Frédéric Sor

Linear mitochondrial DNAs of yeasts: frequency of occurrence and general features.

In most yeast species, the mitochondrial DNA (mtDNA) has been reported to be a circular molecule. However, two cases of linear mtDNA with specific termini have previously been described. We examined the frequency of occurrence of linear forms of mtDNA among yeasts by pulsed-field gel electrophoresis. Among the 58 species from the genera Pichia and Williopsis that we examined, linear mtDNA was found with unexpectedly high frequency. Thirteen species contained a linear mtDNA, as confirmed by restriction mapping, and labeling, and electron microscopy. The mtDNAs from Pichia pijperi, Williopsis mrakii, and P. jadinii were studied in detail. In each case, the left and right terminal fragments shared homologous sequences. Between the terminal repeats, the order of mitochondrial genes was the same in all of the linear mtDNAs examined, despite a large variation of the genome size. This constancy of gene order is in contrast with the great variation of gene arrangement in circular mitochondrial genomes of yeasts. The coding sequences determined on several genes were highly homologous to those of the circular mtDNAs, suggesting that these two forms of mtDNA are not of distant origins.

Structure of a linear plasmid of the yeast Kluyveromyces lactis; Compact organization of the killer genome

SummarySome strains of the yeast Kluyveromyces lactis contain a pair of linear DNA plasmids, k1 and k2, 8.8 and 13.8 kilobase pairs long, respectively. Simultaneous presence of the two plasmids confer a killer phenotype on the cell by producing a toxin which blocks the growth of sensitive yeast species. Previous genetic studies have suggested that the toxin protein is coded by the k1 plasmid. We have now determined the total nucleotide sequence of k1 DNA. The genome is 8,874 base pairs in length. It contains four protein-coding reading frames, three transcribed from one strand and the fourth transcribed from the complementary strand and has terminal inverted repeats of 202 base pairs. Nuclease S1 mapping confirmed this arrangement and showed that these genes are transcribed. The terminal repeats and the four genes form an extremely compact genome, with some overlapping of genes. All four genes use highly biased codons, 86% of them having A or T at the wobble position, reminiscent of yeast mitochondrial genes. Three genes share a very similar 5′ leader sequence. The nature of gene products is discussed in the light of what is known of the excreted toxin protein.

Linear mitochondrial DNAs of yeasts: closed-loop structure of the termini and possible linear-circular conversion mechanisms.

The terminal structure of the linear mitochondrial DNA (mtDNA) from three yeast species has been examined. By enzymatic digestion, alkali denaturation, and sequencing of cloned termini, it was shown that in Pichia pijperi and P. jadinii, both termini of the linear mtDNA were made of a single-stranded loop covalently joining the two strands, as in the case of vaccinia virus DNA. The left and right loop sequences were in either of two orientations, suggesting the existence of a flip-flop inversion mechanism. Contiguous to the terminal loops, inverted terminal repeats were present. The mtDNA from Williopsis mrakii seems to have an analogous structure, although terminal loops could not be directly demonstrated. Electron microscopy revealed the presence, among linear molecules, of a small number of circular DNAs, mostly of monomer length. Linear and circular models of replication are considered, and possible conversion mechanisms between linear and circular forms are discussed. A flip-flop inversion mechanism between the inverted repeat sequences within a circular intermediate may be involved in the generation of the linear form of mtDNA.

Diphthamide synthesis in Saccharomyces cerevisiae : structure of the DPH2 gene

A gene involved in diphthamide biosynthesis, DPH2, was cloned from Saccharomyces cerevisiae by complementation of a diphthamide mutant. DPH2 exists as a single-copy gene in the yeast genome and is located on the left arm of chromosome XI. Sequence analysis of the DPH2 locus predicts that the DPH2 gene product is a 534-amino acid (aa) protein, with a calculated M(r) of 59,772. This conclusion was supported by Northern blot analysis of the DPH2 transcript and gel analysis of the DPH2 protein overproduced in Escherichia coli. Gene disruption studies indicate that the DPH2 gene is not essential for viability of yeast. The role of DPH2 in diphthamide biosynthesis is discussed.

Analysis of mitochondrial ribosomal proteins of Saccharomyces cerevisiae by two dimensional polyacrylamide gel electrophoresis.

SummaryProteins from mitochondrial ribosomes of Saccharomyces cerevisiae were analysed by a two dimensional gel electrophoresis method. Each ribosomal subunit revealed a reproducible characteristic pattern of protein components. The 37S small subunit contained 33 protein species with an average molecular weight of 27,300 daltons (ranging from 60,000 to 9500 daltons). The 50S large subunit showed 38 protein species with an average molecular weight of 23,000 (ranging from 41,000 to 10,000 daltons). Ribosomes from various sources were compared on the basis of protein composition.

Mapping of murine Interferon-α genes to chromosome 4

Abstract A cDNA library was constructed from polysomal poly(A) + RNA from Newcastle disease virus (NDV)-induced mouse C243 cells, and screened with a human interferon-a (HuIFN-α) cDNA probe. A cDNA clone for one of the murine interferon-a (MuIFN-α) genes was isolated, and sequencing analysis revealed that it was a partial copy which is almost identical to the published sequence for the MuIFN-α2 gene. This partial cDNA clone represents a virus-induced message as seen by Northern blot analysis of RNA from NDV-induced C243 cells, and Southern blot analysis of DNA from BALB/c mouse revealed the presence of a multiple IFN-α gene family. The MuIFN-α genes were mapped to chromosome 4 by Southern blot analysis of hamster/mouse somatic cell hybrid DNAs.

Unequal excision of complementary strands is involved in the generation of palindromic repetitions of rho− mitochondrial DNA in yeast

In the rho- mutants of yeast, the mitochondrial genome is made up of a small segment excised from the wild-type mitochondrial DNA. The segment is repeated either in tandem or in palindrome to form a series of multimeric DNAs. We have asked how the palindromic organization arises. From several palindromic rho- mitochondrial DNAs, we have isolated the restriction fragments that contained the head-to-head or tail-to-tail junction of the repeating units, and have determined their nucleotide sequences. We found that the palindromes were not symmetrical right up to the junction points: at the junction, there was always an asymmetrical sequence of variable length. At both ends of this junction sequence, we found inverted oligonucleotide sequences that were variable in each mutant and that were present in the wild-type DNA. At the moment of excision, a single-strand cut seems to occur at each of these short inverted repeats, in such a way that the two complementary strands of the genome are cut unequally and the single-stranded overhangs become the junction sequences between the palindromic repeating units. This scheme may account for the complex structures of many rho- mitochondrial DNAs.

A fine restriction map of the linear mitochondrial DNA of Tetrahyemena pyriformis: genome size, map locations of rRNA and tRNA genes, terminal inversion repeat, and restriction site polymorphism

SummaryA fine restriction map of the linear mitochondrial DNA of Tetrahymena pyrifonnis strain ST is presented. 1. Based on agarose gel electrophoresis data together with limited nucleotide sequences available on some restriction fragments, we estimate the actual size of this genome to be about 55,000 base pairs. 2. Seven tRNA gene locations have been assigned, which are scattered along the genome length. Six of these locations encode the genes for tRNAPphe, tRNAhis, tRNAtrp, and tRNAglu, and the duplicate tRNAtyr genes which are located at the inverted terminal repeat segments. The tRNA gene(s) encoded in one location has not been identified. We have not yet found the tRNAleu and tRNAmet genes, which were previously shown to be encoded in the genome (Chiu et al. 1974; Suyama 1982). 3. We have mapped the 14S rRNA gene by sequencing the 170 bp segment of EcoRl fragment 8 and by aligning its sequence with E. coli 16S rRNA. From our recent complete sequence data the gene size was found to be about 1,650 bp, which is unexpectedly large for the 14S rRNA which has an estimated size of 1,300 bp. The 14S rRNA is probably a cleavage product of the larger primary transcript of which 200–300 bases of the 5′ end are missing. 4. The duplicate copies of the 21S rRNA gene at the terminal duplication inversion segments were analyzed. ClaI fragment 7 (1,500 bp) corresponds in sequence from base position 850 to 2,390 of the 20S rRNA gene of Paramecium mitochondrial DNA (Seilhamer et al. 1984b). The 21S gene is approximately 2,500 by long. The presence of some restriction site polymorphism is apparent in this segment. 5. Each of the 21S gene copies precedes the tRNAtyr gene, but the space flanking one tRNAtyr gene differs in size and restriction sites from the space flanking another tRNAtyr gene. Thus, this space corresponds to the segment of an imperfect match in the terminal duplication inversion of Goldbach et al. (1978a). 6. Saccharomyces cerevisiae mitochondrial probes including Cob, ATPase VI and IX, and cytochrome oxidase I gene sequences, 21S and 15S rRNAs, and mouse mitochondrial DNA showed no significant hybridization with any restriction fragments of Tetrahymena mitochondrial DNA. The results are in accordance with an extensive sequence divergence previously found in the Tetrahymena mitochondrial genome (Goldbach et al. 1977).

Mitochondrial ribosomal RNA genes of yeast: Their mutations and a common nuclear suppressor

SummaryDue to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far.In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca2+ in place of Mg2+. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg2+.The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU101. The SU101 mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene.These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.

Mitochondrial and nuclear mutations that affect the biogenesis of the mitochondrial ribosomes of yeast

Summary1.Several nuclear mutants have been isolated which showed thermo-or cryo-sensitive growth on non-fermentable media. Although the original strain carried mitochondrial drug resistance mutations (CR, ER, OR and PR), the resistance to one or several drugs was suppressed in these mutants. Two of them showed a much reduced amount of the mitochondrial small ribosomal subunit (37S) and of the corresponding 16S ribosomal RNA. Two dimensional electrophoretic analysis did not reveal any change in the position of any of the mitochondrial ribosomal proteins. However one of the mutants showed a striking decrease in the amounts of three ribosomal proteins S3, S4 and S15.2.Four temperature-sensitive mitochondrial mutations have been localized in the region of the gene coding for the large mitochondrial ribosomal RNA (23S). These mutants all showed a marked anomaly in the mitochondrial large ribosomal subunit (50S) and/or the corresponding 23S ribosomal RNA.