Var1 Gene Research Articles

The translational activator Sov1 coordinates mitochondrial gene expression with mitoribosome biogenesis.

Mitoribosome biogenesis is an expensive metabolic process that is essential to maintain cellular respiratory capacity and requires the stoichiometric accumulation of rRNAs and proteins encoded in two distinct genomes. In yeast, the ribosomal protein Var1, alias uS3m, is mitochondrion-encoded. uS3m is a protein universally present in all ribosomes, where it forms part of the small subunit (SSU) mRNA entry channel and plays a pivotal role in ribosome loading onto the mRNA. However, despite its critical functional role, very little is known concerning VAR1 gene expression. Here, we demonstrate that the protein Sov1 is an in bona fide VAR1 mRNA translational activator and additionally interacts with newly synthesized Var1 polypeptide. Moreover, we show that Sov1 assists the late steps of mtSSU biogenesis involving the incorporation of Var1, an event necessary for uS14 and mS46 assembly. Notably, we have uncovered a translational regulatory mechanism by which Sov1 fine-tunes Var1 synthesis with its assembly into the mitoribosome.

Transcriptional up-regulation of genes involved in photosynthesis of the Zn/Cd hyperaccumulator Sedum alfredii in response to zinc and cadmium

Zinc (Zn) and cadmium (Cd) are two closely related chemical elements with very different biological roles in photosynthesis. Zinc plays unique biochemical functions in photosynthesis. Previous studies suggested that in some Zn/Cd hyperaccumulators, many steps in photosynthesis may be Cd tolerant or even Cd stimulated. Using RNA-seq data, we found not only that Cd and Zn both up-regulated the CA1 gene, which encodes a β class carbonic anhydrase (CA) in chloroplasts, but that a large number of other Zn up-regulated genes in the photosynthetic pathway were also significantly up-regulated by Cd in leaves of the Zn/Cd hyperaccumulator Sedum alfredii. These genes also include chloroplast genes involved in transcription and translation (rps18 and rps14), electron transport and ATP synthesis (atpF and ccsA), Photosystem II (PSBI, PSBM, PSBK, PSBZ/YCF9, PSBO-1, PSBQ, LHCB1.1, LHCB1.4, LHCB2.1, LHCB4.3 and LHCB6) and Photosystem I (PSAE-1, PSAF, PSAH2, LHCA1 and LHCA4). Cadmium and Zn also up-regulated the VAR1 gene, which encodes the ATP-dependent zinc metalloprotease FTSH 5 (a member of the FtsH family), and the DAG gene, which influences chloroplast differentiation and plastid development, and the CP29 gene, which supports RNA processing in chloroplasts and has a potential role in signal-dependent co-regulation of chloroplast genes. Further morphological parameters (dry biomass, cross-sectional thickness, chloroplast size, chlorophyll content) and chlorophyll fluorescence parameters confirmed that leaf photosynthesis of S. alfredii responded to Cd much as it did to Zn, which will contribute to our understanding of the positive effects of Zn and Cd on growth of this plant.

Mitochondrial Genome Evolution in a Single Protoploid Yeast Species

Mitochondria are organelles, which play a key role in some essential functions, including respiration, metabolite biosynthesis, ion homeostasis, and apoptosis. The vast numbers of mitochondrial DNA (mtDNA) sequences of various yeast species, which have recently been published, have also helped to elucidate the structural diversity of these genomes. Although a large corpus of data are now available on the diversity of yeast species, little is known so far about the mtDNA diversity in single yeast species. To study the genetic variations occurring in the mtDNA of wild yeast isolates, we performed a genome-wide polymorphism survey on the mtDNA of 18 Lachancea kluyveri (formerly Saccharomyces kluyveri) strains. We determined the complete mt genome sequences of strains isolated from various geographical locations (in North America, Asia, and Europe) and ecological niches (Drosophila, tree exudates, soil). The mt genome of the NCYC 543 reference strain is 51,525 bp long. It contains the same core of genes as Lachancea thermotolerans, the nearest relative to L. kluyveri. To explore the mt genome variations in a single yeast species, we compared the mtDNAs of the 18 isolates. The phylogeny and population structure of L. kluyveri provide clear-cut evidence for the existence of well-defined geographically isolated lineages. Although these genomes are completely syntenic, their size and the intron content were found to vary among the isolates studied. These genomes are highly polymorphic, showing an average diversity of 28.5 SNPs/kb and 6.6 indels/kb. Analysis of the SNP and indel patterns showed the existence of a particularly high overall level of polymorphism in the intergenic regions. The dN/dS ratios obtained are consistent with purifying selection in all these genes, with the noteworthy exception of the VAR1 gene, which gave a very high ratio. These data suggest that the intergenic regions have evolved very fast in yeast mitochondrial genomes.

Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe.

In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomzyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana(r)-6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana(r)-6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity.

Relocation of the unusual VAR1 gene from the mitochondrion to the nucleus.

The Var1 protein (Var1p) is an essential, stoichiometric component of the yeast mitochondrial small ribosomal subunit, and it is the only major protein product of the mitochondrial genetic system that is not part of an energy transducing complex of the inner membrane. Interestingly, no mutations have been reported that affect the function of Var1p, presumably because loss of a functional mitochondrial translation system leads to an instability of mtDNA. To study the structure, function and synthesis of Var1p, we have engineered yeast strains for the expression of this protein from a nuclear gene, VAR1U, in which 39 nonstandard mitochondrial codons were converted to the universal code. Immunoblot analysis using an epitope-tagged form of Var1Up showed that the nuclear-encoded protein was expressed and imported into the mitochondria. VAR1U was tested for its ability to complement a mutation in mtDNA, PZ206, which disrupts '3-end processing of the VARI mRNA, causing greatly reduced synthesis of Var1p and a respiratory-deficient phenotype. Respiratory growth was restored in PZ206 mutants by transformation with a centromere plasmid carrying VAR1U under ADH1 promoter control, thus proving that VAR1 function can be relocated from the mitochondrion to the nucleus. Moreover, epitope-tagged Var1Up co-sedimented specifically with small ribosomal subunits in high salt sucrose gradients. The relocation of VAR1 from the mitochondrion to the nucleus provides an excellent system for the molecular genetic analysis of structure-function relationships in the unusual Var1 protein.

The complete mitochondrial DNA sequence of Hansenula wingei reveals new characteristics of yeast mitochondria

The complete 27,694-bp mitochondrial (mt) DNA sequence of Hansenula wingei, which is a typical budding yeast and contains circular mitochondrial DNA, has been determined. The mt sequence contains genes encoding large and small ribosomal RNAs, 25 tRNAs, three subunits of cytochrome c oxidase (subunits 1, 2 and 3), three subunits of ATPase (subunits 6, 8 and 9), apocytochrome b, seven subunits of NADH dehydrogenase (subunits 1, 2, 3, 4, 4L, 5 and 6), and a ribosomal protein, VAR1. The VAR1 gene is considered to be a typical yeast type. This is consistent with data on DNA and the deduced amino-acid sequence homology comparisons of genes ubiquitous in yeast and fungi. However, we have identified seven genes encoding NADH dehydrogenase subunits, which are not found in other yeast mitochondrial genomes, thus placing the H. wingei mitochondrial genome in a unique position. In addition the H. wingei mitochondrial genome also encodes one tRNA pseudogene and one short unidentified ORF. The genome is compact with only two introns both of which contain an ORF. One intron lies in the large rRNA gene while the other is situated in the cytochrome c oxidase subunit-1 gene. The conserved nonanucleotide motif (A/T)TATAAG (T/A)(A/T), which is a transcription start signal in Saccharomyces cerevisiae mitochondria, has also been found in the H. wingei mitochondrial genome. The codon assignments for ATA and CTN in H. wingei mitochondria are different from those in S. cerevisiae mitochondria. These results indicate a unique and novel structure for the H. wingei mitochondrial genome in terms of characteristics which are typical for both yeast and for filamentous fungi. This is the first complete mt DNA sequence report in yeast.

Yeast sequencing reports. Genes of the linear mitochondrial DNA of Williopsis mrakii: Coding sequences for a maturase‐like protein, a ribosomal protein VAR1 homologue, cytochrome oxidase subunit 2 and methionyl tRNA

The mitochondrial DNA (mtDNA) in some yeasts has a linear structure with inverted terminal repeats closed by a single-stranded loop. These mtDNAs have generally a constant gene order, beginning with a small ribosomal RNA gene at the right end and terminating with a cytochrome oxidase subunit 2 gene (COX2) at the left end, independently of the wide variation in genome size. In the mtDNAs from several species of the genus Williopsis, we found an additional open reading frame, ORF1, which was homologous to the Saccharomyces cerevisiae RF1 gene encoding a group I intron maturase-like protein. ORF1 genes from W. mrakii and W. suaveolens were mapped and sequenced. Next to ORF1, COX2 and methionyl tRNA genes were present on the opposite strand. The same relative positions of genes in the mtDNAs so far examined suggests that the constancy of gene order is generally conserved also at the level of individual tRNA genes. We identified another open reading frame, ORF2, in W. mrakii mtDNA. It was mapped next to the cytochrome oxidase subunit 3 gene. Rich in adenine-thymine bases, ORF2 appears to be a homologue of the VAR1 gene which codes for a small ribosomal subunit protein in S. cerevisiae mitochondria. Nucleotide sequences data have been deposited in the EmBL data library under the following Accession Numbers: X66594 (Apocytochrome b and ORF2 genes of W. mrakii), X66595 (ORF1, tRNA-Met and COX2 genes of W. mrakii), X73415 (tRNA-Met and COX2 genes of W. suaveolens), X73416 (ORF1 gene of W. suaveolens) and X73414 (tRNA-Met and COX2 genes of P. jadinii).

Suppression of a yeast mitochondrial RNA processing defect by nuclear mutations.

The S. cerevisiae strain h56 is a temperature-sensitive mit- mutant containing a single nucleotide substitution in the region 5' to the reading frame of the mitochondrial var1 gene. The mutation decreases the efficiency of processing of a precursor RNA such that little var1 mRNA is produced at the restrictive temperature, 36 degrees C. This communication reports the isolation and characterization of several strains carrying nuclear mutations which suppress the temperature-sensitivity of h56. Both dominant and recessive suppressor mutations were isolated. One dominant suppressor strain (h56-S4) was characterized biochemically, and the mechanism of suppression shown to involve a restoration of precursor RNA processing at the restrictive temperature, with a concomitant increase in the synthesis of the var1 protein. It appears likely that the suppressing allele encodes a component of an RNA processing endoribonuclease active on var1 transcripts. A genomic library was constructed from the h56-S4 strain, and several plasmids showing suppressed activity were isolated. A preliminary analysis of these plasmids is presented.

Analysis of the role of the NUC1 endo/exonuclease in yeast mitochondrial DNA recombination.

Mitochondrial DNA recombination was reduced in an yeast mutant lacking the NUC1 endo/exonuclease. Between linked markers in either the omega or cob region the frequency of recombination decreased nearly 50% compared to wild-type. Gene conversion frequencies in the var1 gene and in the omega region were also lower in the mutant strain. In particular, the gradient of gene conversion at omega was most affected by the absence of the NUC1 nuclease. In crosses between nuclease-deficient and wild-type strains, gene conversion frequencies at omega were reduced only when the omega+ allele was contributed to the zygote by the nuclease-deficient parent. We propose that the 5' exonuclease activity of the NUC1 nuclease functions during recombination to enlarge heteroduplex tracts following a double-strand break in DNA. In crosses between nuclease-deficient and wild-type strains, the anisotropy in gene conversion frequencies at omega is hypothesized to be due to the slow mixing of parental mitochondrial membranes as they fuse in the zygote.

Mobility of two optional G + C-rich clusters of the var1 gene of yeast mitochondrial DNA.

Yeast mtDNA contains two different kinds of mobile optional sequences, two group I introns and a short G + C-rich insertion to some var1 genes. Movement of each element in crosses has been called gene conversion though little is known about the mechanism of G + C cluster conversion. A new allele of the var1 gene found in mtDNA of Saccharomyces capensis is described that permitted a more detailed comparison between intron mobility and G + C cluster conversion. The S. capensis var1 gene lacks the cc+ element present in all S. cerevisiae var 1 genes and the previously described optional a+ element. In crosses with cc+ a- and cc+ a+ S. cerevisiae strains, both clusters were found to be mobile and, in the latter cross, appear to convert independently and only to homologous insertion sites. No evidence for flanking marker coconversion (a hallmark feature of intron conversion) was obtained despite the availability of nearby physical markers on both sides of cluster conversion sites. These data indicate that G + C cluster conversion has only a superficial resemblance to intron mobility; analogies to procaryotic transposition mechanisms are considered.

The nuclearSUV3-1mutation affects a variety of post-transcriptional processes in yeast mitochondria

The SUV3-1 mutation was isolated earlier as a suppressor of a deletion of a conserved RNA processing site (dodecamer) near the 3' end of the var1 gene. Previous studies indicate that the suppressor enhances translation of mutant var1 messages; unexpectedly, it also causes over-accumulation of excised intron RNA of the large rRNA gene intron and blocks cleavage at the dodecamer site within that intron. In this study most mitochondrial genes in SUV3-1 and suv3 nuclear contexts are surveyed for changes in levels of mRNA, for interference with dodecamer cleavage and splicing and for levels of excised intron RNAs. SUV3-1 has little or no effect on the size or abundance of unspliced RNAs tested. It results, however, in a marked increase in the abundance of seven of eight excised group I intron RNAs tested, most of which are not detectable in wild-type (suv3) strains. The suppressor lowers levels of the cob and coxl mRNAs about 2-5 and 20-fold, respectively. The effect on coxl mRNA results from a decrease in the splicing of its intron 5 beta. Despite the reduction in these mRNA levels, the amounts of coxl and cyt b polypeptides were close to wild-type levels in SUV3-1 cells. These data show that the suv3 gene plays a prominent role in post-transcriptional and translation events in yeast mitochondria.

Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

The role of a conserved dodecamer sequence in yeast mitochondrial gene expression.

All mRNAs on the yeast mitochondrial genome terminate at a conserved dodecamer sequence 5'-AAUAAUAUUCUU-3'. We have characterized two mutants with altered dodecamers. One contains a deletion of the dodecamer at the end of the var1 gene, and the other contains two adjacent transversions in the dodecamer at the end of the reading frame of fit1, a gene within the omega+ allele of the 21S rRNA gene. In each mutant, expression of the respective gene is blocked completely. A dominant nuclear suppressor, SUV3-1, was isolated that suppresses the var1 deletion but is without effect on the fit1 dodecamer mutations. Unexpectedly, however, we found that SUV3-1 blocks expression of the wild-type fit1 allele by blocking processing at its dodecamer. SUV3-1 has pleiotropic effects on mitochondrial gene expression, affecting RNA processing, RNA stability, and translation. Our results suggest that RNA metabolism and translation may be part of a multicomponent complex within mitochondria.

In vivo double-strand breaks occur at recombinogenic G + C-rich sequences in the yeast mitochondrial genome.

An optional 46-base-pair G + C-rich element (GC cluster) in the coding region of the yeast mitochondrial var1 gene inserts preferentially in crosses into recipient alleles that lack the sequence. Unlike a similar gene conversion event involving the insertion of an optional 1143-base-pair intron, the mitochondrial 21S rRNA gene, which requires the action of a protein encoded by a gene within that intron, conversion of the var1 GC cluster does not require any protein product of the mitochondrial genome. We have detected double-strand breaks in the var1 gene in mitochondrial DNA isolated from unmated haploid rho+ and rho- strains at or near the boundaries of the optional GC cluster, as well as at a conserved copy of that sequence 160 base pairs upstream. No double-strand breaks were detected in the recipient var1 DNA molecules in the vicinity of the optional GC cluster target sequence. This contrasts with 21S rRNA-encoding DNA (rDNA) intron conversion where the recipient, but not the donor DNA, is cleaved at the element insertion site. These results suggest that although the 21S rDNA intron and the var1 GC cluster are preferentially inserted into their respective short alleles, these conversions probably occur by different mechanisms.

A mitochondrial intergenic mutation affecting processing of specific yeast mitochondrial transcripts.

The mutation in the temperature-conditional mit- mutant h56, mapped previously to the var1 gene region of Saccharomyces cerevisiae mitochondrial DNA, results in a specific inhibition of var1 protein synthesis in cells incubated at the non-permissive temperature, 36 degrees C (1). We have now characterized the mutation present in mutant h56 by DNA sequencing and found it to be an A to T transversion located 109 nucleotides upstream of the var1 reading frame. Two spontaneous revertants of mutant h56 restore the parental strain sequence at residue -109, confirming that this single base change within the 5'-untranslated region of the var1 mRNA is responsible for defective synthesis of the var1 protein. A comparison of var1 transcripts in the parental and mutant strains has shown that the mutation specifically blocks formation of var1 mRNA at 36 degrees C and leads to accumulation of precursor transcripts. Expression of the oli1 gene, co-transcribed with the var1 gene in primary transcripts, is not affected. It is concluded that the mutation in mutant h56 alters the secondary structure of the precursor RNA, inhibiting an endonucleolytic cleavage required to generate the 5' end of var1 mRNA.

The AT spacers and the var1 genes from the mitochondrial genomes of Saccharomyces cerevisiae and Torulopsis glabrata: evolutionary origin and mechanism of formation

Intergenic sequences represent 63% of the mitochondrial ‘long’ (85 kb) genome of Saccharomyces cerevisiae. They comprise 170–200 AT spacers that correspond to 47% of the genome and are separated from each other by GC clusters, ORFs, ori sequences, as well as by protein-coding genes. Intergenic AT spacers have an average size of 190 bp, and a GC level of 5%; they are formed by short (20–30 nt on the average) A/T stretches separated by C/G mono- to trinucleotides. An analysis of the primary structures of all intergenic AT spacers already sequenced (32 kb; 80% of the total) has shown that they are characterized by an extremely high level of short sequence repetitiveness and by a characteristic sequence pattern; the frequencies of A/T isostichs conspicuously deviate from statistical expectations, and exponentially decrease when their (AT + TA)/(AA + TT) ratio, R, decreases. A situation basically identical was found in the AT spacers of the mitochondrial genome (19 kb) of Torulopsis glabrata. The sequence features of the AT spacers indicate that they were built in evolution by an expansion process mainly involving rounds of duplication, inversion and translocation events which affected an initial oligodeoxy-nucleotide (endowed with a particular R ratio) and the sequences derived from it. In turn, the initial oligodeoxy-nucleotide appears to have arisen from an ancestral promoter-replicator sequence which was at the origin of the nonanucleotide promoters present in the mitochondrial genomes of several yeasts. Common sequence patterns indicate that the AT spacers so formed gave rise to the var1 gene (by linking and phasing of short ORFs), to the DNA stretches corresponding to the untranslated mRNA sequences and to the central stretches of ori sequences from S. cerevisiae.

Var1 gene on the mitochondrial genome of Torulopsis glabrata

We have cloned and sequenced a region of the Torulopsis glabrata mitochondrial genome homologous to the Saccharomyces cerevisiae var1 gene ( var1 Sc). An open reading frame that could encode a protein of 339 amino acids was found with 72·7% amino acid and 85·3% nucleotide sequence homology to the S. cerevisiae var1 gene. The T. glabrata gene ( var1 Tg) is transcribed yielding two stable RNAs, a more abundant 13·5 S RNA and a less abundant 18 S species. We have also identified a candidate for a T. glabrata var1 protein among mitochondrial translation products labeled in isolated mitochondria. The var1 Tg gene is even more A + T-rich (93%) than var1 Sc (89·6%) and has conserved the strong codon bias of var1 Si. Major differences between the two sequences were found. Significant among these are that no GC clusters are found in var1 Tg and the sequences surrounding each of the sites where known polymorphisms exist in var1 Sc have deletions at the corresponding sites in var1 Tg. These data are discussed with respect to possible origins of these var1 genes and translocation of GC clusters in S. cerevisiae mitochondrial DNA.

The unusual varl gene of yeast mitochondrial DNA.

The var1 gene specifies the only mitochondrial ribosomal protein known to be encoded by yeast mitochondrial DNA. The gene is unusual in that its base composition is nearly 90 percent adenine plus thymine. It and its expression product show a strain-dependent variation in size of up to 7 percent; this variation does not detectably interfere with function. Furthermore, var1 is an expandable gene that participates in a novel recombinational event resembling gene conversion whereby shorter alleles are preferentially converted to longer ones. The remarkable features of var1 indicate that it may have evolved by a mechanism analogous to exon shuffling, although no introns are actually present.

A new putative gene in the mitochondrial genome of Saccharomyces cerevisiae

The 2200-bp ori2- ori7 region of the mitochondrial (mt) genome of Saccharomyces cerevisiae has been sequenced on the genome of a petite, b7, excised at those ori sequences from wild-type strain B. The region contains an open reading frame, ORF5, which is transcribed into a 900-nucleotide (nt) RNA in both the parental wild-type strain and its derived petite, b7. This RNA uses as a template the strand used by most mt transcripts. Its start point is located 337 nt upstream of ORF5; and a messenger termination site has been found 900 nt downstream of the initiation site. These data suggest that ORF5 is a new mitochondrial gene. The G + C content of ORF5 is only 15.7%; 90% of the G + C base pairs of ORF5 are comprised in a palindromic G + C cluster similar to that present in the var1 gene. The coding capacity of ORF5 is 46 amino acids (aa), mainly represented by methionine, phenylalanine, arginine, valine, asparagine, isoleucine and tyrosine. The aa composition and the codon usage of ORF5 are reminiscent of those of var1 and of other intergenic ORFs.