Richard Morimoto

Restriction enzyme analysis of mitochondrial DNAs of petite mutants of yeast: Classification of petites, and deletion mapping of mitochondrial genes

SummaryWe have analyzed the restriction digest patterns of the mitochondrial DNA from 41 cytoplasmic petite strains of Saccharomyces cerevisiae, that have been extensively characterized with respect to genetic markers. Each mitochondrial DNA was digested with seven restriction endonucleases (EcoRI, HpaI, HindIII, BamHI, HhaI, SalI, and PstI) which together make 41 cuts in grande mitochondrial DNA and for which we have derived fragment maps. The petite mitochondrial DNAs were also analyzed with HpaII, HaeIII, and AluI, each of which makes more than 80 cleavages in grande mitochondrial DNA. On the basis of the restriction patterns observed (i.e., only one fragment migrating differently from grande for a single deletion, and more than one for multiple deletions) and by comparing petite and grande mitochondrial DNA restriction maps, the petite clones could be classified into two main groups: (1) petites representing a single deletion of grande mitochondrial DNA and (2) petites containing multiple deletions of the grande mitochondrial DNA resulting in rearranged sequences. Single deletion petites may retain a large portion of the grande mitochondrial genome or may be of low kinetic cimplexity. Many petites which are scored as single continuous deletions by genetic criteria were later demonstrated to be internally deleted by restriction endonuclease analysis. Heterogeneous sequences, manifested by the presence of sub-stoichiometric amounts of some restriction fragments, may accompany the single or multiple deletions. Single deletions with heterogeneous sequences remain useful for mapping if the low concentration sequences represent a subset of the stoichiometric bands. Using a group of petites which retain single continuous regions of the grande mitochondrial DNA, we have physically mapped antibiotic resistance and mit- markers to regions of the grande restriction map as follows: C (99.3-1.4 map units)-OXI-1 (2.5-15.7)-OXI-2 (18.5-25)-P (28.1-34.2)-OXI-3 (32.2-61.2)-OII (60-62)-COB (64.6-80.8)-OI (80.4-85.7)-E (95-98.9).

Physical mapping of the yeast mitochondrial genome: derivation of the fine structure and gene map of strain D273-10B and comparison with a strain (MH41-7B) differing in genome size.

Summary(1)We have derived a fine-structure map of the 70 kb mitochondrial genome of the yeast S. cerevisiae, strain D273-10B, and compared it with our previous maps for strain MH41-7B. Restriction fragment maps for 56 enzyme recognition sites for 13 endonucleases, Eco RI, Hpa I, Bam HI, Hha I, Hinc II, Xba I, Hind III, Bgl II, Pvu II, Sal I, Pst I, Sst I, and Xho I, have been derived. We have used several methods to obtain these maps: (a) Four enzymes (Sal I, Sst I, Xho I, Pst I), each of which cuts D273-10B mtDNA at a single site, were employed to localize and orient fragments from multi-site enzyme digests that are cleaved by the single-site enzyme. (b) Radioactively labeled probes (rRNA or copy RNA [cRNA] transcribed from simple-sequence petite mtDNA) were hybridized to restriction fragments from different digests for identification of fragments which share common sequences. (c) The products of double or triple enzyme digests were identified for mapping and confirmation of the localization of restriction sites.(2)The antibiotic-resistant (antR) loci for erythromycin (E), chloramphenicol (C), paromomycin (P), and oligomycin (OI, OII) were positioned on the physical restriction map by hybridization of 3H-labeled cRNA transcribed from simple-sequence petite mtDNAs that retain a single genetic antR marker to appropriate restriction fragments bound to nitrocellulose filters(3)Mitochondrial transcripts (21s rRNA, 14s rRNA, and tRNAs) labeled with 125I were hybridized to restriction fragments for identification of the corresponding coding sequence.(4)The gene order and localization of the antR loci and mitochondrial transcripts are as follows: C(0-1.5u)-tRNA I(0-21.5u)-P(29-36.6u)-tRNA II(29-46.4u)-14s rRNA(36-38.3u)-OII(60.3-62.5u)-tRNA III(73-76u)-OI(78.6-83.0u)-tRNA IV(82.5-83.0u)-E(94.2-98.6u)-21s rRNA (94.2-99.4u).(5)The DNA fine structure and gene map of the 70 kb D273-10B mtDNA were compared to the map of the larger MH 41-7B (76 kb) mtDNA. There are 56 restriction sites on D273-10B and 67 sites on MH41-7B for the 13 enzymes studied. The additional restriction sites are largely accounted for by the presence, in MH 41-7B, of two sets of sequences, “A” (2.7 kb) and “B” (3.0 kb), located on either side of the OII marker. The remainder of the fragment map is remarkably similar for the two strains. The distances separating the antR loci and the mitochondrial transcripts are very similar except in the two regions surrounding OII.

Physical mapping of genes on yeast mitochondrial DNA: Localization of antibiotic resistance loci, and rRNA and tRNA genes

SummaryWe have physically mapped the loci conferring resistance to antibiotics that inhibit mitochondrial protein synthesis (erythromycin, chloramphenicol and paromomycin) or respiration (oligomycin I and II), as well as the 21s and 14s rRNA and tRNA genes on the restriction map of the mitochondrial genome of the yeast Saccharomyces cerevisiae. The mitochondrial genes were localized by hybridization of labeled RNA probes to restriction fragments of grande (strain MH41-7B) mitochondrial DNA (mtDNA)1 generated by endonucleases EcoRI, HpaI, BamHI, HindIII, SalI, PstI and HhaI. We have derived the HhaI restriction fragment map of MH41-7B mit DNA, to be added to our previously reported maps for the six other endonucleases.The antibiotic resistance loci (antR) were mapped by hybridization of 3H-cRNA transcribed from single marker petite mtDNAs of low kinetic complexity to grande restriction fragments. We have chosen the single Sal I site as the origin of the circular physical map and have positioned the antibiotic loci as follows: C (99.5-1.Ou)-P(27-36.Ou)-OII (58.3-62u)-OI (80-84u)-E (94.4-98.4u). The 21s rRNA is localized at 94.4-99.2u, and the 14s rRNA is positioned between 36.2-39.8u. The two rRNA species are separated by 36% of the genome. Total mitochondrial tRNA labeled with 125I hybridized primarily to two regions of the genome, at 99.5-11.5u and 34-44u. A third region of hybridization was occasionally detected at 70-76u, which probably corresponds to seryl and glutamyl tRNA genes, previously located to this region by petite deletion mapping.

Physical mapping and characterization of the mitochondrial DNA and RNA sequences from mit- mutants defective in cytochrome oxidase peptide 1 (OXI 3)

SummaryYeast mitochondrial mutants defective in cytochrome c oxidase have been isolated and genetically characterized by Slonimski and Tzagoloff (1976). Three genetic loci, designated OXI 1, OXI 2, and OXI 3, each affect the synthesis of a separate mitochondrially coded subunit of cytochrome oxidase (Cabral et al., 1977). We have analyzed the mitochondrial DNA and RNA of mutants with defects at the locus which impair the synthesis of the 43,000 subunit I peptide, in order to map the structural gene and identify the RNA species transcribed from this region.Mitochondrial DNA (mtDNA) from nine OXI 3 mutants derived from the grande strain D273-10B was digested with the endonucleases Eco RI, Hpa I, Hha I, Hine II, Xba I, and Hpa. II. Two mutants, M10-150 and M5-16, were found to have large contiguous deletions corresponding, respectively, to 7500 and 5400 base pairs (bp). In strain M11-125, two mutations separated by 8500 bp were detected; one results in the deletion of a Hha I site and the other introduces a new Hpa I (Hinc II) site. Based on the size of the lesion present in M5-16, the OXI 3 gene is mapped within 47.5 to 57.0 map units.No detectable changes in the restriction patterns of fragments in the OXI 3 region were observed in the remaining seven mutants. Mutant M11-82, in which no deletionwas detected, is genetically similar to the 5400 bp deletion mutant M5-16 (Slonimski and Tzagoloff, 1976). Since our gel resolves fragments that differ by 10–50 bp, the data indicate that a deletion present in this strain must be smaller than 50 bp.Mitochondrial RNA was isolated from OXI 3 mutants and analyzed by gel electrophoresis containing the denaturing agent methylmercury hydroxide. Three species, corresponding to 2800, 2550, and 620 nucleotides, are absent in mitochondrial RNA from M5-16 and M10-150. These three transcripts appear to be specific to the sequences absent in the deletion mutants M5-16 and M10-150, since they are present in a OXI 3 point mutant, M11-224, and in mit- mutants affecting cytochrome b or subunits II and III of cytochrome oxidase.

Stable heterogeneity of mitochondrial DNA in grande and petite strains of S. cerevisiae

Abstract Several instances of mitochondrial DNA heterogeneity in grande and petite strains of Saccharomyces cerevisiae were examined. We have detected heterogeneity in the mtDNA from some of the progeny strains of a cross between two grande strains (D273-10B, MH41-7B) which differ in genome size and restriction cleavage pattern of their mtDNA. The progeny strains transmit restriction fragments characteristic of both parental strains from homologous regions of the mitochondrial genome, and this sequence heterogeneity is not eliminated by additional subcloning. Sequence diversity is more common in the mtDNA of petite than of grande strains of yeast. We have examined subclones of one petite strain to identify the origin of this variability. Many of the submolar restriction fragments persist in independent subclones of this petite after 15 and 30 cell divisions; some submolar fragments disappear, and some new fragments appear. We conclude that the observed sequence heterogeneity is due to molecular heterogeneity, i.e., to differences in the multiple copies of the petite mitochondrial genome, as well as to clonal heterogeneity. It is likely that tandem repeats on the same mtDNA molecule also differ, i.e., that there is intramolecular heterogeneity, and that this accounts for the stability of the heterogeneity. Continuing deletion is probably responsible for the appearance of “new” fragments in petite subclones.

Analysis of mitochondrial RNA in Saccharomyces cerevisiae

SummaryMitochondrial RNA from grande yeast was analyzed by electrophoresis on agarose-urea, acrylamide-urea, and methyl mercuric hydroxide-agarose gels. These gel systems display more than 40 RNA bands that copurify with mitochondria; these bands are not present in cytoplasmic RNA preparations. Analysis of molecular weight on methyl mercuric hydroxide gels indicates a size range of 200 to 9,500 nucleotides, including 11 species larger than 21S rRNA (3,700 nucleotides). The mitochondrial origin for many of these species was further verified by transfer of RNA from gels to diazobenzyl oxymethyl paper and hybridization to 32P-labeled mitochondrial DNA. The total molecular weight of the catalogued RNA species was approximately 110,000 nucleotides, considerably greater than the size of the 76,000-base-pair genome. These results suggest that large primary transcripts are processed by multiple cleavages to mature RNA species.

Physical mapping of the Xba I, Hinc II, Bgl II, Xho I, Sst I, and Pvu II restriction endonuclease cleavage fragments of mitochondrial DNA of S. cerevisiae

SummaryA detailed molecular dissection of the yeast mitochondrial genome can be made with restriction endonucleases that generate site-specific cuts in DNA. The ordering of restriction fragments provides the basis of the physical mapping of mitochondrial transcripts and antibiotic resistance (ant®) loci, and is a means of analyzing the molecular organization of mtDNA of petite and mit- deletion mutants.We have previously mapped the sites in the mtDNA of yeast strain MH41-7B recognized by the endonucleases Eco RI, Hpa I, Hind III, Bam HI, Sal I, Pst I, and Hha I, providing a total of 41 cleavage sites. We have now mapped the sites recognized by the endonucleases Xba I, Hinc II, Bgl II, Pvu II, Xho I, and Sst I, which make 6, 13, 5, 6, 2, and 2 cuts, respectively. Fragment maps for each of these endonuclease sites were derived by analysis of the products of double-enzyme digests and by hybridization of 3H-cRNA probes transcribed from low-kinetic-complexity petite mtDNAs to restriction fragments generated by various combinations of enzymes.

TRANSCRIPTION OF YEAST MITOCHONDRIAL DNA

ABSTRACT Yeast mtRNA was analyzed by means of 6M ureaagarose, 6M ureaacrylamide, and 10 mM methyl mercuric hydroxide-agarose gels, and RNA was transferred from gels to diazebenzyloxymethyl paper for subsequent hybridization to labeled DNA. In grande yeast, more than forty different species of RNA, ranging from 200 to 9500 nucleotides, were identified, including 11 species larger than 21S rRNA. Petites contain many of these RNA species, making deletion mapping possible. Processed tRNA, however, is identified only in petites that contain a region of the genome near P. Petites were thus used not only for deletion mapping of mitochondrial RNA species, but also for identification of different types of mtRNA processing. Hybridization studies on RNA from petite Fll appear to identify precursors to 21S rRNA. Overall, the number and size of these mitochondrial transcripts imply the existence of large precursor molecules which are subsequently processed by cleavage to a mature form. The promoter function of yeast mtDNA has been examined by analysis of a mitochondrial transcription complex, and by the use of purified yeast mitochondrial RNA polymerase. The transcription complex primarily synthesizes RNA from the same regions and from the same strand that encodes the in vivo rRNAs. Since the 21S and 14S rRNA genes are widely separated, transcription must occur from at least two promoters. When template-dependent RNA polymerase was isolated either from the transcription complex or from soluble protein fractions, we found that the two enzyme fractions expressed similar properties. The soluble polymerase has been extensively purified by a variety of chromatographic techniques; this purified enzyme is associated with a 45,000 D peptide. Prior to a final glycerol gradient centrifugation, a 65,000 D peptide is associated with the enzyme activity in a molar ratio. The enzyme from the glycerol gradient was found to be extremely labile. In further experiments, antibodies to the 45,000 D band precipitated that peptide and inhibited RNA polymerase activity. Promoter function and mapping have been studied by means of transcription in vitro of mtDNA as well as binding of the purified RNA polymerase to restriction fragments. The results show the purified RNA polymerase binds to and transcribes mitochondrial DNA in a non-random fashion.