Volume 4 Number 7 July 1977

Nucleic Acids Research

Restriction cleavage map of mitochondrial DNA from the yeast Saccharomyces cerevisiae l~~~~~~~~~~~~~~~

Richard Morimoto, Alfred Lewin, and Murray Rabinowitz

Departments of Medicine, Biochemistry, and Biology, and The Franklin McLean Memorial Research Institute, University of Chicago, Chicago, IL 60637, USA Received 3 May 1977

ABSTRACT

Mitochondrial DNA (mtDNA) from the yeast SacchaWomyce6 ceAev4sLae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 x 107 d or 75 kb. A fifth grande strain, D273-1OB from S. ceAevL&iae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.

INTROIXCTION Electron-microscopic examination of yeast mitochondrial DNA (mtDNA) released from osmotically shocked mitochondria has detected rare 25i circular duplex molecules.1 The size of yeast mtDNA was subsequently confirmed by renaturation kinetic analysisl"2 and restriction endonuclease analysis.3-5 Yeast mtDNA contains one cistron for each of the two mitochondrial ribosomal Nomenclature: Fragments generated by restriction enzymes and separated by agarose gel electrophoresis are designated by an abbreviation of the enzyme used (i.e., R for Eco RI, Hpa for Hpa I, Hind for Hind III, Bam for Bam HI, and Hha for Hha I), and numbered according to their mobility on the gels.

Abbreviations: C, chloramphenicol; E, erythromycin; 0, oligomycin; P, paromomycin; kb, kilobases.

C) Information Retrieval Limited I Falconberg Court London W1 V 5FG England

2331

Nucleic Acids Research 20 to 25 tRNAs8-l1 and for a number of mRNAs.12913 Yeast mtDNA probably specifies the known mitochondrial translation products, i.e., three of the seven peptides of cytochrome oxidase,14,15 four of the nine components of the mitochondrial oligomycin-sensitive ATPase complex,16 and the cytochrome b component of coenzyme Q cytochrome c reductase. 17,18 Our interest in the organization of yeast mtDNA is directed primarily toward the identification and localization of genes. A precise knowledge of the organization of yeast mtDNA will facilitate the study of the regulation of transcription. A restriction fragment map also provides a framework for the analysis of mitochondrial mutations. Restriction endonucleases make a limited number of duplex cleavages in DNA by recognizing specific nucleotide sequences, thus providing DNA fragments useful in physical mapping studies. Restriction fragments may be ordered by a variety of procedures. Several of the methods currently in use, however, cannot be applied directly to yeast mtDNA because the DNA can only be isolated as randomly cleaved molecules having 1/3 to 1/2 the size of the intact genome.2 Furthermore, the DNA contains many nicks and gaps, making end labeling techniques of restriction fragments difficult to interpret for mapping purposes. The random cleavage of the mtDNA results in considerable DNA background fluorescence in the gels due to molecules having no, or only one, restriction site. In addition, the larger fragments are present in submolar quantities, since they are more likely to be randomly cleaved. Despite these difficulties, we have been able to construct a restriction map from fragments generated by Eco RI, Hind III, Hpa I, Bam III, Pst I, and Sal I. This work has been reported in preliminary form.19

RNAS6,7; it codes for

MATERIALS AND METHODS Yeast strains, growth conditions, and preparation of mitochondria

The haploid respiratory sufficient (grande) strains of Sacchacumyce2 ceevizae used in this study were MH41-7B (C R B , 01R OiS PR), MH32-12D OIIRI E OI, TR3-1SA (C, E 19D (CS, ES,' 0Is, OII, PR), and D273-1OB. The derivation of these strains has been described by Fukuhara.20o,21 The yeasts were grown to the middle logarithmic stage in 2% galactose, 0.1% glucose, 1% bactopeptone, and 1% yeast extract. Mitochondria were isolated from glusulase-prepared protoplasts according to the method of Casey ,

et at.9

2332

OIs,

pR),

0IIs$, P)2

(CR,9

I

Nucleic Acids Research Preparation of mitochondrial DNA fragments

Purified mitochondria were suspended in 10 mM Tris, pH 7.5, 2 mM EDTA, and lysed in 1% sarkosyl. The mtDNA was separated from nuclear DNA by preparative CsCl density gradient centrifugation as described by Morimoto et at.4 The mtDNA was dialyzed extensively against 10 mM Tris, pH 7.5, 2 mM EDTA, in the cold. Preparations of DNA were intermittently checked for purity by analytical isopycnic centrifugation and were found to have less than 5% nuclear DNA contamination. Eco Rl was prepared from E. coti RY13 as described by Yoshimori,22 and was later purchased from New England Biolabs, Beverly, Massachusetts. Hind III was purified from Hemophitul £ntuenzae d through one phosphocellulose column, following a modification of the method of Smith and Wilcox.23 Hpa I was purified from HemophiZwz pa4ain6tuenzae as described by Sharp et at.2 and was later purchased from New England Biolabs. Bam HI was purified from BacLQ2uz amyQotique6acien through phosphocellulose by a modification of the procedure of Wilson and Young.25 Endonucleases Sal I from StAeptomyce6 a2bu4,26 Pst I from PtoLwvidencia taXtjlj27 o and Hha I from Haemophituw haemoZyticu628 were purchased from New England Biolabs. Incubations of mtDNA with Eco RI or Sal I were performed at 370C in 50 mlM Tris, pH 7.5, 30 mM MgCl2, with sufficient enzyme to yield limit digests. Hind III, Hpa I, Bam HI, and Pst I were incubated at 370C in 6 mMrF Tris, pH 7.5, 6 lMM MgCl2, 6 mM 2-mercaptoethanol, with sufficient enzyme to ensure complete digestion of the DNA. Digested DNAs were fractionated by electrophoresis on 0.3 or 0.4% agarose (Sea Kem) tube gels (1.3 x 20 cm) for preparative and analytical purposes, or on 1% slab gels as described by Sugden t at.,29 in 40 mM Tris, 36 mM NaH2PO4, 1 mM EDTA (pH 7.4) buffer containing 0.5 ag/ml ethidium bromide. The restriction patterns of DNA were visualized by fluorescence under UV light. Calibration of fragment molecular size was obtained by use of internal standards, by co-migration with Eco RI or Hind III digests of T530 or X DNA. 31 Fragments of DNA were eluted from agarose gels by a modification of the procedure described by Wilkie.32 Gel slices were solubilized at 570C in 2.5 M NaCl04, 0.4 M NaCl, 0.1 M sodium phosphate buffer, pH 6.8, and passed over a Dowex-SOW column at 570C. The DNA fragments were adsorbed to a 1.0 ml hydroxylapatite column (BioRad) at 570C. The columns were washed sequentially with 10 ml 2.5 M NaCl04, 0.4 M NaCl, 0.1 M sodium phosphate buffer, pH 6.8, and with 10 ml 0.2 M sodium phosphate buffer, pH 6.8; then the DNA was eluted with 4 ml of 0.4 M sodium phosphate buffer at room temperature. Sheared

2333

Nucleic Acids Research salmon sperm DNA (5 wg) was added, and the samples were concentrated by extraction with 2-butanol, 1% isoamyl alcohol.33 The phosphate and organic solvents were removed by extensive dialysis against 10 mM Tris, pH 7.5, 2 mM EDTA. RESULTS

Special problems in mapping of yeast mitochondrial DNA The fragment map of yeast mtDNA was derived primarily by redigestion of isolated restriction fragments generated by one endonuclease, with other restriction enzymes. As illustrated in detail below, analysis of such reciprocal digestions can establish the order of fragments derived from each enzyme and the exact overlap between the fragment maps for each restriction endonuclease. Some fragments, however, may not be localized conclusively by this procedure; for example, when digestion of a fragment yields more then three subfragments, only the two terminal fragments may be ordered. We were able to carry out complete reciprocal redigestion analysis for fragments generated by Eco RI and Hpa I, even though yeast mtDNA is isolated as randomly cleaved molecules with an average molecular weight of about 1.5 x 107 daltons. The random degradation of isolated mtDNA resulted in considerable contamination of some of the fragments with randomly cleaved larger fragments (e.g., R2 contaminated with randomly cleaved R1). By comparison of molar ratios of products, however, it was simple to distinguish true digestion products from lower-concentration contaminating bands. For limit digests, the largest fragment, i.e., Eco Rl or Hpa 1, was uncontaminated even though present in low concentration, because all randomly cleaved molecules migrated more rapidly. The fragmented nature of the mtDNA prevented us from obtaining complete redigestion studies with Bam HI and Hind III. Fragment Bam 1 (Molecular size, 33.0 kb) could not be isolated, and the relatively large Hind 1-4 (24.0 15.0 kb) fragments could not be separated effectively without significant cross-contamination. The data which we were able to obtain, however, served to o#der several of the other Eco RI and Hpa I fragments and formed the basis of the Bam HI and Hind III maps. The remainder of the Bam HI and Hind III maps was derived mainly from the use of two additional endonucleases, Pst I and Sal I, each of which recognizes only a single site on yeast mtDNA. Codigestion by Sal I or Pst I with one of the multiple-site endonucleases provides independent information about the overlap of fragments in the different maps. Analysis of double and triple enzyme digests supplied additional information about the orientation of the fragments cleaved and confirmed conclusions drawn from previous procedures. 2334

Nucleic Acids Research With these methods, fragment maps for Eco RI, Hpa I, Hind III, and Bam HI were derived, except for the precise location of one of the Eco RI fragments, which was done by partial digest analysis. Neither the isolation of partial digest bands nor the establishing of linkage by redigestion with the same enzyme was possible because of the high background of randomly cleaved molecules. Since this results in substantial contamination of the visible partial digest bands, it prevented a clear-cut interpretation of the results. Contamination of an isolated fragment is more severe in partial than in complete digests because it is derived, in the former, from all possible combinations of fragments whereas in the complete digests it is limited to species which, when intact, migrate more slowly. Therefore, analysis was limited to the less rigorous procedure of measuring the molecular weights of partial digest bands, with internal molecular standards used for calibration. These data were used for positioning of the smallest Eco RI fragment. Fragment patterns of grande mtDNA obtained with various endonucleases.

Although isolated yeast mtDNA has a heterogeneous size distribution (25-40 kb), specific restriction fragments can readily be identified above a background of randomly cleaved molecules as long as sufficient numbers of DNA molecules larger than the largest restriction fragment are present. Limit digests of grande mtDNA MH41-7B by Eco RI, Hind III, Bam HI, and Hpa I endonucleases yielded 10, 6, 5, and 7 fragments, respectively (Fig. 1). Fragment

I02

;2

2 15 times. striction patterns were observed in some strains; these were interpreted as alterations of individual restriction sites. Eco RI digests of mtDNA from strain 19d lack fragments Ri (23.7 kb) and R4 (8.3 kb) present in MH41-7B, and are replaced by a single fragment (32.2 kb) with a size equal to the sun of the two missing bands. Similarly, Eco RI digests of strain MH32-12D mtDNA differ from those of MH41-7B in that fragments R2 (17.3 kb) and R4 (8.3 kb) are missing and a new 23. S kb fragment appears. These changes at the Eco RI

-278

- 15

20 3

-71

20 0

12 6J 'Z 6

14 7

-

-

7Z2 69

71

6 0

-53

-51

..

-

- 35

-

42 6

-'33

F;

4 2 3.9

- 32

24

-

23

-2 3

-1 7

11

09

EcoRI

2348

HpoI

BomHl

Hno I

Figure 15. Eco RI, Hpa I, Bam HI, and Hha I digestion patterns of D273-1OB mtDNA. Fragments were separated on a 1.2% agarose slab gel.

Nucleic Acids Research sites flanking fragment R4 are consistent with a fragment order of 1-4-2 derived for M[H41-7B mtDNA. Hpa I digestion of mtDNA of strains 19d, MH32-12D, and TR3-15A shows that Hpa 2 (20.6 kb) and Hpa 6 (3.2 kb) are missing, and that a new additive fragment of 24 kb is present. Linkage of Hpa 6 and 2 is again consistent with the mapping of the Hpa I fragments from N1H41-7B. We may conclude that restriction sites are similar for these four grande strains, although they differ from MH41-7B and from each other with respect to single Eco RI or Hpa I sites. Mitochondrial DNA from strain D273-1OB of S. ceAeviziae, cleaved by the various restriction endonucleases, revealed a sum molecular weight of 67.5 kb. The restriction patterns were generally quite similar to those observed in MH41-7B, with the most striking changes in the large fragments (Fig. 15). These changes in the number and size of fragments have no simple interpretation like that presented for the four similar grande mtDNAs. A fragment map was derived for Eco RI, Hpa I, and Bam HI (Morimoto, unpublished data).

DISCUSSION We have derived a physical fragment map of mtDNA from the yeast SacchatomyceQ5 ccvis4iae for the restriction endonucleases Eco RI, Hpa I, Bam HI, and Hind III. Our attempts to define the restriction fragment map of yeast mtDNA were hindered by our inability to isolate intact mtDNA. The major framework for the map was provided by reciprocal redigestion of isolated Eco RI, Hpa I, and Bam HI fragments. Two enzymes which make unique cuts, Sal I and Pst I, were useful for localization and positioning of the fragments generated by multisite enzymes that were cleaved by the single-site enzyme. Partial digest analyses were of limited value because of the impossibility of isolating incompletely digested DNA fragments that were not grossly contaminated with randomly cleaved fragments. Partial digest analysis was used only to order the smallest Eco RI fragment (R10). The Eco RI fragment map which we have obtained is identical to that reported by Sanders et at. for their S. ceAteviiae strain KL14-14A.34 The fragment patterns of the five strains of S. ceAeviia that we studied could be separated into two major classes. Four strains (MI41-7B, 19d, MH32-12D, and TR3-15A) had few differences in the restriction maps; the variability could be accounted for by the alterations at a single restriction site. Strain D273-1OB, which has a smaller genome (70.0 kb) revealed more changes than could be explained by changes at sites of endonuclease cleavage. Similarly, Sanders et aR. 34 have examined three grande strains which have vari2349

Nucleic Acids Research able size, i.e., S. coubeAgenia (68.5 kb), S. ceAevisiae JSl-3D (71.5 kb), and KL14-4A (76.5 kb). These authors suggest that one region of the genome may be susceptible to insertion or deletion of sequences. The availability of the fragment map has allowed the physical mapping of a variety of mitochondrial genes. As will be reported in detail elsewhere, mitochondrial 21S and 14S rRNA, antibiotic resistance narkers C, E, P, O0, and OII, and tRNA genes have been localized on the physical map by hybridization to restriction fragments.'9'35 The fragment map has also allowed us to analyze the sequence rearrangements that occur in some cytoplasmic petite deletion mutations. 19,36 Our restriction map provides 30 sites for analysis and mapping. For fine structural analysis of the DNA additicaml sites are necessary. We have used the fragment maps derived here to obtain the physical maps for Hha I, Xba I, Bgl II, and Xho I, which make 11, 6, 5, and 2 cuts respectively.35

ACKNWLEDGIE?TIS We thank Dr. Gary Hayward and Dr. Richard Roberts for generous gifts of bacterial strains and restriction endonucleases, and for many helpful discussions. We also wish to thank J. Sanders for the suggestion to use Pst I. This study was supported in part by Gaants HL 09172 and HL 04442 from the National Institutes of Health and the Louis Block Fund of the University of Chicago. The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U. S. Energy Research and Development Administration under Contract EY-76-C-02-0069. R.M. was supported by USPHS Training Grant T32-GM07197 and A.L., by USPHS Training Grant 5-T0l-GM00090-19.

*Author to whom reprint requests should be addressed. REFERENCES 1. Hollenberg, C.P., Borst, P., and van Bruggen, E.F.J. (1970) Biochim. Biophys. Acta 209, 1-15. 2. Locker, J., Rabinowitz, M., and Getz, G.S. (1974) J. Mol. Biol. 85, 393-410. 3. Sanders, J.P.M., Borst, P., and Weijers, P.J. (1975) Mol. Gen. Genet. 143, 53-64. 4. Morimoto, R., Lewin, A., Hsu, H.J., Rabinowitz, M., and Fukuhara, H. (1975) Proc. Nat. Acad. Sci. U.S.A. 72, 3868-3872. 5. Bernardi, G., Prunell, A., and Kopecka, H. (1975) in Molecular Biology of Nucleocytoplasmic Relationships (Puisex-Dao, ed.), pp. 85-90. Elsevier, Amsterdan. 6. Borst, P., and Grivell, L.A. (1971) FEBS Letters 13, 73-88. 7. Faye, G., Kujawa, C., and Fukuhara, H. (1974) J. Mol. Biol. 88, 185-203. 8. Cohen, M., and Rabinowitz, M. (1972) Biochim. Biophys. Acta 281, 192-201. 2350

Nucleic Acids Research 9. 10.

11. 12. 13.

14. 15. 16. 17. 18.

19.

20. 21.

22.

23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

Casey, J., Hsu, H.J., Rabinowitz, M., Getz, G.S., and Fukuhara, H. (1974) J. Mol. Biol. 88, 717-733. Martin, N., Rabinowitz, M., and Fukuhara, H. (1976) J. Mol. Biol. 101, 285- 296. Martin, N., and Rabinowitz, M. (1976) in The Genetics and the Biogenesis of Mitochondria and Chloroplasts (Biuchler, T., Neupert, W., Sebald, W., and Werner, S., eds.). North Holland Publ., Amsterdam, pp. 749-754. Hendler, F.J., Padmanaban, G., Patzer, J., Ryan, R., and Rabinowitz, M. (1975) Nature 258, 357. Padmanaban, G., Hendler, F., Patzer, J., Ryan, R., and Rabinowitz, M. (1975) Proc. Nat. Acad. Sci. U.S.A. 72, 4293-4397. Mason, T.L., Poyton, R.O., Wharton, D.C., and Schatz, G. (1973) J. Biol. Chem. 248, 1348-1354. Tzagoloff, A., Akai, A., Needlanan, R.B., Zulch, G. (1975) J. Biol. Chem. 250, 8236-8242. Tzagoloff, A., Rubin, M.S., and Sierra, M.F. (1973) Biochim. Biophys. Acta 301, 71-77. Weiss, H. (1972) Eur. J. Biochem. 30, 469-478. Katan, M.B., and Groot, G.S.P. (1976) in The Genetics and the Biogenesis of Mitochondria and Chloroplasts (Biuchler, T., Neupert, W., Sebald, W., and Werner, S., eds.). North Holland Publ., Amsterdam, pp. 273-280. Morimoto, R., Lewin, A., Merten, S., and Rabinowitz, M. (1976) in The Genetics and the Biogenesis of Mitochondria and Chloroplasts (Biichler, T., Neupert, W., Sebald, W., and Werner, S., eds.). North Holland Publ., Amsterdam, pp. 519-524. Fukuhara, H., Bolotin-Fukuhara, M., Hsu, H.-J., and Rabinowitz, M. (1976) Mol. Gen. Genet. 145, 7-17. Bolotin-Fukuhara, M., and Fukuhara, H. (1976) Proc. Nat. Acad. Sci. U.S.A. 73, 4608-4612. Yoshimori, R.N. (1971) Ph.D. Thesis, University of California, San Francisco Medical School, San Francisco, California. Smith, H.O., and Wilcox, K. (1970) J. Mol. Biol. 51, 379-391. Sharp, P.A., Sugden, B., and Sambrook, J. (1973) Biochemistry 12, 3055-3063. Wilson, G.A., and Young, F.E. (1975) J. Mol. Biol. 97, 123-125. Arrand, J.R., Meyers, P.A., and Roberts, R.J. Unpublished observations. Smith, H.O., Blattner, F., and Davies, J. (1976) Nucleic Acid Res. 3, 343- 358. Roberts, R.J., Myers, P.A., Morrison, A., and Murray, K. (1976) J. Mol. Biol. 103, 199-208. Sugden, B., De Troy, B., Roberts, R.J., and Sambrook, J. (1975) Anal. Biochem. 68, 36-46. Von Gabain, A., Hayward, G.S., and Bujard, H. (1976) Mbl. Gen. Genet. 143, 279-290. Thomas, M., and Davis, R.W. (1975) J. Mol. Biol. 91, 315-328. Wilkie, N.M., and Cortini, R. (1976) J. Virol. 20, 211-221. Stafford, D.W., and Bieber, D. (1975) Biochim. Biophys. Acta 378, 18-21. Sanders, J.P.M., Heyting, C., DiFranco, A., Borst, P., and Slonimski, P.P. (1976) in The Genetic Fuction of Mitochondrial DNA (Saccone, C., and Kroon, A.M., eds.). North-Holland Publ., Amsterdam, pp. 259-272. Morimoto, R., Meitten, S., Lewin, A., Martin, N., and Rabinowitz, M. (manuscript in preparation). Lewin, A., Merimoto, R., Fukuhara, H., and Rabinowitz, M. (manuscript in

preparation).

2351