Physical mapping of the yeast mitochondrial genome

Abstract

(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 MH41-7B (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 MH41-7B, 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 fragments 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.