Current Genetics

Current Genetics, 1,163-172 (1980)

©

by Springer-Verlag 1980

Analysis of Mitochondrial RNA in Saccharomyces cerevisiae* Joseph Locker, Richard Morimoto**, Richard M. Synenki, and Murray Rabinowitz Departments of Medicine, Pathology, Biochemistry, and Biology, and The Franklin McLean Memorial Research Institute***, The University of Chicago, 950 East 59th Street, Chicago,Illinois 60637, USA

Summary. Mitochondrial RNA from grande yeast was analyzed by electrophoresis on agarose-urea, acrylamideurea, and methyl mercuric hydroxide-agarose gels. These gel systems display more than 40 RNA bands that copurify with mitochondria; these bands are not present in cytoplasmic RNA preparations. Analysis of molecular weight on methyl mercuric hydroxide gels indicates a size range of 200 to 9,500 nucleotides, including 11 species larger than 21S rRNA (3,700 nucleotides). The mitochondrial origin for many of these species was further verified by transfer of RNA from gels to diazo, benzyloxymethyl paper and hybridization to 32P4abeled mitochondrial DNA. The total molecular weight of the catalogued RNA species was approximately 110,000 nucleotides, considerably greater than the size of the 76,000-base-pair genome. These results suggest that large primary transcripts are processed by multiple cleavages to mature RNA species.

Key words: RNA electrophoresis -Transfer-hybridization of mtDNA - Precursor RNA.

Introduction Yeast (Saccharomyces cerevisiae) has been used extensively for the study of mitochondrial biogenesis because

Offprint requests to: M. Rabinowitz *This study was supported in part by Grants HL 04442 and HL 09172 from the National Institutes of Health and the Louis Block Fund of The Universityof Chicago. **Present address:The Biological Laboratories, Harvard University, Cambridge, MA 02138, USA. ***The Franklin McLean Memorial Research Institute is operated by The University of Chicago for the United States Department of Energy under Contract No. EY-76C-02-0069.

of the highly developed genetic and physical analysis of its mitochondrial genome. Recent examination of the structure of some yeast mitochondrial genes and characterization of their transcripts suggest that this s y s t e m may, moreover, provide insight into general problems in eukaryote molecular biology, such as the functional role of intervening sequences in genes and the manner in which transcripts are processed. Mitochondrial biogenesis is controlled by both the nuclear and the mitochondrial genomes. Most mitochondrial proteins are encoded in the nuclear genome. The yeast mitochondrial genome specifies only a small number of components, including the large and small mitochondrial ribosomal RNAs; about 25 transfer RNAs; 4 ATPase, 3 cytochrome oxidase, and 1 cytochrome b peptides; and the "Var 1" protein which may be associated with ribosomes (for recent reviews, see Locker and Rabinowitz, 1979; Borst and Grivell, 1978). It should be noted that the 21S rRNA gene of many strains contains a 1,O00-hase intervening sequence (Borst and Griveil, 1978; Bos et al., 1978; Faye et al., 1979), and that there are several intervening sequences in the gene for cytochrome b (COB) (Slonimski et al., 1978; Mahler et al., 1978). The known mitochondrial gene products account for about 75% of the information carried by the mitochondrial genome of animals (1 x 107 daltons), provided that meaningful transcription is asymmetrical. On the other hand, yeast (Saccharomyces cerevisiae, baker's yeast) has a mitochondrial genome of 5 x 107 daltons, five times more complex than that of animal mitochondria, but the number of known transcription and translation products is not substantially greater. Thus, the established transcription and translation products of the yeast mitochondrion account for a relatively small fraction of the 5 x 107 dalton genOme. 0172-8083/80/0001/0163/$ 02.00

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J. Locker et al.: Analysis of Mitochondrial RNA in Saceharomyces cerevisiae

In HeLa cells, complete transcription o f b o t h strands o f mitochondrial DNA (mtDNA) has been shown b y a variety o f techniques (Murphy et al., 1975). Available data suggest that transcription of yeast m t D N A is asymmetric (Jakovcic et al., 1979; Levens et al., 1979), b u t the possibility o f symmetrical transcription has not been ruled out. Hybridization o f labeled yeast m t D N A with a large excess o f mitochondrial RNA ( m t R N A ) saturates at 3 5 - 4 0 % , which corresponds to at least 70% transcription o f one DNA strand equivalent (Jakovcic et al., 1979). Electrophoretic analysis of yeast m t R N A on nondenaturing acrylamide-SDS gels shows three major bands (21 and 14S r R N A and 4S t R N A ) and about 7 minor bands, the latter with approximate molecular weights o f 2.1, 1.7, 1.4, 0.8, 0.8, 0.7, and 0.4 x 106 daltons ( F a y e et al., 1974; Van Ommen and Groot, 1977). Van Ommen and Groot (1977), however, were able to map at least 13 distinct transcripts other than t R N A b y hybridization to restriction endonuclease fragments, although most o f the transcripts did not resolve on acrylamide gels. Studies on the smaller mammalian mitochondrial genome have shown a much more complex pattern o f transcription, with about 32 distinct m t R N A species having a total molecular weight o f about 20 x 106 daltons (Murphy et al., 1975; Amalric et al., 1978); these results suggest that m a n y o f the mammalian m t R N A molecules are high-molecular-weight precursors that are subsequently processed to smaller functional R N A species. In the present study, we have analyzed m t R N A from grande yeast b y electrophoresis in partially denaturing agarose-urea (Locker, 1979) and acrylamide-urea (Gross et al., 1976) gels, and in completely denaturing methyl mercuric hydroxide-agarose gels (Bailey and Davidson, 1976). More than 40 species o f m t R N A were discernable. Our purpose in this study was to identify and catalogue the transcripts o f grande yeast, to distinguish the transcripts from molecular aggregates, and to verify that t h e y originate from t h e mitochondrial genome. The large number o f m t R N A species identified and thefpresence o f large non-translated inserts in yeast mitochondrial genes (Borst and Grivell, 1978; Bos et al., 1978; F a y e et al., 1979; Slonimski et al., 1978; Mahler et al., 1978) suggest a " e u k a r y o t i c " t y p e o f RNA processing for the yeast mitochondrion. This system should be an excellent one for the study o f R N A processing, since most o f the RNA intermediates can be identified from a relatively simple genome that has been thoroughly investigated b y restriction endonuclease mapping and genetic analysis. Analysis o f m t R N A from petite mutants and transcript mapping are presented separately (Mofimoto et al., 1979). Some o f the work presented here has appeared in preliminary form (Levens et al., 1979; Morimoto et al., 1978; Morimoto, 1978).

Materials and Methods Yeast Strains, Growth Conditions, and Mitochondrial Isolation. The haploid grande strains MH41-7B (mitoehondrial genotype cRI~RoRoSIPR), IL8-8C (cRERoSo,,pS), MH32-12D (CRE ROISoIRpR), and 19D (cSESoSoISpS) ~ere used (Bolotin-Fukuhara and Fukuhara, 1976). Strains R1 (Mounolou et al., 1966) and D273-10B (Tzagoloff et al., 1975), also haploid grandes, were 'not characterized with respect to mitochondrial antibiotic resistance markers. Yeast growth was carried out in 2% yeast extract, 1% bactopeptone, 2% galactose, and 0.1% glucose. Cells were collected at the midexponenfial phase of growth, and mitochondria were isolated from washed glusulase-treated protoplasts (Locker et al., 1974). Protoplasts were disrupted in 0.7 M sorbitol, 10 mM Tris, pH 7.5, 1 mM EDTA, and 0.1% bovine serum albumin; nuclei were removed by centrifugation at 3,000 x g for 10 rain, and mitochondria were pelleted at 18,000 x g for 10 rain. The postmitochondrial supernatant was saved as a source of cytoplasmic RNA. Mitoehondfia were then washed five times by resuspension in the same buffer and recentfifugation. RNA Preparation. Mitochondfia were suspended in 10 mM Tris, pH 7.5, 10 mM EDTA, in the proportion of 1 ml/10 g wet weight of yeast. Sarkosyl, 10%, was added to a final concentration of 1%, and lysis was achieved in 15-30 s. An equalvolume of distilled phenol (equilibrated with the same buffer) was immediately added at room temperature, and the mixture was homogenized by inversion at 30 rpm for 10 rain. The phases were then separated by centrifugafion at 9,000 x g for 10 rain, and the upper aqueous phase was saved and stored in the dark at 4 ° C. On storage, additional precipitate formed which was left undisturbed at the bottom of the tube. A similar phenol extraction was performed on the post-mitochondrial supematant. For most experiments, the phenol-saturated extract was applied directly to gels. This mixture was stable for several months. In some cases, the phenol was removed by ether extraction, dialysis, or by ethanol precipitation of the RNA. A typical cell extract had an A260nm of about 20. DNA Preparation. Mitochondrial DNA was purified by repeated banding of mtDNA extracts on CsC1 gradients until contaminating nuclear DNA was no longer detectable (Locker et al., 1974). Enzymes. Ribonuclease A (Worthington) was dissolved at a concentration of 1 mg/ml in 10 mM Tris, pH 7.4, and heated to 100°C for 10 rain to remove DNase activity. Deoxyribonuclease I (Worthington, RNase "flee") was purified free of ribonuclease activity by iodoacetate treatment (Zimmerman and Sandeen, 1966), followed by chromatography on G-50 Sephadex (Henikoff, 1977). Agarose-Urea Gel Eleetrophoresis. Electrophoresis of RNA in agarose-urea was carried out as described by Locker (1979). Agarose was dissolved in 6 M urea and buffer, boiled briefly, and poured into gel tubes and slabs while hot. The mixture was then cooled to room temperature and placed at 4 °C for several hours in order to gel. Agarose gels (0.6% and 1%) were formed in cylindrical gel tubes measuring 10 x 0.4, 20 x 0.4, or 20 x 0.8. Agarose gels (1.5%) were formed in similar tubes, or as vertical slabs measuring 20 x 20 x 0.15 cm. Gels were run either in Tris-phosphate (0.04 M Tris, 0.036 M NaH2PO 4, 0.001 M EDTA, pH 7.4) or Tris-acetate (0.04 Tris, 0.02 M Na acetate, 0.033 M acetic acid, 0.001 MEDTA, pH 7.4).

J. Locker et al.: Analysis of Mitochondrial RNA in Saceharomyees cerevisiae Electrophoresis was carried out at room temperature at high field strength (5V/cm) for 3-6 h. Bands are considerably sharpened and resolution is increased when compared with gels subjected to a lower voltage for longer times.

165

by extraction with equilibrated phenol. The labeled DNA was then purified free of labeled nucleotides by chromatography on a 5 ml G-50 Sephadex column equilibrated with 0.01 M NaC1, 0.01 M Tris, pH 7.4, and 0.001 M EDTA. The excluded volume was collected.

AcrylamMe-6M Urea Gels. Acrylamide urea gels (Gross et al., 1976) were formed from 6M urea, 3.8% acrylamide, 0.2% bisacrylamide, 0.04% TEMED, and 0.1% ammonium presulfate in Tris-phosphate or Tris-acetate electrophoresis buffer. Electrophoresis was carried out at 5V/cm for 3-6 h.

Agarose-Methyl-Mercuric Hydroxide Gels. Agarose methyl mercuric hydroxide gels (1 or 1.5%) were run in borate buffer (O. 05 M boric acid, 0.005 M Na2B407. 10H20, 0.01 M sodium acetate, and 0.001 M EDTA, pH 8.2) according to the method of Bailey and Davidson (1976). RNA was ethanol-precipitated and dissolved in borate buffer containing 10% sucrose and a concentration of methyl mercuric hydroxide equal to that of the gel (10 mM unless otherwise specified). For comparative electrophoresis, samples were always loaded in identical volumes.

Hybridization. The DNA was denatured for 10 min at 100 °C and the hybridization carried out in 2 ml of hybridization buffer containing 1.5 x 106 c/rain of DNA per transfer strip. The hybridization buffer contained 0.3 M NaC1, 0.03 M Na citrate (2X SSC), 0.02% Ficoll (Sigma), 0.02% polyvinylpyrrolidone (Sigma), 0.02% bovine serum albumin, 2 mg/ml sonicated denatured calf thymus DNA, and 15% formamide. Hybridization was carried out for 18 h at 45 °C (approximately T m - 25 °C).

Results Analysis o f mtRNA on Gels Containing Urea

Staining of Gels. Agarose gels were stained for 30 min, and acrylamide gels for 1-2 h, in ethidium bromide, 0.5 ~g/ml. Agarose urea gels were stained in water, and methyl mercuric hydroxideagarose gets were stained in 0.5 M ammonium acetate. Gels were photographed under ultraviolet light with high-speed Polaroid and Kodak Plus X film. Bands were analyzed from negatives.

Molecular Weight Calibration. The molecular weight of RNA molecules was measured by comparison to internal standards on methyl mercury gels. Molecular weight was calculated from the standard plot of log molecular weight versus relative mobility. The standard RNAs and the molecular weight values used are: Rabbit 28S rRNA (1.74 x 106) (Wellauer et al., 1974), 18S rRNA (7.1 x 105) (McKonkey and Hopkins, t969), and a- and t3-globinmRNA (2.09 +-0.44 x 105 and 2.3 -+ 0.28 x 105) (Spohr et al., 1976); E. coli 23S (1.05 x 106) (Morrison and Lingrel, 1976), and 16S rRNA (0.56 x 106) (Bailey and Davidson, 1976). These selected values appear to conform most consistently to a linear relationship in 10-20 mM methyl mercuric hydroxide agarose gels. The partially purified giobin mRNA was a gift of Dr. Martin Gross (The University of Chicago), and the E. coli RNA was purchased from Miles Laboratories.

Transfer of RNA to Diazobenzyloxymethyl Paper. Diazobenzyloxymethyl paper (Alwine et al., 1977) (Enzo Biochemieals, "Enzobond") was diazotized in fresh HNO2 (1.2 N HC1 + 1% NaNO2) for 30 min at 4 °C and washed briefly in severn changes of water and three times in 0.05 M sodium phosphate buffer, pH 6.0, immediately before transfer. A gel containing RNA was applied to the paper and blotted with an excess of the same buffer over night. The paper was incubated for 4 h in hybridization buffer containing 1% glycine, and hybridization to radioactive 32pqabeled DNA was carried out. The filters were washed in several changes of 2% sodium dodecyl sulfate, 0.3 M NaC1, 0.03 M Na citrate for a total of 2 h, dried, and autoradiographed with x-ray film. Nick Translation ofDNA. Mitoehondrial DNA from grande MH41 was labeled by nick translation (Rigby et al., 1977). To the 50 tzl reaction mixture containing 1 t~C each of 32p-dATP, 32p-dTTP, 32p-dCTP, and 32p-dGTP (each ~300 Ci/mMol), as well as 10 mM MgC12, 10 mM mercaptoethanol, 100 ~zg/mlgelatin, and 1 /~g DNA, we added 0.125 ng deoxyribonuclease I and 2.5 units DNA polymerase I (Boehringer-Mannheim, FRG). The mixture was incubated for 1 h at 13 °C, and the reaction was terminated

Mitochondrial RNA was analyzed on agarose-6M urea, acrylamide-6M urea, and agarose-methyl mercuric hydroxide gels (see the following section). Use of different gel densities optimized resolution of different sizes of RNA molecules. A comparison of the resolution of mtRNA in 0.6% agarose-6M urea, 1.5% agarose-6M urea, and 4% acrylamide-6M urea is shown in Fig. 1. To make the band nomenclature convenient, we divided the RNA molecules into three size classes, each of which is best resolved in one o f these three gel formulations: A (bands larger than 21S rRNA), B (bands from 21S rRNA to 14S rRNA), and C (bands smaller than 14S rRNA). Differential hydrolysis with ribonuctease and deoxyribonuclease (Locker, 1979) demonstrates that mitochondrial DNA migrates as a condensed band at the top of the gel and can be distinguished readily from RNA bands even without nuclease digestion. As is expected, this m t R N A preparation, from strain MH41, shows moderate contamination with cytoplasmic RNA bands.

Analysis o f High-Molecular-Weight mtRNA and R N A Denaturation To evaluate whether the numerous high-molecular-weight bands represent aggregates or single RNA chains, we used two methods. (1) RNA was heated to 65 °C in 75% formamide before electrophoresis on agarose-urea gels (Fig. 2); and (2) RNA was treated with methyl mercuric hydroxide, and electrophoresis was carried out on methyl mercuric hydroxide-agarose gels (Fig. 3). When the RNA is heated to 65 °C for 10 min, with or without the addition of 6M urea, a small number of bright, sharp new bands appear in the high-molecular-weight region C~ 21s) of the gel (Fig. 2). These new bands are presumed to be aggregates that form during incubation at 65 °C. Treatment with 75% formamide or heating to 65 °C for 10

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J. Locker et al.: Analysis of Mitochondrial RNA in Saccharomyces cerevisiae

Fig. 1. Electrophoresis of yeast mtRNA in gels containing 6M urea. Eleetrophoresis was carried out as described in Materials and Methods. For convenience, the pattern of RNA bands was divided into three regions, each of which is best resolved in a gel of a particular composition. Region A, best resolved in 0.6% agarose-6M urea, contains RNA species larger than 21S rRNA (3,700 nucleotides); region B, best resolved in 1.5% agarose-6M urea, contains RNA species from 21S rRNA to 14S rRNA (2,000 nucleotides); and region C contains RNA species smaller than 14S rRNA and is best resolved on 4% aclylamide-6M urea gels

Fig. 2. A-D. Effects of heat and formamide on RNA electrophoresis. All gels contained 5 #g of RNA, and electrophoresis was for 3 h at 5 V/era. The RNA on the gels was treated as follows: (A) untreated; (B) incubated at 65 °C for 10 rain; (C) incubated in 75% formamide at room temperature for 10 rain; and (D) incubated in 75% formamide at 65 °C for 10 min. Arrows denote two new bands formed in gel B only

min in 75% formamide removes these bands, but otherwise has no effect on the gel pattern. Figure 3 shows the electrophoresis of m t R N A from strain MH41 on 10 mM methyl mercuric hydroxide - 1 % agarose gels after pretreatment with .10 mM methyl mercuric hydroxide. Although RNA fluorescence is less intense on methyl mercuric hydroxide than on agarose-urea gels, the high-molecular-weight RNA bands are present and the pattern is similar to those observed on agarose urea gels. The band pattern becomes brighter, but does not otherwise change with increasing RNA concentration. Such high-molecularweight bands are not present in cytoplasmic RNA. To insure that full denaturation was achieved, we compared the effects of methyl mercuric hydroxide at concentrations ranging from 0-20 mM (not shown). The electrophoretic patterns of m t R N A on gels o f 10 and 20 mM methyl mercuric hydroxide are virtually indistinguishable. A significant transition occurs between 2 and 5 mM for all o f the RNA bands; the 5 and 10 mM gels show identical mitochondrial RNA patterns, but in the 5 mM gel there is incomplete denaturation o f a double-

stranded RNA present as a contaminant from the cytoplasm (Hastie et al., 1978). Although there was variation in the relative amounts, the electrophoretic patterns of high-molecular-weight RNA from all yeast strains analyzed were indistinguishable, except for strain D273, which showed altered migration of two bands (designated A3 and A l l ) on methyl mercuric hydroxide gels (Fig. 3 and 4a). The electrophoretic patterns were similar, but not identical, for agarose-urea and agarose-methyl mercuric hydroxide. In agarose-urea, band A9 is split into two bright bands (A9 and A10), but band A10 cannot be detected on methyl mercuric hydroxide gels (Fig. 4b). Bands A9, A10, and A l l from strains MH41 and D273 comigrate on agarose-urea, but not on methyl mercuric hydroxideagarose. Although the patterns are similar, an exact correlation between all of the high-molecular-weight bands present in the two gel systems may not be possible. Probable correlations, and the extrapolated and apparent molecular weights of these bands, are shown in Table 1.

J. Locker et al.: Analysis of Mitoehondrial RNA in Saccharomyces cerevisiae

167

Fig. 3. Electrophoresis of MH41 mtRNA on methyl mercuric hydroxide-l% agarose gels. Electrophoresis was for 3 h at 5 V/ cm on 1% agarose gels containing 10 mM methyl mercuric hydroxide. A print of the high-molecttlar-weight region of the gels was underexposed to enhance the faint RNA bands, which are numbered on the figure. A cytoplasmic band (double-stranded RNA) is designated by C Fig. 4 A and B. Higher resolution of some mtRNA species. Electrophoresis was carried out for 6 h at 5 V/cm on 20 cm gels. (A) Methyl mercuric hydroxide gels. Bands 9 and 11 show altered migration in strain D273, and band 10 is not apparent in either strain D273 or strain MH41. (B) Agarose urea gels. Bands 9, 10, and 11 from strains MH41 and D273 comigrate

Fig. 5. 1.5% agarose-6M urea gels of mtRNA. Electrophoresis was carried out over a length of 20 cm for 6 h at 5V/cm. No bands above A7 could be resolved (2.2 x 106 daltons). Band C1 from strain D273 (arrow) migrates more slowly than the equivalent band in the other strains (0.34 vs. 0.32 x 106 daltons)

J. Locker et al.: Analysis of Mitochondrial RNA in Saceharomyeescerevisiae

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Table 1. Calculated molecular weights of mitoehondrial RNA

species. Molecular weights were calculated as follows: 21S (1.28 x 106 daltons, 3,700 nucleotides) and 14S (0.68 x 106 daltons, 2,000 nucleotides) rRNA were used as internal standards. For methyl mercuric hydroxide gels, molecular weights of species larger than 21S rRNA were extrapolated from 1% agarose-10 mM methyl mercuric hydroxide gels and for smaller RNAs, measurements weIe interpolated and extrapolated from 1.5% agarose methyl mercuric hydroxide gels. Apparent molecular weights from agarose-urea gels were obtained similarly, from 0.6% agarose-6M urea and 1.5% agarose-6M urea gels. Apparent molecular weights from 4% acrylamide-6M urea gels were obtained by construction of a linear semi-logarithmic plot from band C4 (0.27 x 106 daltons, 770 nucleotides) and an arbitrary tRNA midpoint (0.028 x 106 daltons, 70 nucleotides). Differing values from strain D273 are given in parentheses

Region

A

B

Band number

1 2 3 4 5 6 7 8 9 10 11 1(21S) 2 3 4 5 6 7 8 9(14S)

Methyl mercuric hydroxide M x 106 N 3.25 3.10 2.95 2.75(2.70) 2.50 2.35 2.20 1.90 1.75

6M urea M x 106

1.54(1.59)

3.75 3.50 3.05 2.95 2.83 2.65 2.50 2.10 1.93 1.85 4,500(4,600) 1.56

1.28 1.25 1.20 1.12 1.07 1.03 0.95 0.91 0.68

3,700 3,600 3,500 3,200 3,100 3,000 2,700 2,600 2,000

1 0.32 2 0.315 3 0.29 4 0.265 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(tRNA)

N

9,400 9,000 8,500 7,900(7,800) 7,200 6,800 6,400 5,500 5,100

920 910 840 770

1.28 1.26 1.24 1.04 1.02 1.00 0.92 0.89 0.68 0.37 0.36 0.34 0.23 0.22 0.22 0.21 0.21 0.19 0.17 0.15 0.15 0.14 0.14 0.13 0.13 0.12 0.070 0.030 0.025

Analysis of Intermediate-Molecular-Weight RNA RNA species smaller than 21S RNA are better resolved on gels o f higher agarose content. Figure 5 shows the electrophoresis o f several grande m t R N A s on 1.5% agarose-6M urea. The agarose-urea and agarose-methyl mercuric hydroxide band patterns are similar. Molecular weight was calibrated on 1.5% agarose-10 mM CH3HgOH gels (Fig. 6). The interpolated and apparent molecular weights o f bands from this region are shown in Table 1.

Analysis ofLow-Molecular-Weight RNA R N A with molecular weights below 0.3 x 106 daltons (most o f the C region) is best resolved on 4% acrylamideurea gels (Fig. 7). Essentially the same pattern is observed with 2.7% acrylamide urea gels, but these are more difficult to handle. The C region shows 19 presumptive mitochondrial bands, excluding mitochondrial tRNA (C20). The apparent molecular weights o f these bands (Table 1) have been interpolated from the main t R N A band (2.5 x 104 daltons) and band C3 (2.9 x 105 daltons). In 6M urea gels, mitochondrial t R N A can easily be distinguished from cytoplasmic tRNA b y its slower migration. Band C 1 9 , j u s t above the main t R N A band, is prominent in a l l strains and represents mitochondrial t R N A asp (Merten, 1979). The faint unnumbered bands between bands B9 and C1 (from 0.32 - 0.68 x 106 daltons) and between bands C3 and C4 (from 0.26 - 0.29 x 106 daltons) are most prominent in strain MH41 and least prominent in strain MH32. Conversely, the lowermolecular-weight bands from C10-C17 (0.12 - 0.17 x

2-

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z xtO~_640 630 610 590 550 490 430 420 400 390 380 360 350 200 90 70

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Fig. 6. RNA molecular weight calibrations on 1.5% agarose-10 mM methyl mercuric hydroxide gels. Various RNA species were run together in single eleetrophoretic channels for calibration. The molecular weights for standards (o) are given in Materials and Methods, and the mitoehondrial RNA species calibrated (I) are indicated in italics

J. Locker et al.: Analysis of MitochondrialRNA in Saccharomyces cerevisiae 106 daltons) are most prominent in MH32 and least prominent in MH41. The RNAs of s~rains MH41, MH32, R1, 19D, and IL8 appear to be identical, except for variable proportions of bands in the C region. Strain D 273 shows two significant differences from the above strains in the C region pattern: band C1 has a higher molecular weight (0.34 vs. 0.32 x 106 daltons) band C6 (0.22 x 106 daltons) is absent in D273 and appears to be replaced by a new band (0.23 x 106 daltons) between bands C4 and C5. The resolution of mtRNA species in the three different gel formulations is summarized in Fig. 8.

169

Transfer-Hybridization of mtRNA Although it is clear that these RNA bands purify with mitochondria and are not present in the post-mitochondrial supernatant cytoplasm, the mitochondrial origin of these transcripts was studied further by hybridization to purified mitochondrial DNA. Figure 9 illustrates an experiment in which mitochondrial and cytoplasmic RNA were transferred to diazobenzyloxymethyl paper and then hybridized to total 32P-labeled MH41 DNA. The cytoplasmic RNA does not hybridize, but virtually every stainable band in the 1.5% agarose-6M urea gel that has been identified as mitochondrial is, as expected, labeled by hybridization to labeled mtDNA. Discussion

Fig. 7. 4% aerylamide-6Murea gels of mtRNA. 20 cm gels were run for 6 h at 5 V/cm. In this medium, resolution is optimum for bands below 0.3 x 106 daltons. Arrows indicate two bands in strain D273 that migrate differently from the other strains and correspond to RNA species C1 and C6

The 68 to 76 kb yeast mitochondrial genome (Borst and GriveU, 1978; Morimoto et al., 1979a)codes for a limited number of products which account for only 30-40% of a single-strand equivalent of mtDNA. Since the genome size is relatively small, it has been possible to visualize directly the individual mtRNA transcripts by the use of highly discriminating agarose-urea and methyl mercuric hydroxide-agarose gels. With these procedures, we were able to detect more than forty different RNA species other than tRNA, which vary in molecular weight from 200 to 9,400 nucleotides. We have demonstrated that these transcripts are indeed mitochondrial since they do not appear in purified cytoplasmic RNA, and since they are selectively present in deletional petites which retain different segments of the mitochondrial genome (see Morimoto et al., 1979b). This conclusion was confirmed by hybridization of mtDNA with RNA bands transferred from gels to diazolbenzyloxymethyl paper, which demonstrates that virtually every visible RNA band (except for contaminating cytoplasmic 28 and 18S RNA) hybridizes with mtDNA and is thus a transcript of the mitochondrial genome. These RNA-DNA hybridization studies have thus far been limited to 1.5% agarose urea gels, so that high-molecular-weight species visualized on 0.6% agarose-urea gels, and the low molecular species observed on 4% acrylamide-6M urea gels, have been characterized as mitochondrial products only by their co-purification with mitochondria and by their absence in cytoplasmic extracts. Of particular note are the large number of mitochondrial transcripts observed and the presence of high-molecular-weight species having molecular sizes greater than that of 21S RNA. The aggregate molecular weight of the observed transcripts adds up to 110,000 bases, 30% larger than the entire mitochondrial genome, suggesting that many of the RNA species are processing intermediates. The large-molecular-weight species observed do not appear to be aggregates, since they persist in fully denatur-

170

J. Locker et al.: Analysis of Mitochondrial RNA in Saceharomyees cerevisiae

0.6% AGAROSE

1.5%AGAROSE

4% ACRYLAMIDE

Mi--tCy--C-~

Mit Cyt

B C

A

A

9. 43 65



I

B

7 S 109 11

B

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................ I

9 ........

C

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1 ::::::::5::::: :.:.:.:.:.:.:.: .-.:.:.:.:.:.:.:.-.

Fig. 8. Summary of mtRNA gel anaysis. The patterns are composites based on electrophoresis of RNA from strains MH41, MH32, IL8, and 19d

Fig. 9. Hybridization of total mtDNA to mtRNA transferred to diazobenzyloxymethyl paper. Left: the 1.5% agarose-6M urea gel channels which were transferred; right: autoradiograms of the hybrids

ing gels containing methyl mercuric hydroxide. Furthermore, two high-molecular-weight species have been shown (Levens et al., 1979; Morimoto et al., 1979b) to hybridize with petite E41 DNA, which contains the gene for 21S rRNA, and these transcripts thus appear to be precursors of this ribosomal RNA. Also, analysis of RNA of petite deletion mutants, and the use of the RNA transfer hybridization system, have made possible partial mapping of some of these transcripts, and of their presumptive precursors, to specific regions in the mitochondrial genome (Morimoto et al., 1979b). In this study, mtRNA species were analyzed on both agarose-urea (Locker~ 1979) andmethyl mercuric hydroxide-agarose gels (Bailey and Davidson, 1976). Although the agarose-6M urea electrophoresis mixture is not fully denaturing to RNA, the system proved to be highly effective because of the resolution achieved. The sharp-

ness of bands is about the same as in methyl mercuric hydroxide gels; the ethidium bromide staining of agaroseurea gels is considerably brighter, however, so that equivalent patterns may be seen with about 1/3 to 1/4 as much RNA. Agarose-urea gels are thus particularly valuable for the detection of transcripts present in low concentration. Since the agarose-urea system is only partially denaturing, fully denaturing methyl mercuric hydroxide gels were required for an accurate calibration of molecular weight. Comparison of the two types of electrophoresis revealed very similar RNA patterns. The small differences observed with the two gel systems probably result from the presence of short undenatured regions in the mtRNA mn on agarose-urea gels. The much larger changes in the relative migration of cytoplasmic RNA species results from their higher G + C content.

J. Locker et al.: Analysis of Mitochondrial RNA in Saceharomyces cerevisiae Some interesting phenomena have been observed as a result of this analysis on dual gel systems. For example, bands A9, AI0, and A l l from strain MH41 comigrate with the same bands from strain D273 on agarose-urea gels. On methyl mercuric hydroxide gels, band A10 cannot be resolved (and apparently has the same molecular weight as A9), and A l l of MH41 and A l l of D273 no longer comigrate, indicating that A11 has a slightly different molecular weight in the two strains. The fact that A11 from MH41 and D273 does comigrate on the partially denaturing gels, however, seems to indicate that each RNA species folds up into species which have the same radius of gyration in 6M urea at room temperature. One possible explanation may -be the presence of a secondary structure that forms internal loops within the strand, so that the RNA regions external to the loops are of constant size and only the size of the loops varies. Methyl mercuric hydroxide-agarose gels were used for molecular weight calibration of intermediate- and high-molecular-weight RNA molecules. We titrated the amount of methyl mercuric hydroxide necessary to give complete denaturation by observing the change in gel pattern. Calibration was carried out with a mixture of RNAs comigrating in the same well or tube to minimize variation. The calibration was complicated by the different molecular weight values cited for standard RNAs; a set of values was chosen which gave the most nearly linear molecular weight plot. The relative migration of high-molecular-weight RNA diverges from linearity at different molecular weights, depending on the amount of agarose in the gel. In 1.5% agarose, deviation from linearity seems to occur near the position of mammalian 28S RNA. In 1% agarose, the migration is linear to somewhat above the position of this species, but we have not calibrated the point where this transition takes place. Therefore, the extrapolated molecular weight probably deviates from the true molecular weight, which will be higher than the calculated values. This discrepancy will increase with increasing molecular weight. Low-molecular-weight RNAs were studied on acrylamide-6M urea only, so that molecular weight calibrations for these species were not obtained in a fully denaturing gel system. As a result, these low-molecular-weight values must be considered as estimates, which may vary considerably from the true molecular weights. Recent calibrations of 21S rRNA by electron microscopy (Merten, 1979) and by free-structure restriction enzyme mapping (Heyting et al., 1979) indicate that this transcript is ordy 3,100-3,200 nucleotides long. Thus, there is a substantial discrepancy between these results and the calibration obtained in this study and earlier studies (e.g., Reijnders et al., 1973) with denaturing gels. This discrepancy may be attributed to the anomalous behavior of high A + T nucleic acids in one or all of these different experimental systems, or to specific

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sequence anomalies of these molecules, and it is quite possible that denaturing gels overestimate the length of these RNA species by 10-15%. Resolution of the discrepancy will probably require determination of RNA sequences. Further, it is not clear whether this anomolous behavior is true only for rRNA or for all mtRNA species, since the A + T content is expected to vary considerably from species to species. The differences between the banding pattern of strain D273 and that of the other strains are possibly explained by the altered mitochondrial genome of D273. Except for strain D273, which has a 70,000-base-pair genome (Morimoto, 1978; Morimoto et al., 1979b) and a different lineage (Tzagoloff et al., 1975), all of the grande strains studied in this paper have a 76,000-base-pair genome and are of similar lineage (Morimoto, 1978; Bolotin-Fukuhara and Fukuhara, 1976; Morimoto et al., 1979b). The RNA band patterns may be somewhat more complex than those demonstrated by fluorescence. One conclusion is that the forty-odd species of RNA molecules characterized in the present study are an underestimate; they are the most abundant species, but others may be detected by more sensitive methods of analysis; and the number of species Finally detected will increase as specific hybridization probes are used. The data presented here indicate the large number of transcripts that can be characterized from the yeast mitochondrial genome. The relatively small number of genes that the genome actually contains implies that considerable RNA processing takes place. The recent discovery, in yeast mitochondria, of split genes containing non-translated intervening sequences suggests that some of the RNA species may remit from the processing of intervening sequences. The first step toward resolving this question is the description of the individual transcripts. The yeast mitochondrial genome appears to be the simplest system in which intervening sequences and their processing have been discovered, making it optimal for study of the function of split genes and the processing of their transcripts.

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Communicated by F. Kaudewitz Received November 2, 1979

Analysis of mitochondrial RNA in Saccharomyces cerevisiae.

Mitochondrial RNA from grande yeast was analyzed by electrophoresis on agarose-urea, acrylamide-urea, and methyl mercuric hydroxide-agarose gels. Thes...
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