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.

The yeast nuclear gene DSS1, which codes for a putative RNase II, is necessary for the function of the mitochondrial degradosome in processing and turnover of RNA.

The yeast nuclear gene DSS1 codes for a mitochondrial protein containing regions of homology to bacterial RNase II and can act as a multicopy suppressor of a deletion of the SUV3 gene, which encodes an RNA helicase. In order to establish the function of the DSS1 gene in mitochondrial biogenesis we studied RNA metabolism in yeast strains disrupted for SUV3 or DSS1. The results indicate that in the absence of DSS1 the in vitro activity of 3'-5' exoribonuclease is abolished and mitochondrial translation is blocked. In disruption strains harboring intronless mitochondrial genomes steady-state levels of COB mRNA and 16S rRNA were very low, while in the presence of a mitochondrial genome containing the omega intron in the 21S rRNA gene the excised intron accumulates. Moreover we observed an accumulation of precursors of 21S rRNA and the VAR1 mRNA. All these phenotypes are virtually identical to those of strains in which SUV3 is disrupted. We suggest that the DSS1 gene product, like the SUV3 gene product, is a subunit of the yeast mitochondrial degradosome (mtEXO), and that this protein complex participates in intron-independent turnover and processing of mitochondrial transcripts. In addition our studies exclude any role for the NUC1 nuclease in these phenomena.

Yeast nuclear PET127 gene can suppress deletions of the SUV3 or DSS1 genes: an indication of a functional interaction between 3' and 5' ends of mitochondrial mRNAs.

Saccharomyces cerevisiae nuclear genes SUV3 and DSS1 encode putative RNA helicase and RNase II, respectively, which are subunits of the mitochondrial degradosome (mtEXO): a three-protein complex which has a 3' to 5' exoribonuclease activity and plays a major role in regulating stability of mitochondrial RNA. Lack of either of the two gene products results in a respiratory negative phenotype, while on the molecular level it causes a total block of mitochondrial translation, loss of the in vitro exoribonuclease activity and changes in stability and processing of many mtRNAs. We have found that the yeast nuclear gene PET127 present on a low or high copy number vector can effectively suppress the effects of the SUV3 or DSS1 gene disruptions. Since the product of the PET127 gene is involved in processing of the 5' ends of mitochondrial mRNAs, we suggest that there is a functional coupling between the 5' and 3' ends of mitochondrial mRNAs.

The NAM9-1 suppressor mutation in a nuclear gene encoding ribosomal mitochondrial protein of Saccharomyces cerevisiae.

The nuclear gene NAM9 from Saccharomyces cerevisiae (Sc) codes for a protein which, on the basis of sequence homology, was previously postulated to be a mitochondrial (mt) equivalent of the Escherichia coli (Ec) S4 ribosomal protein (r-protein) [Boguta et al., Mol. Cell. Biol. 12 (1992) 402-412]. The mt-r character of the NAM9 product is now confirmed by cross-reaction with the antisera for the Sc mt r-proteins. The NAM9-1 mutation, characterized previously as the nuclear suppressor of some ochre mt mit- mutants, is found to be a single nucleotide substitution changing Ser82 to Leu within the part of NAM9 corresponding to the S4 region involved in interaction with the 16S rRNA. This indicates that the mechanism of NAM9-1 suppression could be analogous to the suppression due to ram (ribosomal ambiguity) mutations in the Ec structural gene encoding r-protein S4. The NAM9-1 mutation leads also to defect in respiratory growth in the background of the wild-type mit+ genome.

The novel nuclear gene DSS-1 of Saccharomyces cerevisiae is necessary for mitochondrial biogenesis.

A previously unknown nuclear gene DSS-1 from Saccharomyces cerevisiae was cloned and sequenced. The gene was isolated as a multicopy suppressor of a disruption of the SUV-3 gene coding for a DEAD/H box protein involved in processing and turnover of mitochondrial transcripts. The DSS-1 gene codes for a 970 amino-acid protein of molecular weight 111 kDa and is necessary for mitochondrial biogenesis. Amino-acid sequence analysis indicates the presence of motifs characteristic for Escherichia coli RNase II, the dis3 protein from Schizosaccharomyces pombe, the cyt4 protein participating in RNA processing and turnover in Neurospora crassa mitochondria, and the vacB protein from Shigella flexneri. We suggest that the DSS-1 protein may be a component of the mitochondrial 3'-5' exoribonuclease complex.

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.

Structure and transcriptional control of the Saccharomyces cerevisiae POX1 gene encoding acyl-coenzyme A oxidase.

We have cloned the Saccharomyces cerevisiae gene coding for the peroxisomal enzyme: fatty acyl-CoA oxidase (POX). The gene (named POX1) is unique in S. cerevisiae and has been identified through homology with the POX4 and POX5 genes of Candida tropicalis. The POX1 gene encodes a 84-kDa POX protein composed of 748 amino acids. The identity between the S. cerevisiae and C. tropicalis enzymes is about 40%, and there is a greater degree of similarity between the N termini than the C termini. A disruption of the POX1 coding sequence diminishes the ability of yeast cells to grow on oleic acid as a sole carbon source. The expression of the POX1 gene is regulated at the level of transcription, and is induced more than 25-fold by the addition of oleic acid to the medium.

Yeast KEX1 gene encodes a putative protease with a carboxypeptidase B-like function involved in killer toxin and alpha-factor precursor processing.

The yeast KEX1 gene product has homology to yeast carboxypeptidase Y. A mutant replacing serine at the putative active site of the KEX1 protein abolished activity in vivo. A probable site of processing by the KEX1 product is the C-terminus of the alpha-subunit of killer toxin, where toxin is followed in the precursor by 2 basic residues. Processing involves endoproteolysis following these basic residues and trimming of their C-terminal by a carboxypeptidase. Consistent with the KEX1 product being this carboxypeptidase is its role in alpha-factor pheromone production. In wild-type yeast, KEX1 is not essential for alpha-factor production, as the final pheromone repeat needs no C-terminal processing. However, in a mutant in which alpha-factor production requires a carboxypeptidase, pheromone production is KEX1-dependent.

Transformation of Aspergillus nidulans by the argB gene.

Aspergillus nidulans argB mutant was transformed with the plasmid DNA containing the argB gene. Analysis of transformants revealed that transformation was due to integration of either argB gene alone or the whole plasmid DNA into the A. nidulans genome. In 5 out of 23 transformants studied, integration took place in the locus different than the original argB locus. The amplification of integrated sequences was often observed. Integrated DNA was found to be mitotically stable, while the meiotic stability depends on the mode of integration. The activity of the ornithine carbamoyltransferase (the argB gene product) was measured and in some transformants bearing the amplified argB sequence was found to be strongly elevated.

Expression of the Aspergillus nidulans argB gene in Escherichia coli.

The Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OTCase) is not expressed in Escherichia coli. However, E. coli OTCase-deficient strains transformed with plasmids carrying the argB gene from A. nidulans reverted to prototrophy at a high frequency. In these derivatives the argB gene became functional due to DNA rearrangements upstream of the coding sequence. Two types of rearrangement were characterized. One was identified as an insertion of IS2. The second was a deletion that resulted in transcription of the argB gene from the TcR gene promoter and translation from a newly created ribosome-binding site formed at the junction between the A. nidulans and vector DNA sequences.

Intracellular localization of Aspergillus nidulans ornithine carbamoyltransferase in native host cells and in Saccharomyces cerevisiae cells harbouring its cloned structural gene.

Differential centrifugation of the Aspergillus nidulans cell lysate shows that ornithine carbamoyltransferase (EC 2.1.3.3) appears mainly in the particulate (organellar) fraction. The enzyme was located to the mitochondria by co-sedimentation with cytochrome oxidase in isopycnic density gradient and by cytochemical-electron microscopic means. Arginase (EC 3.5.3.1) and ornithine delta-aminotransferase (E.C. 2.6.1.13) were found to reside in cytosol. The release of ornithine carbamoyltransferase from the organellar fraction by various agents indicates that the enzyme resides in the mitochondrial matrix. In Saccharomyces cerevisiae the plasmid pSAL43, carrying cloned Aspergillus nidulans ornithine carbamoyltransferase gene, directs the synthesis of the enzyme partially associated with yeast mitochondria even though the homologous yeast enzyme is exclusively cytosolic. The implications of these findings are discussed.

Cloning and characterization of the ornithine carbamoyltransferase gene from Aspergillus nidulans.

An Aspergillus nidulans DNA fragment composed of two adjacent SalI subfragments (1.8 and 0.85 kb) that carries an argB gene complementing the yeast arg3 mutation has been isolated from two different gene libraries. Hybridization results and immunological tests indicate that the cloned fragment contains the A. nidulans structural gene coding for ornithine carbamoyltransferase (OTCase). Using the cloned gene as a probe, the specific mRNA was identified. The level of this RNA observed in A. nidulans strains grown under various conditions correlated with the level of the OTCase activity, suggesting transcriptional control of OTCase synthesis. Expression of the cloned gene in Saccharomyces cerevisiae does not depend on its orientation in the vector. In Escherichia coli, the cloned gene does not function; however arg- transformants revert to prototrophy with high frequency possibly due to DNA rearrangements within the recombinant plasmid.

Cloning of Aspergillus nidulans DNA. I. Selection and analysis of recombinant plasmids capable of complementing pyrF, argIF and proAB mutations in Escherichia coli.

Fragments of Aspergillus nidulans DNA obtained after digestion with EcoRI, BamHI and HindIII endonucleases were cloned in Escherichia coli in plasmid pBR322. These gene banks were used for transformation of 15 E. coli auxotrophic mutants and in 5 cases prototrophic clones containing recombinant plasmids were selected. Three different recombinant plasmids conferring prototrophy to pyrF, proAB and argIF mutants were analyzed. Hybridization experiments indicated that in two of these plasmids the inserted fragment of DNA hybridized not only to the A. nidulans but also to the E. coli DNA.