Some yeast mitochondrial RNAs are circular

We have compared mitochondrial transcripts from yeast Saccharomyces cerevisiae strain D273-10B grown in the presence of 2 % galactose (non-repressed cells) or IS "/, glucose (glucose-repressed cells).The ethidium-bromide-stained electrophoretic pattern of mitochondrial RNAs from glucose-repressed cells shows a clear decrease of tRNAs. In addition, some RNA bands appear to be specific for a single growth condition. To identify these RNA species we have performed hybridization experiments with 32P-labelled mitochondrial DNA from petite mutant cells. The mitochondrial repeat units of the mutants retained only one of the following genes: oxil, oxi2, oxi3, oli2, cob and o l d . Unchanged amounts of oxi2 and 01i2 transcripts and reduced concentrations of olil and oxil putative mRNAs are present in glucose-repressed cells. In the same growth condition we observe a decreased processing of a precursor RNA species from the split cob gene and reduced amounts of transcripts corresponding to the first, second and fifth intron of the split oxi3 gene. The oxi3 first and second introns, whose transcripts are the most variable, include long open reading frames in their nucleotide sequence, but at present it is not known whether the corresponding RNA species have a functional role. Our results show that their concentrations are related to the growth condition.The growth of yeast Saccharomyces cerevisiae in a medium containing a high concentration of glucose results in a very strong decrease of respiration [l]. Many enzymes involved in the respiratory catabolism of glucose are actually inactivated and their synthesis is absent or reduced to undetectable levels [2,3]. Morphological changes of yeast mitochondria related to the glucose-repressed growth condition have also been described [4]. The mitochondrial protein synthesis [ 5 ] and the level of mitochondrial RNA polymerase [6] are lowered; however, all the mitochondrially coded proteins are synthesized [7]. In addition, the acylatable form of mt-tRNAs"' is not detectable in resting repressed cells [8]. The release from glucose repression results in the increase of mtRNA polymerase concentration [6] and of mitochondrial protein synthesis [ S ] ; the respiratory catabolism of the carbon source is also restored [2].Glucose repression (and the release from it) in S. cerevisiue involves a coordinated expression of nuclear and mitochondrial genomes IS].In order to understand better the molecular events involved in this phenomenon, we compared the mitochondrial transcripts obtained from glucose-repressed cells with those observed in non-repressed conditions [lo]. The physiological meaning of the observed differences is also discussed. MATERIALS A N D METHODS Strains and Culrure ConditionsThe haploid strain D273-10B was grown aerobically at 28 "C in a medium containing 1 "/, yeast extract, 1 peptone and 2 % galactose or 15% glucose. Mid-exponential phase cells were harvested and used to prepare mtRNAs (see below).The rho-strains, kindly provided

Section: Discussionmentioning

confidence: 99%

Mitochondrial Transcripts in Glucose‐Repressed Cells of Saccharomyces cerevisiae

Baldacci

Zennaro

1982

“…2b), we used two probes retaining different parts of the gene: DS6/A407 mtDNA (containing the A4-AS-A6 exons and a14-a15 introns) was used as an exonic probe and DS6/A400 mtDNA (containing a11 intron and part of a12 intron), was used as an intronic probe; the mini-exon A2 (36 bases) present in the second probe is too small to allow hybridization to oxi3 mRNA. Using the first probe, the hybridization pattern of mtRNA from repressed cells showed only high-molecular-mass precursors and a strong 1 .O x 103-base band corresponding to the a15 intron, which is a stable circular RNA species [22,23].…”

Section: Release From Glucose Repression In Resting Cellsmentioning

confidence: 99%

Mitochondrial transcription and processing of transcripts during release from glucose repression in ‘resting cells’ of Saccharomyces cerevisiae

Zennaro¹,

Grimaldi²,

Baldacci³

et al. 1985

Mitochondria1 transcription and processing of transcripts have been investigated at different stages of release from glucose repression in resting cells of Saccharomyces cerevisiae.Transcripts were identified by hybridization with nick-translated or terminally labelled gene-specific probes. This allowed the determination of the steady-state levels of individual transcripts in the mitochondrial RNA population.Results showed different gene-specific patterns of response to respiratory induction: no increase in the level of transcripts (oxi2); a rapid increase in the steady-state levels of all transcripts (cob); a very strong increase in the processing of the high-molecular-mass precursors (oxi3 and oli2); an increase in the level of stable circular transcripts (oxi3). As a whole the results indicate specific and differentiated effects of release from glucose repression on the expression of the different mitochondrial genes and demonstrate the importance of processing events in mitochondrial regulation.In Saccharomyces cerevisiae, release from glucose repression and the consequent increase in respiratory activity [I -31 involve important changes in mitochondrial morphology [4] and strong increases in the level of mitochondrial RNA polymerase [5] and in the rates of synthesis of mitochondrial respiratory enzymes [6]. In an attempt to elucidate the mitochondrial events accompanying glucose repression, we had previously compared the mitochondrial transcripts in growing repressed and derepressed cells; differences specific for the transcripts of various mitochondrial genes were noted [7].Recovery of respiratory capacity after release from glucose repression can be achieved in resting cells, thus allowing an examination of the time course of respiratory induction under conditions which are not affected by variations in growth rate [8]. We have, therefore, analyzed the steady levels of the transcripts of various mitochondrial genes at different stages after release from glucose repression in resting cells. The results allowed the identification of some steps that are specifically affected by glucose repression. MATERIALS AND METHODS Strains and growth conditionsThe wild-type haploid strain D273-1 OB was grown aerobically at 28 "C in YP medium (1 % yeast extract, 1 YO peptone) containing 15% glucose or 2% galactose as the carbon source. Respiratory induction in resting cells was obtained under the following conditions: cells grown on 15% glucose were collected at the mid-exponential growth phase (about 8 -9 A600 n m units), washed three times and suspended at the same absorbance in the derepression buffer (67 mM phosphate pH 6.5, 2% lactate, 0.1 % glucose). As a control, a portion of the cells was suspended in the same buffer Abbreviation. DBM-paper, diazobenzyloximethyl-paper.containing 15% glucose as carbon source. Cultures were shaken vigorously at 28 "C. At various time intervals, samples were withdrawn for cell counting, respiration measurements and mitochondrial RNA (mtRNA) preparations.Respiration was measured with ...

“…The protein/nucleic acid films was picked up with copper grids covered with parlodion films. Further treatments and electron microscopy with a Philips 201 instrument were as described [15].…”

Section: Electron Microscopymentioning

confidence: 99%

Genes coding for the elongation factor EF‐1_α in Artemia

Lenstra

Vliet

Arnberg

et al. 1986

The elongation factor EF-1, is one of the most abundant proteins in eukaryotic cells, where it catalyzes the binding of aminoacyl-tRNA to ribosomes. The genes coding for this protein in the brine shrimp Artemia were analyzed by gene cloning, electron microscopy and chromosomal blot hybridization. There arc only a few (about four) copies of one type of gene per haploid genome. These genes contain five exons divided over lo4 base pairs. Local rearrangements give rise to a number of gene variants. Cross-hybridizations of Artemia cDNA probes with yeast and Drosophila DNA revealed two different yeast EF-1, genes and one or two different Drosophila genes, respectively.Nucleotide sequencing revealed signals for synthesis and processing of EF-1, transcripts as well as the exact location of exons. One interruption in the coding sequence corresponds closely to a splice junction in the gene coding for the homologous chloroplast protein EF-Tu from Euglena gracilis, presumably of prokaryotic origin. The first exon in the chloroplast gene codes for the region of EF-Tu that is homologous to regions of the elongation factor EF-G and of the initiation factor IF2, respectively.During the evolution of the diverse forms of life, basic principles of protein biosynthesis have been conserved. In eukaryotes, the attachment of aminoacyl-tRNA to ribosomes is catalyzed by the elongation factor EF-la, which is homologous to prokaryotic EF-Tu [l]. Both EF-1, and EF-Tu are present in much larger amounts than other protein components of the protein biosynthesis machinery [2, 31. In fact, EF-Tu in Escherichia coli accounts for 5% of the cellular proteins [3, 41, while the amount of EF-1, in eukaryotic cells is comparable to the abundancy of the major cytoskeletal proteins [5 -71.To gain insight into how the active biosynthesis of EF-1, is achieved we describe here the analysis of genomic EF-1, clones and hybridizations of EF-1, cDNA probes to genomic DNA. Previously, we proposed that EF-1, from the brine shrimp Artemia was coded for by between one and four different genes [8] MATERIALS AND METHODS MaterialsArtemia cysts were purchased from Mountain Hartz Corporation (Ontario, Canada). For the isolation of Artemia DNA, free swimming nauplii were homogenized in 4 vol. 10 mM Tris/Cl, 5 mM MgC12, pH 8.0, at 4°C and filtered. Subsequently, glycogen was removed by repeated centrifugation of nuclei through l .5 M sucrose in the same buffer. DNA was purified from nuclei by lysis in 10 mM Tris/Cl, 10 mM EDTA, 2% (w/v) SDS, repeated phenol and phenol/ chloroform extractions, isopropanol precipitation at room temperature, RNase and proteinase K treatment, followed by a second round of phenolic extractions and isopropanol precipitation. Drosophila DNA was a gift of Dr T. J. M.Hulsebos. Nitrocellulose Southern blots of yeast DNA were donated by Dr C. M. T. Molenaar. Construction and screening ojan Artemia gene libraryArtemia DNA (0.5 mg) was digested with 130U MboI (Biolabs) for 2 h and fractionated on a sucrose gradient as described [12]. After dephospho...