Restriction cleavage map of mitochondrial DNA from the yeast Saccharomyces cerevisiae

volume 4 Number 7 July 1977 Nucleic Acids Research Restriction cleavage map of mitochondrial DNA from the yeast Saccharomyces cerevisiae Richard Mori mot o, 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 Mitochondrial DNA (mtDNA) from the yeast Sacchawmycu ceA.e.viA<La.z 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 1 0 7 d or 75 kb. A fifth grande strain, D273-10B from S. c&ie.v.ib.ia.e., revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb. INTRODUCTION Electron-microscopic examination of yeast mitochondrial DNA (mtDNA) released from osmotically shocked mitochondria has detected rare 25u circular duplex molecules.1 The size of yeast mtDNA was subsequently confirmed by renaturation kinetic analysis 1 ' 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. © Information Retrieval Limited 1 Falconberg Court London W 1 V 5 F G England 2331 ABSTRACT Nucleic Acids Research MATERIALS AND METHODS Yeast strains, growth conditions, and preparation of mitochondria The haploid respiratory sufficient (grande) strains of ZQAz\ilb.LoLz used in this study were MH41-7B (CR, E R , O j R , 0 n S , P1*), NH32-12D ((*, E*, 0 : S , 0 n R , P*), 19D (CS, E S , 0 T S , 0 n S , P S ) , TR3-15A (CR, E R , 0 j R , °II ' ) ' a n d D 2 7 3 ~ 1 ° B - Th e derivation of these strains has been described by Fukuhara. 20 ' 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 2332 RNAs s > 7 ; it codes for 20 to 25 tRNAs 8 " 11 and for a number of mRNAs. 12 . 13 Yeast mtDNA probably specifies the known mitochondrial translation products, i.e., three of the seven peptides of cytochrome oxidase,11*'15 four of the nine components of the mitochondrial oligomycin-sensitive ATPase complex,16 and the cytochrome b_ component of coenzyme Q cytochrome £ 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 Nucleic Acids Research Preparation of mitochondrial DNA fragments Fragments of DNA were eluted from agarose gels by a modification of the procedure described by Wilkie. 32 Gel slices were solubilized at 57 C in 2.5 M NaClOi,, 0.4 M NaCl, 0.1 M sodium phosphate buffer, pH 6.8, and passed over a Dowex-50W column at 57°C. The DNA fragments were adsorbed to a 1.0 ml hydroxylapatite column (BioRad) at 57°C. The columns were washed sequentially with 10 ml 2.5 M NaC10 4 , 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 Purified mitochondria were suspended in 10 mM Tris, pH 7.5, 2 mM EDTA, and lysed in II sarkosyl. The mtDNA was separated from nuclear DNA by preparative CsCl density gradient centrifugation as described by Morimoto et aJL.k 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 HmophiZuA -inihiznzae. d through one phosphocellulose column, following a modification of the method of Smith and Wilcox. 23 Hpa I was purified from Hemophilia paMxu.nhhLe.nzaz as described by Sharp et al.2k and was later purchased from New England Biolabs. Bam HI was purified from SacAlZm amylotique-hade-n!) through phosphocellulose by a modification of the procedure of Wilson and Young. 25 Endonucleases Sal I from SttzptomyceA albai,26 Pst I from VtiovJAendia. iiwvULL,27 and Hha I from Hae/nopkULuA ha. (...truncated)