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.     

The novel function of the Saccharomyces cerevisiaeCBP2 gene as a splicing factor essential to excision of the Saccharomyces douglasiiLSU 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. cerevisiaeCBP2 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. cerevisiaeCBP2 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.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. 2. Localization of genes by interspecific hybridization in strain ade7-50h- and cloning of the genome in small fragments

A series of 18 small overlapping restriction fragments has been cloned, covering the complete mitochondrial genome of Schizosaccharomyces pombe. By hybridizing mitochondrial gene probes from Saccharomyces cerevisiae and Neurospora crassa with restriction fragments of Schizosaccharomyces pombe mitochondrial DNA, the following homologous genes were localized on the mitochondrial genome of S. pombe: cob, cox1, cox2 and cox3, ATPase subunit 6 and 9 genes, the large rRNA gene and both types of open reading frames occurring in mitochondrial introns of various ascomycetes. The region of the genome, hybridizing with cob exon probes is separated by an intervening sequence of about 2500 bp, which is homologous with the first two introns of the cox1 gene in Saccharomyces cerevisiae (class II introns according to Michel et al. 1982). Similarly, in the cox1 homologous region, which covers about 4000 bp, two regions were detected hybridizing with class I intron probes, suggesting the existence of two cox1 introns in Schizosaccharomyces pombe. Hybridization with several specific exon probes with a determined order has revealed that cob, cox1, cox3 and the large rRNA gene are all transcribed from the same DNA strand. The low intensities of hybridization signals suggest a large evolutionary distance between Schizosaccharomyces pombe and Saccharomyces cerevisiae or Neurospora crassa mitochondrial genes. Considering the length of the mitochondrial DNA of Schizosaccharomyces pombe (about 19.4 kbp) and the expected length of the localized genes and intron sequences there is enough space left for encoding the expected set of tRNAs and the small rRNA gene. The existence of leader-, trailer-, ori- and spacer sequences or further unassigned reading frames is then restricted to a total length of about 3000 bp only.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene

The DNA sequence of the cob region of the Schizosaccharomyces pombe mitochondrial DNA has been determined. The cytochrome b structural gene is interrupted by an intron of 2526 base-pairs, which has an open reading frame of 2421 base-pairs in phase with the upstream exon. The position of the intron differs from those found in the cob genes of Saccharomyces cerevisiae, Aspergillus nidulans or Neurospora crassa. The Sch. pombe cob intron has the potential of assuming an RNA secondary structure almost identical to that proposed for the first two cox1 introns (group II) in S. cerevisiae and the p1-cox1 intron in Podospora anserina. It has most of the consensus nucleotides in the central core structure described for this group of introns and its comparison with other group II introns allows the identification of an additional conserved nucleotide stretch. A comparison of the predicted protein sequences of group II intronic coding regions reveals three highly conserved blocks showing pairwise amino acid identities of 34 to 53%. These regions comprise over 50% of the coding length of the intron but do not include the 5' region, which has strong secondary structural features. In addition to the potential intron folding, long helical structures involving repetitive sequences can be formed in the flanking cob exon regions. A comparison of the Sch. pombe cytochrome b sequence with those available from other organisms indicates that Sch. pombe is evolutionarily distant from both budding yeasts and filamentous fungi. As was seen for the Sch. pombe cox1 gene (Lang, 1984), the cob exons are translated using the universal genetic code and this distinguishes Sch. pombe mitochondria from all other fungal and animal mitochondrial systems.     

The intron of the mitochondrial 21S rRNA gene: distribution in different yeast species and sequence comparison between Kluyveromyces thermotolerans and Saccharomyces cerevisiae

We have screened numerous different yeast species for the presence of sequences homologous to the intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (intron r1) and found them in all Kluyveromyces species, some of the Saccharomyces species and none of the other yeasts tested. We have determined the nucleotide sequence of the r1-intron in K. thermotolerans and compared it with that of S. cerevisiae. The two introns are inserted at the same position within the 21S rRNA gene. They contain homologous internal open reading frames (ORFs) initiated at the same AUG codon which can be aligned over their entire length. Several silent multi-substitutions indicate that these intronic ORFs represent selectively conserved functional genes. Other intron segments, on the contrary, reveal short blocks of extensive homology separated by non-homologous stretches and/or additions-deletions. Comparison of our two yeast r1-introns with equivalent introns of N. crassa and A. nidulans mitochondria reveals that introns with very similar RNA secondary structures can accommodate different types of ORFs.     

TheCBP2 gene fromSaccharomyces douglasii is a functional homologue of theSaccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome

InSaccharomyces cerevisiae the only known role of theCBP2 gene is the excision of the fifth intron of the mitochondrialcyt b gene (bI5). We have cloned theCBP2 gene fromSaccharomyces douglasii (a close relative ofS. cerevisiae). A comparison of theS. douglasii andS. 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 theS. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-typeS. douglasii mitochondrial genome, but not in the presence of an intronlessS. cerevisiae mitochondrial genome. Also theS. douglasii andS. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from theS. douglasii mitochondrial genome.     

The efficiency of functional mitochondrial replacement in Saccharomyces species has directional character

Optimal interactions among nuclear and mitochondria-coded proteins are required to assemble functional complexes of mitochondrial oxidative phosphorylation. The communication between the nuclear and mitochondrial genomes has been studied by transplacement of mitochondria from related species into mutants devoid of mitochondrial DNA (rho0). Recently we have reported that the mitochondria transferred from Saccharomyces paradoxus restored partially the respiration in Saccharomyces cerevisiae rho0 mutants. Here we present evidence that the S. cerevisiae mitochondria completely salvage from respiration deficiency, not only in conspecific isolates but also in S. paradoxus. The respiratory capacity in less-related species can be recovered exclusively in the presence of S. cerevisiae chromosomes. The efficiency of the re-established oxidative phosphorylation did not rely on the presence of introns in the S. cerevisiae mitochondrial DNA. Our results suggest that, apart from evolutionary distance, the direction of mitochondrial replacement could play a significant role in installing the complete (wild-type-like) interaction between mitochondria and nuclei from different species.     

TheCBP2 gene fromSaccharomyces douglasii is a functional homologue of theSaccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome

InSaccharomyces cerevisiae the only known role of theCBP2 gene is the excision of the fifth intron of the mitochondrialcyt b gene (bI5). We have cloned theCBP2 gene fromSaccharomyces douglasii (a close relative ofS. cerevisiae). A comparison of theS. douglasii andS. 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 theS. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-typeS. douglasii mitochondrial genome, but not in the presence of an intronlessS. cerevisiae mitochondrial genome. Also theS. douglasii andS. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from theS. douglasii mitochondrial genome.     

The novel function of the Saccharomyces cerevisiaeCBP2 gene as a splicing factor essential to excision of the Saccharomyces douglasiiLSU 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. cerevisiaeCBP2 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. cerevisiaeCBP2 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. Received: 1 October 1997 / Accepted: 18 November 1997     

Two homologous mitochondrial introns from closely related Saccharomyces species differ by only a few amino acid replacements in their Open Reading Frames: one is mobile, the other is not.

We have undertaken a comprehensive study of the gene conversion of all the mitochondrial introns of Saccharomyces capensis. The approach used involved the measurements of intron transmission amongst the progeny of crosses between a recipient strain (Saccharomyces cerevisiae intronless mitochondria) and various donor strains (Saccharomyces capensis, with various combinations of mitochondrial introns). We have shown that the S. capensis second intron (bi2 of cytochrome b gene) is extremely active as a donor in gene conversion whereas its homologous S. cerevisiae intron is not. Determination of sequence of the S. capensis intron demonstrates that it differs from that of the homologous S. cerevisiae intron (bi2) by a very small number of nucleotide substitutions.     

Molecular basis of the 'box effect', A maturase deficiency leading to the absence of splicing of two introns located in two split genes of yeast mitochondrial DNA

In the mitochondrial DNA of Saccharomyces cerevisiae, the genes cob-box and oxi3, coding for apocytochrome b and cytochrome oxidase subunit I respectively, are split. Several mutations located in the introns of the cob-box gene prevent the synthesis of cytochrome b and cytochrome oxidase subunit I (this is known as the 'box effect').-We have elucidated the molecular basis of this phenomenon: these mutants are unable to excise the fourth intron of oxi3 from the cytochrome oxidase subunit I pre-mRNA; the absence of a functional bI4 mRNA maturase, a trans-acting factor encoded by the fourth intron of the cob-box gene explains this phenomenon. This maturase was already known to control the excision of the bI4 intron; consequently we have demonstrated that it is necessary for the processing of two introns located in two different genes. Mutations altering this maturase can be corrected, but only partially, by extragenic suppressors located in the mitochondrial (mim2) or in the nuclear (NAM2) genome. The gene product of these two suppressors should, therefore, control (directly or indirectly) the excision of the two introns as the bI4 mRNA maturase normally does.     

Diversity in organization and the origin of gene orders in the mitochondrial DNA molecules of the genus Saccharomyces

Sequencing of the Saccharomyces cerevisiae nuclear and mitochondrial genomes provided a new background for studies on the evolution of the genomes. In this study, mitochondrial genomes of a number of Saccharomyces yeasts were mapped by restriction enzyme analysis, the orders of the genes were determined, and two of the genes were sequenced. The genome organization, i.e., the size, presence of intergenic sequences, and gene order, as well as polymorphism within the coding regions, indicate that Saccharomyces mtDNA molecules are dynamic structures and have undergone numerous changes during their evolution. Since the separation and sexual isolation of different yeast lineages, the coding parts have been accumulating point mutations, presumably in a linear manner with the passage of time. However, the accumulation of other changes may not have been a simple function of time. Larger mtDNA molecules belonging to Saccharomyces sensu stricto yeasts have acquired extensive intergenic sequences, including guanosine-cytosine-rich clusters, and apparently have rearranged the gene order at higher rates than smaller mtDNAs belonging to the Saccharomyces sensu lato yeasts. While within the sensu stricto group transposition has been a predominant mechanism for the creation of novel gene orders, the sensu lato yeasts could have used both transposition- and inversion-based mechanisms.     

Mitochondria--tool for taxonomic identification of yeasts from Saccharomyces sensu stricto complex

Mitochondrial genomes of Saccharomyces and close relatives previously used for transplacement of mitochondria to S. cerevisiae were examined. The origins of replication in mitochondrial DNA, the presence of nuclear and mitochondrial polymorphic loci and the ability to produce mitochondrial respiration-deficient mutants were used to reclassify some collection yeasts and to assign others into four separate subgroups. The first included isolates identical to Saccharomyces cerevisiae (S. italicus, S. oviformis, S. chevalieri and S. capensis) which possess 5 or more replication origins. The second group consists of S paradoxus (var douglasii) mitochondrial genome with the equal number of ori sequences but incompatible mitochondria. The third group represents Saccharomyces sensu stricto petite-positive species (S. carlsbergensis, S. heterogenicus, S. uvarum, S. willianus) with 1-2 origins of replication significantly different from S. cerevisiae. In addition, the locus between tRNA(fMet) and tRNA(Pro) is about one-half of the 1400 bp members of S. cerevisiae complex. The last group includes isolates that do not belong to Saccharomyces sensu stricto group as they are petite-negative and devoid of any S. cerevisiae-like replication origins.     

Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae.

The genes of Saccharomyces cerevisiae coding for the mitochondrial threonine and tryptophan tRNA synthetases and for a putative mitochondrial ribosomal protein have been cloned. These, and the previously cloned gene for a mitochondrial elongation factor, were used to disrupt or partially delete the wild-type chromosomal copies of the genes in the respiratory-competent strain W303. In each case, inactivation of a gene whose product is required for mitochondrial protein synthesis causes an instability in mitochondrial DNA. Although intact mitochondrial genomes are rapidly and quantitatively eliminated in the protein synthesis defective strains, specific rho- genomes can be maintained stably over many generations. These results indicate that mitochondrial protein synthesis is required for the propagation of wild-type mitochondrial DNA in yeast.