sign in sign up
<<
Home , Primary transcript

Proc. Nati. Acad. Sci. USA Vol. 80, pp. 5564-5568, September 1983 Biochemistry

Identification of a single transcriptional initiation site for the glutamic tRNA and COB in yeast mitochondria- (Saccharomyces cerevwiae/guanylyltransferase/in-vitro capping/in vitro /cytochrome b). THOMAS CHRISTIANSON*, JOHN C. EDWARDSt, DAVID M. MUELLERt, AND MURRAY RABINOWIZT§ Departments of tMedicine, tBiochemistry, and *Biology, The University of Chicago, Chicago, Illinois 60637 Contributed by Murray Rabinowitz, June 20,.1983 ABSTRACT We have identified a single transcriptional ini- their sequences to known DNA sequences-11-13). In recent tiation site for the glutamic tRNA and COB.(cytochrome b) genes experiments, we mapped 20 unique initiation sites and iden- by using the complementary techniques of in vitro capping of RNA tified the precise position on the genome for; 13 of them (15). and in vitro transcription-ln the capping reaction, mitochondrial The second procedure involves the study of. initiation with a RNA is labeled with [a-32P]GTP by vaccinia virus guanylyltrans- homologous in vitro transcription system using purified mi- ferase. This reaction is specific for the 5' ends of RNA retaining tochondrial RNA polymerase and cloned DNA templates (14). the terminal triphosphate of transcriptional initiation. Exploiting We have found that the purified polymerasesis capable of spe- the extremely low G+C content (18%) of yeast mitochondrial DNA, cific initiation-at the rRNA initiation sites identified by cap- we digested in vitro capped transcripts from various petite dele- tion mutants with the G-specific RNase Ti. By petite deletion ping. mapping, a capped transcript giving rise to a 51-base RNase T1- About 25 homologous bases are found at the sites of tran- generated was localized near the glutamic tRNA scriptional initiation for the two ribosomal genes (12, 16). A . When the sequence of this oligonucleotide was determined, nonanucleotide conserved sequence immediately upstream from it perfectly matched the DNA sequence 391 bases upstream of the each rRNA initiation site is revealed by comparison of the DNA glutamic tRNA. Purified yeast mitochondrial RNA polymerase in- sequences in Saccharomyces cerevisiae with a distantly related itiated transcription in vitro at the same site as shown by the se- yeast species, Kluyveromyces lactis (13). Copies of this non- quence of the 33-base oligonucleotide product of the reaction per- anucleotide appear at a few other scattered sites in the genome formed in the absence of CTP. Initiation starts at a nonanucleotide (13, 17). Exact identification of initiation sites for other genes sequence previously implicated in yeast mitochondrial transcrip- is necessary to evaluate the role of these sequences in tran- tional initiation. Because there is no evidence of an initiation site scriptional initiation. in the 1,050 bases between the glutamic tRNA and COB genes, We therefore examined the initiation site for the COB gene. the two genes are likely to be transcribed together. Further evi- The entire COB gene region, including about 3,000 bases up- dence of a long common transcript was provided by RNA blot hy- stream from the translational initiation codon, has been sub- bridization. jected to sequence analysis (5, 6, 18, 19). S1 nuclease mapping has shown that the major COB transcript extends about 900 The COB gene, coding for a subunit of the cytochrome bcl bases upstream the gene (18, 20). The next gene upstream from complex of yeast mitochondria, has been the subject of inten- COB codes for glutamic tRNA and is separated from the COB sive study. Production of a mature COB mRNA involves com- coding region by about 1,050 (Fig. 1). plex processing including splicing of several (1-4), the In this study, we identify a single transcriptional initiation exact number of which is strain -dependent (5, 6). Some of these site for the glutamic tRNA and COB genes. We find that the introns are unusual in that they code for maturases-that is, sequence of an in vitro capped mitochondrial transcript that that appear to be required for splicing (6-10). maps to this region is identical to the sequence of RNA prod- However, although processing mutants and intermediates in- ucts transcribed in vitro from a cloned DNA template. The se- volved in formation of mature COB message have been exten- quences match the DNA sequence (19) 391 bases upstream from sively analyzed, the nature of the of this gene the glutamic tRNA. Initiation starts at the last base of a nonanu- has not been characterized. To understand more fully the con- cleotide sequence that is homologous-to the DNA segment pre- trol of expression of the COB gene, it is necessary to know the ceding the rRNA genes and has been implicated in site of transcriptional initiation for COB transcripts. function. The glutamic tRNA and COB genes appear to share We have recently defined.transcriptional initiation sites on a common primary transcript because neither nonanucleotide the mitochondrial genome (11-14) by using two new proce- sequences nor other capped transcripts are observed between dures. In one, vaccinia virus guanylyltransferase is used in an the identified initiation site and the COB coding sequence. in vitro capping reaction to label RNA which retains its initi- ating (11). This reaction is specific for 5'-polyphos- MATERIALS AND METHODS phate-terminated RNA molecules. By hybridization of capped transcripts to restriction fragments of mitochondrial DNA im- Strains. Grande strains MH41-7B and D273-10B were used mobilized on nitrocellulose,' we previously demonstrated a in this study and have been described by Morimoto and Ra- minimum of four or five widely separated initiation sites (11). binowitz (23). The petite strain DS400/A12 was generously In addition, the exact position of the initiation sites for the large provided by A. Tzagoloff (Columbia University, New York). The and small ribosomal genes was determined by sequence anal- petites Q3122, 01011, 0,10, and 01-2 were described by Lewin ysis of the guanylyltransferase-labeled mRNAs and matching et al. (22). Isolation. Mitochondrial RNA was prepared from The publication costs of this article were defrayed in part by page charge yeast cells as described (12). Mitochondrial DNA was purified payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. § To whom reprint requests should be addressed. 5564 Biochemistry: Christianson et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5565 from similar preparations by cesium chloride density gradient vitro transcription reaction were extracted-from polyacrylamide centrifugation in the presence of bisbenzimide (8). Cloned DNA gels as described (14). was isolated from bacterial cells by NaDodSO4 lysis and poly- Agarose/Urea Electrophoresis and RNA Blotting. Electro- ethylene glycol precipitation as described (24, 25). phoresis of mitochondrial RNA was performed on 1.5% agar- Cloning:DNA. MH41-7B mitochondrial DNA. was digested ose/6 M urea gels (30). RNA was blotted onto nitrocellulose by with Hha I and separated on a 0.8% agarose gel. Hha I fragment the procedure of Thomas (31) with the addition of a 45-minute 8 was isolated and digested with Sau3A. The products were li- soak of the gels in 3 M NaCl/0.3 M sodium citrate before trans- gated at the BamHI site of pUR222 and used to trans- fer. form Escherichia coli strain FZ-AM15recA- as described by Ruther et al. (26). All experiments with recombinant RESULTS were conducted in accordance with the National Institutes of Health guidelines for recombinant DNA research. Mitochondrial RNA from the grande strains MH41-7B and D273- Guanylyltransferase Reactions. Guanylyltransferase was lOB and from the petite strains DS400/A12, Q3122, OIOII, 0110, isolated from vaccinia virions according to Levens et al. (11). and OII-2 was subjected to the in vitro capping reaction with. The in vitro capping reactions were performed and stopped as guanylyltransferase and [a-32P]GTP. Because the G+C content described (11, 12). The reaction mixture was extracted twice *of yeast mitochondrial DNA is only 18%, primary transcripts with phenol and once with chloroform and resolved by rapid vary greatly in the number of bases from the initiating nucleo- column chromatography on Bio-Gel P-6 (14, 27). The excluded tide to. their first G. By exploiting this feature, the capped mi- volume was precipitated with 3 vol of ethanol. -tochondrial from each strain were completely digested In Vitro Transcription. Yeast mitochondrial RNA polymer- with the G-specific RNase T1. Afterward, the oligonucleotide ase was purified as described by Levens et al. (28), except that products were subjected to electrophoresis and autoradiogra- DEAE-Sephacel was substituted for DEAE-Sephadex and the phy. An example of this type of digest of capped RNA from the entire purification was carried out in 0.1 mM dithiothreitol. petite 0110 is presented in Fig. 2. Transcription reactions were carried out as described by Ed- wards et al. (14) for [y-32P]ATP labeling in the absence of CTP. A. B. After rapid column chromatography, the products were sepa- LG G -C IPy G Pyi A, rated. on an 8% polyacrylamide/8.3 M urea gel. :\~~~~~~~~~- Limit RNase TI Digestion and Extraction of Oligonucleo- tides from Gels. Mitochondrial RNA labeled at the 5' end by -50- 051 in vitro capping was digested to. completion with RNase Ti 40 - (Calbiochem-Behring) which cleaves specifically after G. These were separated on polyacrylamide gels (29), excised, and extracted (12). The products of the minus CTP in 20- 4kb -2 0 2 4 6 8 10 12 ! 10-- MH41 tRNA9'U COB OLI-I 10--0 00

dw D273 tRNA C OLI-I _*--A sequenced DNA --_o -UA DS400/A12 _ Q3122 d * - u

0,0 *- A

PC8 - _ pLT727 FIG. 2. Analysis of the 51-base oligonucleotide. (A) In lane G, mi- FIG. 1. Genetic and physical maps ofthe glutamic tRNA and COB tochondrial RNA from petite Od10 was capped in vitro, digested to com- region. Grande strain MH41 has the long version ofthe CORgene, and pletion with the G-specific RNase T1, and electrophoresed on a 20% strain D273 has the short version which lacks three introns. Open read- polyacrylamide sequencing gel. The T1 products are sized by compar- ing frames in the introns are stippled. Above the two grande maps is ison to the-adjacent lane L, a reference ladder made by partial alkaline a kilobase scale, and the heavy line delineates the extent of the pub- digestion (29) oftotal cappedRNA. Two oligonucleotides are visualized, lished DNA sequences (5, 6, 18, 19, 21). The regions of the grande ge- one of 11 bases and another of approximately 51 bases. Several addi- nome retained in the five petites-used in the deletion mappingare shown tional faint oligonucleotides appeared after longer exposure ofthe com- as crosshatched bars (5, 18, 22); four-of the .petites extend beyond the plete T1 digest of petite Oi10. (B) The 51-base oligonucleotide was iso- figure. Deletion mapping with the petites suggests that the initiation lated and its-sequence was determined by partial digestion with base- site (seeResults) that gives riseto the 51-base T1 oligonucleotide prod- specific RNases. Conditionsfor all enzymatic digestions were essen- uct is located in a 2.5-kilobase'region;this region is shown by a bracket. tially as described by Silberklang et al. (32), except that the U2 diges- The two black bars at the bottom represent the mitochondrial se- tion was performed at pH 3.5. The lanes ofthe 20%polyacrylamide se- quences. present in the two plasmids used in this. study; pBLT727 is .quencing gel (29) were: A, RNA digested with the A-specific RNase U2; aligned with the D273 map. The arrows mark the initiation-site for the Py, with the U- and C-specific RNase from Bacillus cereus; -C, with glutamic tRNA and COB. genes. the G-, A-, U-specific RNase Phy I; G, with the G-specific RNase T1. 5566 Biochemistry: Christianson et al. Proc. Natl. Acad. Sci. USA 80 (1983) We observed a number of unique capped oligonucleotides in act length of the capped T1 oligonucleotide. the digests from both grande strains and from four of the petite An imperfect nonanucleotide initiation sequence (in which strains but not in the small (5-kilobase) petite 011-2. Because G replaces an A at position -3), previously noted by Baldacci the petites retain different regions of the mitochondrial ge- and Bernardi (17), was at the initiation site of the 51-base oli- nome (Fig. 1), the transcriptional initiation sites can be local- gonucleotide. Transcription started at the last A of the nine-base ized by deletion mapping. We have mapped a 50-base oligo- sequence, which is also the case for the initiation sites of the nucleotide to a 2.5-kilobase region upstream of the glutamic ribosomal genes (11-13). No other copies of the nonanucleotide tRNA (Fig. 1). The oligonucleotide was present in the digests initiation sequence are present in the DNA downstream from from petites Q3122, 01011, and 0110 and also was observed in this site to the glutamic tRNA gene, nor are copies present in RNA from the grande strains (data not shown). The transcript the 1,050 bases of DNA between the tRNA and COB genes. from which the 50-base oligonucleotide arises must originate in Furthermore, none of the sequences of other capped oligo- the region of the overlap between petites Q3122 and 0101I. nucleotides derived from petite or grande mitochondrial RNA The absence of the 50-base oligonucleotide from limit T1 di- are found in this region (15). These results indicate that the glu- gests of capped RNA from 011-2 and DS400/A12 further sug- tamic tRNA and COB genes are cotranscribed as a multigene gests that the transcript originates within the 2.5-kilobase re- precursor from the same initiation site. gion indicated in Fig. 1. The second approach to study of the initiation of the glu- The oligonucleotides arising from the petites were excised tamic tRNA and COB genes was by in vitro transcription. We from polyacrylamide gels, eluted, and subjected to enzymatic previously showed that purified yeast mitochondrial RNA poly- sequence analysis reactions. The approximately 50-base oli- merase initiates transcription in vitro with high fidelity at the gonucleotides from petites Q3122, 01011, and 0110 proved to rRNA genes (14). In order to study the glutamic tRNA tran- have the same sequence. On a 20% polyacrylamide gel, the se- scriptional initiation site with this system, a 700-nucleotide Sau3A quence could be read clearly from the 1st to about the 36th base fragment containing the initiation site was cloned into the BamHI (Fig. 2). A perfect match for the 36 bases was found in DNA site in the vector pUR222 (26). This clone was designated pC8, sequenced by Goursot et al. (19), starting 391 bases upstream and the region it contains is shown in Fig. 1. RNA was syn- of the glutamic tRNA gene. The first G in the transcript is 51 thesized from the supercoiled pC8 template by using purified bases downstream from the initiation site, establishing the ex- mitochondrial RNA polymerase with [y-32P]ATP in the absence of CTP. Correct initiation would be expected to yield a tran- A. B. script, uniquely labeled at its 5' initiating end, that terminates at the first C (position 34) in the transcript. T Polyacrylamide gel electrophoresis of the products of in vi- tro transcription from pC8 in the absence of CTP reveals that -U,. the major product has approximately the expected size of 33 m ....U nucleotides (Fig. 3). To confirm the identity of this transcript, the 33-nucleotide band was purified from the gel and its se- quence was determined (Fig. 3). The sequence was the same O.-* -- A as that obtained for the capped 51-base oligonucleotide. The l- U complementary results from the two techniques further prove - A -A A B C A 75 - 31 - A A 10000- (DNA)- (b' _ -U 5000-4. .4 _W -A 21 S- b _ _ -U )-" UL 2000- (28 I 0 -A 1S-iss6- (18S) 0 37 - 1000 - u °i e -33 z 500- -A

19 a 2004 a

FIG. 4. RNA blot hybridizations. Lanes: A, ethidium bromide flu- orescence pattern of MH41 mitochondrial RNA separated on a 1.5% FIG. 3. Analysis of the 33-base oligonucleotide transcribed in vitro agarose/6 M urea gel; B, nitrocellulose blot of A probed with the 700- in the absence of CTP. (A) In lane.T, the products of in vitro transcrip- .base Sau3A fragment carrying the glutamic tRNA initiation site iso- tion.in the absence ofCTP were separated on an 8% polyacrylamide gel. lated from clone pC8; C, nitrocellulose blot of A probed with the COB- The products were sized by referenceto labeled markerDNA fragments specific clone pBLT727. A high molecular weighttranscript common to in lane M. An oligonucleotide of about 33 bases was visualized. (B) The both hybridizations is marked with an arrow. The labels for the con- 33-base oligonucleotide was isolated and analyzed by partial digestion taminating cytoplasmic 28S and 18S RNA species are enclosed in pa- with base-specific RNases. The code used for the lanes is as in Fig. 2, rentheses. Hybridizations wereperformed asdescribed(12),exceptthat except that U refers to the digest with the pyrimidine-specific RNase DNA from petites F11 and P9, which do not contain the COB region from B. cereus. (22), were used as carrier to decrease possible cross hybridization. Biochemistry: Christianson et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5567 4iwfioting nuceofide -/0 ATATAAGT A /0 20 30 40 50 DM4 ATA'TTATATAGGT AATATATAAA'AATAATATAA'AATAATTATAATTCAATTTATATATTAATAMTTCC-* RM AAUAUAUAAAAAUAAUAUAAAAUAAUUAUAAUUCAA- - -6 FIG. 5. The glutamic tRNA initiation site. The sequence at the 5' end of the glutamic tRNA-COB transcript, as determined from the in vitro capped oligonucleotide, is compared to the homologous strand ofthe DNA (21). The initiation site is markedwith an arrow. Above the two sequences is the consensus nonanucleotide initiation sequence; note that in the DNA sequence there is a single base alteration from the consensus at position -3. the precision of the in vitro transcription system and ensure to be necessary for transcription in yeast mitochondria because that both techniques are reliable probes for transcriptional ini- it occurs not only at the ribosomal genes and the glutamic tRNA tiation. but also at each of 10 other identified origins of transcription Because neither the nonanucleotide initiation sequence nor (15). In the case of the glutamic tRNA-COB transcriptional unit, the sequences of any in vitro capped transcript can be found the nonanucleotide sequence is imperfect, with a G replacing between the initiation site we have identified and the COB gene, an A at position -3 from the initiating nucleotide (Fig. 5). This it is likely that the glutamic tRNA and the COB genes are tran- indicates that the nonanucleotide initiation sequence need not scribed as a common transcript from this initiation site. We be perfectly conserved. The single base substitution in the therefore sought to identify such a common precursor in grande nonanucleotide initiation sequence may be involved in mod- mitochondrial RNA by blot hybridization. MH41 mitochondrial ulating rates of transcription because the steady-state level of RNA was electrophoresed on agarose/urea gels and blotted onto cappable ends from the glutamic tRNA-COB initiation site is nitrocellulose. Identical strips were hybridized with probes much lower than the level of cappable ends from the rRNAs. specific either for the initiation site (pC8) or for the 3' end of The common presence of the nonanucleotide initiation se- the COB coding region (cloned EcoRI fragment 6, pBLT727) quence before all the demonstrated transcriptional units and (33). These hybridizations were visualized by autoradiography the existence of multigenic transcripts with tRNA genes and (Fig. 4). A number of transcripts are seen, especially with the or rRNA genes show that the same RNA polymerase COB-specific probe. A long transcript is seen to be common to is used for all mitochondrial transcripts. This conclusion is both probes. This transcript is at least 9,000 nucleotides long, strengthened by the fact that the same purified RNA poly- adequate to contain the initiation site and the 3' end of the COB merase transcribes the ribosomal RNA and the glutamic tRNA- gene (long version of the gene). Conclusions based on blot hy- COB genes. Both the capping reaction and the results from in bridization must be treated with some caution because cross vitro transcription show that the polymerase is highly specific hybridization has been observed with yeast mitochondrial DNA for the transcriptional initiation sites. probes (34). However, the data support the view that a common transcript derived from the glutamic tRNA and COB genes is We thank Drs. Lucia Rothman-Denes and Joseph Locker for their present in the mitochondria. critical review of the manuscript. This study was supported in part by National Institutes of Health Grants HL-04442 an HL-09172, by Grant NP-281 from the American Cancer Society, and by a grant from the DISCUSSION Louis Block Fund of the University of Chicago. J.C.E. and D.M.M. With the complementary techniques of in vitro capping of were supported by training grants from the National Institutes of Health. transcripts isolated from mitochondria and in vitro transcrip- tion 1. Church, G. M., Slonimski, P. P. & Gilbert, W (1979) Cell 18, 1209- with purified RNA polymerase, we have found a tran- 1215. scriptional initiation site preceding the glutamic tRNA gene. 2. Van Ommen, G.-J. B., Boer, P. H., Groot, G. S. P., DeHaan, M., Because no other capped transcripts nor the nonanucleotide Roosendaal, E., Grivell, L. A., Haid, A. & Schweyen, R. J. (1980) consensus sequence could be found between the glutamic tRNA Cell 20, 173-183. gene and the COB gene, these genes apparently share a com- 3. Halbreich, A., Pajot, P., Foucher, M., Grandchamp, C. & Slon- mon initiation site and transcript. This primary transcript ex- imski, P. (1980) Cell 19, 321-329. tends 391 nucleotides upstream from the tRNA gene. Because 4. Schmelzer, C., Haid, A., Grosch, G., Schweyen, R. J. & Kau- dewitz, F. (1981) J. Biol. Chem. 256, 7610-7619. by RNA blot hybridization we find a low abundance of putative 5. Nobrega, F. G. & Tzagoloff, A. T. (1980)J. Biol. Chem. 255, 9828- precursor which hybridizes to probes from the COB coding re- 9837. gion and from the region of the initiation site, the expected pri- 6. Lazowska, J., Jacq, C. & Slonimski, P. P. (1980) Cell 22, 333-348. mary transcript appears to be present in yeast mitochondria. 7. Lamb, M. R., Anziano, P. Q., Glaus, K. R., Hanson, D. K., There have been several other reports of multigene tran- Klapper, H. J., Perlman, P. S. & Mahler, H. R. (1983)J. Biol. Chem. scripts in yeast mitochondria. At least one tRNA gene is in- 258, 1991-1999. 8. De la Salle, H., Jacq, C. & Slonimski, P. P. (1982) Cell 28, 721- cluded on the 3' end of the precursors of the 21S rRNA gene 732. (35, 36), and multigene tRNA transcripts have also been dem- 9. Weiss-Brummer, B., Rodel, G., Schweyen, R. J. & Kaudewitz, onstrated (37). Like the common transcript for the glutamic tRNA F. (1982) Cell 29, 527-536. and COB genes, a tRNA (valine tRNA) and protein gene (OXI- 10. Grivell, L. A. & Borst, P. (1982) Nature (London) 298, 703-704. 2) have been shown to be transcribed together (38). 11. Levens, D., Ticho, B., Ackerman, E. & Rabinowitz, M. (1981)1. Although multigene transcripts are present in Biol. Chem. 256, 5226-5232. yeast mito- 12. Christianson, T., Edwards, J., Levens, D., Locker, J. & Rabi- chondria, the yeast mitochondrial DNA is not transcribed into nowitz, M. (1982) J. Biol. Chem. 257, 6494-6500. genome-length transcripts. In this regard, yeast mitochondria 13. Osinga, K. A., De Haan, M., Christianson, T. & Tabak, H. F. (1982) differ from animal mitochondria. In addition to the separate Nucleic Acids Res. 10, 7993-8006. transcriptional initiation sites for the ribosomal genes (11-13), 14. Edwards, J. C., Levens, D. & Rabinowitz, M. (1982) Cell 31, 337- we have evidence for more than 20 other initiation sites (11, 346. 15). The existence of units that 15. Christianson, T. & Rabinowitz, M. (1983)J. Biol. Chem., in press. multiple transcriptional suggests 16. Osinga, K. & Tabak, H. F. (1982) Nucleic Acids Res. 10, 3617-3626. a high degree of coordination and regulation is involved in the 17. Baldacci, G. & Bernardi, G. (1982) EMBO J. 1, 987-994. expression of the yeast mitochondrial genome. 18. Bonitz, S. G., Homison, G., Thalenfeld, B. E., Tzagoloff, A. & The conserved nonanucleotide initiation sequence is likely Nobrega, F. G. (1982)J. Biol. Chem. 257, 6268-6274. 5568 Biochemistry: Christianson et al. Proc. Nati. Acad. Sci. USA 80 (1983)

19. Goursot, R., Mangin, M. & Bernardi, G. (1982) EMBO J. 1, 705- 29. Donis-Keller, H., Maxam, A. M. & Gilbert, W. (1977) NucleicAcids 711. Res. 4, 2527-2538. 20. Grivell, L. A., Hensgens, L. A. M., Osinga, K. A., Tabak, H. F., 30. Locker, J. (1979) Anal. Biochem. 98, 358-367. Boer, P. H., Crusius, J. B. A., Van der Laan, J. C., DeHaan, M., 31. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205. Van der Horst, G., Evers, R. F. & Arnberg, A. C. (1982) in Mi- 32. Silberklang, M., Gillum, A. M. & RajBhandary, U. L. (1979) tochondrial Genes, eds. Attardi, G., Borst, P. & Slonimski, P. (Cold Methods Enzymol. 59, 58-109. Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 225-239. 33. Berg, P. E., Lewin, A., Christianson, T. & Rabinowitz, M. (1979) 21. de Zamaroezy, M., Marotta, R., Faugeron-Fonty, G., Goursot, Nucleic Acids Res. 6, 2133-2150. R., Mangin, M., Baldacci, G. & Bernardi, G. (1981) Nature (Lon- 34. Butow, R. A., Lopez, I. C., Chang, H.-P. & Farrelly, F. (1980) in don) 292, 75-78. Organization and Expression of the Mitochondrial Genome, eds. 22. Lewin, A., Morimoto, R., Rabinowitz, M. & Fukuhara, H. (1978) Kroon, A. M. & Saccone, C. (Elsevier/North-Holland Biomedi- Mol. Gen. Genet. 163, 257-275. cal, Amsterdam), pp. 195-205. 23. Morimoto, R. & Rabinowitz, M. (1979) Mol. Gen. Genet. 170, 25- 35. Locker, J., Synenki, R. M., Merten, S. & Rabinowitz, M. (1981) 48. Ann. N.Y. Acad. Sci. 361, 105-118. 24. Guerny, P., LeBlanc, D. J. & Falkow, S. (1973)J. Bacteriol. 116, 36. Locker, J. & Rabinowitz, M. (1981) Plasmid 6, 302-314. 1064-1066. 37. Frontali, L., Palleschi, C. & Francisci, S. (1982) Nucleic Acids Res. 25. Clewell, D. B. (1972)1. Bacteriol. 110, 667-676. 10, 7283-7293. 26. Ruther, U., Koenen, M., Otto, K. & Muller-Hill, B. (1981) Nu- 38. Thalenfeld, B. E., Hill, J. & Tzagoloff, A. (1983)J. Biol. Chem. 258, cleic Acids Res. 9, 4087-4098. 610-615. 27. Penefsky, H. S. (1977)J. Biol. Chem. 252, 2891-2899. 28. Levens, D., Lustig, A. & Rabinowitz, M. (1981)J. Biol. Chem. 256, 1474-1481.