Saccharomyces Douglasii Research Articles

Molecular phylogenetics of the genus trichosporon inferred from mitochondrial cytochrome B gene sequences.

Mitochondrial cytochrome b (cyt b) genes of 42 strains representing 23 species of the genus Trichosporon were partially sequenced to determine their molecular phylogenetic relationships. Almost half of the 22 strains investigated (from 11 different species) contained introns in their sequences. Analysis of a 396-bp coding sequence from each strain of Trichosporon under investigation showed a total of 141 (35.6%) variable nucleotide sites. A phylogenetic tree based on the cyt b gene sequences revealed that all species of Trichosporon except Trichosporon domesticum and Trichosporon montevideense had species-specific cyt b genes. Trichosporon sp. strain CBS 5581 was identified as Trichosporon pullulans, and one clinical isolate, IFM 48794, was identified as Trichosporon faecale. Analysis of 132-bp deduced amino acid sequences showed a total of 34 (25.75%) variable amino acid sites. T. domesticum and T. montevideense, Trichosporon asahii and Trichosporon asteroides, and Trichosporon gracile and Trichosporon guehoae had identical amino acid sequences. A phylogenetic tree constructed with the ascomycetes Saccharomyces douglasii and Candida glabrata taken as outgroup species and including representative species from closely related genera species of Trichosporon clustered with other basidiomycetous yeasts that contain xylose in their cell wall compositions. These results indicate the effectiveness of mitochondrial cyt b gene sequences for both species identification and the phylogenetic analysis of Trichosporon species.

Arsenical resistance genes in Saccharomyces douglasii and other yeast species undergo rapid evolution involving genomic rearrangements and duplications.

We have isolated and characterized three adjacent Saccharomyces douglasii genes that share remarkable structural homology (97% amino acid sequence identity) with Saccharomyces cerevisiae ARR1 (ACR1), ARR2 (ACR2) and ARR3 (ACR3) genes involved in arsenical resistance. The ARR2 and ARR3 genes encoding the cytoplasmic arsenate reductase and the plasma membrane arsenite transporter are functionally interchangeable in both yeast species. In contrast, a single copy of S. douglasii ARR1 gene is not sufficient to complement the arsenic hypersensitivity of a S. cerevisiae mutant lacking the transcriptional activator Arr1p. This inability may be related to a deletion of a 35-bp sequence including the putative Yap-binding element in the ARR1 promoter of S. douglasii. Different mechanisms of regulation of ARR1 genes expression may therefore explain the increased tolerance of S. douglasii to arsenic in comparison with S. cerevisiae. The apparent duplication of the ARR gene cluster in the S. douglasii genome may constitute another factor contributing to the observed differences in arsenic sensitivity. Comparison of ARR genes from the genomes of several yeast species indicates that they are located in subtelomeric regions undergoing rapid evolution involving large-scale genomic rearrangements.

The SUV3 gene from Saccharomyces douglasii is a functional equivalent of its Saccharomyces cerevisiae orthologue and is essential for respiratory growth.

In the yeast Saccharomyces cerevisiae the product of the nuclear gene SUV3 has been shown to be involved in a variety of mitochondrial post-transcriptional processes. We have cloned and sequenced the SUV3 gene from Saccharomyces douglasii, a close relative of S. cerevisiae which has important changes in the organization of its mitochondrial genome and concomitant changes in nucleo-mitochondrial interactions. We show that the S. douglasii SUV3 gene shares considerable structural homology (92% amino acid sequence identity) with its S. cerevisiae counterpart and that their nucleotide sequences display evidence of recent divergence. To determine the function of the S. douglasii SUV3 gene we have constructed a strain carrying an inactive SUV3 gene and analyzed the effect of this inactivation on the integrity of the mitochondrial genome and on the stability of mitochondrial transcripts. We have demonstrated that the S. douglasii SUV3 gene, like the S. cerevisiae gene, is essential for respiratory growth and for stability of the intron-containing mitochondrial transcripts, thus the two genes are functionally equivalent. Also the S. douglasii and S. cerevisiae SUV3 genes are completely interchangeable, despite the differences in the structure of the mitochondrial chromosome in the two yeasts.

The Rieske FeS protein encoded and synthesized within mitochondria complements a deficiency in the nuclear gene.

The Rieske FeS protein, an essential catalytic subunit of the mitochondrial cytochrome bc1 complex, is encoded in yeast by the nuclear gene RIP1, whose deletion leads to a respiratory-deficient phenotype. By using biolistic transformation, we have relocated the nuclear RIP1 gene into mitochondria. To allow its expression within the organelle and to direct its integration downstream of the cox1 gene, we have fused the 3' end of the Saccharomyces douglasii cox1 gene upstream of the mitochondrial copy of RIP1 (RIP1m) flanked by the Saccharomyces cerevisiae cox1 promoter and terminator regions. We show that RIP1m integrated between the cox1 and atp8 genes is mitotically stable and expressed, and it complements a deletion of the nuclear gene. Immunodetection experiments demonstrate that the mitochondrial genome containing RIP1m is able to produce the Rip1 protein in lower steady-state amounts than the wild type but still sufficient to maintain a functional cytochrome bc1 complex and respiratory competence to a RIP1-deleted strain. Thus, this recombined mitochondrial genome is a fully functional mitochondrial chromosome with an extended gene content. This successful mitochondrial expression of a nuclear gene essential for respiration can be viewed at the evolutionary level as an artificial reversal of evolutionary events.

Expression studies and promoter analysis of the nuclear gene for mitochondrial transcription factor 1 (MTF1) in yeast.

The basal mitochondrial transcription apparatus of Saccharomyces cerevisiae consists of the core enzyme for mitochondrial RNA polymerase and the specificity factor. The core enzyme is homologous to those of bacteriophages T3, T7 and SP6 whereas the specificity factor shows similarities with bacterial sigma factors. Recently it was shown that the bacteriophage-type core enzyme is widespread among the eukaryotic lineage and a common picture for the mitochondrial transcription apparatus in eukaryotic cells is now emerging. In contrast to the situation for the core enzyme, the gene for the specificity factor has only been identified from S. cerevisiae and more recently from two other yeast species. As the specificity factor is the key component for initiation of transcription at the mitochondrial promoter we wanted to study in more detail gene expression, regulation, and the function of the promoter of the nuclear MTF1 gene. For this purpose the messenger RNA level for scMTF1 was investigated under a large number of different growth conditions and thereby exhibited a very low, but regulated and carbon source-dependent, expression. Deletion experiments identify the minimal promoter for functional complementation in yeast. To evaluate the functional conservation of the promoter elements the homologous MTF1 gene from the closely related yeast Saccharomyces douglasii was isolated and tested in heterologous complementation experiments. In spite of a highly conserved protein sequence these studies demonstrate that at low-copy number sdMTF1 is not able to substitute for scMTF1 in S. cerevisiae. Promoter exchange experiments with MTF1 from S. cerevisiae and S. douglasii demonstrate that differences in gene expression are responsible for the failure in heterologous complementation. This finding prompted us to compare the promoter regions of MTF1 from four different yeast species. For this purpose the sequences of the 5' regions from S. douglasii, S. kluyveri and Kluyveromyces lactis were determined. A comparison of these sequences identifies significant differences and rapid changes in the intergenic regions, even between closely related yeast species.

The novel function of the Saccharomyces cerevisiae CBP2 gene as a splicing factor essential to excision of the Saccharomyces douglasii LSU intron in vivo.

In the yeast Saccharomyces cerevisiae, the product of the nuclear gene CBP2 is required exclusively for the splicing of the terminal intron of the mitochondrial cytochrome b gene. The homologous gene from the related yeast, Saccharomyces douglasii, has been shown to be essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome and dispensable in the presence of an intronless mitochondrial genome. The two CBP2 genes are functionally interchangeable although the target intron of the S. cerevisiae CBP2 gene is absent from the S. douglasii mitochondrial genome. To determine the function of the CBP2 gene in S. douglasii mitochondrial pre-RNA processing we have constructed and analyzed interspecific hybrid strains between the nuclear genome of S. cerevisiae carrying an inactive CBP2 gene and S. douglasii mitochondrial genomes with different intron contents. We have demonstrated that inactivation of the S. cerevisiae CBP2 gene affects the maturation of the S. douglasii LSU pre-RNA, leading to a respiratory-deficient phenotype in the hybrid strains. We have shown that the CBP2 gene is essential for excision of the S. douglasii LSU intron in vivo and that the gene is dispensable when this intron is deleted or replaced by the S. cerevisiae LSU intron.

Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum.

We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum. Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii. Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified. The gene order of S. uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S. uvarum, through specific genome rearrangements.

The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria.

The Cbp2 protein is encoded in the nucleus and is required for the splicing of the terminal intron of the mitochondrial COB gene in Saccharomyces cerevisiae . Using a yeast strain that lacks this intron but contains a related group I intron in the precursor of the large ribosomal RNA, we have determined that Cbp2 protein is also required for the normal accumulation of 21S ribosomal RNA in vivo . Such strains bearing a deletion of the CBP2 gene adapt slowly to growth in glycerol/ethanol media implying a defect in derepression. At physiologic concentrations of magnesium, Cbp2 stimulates the splicing of the ribosomal RNA intron in vitro . Nevertheless, Cbp2 is not essential for splicing of this intron in mitochondria nor is it required in vitro at magnesium concentrations >5 mM. A similar intron exists in the large ribosomal RNA (LSU) gene of Saccharomyces douglasii . This intron does need Cbp2 for catalytic activity in physiologic magnesium. Similarities between the LSU introns and COB intron 5 suggest that Cbp2 may recognize conserved elements of the these two introns, and protein-induced UV crosslinks occur in similar sites in the substrate and catalytic domains of the RNA precursors.

The CBP2 gene from Saccharomyces douglasii is a functional homologue of the Saccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome.

In Saccharomyces cerevisiae the only known role of the CBP2 gene is the excision of the fifth intron of the mitochondrial cyt b gene (bI5). We have cloned the CBP2 gene from Saccharomyces douglasii (a close relative of S. cerevisiae). A comparison of the S. douglasii and S. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that the S. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome, but not in the presence of an intronless S. cerevisiae mitochondrial genome. Also the S. douglasii and S. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from the S. douglasii mitochondrial genome.

Sequence of the mitochondrial gene encoding subunit I of cytochrome oxidase in Saccharomyces douglasii

We have determined the complete sequence of the mitochondrial (mt) gene ( COXI) coding for cytochrome oxidase subunit I of Saccharomyces douglasii. This gene is 7238 bp long and includes four introns. The salient feature of the S. douglasii COXI gene is the presence of two introns, Sd.ai1 and Sd.ai2, which have not been observed in S. cerevisiae genes. Both are group-I introns and are located at novel positions compared with the S. cerevisiae COXI. Interestingly, one of these introns (the second one) is inserted at the same position as intron 2 of COXI of Kluyveromyces lactis and also as intron 8 of the same gene in Podospora anserina. The ORFs contained in these three introns display a high degree of similarity. Comparison of exonic and intronic sequences of the COXI of two Saccharomyces species reinforces our previous conclusions: the evolution of mt genes in yeast obeys different rules to those found in vertebrates.

Characterization and mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in Saccharomyces cerevisiae.

The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S. cerevisiae A49 subunit and Saccharomyces douglasii subunits based on a polymorphism existing between the subunits of RNA polymerase A in these two species. The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit. No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found. Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies. Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase. The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant.

Incipient mitochondrial evolution in yeasts: I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.

Incipient mitochondrial evolution in yeasts: II. The complete sequence of the gene coding for cytochrome b in Saccharomyces douglasii reveals the presence of both new and conserved introns and discloses major differences in the fixation of mutations in evolution

We have determined the complete sequence of the mitochondrial gene coding for cytochrome b in Saccharomyces douglasii. The gene is 6310 base-pairs long and is interrupted by four introns. The first one (1311 base-pairs) belongs to the group ID of secondary structure, contains a fragment open reading frame with a characteristic GIY … YIG motif, is absent from Saccharomyces cerevisiae and is inserted in the same site in which introns 1 and 2 are inserted in Neurospora crassa and Podospora anserina, respectively. The next three S. douglasii introns are homologous to the first three introns of S. cerevisiae, are inserted at the same positions and display various degrees of similarity ranging from an almost complete identity (intron 2 and 4) to a moderate one (intron 3). We have compared secondary structures of intron RNAs, and nucleotide and amino acid sequences of cytochrome b exons and intron open reading frames in the two Saccharomyces species. The rules that govern fixation of mutations in exon and intron open reading frames are different: the relative proportion of mutations occurring in synonymous codons is low in some introns and high in exons. The overall frequency of mutations in cytochrome b exons is much smaller than in nuclear genes of yeasts, contrary to what has been found in vertebrates, where mitochondrial mutations are more frequent. The divergence of the cytochrome b gene is modular: various parts of the gene have changed with a different mode and tempo of evolution.

Natural variation in yeast RNA polymerase A. Formation of a mosaic RNA polymerase A in a meiotic segregant from an interspecific hybrid.

There is a natural variation in the molecular structure of RNA polymerase A isolated from several genetically distant yeast species, Saccharomyces cerevisiae, Saccharomyces douglasii, Schizosaccharomyces pombe, and Candida tropicalis. Several biochemical criteria were used to identify their homologous polypeptide components. Based on these correlations, the minimal subunit composition of S. cerevisiae (and Saccharomyces carlsbergensis) RNA polymerase A was tentatively defined as A190, A135, A40, A27, A23, A19, and A14.5. Without the two Saccharomyces species, S. cerevisiae and S. douglasii, 7 of 13 polypeptides of enzyme A(A49, A43, A40, A34.5, A19, A14.5, and A14) differ slightly in molecular weight and can be resolved by electrophoresis on polyacrylamide gel. The RNA polymerase A isolated from the diploid interspecific hybrid contains all the polypeptides characteristic of the two parents. One meiotic segregant had a hybrid RNA polymerase A with five of the polymorphic polypeptides (A49, A43, A19, A14.5, and A14) coming from S. douglasii and two (A40 and A34.5) from S. cerevisiae. In three successive backcrosses with S. cerevisiae, all the genes for S. douglasii polypeptides were shown to recombine although parental ditype tetrads predominated in the four four-spored asci examined. Thus, the genes for the seven polymorphic polypeptides are not clustered: they lie on at least three different chromosomes.