNA as determined by analytical density gradient centrifugation.

RNA-DNA Hybridization 3H-labelled mitochondrial 23S rRNA (specific activity :12,000 cpm per gg) was prepared from the mitochondria of the rho + strain IL8 8C as already described (Faye et al., 1974). RNA-DNA filter hybridization was performed in 8 M urea, 0.3 M NaC1 and 0.03 M trisodium citrate at 37°C for 48 h. A constant amount (about 0.4 0.6 gg) of 23S rRNA was incubated with increasing amounts of filterbound mitDNA of rho + or rho- strains (10, 20, 30 and 40 gg per filter).

Restriction Enzyme Digestion and Purification of Restriction Fragments The restriction endonucleases Alu I and Hae III were purified according to published procedures (Yang et al., 1976; Middleton et al., 1972); Hha I was purified by an unpublished method by R.J. Roberts. Hind II and Hind III were purchased form Miles. The restriction endonuclease digests were analysed on a 6% acrylamide gel (Jeppsen, 1974) calibrated with Hae III fragments of SV 40 DNA. The elution of the restriction fragments from electrophoresis gets was carried out as described by Galibert et al. (1974).

Preparation of Mitochondrial 23S rRNA for Electron Microscopy Mitochondria of the rho ÷ strain IL8-8C were purified and total mitochondrial RNA was extracted as previously described (Faye et al., 1974). The two rRNA species were separated by sedimentation in isokinetic sucrose gradients which contained 0.02 M sodium acetate-HC1 (pH 6.0), 0.1 M NH4C1 and 0.002 sodium EDTA. Centrifugation was performed in a rotor SW 27 at 26,000 rpm for 17h at 4 ° C. Purity and molecular size of the 23S rRNA recovered were checked by polyacrylamide gel electrophoresis (Faye et al., 1974).

Electron Microscopy Merck's formamide was crystallized under continuous stirring in a beaker refrigerated at 0°C in a Colora low-temperature bath. The crystals were collected by centrifugation as described by Casey and Davidson (1977). The process was repeated 3 times. Formamide was stored at - 7 0 ° C. Hybrids between 23S rRNA and Hae III mitDNA fragments were formed according to Wellauer and Dawid (1977). Final concentrations of rRNA were 25 to 30 gg/ml and mitDNA fragments, 1 to 5 ~tg/ml. The hybridization solutions (50 ~tl) were heated at 65°C for 5 mn to denature the DNA then incubated at 50 ° C for 3 h. This temperature was chosen from an extrapolation of the data obtained by Thomas et al. (1976). Hybridization solutions were diluted 30 fold in the spreading buffer described by Wellauer and Dawid (1977) and then spread onto a distilled water hypophase. The DNA-protein films were adsorbed on carbon-coated mica disks according to Inman and Schn6s (1970). The rotary shadowing was done with platinum-palladium. The grids were viewed with a Philips 300 electron microscope (magnification 25,000).

G. Faye et al. : Mitochondrial 23S Ribosomal R N A Gene of the Yeast Saccharomyces cerevisiae

Results

1978) with the enzyme Hind II. We verified the positions of the Hind II sites relatively to the Alu I sites by using the rho R53 which covers the entire sequence of this region. This detailed restriction map has allowed us to locate two r h o - genomes called CO and B8, with good accuracy. The CO repeat unit is 1,080 bp long and contains only one Hind II site, one Hha I site, one Hind III site and two Alu I sites. The location of the CO m i t D N A sequence is thus unambiguous (Fig. 1). It has kept a large portion of the A sequence and a fragment of m i t D N A 100 to 150 bp long, on the left of A. This short fragment of D N A includes the loci R I and R II (Jacq et al., 1977). The other mutant, B8, has a 1,460 bp long repeat unit and includes all the A sequence. The extremities of the sequence of B8 could not be determined precisely because of a lack of known restriction sites (Fig. 1). However for the purpose of this work, it suffices to say that B8 has, in addition to the A sequence, a sequence 100 to 150 bp long to the immediate left of A and a sequence 200 to 300 bp long to the immediate right of A. Two other r h o - R53 and C721 have been used in this study. They have both kept the three loci R I, R II and R I I I . R53

Location of Rho- Repeat Units on the Restriction Map of the 09 Region Rho mutants (mitochondrial petite mutants) are generated by large deletions of the wild type m i t D N A sequence. They retain only a fragment (or a few fragments) of m i t D N A in the form of repetitive sequences. There are many r h o - mutants which have retained the region of the 23S r R N A gene with Rib loci. In order to examine whether the R I R II segment has a sequence homologous to a part of the 23S r R N A we have chosen to study the r h o - mutants which have lost all the mitochondrial genome but the R I R II segment. The prerequisite for this approach was to know how the repeat units of these r h o - are located with respect to the sequence of the R I - R II region. A detailed restriction map of this region including co, is presented on Fig. 1. This map was derived from our previous determination (Jacq et al., 1977) obtained with the restriction enzymes Alu I, Hae III, Hind III and Hha I and from the results of others Borst et al., 1977; Morimoto et al.,

lmoxt

1

Rn >

R 111




103

OLI 1 U

>




B

........

Fig. 2A and B. Electron micrographs and drawings of heteroduplexes between 23S rRNA and the Hae III co+ mitDNA fragment carrying the Rib II and Rib I loci. C. Electron micrograph and drawing of a heteroduplexe between 23S rRNA and the Hae III co- mitDNA fragment carrying the Rib II and Rib I loci. Arrows indicated the extremities of the double-stranded segments. Bar represent 0.2 ~tm. . . . . . DNA, . . . . . RNA

d o u b l e s t r a n d e d long arm, 685 bp (calculated with 26 molecules), we o b t a i n 1,195 bp. Therefore we t h i n k that the H a e III digestion at the H a e IIIA site h a d b e e n i n c o m p l e t e a n d the 4,500 + 510 bp f r a g m e n t was isolated together the m a j o r 4,500 b p fragment. F o r

the two other extreme molecules with a long a r m of a b o u t 1,800 a n d 1,900 b p b o t h two H a e III sites (Hae IIIA a n d H a e IIIB) h a d p r o b a b l y escaped from the H a e I I I digestion. Let us r e m a r k that the two values 1,800 a n d 1,900 are n o t i n c l u d e d in 95% confi-

G. Faye et al. : Mitochondrial 23S Ribosomal R N A Gene of the Yeast Saccharornyces cerevisiae

106

(D

I!tA_. 1!t X__

zl0 it .0

.1

.2

.3 A LENGTH in gm

.5

0.6

Fig. 3 A - D . Distributions of the lengths of different segments of the observed heteroduplexes. A Double stranded long arm of the heteroduplexes between 23S r R N A and the 4,500 bp Hae III ~o+ m i t D N A fragment. B Single stranded loop of the heteroduplexes between 23S r R N A and the 4,500 bp Hae III o) + m i t D N A fragment. C Double stranded short a r m of the heteroduplexes between 23S r R N A and the 4,500 bp Hae III o~÷ mit D N A fragment. D D o u ble stranded heteroduplexes between 23S r R N A and the 3,500 bp Hae III co- m i t D N A fragment

dence limits: 790+ 2 x SD i.e. 790+640 bp. From these considerations we conclude that for the double stranded long arm the most likely average length is 685 bp and not 790 bp. For the lengths of the single stranded loop and of the short double stranded arm, no exceptional molecules were found. Figure 4 shows a schematic drawing of the co+ R N A - D N A heteroduplex molecule. The length of the single stranded loop is 1,140 + 100 bp and the double stranded long arm is 685 + 80 bp long. These values are consistent with the data obtained either by restric-

Table 3. Lengths of R N A - D N A heteroduplexes formed between 23S r R N A and the 4500 bp co+ or 3500 bp co Hae III m i t D N A fragments carrying the Rib II and Rib I loci. We have considered that 1 g m = 3000 bp Long arm co+ co

Loop

6 8 5 ± 8 0 b p (26) a 790_+ 320 bp (30)

ll40+100bp

Short arm (30)

3 9 0 + 6 0 b p (30)

1040_+ 145 bp (30)

Results are expressed as average _+standard deviation. The number of molecules used in the calculations is indicated in parentheses a Not included 4 exceptional molecules, see text

tion mapping of this region or by electron microscope observations of D N A - D N A heteroduplexes between the co+ 4,500bp H a e l I I fragment and the co3,500 bp Hae III fragment (Jacq et al., 1977). The double stranded short arm is 390 +60 bp long, that is, the 23S r R N A gene extends over 390 bp to the left of the A sequence. When the Hae III co m i t D N A fragment was hybridized with the 23S rRNA, we observed R N A - D N A heteroduplexes with no loop. We have analysed 30 heteroduplex molecules with two single stranded tails (Fig. 2 C). The average length of the double stranded segment is 1,040 + 145 bp (Table 3). This value is very similar to the sum of the double stranded long arm and short arm of heteroduplexes obtained with the Hae III co+ mitDNA fragment: 685 + 3 9 0 = 1,075 bp. In the rRNA-co m i t D N A heteroduplexes, no single stranded loops could be observed. If such structures exist, their size would be below a few dozens of base pairs, considering the practical limit of resolution of our electron microscope observations.

Discussion

The 23S r R N A gene is, so far, one of the best characterized yeast mitochondrial gene. The locus R III has already been shown to be located in the 23S r R N A gene. It is clear from our work that the genetic loci R I and R II are in the 23S r R N A coding sequence. Mutations at these three loci bring about structural

Hae I11 mit DNA I 390_+60

!

6155 + 80 bp

I

2 3 5 rRNA

Fig. 4. Schematic picture of heteroduplex molecules obtained with the 23S r R N A and the Hae III co + m i t D N A fragment (1 g m = 3,000 bp)

G. Faye et al. : Mitochondrial 23S Ribosomal RNA Gene of the Yeast Saccharomyces cerevisiae modifications of the 23S r R N A which can change the antibiotic sensitivity of mitoribosomes. The R I and R II loci are located fewer than 150 bp from the left extremity of the A sequence (Fig. 1) (R I being very close to this extremity) and fewer than 390 bp from the left end of the 23S r R N A coding sequence (Fig. 4). This work establishes the presence of an insertion interrupting the mitochondrial 23S r R N A structural gene of the co+ mitDNA. This insertion is 1,140_+ 100 bp long and is located at 685 _+80 bp from the Hae III site delimiting the right end of the Hae III co+ m i t D N A fragment and at 390+60 bp from one extremity of the 23S r R N A coding sequence (Fig. 4). The size and location of this insertion seem to be identical to those of the A sequence which was shown to differentiate the co+ and co- alleles (Jacq et al., 1977). The absence of any insertion detectable with the electron microscope, in the co- genome when t h e 23S r R N A (co+ rRNA) was hybridized with the Hae III co- m i t D N A fragment, strongly favours the identity of the insertion observed in the R N A - D N A heteroduplexes and the A sequence. In the rest of this discussion we will refer to these two sequences as being identical. It might be worth while to reinterpret some results we had obtained a few years ago (Faye et al., 1974). Three r h o - mutants C42, D61, and D41 derived from the co+ IL8-8C strain, had lost the R I I I locus but had retained the R I and R II loci. By quantitative R N A - D N A hybridization studies with labelled r R N A and the r h o - mitDNAs, we had found that these rho had a 300 to 600 bp m i t D N A sequence homologous to a part of the 23S rRNA. Recently Lewin et al. (Lewin et al., 1978) have presented the restriction maps of these mutants and it appeared that the right extremities of their repeat units ended in the A sequence. In other words, these early results retrospectively argue in favour of our present electron microscopy studies which establish that a 390_+60 bp long sequence is homologous to the 23S r R N A at the left of the A sequence. Intervening sequences have now been found in a number of eukaryotic genes. The first example described was that of the Drosophila r R N A (Wellauer and Dawid, 1977; Glover and Hogness, 1977; White and Hogness, 1977; Pelegrini et al., 1977). In that case, two-thirds of the Drosophila 28S r R N A genes were shown to contain intervening sequences and the question was asked whether these interrupted genes were functional. In contrast, it seems that all the 23S r R N A genes from yeast mitochondria of co+ strains are interrupted by the 1,000-1,100 bp sequence and necessarily code for functional 23S rRNA, since there is only one single copy of this gene per m i t D N A

107

molecule. Our results are to be compared with those recently found by Rochaix and Malnoe (1978) in the genome of Chlamydomonas rhernardii chloroplast. They demonstrate that all chloroplast 23S r R N A sequences are interrupted by a 940 bp sequence which is not homologous to the rRNA. Moreover this chloroplast intervening sequence appeared to be located 270 + 40 bp from the 5' end of the 23S r R N A coding strand. Though we do not yet know the 5'-3' orientation of the yeast mitochondrial 23S r R N A coding strand, it is interesting to note that the 1,000-1,100 bp insert is also located close to one extremity of the r R N A gene. The function of these intervening sequences is still a challenging question. In the case of the globin insert (Tilghman et al., 1978) they seem to be transcribed and then processed to mature messenger RNA. Such a processing may also be true for many other systems. It is tempting to speculate on the possible implications of the splicing process, if any, in our system. The case of the mitochondrial 23S r R N A insert is remarkable in the sense that this sequence is also responsible for the polarity of recombination in the R I, R II, R I I I region. The phenomenon of recombination between two different molecules, one differing from the other by a large insertion, is not well understood. Dujon (Dujon et al., 1974) has explained this polarity by an obligatory conversion of the allele coto the allele co+ with, consequently, a dissymmetrical co-conversion of the alleles of the flanking loci R I, R II and R I I I . We will not discuss here a detailed model of the genetic mechanism(s) which could take place at the co locus in a heterologous cross co+ x co-. We will only suppose that there is at least one site-specific process governing the recombination at the co locus, that is to say this process requires the integrity of a defined sequence (that we will name S sequence) at the co locus (for example a small palindromic sequence). This sequence will be entire in the co- strains (and will belong also to the r R N A sequence) and cleft in two parts by the A sequence in the co+ strains. In the crosses co+ × co the majority of the mitochondrial genomes obtained in the progeny carry the locus co+. This property should lead to the disappearance of the co- allele in nature. Actually both alleles have been more or less equally found in the laboratory strains examined. This paradox may be explained by the current views concerning the processing of the intervening sequences. It has been suggested that this processing, acting on a R N A precursor, involves enzymatic mechanisms which might not be 1(I0% accurate and could be subject to mutational alterations (Gilbert, 1978). In our system, if we admit that the 23S r R N A trancribed from an co+ strain has to be

108

G. Faye et al. : Mitochondrial 23S Ribosomal RNA Gene of the Yeast Saccharornyces cerevisiae

processed, whereas the 23S r R N A transcribed from an co- strain does not require this processing, we can imagine that his situation confers to the costrains a selective advantage over co+ strains, this advantage being in balance with the drawback of the polarity effect. Mutations affecting the co locus have been described (Dujon et al., 1976). These mutations which have been always isolated from co strains have been named coN (for neutral) because they lead to a loss of the polarity effect in the crosses co+ x coN or co- x coN. These CON mutations are associated with mutations at the R I locus which change the sensitivity of mitoribosomes to chloramphenicol (but a functional r R N A can still be made). Since the R I locus is very close to the co locus it can be thought that the same mutational event affects the S sequence at the locus co and the R I locus (Dujon et al., 1976). The modification of the S sequence would thus hinder the site-specific process mentioned before, in the coN X co+ crosses. On the other hand it has never been possible to obtain coN mutants from co+ strains. One reason might be that the potential CONmutants screened from co+ strains would be modified simultaneously in the R I locus and in the left part of the S sequence located at the left extremity of the A sequence (Fig. 1). Thus these mutants would be altered in the processing of the r R N A and would synthesize a non-functional 23S rRNA, the A transcript being unexcised for example. Eventually with respect to the general aspect of intervening sequences, we may say, as already noticed for the Chlamydomonas chloroplast genome (Rochaix and Malnoe, 1978), that these organelle DNAs resemble a eukaryotic rather than prokaryotic genome. This has been already suggested for yeast mitochondrial genome by Prunell and Bernardi (1974) from different biochemical approaches. Indeed, it has been strongly suggested (Slonimski etal., 1978) that other yeast mitochondrial genes seem to be split by intervening sequences.

Acknowledgements. We thank Hiroshi Fukuhara for support and critical remarks, P.P. Slonimski for advices and fruitful discussions, P. Netter for generous gift of strains, G. Brun and F. Pochon for laboratory facilities, D. Th~ninges and J. Laporte to initiate us in electron microscopy, F. Mignotte for technical assistance, J.D. Rochaix, P. Malnoe, A. Lewin, R. Morimoto, M. Rabinowitz and H. Fukuhara for unpublished information, M. Bolotin-Fukuhara and G. Leblon for useful discussions and I. Stroke for her critical reading of the manuscript. G.F. gives a special thank to Calixtina and Firmin. This work was supported by ATP 3178 and A1-3067 and DGRST grants N ° 77.7.0306 and N ° 77.0307 and N ° 75.7.0748.

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b y F. K a u d e w i t z

Received September 8, 1978