Current Genetics

Current Genetics (1982) 5:97-107

© Springer-Verlag 1982

Biogenesis of Mitochondria: Mapping of Transcripts from the oli2 Region of Mitochondrial DNA in Two Grande Strains of Saccharomyces cerevisiae 1 Gary S. Cobon 2, Manfred W. Beilharz, Anthony W. Linnane, and Phillip Nagley Department of Biochemistry,Monash University,Clayton, Victoria, 3168, Australia

Summary. A series of 14 contiguous restriction fragments of yeast mitochondrial DNA that cover a 5.2 kb segment including the oli2 gene provided a set of hybridization probes that were used to analyze gel fractionated mitochondrial RNA immobilized on diazotized paper. Of the nine oli2 region transcripts in grande strain J69-1B (class I), the five largest (5,100, 4,500 (most prominent), 3,900, 3,600 and 3,000 nucleotides) contain the oli2 coding sequence; all appear to have the same 3' end. The four smallest J69-1B transcripts (1,900, 1,700, 800 and 600 nucleotides) contain only sequences downstream of the oli2 gene. A 1.8 kb DNA region specifying these four transcripts is deleted from grande strain JM6 (class II); these transcripts are thus dispensible for respiratory function. The five oli2 transcripts of JM6 (3,000, 2,400 (most prominent), 2,000, 1,700 and 1,150 nucleotides) all contain the oli2 coding sequence and are each related to a corresponding J69-1B transcript; the 5' and 3' ends match in each case, but the JM6 transcripts are about 2,000 l~ucleotides shorter. In particular, the putative oli2 mRNA has a 5' leader of about 1,400 nucleotides, a coding region of 780 nucleotides, and a variant 3' tail that is about 2,300 nucleotides in J69-1B, and about 200 nucleotides in JM6. Key words: Yeast mitochondria - Transcript mapping Hybridization probes - Genome variation

1 This is paper 58 in the series Biogenesis of Mitochondria. Paper 57 is Stephenson et al. (1981) 2 Present address: Biotechnology Australia Pry. Ltd., P.O. Box N109, Petersham North, New South Wales, 2049, Australia Offprint requests to: P. Nagley

Introduction

Mitochondrial mutations which affect the function of the ATPase complex have been shown to map in two widely separated regions (olil and oli2) of the mitochondrial genome of Saccharomyces cerevisiae. The olil region has been shown to code for the proteolipid component (subunit 9) of the ATPase complex, by direct comparison of the nucleotide sequence of the olil gene (Macino and Tzagoloff 1979; Hensgens et al. 1979) with the amino acid sequence of the proteolipid (Wachter et al. 1977). The proteolipid subunit is the oligomycin binding protein which is involved in the activity of the ATP-synthetase associated proton channel across the mitochondria! inner membrane (Fillingame 1980). The oli2 gene has been shown to code for subunit 6 of the ATPase complex. It has been found that a number of mitochondrial m i t - mutants with lesions in the oli2 gene did not synthesize subunit 6. However, these mit- mutants did synthesize mitochondrial translation products which were immunoprecipitated with rabbit anti-ATPase antibody but which were shorter than the 20,000 dalton subunit 6 (Roberts et al. 1979; Linnane et al. 1980). One function ofsubunit 6 is thought to be associated with the coupling of respiration to ATP synthesis (Murphy et al. 1980). It may also have a role in binding the F1 sector of the ATPase to the membrane associated proteolipid (Stephenson et al. 1981). The oli2 gene maps between the genes coding for subunit I of the cytochrome oxidase complex (oxi3 locus) and the cytochrome b apoprotein (cyb) (see Borst and Grivell t 981, for a recent map). DNA sequencing studies (Macino and Tzagoloff 1980) have led to the assignment of the coding region of the oli2 gene to a 780 bp open reading frame that crosses the boundary between two EcoRI fragments of mitochondrial DNA (mtDNA), 0172-8083/82/0005/0097/$ 02.20

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Fig. 1. Physical maps of mitochondrial genomes. The uppermost map is that of the oli2 region in the class I grande strain J69-1B; R7, R8, R3 signify particular EcoRI fragments of J69-1B mtDNA (see Nagley et al. 1981). Below the J69-1B map are shown the fight end of the oxi3 gene and the: entire oli2 coding region (boxed). Fourteen contiguous fragments of this region of mtDNA used as hybridization probes are indicated; the size of each fragment (shown below the line) was taken from the sequence where available, or from the mobility of the fragment on gels. At the bottom of the Figure are the maps of the mtDNA genomes of petites DS14 and G4 aligned with the J69-1B map. Restriction enzyme sites are indicated as follows: EcoRI, o; HpaII, c; HinfI, •; MboI, zx;PstI, •

probes used is a series of fourteen contiguous restriction enzyme fragments derived from the oli2 gene and surrounding sequences. We now identify a total of nine discrete transcripts from the oli2 region in class I strains, and five in class II strains. The positions at which the 5' and 3' ends of these transcripts map are determined and the results lead to a scheme for the transcription of the oli2 region. Class I strains contain a series of five transcripts carrying oli2 gene coding sequences; the four other transcripts contain sequences exclusively derived from downstream of the oli2 gene. The segment of mtDNA not found in class I strains downstream of the oli2 region is mapped with more precision than previously and is shown to include the region of mtDNA from which the four shorter class I transcripts are derived. Thus the pattern of transcription in class II strains can now be accounted for in terms of the mtDNA sequences missing from these strains.

Materials and Methods denoted R7 and R8 in Fig. 1. The protein predicted from this reading frame has a molecular weight of 28257 which can be compared with the apparent Mr of 20000 estimated from the mobility of ATPase subunit 6 on sodium dodecyl sulphate-polyacrylamide gels (Roberts et al. 1979; Linnane et al. 1980). Nagley et al. (1981) studied RNA species transcribed from mtDNA in the region of the oli2 gene and characterised two classes of respiratory competent strains on the basis of the different patterns of transcription in this region and the organization of the mtDNA near the oli2 gene. Using a petite mtDNA hybridization probe (DS14) that contained sequences from the oli2 gene and flanking regions (see Fig. 1), it was found that class I strains contained transcripts measured to be 5,t00, 4,500, 1,900 and 800 nucleotides in length. The 5,100 and 4,500 base transcripts had been described previously (Van Ommen et al. 1979; Grivell et al. 1980 ;Morimoto et al. 1979). The oli2 transcripts of class II strains were found to be 3,000, 2,400, 2,000 and 1,150 nucleotides in size. The class II strains were shown to lack a segment of mtDNA about 1800 base pairs in length that is present in class I strains in the A+T-rich spacer downstream from the oli2 gene (Nagley et al. 1981). No clear relationship between the absence of these mtDNA sequences and the altered transcription pattern in class II strains could be deduced without knowledge of the positions on mtDNA at which the 5' and 3' ends of individual transcripts map. In this paper we describe experiments using a number of different hybridization probes in order to define in more precise terms the patterns of transcription of the oli2 region in class I and class II strains. Amongst the

Yeast Strains. Saccharomyees cerevisiae strains used here are the grandes J69-1B (Atchison et al. 1980) and JM6 (Vodkin 1977), and petites DS14 (Macino and Tzagoloff 1980) and G4 (Linnane et al. 1980). Extraction and Analysis of Mitoehondrial RNA and DNA. RNA was extracted from isolated mitochondria by a phenol method at 60 °C (Nagley et al. 1981) with the modification that the RNA was not ethanol precipatated, but was stored as a phenol-saturated aqueous phase at -20 °C (Locker 1979). This extract was electrophoresed in 1.5% agarose/6 M urea gets (except where indicated) and transferred to diazotized paper as in the previous work (Nagtey et al. 1981), except that diazotized aminothiophenol-paper was used (B. Seed personal communication), and the gels were not treated with NaOH before transfer. Purified mtDNA (Vaughan et al. 1980) was digested with restriction enzymes using standard procedures. For hybridization studies, the digested DNA was electrophoresed in 1.5% agarose slab gels and transferred to nitrocellulose sheets (Schleicher and Schuell BA85) as before (Nagley et al. 1981). Preparation of 32p-Labelled DNA Fragments. The fourteen contiguous restriction enzyme fragments used as hybridization probes are shown in Fig. 1. For probes 1-6, a recombinant DNA plasmid (pJE7), containing EcoRI fragment R7 of strain J691B cloned in pBR325 (Bohvar 1978) and propagated in E. eoli ED8654, was digested with EcoRI. The 2.6 kb mtDNA fragment was separated from the pBR325 sequences on a 1% agarose gel. The R7 band was sliced out, recovered by electroelution and digested with a combination of HpalI and HinfI. To obtain probes 7 to 14, mtDNA isolated from petite DS14 was first digested with EcoRI and the two fragments (2.45 + 1.75 kb) produced were separated on a 5% polyacrylamide gel, electroeluted and each was digested with HpaII. The fragments derived from R7 or from DS14 EcoRI fragments were incubated under conditions in which the 3' ends of all EcoRI, HinfI and HpalI termini would become labelled by incorporation of either [c~-32p]dATP or [a-32p]dCTP (Amersham, >2,000 Ci/mmole). The reaction mixture (50~1) contained 10 mM Tris-HClpH 7.4, 7 mM MgC12,

G. S. Cobon et aL: Transcripts of oli2 Region of Yeast Mito chondrial DNA

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described above. The end labelled fragments were denatured and the complementary strands were resolved by electrophoresis on 5% polyacrylamide gels (Maxam and Gilbert 1980). A portion of each single stranded fragment was subjected to the chemical DNA sequencing reactions as described (Maxam and Gilbert 1980). DS14 mtDNA was labelled in a nick translation reaction essentially as described (Rigby et al. 1977). Labelling of the HpalI plus EcoRI digested DS14 mtDNA was carried out as described above except that the fragments were not separated by electrophoresis either before or after labelling.

Hybridization Conditions. Strips of nitrocellulose or diazotized

Fig. 2. Families of oli2 transcripts in strains J69-1B and JM6. RNA extracted from strains J69-1B (lanes A, C, E) or JM6 (lanes B, D, F) was electrophoresed in 1.5% agarose/6 M urea gels, stained with ethidium bromide (lanes A, B), transferred to diazotized paper and hybridized to DS14 mtDNA which had been labelled by nick translation (lanes C, D) or digested with HpaII plus EcoRI then 3' end labelled (lanes E, F). For lane A, the following bands are indicated: dsRNA, killer-related double stranded RNA; 21S, mitochondrial 21S rRNA; 15S mitochondrJal 15S rRNA. The latter two species (with sizes taken to be 3,700 and 2,000 nucleotides respectively were used as size markers for the determination of the size ofoli2 transcripts detected by autoradiography (see also Discussion herein). The measured size of each transcript in nucleotides is shown for each autoradiogram

50 mM NaC1, 1 mM dithiothreitol, 2 units of DNA polymerase I Klenow fragment (Boehringer), restriction enzyme fragments of mtDNA (1-10 ~zg) and [c~-32PIdATP and [~-32p]dCTP. The concentrations of labelled nucleotides were adjusted so that the number of moles of each nucleoside triphosphate in the reaction mixture was equivalent to that required for quantitative incorporation into the particular 3' ends of the restriction enzyme fragments to be labelled. This was usually of the order of 50 pmol per incubation. Incorporation was carried out at 10 °C for 10 rain. The labelled double stranded DNA fragments were precipitated three times at - 7 0 °C with ethanol in the preser~ce of 2 M ammonium acetate and 20 ~zg of carrier tRNA, and once with ethanol in the presence of 0.3 M sodium acetate. The fragments were then separated by electrophoresis in 5% polyacrylamide gels, and recovered by electroelution. For the preparation of strand-specific probes, mtDNA isolated from petite G4 was digested with either HinfI (yielding a single 1.6 kb fragment) or MboI (two fragments produced; 1.2 kb and 381 bp). The two MboI fragments were separated on a 2% agarose gel and electroeluted. For 3' end labelling the HinfI digested G4 mtDNA or the 381 bp MboI fragment were incubated with DNA polymerase I and [a-321dATP (with 100 ~M non-radioactive dGTP in the case of the MboI fragment) as

paper were incubated in heat sealable potythene b ~ s containing hybridization solution (50% formamide, 0.04% ficoll, 0.04% polyvinylpyrrolidone, 0.0~4% bovine serum albumin (Denhardt 1966), 0.45 M NaC1, 0.045 M Na citrate, 10% Dextran sulphate (Wahl et aI. 1979), 0.1% sodium dodecyl sulphate, 2 mg/ml sonicated denatured calf thymus DNA) for 3-4 h at 37 °C. This mixture was removed and the heat denatured probe was added in 2.5 ml of hybridization solution. Hybridization was at 41 °C for 16 h with the following exceptions. Probe 14 (107 bp) was hybridized at 36 °C for 16 h; probes 3 (31 bp] and 8 (15-20 bp) were hybridized at 20 °C for 16 h in hybridization mixture without formamide. Following hybridization, the strips of nitrocellulose or diazotized paper were removed from the bags and washed with four changes of 0.3 M NaC1, 0.03 M Na citrate, 0.1% sodium dodecyI sulphate at 25 °C for at least 15 rain per wash.

Results Transcripts o f the oli2 Region in Grande Strains In these experiments, two grande strains, representative o f class I U69-1B) and class II (JM6) (Nagley et al. 1981) were used. Mitochondrial R N A ( m t R N A ) extracted f r o m these t w o strains was electrophoresed in 1.5% agarose/6 M urea gels and stained with ethidium b r o m i d e (Fig. 2, lanes A and B). The t w o major R N A species in each track were the t w o m i t o c h o n d r i a l r R N A species. In addition to the absence o f killer double stranded R N A f r o m JM6 (cf. V o d k i n 1977), other differences in less abundant transcripts are apparent. To visualize the transcripts o f the oli2 region, gel-fractionated R N A o f each strain was transferred to diazotized paper and hybridized to the m t D N A o f petite D S 1 4 (Fig. 1 ) w h i c h had b e e n labelled to a specific activity o f 3 x 106 c p m / #g by nick translation. The resultant autoradiograms (Fig. 2, lanes C, D) displayed major h y b r i d i z a t i o n to the f o u r transcripts in each strain that we described previously (Nagley et al. 1981). F o r J69-1B (Fig. 2, lane C) these transcripts were measured to be 5,100, 4,500 (most p r o m i n e n t ) , 1,900 and 800 nucleotides in length and for JM6 (Fig. 2, lane D), 3,000, 2,400 (most prominent), 2,000 and 1,150 nucleotides. When a m i x t u r e o f 3' end labelled D N A fragments prepared f r o m HpaII plus E c o R I digestion o f D S 1 4 m t D N A (specific activity 2 x 107 c p m / g g total D N A ) was used as a h y b r i d i z a t i o n probe, a further five tran-

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G.S. Cobon et al.: Transcripts of oli2 Region of Yeast Mitochondrial DNA

Direction of Transcription and Identification of Coding Strand

Fig. 3. Hybridization of strand-specific mtDNA probes to oli2 transcripts. Mitochondrial nucleic acids extracted from strain J69-1B (lanes A, B, E, F) or JM6 (lanes, C, D, G, H) were electrophoresed in 1.5N agarose/6 M urea gels, transferred to diazotized paper and hybridized to separated strands of 3' end labelled Hinfl digested mtDNA from petite G4 (fast strand, lanes A, C; slow strand, lands B, D), or to the separated strands of the 3' end labelled 381 bp MboI fragment from petite G4 (fast strand, lanes E, G; slow strand, lanes F, H). The measured sizes of the transcripts in nucleotides are indicated

scripts were observed in J69-1B mtRNA at 3,900, 3,600, 3,000, 1,700 and 600 bases (Fig. 2, lane E), while for JM6 mtRNA (Fig. 2, lane F) one further transcript (1,700 bases) was observed. These extra transcripts were just visible above the background when prolonged autoradiography was carried out of the transfers in which the nick translated probe was used (data not shown). Tire ability to detect these additional transcripts more clearly in lanes E and F is considered to be due to at least two factors. These are the higher specific activity of the 3' end labelled fragments, and the different distribution of radioactive label in the DS14 sequences. Specifically, the nick translated probe has the 32p label spread more or less evenly through the DS14 sequences, probably enriched somewhat in A+T-rich sequences. In contrast, the mixture of fragments constituting the end-labelled probe has the label concentrated at specific sites within the DS14 sequence. These sites lie in relatively G+C-rich regions, which would hybridize efficiently to their complementary sequences in oli2 transcripts. Close inspection of the autoradiogram depicted in Fig. 2, lanes E and F indicated the presence of a further three faint bands (4,300, 2,100 and 1,200 bases); these are not transcripts of the oli2 gene (see description of hybridization of probe 8, below).

In view of the recent demonstration by Beilharz et al. (1982) that extensive regions of both strands of yeast mtDNA are transcribed in vivo, it was important to establish the particular strand on which the oli2 region transcripts in class I and class II strains are coded. Strand specific probes were prepared from the mtDNA of a petite mutant G4 (see Fig. 1). The G4 mtDNA genome is 1.6 kb in length and contains about 70% of the coding region of the oli2 gene together with sequences derived from R8. G4 mtDNA was digested with HinfI (which cuts this DNA once within the oli2 coding region) and 3' end labelled. The single-strands were resolved in a polyacrylamide gel into a fast and a slow strand. Each labelled strand was used as a hybridization probe against gel fractionated mtRNA from strains J69-1B and JM6 bound to diazotized paper. The results (Fig. 3) show that the fast strand of G4 hybridized to all nine transcripts of J69-1B (lane A) and to all five transcripts of JM6 (lane C), but the slow strand did not hybridize to any abundant transcripts (lanes B and D). Thus all transcripts are derived from the same strand of mtDNA. To identify the particular strand that is used as a template for transcription, the nucleotide sequence of a portion of each single stranded 3' end labelled probe was determined. From the sequence of the first 90 nucleotides read in each case (data not shown), the identity of each strand was established. From the orientation of the G4 mtDNA segment (Fig. 1), the direction of transcription was deduced to be from left to right in the context of Fig. 1 (i.e. from oxi3 to cyb). This represents an empirical demonstration of the direction of transcription of the oli2 region. The sequence of the slow (non-hybridizing) strand was identical to that shown for the oli2 sequence (inferred mRNA sequence) of Macino and Tzagoloff (1980). An analogous experiment was carried out using the 381 bp MboI fragment of G4 containing exclusively sequences derived from within the coding region of the oli2 gene (Fig. 3, lanes E-H). The results showed that in the case of J69-1B, the hybridizing strand detected only five transcripts (lane E). The failure to detect the four smallest transcripts with this probe suggests that these RNAs do not include the entire oli2 structural gene sequences. Furthermore, these transcripts are encoded by mtDNA sequence to the fight of the oli2 gene since they hybridize to the G4 probe (lane A). On the other hand, the five JM6 transcripts were all detected by this 381 bp MboI fragment (lane G). The determination of the nucleotide sequence of each 3' end labelled single strand confirmed the direction of transcription to be from left to right for both J69-1B and JM6.

G. S. Cobon et al.: Transcripts of oli2 Region of Yeast Mitochondrial DNA

Fig. 4. Mapping of the oli2 transcripts of J69-1B. RNA extracted from J69-1B mitochondria was electrophoresed in 1.5% agarose/6 M urea gels, stained with ethidium bromide (lane S), transferred to diazotized paper and strips of the paper were hybridized to each of the fourteen 3' end labelled DNA fragments (lanes 1-14) derived form oli2 gene and the surrounding sequences shown in Fig. 1. One strip was hybridized (lane D) to DS14 mtDNA which had been digested with HpaII plus EcoRI and 3' end labelled. The measured sizes of the transcripts in nucleotides are indicated

Mapping o f the 5' and 3' Ends o f the oli2 Transcripts o f J69-IB In order to determine the positions of the 5' and 3' ends of the transcripts of the oli2 region, we prepared a series of fourteen a2P-labelled contiguous restriction enzyme fragments of mtDNA that cover the oli2 region and surrounding sequences. These hybridization probes, numbered for convenience 1 - 1 4 (Fig. 1), extend from the left end of R7 (1.9 kb upstream of the oli2 gene) through into R3 (2.5 kb downstream of the oli2 gene). Probes 1 - 6 were prepared from a recombinant plasmid carrying the J69-1B fragment R7 cloned into pBR325. The order of the restriction enzyme sites has been derived from a series of restriction enzyme digests and DNA sequencing studies (C. Novitski et al. manuscript in preparation). Probes 7 - 1 4 were prepared from petite DS14 mtDNA; the identity and order of the restriction enzyme fragments was obtained from the published sequence of this mtDNA (Macino and Tzagoloff 1980). The organization of HpalI fragments in the corresponding region of J69-1B closely resembles that of DS14 (Nagley et al. 1981; see also following section herein). Each fragment was labelled at the 3' ends by incorporation of [32p]-nucleoside triphosphates. The probes were incubated under hybridization conditions with strips of diazotized paper on which gel-fractionated J69-1B mtRNA had been immobilised. The resulting autoradio-

101

grams (Fig. 4) permit the determination of the mtDNA segment that is complementary to each of the nine transcripts previously identified with the mixture of 3' end labelled DS14 fragments (cf. Fig. 4, lane D). These data lead to the oli2 transcription map shown in Fig. 5. Probe 1 hybridized to a complex set of transcripts including two which corresponded in size (5,100 and 4,500 nucleotides) to the largest two transcripts that had been detected by the DS14 probe. Since the coding region of the oxi3 gene extends 54 bp into the left end of R7 (Bonitz et al. 1980), probe 1 would be expected to hybridize to oxi3 transcripts which themselves constitute a complex family (Grivelt et al. 1980). Probe 2 (which lies 750 bases from the left end of R7) and probe 3 hybridized only to the 5,100 and 4,500 nucleotide transcripts, but did not detect any of the oxi3 transcript. Probe 4 detected the 5,100 and 4,500 transcripts as well as the 3,900 and 3,600 nucleotide oli2 specific transcripts. Probes 5, 6 and 7 cover the oli2 gene itself (Fig. 1), and these probes detected the above four transcripts as well as the 3,000 nucleotide transcript. Probes 9, 12 and 13 detected the preceding five transcripts as well as the 1,900 and 1,700 base species. Only with probes 10 and 11 were all nine transcripts observed, including the 600 and 800 nucleotide species. Probe 14 failed to hybridize within the limits of detection to any RNA species present in the phenol extract. Prolonged exposure of the autoradiogram in lane 14 showed that this probe hybridized to the DNA present at the uppermost regions of the gel track; this probe also hybridized strongly to mtDNA fragments on nitrocellulose paper under the same hybridization conditions used for the transcript mapping (see next section). Anomalous results were obtained with probe 8. This probe has a size of approximately 15-20 bp (measured from gel mobility) with a HpaII site at each end and a HaeIII site within; its full sequence is not known (Macino and Tzagoloff 1980). This probe showed prominent hybridization to three transcripts (4,300, 2,100 and 1,200 nucleotides which were not detected by the neighbouring probes 7 and 9; the expected five largest oli2 transcripts hybridized relatively weakly to probe 8, and could be seen only on prolonged exposure to the autoradiogram. The probable explanation for this anomaly is that the particular highly G+C-rich sequence in probe 8 is represented in one or more other regions of the mitochondrial genome (cf. Cosson and Tzagoloff 1979); these sequences are found in at least three transcripts that are present in mtRNA at greater abundance than the oli2 transcripts. In support of this conclusion, we show here below that the same three transcripts (4,300, 2,100 and 1,200 nucleotides) in JM6 mtRNA are found to hybridize to probe 8, yet the sequences of probe 8 lie within a segment of mtDNA in J69,1B which is

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Fig. 5. Transcription map of the eli2 gene and surrounding regions in strain J69-1B. The uppermost portion of the Figure corresponds so that of Fig. 1, except that the DNA intervals corresponding to each of the fourteen hybridization probes are indicated directly the simplified restriction map of J69-1B mtDNA. The hybridization data presented in Fig. 4 were used to map the 3' and 5' ends of the transcripts detected by each of the fourteen probes used. The DNA region coding each transcript (marked here by its size in nucleotides) is indicated by a solid line; the broken lines represent the uncertainty in mapping a particular transcript (see text). The restriction enzyme sites shown are: Eco RI, o HpaII, o; Hinfl,

within the 3' portion of the coding region of the oxi3 gene. Considering the seven longest transcripts in Fig. 5, it is apparent that they may all have the same 3' end. Since all seven show strong hybridization to probe 13, it is likely that they have substantial homology with this probe. Since the 3,900 and 1,900 transcripts were judged to show at least 150 bases in common to probe 13 (see above), the possibility is raised that all seven terminate at a point close to the HpaII site at the 13/14 junction. It this is so, the differences in the length of this family of seven longest transcripts would be determined solely by differences at the 5' end of each molecule. The 600 and 800 nucleotide transcripts are drawn within limits which include the sequences of probes 10 and 11. Since they both hybridize to probe 11 much more strongly than to probe 10, it is inferred that both have major sequence homology with probe 11 ; the solid lines representing these transcripts are thus drawn mainly across probe 11.

Mapping of DNA Sequences Found in J6 9-1B but Absent from Strain JM6 missing from JM6 mtDNA downstream of the eli2 gene. When total DS14 mtDNA is used to probe J69-1B mtRNA, these three non-eli2 transcripts are found to hybridize weakly (Fig. 2, lane E). The transcription scheme (Fig. 5) derived from the autoradiograms in Fig. 4 was drawn by analysis of the various mtDNA fragments to which a particular transcript hybridizes, and the measured size of the transcripts. The 3' end in each of the seven largest transcripts is positioned just to the left of, but not including probe 14; each of the seven transcripts hybridize to probe 13 but not to probe 14. As it is not known how much of the 250 bp probe 13 has sequence homology with each transcript the uncertainty (discontinuous lines) is represented formally as 250 bases on the 3' end of each (except for the 3,900 and 1,900 nucleotide species, see below). Measuring the length of each transcript leftwards from the position of the probe 13/14 junction determines the positions of the 5' end of each transcript; the uncertainty indicated at this end is also about 250 bases. In all cases the transcripts show hybridization to the expected contiguous set of probes within their length. In two cases (the 3,900 and 1,900 nucleotide transcript) the uncertainty of both ends is judged to be about 100 nucleotides as summation of the sizes of the fragments to which these transcript hybridize are 4,010 and 2,010 bp respectively. The end of the 5,100 nucleotide transcript is positioned within a DNA segment that includes the left hand end of R7. This segment includes the 3' end of the oxi3 gene, which raises the possibility that the 5,100 nucleotide transcript has its 5' end located

Im developing a coherent scheme to account for JM6 transcripts, it was convenient to study further the altered sequence organization of the JM6 mtDNA in the vicinity of the eli2 gene (Nagley et al. 1981)using the restriction fragment probes (7-14) derived from DS14. Concurrently with the transcription studies, these probes derived from DS14 were hybridized to strips of nitrocellulose paper on which HpaII digests of J69-1B mtDNA were immobilized. The data obtained from the resultant autoradiographs (Fig. 6) confirm the identity of the probes used, demonstrate their purity, and allow a clear definition of the DNA sequences in this area which are missing from strain JM6. Probe 8 was not used in this analysis as it was considered that it would not detect its complementary 15-20 bp fragment, which would be unable to bind to the nitrocellulose filter. When the mixture of 3' end labelled fragments of DS14 mtDNA derived by EcoRI plus HpaII digestion was hybridized to J69-1B mtDNA (Fig. 6, panel A), four major bands and several minor bands of hybridization were observed (lane D). Using the nomenclature indicated in Fig. 6 (panels A and C) these bands are identified as H1, H2, H3 and a doublet of H5 and H6. Note that fragments H7 (107 bp) and the 15-20 bp HpaII fragment lying between H1 and H5 are not detected by this Southern blot analysis. Several minor bands of hybridization are also seen in lane D of Fig. 6, panel A. These include the 830 bp HpalI fragment (labelled HL) lying to the left of H1 (see panel C) which has sequences in common with H4 of DS14. Fragment

G. S. Cobon et al.: Transcripts of oli2 Region of Yeast Mitochondrial DNA

Fig. 6. Mapping of the mtDNA sequences downstream of the oli2 gene which are in J69-1B but absent from JM6. Purified

mtDNA from strain J69-1B (Panel A) or JM6 (panel B) was dNested with HpaII, electrophoresed in 1.5% agarose gels and transferred to nitrocellulose sheets. Strips of the nitrocellulose were hybridized to the 3' end labelled probes (7-14) derived from DS14 (with the exception of probe 8). Strips were also hybridized to DS14 mtDNA which had been digested with HpaII plus EcoRI then 3' end labelled (lane D). The position on the strips are shown of the HpaII fragments of DS14 (Macino and Tzagoloff 1980) that correspond exactly to those of J69-1B (Nagley et al. 1981) namely H1 (1,470 bp), H2 (1,050 bp), H3 (470 bp), H5 (245 bp, H6 (250 bp), as well as the HpaII fragment of J69-1B that includes the left end of the DS14 mtDNA genome, namely HL (830 bp). The corresponding J69-1B HpaII fragment at the right end of DS14 is denoted HR (see text). In addition, the positions are shown of JM6 HpaII fragments 1,600 bp and 350 bp in size. Panel C shows the EcoRI, % and HpaII, o, map of DS14 and J69-1B mtDNA together with the map for JM6 derived from this analysis, The broken lines in the JM6 map represent the formal uncertainties in the mapping of sequences missing from JM6. The arrowhead symbols • represent the most probable positions at which the ends of the missing sequences are considered to map (see text). The symbol? on the JM6 map indicates the uncertainty in the existence of a HpaII site at this position in JM6

H4 was identified by Macino and Tzagoloff (1980) as the DS14 fragment carrying sequences from both HL and HR. Fragment HR (Lying to the right of H7) is judged to be about 450 bp in length (cf. data of Macino and Tzagoloff 1980, for petite DS19), and the hybridization of this fragment to the total DS14 probe would be obscured by that o f H3 (470 bp long). Other minor

103

bands in lane D of panel A are attributed to the hybridization of sequences of H7 to other regions of mtDNA (see discussion below of hybridization pattern of probe 14). Considering the hybridization of the individual restriction fragment probes to HpaII digests of J69-1B mtDNA (Fig. 6, panel A) it can be seen that probes 7, 9, 10, 11, 12 and 13 hybridize to the single HpaII restriction fragment predicted from the map in Fig. 1. Probe 14 did not detect its homologous fragment H7 as this was too small (107 bp) to bind efficiently to the mitrocellulose filter under the conditions used here. However, hybridization was observed with this probe (lane 14 of panel A)~to three HpalI fragments (about 1,400, 950 and 700 bp) that are barely visible when total DS14 probe was used (lane D). These bands are ascribed to the presence of sequences in H7 that are denoted ori7 by de Zamoroczy et al. (1981), which have homology to other ori sequences located elsewhere on the mitochondrial genome. These ori sequences are thought to be origins of mtDNA replication (see de Zamoroczy et al. 1981). When HpaII digests of strain JM6 (Fig. 6, panel B) were hybridized to the total DS14 probe (lane D) the major hybridization occurred to a 1,600 bp fragment. Several weaker bands were also observed, which include the 800 bp HpalI fragment (HL) detected in J69-1B and a 350 bp HpalI fragment not seen in J69-1B. None of the fragments corresponding to H1, H2, H3, H5 or H6 are seen. When the individual probes were used, only probes 7, 12, 13 and 14 showed significant hybridization (Fig. 6, panel B). Probes 7 and 12 both hybridized to the 1,600 bp HpaII fragment;probes 9, 10 and 11 did not hybridize to any detectable fragment, while probes 13 and 14 hybridized to the 350 bp fragment. Since the sizes of probe 13 (250 bp) and 14 (107 bp) add up almost exactly to 350 bp, the most likely explanation of the results is that the sequences corresponding to probes 13 (H6) and 14 (H7) are present in strain JM6 but the HpaII site between them is absent (see map in panel C). Probe 14 also hybridized weakly to three DNA bands other than the 350 bp fragment; these bands are similar to these seen in J69-1B mtDNA which were ascribed to the hybridization of oril sequences to ori sequences elsewhere in mtDNA. Some of the unassigned bands in the JM6 mtDNA probed with total DS14 mtDNA (lane D) may correspond to these on'-bearing fragments. From this analysis we conclude that the sequences found in J69-1B that are missing from JM6, include some of probe 7, all of probes 8, 9, 10 and 11 as well as some of probe 12 (Fig. 6, panel C). To position the ends of missing sequences more precisely consideration was given to the length of the missing sequences, namely 1,800 bp (Nagley et al. 1981), and the relative intensities

G.S. Cobon et al.: Transcripts of oli2 Region of Yeast Mitochondrial DNA

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the sequences present in R8 and extends a short distance (probably no more than 200 nucleotides) into R3. This revises our previous estimation of the location of the sequences missing from JM6 (Nagley et al. 1981).

Mapping of the oli2 Transcripts of Strain JM6

Fig. 7. Mapping of the oli2 transcripts of strain JM6. RNA extracted from strain JM6 mitochondria was treated and hybridized to mtDNA probes. All indications are as in Fig. 4, except that data for probes 9, 10, 11 are not shown here as these contain sequences not found JM6 mtDNA (see Fig. 6, panel B)

4 OXI3 )

c

5

[] 6

[

OLIZ

07

1WD1310m24

JM6 MTDNA

1

TRANSCRIPTS 3000 N 2400 N 2000 N 1700 N 1150 N

TRANSCRIPTION y

Fig. 8. Transcription map of the oli2 gene and surrounding regions in strain JM6. The hybridization data presented in Fig. 7 were used to map the 3' and 5' ends of the transcripts detected in JM6 nucleic acid extracts. All indications are as in Fig. 5. Note that JM6 lacks mtDNA sequences corresponding to probes 8, 9, 10, 11; owing to this deletion, sequences in JM6 homologous to parts of probes 7 and 12 lie in one DNA interval on this map

of hybridization of probes 7 and 12 to the JM6 HpalI digest (panel B, lanes 7 and 12). Probe 7 hybridized comparatively weakly to the 1,600 bp HpaII fragment. Although both DNA molecules share 80 nucleotides of the 3' end of the oli2 coding region, there may be few other sequences in common between these DNA segments. By contrast, probe 12 hybridized comparatively strongly, and it is concluded that much of its 460 bp length is shared with the JM6 1,600 bp HpaII fragment. The arrowheads at the bottom of panel C represent the most likely positions of ends of the sequences in J69-1B missing from JM6. The deletion has its left end just past the 3' end of the oli2 gene, includes the remainder of

Hybridization of RNA isolated from strain JM6 to the 14 hybridization probes (Fig. 7) showed that probes 1 - 7 , 12 and 13 all hybridized to the transcripts of 3,000 and 2,400 nucleotides. Probe 1 visualized the family of oxi3 transcripts of JM6 as seen in J69-1B mtDNA (Fig. 4). Probe 4 detected the 2,000 and 1,700 nucleotide oli2 transcripts and probes 5, 6, 7, 12 and 13 hybridized to all five oli2 transcripts, visualized by the total end labelled DS14 probe (Fig. 7, lane D). Probes 8 - 1 1 (data not shown) and 14 (Fig. 7)were not observed to hybridize to oli2 transcripts. Nevertheless, probe 8 did hybridize to the three non-oli2 transcripts similar to those detected by probe 8 in J69-1B (Fig. 4, lane 8), and which are presumed to be derived from elsewhere on the mtDNA genome. These transcripts are also weakly detected by the DS14 probe in strain JM6 (Fig. 7, lane D). The transcription map for JM6 derived from this analysis (Fig. 8) was constructed in the same way as that for J69-1B (Fig. 5). The map shows that the five oli2 transcripts of strain JM6 are analogous to the five largest transcripts of strain J69-1B (Fig. 5) in that their respective 5' and 3' ends map within a similar segment of mtDNA. Each transcript in JM6 is about 2,000 nucleotides shorter than its J69-1B counterpart. This can be directly correlated with the absence of 1,800 bp from JM6 mtDNA downstream of the oli2 gene (Fig. 6). The four smallest transcripts in J69-1B (1,900, 800 and 600 bases) are not found in strain JM6. Each of these transcripts is specified by a J69-1B mtDNA segment (Fig. 5) which is deleted from JM6 either in its entirety (800 and 600 base species) or for most of its length, including the 5' end (1,900 and 1,700 base species).

Detection of Larger Transcripts of oli2 Region The transcriptional analyses presented to date have led to the identification and mapping of the most abundant oIi2 transcripts which are resolved in 1.5% agarose/6 M urea gels. There was some evidence for hybridization above the 5,100 transcript in prolonged exposure of the autoradiograms of 1.5% agarose gels presented above. To improve the resolution of these large transcripts of the oli2 region, RNA extracted from strains J69-1B and JM6 was electrophoresed in 0.6% agarose/6 M urea gels and stained with ethidium bromide (Fig. 9, lanes

G. S. Cobon et al.: Transcripts of oli2 Region of Yeast Mitochondrial DNA

105

a low recovery of DNA in the aqueous phase (Markov and Arion 1973) which is consistent with the absence of a DNA staining band in the uppermost regions of all agarose gels here. Second, before transfer to the diazotized paper, these agarose gels were not treated with NaOH which would result in the DNA present in the gel retaining its double stranded nature and thus being unable to bind efficiently to the diazotized paper (Noyes and Stark 1975).

Discussion

Fig. 9. Analysis of oli2 transcripts on low concentration agarose gels. RNA extracted from strains J69-1B (lanes A, B, C) or JM6 (lanes D, E, F) was electrophoresed in 0.6% agarose/6 M urea gels, stained with ethidium bromide (lanes A, D), transferred to diazotized paper and hybridized to DS14 mtDNA that had been digested with HpalI plus EcoRI then 3' end labelled (lanes B, E). The samples in lanes C and F correspond to those in lanes B and E, respectively, except that the RNA was treated with DNase I before electrophoresis (see Beilharz et at. 1982). Indications at left of Figure are as for Fig. 2

A and D). The RNA in these lower concentration agarose gels was then transferred to diazotized paper and hybridizied to a probe of a DS14 mtDNA that had been digested with HpalI plus EcoRI then 3' end labelled. In both J69-1B (Fig. 9, lane B) and JM6 (lane E), the discrete oli2 transcripts are found in similar positions relative to the 21S and 15S mitochondrial rRNA (cf. Fig. 2, lanes E, F). In addition, a distinct region of hybridization is observed in both lanes B and E to nucleic acids with a low electrophoretic mobility. This zone of hybridization is broad, and corresponds to material which is clearly larger than 6 kb in length. The size of these transcripts was not able to be determined accurately from these gels. Treatment of the nucleic acids with DNase I prior to electrophoresis (Fig. 9, lanes C and F) resulted in only a minor reduction in the extent of hybridization to this region indicating that the majority of the hybridizing species are RNA and not DNA. That there was little DNA detected by this analysis is considered to be due to two major factors. First extraction of nucleic acids with phenol at 60 °C has been shown to lead to

The data presented in this paper indicate that nine distinct transcripts derived from the oli2 region of mtDNA can be observed in class I strains, while five are found in class II strains of S. cerevisiae. Other larger transcripts were also observed. The positions of the 5' and 3' ends of all fourteen distinct transcripts have b e e n determined by use of a series of contiguous hybridization probes covering a region of mtDNA some 5.2 kb long that spans the oli2 gene. All transcripts are empirically demonstrated to be transcribed in the same direction, which is in the same sense as that found for almost all other abundant transcripts of yeast mtDNA (see Borst and Grivell 1981). The precision of 5' and 3' end mapping of oli2 transcripts was influenced by two factors in this study. The first of these is that the use of hybridization probes only allows ends to be positioned within the region covered by the terminal probe in a contiguous set that hybridizes to the transcript in question. A second factor is the accuracy with which the lengths of particular transcripts can be measured in the agarose/urea gel system employed here. The internal standards employed for calibrating the gels were the two mitochondrial rRNAs, with apparent sizes taken to be 3,700 and 2,000 nucelotides for the 21S and 15S rRNA, respectively (Locker et al. 1980). Other studies on these RNAs or on the genes which specify them indicate the rRNA sizes to be 3,100 (Heyting et al. 1989; Atchison et al. 1979; Dujon 1980) and 1,600 nucleotides (Tabak et al. 1979), for the 21S and 15S rRNA, respectively. It is not known to what extent the agarose/urea gels overestimate the sizes of the oli2 transcripts studied here. The main consequence of possible size overestimation would be that the 5' ends of many transcripts would be further to the right than shown in Figs. 5 and 8, since the 3' end positionings are reasonably good, having regard to the relatively short size of the probe 13 in which this end lies (see Results). Nevertheless, the hybridization data does not permit the 5' end to be further to the right than the rightmost 100 nucleotides in the first hybridizing probe in each case. For most transcripts in Figs. 5 and 8, the DNA interval which covers the probes

106

G.S. Cobon et al.: Transcriptsof oli2 Region of Yeast MitochondrialDNA

that hybridize to each transcript is consistent with the measured length of the transcript. The only transcripts for which the 5' end mapping cannot be corroborated by comparing DNA intervals with measured RNA sizes are the two largest transcripts in the class I and class II strains, namely the 5,100 and 4,500 base transcripts, and the 3,000 and 2,400 base transcripts, respectively. The reason is that these four transcripts all hybridized to probe 1 (the leftmost probe) and we have not identified a discriminating probe near their 5' ends to which they do not hybridize. For these transcripts in particular, further work is required to locate their 5' ends more precisely. A corollary of the general coincidence between the DNA intervals over which hybridization occurs, and the measured length of the RNA transcript is that there do not appear to be any major introns removed from the oli2 transcripts studied here. This is supported by the hybridization of all contiguous probes in a set to a given transcript. This would include the two largest transcripts of class I and class II strains, We cannot exclude the possibility of small introns that lie wholly within the sequences of one probe. Nevertheless, S1 nuclease mapping studies haves been carried out on DNA-RNA hybrids formed between EcoRI fragments of mtDNA and total phenol extracted mtRNA of the class I strain J69-1B Alkaline gel electrophoresis of the S1 spared mtDNA fragments revealed that R8 was protected in its entirety, while R7 was some 200-400 bases shorter (Bellharz 1982). The results are interpreted to indicate that the most abundant oIi2 transcript, namely the 4,500 base species, carries all sequences of R7 and R8 except for 200-400 nucleotides at the left end of R7, which is where its 5' end is presumed to lie. The most likely candidate for the mRNA of the oli2 gene in class I strains is the most abundant 4,500 nucleotide species, although it cannot be excluded that other RNA transcripts which include the entire oil2 coding region can also be translated in vivo. It is noteworthy that in some m i t - mutants (oxi3 and cyb) which still continue to synthesize a functional ATPase subunit 6, the only two oli2 transcripts detectable are the 5,100 and 4,500 base species (Beilharz et al. manuscript in preparation). If the 4,500 base species is the mRNA, the mapping shown in Fig. 5 indicates that it has a 5' leader of some 1,400 nucleotides, a coding region of 780 nucleotides, and 3' sequences 2,300 nucleotides in length. The equivalent abundant mRNA candidate in class II strains (2,400 bases) has the same 5' leader and coding region, but its 3' tail is only about 200-300 nucleotides long. Indeed all class II transcripts have this short 3' tail following the coding region (Fig. 8). In the light of observations that class II strains make an ATPase subunit 6 indistinguishable from that of class I strains (Beilharz 1982) it is clear that the variation in

the 3' sequences does not affect translation of the oli2 mRNA. Another example of this situation has recently been reported by Setzer et al. (1980) who identified four distinct transcripts of the mouse dihydrofolate reductase gene with properties of mRNA. These four transcripts are polyadenylated, polysomal in location, and translatable in vitro, but each differs in the length of the 3' untranslated region, ranging from about 80 to 930 nucleotides. Four transcripts of the oli2 region (1,900, 1,700, 800 and 600 nucleotides) were found not to contain structural gene sequences. They were mapped to a region of the mtDNA covered by the 3' tail regions of the longer structural gene-containing transcripts. This region consists of highly A+T-rich sequences characteristic of the long "spacers' between yeast mitochondrial genes. However, there are two short regions of relatively high G+C content that lie within the segments H2 and H3 depicted in Fig. 6, panel C. These two regions were noted by Macino and Tzagoloff (1980) in the sequence of DS14 as other possible coding domains besides the oli2 structural gene. It is interesting that the four transcripts that do not contain structural gene sequences were mapped to the region within which these unassigned reading frames (URFs) are located (see Fig. 5). The absence of the sequences carrying the downstream URFs from class II strains (cf. Fig. 6) indicate that whatever functions may be encoded by these URFs, they are not required for respiratory competence. The relationship between the mtDNA segment missing from JM6 and its altered transcription pattern relative to J69-1B can now be stated. Comparing Figs. 5 and 8, it can be seen first of all that JM6 lacks the four smallest transcripts of J69-1B that are encoded in full or for their major portion, by the DNA segment that JM6 lacks. Secondly, the 5' and 3' ends of each of the five JM6 transcripts correspond to the 5' and 3' ends of each of the five largest J69-1B transcripts, the difference between the class I and II transcripts being a 2,000 nucleotide segment missing from the interior of the transcript in each case. These findings suggest that the particular sequences deleted from class II strains do not play a role in the metabolism of the transcripts (processing, or termination of transcription). The biosynthetic relationship between the sets of transcripts within one class still remains to be established. Two basic schemes can be considered. In the first case, members of a family to transcripts may be related to each other by a series of post transcriptional processing or cleavage events involving common precursor RNA molecules. In the second case, several sequences in mtDNA may act as promoters for transcription with varying efficiencies thus giving rise to the families of oli2 transcripts with different 5' termini and varying relative abundances. The available evidence does not

G. S. Cobon et al.: Transcripts of oli2 Region of Yeast Mitochondrial DNA support either seheme in its entirety. Studies in which promoters for transcription were identified by analysis o f transcripts still carrying the terminal 5' triphosphate residue failed to locate any promoters in the region o f the oli2 gene (Levens et al. 1981). Of the five putative promoters identified this study, the closest which could serve as a promoter for the oli2 gene lay some 1 5 - 2 0 kb away. The oxi3 gene and the 15S rRNA gene lie between this promoter and the oli2 gene. Wherever the promoter for transcription of the oli2 region lies, large transcripts containing gene sequences identified here (Fig. 9, see also Beilharz et al. 1982), may be the precursor RNAs from which the abundant oli2 transcripts are derived by processing events. Indeed, two versions o f the processing scheme can account for the families of oli2 transcripts observed. The transcripts may be produced by a series o f successive cleavage events, or alternate processing pathways may exist for each transcript to be produced from one original precursor molecule. On the "other hand, there is evidence for the use by the mitochondrial transcription machinery of a number o f possible promoters in one region o f the genome. Petite mutants which retain short segments of the grande mitochondrial genome often contain m t R N A (Morimoto et al. 1979). In the particular petites that we have shown to contain a number of discrete mitoclmndrial transcripts, namely G4 and DS14 (Beilharz 1982), the petite genome does not include the segment of m t D N A which is complementary to the 5' end o f the abundant transcripts arising from this region in the parental grande strain. Thus there are sequences in G4 and DS14 that act as promoters for transcription in these petites, but which clearly are not used for the production of the 5,100 and 4,500 nucleotide transcripts of the parent class I grande strains.

Acknowledgements. This work was supported by the Australian Research Grants Committee. We thank Mr. Ulrik John for his competent technical assistance.

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Communicated b y F. Kaudewitz Received March 16, 1982