Cytoplasmic splicing of tRNA in Saccharomyces cerevisiae

The splicing of nuclear encoded RNAs, including tRNAs, has been widely believed to occur in the nucleus. However, we recently found that one of the tRNA splicing enzymes, splicing endonuclease, is localized to the outer surface of mitochondria in Saccharomyces cerevisiae. These results suggested the unexpected possibility of tRNA splicing in the cytoplasm. To investigate this possibility, we examined whether cytoplasmic pre-tRNAs are bona fide intermediates for tRNA maturation in vivo. We isolated a new reversible allele of temperature-sensitive (ts) sen2 (HA-sen2-42), which encodes a mutant form of one of the catalytic subunits of yeast splicing endonuclease. The HA-sen2-42 cells accumulated large amounts of pre-tRNAs in the cytoplasm at a restrictive temperature, but the pre-tRNAs were diminished when the cells were transferred to a permissive temperature. Using pulse-chase/hybrid-precipitation techniques, we showed that the pre-tRNAs were not degraded but rather converted into mature tRNAs during incubation at the permissive temperature. These and other results indicate that, in S. cerevisiae, pre-tRNAs in the cytoplasm are genuine substrates for splicing, and that the splicing is indeed carried out in the cytoplasm.     

Isolation of a mutant allele that deregulates the threonine biosynthesis in Saccharomyces cerevisiae

We have cloned the yeast allele HOM3-R2, that codes for a mutant aspartate kinase which is insensitive to feedback inhibition by threonine, by gap-repair. A strain carrying this allele in a multicopy plasmid, or integrated into the genome, accumulates 14-times and 8-times more threonine than the wild-type, respectively. The sequence of the mutant allele differs from that of the wild-type in a single base pair change, namely a G by an A, at position 1355 in the open reading frame. The fact that the presence of this mutant allele in a cell induces threonine overproduction points to aspartate kinase as the key enzyme in the regulation of threonine biosynthesis in yeast.     

Regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae

Summary The threonine deaminase gene (ILV1) of Saccharomyces cerevisiae has been designated multifunctional since Bollon (1974) indicated its involvement both in the catalysis of the first step in isoleucine biosynthesis and in the regulation of the isoleucine-valine pathway. Its role in regulation is characterized by a decrease in the activity of the five isoleucine-valine enzymes when cells are grown in the presence of the three branched-chain amino acids, isoleucine, valine and leucine (multivalent repression). We have demonstrated that the regulation of AHA reductoisomerase (encoded by ILV5) and branched-chain amino acid transaminase is unaffected by the deletion of ILV1, subsequently revealing that the two enzymes can be regulated in the absence of threonine deaminase. Both threonine deaminase activity and ILV1 mRNA levels increase in mutants (gcd2 and gcd3) having constitutively derepressed levels of enzymes under the general control of amino acid biosynthesis, as well as in response to starvation for tryptophan and branched-chain amino acid imbalance. Thus, the ILV1 gene is under general amino acid control, as is the case for both the ILV5 and the transaminase gene. Multivalent repression of reductoisomerase and transaminase can be observed in mutants defective in general control (gcn and gcd), whereas this is not the case for threonine deaminase. Our analysis suggests that repression effected by general control is not complete in minimal medium. Amino acid dependent regulation of threonine deaminase is only through general control, while the branched-chain amino acid repression of AHA reducto isomerase and the transaminase is caused both by general control and an amino acid-specific regulation.     

Characterization of a mutation in Saccharomyces cerevisiae that produces mutant isoaccepting tRNAs for several of its tRNA species

Summary Genetic data presented in Bell et al. (1977) demonstrated that a mutation in Saccharomyces cerevisiae (designated mia) is responsible for the production of mutant isoaccepting tRNA molecules for some tRNA species. Besides extending this phenotype to other tRNAs, we have shown that mutant isoacceptors are produced at the expense of the normal levels of wild type tRNA and that the presence of mutant isoacceptors has no adverse effects on strains harbouring the mutation. The observation that mia strains have mutant isoacceptors as the predominant tRNA species under certain growth conditions suggests that mutant isoacceptors are biologically active molecules. Pulse-label and chase experiments indicate that mutant isoacceptors are slowly converted to wild type tRNA molecules in vivo, suggesting that they could be precursor molecules. This is consistent with the hypothesis that mia is defective in a modification process in the maturation of tRNA molecules. Analysis of a double mutant that produces mia isoacceptors which also lack N²-dimethylguanine shows that some of the modifications to tRNA molecules need not follow a specific sequence.     

Identification of programmed translational -1 frameshifting sites in the genome of Saccharomyces cerevisiae

Frameshifting is a recoding event that allows the expression of two polypeptides from the same mRNA molecule. Most recoding events described so far are used by viruses and transposons to express their replicase protein. The very few number of cellular proteins known to be expressed by a -1 ribosomal frameshifting has been identified by chance. The goal of the present work was to set up a systematic strategy, based on complementary bioinformatics, molecular biology, and functional approaches, without a priori knowledge of the mechanism involved. Two independent methods were devised. The first looks for genomic regions in which two ORFs, each carrying a protein pattern, are in a frameshifted arrangement. The second uses Hidden Markov Models and likelihood in a two-step approach. When this strategy was applied to the Saccharomyces cerevisiae genome, 189 candidate regions were found, of which 58 were further functionally investigated. Twenty-eight of them expressed a full-length mRNA covering the two ORFs, and 11 showed a -1 frameshift efficiency varying from 5% to 13% (50-fold higher than background), some of which corresponds to genes with known functions. From other ascomycetes, four frameshifted ORFs are found fully conserved. Strikingly, most of the candidates do not display a classical viral-like frameshift signal and would have escaped a search based on current models of frameshifting. These results strongly suggest that -1 frameshifting might be more widely distributed than previously thought.     

A calcium-dependent ergosterol mutant of Saccharomyces cerevisiae

ERG24 is the structural gene for the C14-sterol reductase in yeast. A lack of activity in that enzyme, mediated either by the morpholine fungicides or the insertional inactivation of ERG24, causes the accumulation of the aberrant sterol ignosterol. Cells producing this sterol are unable to grow aerobically in the routine laboratory medium, YPD. However, growth does occur on a synthetic defined medium. A novel calcium-dependent phenotype associated with alterations in the ergosterol biosynthetic pathway in yeast is described. In addition, reduction of yeast growth with an azole inhibitor of the C-14 sterol de-methylase was also modulated by an excess of calcium ions in the culture medium. These results define a new effect of ergosterol deficiency and provide important practical implications for utilizing morpholine and azole sterol biosynthetic-inhibiting fungicides. Received: 19 February / 25 May 1998     

6-Azauracil inhibition of GTP biosynthesis in Saccharomyces cerevisiae

Summary The addition of 6-azauracil to the growth medium causes a strong reduction of the GTP level in the nucleotide pool of Saccharomyces cerevisiae. In-vitro experiments show a strong inhibition of IMP dehydrogenase activity by 6-azaUMP explaining the preceeding effect. PPR2 mutants, previously characterized by an increased sensitivity to 6-azauracil compared to the wildtype, are specifically susceptible to the lowering of the GTP pool, and are able to grow in presence of 6-azauracil when guanine is added to the medium.     

General and specific controls of lysine biosynthesis in Saccharomyces cerevisiae

Summary Six of the eight enzymes of the -aninoadipate pathway for the biosynthesis of lysine in Saccharomyces cerevisiae were examined for repressibility to lysine and for susceptibility to the general control of amino acid biosynthesis. All of the enzymes exhibited a 2 to 4 fold lower level of specific activity in the wildtype strain X2180 when grown in lysine supplemented medium as compared to minimal medium. However, levels of only three of the enzymes, -aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase, were derepressed in the leaky lysine mutant 7305d and leaky arginine mutant 7853-6c when grown in minimal medium. These observations are characteristic of enzymes under general control of amino acid biosynthesis. The remaining three enzymes, homocitrate synthease, homoaconitase and homoisocitrate dehydrogenase were repressed in 7305d cells grown in minimal or lysine supplemented medium.     

Flow microcalorimetry of a respiration-deficient mutant of Saccharomyces cerevisiae

In aerobic batch cultures in mineral medium with glucose of a respiration-deficient mutant of Saccharomyces cerevisiae, growth parameters were estimated and the heat evolved was measured by a flow microcalorimeter. A growth enthalpy of – 163.6 joule per mole of glucose consumed was measured. Under anaerobic conditions, the value was – 134.6 joule, closer to the expected for alcoholic fermentation alone. The difference was found to be due to cyanide-resistant respiration under aerobic conditions.     

Some aspects of the phenylacetylcarbinol biosynthesis by Saccharomyces cerevisiae

The biosynthesis of phenylacetylcarbinol (PAC) by Saccharomyces cerevisiae in media containing benzaldehyde and fermentable sugars was studied under different experimental conditions. In shaking cultures with small volumes of medium a sigmoidal production curve for PAC was obtained. During the first and second part of the fermentation period (5 to 10 hours), the number of living yeast cells increased. In the third period (from the 10th to the 20th hour), the number of living yeast cells decreased rapidly. In agitated batch cultures the PAC produced during the first 5 hours of fermentation decreased only slowly on further incubation. In agitated but non aerated batch cultures, the PAC production increased rapidly, but the yields were much lower than in aerated batches. Acetone dried cells of baker's yeast produced PAC from benzaldehyde but no benzylalcohol. Besides PAC and acetylbenzoyl, a new biosynthetic product, trans-cinnam-aldehyde was determined.     

The secretion and post translational modification of interferons from Saccharomyces cerevisiae

Summary Studies with three interferon molecules, IFN-₂, IFN- ₁, and a hybrid interferon, IFNX-430 are described which illustrate that both the expression and secretion characteristics of heterologous proteins in yeast cells reflect properties of the proteins themselves.Recombinant DNA techniques have also been used to demonstrate that the efficient processing of mature heterologous proteins from the yeast factor secretion leader can be affected by sequences on the carboxyl side of the initial cleavage site.Secretion studies with heterologous proteins in S. cerevisiae are aimed at maximising yield, the percentage of extracefular product and correct amino terminus sequence. The results presented here show that all three factors are susceptible to currently unpredictable properties of the foreign sequence. This situation, in turn, means that heterologous proteins can be used as tools in the biochemical dissection of the yeast secretion process.     

A galactose-dependent cmd1 mutant of Saccharomyces cerevisiae: involvement of calmodulin in nuclear division

Summary The coding region of a yeast calmodulin gene was fused to a galactose-inducible GAL1 promoter, and a conditional-lethal mutant of Saccharomyces cerevisiae, in which the expression of calmodulin was regulated by galactose, was constructed. The mutant grew normally in galactose medium, but in glucose medium, in which the promoter was repressed, it ceased growing after 12–15 h. The growth arrest was associated with a decrease in intracellular calmodulin levels: after 12h, no intracellular calmodulin protein was detectable. Analysis of the terminal phenotype showed that when the cell stopped growing, it had a bud, a nucleus after S-phase and a short mitotic spindle. Thus, the defect was mainly in nuclear division. Bud growth was partially inhibited in these cells: 27% of the cells stopped growing with a small bud. Furthermore, calmodulin-deficient cells showed elevated rates of chromosome loss, possibly as the result of a defect in the precise segregation of chromosomes.     

A novel mutation that affects utilization of galactose in Saccharomyces cerevisiae

Summary A novel type of regulatory mutation for galactose metabolism in Saccharomyces cerevisiae is described. The mutation named gal11 was recessive, non-allelic to GAL4, GAL80, GAL2, or GAL3, and unlinked to the gene cluster of GAL1, GAL10, and GAL7. It caused a coordinate reduction of galactokinase, galactose-1-P uridylyl transferase, and UDP-glucose 4-epimerase by a factor of more than 5, rendering the mutant cells galactose-nonfermenting. The effect of the mutation was manifested not only in cells grown on galactose but also in cells constitutively synthesizing the galactose-metabolizing enzymes.This work was supported in part by a grant (Project No. 410712) from the Ministry of Education, Culture, and Science or Japan     

The GPI1 homologue from Plasmodium falciparum complements a Saccharomyces cerevisiae GPI1 anchoring mutant

Glycosylphosphatidylinositol (GPI) represents an important anchoring molecule for cell surface proteins. The first step in its synthesis is the transfer of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine to phosphatidylinositol (PI). This chemically simple step is genetically complex because three or four genes are required in both yeast (GPI1, GPI2 and GPI3) and mammals (GPI1, PIG A, PIG H and PIG C), respectively. Here, we report cloning of a Plasmodium falciparum (P. falciparum) homologue of GPI1 (PfGPI1). Analysis showed that P. falciparum Gpi1p is somewhat more similar to the yeast proteins than human Gpi1p, showing 26 and 20% amino acid sequence identity with the Saccharomyces cerevisiae and Homo sapiens proteins, respectively. Multiple sequence alignment demonstrates also that the C-terminal half GPI1 proteins is much better conserved than the N-terminal half. The P. falciparum Gpi1p has a calculated molecular weight of 65 kDa and a predicted potential tyrosine phosphorylation site. The potential tyrosine phosphorylation site seems to occur in all other known Gpi1 proteins. Like the other GPI1 proteins, the predictive software revealed the absence of targeting signals such as organelle transit peptides, DNA binding sites, or N-terminal secretory signals. Hydrophobicity plots revealed multiple hydrophobic regions that could function as transmembrane segments. The cloned P. falciparum GPI1 gene complemented a gpi1 yeast mutant.     

Substrate-induced cAMP signals in wild-type and mutant strains of Saccharomyces cerevisiae

Addition of glucose or other substrates to starved Saccharomyces cerevisiae cells triggers a cyclic AMP signal which induces the protein phosphorylating cascade. Before the addition of various substrates the wild-type and mutant yeast strains were arrested at the G1 phase of the cell division cycle by transferring the cells, grown at 26 degrees C to 36 degrees C in a synthetic medium without any substrate. After the temperature shift back to 26 degrees C different substrates were added and the cAMP levels were measured. The highest cAMP levels were observed immediately after the addition of the substrates. A relationship between the maximum growth rate of the individual strains or mutants at a given substrate and the intracellular cAMP level is discussed.