Enhanced expression of the DNA damage-inducible gene DIN7 results in increased mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae.

We reported previously that the product of DIN7, a DNA damage-inducible gene of Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are involved in DNA repair and replication. This family includes the S. cerevisiae protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG family which specifically functions in mitochondria. We reported previously that overexpression of DIN7 results in a mitochondrial mutator phenotype. In the present study we wished to test the hypothesis that this phenotype is dependent on the nuclease activity of Din7p. For this purpose, we constructed two alleles, din7-D78A and din7-D173A, which encode proteins in which highly conserved aspartates important for the nuclease activity of the XPG proteins have been replaced by alanines. Here, we report that overexpression of the mutant alleles, in contrast to DIN7, fails to increase the frequency of mitochondrial petite mutants or erythromycin-resistant (Er) mutants. Also, overproduction of din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit gross rearrangements of mtDNA, and that this correlates with enhanced recombination within the mitochondrial cyt b gene. These results suggest that the stability of the mitochondrial genome of S. cerevisiae is modulated by the level of the nuclease Din7p.

Critical base substitutions that affect the splicing and/or homing activities of the group I intron bi2 of yeast mitochondria.

The second intron (bi2) of the cyt b gene from related Saccharomyces species has an extraordinarily conserved sequence and can have different functions in wild-type cells. The protein encoded by the S. cerevisiae intron functions as a maturase to promote intron splicing, while the homologous S. capensis intron encodes a bifunctional protein that acts both as a maturase and as a homing endonuclease (I-ScaI) promoting intron mobility. The protein encoded by intron bi2 belongs to a large gene family characterized by the presence of two conserved LAGLIDADG motifs (P1 and P2). In this study, we analysed a set of splicing-deficient mutants of the S. cerevisiae intron bi2 that carry non-directed mutations affecting the maturase activity, and a set of directed missense mutations introduced into the bifunctional protein encoded by the S. capensis intron. Analysis of these mutations has allowed identification of the residues in the conserved P1 and P2 motifs which are crucial for splicing and homing activities. Moreover, several mutations which are located in the C-terminal part of the protein have been found to affect both functions.

Purification and characterization of the DNA cleavage and recognition site of I-ScaI mitochondrial group I intron encoded endonuclease produced in Escherichia coli.

The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs. Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain. It acts as a specific homing endonuclease I- Sca I promoting intron mobility and as a maturase promoting intron splicing. Here we describe the synthesis of a universal code equivalent to the mitochondrial sequence coding for this protein and the in vitro characterization of I- Sca I endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli. We have also determined the cleavage pattern as well as the recognition site of p28bi2. It was found that p28bi2 generates a double-strand cleavage downstream from the intron insertion site with 4 nt long 3'-overhangs. Mutational analysis of the DNA target site shows that p28bi2 recognizes a 16-19 bp sequence from positions -11 to +8 with respect to the intron insertion site.

Intragenic suppressors that restore the splicing and homing activities of the protein encoded by the second intron of the Saccharomyces capensis cyt b gene.

The second (bi2) intron of the mitochondrial cyt b gene from Saccharomyces capensis encodes a bifunctional protein which acts both as a maturase, promoting intron splicing, and as a homing-endonuclease, I-ScaI, promoting intron mobility. In this work we isolated and characterized revertants from a respiratory-deficient mutant in which both functions of the protein have been lost. Intragenic revertants resulted mainly from monosubstitutions in the mutated codon and in one case from a distant second site mutation. All novel variants of the S. capensis bi2 intron-encoded protein are competent for the maturase activity but only two of them can partially complement the homing function.

MitBASE : a comprehensive and integrated mitochondrial DNA database. The present status.

MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects, under a single interface, databases for Plant, Vertebrate, Invertebrate, Human, Protist and Fungal mtDNA and a Pilot database on nuclear genes involved in mitochondrial biogenesis in Saccharomyces cerevisiae. MitBASE reports all available information from different organisms and from intraspecies variants and mutants. Data have been drawn from the primary databases and from the literature; value adding information has been structured, e.g., editing information on protist mtDNA genomes, pathological information for human mtDNA variants, etc. The different databases, some of which are structured using commercial packages (Microsoft Access, File Maker Pro) while others use a flat-file format, have been integrated under ORACLE. Ad hoc retrieval systems have been devised for some of the above listed databases keeping into account their peculiarities. The database is resident at the EBI and is available at the following site: http://www3.ebi.ac.uk/Research/Mitbase/mitbas e.pl. The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecies diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme.

A human putative Suv3-like RNA helicase is conserved between Rhodobacter and all eukaryotes.

We have cloned and sequenced a cDNA of the human homologue of the Saccharomyces cerevisiae Suv3 putative RNA helicase which is indispensable for mitochondrial function in yeast. The human Suv-3-like protein has a typical mitochondrial leader sequence. Northern blot data and analysis of ESTs in the data banks indicate that this human gene (SUPV3L1) is expressed in practically all tissues, though at different levels. Sequence homology analysis has shown a strong conservation of the protein in a number of eukaryotic organisms -- plants, mammals and fungi, but no close homologues exist in bacteria with the exception of the purple bacterium Rhodobacter sphaeroides. This gene is thus ubiquitously present in all eukaryotic organisms.

MitBASE: a comprehensive and integrated mitochondrial DNA database.

MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects all available information from different organisms and from intraspecie variants and mutants. Research institutions from different countries are involved, each in charge of developing, collecting and annotating data for the organisms they are specialised in. The design of the actual structure of the database and its implementation in a user-friendly format are the care of the European Bioinformatics Institute. The database can be accessed on the Web at the following address: http://www.ebi.ac. uk/htbin/Mitbase/mitbase.pl. The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecie diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme.

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.

A novel nuclear gene, CBT1, essential for mitochondrial cytochrome b formation: terminal processing of mRNA and intron dependence.

We describe a new nuclear gene, CBT1 (Cytochrome B Termination), specifically involved in the generation of mature mRNA of cytochrome b in yeast mitochondria. Disruption of CBT1 (corresponding to ORF YKL 208W) results in a respiratory deficiency (no growth on acetate and ethanol, a reduced growth on glycerol, and a moderate growth on lactate). Cytochrome b is practically undetectable spectrally, while cytochromes a and a3 (cytochrome oxidase) appear unaffected by the disruption. Analysis of mitochondrial transcripts shows a reduced abundance of cytb mRNA, which in addition is approximately 200 nucleotides longer than that of the wild-type. Sequencing of the 3' region of the mutant cytb mRNA with an oligonucleotide primer positioned 148 nt downstream from the dodecamer sequence ("end-of-messenger" signal), demonstrates that the mutant transcript is extended beyond this position and is not processed at the conserved dodecamer cleavage site. The CBT1 gene product may be one of the components required for the exact 3' cleavage of the cytb messenger and may also be related to RNA splicing, since the intron-containing cytb gene is not as well expressed as the intron-less gene and the respiratory deficiency is more severe. We propose, that the CBT1 protein is necessary for the correct trimming of the end of cytb pre-mRNA and may be a part of the multi-component complex involved in this process.

Replacement of two non-adjacent amino acids in the S.cerevisiae bi2 intron-encoded RNA maturase is sufficient to gain a homing-endonuclease activity.

Two homologous group I introns, the second intron of the cyt b gene, from related Saccharomyces species differ in their mobility. The S.capensis intron is mobile and encodes the I-ScaI endonuclease promoting intron homing, whilst the homologous S.cerevisiae intron is not mobile, but functions as an RNA maturase promoting splicing. These two intron-encoded proteins differ by only four amino acid substitutions. Taking advantage of the remarkable similarity of the two intron open reading frames and using biolistic transformation of mitochondria, we show that the replacement of only two non-adjacent residues in the S.cerevisiae maturase carboxy-terminal sequence is sufficient to induce a homing-endonuclease activity without losing the splicing function. Also, we demonstrate that these two activities reside in the S.capensis bi2-encoded protein which functions in both splicing and intron mobility in the wild-type cells. These results provide new insight into our understanding of the activity and the evolution of group I intron-encoded proteins.

The S. cerevisiae nuclear gene SUV3 encoding a putative RNA helicase is necessary for the stability of mitochondrial transcripts containing multiple introns.

The product of the nuclear gene SUV3 is implicated in a variety of post-transcriptional processes in yeast mitochondria. We have analysed the effect of SUV3 gene-disruption on the expression of intron-containing alleles of the mitochondrial cytb and cox1 genes. We have constructed several strains with mitochondrial genomes containing different combinations of cytb and cox1 introns, and associated these genomes with the disruption of SUV3. The resulting strains were tested for their respiratory competence and spectral cytochrome content. All the strains containing only two or three introns showed normal expression of cytb and cox1, whereas the strains containing more introns were unable to express the appropriate gene. The analysis of mitochondrial RNAs by Northern hybridisation showed that the loss of respiratory competence in the strains containing more introns is due to the decrease of mRNA level with no over-accumulation of high-molecular-weight precursors. However, the transcription of the genes was not affected. These results led us to the notion that SUV3 is required for the stability of intron-containing cytb and cox1 transcripts in a cumulative way, not dependent on any particular intron.

Homing of a group II intron in yeast mitochondrial DNA is accompanied by unidirectional co-conversion of upstream-located markers.

Group II introns ai1 and ai2 of the Saccharomyces cerevisiae mitochondrial COXI gene encode proteins having a dual function (maturase and reverse transcriptase) and are mobile genetic elements. By construction of adequate donor genomes, we demonstrate that each of them is self-sufficient and practises homing in the absence of homing-type endonucleases encoded by either group I introns or the ENS2 gene. Each of the S. cerevisiae group II self-mobile introns was tested for its ability to invade mitochondrial DNA (mtDNA) from two related Saccharomyces species. Surprisingly, only ai2 was observed to integrate into both genomes. The non-mobility of ai1 was clearly correlated with some polymorphic changes occurring in sequences flanking its insertion sites in the recipient mtDNAs. Importantly, studies of the behaviour of these introns in interspecific crosses demonstrate that flanking marker co-conversion accompanying group II intron homing is unidirectional and efficient only in the 3' to 5' direction towards the upstream exon. Thus, the polar co-conversion and dependence of the splicing proficiency of the intron reported previously by us are hallmarks of group II intron homing, which significantly distinguish it from the strictly DNA-based group I intron homing and strictly RNA-based group II intron transposition.

Two homologous introns from related Saccharomyces species differ in their mobility.

We have studied gene conversion initiated by the ai3 intron of the Saccharomyces cerevisiae mitochondrial (mt) COXI gene and its homologous intron (S.cap.ai1) from Saccharomyces capensis. The approach used involved the measurement of intron transmission amongst the progeny of crosses between constructed recipient and donor strains. We found that the S. cerevisiae ai3 intron is extremely active as a donor in gene conversion, whereas its homologous S. capensis intron is not. We have established the sequence of S.cap.ai1 and compared its open reading frame (ORF) with that of I-SceIII encoded by the homologous S. cerevisiae intron. The two protein-coding intron sequences are almost identical, except that the S. capensis ORF contains an in-frame stop codon. This finding provides a strong indication that the 3' part of the S. cerevisiae intron ORF encoding I-SceIII (which should not be translated in the S. capensis intron) must be critical for function of mtDNA endonucleases to mediate intron mobility.

The C-terminal domain of yeast cytochrome b is essential for a correct assembly of the mitochondrial cytochrome bc1 complex.

Yeast mutants modifying the C-terminal region of mitochondrial cytochrome b were isolated and characterized. A nonsense mutation of the leucine codon 335 (TTA-->TAA), 50 residues before the normal C-terminus, blocks incorporation of heme into the apocytochrome b and prevents growth on non-fermentable substrates. The same defects were observed in a frameshift mutant (after codon 348, TAT-->TATT) in which the last 37 C-terminal residues are predicted to be replaced by a novel sequence of 33 amino acids. Function was regained in the nonsense mutant only by true back mutations restoring a protein of the wild-type sequence. The respiratory capacity was restored to wild-type levels in the frameshift mutant by a variety of single base subtractions located within a window of 24 bases before or after the original +T addition, these pseudo-reversions resulted in single or multiple (up to five) consecutive amino acid replacements between positions 346 and 354 and restored the wild-type sequence from position 355 to 385. These data, combined with hydropathy calculations and sequence comparisons, suggest that the C-terminal domain of cytochrome b forms a transmembrane segment essential for the correct assembly of the cytochrome bc1 complex.

LISTA, a comprehensive compilation of nucleotide sequences encoding proteins from the yeast Saccharomyces.

The amount of nucleotide sequence data is increasing exponentially. We therefore made an effort to make a comprehensive database (LISTA) for the yeast Saccharomyces cerevisiae. Each sequence has been attributed a single genetic name and in the case of allelic duplicated sequences, synonyms are given, if necessary. For the nomenclature we have introduced a standard principle for naming gene sequences based on priority rules. We have also applied a simple method to distinguish duplicated sequences of one and the same gene from non-allelic sequences of duplicated genes. By using these principles we have sorted out a lot of confusion in the literature and databanks. Along with the genetic name, the mnemonic from the EMBL databank, the codon bias, reference of the publication of the sequence and the EMBL accession numbers are included in each entry.

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. Comparisons 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.

A comprehensive compilation of 1001 nucleotide sequences coding for proteins from the yeast Saccharomyces cerevisiae (= ListA2)

The amount of nucleotide sequence data is increasing exponentially. We therefore continued our effort to make a comprehensive database for the yeast Saccharomyces cerevisiae. In this database (ListA2) we have compiled 1001 protein coding sequences from this organism. Each sequence has been attributed a single genetic name and in the case of allelic duplicated sequences, synonyms are given, if necessary. For the nomenclature we have introduced a standard principle for naming gene sequences based on priority rules. We have also applied a simple method to distinguish duplicated sequences of one and the same gene from non-allelic sequences of duplicated genes. By using these principles we have sorted out a lot of confusion in the literature and databanks. Along with the genetic name, the mnemonic from the EMBL databank, the codon bias, reference of the publication of the sequence and the EMBL accession numbers are included for each entry. The database is available on request.

The MRS1 gene of S. douglasii: co-evolution of mitochondrial introns and specific splicing proteins encoded by nuclear genes.

We have developed a rapid and simple methodology to locate yeast genes within cloned inserts, obtain partial sequence information, and construct chromosomal disruptions of these genes. This methodology has been used to study a nuclear gene from the yeast S. douglasii (a close relative of S. cerevisiae), which is essential for the excision of the mitochondrial intron aI1 of S. douglasii (the first intron in the gene encoding subunit I of cytochrome oxidase), an intron which is not present in the mitochondrial genome of S. cerevisiae. We have shown that this gene is the homologue of the S. cerevisiae MRS1 gene, which is essential for the excision of the mitochondrial introns bI3 and aI5 beta of S. cerevisiae, but is unable to assure the excision of the intron aI1 from the coxI gene of S. douglasii. The two genes are very similar, with only 13% nucleotide substitutions in the coding region, transitions being 2.5 times more frequent than transvertions. At the protein level there are 86% identical residues and 7% conservative substitutions. The divergence of the MRS1 genes of S. cerevisiae and S. douglasii, and the concomitant changes in the structure of their mitochondrial genomes is an interesting example of the co-evolution of nuclear and mitochondrial genomes.

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.

NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes.

We report the genetic characterization, molecular cloning, and sequencing of a novel nuclear suppressor, the NAM9 gene from Saccharomyces cerevisiae, which acts on mutations of mitochondrial DNA. The strain NAM9-1 was isolated as a respiration-competent revertant of a mitochondrial mit mutant which carries the V25 ochre mutation in the oxi1 gene. Genetic characterization of the NAM9-1 mutation has shown that it is a nuclear dominant omnipotent suppressor alleviating several mutations in all four mitochondrial genes tested and has suggested its informational, and probably ribosomal, character. The NAM9 gene was cloned by transformation of the recipient oxi1-V25 mutant to respiration competence by using a gene bank from the NAM9-1 rho o strain. Orthogonal-field alternation gel electrophoresis analysis and genetic mapping localized the NAM9 gene on the right arm of chromosome XIV. Sequence analysis of the NAM9 gene showed that it encodes a basic protein of 485 amino acids with a presequence that could target the protein to the mitochondrial matrix. The N-terminal sequence of 200 amino acids of the deduced NAM9 product strongly resembles the S4 ribosomal proteins from chloroplasts and bacteria. Significant although less extensive similarity was found with ribosomal cytoplasmic proteins from lower eucaryotes, including S. cerevisiae. Chromosomal inactivation of the NAM9+ gene is not lethal to the cell but leads to respiration deficiency and loss of mitochondrial DNA integrity. We conclude that the NAM9 gene product is a mitochondrial ribosomal counterpart of S4 ribosomal proteins found in other systems and that the suppressor acts through decreasing the fidelity of translation.