Functional nuclear suppressor of mitochondrialoxi2 mutations in yeast

Summary A semidominant nuclear suppressor, callednam6, ofoxi2-V276 mitochondrial mutation has been isolated and characterized. The nuclear character ofnam6 was proved by its retention inrho° strains, lack of mitotic segregation in diploids and meiotic 2:2 segregation in tetrads. The specificity ofnam6 was tested on 315mit ^– mutations of four mitochondrial genes (oxi1, oxi2, oxi3, andcob-box). It suppresses clearly only three mutations in theoxi2 gene, restoring partially or completely cytochrome aa₃ formation. The results suggest a functional character of the suppression.     

Contrasting mutation rates in mitochondrial and nuclear genes of yeasts versus mammals

Summary Base substitutions have been compared in two mitochondrial and two nuclear genes from three yeasts and three mammals. In yeasts, the two mitochondrial genes, cytochrome oxidase subunit 2 (COX2) and apocytochrome b (CYB), have fewer changes on a percentage basis than the nuclear-encoded cytochrome c (CYC) gene. By contrast, in mammals, the same mitochondrial genes have more mutations than CYC on a percentage basis. Sequence comparisons of the nuclear small-subunit ribosomal RNA (nSSU) gene shows that there are more substitutions per unit length in the three yeasts than in the three mammals. This result suggests that although the yeasts are more distantly related than the mammals, their mitochondrial genes have accumulated fewer changes.     

Either one of the two yeast EF-1α genes is required for cell viability

Summary Two genes,TEF1 andTEF2, encode the protein elongation factor EF-1 in the yeastSaccharomyces cerevisiae. We have generated yeast haploid strains containing eitherTEF1 orTEF2 interrupted by insertion of a large piece of foreign DNA. Cells which contain either one functional copy of the EF-1 genes are viable. In contrast, attempts to isolate a yeast haploid strain with bothTEF1 andTEF2 inactivated have failed suggesting that the double gene disruption is a lethal event.     

Molecular cloning and analysis of the nuclear gene MRP-L6 coding for a putative mitochondrial ribosomal protein from Saccharomyces cerevisiae

The Saccharomyces cerevisiae nuclear gene MRP-L6 was cloned by complementation of the respiratory-deficient mutant pet-ts 2523 with a library of wildtype yeast genomic DNA. The isolated gene was part of a 3.8-kb sequenced DNA fragment containing, in addition to MRP-L6, two unassigned reading frames, ORF1 and ORF2. MRP-L6 codes for a basic protein of 205 amino acids and a molecular mass of 22.8 kDa. The protein exhibits significant sequence similarity to the ribosomal protein L6 of bacteria and chloroplasts. Unlike the corresponding bacterial proteins, however, the MRP-L6 protein (MRP-L6p) contains at its N-terminus a 16 amino-acid leader sequence exhibiting the known characteristics of mitochondrial import signals. Disruption of MRP-L6 leads to the phenotype of a mitochondrial translation-defective, rho-negative yeast mutant. The results are consistent with MRP-L6p representing an essential component of yeast mitochondrial ribosomes. Expression of MRP-L6 was examined, under conditions of glucose repression and derepression, in wild-type cells and in a series of catabolite repression-defective yeast mutants. In most cases, a distinct though small influence of the carbon source on the expression of an MRP-L6/lacZ reporter construct was observed.     

Construction of new yeast vectors and cloning of the nif (nitrogen fixation) gene cluster of Klebsiella pneumoniae in yeast

Summary Two vectors, termed pG63.11 (7.6 Kb) and pHCG3 (9.6 Kb), suitable for yeast transformation have been constructed. The pHCG3 vector has cosmid properties. Both vectors contain a single 3.3 Kb EcoRI-HindIII fragment of yeast origin which carries the yeast URA3 gene (1.1 Kb) and the origin of replication of the 2 µm plasmid (2.2 Kb). They confer ampicillin resistance and they contain 5 unique EcoRI,HpaI,HindIII,BamHI and SalI restriction sites. Cosmid pHCG3 was used to clone the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae carried by twoHindIII fragments of 17 and 26 Kb, respectively. The resulting cosmid, termed pGPC875 (53 Kb) which conferred a Nif⁺ phenotype to Escherichia coli, was introduced in yeast by transformation. No acetylene reduction activity was detectable in the transformants. However it was shown that the entire information for nitrogen fixation can be replicated and maintained intact in yeast for more than 50 generations of growth.     

Phenotypic suppression and nuclear accommodation of the mit − oxi1-V25 mutation in isolated yeast mitochondria

Summary Phenotypic suppression by the antibiotic, paromomycin, of the mitochondrial oxi1 ^–-V25 mutation, a mutation which arrests by premature ochre codon the synthesis of the cox 11 subunit, was studied in isolated yeast mitochondria competent in translation. This antibiotic is known to suppress the mutation in vivo (Dujardin et al. 1984) and allowed in vitro, at concentrations of 20–1100 Mg per ml. the synthesis of the cox II subunit. This strongly suggests that phenotypic suppression of mit ^– mutations is due to the direct action of paromomycin on mitochondrial ribosomes. The effect of paromomycin bears a resemblance to the function of the omnipotent nuclear suppressor mutation R705. The nuclear suppression was expressed in isolated mitochondria; suppressor mutation influenced the structure of the mitoribosome. Therefore, it appears that mitoribosomes are indeed the common target in the phenotypical and genetic nuclear suppression of the oxi1-V25 mutation.     

Molecular cloning of Rab-related genes in the yeast Yarrowia lipolytica. Analysis of RYL1, an essential gene encoding a SEC4 homologue

Small GTP-binding proteins of the Rab family are involved in the vesicular traffic inside eukaryotic cells. A gene library from the yeast Yarrowia lipolytica was screened with an oligonucleotide deduced from a highly conserved sequence in the Rab family. Four different genes were isolated. One of them, RYL1, was shown to be essential for cell viability. RYL1p displayed a high similarity with and tight phylogenetic relationships to SEC4p. When placed under the control of the GAL10 promoter, RYL1 was able to specifically relieve the thermosensitivity of a sec4–8 mutant of Saccharomyces cerevisiae. Therefore, it is proposed that RYL1 is a functional homologue of the S. cerevisiae SEC4 gene and is involved in the fusion of secretory vesicles with the plasma membrane in the general protein secretion pathway.     

Cloning and characterisation of the ribosomal RNA genes of the dimorphic yeast,Yarrowia lipolytica

Summary The ribosomal RNA genes of Yarrowia lipolytica have been identified, both in restriction digests of total genomic DNA and in a pBR322 gene bank, by hybridisation with cloned Saccharomyces cerevisiae rDNA. The Y. lipolytica rDNA repeat unit is 8.9 kb in size and contains the genes for the 25S and 18S, but not the 5S, rRNA species. The number of copies of these repeat units is approx. 50 per haploid genome. Several clones were found which did not conform to the standard restriction map due to differences outside the coding region. It appears that there is either heterogeneity of the spacer sequence within a strain or that the Y. lipolytica rDNA genes may be present as a number of separate clusters within this yeast's genome.     

Transfer RNA genes and the genetic code in Chlamydomonas reinhardtii mitochondria

Summary Only three tRNA genes are present within a sequenced 12.35 kbp region of the 15.8 kbp mtDNA of Chlamydomonas reinhardtii, a unicellular green alga. The corresponding tRNAs, whose anticodons are specific for TGG (Trp), CAA/G (Gln) and ATG (Met) codons, all display conventional secondary structures. The tRNA^Met gene encodes an elongator rather than initiator species. The standard genetic code is used in C. reinhardtii mitochondria, but codon distribution is highly biased: in a collection of six identified protein coding genes, nine codons (including TGA) are not used at all, while four other sense codons occur very infrequently. In spite of the absence of certain codons, a minimum of 23 tRNAs (assuming separate initiator and elongator tRNAs^Met are used) is needed to translate the C. reinhardtii mitochondrial genetic code. It appears unlikely that this minimal tRNA set is encoded by C. reinhardtii mtDNA.     

Distribution of mitochondrial r1-type introns and the associated open reading frame in the yeast genus Kluyveromyces

Summary We have sequenced the intron in the large subunit ribosomal RNA gene from the mitochondrion of Kluyveromyces lactis. It is a typical group I intron but, unlike the corresponding intron (r1) in Saccharomyces cerevisiae, it does not contain an open reading frame. This intron is widespread in the genus Kluyveromyces although intron-less strains were also found in some species of this genus. Sequences homologous to the open reading frame of the S. cerevisiae ribosomal intron were detected in some strains of K. waltii, K thermotolerans and K. africanus.