Assembly of the mitochondrial membrane system. Analysis of the nucleotide sequence and transcripts in the oxi1 region of yeast mitochondrial DNA.

The region of yeast mitochondrial DNA between 10.7 and 17.9 map units has been characterized by restriction analysis and DNA sequencing. The DNA sequence was obtained from the partially overlapping genomes of the two rho- mutants DS200/A1 and DS302. Two tRNA genes have been found in the sequence upstream of the oxi1 gene. The deduced secondary structures indicate that the genes code for the methionine (5'-CAU-3') and the asparagine (5'-GUU-3') tRNAs of yeast mitochondria. The region between 10.7 and 17.9 units contains two reading frames. One of these corresponds to the oxi1 gene previously shown to code for subunit 2 of cytochrome oxidase (Coruzzi, G., and Tzagoloff, A. (1979) J. Biol. Chem. 254,. 9324-9330; Fox, T. D. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6534-6538). The second reading frame can potentially code for a basic protein with 386 amino acid residues. It is not known at present if this putative gene is translated in vivo. Northern blots of wild type mitochondrial RNA were hybridized to single-stranded probes from the oxi1 gene and flanking regions. The results of these analyses indicate that the primary transcript of the oxi1 region is a high molecular weight RNA (larger than 3 kilobase pairs) which is processed in discrete steps to a mature 850-nucleotide messenger. The 5' leader of the messenger has been established to be 54 nucleotides long and to have a sequence identical with that of the genomic DNA immediately upstream of the oxi1 gene.


Proc. NatL Acad Sci. U. S. A. 76,6534-6538). The second reading frame can potentially code for a basic protein with 386 amino acid residues. It is not known at present if this putative gene is translated in vivo.
Northern blots of wild type mitochondrial RNA were hybridized to single-stranded probes from the 0 x 2 gene and flanking regions. The results of these analyses indicate that the primary transcript of the oxil region is a high molecular weight RNA (larger than 3 kilobaee pairs) which is processed in discrete steps to a mature 850-nucleotide messenger. The 5' leader of the messenger has been established to be 54 nucleotides long and to have a sequence identical with that of the genomic DNA immediately upstream of the oxil gene.
The oxil locus of yeast mitochondrial DNA (mtDNA) has been shown to code for subunit 2 of cytochrome oxidase (1,2). These earlier studies indicated the gene to be 756 nucleotides long, spanning the wild type mtDNA from 14.2 to 15.3 map units. The agreement of the known amino acid sequence of bovine subunit 2 (3) and the deduced sequence of the yeast protein suggested a co-linear gene lacking intervening sequences (1,2). In the present study, we have extended the DNA sequence analysis to the regions of mtDNA flanking the oxil locus. The new data provide a continuous sequence of the genome from 10.7 to 17.9 map units. The region upstream from oxil has been found to contain the genes of the asparagine and methionine tRNAs. The DNA sequence of the region downstream from oxil reveals the presence of a new reading frame (referred to as RF1) potentially capable of coding for a * This research was supported by Research Grant GM25250 from the National Institutes of Health, United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Recipient of a National Research Service Award from the National Institutes of Health, United States Public Health Service. protein with 386 amino acid residues. This reading frame occurs in the same DNA strand as the subunit 2 gene. Analyses of the transcripts originating from the oxil region have enabled us to characterize the subunit 1 messenger and to identify some of the precursors to the messenger.

MATERIALS AND METHODS
Yeast Strains-The genotype and sources of the strains of Saccharomyces cereuisiae are listed in Table I. The following protocol was used to isolate DS302. The respiratory-competent strain S. cerevisiae D273-10B/A21 was mutagenized with ethidium bromide under nongrowing conditions (10) and the clone DS300 selected for the retention of markers between the cap and par loci (Table 11). This mutant was subjected to two additional cycles of mutagenesis with ethidium bromide to obtain DS302. The marker retention of DS302 was confined to the oxil locus. The details of the genetic manipulations have been described previously (5).
Purification of Mitochondrial DNA and RNA-The p-mutants were grown at 30 "C in 2% glucose medium supplemented with 1% yeast extract and 1% peptone. Cells were harvested in stationary phase and mitochondria were prepared by the method of Faye et al. (11). mtDNA was extracted with 2% Sarkosyl and purified on CsCl density gradients (12). Mitochondrial RNA was prepared by the method of Bonitz et al. (13) from the wild type S. cereuisiae D273-10B/A1.
Restriction Endonuclease Analysis-Restriction enzymes were purchased from New England Biolabs. All digestions were performed at 37 "C in a buffer containing 10 mM Tris-C1, pH 7.5,6 MgC12, 6 mM 2-mercaptoethanol. The digestion products were analyzed on 1 or 2% agarose gels using the Tris/borate buffer system of Peacock and Dingman (14). +X174 RF DNA digested with Hue I11 (15) and A-DNA digested with Hind111 (16) were used as molecular weight standards.
Preparative Isolation of Restriction Fragments-mtDNA of DS200/A1 and DS302 (100-200 pg) were digested with HinfI and Hpa 11, respectively. The restriction fragments were separated on an 0.8cm-thick slab of 1% agarose. The gel was stained with ethidium bromide and sections containing the desired fragments were cut out. The DNA was eluted from the gel electrophoretically and further purified by extraction with phenol and several precipitations with alcohol in the presence of 2 M ammonium acetate.
DNA Sequencing-mtDNA digested with single or combinations of restriction endonucleases was 5'-end-labeled with [y-32P]ATP (2000-3000 Ci/mmol, New England Nuclear Corp.) in the presence of T4 polynucleotide kinase (17). The labeled fragments were separated into the single strands on 4, 6, or 8% polyacrylamide gels and sequenced by the method of Maxam and Gilbert (17).
Northern Blots-Yeast mtRNA was denatured in the presence of 10 mM methyl mercuric hydroxide and 2 pg were loaded on 0.6-cmwide wells of 1.2% agarose, 10 mM methyl mercury gels. After electrophoresis at 5 V/cm for 4-5 h, the gel was treated with 14.3 mM 2mercaptoethanol, stained with ethidium bromide, and photographed. The RNA was transferred to DBM' paper by the method of Alwine et al. (18). Single-stranded DNA fragments 5'-end labeled with 32P were hybridized to DBM strips overnight (19). The strips were exposed for 1-2 days to Kodak XR-1 film with an intensifying screen I The abbreviations used are: DBM, diazobenzyloxymethyl; kbp, kilobase pair. at -80 "C.
Reverse Transcription and Sequencing of cDNA-Total mtRNA (0.5 mg) was applied to 30 ml of a linear 5-207' 0 sucrose gradient containing 25 mM Tris-C1, pH 7.5, 1 M NaCl, and 1 m~ EDTA. The RNA was sized by centrifugation at 22,000 rpm in a Spinco SW27 rotor for 27 h at 15 "C. The gradient was divided into 17 fractions which were analyzed for the sue distribution of the RNA on agarose gels.
total mitochondrial RNA or to the 8-14s RNA fraction obtained Single-stranded restriction fragments were hybridized either to from the sucrose gradient. The hybridizations were done in 50 pl of 1 x SSC. Following incubation at 42 "C for 90 min, the mixture was diluted with 1 volume of 4 M ammonium acetate and the RNA was precipitated with 3 volumes of ethanol. The precipitate was dissolved in 280 pl of a solution containing 50 mM Tris/acetate, pH 8.4, 6 mM Mg acetate, 60 mM NaCl, 10 mM dithiothreitol, 120 p~ concentration of the four deoxynucleoside triphosphates, 10 pg/ml of actinomycin D, and 0.2 mCi of [a-32P)ATP (200-300 or 3000 Ci/mmol, New England Nuclear Corp.). The mixture was divided into 4 equal parts (70 p l ) and to each was added one of the four dideoxynucleoside triphosphates (120 (LM final concentration) to inhibit the reverse transcriptase (20). The reaction was started by the addition of 6 units of reverse transcriptase (J. W. Beard, Life Science, St. Petersburg, FL) and allowed to proceed for 15 min at 42 "C. The reactions were chased to completion by the addition of 15 pl of a solution 250 PM in each of the four deoxynucleoside triphosphates and further incubated for 15 min at 42 "C. The reactions were stopped with an equal volume of 4 M ammonium acetate and the cDNA was precipitated with 3 volumes of ethanol. The cDNA was reprecipitated from 2 M ammonium acetate. After two additional washes with 80% ethanol, the material was dried, dissolved in 90% formamide, heated at 90 "C for 3 min and loaded onto a DNA sequencing gel (21).

RESULTS
Restriction Maps of the DS2OO/Al and DS302 mtDNA Segments-The two p -mutants used in the present study were isolated from the wild type parental strain S. cerevisiae D273-10B/A21. The restriction map of the mtDNA segment retained in the mutant DS200/A1 has been reported previously (1). The mitochondrial genome of this mutant includes the wild type sequence from 10.7-17 map units (Fig. 1). The second mutant DS302 was obtained after several cycles of mutagenesis of the wild type parent with ethidium bromide. Although both DS200/A1 and DS302 have the same genotypes (see Table 11) and substantial sequence overlap, their deletion end points differ. The unit length of the DS302 segment was estimated to be 3.2 kbp based on the cumulative sues of the fragments generated by various restriction endonucleases. The orientation and location of the DS200/A1 and DS302 segments on the wild type mmNA was inferred from their restriction maps and previous assignment of the pvu 11 sites between 14 and 15 units of the wild type map (22). The restriction maps shown in Fig. 1 indicate that the mtDNA segments of the two p-mutants share a common region from approximately 12.8 to 17 map units. The deletion end points were determined from the comparative restriction maps and the DNA sequences of the two segments. The DS302 segment lacked two H p a I1 fragments (fragments 2 and 4 in Fig. 1) and had an extra 620-base pair Hpa I1 fragment (fragment 5) that was mapped at the extreme right hand side of the segment.
Nucleotide Sequence of the DS2OO/Al and DS302 mtDNA Segments-Together the segments of DS200/Al and DS302 cover the region of the wild type mtDNA from 10.7 to 17.9 units. The restriction fragments used for the sequence analysis are shown in Fig. 2. Most of the digestions were done directly on the mitochondrial DNA of each mutant. In some instances, however, Hpa I1 and HinfI restriction fragments were isolated on a preparative scale and subjected to secondary cleavages transcrrptron ______*

TABLE I1
Genotypes of p-mutants Minus and plus signs indicate the loss or retention of the various loci. In addition to the markers shown in the table, the p-mutants were also deleted with respect to oxi3, cobl, cob,?, olil, and pho2. DS301 was derived from DS300 and was used in turn to isolate DS302 by ethidium bromide mutagenesis. The following strains were used to test for the antibiotic resistance and mit-markers  2). The amino acid sequence encoded in the unidentified reading prior to 5'-end labeling. All the sequence data were obtained on 5'-end-labeled single strands. With a few exceptions, most of the restriction sites were sequenced from neighboring sites (Fig. 2).

+801 A A T A A T A A T A A T A A T T A T A A T A A T A T T C T T A A A T A A T A G A
The nucleotide sequence is presented in a linear form starting with the Hae III/Hpa I1 cluster at 10.7 units and ending with the Hpa I1 site at 17.9 units (Fig. 3). The sequence of the oxil gene has already been reported and is not included in Fig. 3. The sequence encompassed by the two p-genomes is complete except for a few nucleotides near one of the Hpa I1 sites and gap of 450-500 nucleotides between 11.7 and 12.4 units. This region could not be sequenced due to the absence of restriction sites in the DNA. Visual analysis of the sequencing gels, however, indicate that the gap occurs in a (A + T)rich stretch of DNA. Aside from the oxil gene (nucleotides +1 to +756), there are three other sequences of interest. This fist is a 72-nucleotide-long sequence (-2182 to -2111) that can be folded to form a cloverleaf structure with a 5'-GUU-3' anticodon. We frame (+998 to +2158) is based on the assignments of the universal code except for UGA which has been shown to code for tryptophan (2, 23) and the GUN family which codes for threonine in yeast mitochondria (24). The sequence enclosed by the brackets may have one or two extra nucleotides.
tentatively identify this to be the gene of the mitochondrial asparagine tRNA. The second tRNA gene is located at 12.4 units (-1075 to -1002). This sequence can also be folded into a tRNA structure with a 5'-CAU-3' anticodon for methionine. Since the sequence of the gene differs from that of the yeast mitochondrial initiator tRNA (25), it most likely codes for the methionine tRNA which has been mapped between the cap and oxi loci (26). The structures of the asparagine and methionine tRNAs deduced from the sequences of their respective genes have fairly conventional features with the correct bases in the invariant positions of the D and T\CC loops and stems (Fig. 4). Based on the orientation of the mtDNA segments, the two tRNAs are transcribed from the same strands as the oxil gene and most other yeast mitochondrial tRNA genes The other potential coding region is an open reading frame (RF1) that starts with an ATG initiation codon at nucleotide +998. RF1 is continuous for at least 879 nucleotides but could be as long as 1161 nucleotides. The uncertainty is due to an (27)(28)(29). ambiguity in the sequence between nucleotides +1880 and  15 only 19%, a value significantly lower than found in other yeast Most notable is the occur-G 3 rence of CGU codons for arginine and the UGG codon for tryptophan (Table 111). Neither of these codons has been have been found within coding sequences (33,34).
The reading frame reported here is highly reminiscent of  suppressor. While the larger transcripts might also function as messengers, this has not been experimentally verified and the most reasonable assumption is that they are incompletely izations are shown in Fig. 5. One of the fragments had a processed precursors of the 11 S RNA. sequence upstream from the gene (probe C ) ; others originated As a first approach to characterizing the onil transcripts, either from within the gene (probes A, B, K) or from RF1 we have prepared a series of single-stranded DNA fragments (probes D-HI. Together, the fragments spanned the region of with defined sequences from within the oxil gene and its DNA from 13.7 to 18.0 units. flanking regions. The probes were hybridized to total mito-A representative autoradiograph of a Northern blot chalchondrial RNA separated on agarose gels and transferred to lenged with the Pure 0 . d probe A is shown in Fig. 6. The DBM paper. The festriction fragments used in these hybrid-probe is seen to hybridize to a transcript with an estimated size of 850 nucleotides. This RNA species corresponds to the   Probe K from the sense strand of the oxil did not detect these (Fig. 6). This negative result confirms the specific-  through J, at most it contains a sequence of 0.7 kb from 16.6 to 17.6 map units. The discrepancy between the length of sequence homology and the size of the transcript cannot be accounted for but could indicate that it is a spliced product.

U. A ' A A U A " U A U C C U U A A U U A A
The following conclusions can be drawn from the Northern blot hybridizations. 1) The p r i m v transcript of the oxil gene is a high molecular weight RNA with a minimal size in excess of 3 kb. 2) The mature messenger of subunit 2 of cytochrome oxidase is most likely formed by stepwise cleavages of a primary transcript at both the 5' and 3' ends. The hybridizations to upstream and downstream probes further suggest that the same precursor may be processed by alternative pathways. At least 9 intermediates are sufficiently stable to be detected as discrete size RNA species. 3) Transcripts with the sequence of RFl are found only in high molecular weight oxil precursor RNAs. Although a unique transcript from the RF1 region does exist, this RNA contains the sequence complementary to the reading frame.

-3
Map Uncts Five of the probes used (D through H) contained RFl sequences. Probe D was complementary to RF1. Probes E through H were complementary to the opposite strand, whose sequence does not contain any reading frame of significant length. Probe D hybridized only to some of the high molecular weight transcripts that were detected by the oxil probes. For example, transcripts 2, 6, 7, 9, and 10 failed to hybridize to probe D, indicating that their 3' termini cannot extend more than 300 nucleotides beyond the end of the oxil gene. It is of interest that probe D did not reveal any new abundant transcript. Unexpectedly, both probes E and F with sequences from the sense strand hybridized to a new transcript of 1.5 kb (Fig. 6). Since this transcript was not detected by probes G The calibration scale used to calculate the sizes of the transcripts is shown next to the ethidium bromide-stained gels.

Transcripts from the oxil Region of Yeast mtDNA
was a HinfI-Rsa I fragment complementary to the gene sequence from nucleotides +434 to +506. The sequence of the DNA copy synthesized from the 3' end of this primer was perfectly complementary to the previously reported sequence of the gene (Fig. 7 and Ref. 1). The second primer was a HinfI-Rsa I fragment with a 3' end 22 nucleotides upstream from the initiation codon and a 5' end a t nucleotide 50 internal to the gene. The cDNA made from this primer is shown in Fig. 8. The cDNA extension is 32 nucleotides long and is complementary to the sequence upstream of the distal HinfI site up to nucleotide -54 (see Fig. 3). The 11 S transcript therefore has a 54-nucleotide leader whose sequence matches the corresponding DNA sequence. The leader combined with the 756-nucleotide-long gene sequence comes close to the measured size of the 11 S transcript. From S1 mapping, Fox and Boerner (39) have concluded that the 3' extension of the messenger must be less than 75 nucleotides in length. Although the 5' and 3' ends of the messenger are generally consistent with the results of the Northern blot experiments, it is not completely clear why probe C which contains part (32 nucleotides) of the leader sequence did not hybridize to the 11 S transcript. A possible explanation is that the conditions used for the hybridizations were too stringent to favor formation of the hybrid, particularly since the complementary region in this probe was confined to 32 nucleotides consisting almost entirely of A + T.

DISCUSSION
The two p-mutants, DS200/A1 and DS302, were selected for the retention of markers in the oxil locus. The mitochondrial genomes of these mutants were established to span the region of the wild type map from 10.7 to 17.9 units. The nucleotide sequences of the mtDNA segments have enabled us to identify the genes of the asparagine and methionine tRNAs. Both genes are located upstream of the oxil locus a t 10.9 (asparagine) and 12.4 (methionine) map units and are transcribed from the same DNA strand as subunit 2 of cytochrome oxidase (1,2).
In addition to the oxil gene, the region sequenced contains a second reading frame (RF1) that has an ATG initiator a t 15.5 units and is probably continuous for 1161 nucleotides. The protein encoded in this putative gene consists of 386 amino acid residues and has a molecular weight of 48,000. Even though this sequence is not part of an intron, it is in many respects similar to a number of reading frames recently found in the intervening regions of yeast mitochondrial genes (13,(35)(36)(37). RF1 has a G + C content of only 19% and is rich in condons for asparagine, lysine, and tyrosine. It also utilizes codons such as the CGU codon for arginine and the UGG codon for tryptophan which up to now have only been found in intron reading frames.
The transcripts copied from the oxil region have been characterized by hybridization of single-stranded DNA probes to total mitochondrial RNA by the Northern blot technique (18). These studies indicate that the mature messenger of subunit 2 is formed by a stepwise cleavage of large precursor transcripts. The processing appears to involve the removal of both 5' and 3' sequences from large precursor transcripts. At least 9 intermediates with discrete sues are detected by probes containing either structural gene or flanking sequences.
Several lines of evidence indicate the mature messenger of subunit 2 to be an 11 S RNA. It is the most abundant and smallest RNA species to hybridize to probes from the oxil gene (38,39). More convincingly still, DeRonde et al. (40) have been able to synthesize subunit 2 in an in vitro wheat germ system programmed with an 11-13 S-enriched fraction from yeast mitochondrial RNA. In the present study, the 5' terminus of the 11 S transcript has been determined from its cDNA sequence. The leader of the 11 S transcript starts 54 nucleotides upstream from the AUG initiator and has a sequence identical with that of the genomic DNA. It is not possible to state whether the 5' terminus is generated by an endonucleolytic cleavage or exonucleolytic trimming of one of the larger precursors.
Probes complementary to RF1 detect only high molecular weight RNAs which also hybridize to probes from the oxil gene. A single unique transcript with an estimated sue of 1.5 kb, however, hybridizes specifically to probes with sequences from the sense strand of RF1. The function of this transcript is not clear to us at present.