A comparison of three ®ssion yeast mitochondrial genomes

The ®ssion yeasts are members of the fungal order Schizosaccharomycetales, a candidate deepdiverging group within Ascomycota. Although a great deal of molecular information is available from Schizosaccharomyces pombe, a model eukaryote, very little is available from other members of this group. In order to better characterize mitochondrial genome evolution in this fungal lineage, the mitochondrial DNA (mtDNA) of two additional ®ssion yeasts, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus var. japonicus, was sequenced. Whereas the mtDNA of S.pombe is only 19 431 bp, the mtDNA of S.octosporus is 44 227 bp, and that of S.japonicus var. japonicus is over 80 kb. The size variation of these mtDNAs is due largely to non-coding regions. The gene content in the latter two mtDNAs is almost identical to that of the completely sequenced S.pombe mtDNA, which encodes 25 tRNA species, the large and small mitochondrial ribosomal RNAs (rnl and rns), the RNA component of mitochondrial RNaseP (rnpB), mitochondrial small subunit ribosomal protein 3 (rps3), cytochrome oxidase subunits 1, 2 and 3 (cox1, cox2 and cox3) and ATP-synthase subunits 6, 8 and 9 (atp6, atp8 and atp9). However, trnI2(cau) (C modi®ed to lysidine) is absent in the S.octosporus mtDNA, as are corresponding ATA codons in its protein-coding genes, and rps3 and rnpB are not found in the mtDNA of S.japonicus var. japonicus. The mtDNA of S.octosporus contains ®ve double hairpin elements, the ®rst report of these elements in an ascomycete. This study provides further evidence in favor of the mobility of these elements, and supports their role in mitochondrial genome rearrangement. The results of our phylogenetic analysis support the monophyly of the Schizosaccharomycetales, but question their grouping within the Archiascomycota. INTRODUCTION The ®ssion yeasts are a small group of unicellular, saprophytic organisms classi®ed within the higher fungal division Ascomycota, order Schizosaccharomycetales (1,2), in the single genus Schizosaccharomyces. The placement of Schizosaccharomyces within Ascomycota is based on a set of fundamental features that delimit this division, including life cycle, mode of ascospore formation and non-centric mitosis (3). Furthermore, the morphology and development of the sexual sporangium of the ®ssion yeasts is similar to the ascus of other ascomycetes (4,5). Phylogenetic studies including a broad range of species from all fungal lineages (6,7) consistently support this assignment. Schizosaccharomyces pombe is an increasingly used organism for research, due largely to the many molecular genetic techniques available (8), and the availability of its complete nuclear genome sequence (9). Several features highlight the utility of this organism as an alternative model system to the budding yeast, Saccharomyces cerevisiae. For example, S.pombe has served as an important model organism in genetic and molecular studies of the cell cycle (10), as it displays features, such as a distinct G2 phase and visible chromosome condensation, typical of other eukaryotes but not found in budding yeasts (11). Of the many ®ssion yeast species described since the initial description of S.pombe, most have been found to be conspeci®c with one of three species: S.pombe Lindner (12), Schizosaccharomyces octosporus Beijerinck (13) and Schizosaccharomyces japonicus Yukawa and Maki (14). Unfortunately, little molecular data are available from the latter two Schizosaccharomyces species. Comparative data from closely related organisms can allow the identi®cation of, for example, regulatory elements or genes in the DNA sequence that would not be recognized by comparing more distantly related organisms (15±17). The mitochondrial DNA (mtDNA) of S.pombe strain 50 h± has been completely sequenced (18,19); however, the only data available from the mitochondrial genomes of other ®ssion yeasts are the sequence of the mitochondrial small ribosomal subunit from S.japonicus var. versatilis (GenBank accession number X72804). In order to provide comparative data from other members of Schizosaccharomyces to better de®ne the mitochondrial genomes in this group, and to better resolve the fungal phylogeny, the mtDNAs of two additional ®ssion *To whom correspondence should be addressed. Tel: +1 514 343 5842; Fax: +1 514 343 2210; Email: [email protected] Nucleic Acids Research, 2003, Vol. 31, No. 2 759±768 DOI: 10.1093/nar/gkg134 at Penylvania State U niersity on M arch 1, 2013 http://narrdjournals.org/ D ow nladed from yeasts, S.octosporus and S.japonicus var. japonicus, were sequenced. The current taxonomic view of Ascomycota, based on molecular phylogenetic analyses of small subunit rRNA sequences (20,21), shows three major lineages: the euascomycetes (®lamentous ascomycetes), the hemiascomycetes (budding yeasts) and the archiascomycetes (a heterogeneous group to which the genus Schizosaccharomyces is considered to belong; `Taphrinomycotina', according to GenBank). The euascomycetes and hemiascomycetes form a monophyletic group, while the archiascomycetes are the most ancestral lineage of the ascomycetes. This basic topology of the phylum Ascomycota is generally consistent throughout most small subunit rRNAand RPB2(beta subunit of RNA polymerase II) (22) based analyses. However, there is no signi®cant statistical support for this topology (23,24). In addition, certain phylogenies based on amino acid sequences (25,26) have placed S.pombe at the base of the budding yeasts, although again with poor support. In contrast, phylogenetic analyses based on multiple concatenated mitochondrial protein sequences have indicated that the position of S.pombe is at the base of the budding yeasts, often with good bootstrap support (6,24). Although both the budding yeasts and S.pombe form long branches in these analyses, maximum likelihood-based analyses that are known to minimize long-branch attraction artifacts (27) increase support for this topology (24). The present study further explores the position of Schizosaccharomyces within the Fungi. MATERIALS AND METHODS Strains, culture conditions and preparation of mtDNA The strains used were S.octosporus (ATCC 2479) and S.japonicus var. japonicus (ATCC 10660). Cells were grown using Yeast Standard Medium, consisting of 1% yeast extract, 1 g/l KH2PO4 and 3% glycerol (for S.japonicus var. japonicus, 3% glucose was used). Cultures were grown for 24±48 h with shaking (100 r.p.m.) at 30°C. Puri®cation of mtDNA was performed using 20±30 g (wet weight) of cells, harvested in the early stationary phase by centrifugation. After resuspension in a sorbitol buffer (0.6 M sorbitol, 5 mM EDTA, 50 mM Tris pH 7.4), the cells were broken mechanically by shaking with glass beads, and a crude mitochondrial fraction was isolated by differential centrifugation. The mitochondrial fraction was lysed in the presence of 1% SDS and 100 mg/ ml proteinase K, at 50°C for 1 h. SDS was subsequently eliminated from the lysate by addition of 1 M NaCl, and after 1 h on ice, the precipitate (SDS±protein complex) was removed by centrifugation. The total nucleic acids were fractionated on a CsCl gradient (1.1 g/ml, 40 000 r.p.m. for 48 h) in the presence of 10 mg/ml bis-benzimide (Hoechst dye 33258). The upper band (A + T-rich DNA) was extracted and recentrifuged in one or two subsequent CsCl gradients. Yields of 0.5±3 mg DNA were typical. Cloning and sequencing of mtDNA mtDNA was physically sheared by nebulization (28), and a size fraction of 500±3000 bp was recovered after agarose gel electrophoresis. DNAs were incubated in the presence of dNTPs, the Klenow fragment of DNA polymerase I and T7 DNA polymerase to generate blunt ends, and cloned into the SmaI site of a modi®ed Bluescript II KS+ vector with a shortened multi-cloning site (pFBS). Recombinant plasmids containing mtDNA inserts were identi®ed by colony hybridization using mtDNA as a probe. DNA sequencing was performed by the dideoxy chain termination method (29), using single-stranded DNA as template and [a-35S]dATP as label. Labeled DNA fragments were subjected to electrophoresis in 4% polyacrylamide gels, dried onto glass plates (30), and autoradiographed. Automated sequencing was performed on a LiCor 4000L apparatus, using an end-labeled primer and a cycle sequencing protocol (Amersham). The mtDNA sequences of S.octosporus and S.japonicus var. japonicus have been deposited in GenBank (accession numbers AF275271 and AF547983, respectively).